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https://f1000research.com/articles/2-62/v1
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27 Feb 13
|
{
"type": "Review",
"title": "Critical care management of patients following transcatheter aortic valve replacement",
"authors": [
"Jesse M Raiten",
"Jacob T Gutsche",
"Jiri Horak",
"John GT Augoustides",
"Jacob T Gutsche",
"Jiri Horak",
"John GT Augoustides"
],
"abstract": "Transcatheter aortic valve replacement (TAVR) is rapidly gaining popularity as a technique to surgically manage aortic stenosis (AS) in high risk patients. TAVR is significantly less invasive than the traditional approach to aortic valve replacement via median sternotomy. Patients undergoing TAVR often suffer from multiple comorbidities, and their postoperative course may be complicated by a unique set of complications that may become evident in the intensive care unit (ICU). In this article, we review the common complications of TAVR that may be observed in the ICU, and different strategies for their management.",
"keywords": [
"Transcatheter aortic valve replacement",
"aortic stenosis",
"ICU",
"comorbidity"
],
"content": "Introduction\n\nAortic stenosis (AS) is a common heart valve disorder that affects 2–9% of the population over age 65 worldwide1. Degenerative-calcific changes represent the most common etiology of AS in the elderly population1. The decision of how to treat AS in the elderly depends largely on patient symptoms. In patients who are asymptomatic, despite having severe AS, the risks of surgery outweigh the benefits, and watchful waiting is prudent2. However, the development of symptoms heralds the need for more aggressive treatment, often requiring surgical intervention.\n\nInvasive therapies for AS range from balloon aortic valvuloplasty (BAV) to aortic valve replacement (AVR) via sternotomy or the transcatheter approach. In the case of traditional AVR via sternotomy, the valve may be replaced with a mechanical or bioprosthetic valve, the choice largely depending on patient age and ability to tolerate systemic anticoagulation. Over the past decade, transcatheter aortic valve replacement via the transfemoral or transapical approach (TF-TAVR and TA-TAVR, respectively) has been studied, with interest bolstered by the results of the Placement of Aortic Transcatheter Valve Trial (PARTNER trial)3.\n\nBAV is the least invasive of therapeutic choices once medical management has been exhausted, typically being performed in the cardiac catheterization laboratory. Prior to success with TAVR, BAV was considered a safe and useful option in patients who were deemed too high risk for surgery4. While BAV may improve hemodynamic parameters, a high recurrence rate of valve stenosis limits the utility of the procedure. Long term survival rates are low and complications are common5,6. In the current practice paradigm, BAV may be an appropriate bridge to TAVR in patients who may be suitable candidates but need medical optimization or do not yet meet criteria for a transcatheter procedure7,8.\n\nEvolving medical practices, including advances in surgical, anesthetic, and perioperative management techniques, have reduced morbidity and mortality associated with AVR. Overall, mortality is under 3% for all patients, but may be even lower in selected patient populations with minimal comorbidities9. However, advancing patient age is creating a higher acuity patient population, many of whom are at greater risk for perioperative morbidity and mortality. A recent retrospective review of high risk patients (defined as Society of Thoracic Surgeons predicted risk of mortality of 10% or greater) undergoing isolated, primary AVR, observed an in-hospital mortality rate of 16.4%10. The same study found a postoperative stroke rate of 4.4%, heart block 5%, multisystem organ failure 6.9%, pneumonia 7.5%, and dialysis 8.2%.\n\nIt is clear that traditional AVR carries considerable risk in patients with major comorbidities. In fact, 30–40% of patients with severe AS never undergo surgery due to coexisting medical conditions, heart failure, or physician and/or patient preference9. Patients deemed too sick, or inoperable, are increasingly becoming candidates for TAVR, either by the transfemoral or transapical approach.\n\nThe postoperative period after cardiac surgery may include pain and mental status changes, hemodynamic instability, cardiac arrhythmias, pulmonary edema and respiratory failure, renal failure, and bleeding and coagulopathy. Advances in perioperative and surgical techniques have led to many patients being “fast-tracked”, with rapid extubation and ICU lengths of stay often less than 24 hours. However, as the patient population has become more elderly, morbidity has increased11 and postoperative intensive care unit (ICU) management has become more complex. In the study previously described of high risk patients undergoing primary AVR, the median ICU length of stay was 3 days10.\n\nTAVR is less invasive than traditional AVR – no sternotomy is performed, cardiopulmonary bypass (CPB) is not necessary, and patients may be extubated in the operating room (OR). Despite its less invasive nature, over the past few years as the number of TAVRs have increased, a unique set of postoperative events and complications have been identified. While some ICU management issues are shared with patients undergoing traditional AVR, TAVR patients are predisposed to ICU concerns that the intensivist needs to recognize and manage appropriately.\n\n\nNeurological issues in the ICU\n\nIn the PARTNER trial, the average age at time of surgery was 83 years3. It is well known that advanced age is a major risk factor for postoperative delirium (POD) after cardiac surgery. POD was not assessed in the original PARTNER trial and there is minimal data about its incidence. A small retrospective chart review found a delirium rate of 51% after TA-TAVR and 16% after TF-TAVR12. In this study, TA-TAVR was associated with a significantly longer ICU length of stay (84 hours) compared to the transfemoral approach (36 hours). ICU length of stay is also an established risk factor for delirium.\n\nPOD is associated with poor outcomes including increased hospital length of stay, increased mortality, and greater nursing home placement13. It is also responsible for a significant financial toll in the ICU14. Given the negative consequences of ICU delirium, it is critical to quickly identify and manage through both pharmacotherapy and other interventions. While delirium may present with agitation or behavior causing self harm, hypoactive delirium is more common and is easier to misdiagnose14. By virtue of their older age, comorbidities, and mandatory ICU courses, patients undergoing TAVR are high risk and should be screened for delirium and managed accordingly. There are multiple screening tools for delirium, including the Confusion Assessment Method for the ICU (CAM-ICU), and the Intensive Care Delirium Screening Checklist (ICDSC). A patient’s CAM-ICU score may be easily calculated and a patient designated as “CAM positive” if delirium is present, or “CAM negative” if they do not exhibit signs of delirium. Some ICUs, such as those at the University of Pennsylvania (Philadelphia, PA, USA), have implemented a nursing-driven protocol whereby every patient in the ICU is assessed daily for delirium as part of the daily nursing assessment. This allows early identification of the delirious patient and rapid intervention.\n\nAntipsychotic drugs that antagonize dopamine receptors in the central nervous system are the mainstay of pharmacotherapy for ICU delirium. While haloperidol is a typical antipsychotic with a proven success record for managing delirium, newer atypical antipsychotic drugs such as quetiapine (Seroquel) and olanzapine (Zyprexa) have a lower incidence of extrapyramidal side effects15. Of particular concern in the TAVR patient population, both the typical and atypical antipsychotics may be associated with a prolonged QT interval on the electrocardiogram, and an increased risk for cardiac arrhythmias. Risk factors for QT interval prolongation include female gender, polypharmacotherapy, cardiovascular disease and bradycardia, and electrolyte disorders15. Unfortunately, these risk factors have a high prevalence in patients undergoing cardiac surgery.\n\nAlthough at least one study in non-cardiac surgery patients has demonstrated a benefit to prophylactically treating patients at high risk for delirium16, prophylactic pharmacotherapy to prevent delirium is not the standard of care in most hospitals. Maneuvers to reduce the risk of delirium, including constant orientation to time and location and maintaining a normal sleep-wake cycle should always be performed in at-risk patients in the ICU. Antipsychotic medication is usually initiated if signs of delirium develop.\n\nPost-operative pain after TAVR may be substantial, particularly after TA-TAVR. While TF-TAVR may be performed entirely via the femoral vessels, TA-TAVR requires a thoracotomy incision. For thoracic surgery procedures requiring thoracotomy, epidural analgesia is the current standard of care. There are numerous reports of using thoracic epidurals for cardiac surgery, with improved outcomes in terms of faster time to extubation, better postoperative analgesia, faster recovery of pulmonary function, improved participation in physiotherapy, and less depression17–19.\n\nThere are few descriptions of using thoracic epidurals for TA-TAVR. In a study of 135 patients who underwent TA-TAVR, the use of general anesthesia and thoracic epidural, compared to GA and intercostal nerve block, was associated with a significant decrease in pulmonary complications (including reintubation postoperatively), and a lower 30 day mortality in the epidural group20. One case report describes the use of epidural anesthesia for TA-TAVR in a patient with severe obstructive lung disease21. The patient was awake throughout the procedure and transferred to the post-anesthetic care unit and step-down unit postoperatively, bypassing the ICU entirely. TF-TAVR has also been performed in awake patients – a comparison of GA versus local/regional anesthesia with ileoinguinal-iliohypogastric blocks found local/regional anesthesia was associated with a shorter operative procedure and hospital length of stay22. The rate of complications was not significantly different between groups.\n\nWhile the minimal data evaluating epidural use during TAVR seems to be associated with favorable outcomes, the risks most notably include bleeding and epidural hematoma formation. Although patients are systemically anticoagulated intraoperatively, the degree of anticoagulation is generally less than that required for CPB. As mentioned above, there are numerous reports of the safe use of epidurals in patients anticoagulated for CPB. The use of other neuraxial monitors, specifically lumbar drains, are routinely employed in patients undergoing thoracoabdominal aneurysm repair, in which a high degree of systemic anticoagulation is necessary.\n\nStroke is probably the most feared neurological complication following aortic valve surgery. The incidence of stroke after traditional AVR in all patients is approximately 1.6%23, but may be greater in high risk patients and those with previous coronary artery bypass surgery24. Stroke is a major source of morbidity in patients following TAVR, with an incidence of 2.4–9.1% after TF-TAVR and 1.5–6.7% after TA-TAVR24. In a recent study of 31 patients who underwent TAVR, diffusion-weighted MRI identified new cerebral infarcts in 77% of patients postoperatively25. The authors identified increased severity of aortic atheroma as a risk factor for new cerebral infarcts.\n\nNeurologic events following TAVR peak in the first week postoperatively. In an analysis of patients enrolled in the PARTNER trial, 12/31 events (stroke or TIA) occurred between postoperative days 0–224. In a study of 253 patients who underwent TAVR (who were not part of the PARTNER trial), the risk of stroke or TIA was greatest in the first 24 hours after surgery26.\n\nWhile perioperative and intraoperative hypotension may be responsible, neurologic events occurring in the first 24 hours are likely related to embolization of calcium and debris, or thrombi that may form on wires and surgical devices intraoperatively24,26. Indeed, a smaller aortic valve area index is associated with a greater risk of early stroke, possibly due to increased propensity for calcium embolization24. New devices are in development to reduce the incidence of cerebral emboli, including the SMT Embolic Deflection Device, which acts as a filter in the aortic arch, reducing emboli to the brain27.\n\nInfarcts affecting the distribution of the middle cerebral artery, the posterior circulation, or involving multiple sites are often embolic in nature, and are characteristic of the cerebral infarcts seen immediately after TAVR26. We believe knowledge of the clinical presentation of embolism to these vessels is critical in order to rapidly diagnose and treat any events. In recognition of the risk of embolic stroke, in the absence of significant postoperative bleeding, anticoagulation is usually initiated on postoperative day 1. Dual antiplatelet therapy (aspirin and clopidogrel) may be used, or aspirin and warfarin if a patient is already taking warfarin for concomitant atrial fibrillation. Although there are no absolute guidelines, anticoagulation is often continued for several months following surgery.\n\n\nCardiac issues in the ICU\n\nPreexisting cardiac conduction system disease is common in patients undergoing AVR, and in one study it was identified in 23% of patients preoperatively28. Baseline conduction abnormalities (particularly bundle branch blocks (BBB)) have been identified as a major risk factor for post-procedural permanent pace maker (PPM)28,29. Depending on the type of valve (Sapien versus CoreValve), post TAVR PPM is reported in 1.8–8.5%, and 19.1–42.5%, respectively9. Anatomical compression on the conduction system by the prosthesis is likely a major contributing factor to postoperative conduction defects.\n\nNew onset atrial fibrillation (NOAF) after TAVR is also common and was identified in 31.9% of patients in one prospective study, at a median time of 48 hours postoperatively30. NOAF was more common with a larger left atrial size and with TA-TAVR. While associated with a higher risk of stroke, NOAF did not increase mortality in this study.\n\nCardiac pacing wires are used intraoperatively to allow rapid cardiac pacing during valve deployment. These wires may be left in place afterwards in patients who experience heart block during the procedure or who have risk factors for arrhythmias postoperatively (preexisting BBB, large left atrial size). While patients who develop heart block during the procedure may be paced in the ICU until PPM can be placed, a backup mode may be useful in patients at risk for arrhythmias. Avoidance of negative chronotropes (beta blockers, digoxin, etc) is prudent in patients with preexisting BBB at risk for worsening arrhythmias.\n\nWhile cardiac complications are common in patients undergoing TAVR, the vast majority are intraoperative events that are diagnosed and treated before the patient arrives in the ICU. Complications seen intraoperatively include coronary artery occlusion from malposition of the graft, myocardial infarction, tamponade, rupture of aortic root or annulus, mitral valve apparatus injury, excessive bleeding, valve migration, and paravalvular leak. Signs of apical myocardial infarction may be seen on electrocardiograms postoperatively and are often related to apical puncture to facilitate valve placement in TA-TAVR.\n\nLabile hemodynamics are common in the immediate postoperative period following TAVR. Left ventricular hypertrophy and diastolic heart disease often make patients very volume responsive, and hypotension is often responsive to volume resuscitation. Hypertension that is not pain related may be managed with nicardipine, a rapidly titratable calcium channel blocker. While there are no well established guidelines for BP management postoperatively, we believe targeting a mean arterial pressure of 60–80 is reasonable.\n\n\nPulmonary considerations in the ICU\n\nPatients undergoing TAVR are considered inoperable by traditional criteria. Coexisting pulmonary disease, most notably chronic obstructive pulmonary disease (COPD), is common in this patient population and was present in 41% of patients randomized to TAVR in the original PARTNER trial3. Of note, 21% of patients had oxygen dependent COPD. The combination of severe lung disease, postoperative pain from sternotomy, and prolonged time under anesthesia in patients undergoing traditional AVR may contribute to difficulty with ventilator weaning in the ICU. In contrast, we believe many patients undergoing TAVR may be fast tracked and extubated at the end of the procedure, appreciable largely to the absence of sternotomy and less postoperative pain, shorter surgical times and less exposure to anesthesia.\n\nThe need for reintubation in the postoperative period may be related to two primary factors-pain, particularly following TA-TAVR, and pulmonary edema. LVH and diastolic heart disease that often accompanies AS may necessitate significant volume resuscitation to maintain stable hemodynamics. As this volume equilibrates in the early postoperative period, pulmonary edema and effusions may develop. This fluid buildup, particularly pleural effusions, may be more clinically significant than following traditional AVR, as chest tubes are not usually placed for TF-TAVR, and only one may be present after the TA-TAVR. Thoracentesis to remove pleural fluid may be necessary as the need for higher cardiac filling pressures to maintain hemodynamics may prevent aggressive diuresis.\n\n\nRenal failure in the ICU\n\nGiven the high acuity of patients undergoing TAVR, it is not surprising that a significant percentage suffer from renal insufficiency. Five percent of patients randomized to TAVR in the PARTNER trial had a baseline creatinine > 2mg/dL3, and almost 50% of patients in another series had preoperative renal failure31. The requirement for intra-arterial contrast medium predisposes patients to acute (ARF), or acute on chronic renal failure postoperatively. In a series of 110 TAVR patients, new onset post-procedure acute kidney injury (AKI) was found in 9% of patients, while 35% of patients with preexisting renal insufficiency experienced acute on chronic failure31. In the majority of cases, ARF was mild and transient, with only one patient requiring hemodialysis. Perioperative red blood cell transfusion has been associated with AKI in TAVR patients32.\n\nNumerous studies have sought to identify ways to prevent and treat contrast-induced nephropathy (CIN). Techniques include ensuring excellent perfusion pressure, administration of N-acetylcysteine, hydration with normal saline or sodium bicarbonate, and post-procedure hemofiltration. While the data is plentiful on the topic, the results are inconsistent regarding the optimal preventive strategy for CIN. Aggressive hydration with sodium bicarbonate carries risks of pulmonary edema and hypercarbia, particularly in the TAVR patient population where heart failure and COPD are common. In patients with significant cardiopulmonary comorbidities, avoiding hypotension and minimizing contrast exposure may be the most prudent ways to decrease the risk of CIN.\n\n\nVascular access complications\n\nVascular complications are common after TAVR and occurred in 30% of patients in the PARTNER trial (16% were major complications)3. In a prospective study of 130 TF-TAVR patients, vascular complications were predicted by center experience, femoral calcification, and the sheath to femoral artery ratio score (SFAR)33. In a review of 101 patients undergoing TF-TAVR, vascular access complications occurred in 32%, and 10% required surgical repair34. Vascular complications include retroperitoneal hemorrhage, femoral or iliac artery dissection, and development of a femoral pseudoaneurysm.\n\nSome complications may become apparent intraoperatively. However, initial recognition of a vascular access complication is often detected in the ICU postoperatively. Proper techniques must be ensured with removal of any femoral arterial sheath. Pressure at the puncture site must be held for an adequate length of time (usually 3–5 minutes for each French size of the catheter), in order to achieve hemostasis. Inadequate pressure can result in pseudoaneurysm or hematoma formation. At our institutions, we remove femoral access sheaths in the OR before the patient is transported to the ICU.\n\nThe presence of a high femoral arterial stick (above the inguinal ligament) may first present in the ICU with the development of a retroperitoneal bleed when the line is removed, despite adequate pressure being held. We believe hypotension presenting in the hours after arterial line removal should trigger a rapid workup for possible retroperitoneal hemorrhage. Non-contrast computed tomography (CT scan) is the study of choice to identify a retroperitoneal bleed, but in the presence of unstable hemodynamics, abdominal tenderness and a newly removed formal arterial catheter, it may be prudent to proceed directly to the OR for surgical exploration.\n\nIn a retrospective review of more than 9000 patients undergoing femoral artery catheterizations, presenting signs of retroperitoneal hematoma included suprainguinal tenderness in 100% of patients, severe back pain in 64%, and femoral neuropathy in 36%35. The diagnosis may be more difficult if the patient is sedated or mechanically ventilated, and a high index of suspicion is needed.\n\n\nConclusion\n\nTAVR is an innovative method to treat aortic valve disease in high risk patients. Its minimally invasive nature eliminates the need for sternotomy, CPB, and reduces procedural and anesthesia time. Nevertheless, TAVR is a major surgical procedure with considerable morbidity and mortality, and intensivists caring postoperatively for these patients must be able to treat the immediate postoperative complications. Prompt recognition of postoperative neurologic events, cardiac arrhythmias, renal failure, vascular complications and hemorrhage are critical to improve patient safety and outcomes.",
"appendix": "Author contributions\n\n\n\nAll authors participated in the writing, research, and preparation of this article, in the initial and all stages of preparation.\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\nFaggiano P, Antonini-Canterin F, Baldessin F, et al.: Epidemiology and cardiovascular risk factors of aortic stenosis. Cardiovasc Ultrasound. 2006; 4: 27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDal-Bianco JP, Khandheria BK, Mookadam F, et al.: Management of asymptomatic severe aortic stenosis. J Am Coll Cardiol. 2008; 52(16): 1279–92. PubMed Abstract | Publisher Full Text\n\nLeon MB, Smith CR, Mack M, et al.: PARTNER Trial Investigators. Transcatheter aortic-valve implantation for aortic stenosis in patients who cannot undergo surgery. N Engl J Med. 2010; 363(17): 1597–607. PubMed Abstract | Publisher Full Text\n\nEltchaninoff H, Cribier A, Tron C, et al.: Balloon aortic valvuloplasty in elderly patients at high risk for surgery, or inoperable. Immediate and mid-term results. Eur Heart J. 1995; 16(8): 1079–84. PubMed Abstract\n\nBen-Dor I, Pichard AD, Satler LF, et al.: Complications and outcome of balloon aortic valvuloplasty in high-risk or inoperable patients. JACC Cardiovasc Interv. 2010; 3(11): 1150–6. PubMed Abstract | Publisher Full Text\n\nOtto CM, Mickel MC, Kennedy JW, et al.: Three-year outcome after balloon aortic valvuloplasty. Insights into prognosis of valvular aortic stenosis. Circulation. 1994; 89(2): 642–50. PubMed Abstract | Publisher Full Text\n\nSaia F, Marrozzini C, Moretti C, et al.: The role of percutaneous balloon aortic valvuloplasty as a bridge for transcatheter aortic valve implantation. EuroIntervention. 2011; 7(6): 723–9. PubMed Abstract\n\nDaly MJ, Monaghan M, Hamilton A, et al.: Short-term efficacy of palliative balloon aortic valvuloplasty in selected patients with high operative risk. J Invasive Cardiol. 2012; 24(2): 58–62. PubMed Abstract\n\nHolmes DR Jr, Mack MJ, Kaul S, et al.: 2012 ACCF/AATS/SCAI/STS expert consensus document on transcatheter aortic valve replacement. J Am Coll Cardiol. 2012; 59(13): 1200–54. PubMed Abstract | Publisher Full Text\n\nThourani VH, Ailawadi G, Szeto WY, et al.: Outcomes of surgical aortic valve replacement in high-risk patients: a multiinstitutional study. Ann Thorac Surg. 2011; 91(1): 49–55. PubMed Abstract | Publisher Full Text\n\nEttema RG, Peelen LM, Schuurmans MJ, et al.: Prediction models for prolonged intensive care unit stay after cardiac surgery: systematic review and validation study. Circulation. 2010; 122(7): 682–9. PubMed Abstract | Publisher Full Text\n\nTse L: Delirium After Transcatheter Aortic Valve Implantation: A Retrospective Chart Review of Associated Risk Factors and Outcomes. (Masters Thesis, University of British Columbia, Canada) 2011. Reference Source\n\nSieber FE: Postoperative delirium in the elderly surgical patient. Anesthesiol Clin. 2009; 27(3): 451–64. PubMed Abstract | Publisher Full Text\n\nPun BT, Ely EW: The importance of diagnosing and managing ICU delirium. Chest. 2007; 132(2): 624–36. PubMed Abstract | Publisher Full Text\n\nCha DS, McIntyre RS: Treatment-emergent adverse events associated with atypical antipsychotics. Expert Opin Pharmacother. 2012; 13(11): 1587–98. PubMed Abstract | Publisher Full Text\n\nWang W, Li HL, Wang DX, et al.: Haloperidol prophylaxis decreases delirium incidence in elderly patients after noncardiac surgery: a randomized controlled trial. Crit Care Med. 2012; 40(3): 731–9. PubMed Abstract | Publisher Full Text\n\nEl-Morsy GZ, El-Deeb A: The outcome of thoracic epidural anesthesia in elderly patients undergoing coronary artery bypass graft surgery. Saudi J Anaesth. 2012; 6(1): 16–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMehta Y, Vats M, Sharma M, et al.: Thoracic epidural analgesia for off-pump coronary artery bypass surgery in patients with chronic obstructive pulmonary disease. Ann Card Anaesth. 2010; 13(3): 224–30. PubMed Abstract | Publisher Full Text\n\nRoyse C, Royse A, Soeding P, et al.: Prospective randomized trial of high thoracic epidural analgesia for coronary artery bypass surgery. Ann Thorac Surg. 2003; 75(1): 93–100. PubMed Abstract\n\nDumont E, Doyle D, Villeneuve J, et al.: Effect of epidural analgesia on outcomes in patients undergoing transapical transcatheter aortic valve implantation. Reference Source\n\nMukherjee C, Walther T, Borger MA, et al.: Awake transapical aortic valve implantation using thoracic epidural anesthesia. Ann Thorac Surg. 2009; 88(3): 992–4. PubMed Abstract | Publisher Full Text\n\nDehédin B, Guinot PG, Ibrahim H, et al.: Anesthesia and perioperative management of patients who undergo transfemoral transcatheter aortic valve implantation: an observational study of general versus local/regional anesthesia in 125 consecutive patients. J Cardiothorac Vasc Anesth. 2011; 25(6): 1036–43. PubMed Abstract | Publisher Full Text\n\nO'Brien SM, Shahian DM, Filardo G, et al.: The Society of Thoracic Surgeons 2008 cardiac surgery risk models: part 2-isolated valve surgery. Ann Thorac Surg. 2009; 88(1 Suppl): S23–42. PubMed Abstract | Publisher Full Text\n\nMiller DC, Blackstone EH, Mack MJ, et al.: Transcatheter (TAVR) versus surgical (AVR) aortic valve replacement: Occurrence, hazard, risk factors, and consequences of neurologic events in the PARTNER trial. J Thorac Cardiovasc Surg. 2012; 143(4): 832–843. PubMed Abstract | Publisher Full Text\n\nFairbairn TA, Mather AN, Bijsterveld P, et al.: Diffusion-weighted MRI determined cerebral embolic infarction following transcatheter aortic valve implantation: assessment of predictive risk factors and the relationship to subsequent health status. Heart. 2012; 98(1): 18–23. PubMed Abstract | Publisher Full Text\n\nTay EL, Gurvitch R, Wijesinghe N, et al.: A high-risk period for cerebrovascular events exists after transcatheter aortic valve implantation. JACC Cardiovasc Interv. 2011; 4(12): 1290–7. PubMed Abstract | Publisher Full Text\n\nOnsea K, Agostoni P, Samim M, et al.: First-in-man experience with a new embolic deflection device in transcatheter aortic valve interventions. EuroIntervention. 2012; 8(1): 51–6. PubMed Abstract | Publisher Full Text\n\nDawkins S, Hobson AR, Kalra PR, et al.: Permanent pacemaker implantation after isolated aortic valve replacement: incidence, indications, and predictors. Ann Thorac Surg. 2008; 85(1): 108–12. PubMed Abstract | Publisher Full Text\n\nBagur R, Manazzoni JM, Dumont É, et al.: Permanent pacemaker implantation following isolated aortic valve replacement in a large cohort of elderly patients with severe aortic stenosis. Heart. 2011; 97(20): 1687–94. PubMed Abstract | Publisher Full Text\n\nAmat Santos IJ, Rodés Cabau J, Urena M, et al.: Incidence, predictive factors, and prognostic value of new-onset atrial fibrillation following transcatheter aortic valve implantation. J Am Coll Cardiol. 2012; 59(2): 178–88. PubMed Abstract | Publisher Full Text\n\nUssia GP, Scarabelli M, Mulè M, et al.: Postprocedural management of patients after transcatheter aortic valve implantation procedure with self-expanding bioprosthesis. Catheter Cardiovasc Interv. 2010; 76(5): 757–66. PubMed Abstract | Publisher Full Text\n\nNuis RJ, Piazza N, Van Mieghem NM, et al.: In-hospital complications after transcatheter aortic valve implantation revisited according to the Valve Academic Research Consortium definitions. Catheter Cardiovasc Interv. 2011; 78(3): 457–67. PubMed Abstract | Publisher Full Text\n\nHayashida K, Lefevre T, Chevalier B, et al.: Transfemoral aortic valve implantation new criteria to predict vascular complications. JACC Cardiovasc Interv. 2011; 4(8): 851–8. PubMed Abstract | Publisher Full Text\n\nKahlert P, Al-Rashid F, Weber M, et al.: Vascular access site complications after percutaneous transfemoral aortic valve implantation. Herz. 2009; 34(5): 398–408. PubMed Abstract | Publisher Full Text\n\nKent KC, Moscucci M, Mansour KA, et al.: Retroperitoneal hematoma after cardiac catheterization: prevalence, risk factors, and optimal management. J Vasc Surg. 1994; 20(6): 905–10.PubMed Abstract"
}
|
[
{
"id": "803",
"date": "04 Mar 2013",
"name": "Hynek Riha",
"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 review paper covering all important issues encountered during postoperative course after transcatheter aortic valve replacement (TAVR). The review is important as the number of these procedures performed worldwide is quickly increasing. Management strategies after TAVR described in the paper are supported by recent references.",
"responses": []
},
{
"id": "804",
"date": "04 Mar 2013",
"name": "Hannah Wunsch",
"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 review article addresses the post-operative management of a relatively new technique for valve replacement. The topic is important and the authors nicely review the range of possible post-operative complications.I have a few small concerns. The first is that the majority of the topics covered are applicable to all cardiac surgery patients. I think it would be helpful for the authors to highlight better which are the issues that are truly specific to these patients (i.e. retroperitoneal hematoma, and possible management of severe COPD) and which are general to valve surgery (e.g. risk of stroke).Second, there are a number of statements that should either be changed, or referenced:-There should be references for the two delirium screening systems described. -The authors state that haloperidol has a “proven success record for mgmt. of delirium”. I am not aware of good studies that prove this; it should either be referenced or removed.-Studies on prophylaxis for delirium are conflicted. The statement in the paper implies that prophylaxis should be standard of care in hospitals. I suggest tempering this statement.-“Epidural anesthesia is the current standard of care” either should be referenced or removed, as, again, this implies that physicians who are not supplying epidurals to these patients are failing to provide appropriate care.-“Dual antiplatelet therapy (aspirin and clopidogrel) may be used, or aspirin and warfarin if a patient is already taking warfarin for concomitant atrial fibrillation” should be referenced, or it should be made clear that there are no guidelines and this is purely the opinion of the authors.-“We believe targeting a mean arterial pressure of 60–80 is reasonable,” would also benefit from a reference, or perhaps include some additional caveat regarding taking into account a patient’s preoperative blood pressure. -Second paragraph on renal failure – data “are” plural. Currently says “data is”",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-62
|
https://f1000research.com/articles/2-61/v1
|
25 Feb 13
|
{
"type": "Research Article",
"title": "Effect of postoperative anemia on functional outcome and quality of life after hip and knee arthroplasties: a long term follow-up",
"authors": [
"Alex Cormier-Lavoie",
"Monique Ruel",
"Marie-Pierre Sylvestre",
"Jean-François Hardy",
"Alex Cormier-Lavoie",
"Monique Ruel",
"Marie-Pierre Sylvestre"
],
"abstract": "Background: Postoperative anemia is frequent in patients undergoing hip and knee arthroplasty. While it is legitimate to think that anemia could decrease postoperative vigor and, consequently, limit the patient’s rehabilitation, our previous study showed that anemia does not impair functional recovery in patients during the immediate postoperative period (10 days). Here we investigate the possible relationship between the postoperative hemoglobin (Hb) concentration and long-term (6 months or more) functional recovery and quality of life (QoL) in patients.Study design and methods: A follow-up, observational study was conducted in the 305 patients 60 years and older who underwent major hip or knee arthroplasty and participated in the Transfusion Requirements in Orthopedic Surgery (TRIOS) study (phase 2). The relationship between postoperative Hb concentration (or variation thereof) and primary outcomes (Functional Status Index (FSI) score, scores in the two categories of the Short Form 36 (SF-36) test and adverse events) was established by linear regression.Results: 160 patients responded to long-term follow-up. There were no significant correlations between the postoperative Hb concentration (or the variation in perioperative Hb) and either the FSI or SF-36 scores or adverse events. Consequently, moderate postoperative anemia does not appear to affect long-term (6 months or more after surgery) functional recovery or QoL in patients undergoing a major arthroplasty.Conclusion: Our results confirm the lack of longer-term effects of anemia on functional recovery observed in the immediate postoperative period in the TRIOS phase 2 study.",
"keywords": [
"postoperative anemia",
"arthroplasties"
],
"content": "Introduction\n\nImmediate postoperative anemia is common in patients who undergo a major hip or knee arthroplasty1. Consequently, clinicians have to decide whether or not a patient should receive a transfusion to counter postoperative anemia that may affect recovery. However, opinions are divided regarding the true need for, and benefit of, transfusions after major orthopedic surgeries. Several studies have suggested that the risk of postoperative morbidity is higher in patients with cardiovascular disease or those with a major illness and anemia after surgery2,3. These results prompted physicians to adopt a more liberal transfusion strategy by regularly transfusing patients to hemoglobin (Hb) concentrations higher than 100 g/L4.\n\nIn contrast, a more conservative transfusion strategy is chosen by many clinicians, including those of the Centre Hospitalier de l’Université de Montréal (CHUM)5, due to the fact that patients are subject to frequent microcirculatory or immunosuppressive complications after a blood transfusion4. Furthermore, the risk of blood-transmitted pathogens (known and unknown) and the limited availability of blood products renders the liberal approach less attractive. A restrictive approach intended to limit transfusions only to patients with an Hb level lower than 70 g/L is, at the moment, favored considering these potential risks associated with blood transfusions5. An important randomized study including 838 intensive care patients showed that the use of a transfusion threshold of 70 g/L (with the aim of keeping the concentration of Hb between 70 and 90 g/L) was at least as safe as the use of a liberal strategy with a transfusion threshold of 100 g/L4. The 30 day postoperative death rate and the rates of cardiovascular complications and organ dysfunction were not significantly different with a restrictive strategy (and were even significantly lower in some subgroups), suggesting that patients who undergo surgery are able to tolerate Hb levels much lower than those previously admitted. According to these results, it seems that the use of a restrictive transfusion strategy does not affect mortality or morbidity in the immediate postoperative period (30 days after surgery). In addition, as described in the meta-analysis by Alvarez et al 2001, many studies have shown that the human body can adapt to lower [Hb] by increasing cardiac output and oxygen extraction along with a decrease in blood viscosity6. These adaptive changes could explain the similar results observed when using restrictive and liberal transfusion strategies.\n\nThe results of the recent Transfusion Requirements in Orthopedic Surgery (TRIOS) phase 2 trial also support a restrictive transfusion strategy7. They suggest, contrary to our initial hypothesis, that anemia does not affect functional recovery and quality of life (QoL) in patients immediately (1 to 10 days) after their surgery. Here we ask whether the lack of effects of anemia observed in our patients during the immediate postoperative period persists in the long-term period (6 months or more after surgery).\n\nThe objective of the present study was to establish the relationship between the postoperative Hb concentration, and functional recovery and QoL in the long-term after a major arthroplasty. Should there be a relationship between anemia and these two outcomes, we would be able to determine a minimal Hb level below which long-term functional recovery and QoL is affected. Should it exist, this threshold could help clinicians and patients decide between a liberal or a restrictive transfusion strategy after major orthopedic surgery.\n\nWe hypothesized that patients with a lower postoperative Hb concentration have a diminished functional recovery and QoL in the long-term after surgery, compared to those who have higher postoperative Hb concentration.\n\n\nMaterials and methods\n\nWe conducted a follow-up, observational study at the CHUM – Hôpital Notre-Dame to evaluate the long-term postoperative evolution of the 305 patients who participated in TRIOS phase 2 trial. The inclusion criteria of the initial TRIOS phase 2 trial were: being over the age of 60 at the time of surgery, candidacy for a knee or hip arthroplasty or a non-urgent prosthesis revision, the ability to walk prior to surgery and to provide informed consent to participate in the study. Additionally, an exclusion criterion implemented in this present follow-up study was that patients should not have had another surgery since the one for which they had been recruited originally. The protocol was evaluated and accepted by the ethics committee of the CHUM. Each patient was sent a letter inviting him/her to participate in the follow-up study by phoning one of the investigators (ACL). Verbal consent from each of the 160 patients who agreed to participate was obtained prior to completing the interview by telephone.\n\nFunctional recovery was measured with the Functional Status Index (FSI), a reliable and valid instrument allowing auto-evaluation of physical function in adults8. This tool informs the clinician on three aspects of the physical function of the patient during a given activity. These three aspects include the need for assistance, the level of pain and the difficulty of performing a given activity. The sum of the results of these three aspects gives a global score describing how a patient perceives his or her physical condition. A high FSI score corresponds to a poor functional status9. Scores range from 54 for fully autonomous patients to 234 for patients experiencing a weak recovery.\n\nEqually, the Short Form 36 (SF-36) was used to evaluate the QoL of patients10. This test provides a physical components score (PCS) and a mental components score (MCS) that evaluate these two functions. Scores range from 0 – 100, with higher scores indicating better health. On the physical components score, low scores indicate many limitations in physical activities; on the mental components score, low scores indicate problems with work or other daily activities as a result of emotional health10.\n\nAt the same time, patients were asked about complications or morbidities associated with postoperative anemia such as: cardiovascular, respiratory or neurological adverse events, major infections and non-specific symptoms of anemia (dizziness, weakness and fatigue). Hb concentrations were recorded on several occasions during the patient’s stay at the hospital during the initial TRIOS phase 2 trial. We selected two of these recordings (taken during the preoperative medical consultation and after surgery, at the time of the 6 Minute Walk Test11,12) for each patient to establish correlations with long-term postoperative recovery.\n\nEach patient’s file was consulted to obtain details on his/her general recovery, postoperative morbidities or mortality using surgery and physiotherapy notes. All this information was noted in a database.\n\nFor the evaluation of functional recovery and QoL using the FSI and the SF-36, each patient was asked to complete these questionnaires during a telephone interview. If a patient had not called back within 10 days after receiving the letter, a member of the team (ACL) called the patient to obtain his/her consent to participate in the study.\n\nDuring the phone call to the patient, a member of the team (ACL) would read every question and note the patient’s answers. Data for FSI, SF-36 and postoperative complications were all obtained during the same phone call. Time from the original TRIOS phase 2 trial to follow-up ranged between 6 months to 3 years. Twenty six to 36 patients completed the questionnaire each week during 7 consecutive weeks, making a total of 160 respondents.\n\nSF-36 scores (PCS and MCS) were calculated using QualityMetric Inc. SF Health Outcomes™ Scoring Software (Lincoln, RI http://www.qualitymetric.com).\n\nData were analyzed in order to determine a Hb concentration below which functional recovery and QoL in patients are impaired. Relationships between Hb concentrations and FSI or SF-36 scores were studied by linear regression using two main models. The first investigated scores in association with postoperative Hb concentration and the second investigated scores in association with individual changes between the preoperative and postoperative [Hb] recordings. An eventual non-linear effect of postoperative Hb on FSI scores was also verified. A p value < 0.05 was considered statistically significant. All analyses were completed using LaTeX and R version 2.13.0 software by the CHUM’s statistician (MPS).\n\n\nResults\n\nFrom February to March 2011, the 305 patients who participated in the TRIOS phase 2 were asked to contact a member of the research team (ACL) to complete FSI and SF-36 questionnaires. Among those 305 patients, 184 were available and accepted to take part in the study at the moment of the phone call but 24 of them were excluded because they had had another surgery since their original operation. As a result, the information of 160 patients was considered.\n\nTable 1 presents the main demographic characteristics of the 160 patients who participated in the follow-up study. The average age of the patients was 72.4 years (range 60 – 91) and 64% of them were women. The average patient’s body mass index (BMI) was 30.5 kg/m2 (range 16.2 – 51.5) and the average (range 1 – 3) American Society of Anesthesiologists’ (ASA) physical status score was 2 (patients with a systemic pathology that does not limit his/her activities) in the majority of patients (61.3%). Eighty-two patients underwent a total knee arthroplasty (TKA) and 78 underwent a total hip arthroplasty (THA).\n\n* Data are reported as mean ± SD for continuous variables and as number (%) for categorical variables. ASA = preoperative physical status according to the American Society of Anesthesiologists; BMI = body mass index.\n\nTable 2 shows average preoperative and postoperative (1 to 10 days after surgery – average 4.6 days) Hb concentrations, average FSI and SF-36 scores and the number of patients who had major complications after surgery. There was a 36.2 g/L difference between mean preoperative and postoperative Hb concentrations. The average postoperative Hb concentration (97.6 ± 12.0 g/L) was below the threshold for anemia as defined by the World Health Organization (120 g/L in women and 130 g/L in men). The percentage of patients who had adverse events of any type (cardiovascular, respiratory or neurological adverse events, major infections and non-specific symptoms of anemia - dizziness, weakness and fatigue) was low, ranging between 1.9 to 8.1%.\n\nData are reported as mean ± SD for continuous variables and as number (%) for categorical variables. *FSI = Functional Status Index; PCS = Physical Component Summary score; MCS = Mental Component Summary score; SF-36 = Short Form 36 quality of life assessment.\n\nTable 3 presents the relationship between the postoperative Hb concentration and each patient’s long-term primary outcome scores. There were no significant associations between postoperative [Hb] and FSI, PCS or MCS. Preoperative Hb concentrations (p = 0.0000) were associated with postoperative [Hb]. Postoperative [Hb] tended to be (but were not significantly) associated with cardiovascular complications (p = 0.0771) and female gender (p = 0.0752). There was no significant association between postoperative Hb concentrations and patients’ other characteristics. The relationships between postoperative [Hb] and FSI and PCS or MCS components of the SF-36 are shown on a scatter plot in Figure 1.\n\nHbPre = Preoperative Hb; FSI = Functional Status Index; PCS = Physical Component Summary score; MCS = Mental Component Summary score; BMI = Body Mass Index; intercept is the value of the outcome when all continuous variables in the model are 0 and all categorical variables are the references categories ; t value is the estimated slope divided by the standard error of the estimated slope.\n\nScatter plots of FSI (A), PCS (B) and MCS (C) scores according to postoperative [Hb]. FSI = Functional Status Index; PCS = Physical Component Summary score; MCS = Mental Component Summary score; HbPost = Postoperative Hb. The lines represent local polynomial regression fitting.\n\nAs shown in Figure 1, there was no statistically significant association between postoperative [Hb] and FSI, PCS or MCS scores. The distribution of the dots in Figure 2 reflects a similar lack of association between Hb concentration and postoperative complications (marked by filled circles).\n\nScatter plots of cardiac adverse events (A), respiratory adverse events (B), major infections (C), non-specific symptoms of anemia (D) and neurological adverse events (E) according to pre and postoperative Hb concentrations. Filled circles represent patients with adverse events. HbPre = Preoperative hemoglobin; HbPost = Postoperative hemoglobin.\n\nTable 4 presents the relationship between the difference between pre and postoperative Hb, FSI and SF-36 components scores, and shows that this difference in Hb concentration was not associated with FSI, PCS and MCS. Only non-specific symptoms of anemia (p = 0.0418) and age (p = 0.0106) were statistically associated with the difference in [Hb]. The difference in Hb concentrations tended to be (but was not significantly) correlated with female gender (p = 0.0684).\n\nFSI = Functional Status Index; PCS = Physical Component Summary score; MCS = Mental Component Summary score; BMI = Body Mass Index; intercept is the value of the outcome when all continuous variables in the model are 0 and all categorical variables are the references categories; t value is the estimated slope divided by the standard error of the estimated slope.\n\nA possible non-linear effect of postoperative [Hb] on FSI scores was investigated (Table 5). To do so, a quadratic transformation of FSI was added I(FSI2). Again, there was no statistically significant relationship between postoperative Hb concentration and FSI scores. Finally, the effect of time between surgery and completion of the follow-up questionnaire on patients’ scores was considered. Time was not statistically associated with PCS and MCS scores but it was statistically correlated with FSI scores (FSI scores worsened as time elapsed; p = 0.0108).\n\nHbPre = Preoperative Hb; FSI = Functional Status Index; intercept is the value of the outcome when all continuous variables in the model are 0 and all categorical variables are the references categories; t value is the estimated slope divided by the standard error of the estimated slope.\n\n\nDiscussion\n\nContrary to our preliminary hypothesis, we were unable to find a relationship between the immediate postoperative Hb concentration (or the difference between pre and postoperative Hb) and functional recovery or QoL in patients in the long-term period after a major arthroplasty. Also, there were no significant correlations between postoperative Hb levels and adverse events or morbidities thought to be associated with postoperative anemia. Consequently, we could not establish a Hb threshold below which functional recovery and QoL in patients are affected.\n\nThe SF-36 and FSI data were analyzed by linear regression using two main models (one studied scores in association with postoperative Hb and the second investigated scores in association with individual differences between pre and postoperative Hb concentrations). There were no significant associations of these two models with SF-36 and FSI. We also considered an eventual non-linear effect of postoperative Hb on FSI scores to ensure that all correlation models were investigated. Again, there were no associations between postoperative Hb and FSI scores.\n\nTwo recent studies support the lack of effects of postoperative anemia on functional recovery and QoL in patients undergoing a major arthroplasty. A randomized clinical trial involving 2016 patients at high cardiovascular risk demonstrated that a liberal transfusion strategy, as compared with a restrictive strategy, did not reduce the death rates, the ability to walk independently or the rates of in-hospital morbidities 60 days after hip-fracture surgery13. Moreover, the results of the TRIOS phase 2 trial also showed that moderate anemia does not affect functional recovery or QoL in patients during the immediate postoperative period7. Considering these outcomes and the fact that recovery from anemia is relatively rapid7, it is not entirely surprising that the long-term effects of anemia on functional recovery and QoL were minimal in the present study (Table 3 and Table 4).\n\nOn the other hand, a recent literature review by Spahn suggests that an increase in postoperative mortality and morbidity could be associated with perioperative anemia1. This review concluded that perioperative anemia is associated with postoperative infections, poorer physical functioning and recovery, and increased length of hospital stay and mortality1. However, prospective clinical studies are required to validate these conclusions. In contrast to Spahn’s review, results of the FOCUS trial demonstrated that the frequency of in-hospital morbidities (including pneumonia, wound infection, thromboembolism, stroke or transient ischemic attack and clinically recognized myocardial infarction), the inability to walk independently on a 60-day follow-up, length of hospital stay and mortality rates were not reduced by using a liberal transfusion strategy (maintaining [Hb] over 100 g/L) as compared with a restrictive strategy (transfusion for symptoms of anemia or at physician discretion for a [Hb] below 80 g/L)13.\n\nSeveral studies have investigated the relationship between postoperative anemia and functional outcomes in patients after surgery. As reviewed in the study by Vuille-Lessard et al, the conclusions of these studies tend to diverge, but in general, most evidence-based studies indicated that the effects of anemia on functional recovery and QoL are limited7.\n\nA major strength of the present study is that we used two different questionnaires to evaluate the patients’ physical and psychological function. This enhances the validity of the results. Another strength of the study is the reliability and validity of the instruments used to collect data. Functional recovery was measured using the FSI, a valid and reliable instrument allowing auto-evaluation of physical function in adults8, while QoL was evaluated using the well-validated SF-3610.\n\nWeaknesses of this study include the inability or the refusal of some patients to report a representative description of their physical and psychological condition. Furthermore, some interviewed patients had concurrent health problems (cancer, diabetes, renal insufficiency and arthritis for example) in addition to their orthopedic surgery that might have also affected their FSI or SF-36 scores. As a result, some patients recovering well from either THA or TKA may have presented low physical function scores due to other health problems. However, this possibility would have affected our results negatively, which was not the case since, overall, FSI scores indicated a good functional recovery. Another weakness of this study is its follow-up observational design and the variable time between surgery and follow-up. Given that total recovery from THA or TKA is relatively rapid7, the difference in time between surgery and the follow-up questionnaire (3 months to 6 years) may have affected the scores to the questionnaires relating to the patient’s recovery and. yet, our results show that this was not the case for PCS and MCS scores and FSI scores were negatively affected by a longer time interval between surgery and the follow-up questionnaire.\n\nIn summary, postoperative anemia does not appear to affect long-term (6 months or more) functional recovery and QoL in patients undergoing THA or TKA. Our follow-up results confirm the lack of effects of anemia on functional recovery and QoL during the immediate postoperative period observed in the TRIOS phase 2 study.",
"appendix": "Author contributions\n\nAlex Cormier-Lavoie interviewed the patients, collected and helped analyze the data, and wrote the manuscript. Monique Ruel helped design and conduct the study. Marie-Pierre Sylvestre analyzed the data and helped write the manuscript. Jean-François Hardy designed the study, helped conduct the study and analyze the data, and reviewed the manuscript. All authors approved the final manuscript for publication.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded solely by departmental sources.\n\n\nAcknowledgements\n\nThe authors would like to thank Ms Sandra Larrivée, M.Sc. Statistics for her assistance with the revision of the statistical analyses and presentation of the results.\n\n\nReferences\n\nSpahn DR: Anemia and patient blood management in hip and knee surgery: a systematic review of the literature. Anesthesiology. 2010; 113(2): 482–95.\n\nCarson JL, Duff A, Poses RM, et al:Effect of anaemia and cardiovascular disease on surgical mortality and morbidity. Lancet. 1996; 348(9034): 1055–60.\n\nHebert PC, Wells G, Tweeddale M, et al:Does transfusion practice affect mortality in critically ill patients? Transfusion Requirements in Critical Care (TRICC) Investigators and the Canadian Critical Care Trials Group. Am J Respir Crit Care Med. 1997; 155(5): 1618–23.\n\nHebert 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.\n\nVuille-Lessard E, Boudreault D, Girard F, et al:Red blood cell transfusion practice in elective orthopedic surgery: a multicenter cohort study. Transfusion. 2010; 50(10): 2117–24.\n\nAlvarez G, Hebert PC, Szick S: Debate: transfusing to normal haemoglobin levels will not improve outcome. Crit Care. 2001; 5(2): 56–63.\n\nVuille-Lessard E, Boudreault D, Girard F, et al:Postoperative anemia does not impede functional outcome and quality of life early after hip and knee arthroplasties. Transfusion. 2012; 52(2): 261–70.\n\nJette AM: The Functional Status Index: reliability and validity of a self-report functional disability measure. J Rheumatol Suppl. 1987; 14(Suppl 15): 15–21.\n\nSo-Osman C, Nelissen R, Brand R, et al:Postoperative anemia after joint replacement surgery is not related to quality of life during the first two weeks postoperatively. Transfusion. 2011; 51(1): 71–81.\n\nBusija L, Pausenberger E, Haines TP, et al:Adult measures of general health and health-related quality of life: Medical Outcomes Study Short Form 36-Item (SF-36) and Short Form 12-ltem (SF-12) Health Surveys, Nottingham Health Profile (NHP), Sickness Impact Profile (SIP), Medical Outcomes Study Short Form 6D (SF-6D), Health Utilities Index Mark 3 (HUI3), Quality of Well-Being Scale (QWB), and Assessment of Quality of Life (AQoL). Arthritis Care Res (Hoboken). 2011; 63(Suppl 11): S383–412.\n\nEnright PL, McBurnie MA, Bittner V, et al:The 6-min walk test: a quick measure of functional status in elderly adults. Chest. 2003; 123(2): 387–98.\n\nEnright PL: The six-minute walk test. Respir Care. 2003; 48(8): 783–5.\n\nCarson JL, Terrin ML, Noveck H, et al:Liberal or restrictive transfusion in high-risk patients after hip surgery. N Engl J Med. 2011; 365(26): 2453–62."
}
|
[
{
"id": "815",
"date": "07 Mar 2013",
"name": "Jacques Lacroix",
"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 easy to understand and their content is a very good summary of what can be found in the full manuscript.The methods section is operational: readers will find enough details to replicate the study.There is no overstatement in the conclusion.The data are original and quite unexpected: I would have guessed that elderly people would suffer from anaemia after a hip or knee surgery, and I am surprised that this is not the case.",
"responses": []
},
{
"id": "1035",
"date": "01 Jul 2013",
"name": "Cynthia So-Osman",
"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 paper contains follow up data from patients analyzed in a previous study (E. Vuille-Lessard et al. 2010). I have great concerns about certain issues that are presented in this paper:1. the proportion of responders was low (52%) and I suspect that selection bias was present. The authors should include the baseline characteristics of this population compared to the original study patients. The mean Hb values seem higher than in the original study. Could this study population be the better, healthier part of the original study population?2. Data analysis was performed by regression analysis to investigate the relationship between postoperative Hb level and functional recovery (by FSI score) and quality of life by SF-36 score. The effect of time between surgery and completion of follow up questionnaires on patients’ scores was evaluated. It would be more informative if the model investigating the relationship of (delta) Hb and scores was corrected for the effect of the time.3. In my opinion p-values are not the only parameter of value. Additionally reporting R or R2 would be more informative, since this parameter will explain the proportion of variation which can be explained by the variable of interest.4. I do not agree with the strengths of the study. Using two different questionnaires is not a way to enhance the validity of the study. Perhaps the used questionnaires are not discriminative enough or are just not suitable to test the hypothesis in this patient population.5. The authors should include possible selection bias as a major weakness, see also point 1.6. A possible error was present in reporting the time of follow up in the weakness paragraph: 3 months to 6 years should be probably 6 months to 3 years.7. Another possible error was present in the number of questionnaires completed. If 26 to 36 were completed during 7 consecutive weeks, > 190 questionnaires should have been available, not 184.In this paper some essential data are lacking and together with the finding that the data was not analyzed appropriately, I conclude that the results do not add to more evidence regarding quality of life or functionality after hip- and knee arthroplasty, even after (long-term) follow up.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-61
|
https://f1000research.com/articles/2-59/v1
|
22 Feb 13
|
{
"type": "Review",
"title": "Is gastroenterology research in decline? A comparison of abstract publication rates from The British Society of Gastroenterology meetings between 1995 and 2005",
"authors": [
"Sarah Prendergast",
"Katharina Mattishent",
"Tom Broughton",
"Ian Beales",
"Sarah Prendergast",
"Katharina Mattishent",
"Tom Broughton"
],
"abstract": "Background: Reports have suggested that academic medicine may be in decline within the UK. Further evidence suggests that rates of subsequent full publication of abstracts presented at major scientific meetings are low and may be declining. We have compared the publication rates of abstracts presented at meetings of the British Society of Gastroenterology (BSG) between 1995 and 2005 and examined factors associated with full paper publication. Methods: Abstracts presented at BSG meetings in 1995 and 2005 were assessed by cross-referencing with multiple databases. Abstract characteristics associated with publication were analysed.Results: There were no differences in overall publication rates, impact factors or time to publication between 1995 and 2005. Overall, basic-science abstracts were twice as likely to achieve full publication than non-basic science. There was a significant fall in the publication rates for case series and audits, and significantly increased rates for fundamental/basic-science abstracts over the study period. There were non-significant increases in publication rates for controlled trials and systematic reviews. In general, publication rates for all predominantly clinically orientated abstracts reduced between the two periods with the most notable fall occurring in nutrition. Conclusions: There was no evidence of a decline in overall abstract publication rates between 1995 and 2005. There seemed to be trend for increased publication rates of abstracts using perceived high-quality study methodologies with a corresponding decrease in those with lower quality methods. The proportion of basic-science abstracts is likely to be a determinant of overall full publication rates following scientific meetings.",
"keywords": [
"bibliometrics",
"biomedical research/trends",
"congresses",
"meeting abstracts"
],
"content": "Background\n\nAbstracts are presented at national and international conferences to rapidly communicate the results of new and original research. This process also allows the researcher to receive preliminary and informal peer review from fellow researchers in the field. This may be an important part of academic development, as it helps authors to identify potential errors and to develop alternative interpretations of their results before proceeding to submission for full publication. It has been suggested that although abstracts submitted to conferences are peer-reviewed; this process may not be as rigorous as that of an indexed journal considering full publication1.\n\nAcceptance of an abstract for presentation at a conference may imply to the researcher that full publication is likely. Presentation of an abstract is certainly a positive sign that publication may be more likely, but this is not certain2. Journal publication rates of submitted abstracts vary widely between 11% and 78% for different medical specialties3–7. A Cochrane review examined abstract publication rates of all medical subspecialties and reported a mean full (peer-reviwed journal) publication rate of 44.5%, with higher rates of 63.1% for randomised or controlled clinical trials5.\n\nCriteria for the acceptance of abstracts for conference presentation include such factors as: evaluation of whether the ideas presented are new, potentially significant, interesting and plausible. However, at the time of presentation, there may be insufficient information concerning data or methodology to assess the true scientific value of the research1. Inherently this acceptance process is inevitably not as rigorous as journal peer-review.\n\nReasons for subsequent non-publication are not entirely clear. Four main reasons have been suggested: (1) lack of time to prepare a manuscript for publication, (2) the study may be still on-going, (3) relationships with co-authors sometimes present a barrier to final publication, and (4) authors’ feelings that pursuit of publication is a low priority8. To these, the possibility of the study being of low scientific quality must be added. It has been suggested that improved preparation prior to the study and stricter guidelines to limit the presentation of abstracts at national and international meetings may help to reduce the number of unpublished studies8.\n\nThe percentage of abstracts that are eventually published is a potentially important clinical issue. Abstract presentations may be deemed to be more credible than is justifiable. For example, The American Academy of Orthopaedic Surgeons suggested that many national meeting attendees may alter their surgical practices, based on informally acquired and unscrutinsed information obtained from abstracts6. This practice has important implications for the development of evidence-based medicine. In addition, cardiology abstracts have been cited as references in journal reference lists, with no difference in citation rates between published and un-published papers9. These findings emphasise the need for caution and restraint in citing abstracts as full references. Other notable sources have discouraged the citation of abstracts, on the grounds that such practices may propagate invalid and erroneous conclusions1. In support of these concerns, many journals will not accept abstract citations10. Clearly, the citing of material that has not been subjected to the full rigour of peer-review is inadvisable and may undermine the scientific value of the work.\n\nAn important factor that appears to influence whether a study described in an abstract proceeds to full publication is the presence of ‘positive’ results. This refers to results that are significant in favour of the experimental treatment11. In a study of research abstracts at an emergency medicine meeting, positive-outcome bias was evident. In addition publication did not relate to study design or quality2. Other factors associated with full publication include oral presentation, acceptance for meeting presentation, randomised controlled study design, and controlled clinical trials and basic research5.\n\nIt also appears that the length of time taken between the presentation of the abstract and full publication is influenced by this so-called ‘publication bias’. The Cochrane review examined the time to publication of a cohort of 196 clinical trials. The investigators found that just over half the trials were published, but that those with positive results (i.e. statistically significant in favour of the experimental arm) were published within 4 or 5 years, whereas those with null or negative results (i.e. not statistically significant or statistically significant in favour of the control arm) were published after 6–8 years12. It has been suggested that these findings had important implications because if trials with positive findings are stopped earlier and published quicker than those with negative findings, then new treatments might be mistakenly assumed to be effective12. These findings also have important implications for the timing of the initiation and updating of a systematic review. The earlier publication of positive results, combined with the tendency to publish ‘significant’ results means that systematic reviews will tend to over-estimate treatment effects5.\n\nIt has also been noted that study type may be an important factor in determining whether a study is published. One study of two UK national paediatric meetings found that most randomised controlled trials were published, but that observational studies submitted were published less often13. Sources have suggested that, because many factors important in a scientific study are impossible to control in a retrospective study, it is not surprising that prospective studies are published at much higher rates than retrospective studies1. Thus the recommendation of well-designed prospective studies with high statistical power should be encouraged.\n\nRecent reports have suggested that academic medicine in the UK may be in decline and that research output is reducing14,15. Possible reasons for this development include the perceived overwhelming bureaucratic processes of research governance16 and lack of funding15,16. This concept is supported by evidence that between 1994 and 2002 the number of publications published by gastroenterology specialist registrar trainees before starting consultant posts, had fallen significantly from a median of 19 in 1993 to a median of 5 by 200217.\n\nFurther supporting evidence includes a downward trend for the percentage of abstract presented at the British Society of Gastroenterology (BSG) annual meetings to reach full publication in the years after 1994. The authors inferred that this observation could be taken as a surrogate marker for a decline in research activity within the UK gastroenterology community, but this study did not further examine whether any study type or subject was more or less likely to reach full publication. They recommended that further work was required to validate their findings18.\n\nTherefore, there appear to be considerable uncertainties as to whether academic medicine and research activity within the United Kingdom's health service are declining and there are very little data concerning predictors of subsequent full publication from abstracts particularly related to gastroenterology, although this latter question may well be more generic. In addition, presenting abstracts at meetings is generally viewed as contributing to career progression for post-qualification doctors in training and presentation of abstracts is used in the scoring of applicants for training posts. There are little data to guide trainees or trainers on how valuable these activities are. In this study we have examined the publication rates from two BSG meetings separted by 10 years to determine if publication rates have changed and also to examine whether there was any relationship between study type, subspeciality within gastroenterology and time-lag to publication.\n\n\nMethods\n\nHard copies of abstracts presented at the BSG scientific meetings were obtained for the years 1995 and 2005. There were two separate BSG meetings in 1995 and one single conference in 2005; data for both meetings in 1995 were combined. Data collection was performed retrospectively starting in 2010, ensuring that a period of at least four years had elapsed since the last meeting chosen to be assessed. This approach was taken to align this study with previous work in the field18 and because it has been reported that the upper time limit to publication after an abstract is presented at a meeting is four years3,4,9. A further repeat search was performed in September 2012 to check for any very delayed publications.\n\nAll abstracts presented at the BSG conference for 1995 and 2005 were collected and cross-referenced with the Ovid, Medline, EMBASE, The Cochrane Library, Web of Science and Wiley Interscience databases to assess for evidence of full publication.\n\nAbstracts were cross referenced using first-author, senior author and at least one key word from the abstract title. Where an abstract appeared to have been published, abstracts and published articles were further examined to ensure that they represented the same study. If two or more abstracts were part of single fully published manuscript, then each one was counted individually as a publication for all aspects of the study.\n\nIn addition, study type, subspeciality within gastroenterology, type of study, citation, impact factor and lag- time to publication were also assessed. The impact factor of the journals for the year in which the paper was published was determined using the Web of Science database. Abstracts from each year were assessed independently by two of the authors; any differences were resolved by consensus under the direction of the senior author (ILPB).\n\nPublication rates and odds ratios with 95% confidence intervals were used to compare abstracts presented in 1995 and 2005 using chi-squared test. Impact factors and time to publication were calculated as mean ± standard deviation and compared using Students t-test. Bonferroni correction was applied for multiple comparisons.\n\n\nResults\n\nFrom the meetings in the 2 years, a total of 938 records were analysed; all records were complete and no records were excluded due to anomalies or missing data. The number of abstracts presented over the two meetings in 1995 was 497, and 441 were presented at the at the single 2005 conference. The number of abstracts which proceeded to full publication was remarkably similar in the 2 years, 88 (17.7%) in 1995 and 77 (17.4%) in 2005; odds ratio (OR) for publication in 2005 compared to 1995 (0.98, 95% CI 0.79 – 1.39).\n\nWe next examined whether study type or overall topic seemed to be associated with publication rates. The data are summarised in Table 1. The most striking changes were a significant fall in the publication rates for case series (OR 0.47, 0.26 – 0.85) and audit articles (with no publications at all from 2005), coupled with a significant increase in publication rates for basic/fundamental science between the 2 years. In light of the latter, the overall chance of publication fell for predominantly “clinical“ (non-basic science) abstracts between 1995 and 2005, (OR 0.74, 0.48 – 1.19). In fact, including data from both years, abstracts focused on basic or fundamental science were more than twice as likely to achieve full publication than non-basic-science abstracts (OR 2.19, 1.51 – 3.16).\n\nWe further examined whether any major area was responsible for the increased publication rates in fundamental science: publication rates increased in all the major areas examined, the greatest rise being in pharmacology but overall rates increased in all areas.\n\nWithin the major subspecialties of gastroenterology, rates of publication generally fell in all areas, the greatest and most significant fall being in nutrition (OR 0.26, 0.01 – 0.97).\n\nThe mean impact factor of the journals that the abstracts were published in from 1995 was 4.67 (± 3.41). The mean impact factor for 2005 was 3.87 (± 2.43). This difference was not statistically significant. The mean time to publication in 1995 was 22.2 months (± 16.9), this was slightly faster for 2005 (mean time 18.6 ± 13.3), but not statistically different.\n\n\nDiscussion\n\nThis study found no significant difference in the percentage of abstracts presented at the BSG conferences in 1995 compared with 2005, that subsequently achieved full publication. These findings are at variance with some earlier studies17,18 that indicated that the abstract publication rates were declining within gastroenterology. The overall publication rate in 1995 was relatively low and it is possible that any decline had already plateaued by 1995, or else 1995 was an outlier year with a low publication rate. Further analysis of surrounding years will be required to address this.\n\nDespite the overall similarities in publication rates between the two different years, 10 years apart, several interesting differences were seen. There was a marked decline in subsequent publication of case series and audit articles, but this was balanced by an increase in publication rates for fundamental science. Overall publication rates did fall for predominantly clinical studies.\n\nThere are two main conclusions that can be drawn from these changes. Firstly that there does seem to be reduction in the rate of full publication of perceived “lower quality” research (case series and audit articles) with an increase in publication rates for basic science, which is usually seen as “higher” quality because of the much greater control of conditions and experimental planning possible19. This trend would seem to be in keeping with increased editorial rigour in peer-reviewed publications. There were only a small number of randomised trials in the dataset but the upward trend for publication rates for both these and systematic reviews over the study period would seem consistent with this concept19. Another factor to be considered is that it is likely that in a greater proportion of basic-science abstracts, the lead author would have been a “professional” scientist (possibly without medical training), whereas “clinical” abstracts would more likely be produced by trainees or consultants in clinical posts. We can speculate that the greater time and focus available, fewer competing clinical commitments and less movement of abstract authors between training posts and regions exhibited by basic-science abstract authors all contributed to the increased publication rate.\n\nThe second conclusion is that crude publication rates are probably an insufficient tool to analyse abstract publication rates from a broadly based scientific meeting such as the BSG annual meetings, which genuinely spans bench to bedside to community, as the proportion of underlying basic-science abstracts may significantly influence publication rates. Further studies comparing publication rates with similar meetings such as cardiology or respiratory societies or basic-science-focused meetings such as the Physiological Society would be interesting.\n\nWithin basic science, there did not seem to be a predominant area that led the increased publication rates; there seemed to be an overall increase. We accept that classification within these areas can be difficult due to considerable overlap between focus and experimental techniques within the fundamental science themes.\n\nThere was a general decline in publication rates in all major subspecialty areas of clinical gastroenterology and hepatology: this probably reflects a general trend for reduced publication rates for primarily clinical studies rather than a shift influenced by topicality or fashion for certain areas within gastroenterology between 1995 and 2005. The most marked fall in publication rates occurred in nutrition, this despite nutritional science and clinical nutrition having a much higher profile over the latter years of the study period. One explanation for this may be that although the BSG has an active nutrition section, there are other scientific meetings and societies, particularly the multidisciplinary British Association for Parenteral and Enteral Nutrition (BAPEN) and perhaps the higher quality nutrition papers from 2005 were presented as such meetings instead of the BSG.\n\nIt has been suggested that certain features of abstracts, such as sample size, study type, methodology and the presence of statistically significant results may influence the chances of full publication20. A fuller examination of the published papers than was possible in this study might reveal more information on these factors. We have considered whether the differences in the number of BSG conferences in 1995 compared with 2005 may have had any effect on the results but feel that this was unlikely and therefore was not pursued further.\n\nOverall publication rates were low, at approximately 17%, but these fall well within the ranges reported from other major meetings3–5 and it must be appreciated that overall publication rates are not only influenced by the vigour with which authors pursue subsequent publication, but also the perhaps competing requirements of journal editors for high-quality, rigorous research papers and conference organisers wishing to maximise attendance at their meetings. Increasing the number of abstracts presented and hence the associated number of presenters is one such way of increasing attendance; theoretically this could lead to increased presentation of abstracts of a lower scientific quality.\n\nThe results obtained from this study suggest that there has not been a decline in abstract publication rates between the years 1995 and 2005. Where publication rates are seen as a surrogate marker for research activity18, this suggests gastroenterology research activity has remained relatively stable, although as discussed above, the crude publication rate may be an inadequate tool to measure this. One other factor to be considered is that although the meetings considered are organised by the British Society of Gastroenterology, these are international meetings and our data cannot be used to specifically imply growth or decline in gastroenterology research activity specifically in the United Kingdom; we did not analyse abstracts for country or origin.\n\nIn conclusion, we have confirmed that abstracts presented at scientific meetings commonly do not result in subsequent full peer-reviewed publications and this should perhaps be considered when appraising the quality of studies published as abstracts only. Within gastroenterology we have shown a decline in publication of case series and audit articles, and an increase in publication of basic/fundamental science. Further studies defining the predictors of subsequent publication as well as comparisons with other meetings and subsequent years would be interesting.",
"appendix": "Author contributions\n\n\n\nSP, KM and ILPB jointly conceived and planned the study. SP, KM, TB and ILPB performed the data extraction and analysis. SP and KM jointly wrote the first draft of the manuscript. ILPB wrote the final version of the manuscript and is the guarantor of the paper. All authors agree to publication.\n\n\nCompeting interests\n\n\n\nILPB is a full member of the British Society of Gastroenterology and undertakes peer-reviewing and editorial activities for several peer-reviewed journals. ILPB and TB have presented abstracts at BSG meetings, which have gone on to subsequent full publication.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nQuencer RM, Grossman R: Unpublished papers perish. Am J Neuroradiol. 1999; 20: 962–3.\n\nCallaham ML, Wears RL, Weber EJ, et al.: Positive-outcome bias and other limitations in the outcome of research abstracts submitted to a scientific meeting. JAMA. 1998; 280(3): 254–7. PubMed Abstract | Publisher Full Text\n\nJuzych MS, Shin DH, Coffey J, et al.: Whatever happened to abstracts from different sections of the association for research in vision and opthalmology? Invest Opthalmol Vis Sci. 1993; 34(5): 1879–82. PubMed Abstract\n\nJuzych MS, Shin DH, Coffey JB, et al.: Pattern of publication of ophthalmic abstracts in peer-reviewed journals. Opthalmology. 1991; 98(4): 553–6. PubMed Abstract\n\nScherer RW, Langenberg P, von Elm E: Full publication of results initially presented in abstracts. Cochrane Database Syst Rev. 2007; (2): MR000005. PubMed Abstract | Publisher Full Text\n\nMurrey D, Wright RW, Seiler JG 3rd, et al.: Publication rates of abstracts presented at the 1993 annual academy meeting. Clin Orthop Relat Res. 1999; (359): 247–53. PubMed Abstract\n\nSanders DS, Carter MJ, Hurlstone DP, et al.: Research outcomes in British gastroenterology: an audit of the subsequent full publication of abstracts presented at the British Society of Gastroenterology. Gut. 2001; 49(1): 154–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSprague S, Bhandari M, Devereaux PJ, et al.: Barriers to full-text publication following presentation of abstracts at annual orthopaedic meetings. J Bone Joint Surg Am. 2003; 85-A(1): 158–63. PubMed Abstract\n\nGoldman L, Loscalzo A: Fate of cardiology research originally published in abstract form. N Engl J Med. 1980; 303(5): 255–9. PubMed Abstract | Publisher Full Text\n\nUniform requirements for manuscripts submitted to biomedical journals. International Committee of Medical Journal Editors. JAMA. 1997; 277(11): 927–34. PubMed Abstract | Publisher Full Text\n\nDickersin K: The existence of publication bias and risk factors for its occurrence. JAMA. 1990; 263(10): 1385–9. PubMed Abstract | Publisher Full Text\n\nHopewell S, Clarke M, Stewart L, et al.: Time to publication for results of clinical trials. Cochrane Database Syst Rev. 2007; (2): MR000011. PubMed Abstract | Publisher Full Text\n\nRiordan FA: Do presenters to paediatric meetings get their work published? Arch Dis Child. 2000; 83: 524–526. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBell J: Resuscitating clinical research in the United Kingdom. BMJ. 2003; 327(7422): 1041–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSewell WA: Thwarted by bureaucracy. BMJ. 2007; 334(7592): 491. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSewell WA: Process of science counteracts enthusiasm. BMJ. 2007; 334: 491.\n\nHopper AD, Atkinson R, Prtak L, et al.: Research trends in British gastroenterology: publication rates in newly appointed NHS consultants over a nine year period. Gut. 2004; 53(6): 913. PubMed Abstract | Free Full Text\n\nHopper AD, Atkinson RJ, Razak A, et al.: Is medical research within the UK in decline? A study of publication rates from the British Society of Gastroenterology from 1994 to 2002. Clin Med. 2009; 9(1): 22–5. PubMed Abstract\n\nAjetunmobi O: Making Sense of Critical Appraisal. Great Britain, Hodder Arnold, London, 2002.\n\nChalmers I, Adams M, Dickersin K, et al.: A cohort study of summary reports of controlled trials. JAMA. 1990; 263(10): 1401–5. PubMed Abstract | Publisher Full Text\n\n\nSupplementary data\n\n"
}
|
[
{
"id": "797",
"date": "25 Feb 2013",
"name": "Penny Neild",
"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": "799",
"date": "26 Feb 2013",
"name": "Jean Crabtree",
"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 would be improved if the following were added:An inclusion in the methods section of the classification criteria for abstracts based on basic/fundamental science.On page 6, the authors state 'proportion of basic science abstracts may significantly influence publication rates'. Data on % abstracts in clinical, audit and basic/fundamental science areas in 1995 and 2005 should be included so changes in patterns of submission/acceptance over the 10 year period can be determined.Information on whether the journal impact factors of clinical versus fundamental/basic science publications differed.Are data available on the funding sources for abstracts published as full papers? Are abstracts of work funded by national research councils and charities more likely to be published than pharmaceutically funded studies?",
"responses": []
},
{
"id": "805",
"date": "04 Mar 2013",
"name": "Harriet 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 is a valid and helpful study",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-59
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https://f1000research.com/articles/2-56/v1
|
20 Feb 13
|
{
"type": "Research Article",
"title": "Glycine-extended gastrin enhances somatostatin release from cultured rabbit fundic D-cells",
"authors": [
"Ian LP Beales"
],
"abstract": "The role of the peptide hormone gastrin in stimulating gastric acid secretion is well established. Mature amidated gastrin is processed from larger peptide precursor forms. Increasingly these processing intermediates, such as glycine-extended gastrin (G-Gly) and progastrin, have been shown to have biological activities of their own, often separate and complementary to gastrin. Although G-Gly is synthesized and secreted by gastric antral G-cells, the physiological functions of this putative mediator are unclear. Gastrin and cholecystokinin (CCK) stimulate the secretion of somatostatin from gastric D-cells as part of the feedback control of gastric acid. In this study the effect of G-Gly and gastrin on the release of somatostatin from rabbit fundic D-cells was examined. D-cells were obtained by collagenase-EDTA digestion and elutriation and cultured for 48 hours. With a 2 hour exposure to the peptides, gastrin but not G-Gly stimulated somatostatin release. Treatment of D-cells for 24 hours with gastrin or G-Gly individually, significantly enhanced subsequent basal as well as CCK- and GLP-1-stimulated somatostatin release. Twenty four hours exposure to gastrin combined with G-Gly synergistically enhanced basal and agonist-stimulated somatostatin release and cellular somatostatin content. Gastrin and G-Gly may be important in the longer term regulation of D-cell function.",
"keywords": [
"gastrin",
"glucagon-like peptides",
"somatostatin"
],
"content": "Introduction\n\nGastrin is initially synthesized as a larger precursor protein and subsequently processed, via a multi-step pathway, to the classical active carboxyl-terminal amidated peptide1. It has become apparent that some of the processing intermediates may have biological activities of their own. Significant biological effects have been reported for both the larger progastrin precursor peptide and the shorter carboxyl-terminal glycine-extended gastrin (G-Gly), suggesting that these are important pathophysiological mediators. The majority of studies have examined the pathophysiological roles of gastrin precursors in gastrointestinal cancers and considerable data have implicated these peptides as stimulants of proliferation and/or inhibitors of apoptosis in a variety of tissues and cell lines, including Barrett’s oeosphagus and oesophageal adenocarcinoma2,3, stomach4,5, pancreas6,7 and normal and malignant colonic epithelium8–12. Growth promoting effects of G-Gly on lung cancer have also been reported13.\n\nAlthough the precise cell signaling pathways activated by gastrin-processing intermediates have not been definitively described, it seems in most cases that mechanisms distinct from the classical gastrin (CCK2) receptor are involved3,6,10. It is not yet clear whether these gastrin-processing intermediates have distinct physiological, as opposed to pathophysiological roles. Glycine-extended gastrin is produced and stored in significant amounts in the gastric antrum, has gastrointestinal trophic effects and interacts with amidated gastrin to modulate gene expression and gastric acid secretion14–16. Gastrin stimulates both acid secretion and somatostatin release as a feedback inhibitory mechanism17. Similarly, release of cholecystokinin (CCK) from duodenal I-cells is believed to be an important negative feedback mechanism, leading to the inhibition of acid secretion via the release of somatostatin from D-cells in the gastric body and fundus17. Somatostatin release is also stimulated by several other peptide hormones released from the proximal and distal small bowel (including glucagon-like peptide-1 (GLP-1), secretin and oxyntomodulin18 and these form part of the physiological feedback mechanisms that decrease gastric acid in the post-prandial period. The current study was designed to assess the effects of G-Gly on somatostatin release from D-cells and compare these effects with those of amidated gastrin.\n\n\nMaterials and methods\n\nNew Zealand White rabbits (2–2.5 kg) (Charles River Ltd, Margate, UK) were housed singly in 120 x 60 x 60 cm cages and fed ad libitum on rabbit chow (Special Diets Services, Witham, UK) with a standard 16/8 hour light/dark cycle according to standard Royal Postgraduate Medical School policy. Rabbits were humanely euthanized with 100 mg/kg pentobarbitone intravenously according to institutional policy. One rabbit was used per cell preparation procedure. Post-mortem, the stomach was removed and primary rabbit fundic D-cells were isolated by EDTA-collagenase digestion and enriched by centrifugal elutriation as described previously18–20. The D-cell enriched fraction was suspended in culture medium (DMEM: Ham’s F12 50:50 containing 4% foetal calf serum (Gibco, Paisley, UK), 10 mM HEPES pH 7.4, 2 mM glutamine, 8 mg/l bovine insulin, 1 mg/l hydrocortisone, 100 mg/l penicillin, 100 mg/l streptomycin, 100 mg/l gentamicin (all from Sigma, Poole, UK) and plated at 1 x 106 cells/well onto 12 well tissue culture plates coated with growth factor-reduced Matrigel (diluted 1:7 with water) (Universal Biologicals, London, UK). Cells were cultured for 48 hours after which either somatostatin release experiments were performed or the culture medium was changed and supplemented with 10 nM gastrin or 10 nM G-Gly as appropriate for a further 24 hours, until release experiments were performed.\n\nSomatostatin release experiments were performed as previously described18–20: the culture medium was removed, the cells washed, with release medium (Earl’s balanced salt solution containing 0.1% bovine serum albumin and 10 mM HEPES, pH 7.4) and basal somatostatin, as well as 10 nM cholecystokinin (CCK) , and 10 nM glucagon-like peptide-1 (7-36 amide) (GLP-1)-stimulated somatostatin release was assessed over 2 hours18–20. Cellular somatostatin was extracted by boiling the adherent cells in 3% (final vol/vol) glacial acetic acid in distilled water20. Both released and cellular somatostatin were assessed by radioimmunoassay using K2 anti-somatostatin serum (kindly provided by Professor SR Bloom and Dr M Ghatei, Royal Postgraduate Medical School, Hammersmith Hospital, using 125I somatostatin-14 as tracer and human somatostatin-14 as standard (Bachem, St Helens, UK)) as previously described18,20. Each experimental condition was tested in duplicate and compared with control, untreated wells on the same plate. Results were compared by analysis of variance and Student’s t-test and represent mean ± SEM of 8 different cell preparations. Gastrin (1–17)-Gly (G-Gly) was purchased from NeoMPS (Strasbourg, France), human gastrin-17, sulfated CCK-8 and GLP-1 (7–36) amide were from Bachem.\n\nCell viability following prolonged gastrin and G-Gly treatment was assessed using the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolinium bromide (MTT) (Sigma) as previously described20.\n\n\nResults\n\nInitial experiments with only the standard 2-hour stimulation period (without any prolonged pretreatment with any peptides) confirmed that gastrin increased basal but not CCK-stimulated somatostatin release. G-Gly over the 2 hour stimulation period did not alter basal, gastrin or CCK-stimulated release (Figure 1 and Table 1). Gastrin alone did stimulate somatostatin release but was less effective than CCK and neither gastrin nor the gastrin plus G-Gly combination had any effect on CCK-stimulated gastrin release.\n\nD-cells were cultured for 48 hours and then stimulated with peptides for 2 hours as shown, Somatostatin-like immunoreactivity released into the media was quantified by radioimmunoassay. Results expressed and mean ± SEM, compared to untreated control cells, n = 8, * p<0.05 compared to basal control, *P<0.01 compared to basal control.\n\nExperimental data from 8 separate stomach preparations showing somatostatin-like immunoreactivity released from cultured rabbit fundic D-cells stimulated for 2 hours with gastrin, glycine-extended gastrin or both peptides (all 10 nM) +/- CCK (10 nM). SLI results expressed as% of basal, unstimulated release in the relevant stomach preparation.\n\nTwenty four hours pretreatment with gastrin enhanced subsequent basal somatostatin release by 13% and CCK-stimulated release by 10% (both P<0.05). G-Gly enhanced basal somatostatin release by 22% and CCK-stimulated release by 24% (both p<0.05) (Figure 2). The combination of gastrin and G-Gly synergistically increased both basal somatostatin release (35%) and subsequent CCK-stimulated somatostatin release (53%) (p<0.05 compared to the effect of either peptide alone) (Figure 2 and Table 2).\n\nD-cells were cultured for 48 hours and then treated for a further 24 hours with peptides as shown, before stimulation with either CCK or control culture medium for 2 hours. Somatostatin-like immunoreactivity released into the media was quantified by radioimmunoassay. Results expressed and mean ± SEM, compared to untreated control cells, n = 8, * p<0.05 compared to relevant basal control, **p<0.05 compared to gastrin or G-Gly as sole pre-treatment.\n\nExperimental data from 8 separate stomach preparations showing somatostatin-like immunoreactivity released from cultured rabbit fundic D-cells stimulated for 2 hours with CCK or GLP-1 (both 10 nM), following a 24-hour pretreatment period with gastrin, glycine-extended gastrin or both peptides (all 10 nM). SLI results expressed as% of basal, unstimulated and untreated release in the relevant stomach preparation.\n\nTo further examine the effects of G-Gly pretreatment on agonist-stimulated release, an alternative direct stimulant of rabbit fundic D-cells, GLP-1, was used18. In keeping with previous studies, GLP-1 did stimulate somatostatin release but was markedly less potent than CCK. As shown in (Figure 3 and Table 2), again 24 hours pretreatment with gastrin alone significantly but relatively modestly potentiated subsequent GLP-1-stimulated somatostatin release (a 25% increase compared to the control GLP-1 treated cells), whilst G-Gly was more effective in enhancing GLP-1-stimulated somatostatin release (a 37% increase compared to the control GLP-1 treated cells). The combination of G-Gly and gastrin was significantly more potent than either peptide alone in enhancing GLP-1 stimulated somatostatin release (a 70% increase compared to the control GLP-1 stimulated cells).\n\nD-cells were cultured for 48 hours and then treated for a further 24 hours with peptides as shown, before stimulation with either GLP-1 or control culture medium for 2 hours. Somatostatin-like immunoreactivity was extracted from cells and quantified by radioimmunoassay. Results expressed and mean ± SEM, compared to untreated control cells, n = 8, * p<0.05 compared to relevant basal control, **p<0.05 compared to gastrin or G-Gly as sole pretreatment.\n\nTwenty four hour pretreatment with both gastrin peptides individually increased D-cell somatostatin content (Figure 4 and Table 3). The dual peptide combination was again synergistic in enhancing cellular somatostatin content, the combination increasing total somatostatin levels by 57% compared to untreated basal levels.\n\nD-cells were cultured for 48 hours and then treated for a further 24 hours with peptides as shown, Somatostatin-like immunoreactivity was extracted from cells and was quantified by radioimmunoassay. Results expressed and mean ± SEM, compared to untreated control cells, n = 8, *p<0.05 compared to untreated basal control. **p<0.05 compared to gastrin or G-Gly as sole treatment.\n\nExperimental data from 8 separate stomach preparations showing cellular somatostatin-like immunoreactivity contained in cultured rabbit fundic D-cells treated for a 24-hour pretreatment period with gastrin, glycine-extended gastrin or both peptides (all 10 nM). SLI results expresses as% of untreated control cells from the relevant stomach preparation.\n\n\nDiscussion\n\nThis study demonstrates that both gastrin and G-Gly enhance the subsequent basal and CCK or GLP-1 stimulated release of somatostatin from rabbit fundic D-cells. No acute stimulatory effects of G-Gly were demonstrated but the 24 hour exposure of D-cells to G-Gly significantly increased somatostatin release. It is clear that hormone release is regulated at multiple points (transcription, translation, processing)21 and that different agents may regulate overall function with different temporal patterns. However, it is not yet clear at which point(s) G-Gly regulates somatostatin release. The increase in cellular somatostatin seen after treatment with G-Gly suggests that upregulation of transcription or translation could be involved. An alternative but not mutually exclusive hypothesis is that the gastrin peptides are specific trophic factors for D-cells and the increased somatostatin release in cultured cells reflects these effects. There was no difference in cell viability, assessed by the modified MTT assay between gastrin or G-Gly treated and non-treated cells, but this does not exclude more subtle enhancement of function. Further studies will be required to elucidate the mechanisms underlying these effects of G-Gly and gastrin.\n\nPrevious studies have suggested that G-Gly has important roles in cell proliferation and regulation of acid secretion14–16, although specific effects on regulation of the gastric endocrine system have not been investigated previously. The effect of G-Gly in increasing acid secretion from cultured parietal cells is only seen with more prolonged exposure15, similar to the results reported here, suggesting this peptide may have longer term regulatory actions on transcription or processing instead of just stimulating hormone release.\n\nIt is known that multiple feedback loops regulate gastric acid secretion. Gastrin stimulates both acid secretion and negative feedback inhibition of acid secretion via the simultaneous release of somatostatin17. G-Gly is also released from G-cells following meals and may enhance acid secretion22. The results reported here suggest that longer term exposure of D-cells to gastrin and G-Gly also stimulates a further negative feedback loop by enhancing subsequent somatostatin release thus providing a means to restrain acid hypersecretion caused by hypergastrinaemia and control longer term acid secretion. Several studies have confirmed that G-Gly is produced in gastric antral G-cells22–25 and this study adds further support to the suggestion that these peptide processing intermediates may have a role in the normal physiological control of the gastric secretions.\n\nThe mechanisms of action of both gastrin and G-Gly in enhancing somatostatin release in these circumstances remain to be elucidated. Interestingly, the combination of these peptides had a synergistic effect on the release of somatostatin as has been noted in the control of acid secretion and cell growth2–4,6,26–28. Gastrin and G-Gly have separate but complimentary actions on cell signaling pathways and gene transcription2,3,14,27. This further supports the notion that both gastrin and G-Gly produced by the gastric antrum and duodenum have important roles in the regulation of gastric homeostasis.",
"appendix": "Author contributions\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by the MRC (Research Training Fellowship awarded to ILPB) and The Royal Society (Small Project Grant Awarded to ILPB).\n\n\nAcknowledgments\n\nThe author would like to acknowledge the late Professor J Calam for helpful discussions and Professor S Bloom and Dr M Ghatei for the gift of the somatostatin antiserum.\n\n\nReferences\n\nDockray GJ, Varro A, Dimaline R, et al.: The gastrins: their production and biological activities. Annu Rev Physiol. 2001; 63: 119–39. PubMed Abstract | Publisher Full Text\n\nOgunwobi OO, Beales IL: Glycine-extended gastrin stimulates proliferation via JAK2- and Akt-dependent NF-kappaB activation in Barrett's oesophageal adenocarcinoma cells. Mol Cell Endocrinol. 2008; 296(1–2): 94–102. PubMed Abstract | Publisher Full Text\n\nBeales IL, Ogunwobi OO: Glycine-extended gastrin inhibits apoptosis in Barrett's oesophageal and oesophageal adenocarcinoma cells through JAK2/STAT3 activation. J Mol Endocrinol. 2009; 42(4): 305–18. PubMed Abstract | Publisher Full Text\n\nIwase K, Evers BM, Hellmich MR, et al.: Regulation of growth of human gastric cancer by gastrin and glycine-extended progastrin. Gastroenterology. 1997; 113(3): 782–90. PubMed Abstract | Publisher Full Text\n\nHe H, Pannequin J, Tantiongco JP, et al.: Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42–dependent pathway. Am J Physiol Gastrointest Liver Physiol. 2005; 289(3): G478–88. PubMed Abstract | Publisher Full Text\n\nSeva C, Dickinson CJ, Yamada T: Growth-promoting effects of glycine-extended progastrin. Science. 1994; 265(5170): 410–2. PubMed Abstract | Publisher Full Text\n\nRengifo-Cam W, Umar S, Sarkar S, et al.: Antiapoptotic effects of progastrin on pancreatic cancer cells are mediated by sustained activation of nuclear factor-{kappa}B. Cancer Res. 2007; 67(15): 7266–74. PubMed Abstract | Publisher Full Text\n\nOgunwobi OO, Beales IL: Glycine-extended gastrin stimulates proliferation and inhibits apoptosis in colon cancer cells via cyclo-oxygenase-independent pathways. Regul Pept. 2006; 134(1): 1–8. PubMed Abstract | Publisher Full Text\n\nBeales IL, Ogunwobi O: Glycine-extended gastrin inhibits apoptosis in colon cancer cells via separate activation of Akt and JNK pathways. Mol Cell Endocrinol. 2006; 247(1–2): 140–9. PubMed Abstract | Publisher Full Text\n\nSingh P, Wu H, Clark C, et al.: Annexin II binds progastrin and gastrin-like peptides, and mediates growth factor effects of autocrine and exogenous gastrins on colon cancer and intestinal epithelial cells. Oncogene. 2007; 26(3): 425–40. PubMed Abstract | Publisher Full Text\n\nFerrand A, Bertrand C, Portolan G, et al.: Signaling pathways associated with colonic mucosa hyperproliferation in mice overexpressing gastrin precursors. Cancer Res. 2005; 65(7): 2770–7. PubMed Abstract | Publisher Full Text\n\nKoh TJ, Dockray GJ, Varro A, et al.: Overexpression of glycine-extended gastrin in transgenic mice results in increased colonic proliferation. J Clin Invest. 1999; 103(8): 1119–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoh TJ, Field JK, Varro A, et al.: Glycine-extended gastrin promotes the growth of lung cancer. Cancer Res. 2004; 64(1): 196–201. PubMed Abstract | Publisher Full Text\n\nTodisco A, Takeuchi Y, Seva C, et al.: Gastrin and glycine-extended progastrin processing intermediates induce different programs of early gene activation. J Biol Chem. 1995; 270(47): 28337–41. PubMed Abstract | Publisher Full Text\n\nKaise M, Muraoka A, Seva C, et al.: Glycine-extended progastrin processing intermediates induce H+, K(+)-ATPase alpha-subunit gene expression through a novel receptor. J Biol Chem. 1995; 270(19): 11155–60. PubMed Abstract\n\nChen D, Zhao CM, Dockray GJ, et al.: Glycine-extended gastrin synergizes with gastrin 17 to stimulate acid secretion in gastrin-deficient mice. Gastroenterology. 2000; 119(3): 756–65. PubMed Abstract\n\nDelValle J, Chiba T, Park J, et al.: Distinct receptors for cholecystokinin and gastrin on canine fundic D-cells. Am J Physiol. 1993; 264(5 PT 1): G811–5. PubMed Abstract\n\nBeales IL, Calam J: Truncated glucagon-like peptide-1 and oxyntomodulin stimulate somatostatin release from rabbit fundic D-cells in primary culture. Exp Physiol. 1996; 81(6): 1039–1041. PubMed Abstract\n\nBeales I, Calam J, Post L, et al.: Effect of transforming growth factor-alpha and interleukin-8 on somatostatin release from canine fundic D-cells. Gastroenterology. 1997; 112(1): 136–43. PubMed Abstract\n\nBeales IL, Calam J: The histamine H3 receptor agonist N alpha-methylhistamine produced by Helicobacter pylori does not alter somatostatin release from cultured rabbit fundic D-cells. Gut. 1998; 43(2): 176–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBate GW, Varro A, Dimaline R, et al.: Control of preprogastrin messenger RNA translation by gastric acid in the rat. Gastroenterology. 1996; 111(5): 1224–9. PubMed Abstract | Publisher Full Text\n\nDelValle J, Sugano K, Yamada T: Glycine-extended processing intermediates of gastrin and cholecystokinin in human plasma. Gastroenterology. 1989; 97(5): 1159–63. PubMed Abstract\n\nDelValle J, Sugano K, Yamada T: Progastrin and its glycine-extended posttranslational processing intermediates in human gastrointestinal tissues. Gastroenterology. 1987; 92(6): 1908–12. PubMed Abstract\n\nSugano K, Aponte GW, Yamada T: Identification and characterization of glycine-extended post-translational processing intermediates of progastrin in porcine stomach. J Biol Chem. 1985; 260(21): 11724–9. PubMed Abstract\n\nMarino L, Muglia B, Dickinson CJ: Glycine-extended post-translational processing intermediates of gastrin and cholecystokinin in the gut. Regul Pept. 1994; 50(1): 73–85. PubMed Abstract | Publisher Full Text\n\nStepan VM, Krametter DF, Matsushima M, et al.: Glycine-extended gastrin regulates HEK cell growth. AM J Physiol. 1999; 277(2 pt 2): R572–81. PubMed Abstract\n\nKaise M, Muraoka A, Seva C, et al.: Glycine-extended progastrin processing intermediates induce H+,K(+)-ATPase alpha subunit gene expression through a novel receptor. J Biol Chem. 1995; 270(19): 11155–60. PubMed Abstract\n\nHigashide S, Gomez G, Greeley GH Jr, et al.: Glycine-extended gastrom potentiates gastrin-stimulated gastric acid secretion in rats. Am J Physiol. 1996; 270(1 pt 1): G220–4. PubMed Abstract"
}
|
[
{
"id": "796",
"date": "25 Feb 2013",
"name": "Denis McCarthy",
"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": "895",
"date": "16 Apr 2013",
"name": "Mark Pritchard",
"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 has assessed the effects of gastrin and glycine-extended gastrin upon somatostatin release from rabbit fundic D cells and this investigation follows on from previous reports by the same author in which he has assessed the effects of other stimulants on somatostatin release using this experimental model. Gastrin (but not G-Gly) had an acute effect following 2h treatment, but both peptides appeared in some way to prime the cells after 20h incubation, so that basal and agonist-stimulated somatostatin release were increased. The concentrations of gastrin peptides used in these experiments were high (10nM) and it would therefore be interesting to investigate whether lower concentrations of Gastrin and G-Gly also exerted similar effects in this experimental system. It would be worth referencing the paper which confirmed high homology between the amino acid sequences of human and rabbit gastrin-17, as human peptides were used in this study. It would also be interesting to investigate whether other gastrin precursors (eg progastrin) caused similar effects.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-56
|
https://f1000research.com/articles/2-55/v1
|
19 Feb 13
|
{
"type": "Review",
"title": "The rise of testicular germ cell tumours: the search for causes, risk factors and novel therapeutic targets",
"authors": [
"Skye C McIver",
"Shaun D Roman",
"Brett Nixon",
"Kate L Loveland",
"Eileen A McLaughlin",
"Skye C McIver",
"Shaun D Roman",
"Brett Nixon",
"Kate L Loveland"
],
"abstract": "Since the beginning of the 20th century there has been a decline in the reproductive vitality of men within the Western world. The declining sperm quantity and quality has been associated with increased overt disorders of sexual development including hypospadias, undescended testes and type II testicular germ cell tumours (TGCTs). The increase in TGCTs cannot be accounted for by genetic changes in the population. Therefore exposure to environmental toxicants appears to be a major contributor to the aetiology of TGCTs and men with a genetic predisposition are particularly vulnerable. In particular, Type II TGCTs have been identified to arise from a precursor lesion Carcinoma in situ (CIS), identified as a dysfunctional gonocyte; however, the exact triggers for CIS development are currently unknown. Therefore the transition from gonocytes into spermatogonia is key to those studying TGCTs. Recently we have identified seven miRNA molecules (including members of the miR-290 family and miR-136, 463* and 743a) to be significantly changed over this transition period. These miRNA molecules are predicted to have targets within the CXCR4, PTEN, DHH, RAC and PDGF pathways, all of which have important roles in germ cell migration, proliferation and homing to the spermatogonial stem cell niche. Given the plethora of potential targets affected by each miRNA molecule, subtle changes in miRNA expression could have significant consequences e.g. tumourigenesis. The role of non-traditional oncogenes and tumour suppressors such as miRNA in TGCT is highlighted by the fact that the majority of these tumours express wild type p53, a pivotal tumour suppressor usually inactivated in cancer. While treatment of TGCTs is highly successful, the impact of these treatments on fertility means that identification of exact triggers, earlier diagnosis and alternate treatments are essential. This review examines the genetic factors and possible triggers of type II TGCT to highlight target areas for potential new treatments.",
"keywords": [
"testicular germ cell tumours",
"seminoma",
"nonseminoma",
"primordial germ cell",
"differentiation",
"CIS"
],
"content": "Introduction\n\nTesticular cancers are generally grouped into three broad categories with type I testicular germ cell tumours (TGCTs) being observed primarily in neonatal boys and young children and consisting of benign teratomas and malignant yolk sac tumours1. Type III TGCT, also called spermatocytic seminomas, affect older men above 50 years of age and are derived from a slow growing expansion of type B spermatogonia2. Type II TGCTs mostly affect men aged between 20 and 40; however, the origins of this tumour type are much earlier during foetal development3. All type II TGCTs develop from a pre-invasive lesion termed Carcinoma in situ (CIS), which has been identified as a dysfunctional foetal germ cell2,4.\n\nIn the developed world the incidence of type II TGCT (seminoma and non-seminoma), but not type I or II, has increased significantly over the last century to become the most common malignancy found in men aged between 20 and 40 years of age5–7. Such findings have led to speculation that environmental factors impact on the tumorigenesis of this cancer8. In addition, several genetic mutations have been discovered that positively impact TGCT rates in both sporadic and familial cases of TGCTs8,9. While the characterisation of risk factors is far from being completed, one of the most promising indicators of TGCT is mutations in c-KIT and KIT ligand10–13.\n\nThe perceived association of TGCTs with infertility combined with evidence that pre-existing subfertility increases in patients with type II tumours, raises the possibility that these lesions may act as indicators of a general reduction in male reproductive health and rising fertility problems within the male population14,15. Type II TGCT is known to develop from dysfunctional gonocytes located within the seminiferous tubules16, indicating that the risk factors predisposing an individual to TGCTs are active early in male foetal development, necessitating the investigation of early germ cell development in an effort to identify causative factors for type II TGCTs. In this review we examine normal germ cell development and potential areas of this differentiation that are modified during the development of type II TGCTs. We also discuss the genetic and environmental contributions to disease specification as well as risk factors and indicators of tumour progression.\n\n\nGerm cell development\n\nPrimordial germ cells (PGCs) are signalled to develop by Bone Morphogenic proteins (BMPs) secreted from the extraembryonic ectoderm and the visceral endoderm17. BMPs (BMP4, BMP2, and BMP8b), in turn activate the expression of Fragilis (IFITMS) genes17,18. In the mouse around six of the Fragilis positive cells begin to express BLIMP1/PRDM1 and PRDM14 at embryonic day (E) 6.5 and thereafter are committed to the germ line (Figure 1)19–23. BLIMP1 is a key component of germ cell specification as studies of the BLIMP1 knockout mouse have revealed that PGCs cluster, but fail to migrate24. The expression of BLIMP represses the expression of somatic genes such as HOX, FDF8 and SNAIL, in addition to DNA methyl transferases25. The combination of the highly proliferative nature of the germ cells i.e. expansion from six cells at E6.5, to 250 cells at E9.5, 1000 cells at E10.5, and 26,000 cells at E13.5, and the lack of maintenance DNA methyltransferases causes general demethylation of the DNA in primordial germ cells25,26. The methylation status, and the repression of somatic cell phenotype, permits the expression of pluripotency factors such as NANOG and OCT 3/4 (but not c-MYC and KLF - required for total pluripotency)26. SOX2, an essential pluripotency factor in mouse, is not expressed in human primordial germ cells. However, it is expressed in nonseminoma germ cell tumours26.\n\nThis is an update and adaption of the classic model of type II TGCT development first proposed by Rajpert-De Meyts93. Current understanding indicates maintained pluripotency combined with incomplete premature differentiation of gonocytes causes the specification of Carcinoma in situ (CIS) cells. Signals caused by puberty cause these cells to proliferate and once additional mutations accumulate CIS cells differentiate into overt type II TGCTs.\n\nOCT 3/4 is expressed in pluripotent cells of the inner cell mass of a blastocyst and, in contrast to the differentiating somatic cells, its expression is maintained in primordial germ cells17,27. OCT 3/4 is believed to control primordial germ cell survival, given that germ cells are lost through an apoptotic pathway in OCT 3/4 null mice17,28. In mice, SOX2 acts in conjunction with OCT 3/4 and its expression is maintained in primordial germ cells until they migrate, colonise the gonad, and become specified as pre-spermatogonia or oogonia, whereupon SOX2 is down regulated17,29. The pattern of NANOG expression tracks that of SOX2, and is required for the maturation of germ cells once they reach the genital ridges17,30. The exact roles of OCT 3/4, SOX2 and NANOG in primordial germ cells is currently unknown, but as they are important for maintaining pluripotency and proliferation in embryonic stem (ES) cells, it is considered likely that they also maintain pluripotency in germ cells and prevent their differentiation17. More recently, several other pluripotency factors have been identified in primordial germ cells, gonocytes and pre-spermatogonia20. One such protein is LIN28, which is involved in maintaining pluripotency and survival. LIN28 is located upstream of, but linked to, both OCT 3/4 and NANOG expression. Additionally LIN28 is not found in postnatal spermatogonia but is expressed in CIS, seminoma, and embryonal carcinoma, further demonstrating the importance of the differentiation of germ cells to maintain normal testis development20,31. Another pluripotency protein identified upstream of the classical pluripotency regulator OCT 3/4, and capable of interacting with NANOG, is SALL432. SALL4 is expressed late primordial germ cells, pre-spermatogonia, and spermatogonial stem cells and is hypothesised to be involved in both the maintenance of primordial germ cells as well as the differentiation of spermatogonial stem cells32,33.\n\nInterestingly, primordial germ cells are not natively pluripotent. Indeed, despite the fact that they express many pluripotency markers, they only differentiate into one cell type i.e. a germ cell26. However, pluripotency can be induced in in vitro cultures of germ cells, if they are isolated before the colonisation of the gonad and incubated in the presence of the growth factors SCF, FGF2, and LIF17. In addition, germ cells are more efficiently transformed into pluripotent cells in the presence of FFG2 in conjunction with MAK2k and GSK3B, as well as TGFB type 1 receptor inhibitors34.\n\nOnce primordial germ cells are specified, germ cell specific genes that promote cell survival, such as STELLA and NANOS3, are up-regulated (Figure 1)17. Other markers of primordial germ cells include SSEA1, PRDM14, DND1, Fragilis, LIN28, c-KIT and MVH26. DND (dead end/Ter) prevents miRNA mediated translational repression and serves as a survival factor for PGCs. Mutations in DND cause testicular teratomas and DND null mice lose their PGCs via apoptosis between E8.5 and E12.59. At E7.5 in the mouse (3 weeks in humans) PLAP (Placental Like Alkaline Phosphatase)-positive PGCs reside in the posterior of the primitive streak and become motile shortly after this time35,36.\n\nPrimordial germ cells initially migrate into the hindgut during its anterior extension (E8-9.5); they then move into the mesoderm (E9.5) and bilaterally travel to the genital ridges to contribute to the formation of the gonads (E10.5-11.5)19,37. This process is complete by E33-37 in humans37,38.\n\nSteel factor (KIT-ligand) has been identified as a key survival and proliferative signal for developing germ cells as well as acting to guide PGCs along the hindgut and towards the genital ridges39,40. The movement of PGCs out of the hindgut and into the gonads (E9.5) is dependent on E-cadherin (CDH1) and β1-integrin (ITGB1)37, and is directed by CXCL1241. On reaching the genital ridges at around E11 to E11.5, the PGCs proliferate and form gonocytes35. At this time, active demethylation continues by UTX (histone demethylase), another pluripotency factor, before the cells then undergo sex specific epigenetic changes required to produce viable germ cells25,42. By E13.5 the gonocytes enter either mitotic arrest in the case of testis, or meiotic arrest in the ovary. Therefore the period of time between the arrival in the gonad and arrest is key to the primordial germ cell proliferation and differentiation43.\n\nMale sex determination is triggered by the expression of SRY (Sex-determining region on the Y chromosome), a high mobility group (HMG) transcription factor which activates SOX9 (SRY related HMG box 9), another transcription factor which in itself is sufficient for sex determination44,45. SOX9-positive pre-Sertoli cells recruit cells from the mesonephros and the coelomic epithelium to form the testicular cords46,47 which occurs in concert with the commitment of male germ cells to the pre-spermatogonia cell fate48. Sertoli cells also secrete paracrine factors (DHH and platelet-derived growth factors) initiating the differentiation of the testosterone producing Leydig cells49.\n\nMale germ cells are maintained in mitotic arrest within the seminiferous tubules by the enzyme CYP26B1 which facilitates degradation of retinoic acid, preventing the expression of STRA8 (stimulated by retinoic acid 8) and hence entry into meiosis50. When the expression of CYP26B1 decreases at E13.5, the RNA binding protein NANOS2 maintains mitotic arrest in male germ cells51. Shortly after birth in mice, and in late gestation in humans, gonocytes (prospermatogonia) migrate from the centre to the basement membrane of the seminiferous tubules and by postnatal day 6 they have begun to divide and are designated single spermatogonia (As) or spermatogonial stem cells (SSCs) and spermatogenesis is initiated52.\n\n\nRisk factors and genetic predisposition of testicular cancer\n\nThe risk factors for type II TGCTs include family predisposition, cryptorchidism, disorders of sexual development, high maternal oestrogen during foetal development, environmental exposures, sub- or infertility, and previous TGCTs3,36,53. Additionally, geographical regions with a high rate of TGCTs have lower sperm quality as well as increased rates of cryptorchidism and hypospadias when compared to regions with low rates of TGCTs54. While not all sexual development disorders lead to an increased risk of TGCTs, they usually occur at higher frequency in less differentiated gonads55. Identified genetic factors, including deletions on the Y chromosome, androgen insensitivity, and KIT mutations; negatively impact germ cell development supporting the notion that the risk of TGCTs is established early3,36,56. It is proposed that the combination of both genetic and environmental factors prevents or delays the maturation of PGCs/gonocytes into pre-spermatogonia by retarding the development of the supporting Sertoli and Leydig cells, thus perturbing the microenvironment required for germ cell development3,57.\n\nPatients lacking the SRY gene on the Y chromosome required for Sertoli cell development, or WT1 required for ovary development, have a high risk of type II TGCTs, because of the lack of correctly differentiated support cells, which in turn inhibit germ cell development3. A partial deletion of the AZoospermia Factor C (AZFC) region (gr/gr mutation) on the short arm of the Y chromosome is also associated with an increased risk of TGCTs56.\n\nMutations in KIT signalling and continued KIT expression are seen in CIS cells, but not more advanced stages of testicular cancer, indicating that this may be an initial feature of CIS cell specification. Furthermore, genetic association studies have identified mutations in genes encoding KIT, KIT ligand and its downstream signalling molecules such as KRAS, SPRY4, and BAK1, as likely predisposition genes for TGCTs3,58–60. It is believed that a region of the Y chromosome encoding TSPY (testis specific protein Y-encoded), with a potential role in germ cell mitotic division, is required for the development of TGCTs. Interestingly, as with KIT, TPSY protein expression is lost when CIS becomes invasive36,55, suggesting that it may have a role in the initiation, but not maintenance, of tumours.\n\nThe switch to meiosis from mitotic amplification is tightly controlled in normal spermatogenesis and there is some indication that this pathway may be prematurely initiated then aborted in CIS and TGCTs61. Several members of the NOTCH signalling pathway control the mitotic to meiotic switch, and mutations in the NOTCH signalling pathway in C. elegans have been shown to elicit tumour-like expansion of germ cells61. This observation stimulated exploration of NOTCH signalling in human TGCTs and revealed that NOTCH1 and NOTCH4 are both over-expressed in CIS and seminoma. Additional genetic screening of affected individuals has identified mutations in members of the NOTCH family as primary risk factors for developing these pathologies61,62. The meiotic protein SYCP3, a component of the synaptonemal complex which aligns sister chromatids in prophase I of meiosis allowing their proper segregation, is also expressed in CIS, seminoma and embryonal carcinoma, thus providing further indication of a premature activation of meiosis in TGCTs development61.\n\nMore recent genetic screens have identified transcription factors DMRT1 and ATF7IP, as well as the telomere regulator TERT, as susceptibility genes for TGCTs58. DMTR1 is required for normal testicular differentiation and DMTR1 knockout mice have been shown to develop teratomas58. ATF7IP is a transcription factor that regulates the expression of subunits of the enzyme complex responsible for maintaining telomere length such as the active component, TERT63. Telomere regulation and length is tightly controlled within cells and usually only immortal cells, i.e. stem cells, express active telomerase. Telomerase is also extensively overexpressed in cancer cells and its activity plays a key role in the transformation process63,64.\n\nHowever, in most cases no known genetic factor contributing to the development of TGCTs is identified and therefore environmental factors may play an essential role in the development of tumours in these individuals. The rates of TGCTs within Western countries have risen sharply since the early 1900s; however, this rise is not consistent across all countries8,65. The most notable differences in TGCT incidences and rates of increase are demonstrated in Europe. For example, increased TGCT rates were documented in England, Wales, and Germany before Denmark and Norway. By contrast, Finland, Eastern Germany, and Poland, still have lower TGCT rates than surrounding countries65,66. The rapid rise in TGCT rates, as well as the variation between geographical locations, implicates exposure to one or more environmental toxicants as a key risk factor65. For instance, it is believed that xeno-oestrogens and anti-testosterones negatively affect the development of Sertoli cells and Leydig cells causing a suboptimal environment for germ cell differentiation leading to reduced fertility and development of CIS cells3. Phthalates represent another possible environmental toxicant that could contribute to the incidence of TGCTs. Indeed, exposure to these toxins is known to be highly variable between different countries and this could, in part, contribute to geographical differences in cancer rates65. Additionally, high levels of phthalates have been documented in the blood of mothers whose sons developed TGCT6. Similarly, rats exposed to phthalates exhibit symptoms similar to testicular dysgenesis, which is also associated with a high risk of TGCT65. The possibility of toxicant exposure leading to TGCT has led to the investigation of detoxification mechanisms in cancer sufferers and their mothers56. While the power of this study is compromised by the small sample size, the analysis did demonstrate a link between specific cytochrome p450 polymorphisms and an increased rate of TGCT development56.\n\n\nThe development of type II testicular germ cell tumours\n\nTGCTs originate from CIS cells, which are found on the basement membrane of abnormal seminiferous tubules that usually lack normal spermatogenesis. CIS cells were first identified in 1972 in biopsies of infertile men67, and a causal link was subsequently established when, during follow-up, the men with this lesion developed tumours while control subjects exhibited no cases of TGCT53. Furthermore, the incidence of CIS is about equal to that of type II TGCTs, suggesting that CIS cells will eventually develop into overt TGCT36.\n\nMorphologically, CIS cells resemble primordial germ cells/gonocytes and express a number of similar markers, in that they lack imprinting and express OCT 3/4, PLAP and c-KIT36,68. In fact, the transcriptome of CIS cells is extremely similar to that of isolated normal human gonocytes, demonstrating a close relationship between these cell types16. CIS cells appear to be blocked from differentiating and entering spermatogenesis3. Instead they accumulate within the seminiferous tubule to become an intratubular lesion termed either seminoma or non-seminoma (embryonal carcinoma)3,36. Interestingly, seminomas can later reprogram to become non-seminomas36. These intratubular lesions proliferate to fill the lumen of the seminiferous tubule and rely on Sertoli cell-derived growth/survival signals and Leydig cell production of testosterone36. Once these cells break their dependence on external signals they become overt testicular cancer36.\n\nInvasive TGCTs are characterised by a gain in copy number of the short arm of chromosome 123,53. Within this chromosomal region there are several genes encoding candidate virulence factors, including NANOG, KRAS2 and BCAT1; however, their role has yet to be confirmed3. BCAT1 for example, is only expressed in embryonal carcinoma and not in other germ cell lesions, indicating that the role of the genes on chromosome 12 may vary depending on the tumour type3. There are several markers which identify seminoma and non-seminoma tumours; for example, OCT 3/4 and NANOG are expressed in both seminoma and embryonal carcinoma, while SOX17 is specific to seminoma tumours and SOX2 is only expressed in non-seminoma69. In extended culture, ES cells gain excess copies of chromosome 12, but they do not seem to exhibit malignant characteristics, indicating that the gain of chromosome 12 is not the sole initiation factor for virulence in TGCTs3.\n\n\nPossible mechanisms of germ cell cancer specification\n\nSeveral risk factors have been identified in a mouse model of teratoma formation (strain 129/Ter)70. These include the continued expression of pluripotency markers and proliferation as well as premature differentiation, e.g. precocious entry into meiosis. Usually pluripotency factors such as NANOG, SOX2, and OCT 3/4, are down-regulated following cell cycle arrest at E13.5, but the germ cells of Ter mice continue to proliferate and express NANOG at E15.570. In this mouse model, PGCs also appear to prematurely differentiate with the detection of cyclin D1 as early as E13.570, compared to its normal expression in spermatogonia at postnatal day 471. It is possible that a similar mechanism occurs for the development of CIS cells considering that CIS cells express markers for primordial germ cells including PLAP, OCT 3/4, and c-KIT, long past embryonic development3. Concurrent with this delayed maturation is the expression of meiotic genes including NOTCH and SYCP3 in both CIS cells as well as seminoma cells61. Therefore maintenance of pluripotency in primordial germ cells while undergoing a defective maturation process, which may include premature activation of meiosis, could underpin the specification of these cells as CIS and tumorigenic (Figure 1).\n\n\nMetastasis\n\nPatients with stage I TGCTs and a concomitant high risk of metastasis are destined to undergo aggressive surgery and chemotherapy3. So far, vascular invasiveness, percentage of embryonal carcinoma and the proliferation index, have been the best predictors of metastasis risk. However, more recently the chemokine-mediated CXCR4 pathway has demonstrated some promise in metastasis prediction - with tumours containing localised high CXCL12 expression being less likely to metastasise3,72. Gilbert and colleagues72 examined TGCTs and found that seminomas expressed higher levels of CXCR4 transcript and protein, than normal testis, but this trend was not maintained in non-seminomas. In addition Gilbert et al.72 demonstrated that a seminoma cell line (TCam2) migrates in response to CXCL12α, via activation of the MAP kinase pathway. However, the non-seminoma cell line 2102EP does not, which is not surprising, as this cell line does not express CXCR4. We have independently looked at the expression of CXCL12 and CXCR4 in TGCTs (McIver SC, Loveland KL, Roman SD, Nixon B, Kitazawa R, McLaughlin EA, unpublished observations) and confirmed that CXCR4 mRNA was overexpressed in seminomas. However, at the protein level we did not find elevated levels of CXCR4 when its expression was examined via immunohistochemistry. Additionally, the tight correlation between CXCL12/CXCR4 with MAP kinase activation that is found in normal testis was abolished in the TGCT samples. We were able to confirm that both CXCL12α and β caused cell migration in the seminoma cell line (TCam2) while no migration response was observed in the CXCR4 positive non-seminoma cell lines 833ke and NTera2/D1. Therefore the expression of CXCL12 and CXCR4 is more likely to be a better indicator of the possibility of seminoma metastasis rather than non-seminoma.\n\n\nTreatment resistance\n\nGenerally seminoma cells (a category that includes CIS cells) are extremely sensitive to irradiation as well as cisplatin-based chemotherapy drugs, while non-seminomas only respond to chemotherapy3. However, the mature teratoma components of non-seminoma tumours are resistant to DNA damage therapies, which is consistent with the loss of embryonal cell characteristics these tumour exhibit3. Although the common chemotherapy and radiotherapy treatments are very effective and maintain quality of life, they can lead to infertility, hypogonadism and retrograde ejaculation, which new treatment options should seek to avoid2,15. Both seminoma and non-seminoma tumours can become resistant to traditional oncology treatments3. Interestingly, evidence suggests that the methylation status of the cells DNA controls the expression of specific genes, such as c-FLAR, which inhibits caspase-dependant apoptosis, thus imparting resistance to cisplatin treatment3. Several other genes have been implicated in cisplatin resistance including Cyclin D13. Cyclin D1 is expressed in murine germ cells from post natal day 3, which coincides with the appearance of spermatogonia in testis and therefore may control the proliferation and differentiation of these cells71. The differentiation and abnormal proliferation of gonocytes is essential to the development of CIS, and therefore Cyclin D1 may play a key role in CIS evolution.\n\n\nRole of miRNA in testicular germ cell tumours\n\nA particularly exciting recent finding is that different types of TGCTs can be distinguished on the basis of their distinct miRNA expression profiles. For example, miR-122a is only expressed in yolk sac tumours69,73. Also the regulator of miRNA maturation, LIN28, has been shown to be expressed in PGCS, gonocytes and pre-spermatogonia CIS, seminoma and non-seminoma, where it regulates totipotency, and functions upstream of the tumour/pluripotency transcription factors OCT1/3 and NANOG31.\n\nThe tumour suppressor p53 generally controls the exit from the cell cycle upon DNA damage, to allow for either repair or apoptosis. It follows that p53 mutation is usually a key step in tumorigenesis; however, in TGCTs p53 is only rarely inactivated74. Voorhoeve et al.75 provided an explanation for this phenomenon when they determined that the presence of microRNAs; miR-372 and miR-373; conveyed a growth advantage and protected against the cellular senescence response to DNA damage, in the presence of wild type p53. Analysis of the molecular basis for this effect revealed that these miRNA molecules act in conjunction with RAS downstream of p53 in order to mimic the phenotype of inactivated p5375. Furthermore, the authors identified LATS2 (Large tumour suppressor homology 2) as a potential target of p53 signalling given that LATS2 deletion accelerates cellular proliferation and tumour development. This can be phenocopied in some seminomas, which do not overexpress miR-372 or miR-373, by mutations in LATS2. The LATS2 protein inhibits cyclin E/CDK2 activity and thereby arrests the cell cycle75. Later studies found that this miRNA cluster is exclusively over expressed in almost all seminomas and most embryonal carcinoma tumours74.\n\nIn addition to their role in the tumorigenesis of TGCTs, miRNAs have an essential role in the development of primordial germ cells and in spermatogenesis76. Therefore we decided to examine the change in the miRNA expression profile between postnatal gonocytes and spermatogonia in mice in the hope of gaining a better understanding of this essential step in both normal spermatogenesis and CIS development. This approach led to the identification of seven differentially expressed miRNA molecules (three of which were up regulated; miR-136, -743a and -463*, and four down regulated miR290-5p, -291a-5p, -294* and -293) during this developmental phase. Analysis of their potential targets indicated that these miRNA molecules were likely to impact the PTEN and Wnt/β catenin signalling pathways77 as well as the CXCR4 signalling pathway previously implicated in TGCT metastasis (McIver SC, Loveland KL, Roman SD, Nixon B, Kitazawa R, McLaughlin EA, unpublished observations). The PTEN tumour suppressor is known to inhibit PI3K signalling to negatively regulate cell growth and therefore its suppression causes increased proliferation and potentially tumourigenesis. The loss of PTEN is also associated with the transformation of CIS cells into overt cancerous tumours78. Furthermore, increased relapse rate in TGCTs has also been found to be associated with the loss of PIK3IP1, an additional negative regulator of PIK kinase79. Both the PTEN and Wnt/β catenin pathways converge to control the function of Cyclin D1, which is expressed in highly proliferative TGCTs where down-regulation instigates cell cycle arrest80, and is associated with chemotherapy resistance in these tumours3. Indeed, on this basis, the control of Cyclin D1 activity has been suggested as a potential treatment target80. Subsequently we selected the three most highly differentially expressed miRNA (Mir-743a, -291a-5p, -293) and instigated a strategy to directly identify their targets. The use of a pull down approach pioneered by Orum and Lund81 in which the miRNAs were used as bait, and bound mRNA analysed by microarray, revealed a significant number of predicted and non-predicted targets bound to each miRNA (McIver SC, Roman SD, Nixon B, McLaughlin EA., unpublished observations).\n\nInterestingly the targets identified were postulated to regulate key germ cell migration and proliferation pathways controlled through PDGF82 and RAC83 signalling (McIver SC, Roman SD, Nixon B, McLaughlin EA, unpublished observations). PDGF signalling is known to play a role in gonocyte migration and proliferation82,84. In addition, PDGF signalling has been shown to work in conjunction with oestrogen signalling84, a mechanism that is easily hijacked by the presence of xeno-oestrogens. In fact gonocytes have been shown to be more sensitive to xeno-oestrogens than endogenous ligands84. This has implications for the development of CIS because high concentrations of xeno-oestrogens are associated with increased TGCT risk3. RAC on the other hand has been shown to be essential for the transmigration of spermatogonial stem cells through the blood-testis barrier during testis colonisation assays83. Using a knockdown assay as an alternate technique to determine the targets of miR-291a-5p, -293 and -743a we identified one promising gene, IGFBP7 (Insulin-like growth factor binding protein 7) (McIver SC, Roman SD, Nixon B, McLaughlin E A, unpublished observations), which is known to act as either a positive (e.g. in glioma85) or negative regulator (e.g. in breast and liver cancer86,87) of cell proliferation and migration depending on the environment in which it is found. Therefore the disrupted expression of IGFBP7 by aberrantly expressed miRNAs during the gonocyte to spermatogonia transition could have implications for their growth and differentiation.\n\nThe miRNA molecules found to be down regulated during gonocyte differentiation into spermatogonia all belong to the miR-290 family. This cluster of miRNA is highly expressed in embryonic stem cells (ES) and is known to be a key regulator of ES cell pluripotency, controlling the expression of OCT4, SOX2 and NANOG88–90. Additionally, upon knockout of the miR-290 cluster, aberrant migration of germ cells has been observed with significantly fewer primordial germ cells reaching the bipotential gonad88. Comparatively less is known about the miRNA molecules that were found to be upregulated during gonocytes differentiation. miR-136 is proposed to be a tumour suppressor in glioma and is capable of targeting the anti-apoptosis genes AEG-1 and BCL-291. miR-743a on the other hand is involved in oxidative stress responses92. On the basis of these data, as well as our own observations of miRNA involvement in key germ cell regulatory pathways77, it appears likely that the changing miRNA profile between postnatal gonocytes and spermatogonia could have a fundamental role in the development of CIS within the testis.\n\n\nConcluding remarks\n\nResearch into both the genetic and environmental factors that predispose an individual to type II TGCTs has been hampered by the lack of a suitable animal model for the study this tumour type. Mouse models are suitable for the study of type I neonatal TGCTs and canines exhibit type III TGCTs caused by an expansion of type B spermatogonia. These models offer a number of advantages in terms of allowing genetic screens and treatments to be conducted to enhance our understanding of these cell types36. In contrast, the study of seminoma and non-seminoma tumours is restricted to cell lines and, in the case of seminoma, these are scarce36, severely limiting explorative experiments to study these tumour types. Type II TGCTs have been extensively characterised at the molecular level resulting in the identification of several predisposition genes, tumour markers, as well as the mode of tumour progression3. However, despite our extensive knowledge of mature tumours, the exact triggers for the development of CIS cells remain unknown. Unfortunately, until these mechanisms are identified, therapeutic interventions are limited. Current treatment techniques are very effective but their side effects often include a loss of fertility, a concerning fact given that these tumours primarily arise in men of reproductive age15. Therefore, a better understanding of the cellular mechanisms underlying germ cell development, are vital to establish novel treatments that are capable of preserving fertility in patients.",
"appendix": "Author contributions\n\n\n\nSM performed the initial research. SM, EM and BN drafted the manuscript. SR and KL performed critical review and editing of the manuscript. All authors provided input to the revision of the article 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 authors gratefully acknowledge the financial assistance to EM, SR and BN by the Australian Research Council, National Health and Medical Research Council and the University of Newcastle. SCM is the recipient of an Australian Postgraduate Award PhD scholarship and a University of Newcastle Vice Chancellors Post Graduate Award. This work was supported by the Australian Research Council Centre of Excellence in Biotechnology and Development (CE0348239) to EAM and SDR and a Fellowship from the NHMRC (ID545916 to KL).\n\n\nAcknowledgements\n\nThe authors would like to thank Jessie Southerland for her helpful and insightful comments on the manuscript.\n\n\nReferences\n\nFrazier AL, Weldon C, Amatruda J: Fetal and neonatal germ cell tumors. Semin Fetal Neonatal Med. 2012; 17(4): 222–230. PubMed Abstract | Publisher Full Text\n\nBahrami A, Ro JY, Ayala AG: An overview of testicular germ cell tumors. Arch Pathol Lab Med. 2007; 131(8): 1267–1280. PubMed Abstract\n\nLooijenga LH, Gillis AJ, Stoop H, et al.: Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance. Int J Androl. 2011; 34(4 Pt 2): e234–e251. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiang W, Xiang C, Cazacu S, et al.: Insulin-like growth factor binding protein 7 mediates glioma cell growth and migration. Neoplasia. 2008; 10(12): 1335–1342. PubMed Abstract | Free Full Text\n\nAmemiya Y, Yang W, Benatar T, et al.: Insulin like growth factor binding protein-7 reduces growth of human breast cancer cells and xenografted tumors. Breast Cancer Res Treat. 2011; 126(2): 373–384. PubMed Abstract | Publisher Full Text\n\nChen D, Yoo BK, Santhekadur PK, et al.: Insulin-like growth factor-binding protein-7 functions as a potential tumor suppressor in hepatocellular carcinoma. Clin Cancer Res. 2011; 17(21): 6693–6701. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMedeiros LA, Dennis LM, Gill ME, et al.: mir-290-295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects. Proc Natl Acad Sci U S A. 2011; 108(34): 14163–14168. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZheng GX, Ravi A, Calabrese JM, et al.: A latent pro-survival function for the mir-290-295 cluster in mouse embryonic stem cells. PLoS Genet. 2011; 7(5): e1002054. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZovoilis A, Pantazi A, Smorag L, et al.: Embryonic stem cell-related miRNAs are involved in differentiation of pluripotent cells originating from the germ line. Mol Hum Reprod. 2010; 16(11): 793–803. PubMed Abstract | Publisher Full Text\n\nYang Y, Wu J, Guan H, et al.: MiR-136 promotes apoptosis of glioma cells by targeting AEG-1 and Bcl-2. FEBS Lett. 2012; 586(20): 3608–3612. PubMed Abstract | Publisher Full Text\n\nShi Q, Gibson GE: Up-regulation of the mitochondrial malate dehydrogenase by oxidative stress is mediated by miR-743a. J Neurochem. 2011; 118(3): 440–448. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRajpert-De Meyts E: Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects. Hum Reprod Update. 2006; 12(3): 303–323. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "790",
"date": "21 Feb 2013",
"name": "Anis Feki",
"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": "966",
"date": "28 May 2013",
"name": "John Schjenken",
"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 is a review discussing testicular germ cell tumours and how they may be triggered with an emphasis on miRNA expression. There are a few minor grammatical and spelling errors throughout this review but these do not impact on the quality of the manuscript. I would like to point out however that my area of expertise is primarily miRNA biology and I do not have a great background in TGCT.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-55
|
https://f1000research.com/articles/2-53/v1
|
15 Feb 13
|
{
"type": "Data Article",
"title": "Cocaine self-administration in mice with forebrain knock-down of trpc5 ion channels",
"authors": [
"Matthew B Pomrenze",
"Michael V Baratta",
"Kristin C Rasmus",
"Brian A Cadle",
"Shinya Nakamura",
"Lutz Birnbaumer",
"Donald C Cooper",
"Matthew B Pomrenze",
"Michael V Baratta",
"Kristin C Rasmus",
"Brian A Cadle",
"Shinya Nakamura",
"Lutz Birnbaumer"
],
"abstract": "Canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that play a crucial role in modulating neuronal excitability due to their involvement in intracellular Ca2+ regulation and dendritic growth. TRPC5 channels a) are one of the two most prevalent TRPC channels in the adult rodent brain; b) are densely expressed in deep layer pyramidal neurons of the prefrontal cortex (PFC); and c) modulate neuronal persistent activity necessary for working memory and attention. In order to evaluate the causal role of TRPC5 in motivation/reward-related behaviors, conditional forebrain TRPC5 knock-down (trpc5-KD) mice were generated and trained to nose-poke for intravenous cocaine. Here we present a data set containing the first 6 days of saline or cocaine self-administration in wild type (WT) and trpc5-KD mice. In addition, we also present a data set showing the dose-response to cocaine after both groups had achieved similar levels of cocaine self-administration. Compared to WT mice, trpc5-KD mice exhibited an apparent increase in self-administration on the first day of cocaine testing without prior operant training. There were no apparent differences between WT and trpc5-KD mice for saline responding on the first day of training. Both groups showed similar dose-response sensitivity to cocaine after several days of achieving similar levels of cocaine intake.",
"keywords": [
"trpc5",
"cocaine",
"knock-down"
],
"content": "Introduction\n\nThe prefrontal cortex (PFC) supports higher order cognitive functions, such as decision-making, reasoning, and working memory1. PFC functioning is impaired in cocaine addicts, which is manifest in their inability to make proper decisions when presented with challenges in their environment2,3. Genetic and environmental factors can influence PFC excitability4–6, and repeated cocaine alters the excitability of the PFC by biasing neurons towards strong inputs, such as those associated with drug cues, which may diminish cognitive function7. Understanding the mechanism underlying how PFC excitability influences the behavioral responses to psychostimulants is fundamental to learning how to reverse these maladaptive alterations in order to treat addiction.\n\nCanonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that play a crucial role in modulating neuronal excitability due to their involvement in intracellular Ca2+ regulation8. The TRPC5 isoform has been shown to play a role in dendritic growth and arborization through CaMKII-mediated mechanisms throughout the brain9,10, as well as the expression of fear conditioning in the amygdala11. TRPC5 channels a) are one of the two most prevalent TRPC channels in the adult rodent brain12; b) are densely expressed in deep layer pyramidal neurons of the PFC12; and c) modulate neuronal persistent activity necessary for working memory and attention13.\n\nSince deep-layer pyramidal neurons of the PFC are known to project to limbic structures that subserve reward, such as the ventral tegmental area, nucleus accumbens, amygdala, laterodorsal tegmentum14, and the rostromedial tegmental nucleus15, TRPC5 channels may influence the ability of cortical networks to exert inhibitory control over these structures. Consequently, motivational and drug reward-seeking behaviors may be affected. In the present data sets, we gathered data from mice that lack functional TRPC5 channels in their forebrain CaMKII-expressing pyramidal neurons to measure their cocaine self-administration behavior as an index of cocaine reward.\n\n\nMaterials and methods\n\n19 adult (25–30 g) male C3H mice were group-housed until surgery. Mice were maintained in a reverse 12 hr light:dark cycle (lights off at 7:00 am) with access to food and water ad libitum. Using the cre-lox system, forebrain specific knock-down of trpc5 was achieved by crossing floxed trpc5 mice with mice that express Cre recombinase under the control of the αCaMKII promoter, where Cre transgene expression was restricted to excitatory neurons in the forebrain. Breeding pairs of the floxed trpc5 mice were initially obtained from Dr. Lutz Birnbaumer at the NIEHS and bred at the Institute for Behavioral Genetics. CaMKII-Cre mice were obtained from UTSW Medical Center, Dallas. All procedures were approved by the Institute for Animal Care and Use Committee of the University of Colorado Boulder.\n\nPrior to behavioral experimentation, mice were anaesthetized with a cocktail of 80 mg/mL ketamine and 6 mg/mL xylazine (Sigma Aldrich) and implanted with intravenous catheters as previously described16. Chronically implanted custom catheters consist of Silastic tubing that is affixed to 23-gauge steel tubing bent at a right angle and inserted into a plastic hypodermic needle hub bound to a circular polyurethane backmount and surgical mesh. Catheters were steam autoclaved and rinsed with 70% ethanol prior to surgery. The skin area on the back and above the jugular vein were shaved and prepared with Betadine scrub, 70% ethanol, and 1% Zephiran to prevent infection. Following incisions and exposure of the right external jugular vein, catheter tubing was channeled subcutaneously from the back out the chest above the exposed vein. Catheter tubing was inserted approximately 7 mm into the vein and secured to the vein and surrounding tissue with sterile suture. Following successful insertion and jugular attachment, the incisions were sutured, stapled, and fixed with Vetbond (3M). The neck of the needle hub contains the 23-gauge tubing that remains capped when animals are not connected to the intravenous self-administration apparatus. Catheters were flushed with heparinized pyrogen-free sterile physiological saline daily to detect resistance to flow and patency. If animals’ nose-poking behavior deviated by >20% of mean responding, catheter integrity and access to the jugular vein was examined using 10 mg/kg Sodium Brevital. If animals did not exhibit sedation within 3 seconds they were omitted from the study. Requests for the customized mouse catheter system used in this study should be directed to http://neuro-cloud.net/nature-precedings/pomrenze16.\n\nSeven days following catheter implantation mice (Saline - trpc5-KD (n = 8), trpc5-WT (n = 8); Cocaine - trpc5-KD (n = 9), trpc5-WT (n = 10)) were individually housed in self-administration operant chambers that contain two identical nose-poke portals (active and inactive). For acquisition and maintenance of cocaine (unit dose = 0.75 mg/kg/infusion; compounded in pyrogen-free sterile physiological saline; NIDA) self-administration, mice received continuous reinforcement (fixed-ratio 1) of cocaine paired with a 10-second LED illumination and 10-second time-out following a nose-poke into the “active” portal. Inactive portals yielded no consequence. All studies were done without prior operant training. Infusions of 50 uL were delivered over a 4-second time period. For the data set presented mice were exposed to a 3-hr saline self-administration pretest on the first day. Subsequent cocaine daily 3-hr sessions continued until stable responding (> 20 infusions, < 20% variability in number of infusions across three daily sessions, > 70% discriminative responding in “active” portal vs “inactive” portal) was achieved in both groups. All genotypes were blind to the investigators.\n\nAfter acquisition of cocaine self-administration and stable maintenance for ≥ 6 days, mice (trpc5-KD (n = 9), trpc5-WT (n = 10)) were challenged with a dose-response schedule of varying unit doses (0.05, 0.1, 0.75, and 2.0 mg/kg/infusion) of cocaine (one unit dose per session). The mean number of cocaine infusions at each unit dose was determined during two separate consecutive sessions.\n\n\nResults\n\nAll mice were exposed to the self-administration chambers to nose-poke for light and saline on their first day of training to establish their baseline levels of exploratory behavior between treatment groups. Cocaine replaced saline thereafter and mice were trained to self-administer cocaine (0.75 mg/kg/infusion) without prior operant training for natural rewards (food pellets). Mice acquired cocaine self-administration by meeting our stated criteria and subsequently exhibited stable self-administration maintenance for ≥ 6 days. WT and trpc5-KD mice demonstrated identical cocaine responding during the maintenance phase. The trpc5-KD mice reached the criteria by the first session and continued stable responding for the duration of the experiments, whereas WT took several days to catch up to KD responding. WT and trpc5-KD mice exhibited similar responding for saline, yet infusions earned by trpc5-KD surpassed WT for cocaine on the first session (Figure 1a). The mouse trpc5-KD nose-poking showed a similar pattern on the first session, surpassing WT nose-pokes (Figure 1b).\n\nA. Mean (± SEM) number of saline (1 session) and cocaine (6 sessions) infusions. Saline responding is similar between genotypes, yet trpc5-KD mice exhibit an increased responding on day 1 for cocaine. Saline-trpc5-KD (n = 8) versus Saline-trpc5-WT (n = 8), Cocaine-trpc5-KD (n = 9) versus Cocaine-trpc5-WT (n = 10). B. Mean (± SEM) number of saline (1 session) and cocaine (6 sessions) nose-poking in active (rewarding) and inactive (non-reinforced) portals. Nose-poking for saline is similar between genotypes, yet trpc5-KD poking is significantly elevated on day 1 of responding for cocaine and continues throughout the sessions. Black circles represent trpc5-KD active poking, open circles represent trpc5-WT active poking, triangles represent trpc5-KD inactive poking, squares represent trpc5-WT inactive poking.\n\nAfter the maintenance phase of self-administration training, separate groups of mice were taken through a dose-response. The sessions leading up to the dose response tests demonstrate similar responding between genotypes (Figure 2a). Dose-response functions demonstrated no difference between genotypes (Figure 2b).\n\nA. Mean (± SEM) number of infusions for the last two self-administration sessions prior to the dose-response challenge are similar between trpc5-KD and WT mice. Cocaine-trpc5-KD (n = 9) versus Cocaine-trpc5-WT (n = 10). B. Mean (± SEM) number of infusions for 2 sessions at each indicated cocaine dose. Dose-response curves reveal similar responding to varying unit doses of cocaine between genotypes.",
"appendix": "Author contributions\n\n\n\nMBP, MVB, and DCC designed the experiments, summarized the data, and wrote the manuscript. KCR conducted the mouse genotyping. BAC constructed the customized self-administration system and contributed to the conceptual design. SN programmed the custom Labview scripts for self-administration and contributed to the conceptual design and contributed to the revision of the manuscript. MBP and MVB performed the experiments and contributed to the conceptual design and contributed to the revision of the manuscript. LB designed and generated the floxed trpc5 mice and contributed to the conceptual design and contributed to the revision of the manuscript. DCC revised the manuscript for critical intellectual content. All authors approve this manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by R01 DA24040 (DCC); T32 DA017637 (MVB); Z01 ES101684 (LB).\n\n\nAcknowledgements\n\nThe authors would like to thank Dr. Melissa Fowler for her contributions to the TRPC5 project, including the characterization of TRPC5 expression in the rodent brain and the development of mRNA probes. We also thank Sam Dolzani for insightful commentary and suggestions for surgical and behavioral procedures.\n\n\nReferences\n\nKesner RP, Churchwell JC: An analysis of rat prefrontal cortex in mediating executive function. Neurobiol Learn Mem. 2011; 96(3): 417–31. PubMed Abstract | Publisher Full Text\n\nTrantham H, Szumlinski KK, McFarland K, et al.: Repeated cocaine administration alters the electrophysiological properties of prefrontal cortical neurons. Neuroscience. 2002; 113(4): 749–53. PubMed Abstract | Publisher Full Text\n\nGoldstein RZ, Volkow ND: Drug addiction and its underlying neurobiological basis: neuroimaging evidence for the involvement of the frontal cortex. Am J Psychiatry. 2002; 159(10): 1642–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUhl GR, Drgon T, Jhonson C, et al.: Molecular genetics of addiction and related heritable phenotypes: genome-wide association approaches identify \"connectivity constellation\" and drug target genes with pleiotropic effects. Ann N Y Acad Sci. 2008; 1141: 318–81. PubMed Abstract | Publisher Full Text\n\nHaile CN, Kosten TR, Kosten TA: Genetics of dopamine and its contribution to cocaine addiction. Behav Genet. 2007; 37(1): 119–45. PubMed Abstract | Publisher Full Text\n\nNiwa M, Yan Y, Nabeshima T: Genes and molecules that can potentiate or attenuate psychostimulant dependence: relevance of data from animal models to human addiction. Ann N Y Acad Sci. 2008; 1141: 76–95. PubMed Abstract | Publisher Full Text\n\nKalivas PW, Volkow N, Seamans J: Unmanageable motivation in addiction: a pathology in prefrontal-accumbens glutamate transmission. Neuron. 2005; 45(5): 647–50. PubMed Abstract | Publisher Full Text\n\nMontell C: The TRP superfamily of cation channels. Sci STKE. 2005; (272): re3. PubMed Abstract | Publisher Full Text\n\nHe Z, Jia C, Feng S, et al.: TRPC5 channel is the mediator of neurotrophin-3 in regulating dendritic growth via CamKIIα in rat hippocampal neurons. J Neurosci. 2012; 32(27): 9383–95. PubMed Abstract | Publisher Full Text\n\nPuram SV, Riccio A, Koirala S, et al.: A TRPC5-regulated calcium signaling pathway controls dendrite patterning in the mammalian brain. Genes Dev. 2011; 25(24): 2659–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiccio A, Li Y, Moon J, et al.: Essential role for TRPC5 in amygdala function and fear-related behavior. Cell. 2009; 137(4): 761–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFowler MA, Sidiropoulou K, Ozkan ED, et al.: Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain. PloS One. 2007; 2(6): e573. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSidiropoulou K, Lu FM, Fowler MA, et al.: Dopamine modulates an mGluR5-mediated depolarization underlying prefrontal persistent activity. Nat Neurosci. 2009; 12(2): 190–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSesack SR, Carr DB, Omelchenko N, et al.: Anatomical substrates for glutamate-dopamine interactions: Evidence for specificity of connections and extrasynaptic actions. Amm N Y Acad Sci. 2003; 1003: 36–52. PubMed Abstract | Publisher Full Text\n\nBarrot M, Sesack SR, Georges F, et al.: Braking dopamine systems: A new GABA master structure for mesolimbic and nigrostriatal functions. J Neurosci. 2012; 32(41): 14094–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPomrenze MB, Baratta MV, Cadle BA, et al.: Cocaine self-administration in the mouse: A low-cost, chronic catheter preparation. Nature Precedings. 2012. Publisher Full Text"
}
|
[
{
"id": "792",
"date": "22 Feb 2013",
"name": "Simon Barak Caine",
"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 nicely conducted study. Note three important reservations regarding interpretation and conclusions: Acquisition studies are notorious for variability, and differences between genotypes were observed only on Day 1 of acquisition and throughout the large majority of the study were identical for the two genotypes; A slight hyperactivity especially under reinforced conditions is a fairly common observation with a variety of manipulations, and nose pokes in Figure 1B are suggestive of such a phenomenon (i.e., higher nose pokes during timeouts as well as during reinforcer availability in the active nose poke hole for KD mice than for WT);There are no data addressing whether similar results would be obtained with other reinforcers, such as food.",
"responses": [
{
"c_id": "375",
"date": "27 Feb 2013",
"name": "Donald Cooper",
"role": "Author Response",
"response": "We thank Dr. Caine for his helpful critique of our Data article. One goal we had for publishing this Data article in F1000Research was to quickly share some of our ongoing behavioral datasets in order to encourage collaboration with others in the field. Our ultimate goal is to publish these datasets together with more complete analysis and conclusions as well as other work from our lab or new collaborators. Because we intend on publishing these datasets with additional data and more complete analyses and conclusions we kept the analyses and conclusions here to a minimum. Reviewer Concern: Acquisition studies are notorious for variability. We certainly agree. To address this issue we took the following approach. 1. The study was conducted with the experimenter blind to the genotype. 2. We reported 9 knock-down and 10 wild-type male age-matched and litter-mate controls that made it through our lengthy testing procedure including dose-response and progressive ratio test (not reported). We have done further analysis on mice that self-administered on Day 1 of cocaine acquisition but whose catheters failed during the next 3 weeks of testing. This brought our numbers up to 12 knock-down and 12 wild-type animals. The Day 1 cocaine means are now trpc5 KD = 27.75 with a standard deviation of 5.3 and wild-type=14.1 with a standard deviation of 2.0. This gives an effect size of 3.39 and a statistical power estimate at 1.0. For behavioral studies a statistical power greater than 0.8 is considered good. So we are confident that there is a difference between groups on Day 1 of cocaine acquisition that is not due to chance alone although ANOVA and posthoc tests are needed and will be presented in a future complete research article. Reviewer Concern:The issue of hyperactivity to a reinforcerNose poking for light in mice is a behavior that is easy to learn because of the naturally reinforcing property of light activation triggered by a beam break in the nose poke hole. To address the issue of general hyperactivity:1. We reported nose poke responding for light and found no differences between genotypes on Day 1 of acquisition (Figure 1). Active nose pokes were higher than inactive nose pokes indicating discrimination in both groups. 2. There were no differences between genotypes in the inactive nose poke port on Day 1 of cocaine acquisition, a general measure of exploratory activity. 3. Other studies in the laboratory have looked at locomotor activation to i.p injections of a locomotor activating dose of cocaine at 10 mg/kg and found no differences between genotypes (data not shown). In addition, a subthreshold dose of 5 mg/kg cocaine or saline also show no differences between genotypes (data not shown). This suggest no group differences in cocaine-induced locomotor activation in response to an initial exposure to cocaine.4. The 4 second duration cocaine infusion time and the 10 second time out likely explains the increase in active nose pokes compared to the number of infusions. However, the ratio of active pokes to infusions was only about 2. Both groups had similar (1.5 -2 active pokes/infusion) differences at steady-state responding (days 3-6) once acquisition criteria was reached. Future analysis will include a within session analysis of nose pokes to determine if the trpc5 KD mice showed differential nose-poke behavior during the 3 hour session (e.g. more nose pokes/infusion during the first part or last part of the session). Furthermore, we plan to examine the ratio of active pokes to infusions across the entire dose response to see if the ratio is dose-dependent. We will include this data set in a revision of the current Data Article.Reviewer Concern: Would we observe similar results with other reinforcers?Although we tested light as an alternative reinforcer it is a very good question whether other consumable natural reinforcers like sucrose would be similar to cocaine on Day 1. These experiments are underway and will be reported with expanded analysis in a future Research Article together with cocaine conditioned place preference, progressive ratio responding and locomotor activity.We welcome any new suggestions from other reviewers or the community at large!"
}
]
},
{
"id": "816",
"date": "07 Mar 2013",
"name": "Michela Marinelli",
"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 study, showing the role of TRPC5 channels in the forebrain, on cocaine self-administration. The experiments are described well. The dose-response curve is convincing, as it shows the characteristic inverted-U-shaped curve. In line with the journal’s guidelines, no statistical results and/or interpretations are provided for any result. However, this makes it difficult to assess the reliability of data, so my comments address this concern.An analysis of drug intake using repeated measures ANOVA with genotype as between-subject factor and days (cocaine 1-6) as the repeated measure does not yield any significant effects (effect of genotype, main effect of days, or interaction genotype x days). Only a t test on day 1 yields a significant genotype effect. I therefore understand that the authors refer to an “apparent” initial increase in drug intake (abstract). It would be important to replicate this finding in the future, to ascertain that it is a true result, rather than variability in initial intake across mice.An analysis of active/inactive hole discrimination using ANOVA with genotype as between-subject factor and days (cocaine 1-6) and hole (active/inactive) as the repeated measure yields a trend for an interaction genotype x hole (p=0.068). It therefore appears that the -/- might have better discrimination. This could be reported. The authors could also consider if there is insufficient power for this difference to be significant.The animals were trained first with saline. Could this have influenced their subsequent intake, and could the initial difference in intake (day 1 of cocaine) be the consequence of differences in the ability to switch across genotypes?From the data, it appears that not all mice were tested for initial saline intake. Could this have influenced the results?The authors should describe (methods) how they ran the dose-response curve (descending doses? Random doses? Etc…)",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-53
|
https://f1000research.com/articles/2-33/v1
|
06 Feb 13
|
{
"type": "Research Article",
"title": "A mutation in negative regulator of basal resistance WRKY17 of Arabidopsis increases susceptibility to Agrobacterium-mediated genetic transformation",
"authors": [
"Benoît Lacroix",
"Vitaly Citovsky",
"Vitaly Citovsky"
],
"abstract": "Agrobacterium is a phytopathogenic bacterium that induces crown gall disease in many plant species by transferring and integrating a segment of its own DNA (T-DNA) into its host genome. Whereas Agrobacterium usually does not trigger an extensive defense response in its host plants, it induces the expression of several defense-related genes and activates plant stress reactions. In the complex interplay between Agrobacterium and its host plant, Agrobacterium has evolved to take advantage of these plant defense pathways for its own purpose of advancement of the infection process. For example, Agrobacterium utilizes the host stress response transcriptional regulator VIP1 to facilitate nuclear import and proteasomal uncoating of its T-DNA during genetic transformation of the host cell. In Arabidopsis, the VIP1 gene expression is repressed by WRKY17, a negative regulator of basal resistance to Pseudomonas. Thus, we examined whether WRKY17 is also involved in plant susceptibility to genetic transformation by Agrobacterium. Using reverse genetics, we showed that a wrky17 mutant displays higher expression of the VIP1 gene in roots, but not in shoots. In a root infection assay, the wrky17 mutant plants were hyper-susceptible to Agrobacterium compared to wild type plants. WRKY17, therefore, may act as a positive regulator of Arabidopsis resistance to Agrobacterium. This notion is important for understanding the complex regulation of Agrobacterium-mediated genetic transformation; thus, although this paper reports a relatively small set of data that we do not plan to pursue further in our lab, we believe it might be useful for the broad community of plant pathologists and plant biotechnologists.",
"keywords": [
"WRKY17",
"Arabidopsis",
"Agrobacterium"
],
"content": "Introduction\n\nThe WRKY protein family is composed of at least 74 members in Arabidopsis thaliana1; they act as transcriptional regulators and participate mainly in the control of gene expression involved in the plant stress response, and, particularly, in the induction of gene expression by pathogen-derived elicitors. Arabidopsis WRKY17, together with another family member WRKY11, is a negative regulator of the basal defense response2. The wrky17 and wrky11 genes are usually induced during the defense response, and Arabidopsis loss-of-function mutants wrky17 and wrky11 display higher expression of numerous stress- or defense-related genes and show increased resistance to infection by Pseudomonas, but not by other pathogens. Thus, wrky17 and wrky11 have been suggested to play a role in the fine-tuning of the defense response, avoiding the effect of excessive reaction2.\n\nAmong the target genes of wrky17/wrky11 is vip1, which is overexpressed in both wrky11 and wrky17 mutants2. VIP1 is a multifunctional bZIP transcription factor that stimulates stress- and defense-related gene expression by binding to a specific DNA hexamer motif present in many promoters that respond to activation of the MPK3 pathway3, including the PR1 pathogenesis-related gene4. VIP1 might also be involved in other stress-dependent regulation pathways, such as osmosensory signaling5. Interestingly, the VIP1-related defense responses are activated during Agrobacterium-host plant interactions, and Agrobacterium has evolved to subvert them to facilitate the infection process4,6.\n\nVIP1, a host protein initially discovered as an interacting partner of the Agrobacterium T-DNA packaging protein VirE27, is involved in several critical aspects of plant genetic transformation by Agrobacterium. Specifically, VIP1 is thought to facilitate nuclear import of the T-DNA-protein complexes7–9, their targeting to the host chromatin10–12, and proteasomal uncoating of the T-DNA molecule from its associated proteins prior to integration13–15. Thus, we investigated one of the VIP1-controlling WRKY mutants, wrky17, in regard to vip1 expression and the potential effects on Agrobacterium infection.\n\n\nResults and discussion\n\nA previous microarray analysis of the wrky17 mutant identified a number of upregulated genes2, one of which, VIP1, represents a major player in plant genetic transformation by Agrobacterium7,10,13. However, microarray analyses of gene expression, although commonly used, often yield divergent data16,17 and, therefore, require direct confirmation by detection of the specific transcripts. Thus, we analyzed the wrky17 mutant for the levels of VIP1 expression.\n\nFirst, we examined three different lines of Arabidopsis plants derived from the wrky17-1 mutant2 for the presence of the WRKY17 transcript using RT-PCR. Figure 1A shows that whereas the wild-type plants produced WRKY17 mRNA, neither of the mutant lines accumulated detectible levels of this transcript. Next, we investigated the effect of the wrky17 mutation on the expression of the VIP1 gene. Using RT-PCR, we analyzed the levels of the VIP1 transcript in plant roots (Figure 1B) and shoots (Figure 1C). The VIP1 transcription activity was substantially higher in the roots of all three wrky17 mutants than in those of wild type plants (Figure 1B). Unexpectedly, we detected no changes in VIP1 expression in the shoots of the same plants, which accumulated VIP1 transcripts in amounts similar to those in the wild-type plants (Figure 1C). Analysis of ACTin2-specific transcripts detected similar amounts of PCR products in all samples, indicating equal efficiencies of the RT-PCR reactions (Figure 1B, C). Collectively, these data suggest that WRKY17 represents one of the transcriptional regulators of the VIP1 gene, but that this regulation is tissue-specific.\n\n(A) WRKY17 expression in whole plants. (B, C) VIP1 expression in roots and shoots, respectively. WT, wild-type plants; 7, 12, and 13 are the three different lines of the homozygous wrky17-1 mutant.\n\nThis is consistent with the previous observations of differential regulation of VIP1 expression during plant development as well as in response to various stimuli. For example, VIP1 transcription is activated upon induction of cell division18, after osmotic stress, and is differentially expressed in different tissues of Arabidopsis5. WRKY17 functions as a transcription inhibitor of several genes involved in plant defense pathways1. Our results suggest that VIP1 is one of the target genes down-regulated, directly or indirectly, by WRKY17 in tissue-specific fashion. Alternatively, VIP1 expression in the shoot tissue could be regulated by additional factors which mask the effect of the WRKY17 knock-out mutation.\n\nOnce we had identified plant tissue showing a clear effect of WRKY17 on VIP1 expression, we investigated whether this effect altered susceptibility to Agrobacterium infection. To this end, we employed the classical Arabidopsis root infection assay19, in which the efficiency of infection is monitored and quantified by measuring the level of transient T-DNA expression, that is early expression of the invading T-DNA molecules before their stable integration in the host genome. Root segments from the wild-type and wrky17 plants were inoculated with Agrobacterium strain EHA105 harboring the binary plasmid pBISN1 with the β-glucuronidase (GUS) gene expression reporter in its T-DNA region. T-DNA expression was quantified based on the percentage of root segments exhibiting GUS histochemical staining. These experiments revealed that T-DNA expression frequencies in roots of all three wrky17 mutant lines were 30–50% higher than those measured in roots of the wild-type plants (Table 1 and Figure 2).\n\nTransformation efficiency is expressed as the percent of GUS-stained roots from the total number of roots tested. All data represent average values of three independent experiments with indicated standard deviations. WT, wild-type plants; 7, 12, and 13 are the three different lines of the homozygous wrky17-1 mutant.\n\nPercentage (number of GUS positive root segments/total number of root segments).\n\nThe increased susceptibility of the wrky17 roots to Agrobacterium infection correlates with elevated transcription levels of the VIP1 gene in this tissue. Considering the known role of VIP1 as an enhancer of Agrobacterium infectivity7–15, it is likely that higher VIP1 expression in roots of the wrky17 mutant is responsible for the increased susceptibility to Agrobacterium. This notion is consistent with our earlier observations that overexpression of VIP1 in tobacco further elevates transformation efficiency8. That we detected this effect of the wrky17 mutation using a transient T-DNA expression assay indicates that increased VIP1 expression affects the early steps of the infection process, i.e., those that occur prior to T-DNA integration and stable expression.\n\n\nConclusion\n\nWe show here that the wrky17 mutant displays elevated VIP1 expression in its roots as well as increased susceptibility to Agrobacterium-induced genetic transformation. This correlation allows a new insight into the interactions between Agrobacterium and its host plants. Specifically, this interaction appears to be affected negatively by WRKY17 such that the infection process is enhanced in the loss-of-function wrky17 mutant. Thus, WRKY17 may represent one of the host factors that elevate resistance to Agrobacterium infection in different plant species and tissues that may vary widely in their susceptibility to Agrobacterium20,21. This is unlike the known role of WRKY17 as a negative regulator of plant resistance to Pseudomonas2. Although this paper reports a relatively small set of data that we do not plan to pursue further in our lab, we believe its publication will be useful for the broad community of plant pathologists and plant biotechnologists.\n\n\nMaterials and methods\n\nArabidopsis thaliana plants, wild-type (ecotype Col0) or wrky17-1 T-DNA insertion mutants (obtained from D. Roby, CNRS Montpellier, France), were grown either in soil or on Gamborg’s B5 medium (20 g.L-1 sucrose, 8 g.L-1 agar), after seed surface sterilization. All plants were grown in an environment-controlled growth chamber at 22°C under long day (16h light/8h dark) conditions. Three lanes of homozygous plants (lanes 7, 12, 13) were isolated from the original wrky17-1 stock.\n\nTotal RNA was extracted from plant tissues using Trizol (Invitrogen), and cDNA synthesis was performed with a RevertAid cDNA synthesis Kit (Fermentas) according to the manufacturer’s instructions. Transcript levels were then estimated by PCR, with 30 cycles of amplification. The resulting cDNA was PCR-amplified for 30 cycles using primers specific for the tested gene or for ACTIN2 as an internal control of a constitutively expressed gene. The following primer pairs were used: 5´ATGACCGTTGATATTATGCGTTTAC3´/5´TCAAGCCGAACCAAACACCAAAC3´ that amplify the full length 966-bp WRKY17 (At2g24570) cDNA, 5´ATGGAAGGAGGAGGAAGAGG3´/5´TCAGCCTCTCTTGGTGAAATCC3´ that amplify the full length 1,026-bp VIP1 cDNA, and 5´ATGGCTGAGGCTGATGATATT3´/5´TTAGAAACATTTTCTGTGAACGATTCC3´ that amplify the full length 1,134 bp ACTIN2 (At3g18780) cDNA.\n\nAll infection assays were performed as described by Gelvin (2006)19 with the Agrobacterium tumefaciens strain EHA105 (from S. Gelvin, Purdue University, USA), harboring a pBISN1 binary plasmid with an intron-containing GUS reporter gene that is not expressed in bacteria22. One-cm long root segments were excised from 3–4 week-old Arabidopsis plants grown on Gamborg’s B5 medium, and bundles of root segments were placed on the MS (Murashige and Skoog) medium. For each experiment, roots were pooled from more than 20 plants and divided into three bundles, each containing more than 100 root segments. Root bundles were overlaid with EHA105 harboring pBISN1 suspension culture at A600 = 0.25 in NaCl 0.9%, and excess liquid was removed by pipette aspiration after 15 min of incubation. Root segments were then incubated for two days at 22°C under the long day conditions, rinsed in water containing 100 mg.L-1 timentine (BioWorld) to eliminate bacteria, and incubated for an additional three days on the MS medium supplemented with timentine. Root segments were then subjected to the GUS histochemical assay23, with overnight incubation at 37°C, and the number of root segments displaying GUS staining was recorded.",
"appendix": "Author contributions\n\n\n\nBL designed experiments, performed experiments, analyzed data and wrote manuscript; VC designed experiments, analyzed data and wrote manuscript. Both authors approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work in our laboratory is supported by grants from United States Department of Agriculture/National Institute of Food and Agriculture (USDA/NIFA) 2008-01012, National Institutes of Health (NIH) R01 GM50224, National Science Foundation (NSF) MCB 1118491, United States-Israel Binational Science Foundation (BSF) 2011070 and United States-Israel Binational Agricultural Research and Development Fund (BARD) IS-4237-09C, to Vitaly Citovsky.\n\n\nAcknowledgments\n\nWe thank Dr Dominique Roby (INRA, Toulouse, France) for the gift of Arabidopsis wrky17 mutant seeds.\n\n\nReferences\n\nEulgem T, Somssich IE: Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol. 2007; 10(4): 366–371. PubMed Abstract | Publisher Full Text\n\nJournot-Catalino N, Somssich IE, Roby D, et al.: The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana. Plant cell. 2006; 18(11): 3289–3302. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPitzschke A, Djamei A, Teige M, et al.: VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression. Proc Natl Acad Sci USA. 2009; 106(43): 18414–18419. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDjamei A, Pitzschke A, Nakagami H, et al.: Trojan horse strategy in Agrobacterium transformation: abusing MAPK defense signaling. Science. 2007; 318(5849): 453–456. PubMed Abstract | Publisher Full Text\n\nTsugama D, Liu S, Takano T: A bZIP protein, VIP1, is a regulator of osmosensory signaling in Arabidopsis. Plant Physiol. 2012; 159(1): 144–155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaltsman A, Krichevsky A, Kozlovsky SV, et al.: Plant defense pathways subverted by Agrobacterium for genetic transformation. Plant Signal Behav. 2010; 5(10): 1245–1248. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTzfira T, Vaidya M, Citovsky V: VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity. EMBO J. 2001; 20(13): 3596–3607. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTzfira T, Vaidya M, Citovsky V: Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIP1. Proc Natl Acad Sci USA. 2002; 99(16): 10435–10440. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCitovsky V, Kapelnikov A, Oliel S, et al.: Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro. J Biol Chem. 2004; 279(28): 29528–29533. PubMed Abstract | Publisher Full Text\n\nLacroix B, Loyter A, Citovsky V: Association of the Agrobacterium T-DNA-protein complex with plant nucleosomes. Proc Natl Acad Sci USA. 2008; 105(40): 15429–15434. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi J, Krichevsky A, Vaidya M, et al.: Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium. Proc Natl Acad Sci USA. 2005; 102(16): 5733–5738. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLoyter A, Rosenbluh J, Zakai N, et al.: The plant VirE2 interacting protein 1. A molecular link between the Agrobacterium T-complex and the host cell chromatin? Plant Physiol. 2005; 138(3): 1318–1321. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTzfira T, Vaidya M, Citovsky V: Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature. 2004; 431(7004): 87–92. PubMed Abstract | Publisher Full Text\n\nZaltsman A, Krichevsky A, Loyter A, et al.: Agrobacterium induces expression of a plant host F-box protein required for tumorigenicity. Cell Host Microbe. 2010; 7(3): 197–209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMagori S, Citovsky V: Agrobacterium counteracts host-induced degradation of its F-box protein effector. Sci Signal. 2011; 4(195): ra69.PubMed Abstract | Publisher Full Text\n\nvan der Spek PJ, Kremer A, Murry L, et al.: Are gene expression microarray analyses reliable? A review of studies of retinoic acid responsive genes. Genomics Proteomics Bioinformatics. 2003; 1(1): 9–14. PubMed Abstract\n\nShi L, Campbell G, Jones WD, et al.: The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models. Nat Biotechnol. 2010; 28(8): 827–838. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAvivi Y, Morad V, Ben-Meir H, et al.: Reorganization of specific chromosomal domains and activation of silent genes in plant cells acquiring pluripotentiality. Dev Dyn. 2004; 230(1): 12–22. PubMed Abstract | Publisher Full Text\n\nGelvin SB: Agrobacterium transformation of Arabidopsis thaliana roots: a quantitative assay. Methods Mol Biol. 2006; 343: 105–113. PubMed Abstract | Publisher Full Text\n\nGelvin SB: Agrobacterium-mediated plant transformation: the biology behind the \"gene-jockeying\" tool. Microbiol Mol Biol Rev. 2003; 67(1): 16–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLacroix B, Tzfira T, Vainstein A, et al.: A case of promiscuity: Agrobacterium’s endless hunt for new partners. Trends Genet. 2006; 22(1): 29–37. PubMed Abstract | Publisher Full Text\n\nLiu CN, Li XQ, Gelvin SB: Multiple copies of virG enhance the transient transformation of celery, carrot, and rice tissues by Agrobacterium tumefaciens. Plant Mol Biol. 1992; 20(6): 1071–1087. PubMed Abstract | Publisher Full Text\n\nJefferson RA, Kavanagh TA, Bevan MW: GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987; 6(13): 3901–3907. PubMed Abstract | Free Full Text"
}
|
[
{
"id": "754",
"date": "07 Feb 2013",
"name": "Herman Scholthof",
"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": "759",
"date": "08 Feb 2013",
"name": "Kiran Mysore",
"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 very short report regarding a finding that may be important for some researchers. Even though the science is acceptable several things need to be changed. Genetic transformation normally refers to stable transformation. The authors have only looked at transient transformation which may or may not be stable (they could have tested this). Therefore, please change the title and the abstract to indicate transient transformation rather than just saying genetic transformation. All wild-type gene names should be in capital (e.g., WRKY17, WRKY 11, VIP1 etc,.). Only the mutants should be in small letters (e.g., wrky17).The authors mention that the VIP1 gene is induced substantially in the wrky17 mutant. However, to my eyes the induction is subtle (probably 2-3 fold). They could have done a better quantification using real-time RT-PCR. Please remove the word “substantial”.Please give the concentration of the Agrobacterium used for infection in CFU.I believe the antibiotic used should be “timetin” and not “timentine”",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-33
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https://f1000research.com/articles/2-50/v1
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14 Feb 13
|
{
"type": "Study Protocol",
"title": "The Perinatal Adverse events and Special Trends in Cognitive Trajectory (PLASTICITY) - pre-protocol for a prospective longitudinal follow-up cohort study",
"authors": [
"Laura Hokkanen",
"Jyrki Launes",
"Katarina Michelsson",
"Jyrki Launes",
"Katarina Michelsson"
],
"abstract": "Prospective follow-up studies on long term effects of pre- and perinatal adverse conditions in adulthood are rare. We will continue to follow the prospective cohort of initially 1196 subjects with predefined at-delivery risk factors out of 22,359 consecutive deliveries during 1971-74 at a single maternity hospital. The risk cohort and 93 controls have been followed up with a comprehensive clinical program at 5, 9, and 16 years of age and by questionnaire at the age of 30 years. Major medical events known to affect the development and growth of the brain, or cognitive functions and personality have been documented. Here we present a pre-protocol for the project, which we will call PLASTICITY, whose aim is to follow consenting subjects and controls into mid-adulthood and beyond, and to explore how the neonatal risk factors modulate neurodevelopmental and neurodegenerative processes such as learning disabilities, ADHD, aging, early onset mild cognitive impairment and even dementia. Our first focus is on the neurological and cognitive outcomes at age 40 years, using detailed neurological, neuropsychological, neuroimaging, genetic, blood chemistry and registry based methods. Results will be expected to offer information on the risk of neurological, psychiatric, metabolic and other medical consequences as well as the need for health and social services at the brink of middle age, when new degenerative phenomena are known to emerge. The evaluation at age 40 years will serve as a baseline for later aging studies. We welcome all comments and suggestions, which we will apply in finalizing details and inviting collaboration.",
"keywords": [
"adhd"
],
"content": "Introduction\n\nWe present here pre-protocol of the \"PerinataL Adverse events and Special Trends In Cognitive TrajectorY\" (PLASTICITY) study. The study aims to test the hypothesis that perinatal adverse events exert an unexpectedly deleterious effect on the brain at middle and older age. This is a prospective longitudinal at-risk cohort study of a 1971–1974 birth cohort that has been prospectively followed to adulthood1. Several papers, theses, and book chapters about the findings in children up to the age of 16 years have been published based on this material2–11. Therefore, we know the individuals that are in different outcome groups and etiologies. All consenting subjects are aimed to be studied now in their 40s and throughout their remaining lives at 10 or 5 year intervals.\n\nThis paper describes our current plans on how this cohort is to be followed-up. An enormous amount of work has been done in the first decades of this follow-up study of individuals from at-risk deliveries. We feel that the unique opportunity of completing the follow-up of such a cohort from birth to death must not be missed. However, we acknowledge that without collaboration, we cannot achieve all that could and should be done. Therefore, we welcome all comments and suggestions, which we will apply in finalizing the study details and inviting collaboration. While a general schedule is outlined here, the exact timetable will depend on methodological, administrative, and financial decisions.\n\n\nBackground\n\nAntenatal and perinatal risks of infant death or disability are well known. These include intrauterine growth restriction, ischemia/hypoxia/asphyxia12 due to many reasons, jaundice13,14, infection, drugs and maternal disorders15,16. There is a wealth of general literature about the diagnosis, etiology, treatment, social consequences and individual outcomes, and the range of conditions reaches from mild defects to cerebral palsy, serious cognitive deficits and death2,12,17–30. The incidence of death or considerable disability is 0.2 to 5 out of 1000 live births in hypoxic-ischemic encephalopathy31,32, intrauterine growth restriction23,33–36, or jaundice21. The outcome has been improving steadily, e.g. according to the Official Statistics of Finland (http://tilastokeskus.fi/til/kuol/tau.html), infant mortality has decreased over 70% from 12.7 by 1000 births in 1971 to 2.7 by 1000 births in 2007.\n\nThe timing, type, and severity of the long term consequences of antenatal and perinatal complications have mostly been studied in relatively short follow-up studies3,8,9,17,19,23,28,32,33,36–41. Most of them cover the period up to early school age, with possibly some overemphasis on cerebral palsy due to its juridical importance in many cases42,37. In studies focusing on cognitive, neuropsychiatric and social performance, the longest follow-up periods currently extend into young adulthood33,43,44. Long term studies report diminished IQ and/or scholarly achievement but these are studies that are either retrospective or rely on data from secondary sources such as tests for conscripts13,45,46. Recent results of a retrospective longitudinal study using more specific cognitive tests indicate impaired psychomotor speed, learning and executive functioning in young adults with very low birth weight43,47. Many very interesting and ambitious longitudinal prospective cohort studies have been started recently (see for instance http://www.birthcohorts.net) but the subjects in the actively followed-up prospective birth risk cohorts we are aware of48–53 will not reach an age when aging-related changes have a serious effect for decades. To the best of our knowledge, no results of prospective follow-up beyond young adulthood exist to date.\n\nThe types and extent of structural abnormalities caused by intrauterine growth restriction and/or asphyxia are well known in seriously disabled children. It has been possible to investigate the more subtle changes in brain structures in vivo only relatively recently. So far there are relatively few MRI studies25,41,54–60 of children who were exposed to adverse conditions in utero or perinatally. However, a wide variety of subtle abnormalities resembling those caused by many diseases in adult life, especially in the brain white matter, have been discovered. The current knowledge about either normal or pathological aging emphasize the role of white matter changes both in degenerative and vascular pathology, e.g. white matter lesions are independent risks in both ischemic stroke, vascular dementia, and also degenerative dementias. The time course of these changes is as yet very poorly known.\n\nThe incidence of Attention Deficit Hyperactivity Disorder (ADHD) is known to be higher among the subjects with pre- and perinatal risks61,62. In a recent study, low birth weight, preterm birth, and low Apgar scores were reported to increase the risk of ADHD up to 5-fold63. Other syndromes of childhood developmental disabilities include reading disorder/dyslexia, non-verbal learning disorder, dyscalculia, and disorders of motor coordination; entities that often overlap and coincide. The etiology of many of these developmental disorders involves the interaction of multiple risk and protective factors, both genetic and environmental64–66. Follow-up studies suggest that in a large number of children ADHD persists in adulthood, but the range in the reported frequencies is wide, 5–66%67. The symptoms may change during lifespan, hyperactivity becoming less common68,69 in adulthood. Prospective follow-up studies spanning into the ages of 30–40 are extremely few and none so far reach beyond 4070,71.\n\nAs people reach adulthood and beyond, they become susceptible to the neuronal effects of ageing. Based on both large population based cohorts and clinical follow-up studies, most cognitive scores appear to decline from the age of 45 onwards, with faster decline in older people72,73. Along with the normal ageing process, pathological processes also start evolving, and the distinction is clinically not easy to make. Mild Cognitive Impairment (MCI) refers to a preclinical stage that converts to dementia as the disease progresses74,75. On a clinical level MCI is a useful concept but neuropathologically it is complex and inadequately understood76. The classical markers of Alzheimer's disease (AD) neuropathology start to appear in 40-year-olds77 with a clear correlation to cognitive functioning78. Terms describing preclinical states of AD (including both \"asymptomatic at-risk state for AD\" and \"presymptomatic AD\") refer to the long asymptomatic stage between the earliest pathogenic events/brain lesions of AD and the first appearance of specific cognitive changes79,80. There is evidence that mid-life levels of cardiovascular risk factors (such as elevated blood pressure, cholesterol and smoking) increase the risk for diseases affecting cognition that emerge 20 years or more after the risk factor is measured81. The role of the vulnerability factors that have been present from the neonatal period in this progression is not known.\n\nApproximately 8% of all dementia cases are in working age (from 30 to 65 years)82 with the estimated prevalence among the 45–64 year age group being 98.1 per 100,00083. In both late and early onset dementias AD is the most common cause but frontotemporal degeneration and hereditary forms of dementia are more prevalent in the early onset group82,84. The significance of rarer and ‘disregarded’ pathologies to late-onset dementias has recently been explored in an epidemiological study85 but the neuropathological mechanisms of early onset dementias are not fully understood. The significance of perinatal and early childhood events in relation to MCI and dementia are unknown, although asphyxia and preterm birth are listed as risk factors. Based on statistics only, approximately 300 subjects in our birth cohort will develop dementia86,87.\n\n\nRationale\n\nOur aim is to identify and study the type and severity of changes that can be revealed by neurological, cognitive, psychiatric, neuroimaging, and neurophysiological techniques as well as metabolic and genetic analyses in a cohort of subject with predefined neonatal adverse events by means of a careful and lifelong follow-up. The results will be compared to those of peers born healthy to test our hypothesis that birth complications may cause undue damage to the central nervous system at a later age.\n\nThe rationale is based on the hypothesis of lowered cognitive reserves following perinatal adverse events which would lead to susceptibility for later damage. The concept of brain reserve is based on the protective potential of anatomical features such as brain size, neuronal density and synaptic connectivity88 and it can be seen as passive, postulating that there is a fixed threshold below which functional impairment will occur88,89. In contrast, behavioral brain reserve, or cognitive reserve, is an active construct, suggesting that the brain actively attempts to cope with brain damage by using pre-existing cognitive processes or by enlisting compensatory processes89,90. Cognitive reserve is not fixed at any point in life; instead, complex interactions exist between genetics, environmental influences, and the ability to actively compensate for the effects of pathology. Further, it has been suggested that the possible neural implementation of cognitive reserve be subdivided into two components that can also be studied using neurophysiological methods: neural reserve, which refers to individual differences in cognitive processing paradigms and neural networks that are in use in the brain, and neural compensation, which refers to alterations in cognitive processing networks that may take place in order to cope with brain pathology89. The risk cohort serves as a model for studying both the expression of cognitive reserve on later neurological conditions, and for evaluating the neural reserve and compensatory mechanisms.\n\nCognitive reserve has typically been estimated by means of autobiographical data such as socioeconomic status, occupational complexity, educational level, and mentally stimulating leisure activity, in addition to specific measures of IQ. A considerable number of cohort studies have shown the protective effects of these variables in incident dementia (see reviews91–93). Both exercise and cognitive stimulation regulate factors that may increase neuronal plasticity, and there is evidence to suggest that environmental enrichment might act directly to prevent or slow neurodegenerative disorders and permit normal cognitive functioning even in the presence of brain pathology94. Similar modulation probably exists in neurodevelopmental disorders, and these factors will be included in the study paradigm. Figure 1 illustrates the rationale of the project PLASTICITY.\n\nPhenotypes of the developmental disorders are suggested to modify the phenotypes of age-related degeneration while individual, genetic and environmental factors interact. Perinatal adverse events may potentially pose a lifelong burden by decreasing the cognitive reserves of the individual. Genetic and epigenetic variables set the framework for both early development and the ageing process. Environmental factors may initiate both a positive and a negative cycle while education and rehabilitation can enhance neural reserves and neural compensation.\n\n\nAims and objectives\n\nIn the present project, several parallel approaches will be applied.\n\n1) Etiology based approach: Study the neurological, neuropsychological and neuropsychiatric outcome in adulthood in relation to antenatal- and perinatal events that have been established in follow-up of our subjects (e.g. asphyxia, low birth weight, hyperbilirubinemia, maternal diabetes).\n\n2) Syndrome based approach: Assess long term effects of commonly recognized neurodevelopmental deficits (such as dyslexia or ADHD), to explore the associated neuropsychological, neurological and neuropsychiatric symptoms in adulthood, and deepen and broaden existing knowledge of such symptoms.\n\n3) MCI at the age of 40: Recognize the potentially elevated risk for cognitive decline in the group of adults with a history of pre- and perinatal risk factors; to study the prevalence of MCI as well as early onset dementia in the working age. Special emphasis will be given to individuals, who have been diagnosed with asphyxia and/or have white matter findings in MRI.\n\n4) Cognitive follow-up: Assess how the different developmental deficits and factors affect and transform into different varieties of MCI/dementia during later age (repeated evaluation cycles at the age of 50, 60, 65, 70, 75 etc. years).\n\n5) Radiological follow-up: Define neuroradiological findings in adults with pre- and perinatal risk factors; to clarify the structural lesions in adulthood and to use this information as a baseline for the MCI/dementia follow-up.\n\n6) Genetic analyses: Analyze the known risk factors of neurological and psychiatric traits (e.g. APOE, DISC1, COMT, ROBO1 to name a few) to be used as covariants in the assessment of disease risk in the whole cohort; genome-wide association study (GWAS) of clearly defined traits, including dyslexia, specific forms of cognitive decline, MRI parameters (leukoaraiosis, regional atrophy etc.). As medical genetics is at the moment the fastest developing of all modalities, this approach will have to be continuously reevaluated.\n\n7) Metabolic and endocrine effects: Measure metabolic and endocrine functions to be used as important cofactors in statistical analysis. Previous data suggests that low birth weight, preterm delivery and asphyxia may cause various metabolic effects e.g. diabetes and other endocrine abnormalities. On the other hand, these are known to influence the normal as well as the pathological aging process independently.\n\n8) Protective brain plasticity: Acknowledge the capacity of the brain for plasticity and neural compensation. The length and type of education, amount of special education and rehabilitation, and participation in cognitively stimulating hobbies, exercise and other activities indicating cognitive enrichment will be assessed. We also aim to test the cognitive reserve hypothesis directly by studying the efficiency of neural networks with evoked potential and functional imaging techniques in a subsample of subjects.\n\n9) Adjustment, lifestyle, psychiatric comorbidities and quality of life: Recognize the potential for psychological and psychiatric concerns. Data from questionnaires will be combined with registry data from national social and health registers as well as with the relevant clinical data.\n\n\nDesign and subjects\n\nThe basis of this study is the 1971–1974 birth cohort from a Helsinki metropolitan area maternity hospital (Kätilöopisto hospital) that has been prospectively studied up to adulthood1. There were 22,359 consecutive births which accounted for approximately 10% of all births in Finland during that time. At birth, 1196 (5.4%) presented with at least one predefined risk, see Table 1.\n\nFirst column (at inclusion) lists the total numbers of cases in each risk category; a case may appear in several categories. Second column (at 5 yrs) lists the numbers of cases with one risk factor only, and the number of cases with multiple risk factors is given separately.\n\n1 Note that 2000 g was considered a low birth weight, but 1500 g is used in some analyses.\n\n2 Number of cases where significant ischemia/hypoxia was diagnosed after inclusion in the study.\n\nOf the 1196 infants, 158 died before the age of 5. Additionally, 25 were severely disabled and were excluded from further analyses. At the first follow-up at 5 years, 67 could not be traced and 101 were unable to participate, therefore 845 children (462 boys, 383 girls) were re-assessed (at least 83% of those alive and not severely disabled)3. Asphyxia is defined as any brain damage caused by direct ischemia or ischemia induced inflammatory response. These are the patients who were included in the study according to the criteria above and who simultaneously had objective findings indicating probable inadequate brain blood supply.\n\nAt the age of 9 years, 748 children were re-assessed and at the age of 16 years, a survey using a mailed questionnaire about neurodevelopmental symptoms was conducted. There were 521 responders. Of these, 142 children were clinically examined (mainly those with observed deficits at the age of 5 or at 9 years). At the age of 30 years, a survey was again conducted and 509 subjects responded.\n\nDetails of clinical examination and other assessments are given in Table 2.\n\nSee footnote for the complete names of the tests. Group sizes (n), RG = risk cohort group, CG = control group.\n\nITPA = Illinois Test of Psycholinguistic Abilities, TOMI = Test of Motor Impairment, WISC = Wechsler Intelligence Scale for Children, WAIS = Wechsler Adult Intelligence Scale, WMS = Wechsler Memory Scale, CBCL = Child Behavior Checklist, YSL = The Youth Self Report.\n\n1 attended the clinical examination.\n\nA control group of 58 children born in uncomplicated deliveries at the same maternity hospital has been followed from the age of 5, and 111 additional children from the age of 9 years of age. Out of the total 169 control cases, 93 returned the questionnaire at the age of 30 years. The control subjects were born at the same maternity hospital during the study period and mostly attended the same primary schools. Hospital records have been reviewed to confirm absence of any perinatal risk factors.\n\nPresently, the youngest subjects in the cohort are 39 and the oldest are 42 years of age. A full clinical follow-up will be performed during 2013–14, before the oldest subjects turn 43.\n\nThe goal is to enroll as many as possible of the original risk cohort, even if they had not been able to participate in some of the earlier follow-up cycles. All surviving cases in the cohort will be contacted with the exception of the severely disabled and those who did not express consent in the survey at 30. We estimate to be able to recruit at least 60% of the entire surviving risk cohort (n = 1038) for the clinical assessment, based on the responses of the 509/845 cases who have already consented to follow-up.\n\nThe control group included 93 cases at the time of the previous survey and we hope to be able to recruit at least 60. This longitudinal control group is important because they have shared the early life experiences, school and social circumstances with the risk cohort.\n\nFor the purposes of future follow-ups, a new control group in addition to the longitudinal controls is needed due to attrition and accumulating differences in social and economic surroundings. The new control group will therefore be recruited from the spouses of the risk cases because they share the current social environment and living conditions.\n\nAttrition is a problem in all longitudinal studies. In Finland, as well as in other Nordic countries, requests to participate in research projects are usually met with a positive attitude and inclusion and dropout rates are known to be quite acceptable95. Also, people to be contacted for recruitment are easily found through national registries. Still, specific measures to encourage subjects’ motivation to continue in the project are needed. Strategies to increase retention will be actively sought and good examples from ongoing projects will be followed (such as49). These include, for instance, lowering the threshold to participate (compensation for travel costs, compensation for lost time, reminders to return questionnaires and flexible schedules for visits), inducing gains and benefits from participating (individual feedback on the medical results), as well as generating a general sense of availability and openness (websites, dissemination of the general results, contact opportunities via phone and email). Subjects will, however, not be paid for participation.\n\n\nMethods and outcomes in childhood\n\nThe database contains detailed data about the family’s social and economic status, maternal risk factors, family genetic traits, medical data about delivery and delivery complications, child’s growth and medical follow up at 5, 9 and 16 years. Additional Child Health Centre information was collected at the ages of 6 months, 12 months, 18 months, 24 months and 4 years. Surveys have included both parents’ and teachers’ questionnaires as well as self-reports, such as the Child Behavior Check List (CBCL)96 and Youth Self Report (YSR)97. A structured neurological assessment of the children was carried out using the Neurodevelopmental Screen developed by Michelsson et al.4, a modification of the test of Bax and Whitmore98 that also includes items from the Berges-Lezine imitation of gestures as well as Gubbay99 test. Other standardized tests and measures used over the course of the follow-up include the Stott Test of Motor Impairment100, Neurological \"Soft signs\" in Adolescence101 Dubowitz developmental screening test102, The Illinois Test of Psycholinguistic Abilities103 (ITPA Finnish version104), Goodenough Draw-a-person test105, Frostig Developmental Test of Visual Perception106, subtests from the Wechsler Intelligence Scale for Children107 (WISC Finnish version108) and subtests from the Wechsler Adult Intelligence Scale109 (WAIS Finnish version110). At the age of 16 part of the cohort was also assessed with more detailed neuropsychological instruments including the Benton Visual Memory Test111 and subtests from the Wechsler Memory Scale112 (WMS Finnish version113). Table 2 shows the various measures categorized by function as well as the age of the subject when tested.\n\nAdult outcome was surveyed with a questionnaire about education and work history, medical and social wellbeing as well as cognitive and psychiatric symptoms at 30 years of age. The questionnaire also included the Barkley Current Symptoms Scale as well as the Childhood Symptoms Scale11.\n\nThe initial purpose of the study was to follow up newborn children with perinatal risk factors into adolescence to estimate the impact of low birth weight, bilirubin etc. on later development. Outcome measures were divided into 1) Medical, e.g. mortality, major disabilities, anomalies, learning disabilities; 2) Psychometric, e.g. development of the linguistic, cognitive and motor skill as assessed by standardized tests; 3) Achievement based, e.g. school performance, education, and work status; and 4) behavioral and social parameters.\n\nThe first results of the prospective follow-up research project were described in 19781. Perinatal mortality in the risk cohort was 5.35% and it accounted for 83% of all perinatal deaths in that hospital during the study period 1971–74. Except for hyperbilirubinemia, which was less frequent in 1974, there was no marked change in the risk profile from 1971 to 19741, indicating that no major breakthrough in treatment success occurred during that time.\n\nThe neurodevelopmental screening test performed at 5 years revealed the highest impairment scores in children with neonatal neurological disorders – which most likely had ischemic etiology – and lowest in those with neonatal septic infections3. After the 5 year assessment, 42% of the children were referred for further assessment and/or rehabilitation measures such as special kindergartens, speech therapy, psychologist assessment or neuropediatric rehabilitation in a specialized centre115.\n\nOf the children with a birthweight of 1500 g or less, 50% died during the first 6 months6. Of those surviving, 12.3% had severe motor, mental or sensory disabilities and even those without were found to have impaired motor function, speech defects and impaired school achievement more often than the controls6. The children with a birth weight of 2000 g or less showed a similar but milder picture: mortality during the first 6 months was 28%, severe disabilities were present in 9%, and those without severe disabilities were found to show more impairment in neurodevelopmental screening examinations and in psychological and articulatory tests at the age of 5 years compared to controls. According to the teachers’ assessment at the age of 9 years, they were more often in need of special education compared to the controls5. Also the children with neonatal hyperbilirubinemia managed less well in neurological and psychological tests at the age of 5 years. They had poorer school grades and more often attended special classes for somatically or mentally disabled at the age of 9 years, but their results were still often better than in the rest of the risk group11. The children born during 1972–73 were analyzed for minor and major congenital anomalies. Those with anomalies were found to perform worse in cognitive and motor tests at the age of 9 compared to the other children in the risk group10. The number of anomalies in the risk group was comparable with the control group but there were more small for gestational age children in the anomaly group than in the non-anomaly group10.\n\nA specific feature in a longitudinal study such as this one is the change in the diagnostic criteria over the years. The initial aim of the prospective study design was to trace children who showed signs of minimal brain dysfunction (MBD), a term which at the time incorporated both behavioral and learning disturbances and various combinations of deficiencies in perception, language, memory, attention, impulse and motor control116,117. The Diagnostic and Statistical Manual of Mental Disorders (DSM)-II in 1968 included a concept of ‘‘hyperkinetic reaction of childhood’’ and in the following version, DMS-III in 1980, this was substituted with attention-deficit with or without hyperactivity. MBD as a diagnostic or even descriptive term was mostly discarded thereafter.\n\nThe prevalence of hyperactivity at the age of 9 was reported to be higher in the study cohort than in the controls10. When the current DSM-IV criteria were retrospectively applied to the childhood data, it was estimated that the cohort includes 122 cases with ADHD (attention deficit hyperactivity disorder) and their long term outcome will be published separately.\n\nThe current DSM-IV recognizes three ADHD subtypes, predominantly inattentive, predominantly hyperactive-impulsive, and a combined subtype. A new version, DSM-V is expected to be published in 2013, and the diagnostic criteria may again change. Old diagnostic groups will be retained in the database but classification of the subjects is constantly updated based on new emerging criteria.\n\n\nMethods and outcomes in adulthood\n\nWith the exception of the subgroup with suspected disorders who were contacted at 16, the majority of the cohort subjects have not been clinically evaluated after the age of 9. None have undergone MRI scans. It is therefore essential to thoroughly assess the whole group in order to have exact data on the adult outcome.\n\nIn the next cycle of assessment at 40 years of age, we are interested in the long term outcome of the developmental disorders dyslexia and ADHD in particular. We are also interested in whether the perinatal risk factors are associated with acquired neurological disorders. Particularly we want to explore the vascular system of the brain, focusing on the subjects with perinatal asphyxia. Later in midlife, at ages 50 to 60 years, the focus will gradually shift towards neurodegenerative disorders, and the study outline will later be updated accordingly.\n\nThe study outcomes, which are considered relevant for the risk group in middle age at 40 years, and also to 50 and 60 years assessment cycles, are outlined in Table 3.\n\nNeurological and medical examination will include e.g. structured neurological history and status, hearing, vision, Mini Mental State Examination (MMSE), cardiovascular status, blood pressure, metabolic indices, measure of head circumference, handedness, dexterity, and body sway.\n\nPsychiatric disorders will be screened by SCID-I118 and SCID-II interviews119. Specific tools for ADHD will include the Conners’ Adult ADHD Diagnostic Interview for DSM-IV (CAADID)120 or the Diagnostic Interview for Adult ADHD (DIVA)121.\n\nNeuropsychological assessment at the age of 40 will include a battery of tests for basic visuospatial and visuoconstructive skills, tests for motor praxis and dexterity; tests for phonological processing, naming (Boston naming122, Rapid alternating stimulus naming123), reading, and arithmetic; executive function measurements (Trails Making Test, Word Fluency, Tower test and Color Word test, either from Delis–Kaplan Executive Function System124 or as stand-alone tests), computerized tests for reaction time and attention (such as Continuous Performance Test125 and Attention Network Test126), tests for memory (such as Rey Auditory Verbal Learning127 and Benton111); as well as subtests of the Wechsler tests (WAIS-IV128 and WMS-III129). There will also be questionnaires for subjective symptoms and mood.\n\nThe same or slightly modified battery of tests will later be used in repeated testing. A particular challenge in planning the battery to use in longitudinal studies is the availability of the tests in years to come. The traditional pen and paper tests will be around but newer computerized methods present the risk of being more short-lived in the ever-evolving technology.\n\nLaboratory assessment and genetic analyses cannot all be anticipated at the moment. Blood samples will be taken and stored until genetic analyses (e.g. for APOE, DISC1, COMT, and ROBO1) can be performed as a batch process. These are open to discussion and collaboration is actively sought.\n\nNeuroradiological imaging including MRI (T1 and T2 weighted and FLAIR T2 imaging, diffusion imaging with diffusion tensor imaging, angiography, and volume measurements of the hippocampi, corpus callosum, relevant nuclei and other relevant structures that have to be defined ad hoc). Ideally, the MMSE would be scheduled the same day or at least within one week of the neuroradiological imaging session. Brain activity will be measured in selected cases using functional MRI (fMRI), recordings of event related potentials (ERP), electroencephalography (EEG) and magnetoencephalography (MEG).\n\nRegistry inquiry In addition to the clinical assessment, health register data will be gathered from the Finnish Social Insurance Institution (Kela) concerning disability benefits, health security, rehabilitation, and unemployment benefits. From the Finnish National Institute for Mental Health and Welfare (THL) register data will be applied concerning diagnoses from the National Hospital Discharge Register.\n\nLongitudinal data will be collected as long as the subjects are willing to participate. We know from Finnish statistics that the estimated life expectancy for someone having reached 30 years of age in 2003 is 46 years for males and 52 for females130. This would mean that the men in this cohort should live up to 76 and the women up to 82 years of age. The project outline will later be updated to include the studies after 65 years of age.\n\nLaura Hokkanen, PhD, professor of clinical neuropsychology, University of Helsinki, is the Principal investigator. Other members of the research group include Jyrki Launes, MD, PhD, specialist in neurology, University of Helsinki, Marja Laasonen, PhD, Helsinki University Central Hospital, Department of Phoniatrics, Anna-Mari Tuulio-Henriksson, PhD, Kela – The Social Insurance Institution of Finland and Maarit Virta, PhD, University of Helsinki, Institute of Behavioral Sciences. Master’s and doctoral level students will be recruited in the project.\n\nCollaborators at this point include Kimmo Alho, Professor of psychology, University of Helsinki, Helsinki Collegium for Advanced Studies and Institute of Behavioural Sciences, Taina Autti, MD, PhD, Professor of radiology, University of Helsinki, Oili Salonen, MD, PhD, Helsinki University Central Hospital, Department of Radiology, Sami Leppämäki, MD, PhD, Helsinki University Central Hospital, Department of Psychiatry, and Pentti Tienari, MD, PhD, Helsinki University Central Hospital, Department of Neurology and Biomedicum, University of Helsinki, Molecular Neurology Research Program.\n\nNational as well as international collaboration is invited. Please send comments and suggestions to Dr Launes at plasticity@live.fi.\n\n\nData analysis and statistical plan\n\nThe original database created in 1971 was non-electronic (punched cards). It was later keyed in and analyzed using the BMDP (Statistical Software, Inc 1983). Currently the database is on PASW Statistics, Release Version 18.0.0 (SPSS, Inc.) and Microsoft Excel 2010 and can thus be converted and transported easily. The integrity of the data has been checked during conversions and will undergo continuous error checking both electronically and manually.\n\nInterestingly, the structure of the database reflects the change in the information processing techniques over the past 40 years. Initially, due to the dichotomous nature of the punched card processing, the variables concerning the neonatal period and the first 5 years are mainly stored in a categorical/discrete format. This limits the statistical approaches as non-parametric statistics must be used. This, however, in no way prevents the use of early perinatal data for creating categories and covariants for later analyses.\n\nAnother common problem in longitudinal birth cohorts is related to repeated psychometric measures. For example it is impossible to use the same psychological/neuropsychological tests for all age groups. Tests of intelligence for pre-school children, school aged children and adults are different and even though they can be scaled in corresponding distributions centering on the mean IQ of 100, they still are not fully comparable.\n\nFor the statistical analysis of new data, commercially available statistical analysis packages will be used. For obvious reasons, the statistical consulting facilities provided by the University of Helsinki will be extensively put to use.\n\n\nEthical considerations\n\nInfants in the original database were enrolled with an informed consent by a parent. All studies have been conducted in accordance with the Helsinki declaration and consent has been given at each phase of the follow-up. In 2001 the subjects gave their written consent for future follow-ups.\n\nThe ethical review was initially done at the Children's Hospital at the Helsinki University Central Hospital for the follow-up visits at 5, 9 and 16.\n\nIn November 2012 the material was handed over to Prof Laura Hokkanen, PhD., by a written agreement by Dr. Katarina Michelsson, MD, PhD. A new ethical review for the current project and the new plan as well as inclusion of a new group of researchers will be applied for from the Review Board of the Helsinki and Uusimaa hospital district during the spring of 2013 (Medical Research Act 488/1999). A new invitation letter will be sent out to all participants for consent.\n\nSpecial care will be taken to respect the autonomy of research subjects, to avoid harm, and to ensure privacy and data protection of the cohort. Identifying information will be handled according to the Finnish Personal Data Act (523/1999).\n\nIf a subject is found to have a condition requiring medical attention, he or she will be referred to proper medical services by the responsible physician.\n\n\nPlans for dissemination of the study outcome\n\nThe results will be published in international peer reviewed scientific journals. Open access electronic publications will be preferred. It is expected that a project of this magnitude will gain publicity in national media.",
"appendix": "Author contributions\n\n\n\nLH is responsible for writing most of the present paper. JL is responsible for the medical details and contributed to writing the present paper. KM is the intellectual owner and main contributor of the original project and the risk cohort data. She has also reviewed the present paper.\n\nAll authors have agreed to the final content of the article and to its submission for publication.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThe first 20 years of the project were supported by the Academy of Finland, Signe and Ane Gyllenberg Foundation, Foundation of Pediatric Research, The Association for Life Insurance Companies, and Rinnekoti Foundation.\n\n\nReferences\n\nMichelsson K, Ylinen A, Saarnivaara A, et al.: Occurrence of risk factors in newborn infants. A study of 22359 consecutive cases. Ann Clin Res. 1978; 10(6): 334–6. 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PubMed Abstract | Publisher Full Text\n\nBraak H, Thal DR, Ghebremedhin E, et al.: Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years. J Neuropathol Exp Neurol. 2011; 70(11): 960–9. PubMed Abstract | Publisher Full Text\n\nNelson PT, Alafuzoff I, Bigio EH, et al.: Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. J Neuropathol Exp Neurol. 2012; 71(5): 362–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDubois B, Feldman HH, Jacova C, et al.: Revising the definition of Alzheimer’s disease: a new lexicon. Lancet Neurol. 2010; 9(11): 1118–27. PubMed Abstract | Publisher Full Text\n\nSarazin M, De Souza LC, Lehéricy S, et al.: Clinical and research diagnostic criteria for Alzheimer’s disease. Neuroimaging Clin N Am. 2012; 22(1): 23–32, viii. PubMed Abstract | Publisher Full Text\n\nLauner LJ: The epidemiologic study of dementia: a life-long quest? 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Publisher Full Text\n\nKirk SA, McCarthy JJ, Kirk WD: The Illinois Test of Psycholinguistic Abilities. Revised edition, University of Illinois Press: Urbana, IL, 1968. Reference Source\n\nKuusinen J, Blåfield L: Illinois Test of Psycholinguistic Abilities. Examiner’s manual. University of Jyväskylä: Jyväskylä, 1974. Reference Source\n\nGoodenough F: Measurement of intelligence by drawings. World Company: New York, 1926.\n\nFrostig M, Lefever W, Whittlesey JRB: Administration and Scoring Manual for the Marianne Frostig Test of Visual Peception. Consulting Psychologists Press: Palo Alto, California, 1966; 40. Reference Source\n\nWechsler D: Wechler Intelligence Scale for Children. Psychological Corporation: New York, 1949. Reference Source\n\nWechsler D: [Wechsler Intelligence Scale for Children. Manual]. Psykologien Kustannus OY: Helsinki, 1971. Reference Sorce\n\nWechsler D: Wechsler Adult Intelligence Scale. The Psychological Corporation: New York, 1955.\n\nWechsler D: [Wechsler adult intelligence scale. WAIS manual]. Psykologien Kustannus OY: Helsinki, 1970.\n\nBenton A: The Revised Visual Retention Test. The Psychological Corporation: New York, 1974; 4.\n\nWechsler DA: A Standardized memory scale for clinical use. J Psychology. 1945; 19(1): 87–95. Publisher Full Text\n\nWechsler D: [Wechsler memory scale. WMS manual]. Psykologien Kustannus OY: Helsinki, 1975.\n\nBarkley RA, Murphy KR: Attention-Deficit Hyperactivity Disorder: A Clinical Workbook (2nd. ed). The Guilford Press: New York, 1998. Reference Source\n\nLindahl E, Michelsson K, Donner M: Prediction of early school-age problems by a preschool neurodevelopmental examination of children at risk neonatally. Dev Med Child Neurol. 1988; 30(6): 723–34. PubMed Abstract | Publisher Full Text\n\nClements SD: Minimal brain dysfunction in children: Terminology and identification. (NINDB monograph no. 3). U.S. Dept. of Health, Education and Welfare, NINDB.: Washington, DC, 1966. Reference Source\n\nHagberg B: [Minimal brain dysfunction–what does it imply in child development and adaptation]. Läkartidningen. 1975; 72(36): 3296–300. PubMed Abstract\n\nFirst MB, Spitzer RL, Gibbon M, et al.: Structured Clinical Interview for DSM-IV Axis I Disorders, Clinician Version (SCID-CV). American Psychiatric Press: Washington DC, 1996.\n\nFirst MB, Gibbon M, Spitzer RL, et al.: Structured Clinical Interview for DSM-IV Axis II Personality Disorders, (SCID-II). American Psychiatric Press: Washington DC, 1997; 41. Reference Source\n\nEpstein JN, Johnson DE, Conners CK: Conners’ Adult ADHD Diagnostic Interview for DSM-IV (CAADID). Multi-Health Systems: Toronto, 2001. Reference Source\n\nKooij JJS: Adult ADHD. Diagnostic assessment and treatment. 3rd ed., Springer, 2013. Publisher Full Text\n\nKaplan E, Goodglass H, Weintraub S: Boston Naming Test. Waverly, Inc., Baltimore 1983.\n\nWolf M: Rapid alternating stimulus naming in the developmental dyslexias. Brain Lang. 1986; 27(2): 360–379. PubMed Abstract | Publisher Full Text\n\nDelis DC, Kaplan E, Kramer JH: Delis Kaplan Executive Function System (D-KEFS). The Psychological Corporation: San Antonio, TX, 2001. Reference Source\n\nConners CK: MHS Staff (Eds.) Conners’ Continuous Performance Test II Computer Program for Windows Technical Guide and Software Manual. Multi-Health Systems: North Tonawanda, NY 2000. Reference Source\n\nFan J, McCandliss BD, Sommer T, et al.: Testing the Efficiency and Independence of Attentional Networks. J Cogn Neurosci. 2002; 14(3): 340–347. PubMed Abstract | Publisher Full Text\n\nLezak MD: Neuropsychological assessment. Oxford University Press: New York 1995; 1056. Reference Source\n\nWechsler D: WAIS-IV - Wechsler Adult Intelligence Scale - IV. Pearson, Psykologien Kustannus OY Helsinki, 2012. Reference Source\n\nWechsler D: Wechsler adult intelligence scale - third edition: Manual. Pearson, Psykologien Kustannus OY Helsinki 2005.\n\nOSF Statistics Finland - Life expectancy in 1983 and 2003.Official Statistics of Finland (OSF): Causes of death, 2003. Reference Source"
}
|
[
{
"id": "820",
"date": "08 Mar 2013",
"name": "Chiadi Onyike",
"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 Perinatal Adverse events and Special Trends in Cognitive Trajectory (PLASTICITY) - pre-protocol for a prospective longitudinal follow-up cohort study is rather intriguing. By observing a well-developed cohort of individuals who suffered perinatal adversity as they enter mid-life and later-life, this study can provide valuable insights into the long-term effects of perinatal adversity on the cognitive, behavioural and functional prospects of the individual. The findings would also be valuable from a neuropsychiatry research perspective, as it offers examination of perinatal and psychosocial risk factors of mid-life and late-life dementia, cognitive decline and psychiatric disorder. Already, some data suggest that learning disabilities are associated with a higher risk for frontotemporal dementia in later life (see Rogalski et al., 2008). Thus this is an excellent and timely study, and the research agenda offers examination of many important questions. As with all major undertakings, the devil is in the details. One would eventually want to see the details, i.e.., the measurement and data analysis procedures. I am especially interested in understanding how the data analysis will manage intervening events such as adverse rearing environment, head trauma, substance abuse, that may have potential for independent or overriding influence on the outcomes.",
"responses": []
},
{
"id": "1274",
"date": "27 Aug 2013",
"name": "Erin Bigler",
"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\nWith the improvements in managing adverse events that occur during the perinatal period, infants that would not have survived given medical and obstetrical care prior to the 1970’s now survive. Although cognitive and neurobehavioral sequelae are commonplace in children who experience adverse perinatal effects, almost all studies that have addressed such problems have been based on select sub-samples, including small N and anecdotal studies. Larger outcome studies often get allocated to one particular type of adverse perinatal even like asphyxia. The uniqueness the study design presented by Hokkanen, Launes and Michelsson is to include all “adverse” perinatal events and prospectively follow this cohort throughout life. In this study from a single maternity hospital, 1196 subjects met inclusion criteria for perinatal adverse events from a consecutive sample of 22, 359. In that these subjects were enrolled from 1971 through 1974 means they are now reaching their 40’s. The other by-product of improved healthcare of the 20th and 21st centuries is that once born, longevity is now the expectation with survival well beyond 78 years of age the norm. Because of this and the potential for those who suffer perinatal adverse events to have increased risk for a host of neurological, neuropsychiatric, cognitive and behavioral effects, it has become very important to better understand how these perinatal influences affect long-term outcome. Much of the past research has merely focused on the transition from surviving the adverse event and its influence on childhood and then the transition to adulthood. This proposed study is truly a lifespan study with a single cohort that experienced adverse perinatal events.In this well thought-out and written study design, Hokkanen and colleagues capitalize on a single cohort of births in Finland where prospectively acquired medical, neurological, developmental, educational and neuropsychological test information has been previously obtained and the current proposed design will capture this cohort in their 40’s. All fields of medicine and psychology have improved in major ways since the 1970s with improved assessment methods and a more comprehensive understanding of how early brain insult may affect function and how it should be measured. As outlined in Figure 1, Hokkanen et al. show the potential interactive nature of numerous relations between neurodevelopmental disorders and later-in-life neurodegenerative disorders. Previously not assumed to be a factor in aging and the development of degenerative diseases like Alzheimer’s, early events are now know to participate in the overall cognitive and brain reserve of the individual. Certain factors may predispose to degeneration yet other factors may relate to plasticity and resiliency. Potentially, the only way some of these factors could be teased out is through long-term prospective studies as proposed by Hokkanen et al. Inexorably this cohort will march on to their 60’s and beyond and tracking their progress may provide immeasurable insights into what are critical vulnerability factors that predispose to mild cognitive impairment and transition to dementia. Hokkanen et al. nicely detail the major factors for data extraction as well as attrition. Table 3 outlines the specific outcome areas that will be targeted and methods to examine outcome. One of the critical improvements since this cohort was first identified is the development in neuroimaging. As outlined in Table 3, MRI including functional MRI studies will be obtained.This manuscript is very well written. The questions raised, the uniqueness of this cohort, the ability to answer not only some long-term developmental questions in terms of outcome, but also aging and risk for dementia merely point out how incredibly unique is this cohort. I very much like the acronym – PLASTICITY. This has been and will be a most important and valuable clinical research cohort to study and will help answer questions about vulnerability as well as resiliency in brain development and aging.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-50
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https://f1000research.com/articles/2-49/v1
|
13 Feb 13
|
{
"type": "Research Article",
"title": "Inpatients’ opinions on a hospital in Portugal",
"authors": [
"Carina C Silva",
"Agripino Oliveira"
],
"abstract": "Background: Little is known about the relationship between the opinions of inpatients and the degree to which hospitals are improving in performance over time. The aim of this study was to determine the personal assessment level of inpatients or their representatives regarding aspects of health care in an internal medicine ward.Methods: We carried out a questionnaire in September 2011 with 284 discharged patients and patient representatives, focusing on their opinions about the department, health professionals and amenities, with response options ranging from 1 (very bad) to 5 (very good). The relationships between domains from the questionnaire and socio-demographic factors were examined using a t-test and one-way ANOVA.Results: The response rate was 78%. The patients showed a slightly higher mean score (m) for factors in the medical care domain than did the patient representatives (m = 4.51 vs. m = 4.27; p = 0.014). The mean score of all the items in all domains was 4.24; this allowed us to determine the difference from the overall mean (DIFM) for medical care (DIFM = 0.18; p = 0.000), foods (DIFM = –0.31; p = 0.000), diagnostic tests (DIFM = –0.15; p = 0.036) and transport (DIFM = –0.41; p = 0.000). Respondents with a medium or higher educational level gave lower scores to the domains food (m = 3.74; p = 0.004), diagnostic tests (m = 3.72; p = 0.04) and transport (m = 3.62; p = 0.025) than those with lower educational levels. The domains facilities (m = 2.4; p = 0.04) and diagnostic tests (m = 3.63; p = 0.009) were given lower scores by those aged <50 years compared with older respondents.Conclusions: Our findings suggest that the evaluation of the responders will allow the hospital management to make improvements in the quality of care.",
"keywords": [
"Questionnaire",
"health services quality",
"health professionals and amenities"
],
"content": "Introduction\n\nPatient satisfaction has been increasingly used as a quality indicator in health care1. The theoretical concept of satisfaction is controversial, but the user relates it with the set of reactions experienced2. Therefore, the measurement of customer satisfaction should be considered as a personal opinion of health care services that are provided. One of the most used measures3 is the difference between the expectations of the user in relation to the care and their perception of the care actually received. Indeed, it is expected that the patients, throughout their experiences, build a set of beliefs about the health system and professionals. The importance of attending to this type of belief has implications for the quality of communication with health professionals, the degree of trust in health care service delivery and customer satisfaction with the care provided. The aim of this study was to determine the personal assessment of inpatients or their representatives regarding aspects of health care in an internal medicine ward.\n\nThe Centro Hospitalar Vila Nova Gaia-Espinho, where the study was conducted, is divided into several units. Patients admitted into the internal medicine department who need diagnostic imaging or invasive procedures are transported by ambulance to the central unit.\n\n\nMaterials and methods\n\nSeveral sources and methods were used to determine the questions to be included in the questionnaire. Firstly, a search was conducted using the Medline database with the aim of evaluating the tools that have been developed so far to assess the satisfaction of patients hospitalized4. Secondly, focus groups of patients, caregivers and health care professionals were used to explore opinions about the positive and negative aspects of care received during hospitalizations. These focus groups were geared towards understanding the issues and expressions that could be used to develop questions to be included in the questionnaire. Thirdly, we developed a pool of items, based on the results of the focus groups and literature search, to be included in the questionnaire. These items were tested with a group of patients and health professionals, and they gave their opinions about the appropriateness of the items and the skills needed to comprehend them and evaluate the content and face validity of the questions.\n\nThe questionnaire was designed with twenty-two closed questions. It captures ten domains selected by their relevance to the study: the department’s image, facilities, medical care, nursing care, health care assistants (HCAs), secretarial services, reception, food, diagnostic tests and transport. Each domain is composed of between one and four discrete items rated on a five-point scale, in which the response options range from 1 (very bad) to 5 (very good) as shown in Appendix 1. The score for each domain represents the mean of the responses to each item within a given domain. The global mean score was determined by the sum of the items divided by the number of items answered. The questionnaire also contained socio-demographic variables, such as gender, age, educational level, occupational status, marital status4 and type of respondent.\n\nThe respondents’ understanding of the questionnaire items can generate different opinions for each one of them. This diversity exposes the problem of internal consistency of the questionnaire, that is, the degree of uniformity between the answers to each item of the questionnaire. This internal consistency can be measured by Cronbach’s alpha5, which varies from 0 to 16, and the higher the count, the greater the reliability of the scale of questionnaire. A value of at least 0.70 reflects an acceptable reliability, between 0.80 to 0.90 moderate to high, and exceeding 0.90 high internal reliability.\n\nFrom 1st to 30th September 2011, the self-administered questionnaire was filled out by the discharged patients or their representatives. All patients admitted more than 48 hours were given the questionnaire and an envelope. Family members or caregivers (referred to as ‘representatives’) replaced patients with severe physical or mental diseases, who would have difficulty in understanding and filling out the questionnaire. The deceased were excluded because their representatives would be in mourning.\n\nAll participants were informed of the study’s objective, and it was explained to them how to fill out the questionnaire. Delivery was carried out in a sealed envelope. To ensure confidentiality, participants were asked to put the completed questionnaires in a closed box at the time of discharge, according to the declaration of Helsinki. The board of directors and ethics committee of the hospital approved the study.\n\nWe describe the frequencies (number), percentages, means (µ), median and standard deviation (σ) of the variables. In univariate analysis, we applied the Student’s t-test and analysis of variance (ANOVA) for the domains addressed in the questionnaire, considering the value of p < 0.05 statistically significant. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 19.\n\n\nResults\n\nA total of 284 inpatients were enrolled for the study, of whom 199 completed the questionnaire (response rate = 78%) and 31 died (10.9%). Respondents had a mean age of 62.9 years with a median of 66.5; 51% were men; 64% were married or cohabitating; 59.3% were retired and 51.7% had a basic (primary) education (Table 1).\n\nCronbach’s alpha measures of internal consistency were computed for each of the ten domains, which showed a reliability for the overall scale of 0.89 (Table 2). In all domains, acceptable values were met, reaching a minimum of 0.868 (for diagnostic tests) and a maximum of 0.880 (for medical care).\n\nAnswers to the items in the ten domains had an overall mean score of 4.24 (Figure 1), out of a maximum of 5. This allowed us to compare with the mean score for each domain. The following domains had significantly more positive scores than the overall mean: department’s image (mean difference (DIFM) = 0.15; p = 0.0001), medical care (DIFM = 0.18; p = 0.0001), nurses (DIFM = 0.21; p = 0.0001) and secretarial services (DIFM = 0.15; p = 0.002). The following domains had significantly more negative scores than the overall mean: reception (DIFM = –0.16; p = 0.016), food (DIFM = –0.31; p = 0.0001), diagnostic tests (DIFM = –0.15; p = 0.036) and transport (DIFM = –0.41; p = 0.0001). In the domains related to the facilities and HCAs, there were no significant differences from with the overall mean.\n\nStudent t test or analysis of variance (ANOVA) with the Tukey’s multiple comparison test performed.\n\nTable 3 shows a univariate analysis of selected variables and their relationship with the mean scores of the questionnaire’s domains. We found that the variables ‘gender’, ‘occupation’, ‘marital status’ and ‘length of hospital stay’ showed no difference in all domains, except for secretarial services, for which higher scores were given by married respondents (µ = 4.49; p = 0.008).\n\nµ-> Mean of distribution; σ-> Standard deviation; HCAs (Health Care Assistants).\n\nFor the variable ‘respondents’, patients gave higher mean scores for the domains: department’s image (µ = 4.49, p = 0.001) and medical care (µ = 4.51; p = 0.014) than patient representatives. In the variable ‘education level’, respondents with a medium/higher level of education (secondary or university education) gave lower mean scores in the domains: HCAs (µ = 4.17; p = 0.013), reception (µ = 3.89; p = 0.002), food (µ = 3.74; p = 0.004), diagnostic tests (µ = 3.72; p = 0.04) and transport (µ = 3.62; p = 0.025). In the variable ‘age group’, respondents < 50 years old, assessed the mean score in the domains: department’s image (µ = 4.17; p = 0.047), facilities (µ = 2.4; p = 0.04), nurse care (µ = 4.15; p = 0.048), HCAs (µ = 4.04; p = 0.046) and diagnostic tests (µ = 3.63; p = 0.009).\n\n\n\n\nDiscussion\n\nThe typical participant was male, married and retired with a low educational level in this convenience sample. Respondents reported good results with the care provided from the professionals, with scores above four points, but the amenities were rated below this score. The present findings seem to be consistent with another study7, which found the following factors to be scored, in descending order: medical performance, nursing staff, amenities and accessibility. In another study in Israel8, the attitudes of nurses and medical care were the most important determinants of patient satisfaction with the care received. In a study performed in Kuwait9, medical care was the most favorably rated domain, followed by admission process and housekeeping, while nursing care was the least favorably rated domain. It can therefore be assumed that the assessment of patient’s satisfaction is based not only on the care received.\n\nThe evaluation instrument used here was a questionnaire developed for this purpose, following a search of the literature on the satisfaction of patients or families to determine the applicability of the questions used. Some studies4–10 have chosen variables related to information provided from the patient or their family, the existing support structures, the services available at the hospital and concerns about the ability to meet the patient’s needs during hospitalization.\n\nTo avoid bias in the questionnaire4, we used two methods: peer review for content validation and prior testing by a group of inpatients. To test the reliability of the questionnaire we used Cronbach’s alpha, and the results showed values indicating internal consistency in all areas. If alpha is too high this may suggest that some items are redundant6 and test the same question but in a different way. A maximum alpha value of 0.90 is recommended6.\n\nThe response rate of 78% for a sample of convenience and a self-administered questionnaire is considered good. This finding is in agreement with Stizia’s11 findings, which showed a response rate of 65% for self-administered questionnaires and in studies with a convenience sample of 25%.\n\nIn this study, we found that gender, marital status, occupation (retired or not) and length of stay did not affect the scores in the domains of the questionnaire. A previous study has shown that older subjects tend to have higher scores12, and this was confirmed in all the areas of our questionnaire.\n\nData from this study showed that the higher the educational level, the lower the scores for the responses in the domains of amenities, with the exception of medical care. A possible explanation for these differences might be better understanding and knowledge of the procedures to be undergone. Our study is in agreement with another4 in which had an inverse relation to educational status, with high educational levels associated with low scores.\n\nWe found that with the variable ‘respondent’, patients provided high scores in all domains, with statistical significance in the medical care. Unlike the inpatients, the representatives tended to be more demanding or critical and reported lower scores in some domains. It is likely that representatives complain about inadequate information and practical advice, especially on how to deal with potential decompensated disease13.\n\nThis study provides valuable information on the effect of all variables in the various fields that constitute our satisfaction tool in hospitalized patients. Therefore, we offer a picture of the determinants of satisfaction in several areas, which have never been studied together. The study may contribute to the understanding of the factors that influence inpatient satisfaction in hospital wards and could be used as a resource for evaluating the quality of care.\n\nThe limitations of this study include that it is a cross-sectional study and inevitably a convenience sample, composed of the patients discharged during one month. However, this convenience sampling is useful when attempting to study the satisfaction of a niche segment such as inpatients in internal medicine. Moreover, the range of possible explanatory variables included in this study, although large, was not as comprehensive as we would have liked. Several other variables, such as previous health status, could also have been assessed. However, when previous mental and physical health is poor, this is associated with lower satisfaction with hospitalization14. These patients have more hospitalizations, more aggressive treatment, and are more likely to suffer from medical complications15.\n\n\nConclusions\n\nThis study allowed us to conclude that respondent satisfaction was above average in all areas of the questionnaire. Although the mean score was high for all domains, amenities had the lowest level of satisfaction, pointing to the need to reassess food, transport and diagnostic tests. Gender, marital status and length of stay at the hospital were not factors that influenced the level of satisfaction of respondents.\n\nDespite the limitations of the study, it was possible to identify the level of satisfaction of patients hospitalized in the internal medicine department, and the influential variables. These findings will allow the hospital management to implement changes in practice and propose actions to improve quality of care, and provide visibility to the teamwork of professionals involved. We conclude that, as in previous studies, there is evidence that the educational level, age and amenities affect the levels of satisfaction. Finally, we must consider that patient representatives are more critical than patients in the evaluation of satisfaction. Therefore, researchers conducting a survey of hospitalized patients’ satisfaction in internal medicine departments should be aware of the effect of variables on the responses to the questionnaire and make the necessary adjustments to provide valid results.",
"appendix": "Author contributions\n\n\n\nThe authors had equal roles in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.\n\n\nCompeting interests\n\n\n\nNo relevant 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 Prof Laura Oliveira, Dra Emilia Moreira and Dr° Claudio Barrios for their useful comments on the English of the manuscript, which substantially contributed to the final shape of this paper.\n\n\nReferences\n\nMainz J: Defining and classifying clinical indicators for quality improvement. Int J Qual Health Care. 2003; 15(6): 523–530. PubMed Abstract | Publisher Full Text\n\nSitzia J, Wood N: Patient satisfaction: a review of issues and concepts. Soc Sci Med. 1997; 45(12): 1829–1843. PubMed Abstract | Publisher Full Text\n\nEsperidião MA, Trad LAB: Avaliação de satisfação de usuários: considerações teórico-conceituais. Cad Saúde Pública. 2006; 22(6): 1267–76. Publisher Full Text\n\nQuintana JM, González N, Bilbao A, et al.: Predictors of patient satisfaction with hospital health care. BMC Health Serv Res. 2006; 6: 102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaberere J, François P, Auquier P, et al.: Development of a French inpatient satisfaction questionnaire. Int J Qual Health Care. 2001; 13(2): 99–108. PubMed Abstract | Publisher Full Text\n\nTavakol M, Dennick R: Making sense of Cronbach’s alpha. IJME. 2011; 2: 53–55. Publisher Full Text\n\nHall MC, Elliot KM, Stiles GW: Hospital patient satisfaction: correlates, dimensionality and determinants. J Hosp Mark. 1993; 7(2): 77–90. PubMed Abstract | Publisher Full Text\n\nHart J, Malinarski Y, Djaldetti M: Survey of patient satisfaction in a community hospital. Isr J Med Sci. 1996; 32(7): 551–4. PubMed Abstract\n\nGuirguis WW, Mokhtar SA, al-Torkey MM, et al.: Patient satisfaction with hospital services: determinants and level in a hospital in Kuwait. J Egypt Public Health Assoc. 1992; 67(1–2): 87–108. PubMed Abstract\n\nNguyen Thi PL, Briancon S, Empereur F, et al.: Factors determining inpatient satisfaction with care. Soc Sci Med. 2002; 54(4): 493–504. PubMed Abstract | Publisher Full Text\n\nSitzia J, Wood N: Response rate in patient satisfaction research: an analysis of 210 published studies. Int J Qual Health Care. 1998; 10(4): 311–317. PubMed Abstract | Publisher Full Text\n\nJaipaul CK, Rosenthal GE: Are older patients more satisfied with hospital care than younger patients? J Gen Intern Med. 2003; 18(1): 23–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStengård E, Honkonen T, Koivisto AM, et al.: Satisfaction of caregivers of patients with schizophrenia in Finland. Psychiatr Serv. 2000; 51(8): 1034–1039. PubMed Abstract | Publisher Full Text\n\nDa Costa D, Clarke AE, Dobkin PL, et al.: The relationship between health status, social support and satisfaction with medical care among patients with systemic lupus erythematous. Int J Qual Health Care. 1999; 11(3): 201–207. PubMed Abstract | Publisher Full Text\n\nPerneger TV: Adjustment for patient characteristics in satisfaction surveys. Int J Qual Health Care. 2004; 16(6): 433–435. PubMed Abstract | Publisher Full Text\n\n\n\n\nQuestionnaire handed out to study participants (English translation).\n\nQuestionnaire handed out to study participants (English translation).\n\nQuestionnaire handed out to study participants (Portuguese translation).\n\nQuestionnaire handed out to study participants (Portuguese translation)."
}
|
[
{
"id": "791",
"date": "22 Feb 2013",
"name": "Erin Aiello Bowles",
"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 only have one major comment, which is that the authors don't provide any information on non-responders. It would be interesting to know more about the characteristics of those people, and whether they differ from responders because this could affect the generalizability of the results.",
"responses": [
{
"c_id": "383",
"date": "14 Mar 2013",
"name": "Antonio Agripino Costa Oliveira",
"role": "Author Response",
"response": "We want to express our appreciation to Erin Aiello Bowles for taking the time and effort necessary to revise our manuscript. We have carefully considered your comment which states that we didn't provide any information on non-responders. We agree that sampling bias is the major problem in patient satisfaction studies; but in our study the response rate was high and the self-administered questionnaire was filled out by patients or their representatives at time of discharge. Indeed we have some information on the variables; sex, age and length of stay, in patient non-responders, but we don´t have any information for the representative non-responders."
}
]
},
{
"id": "976",
"date": "31 May 2013",
"name": "Saravana Kumar",
"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 well conducted research study which sheds light on the patients’ perspectives of aspects of health care in an internal medicine ward. It is a well presented manuscript which is easy to read with logically presented arguments. I have two main comments.1. It would have been worthwhile to expand on the process which led to the development of the survey instrument. While the authors provide a brief overview of the process, such as the conduct of the focus groups etc, it is imperative to provide detailed information about this as the development and the psychometric properties of the instrument can play an important role in the believability of the research findings. 2. I am not sure about some of the items in the survey instrument. For example, what does “is it a reliable department” actually mean? Reliable in term so what? Similarly, what does “is it a department with experience” actually relate to. Is it a question about health practitioners who work there or about how long the department has been in place and hence it reputation? These questions may give rise to ambiguity in the interpretation and as such influence the responses.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-49
|
https://f1000research.com/articles/2-47/v1
|
13 Feb 13
|
{
"type": "Study Protocol",
"title": "Can primary care nurse administered pelvic floor muscle training (PFMT) be implemented for the prevention and treatment of urinary incontinence? A study protocol",
"authors": [
"Sue Child",
"Alice Bateman",
"Joanna Shuttleworth",
"Christian Gericke",
"Robert Freeman",
"Alice Bateman",
"Joanna Shuttleworth",
"Christian Gericke",
"Robert Freeman"
],
"abstract": "Background: We aim to evaluate if Pelvic Floor Muscle Training (PFMT) delivered in primary care results in fewer referrals to secondary care for urinary incontinence (UI), thereby reducing the number and associated costs of surgical procedures for UI.Methods / design: The study will consist of two populations – a prevention group and a treatment group who will both be offered PFMT in primary care. The prevention group will consist of parous women aged 25-64 attending for a routine cervical smear. Their pelvic floor will be assessed using the Modified Oxford Scale (MOS) and a baseline data form will be completed that asks about the frequency and associated bother of urine leakage. From the answers given, the group will be subdivided into two groups. The first (prevention) group will be subdivided into a primary prevention arm (no symptoms of urinary incontinence and pelvic floor strength ≤2 on MOS) and a secondary prevention arm (women reporting symptoms of urine leakage irrespective of MOS). The second (treatment) group will be women of any age who may or may not have had a vaginal birth presenting to their GP with UI. Semi-structured, in-depth interviews will be conducted with a subset of patients and staff with the aim of identifying barriers and facilitators in delivering PFMT in primary care.Discussion: A recently completed community study showed good outcomes with practice nurse delivery of PFMT. We suggest if this were to be implemented more widely it would reduce the need for referral to secondary care. We believe that this study will show whether implementing a package of PFMT delivered in primary care can treat as well as prevent UI and will also be helpful in exploring the benefits / drawbacks of such implementation, thus providing lessons for implementation in other Primary Care Trusts (PCTs).",
"keywords": [
"pelvic floor muscle training",
"nurse",
"urinary incontinence",
"urinary incontinence"
],
"content": "Study rationale\n\nUrinary incontinence (UI) is a distressing and disabling condition as it involves the involuntary leaking of urine from the bladder. It is estimated that 10 million women in the UK suffer with UI1 at a cost to the National Health Service of £594 million per year2. Prevalence rates vary depending on age and severity. Hunskaar et al., estimate prevalence is between 20–30% for young adult women (18–44), 30–40% for middle age women (45–49) and 30–50% for elderly women (≥60 years)1. One of the main forms of UI is stress urinary incontinence (SUI), which can occur with coughing, sneezing or exercise.\n\nThe pelvic floor muscles are often described as a ‘sling’ supporting the pelvic organs and are located beneath the bladder and rectum. They are involved in the process of storing and passing urine. These muscles often become weakened as a result of childbirth and as a result the individual is much more likely to develop some kind of UI. Worldwide, the World Health Organisation (WHO) report that one third of women have urinary incontinence after childbirth3. Supervised pelvic floor muscle training (PFMT) or ‘pelvic floor exercise’ aims to strengthen these muscles to help treat and prevent UI. Evidence suggests the more the pelvic floor is exercised the better the result3.\n\nIt is known that the pelvic floor muscles can be difficult to identify for individual women and that supervision is required to enable them to do pelvic floor exercises properly4. In addition, it has been shown that 30% of women asked to do so are unable to contract their pelvic floor properly4. Even in young, fit, nulliparous women one in ten are unable to contract the pelvic floor to any extent4, and where exercise is undertaken more than a quarter use a technique that could potentially promote incontinence rather than contract the pelvic floor muscle5. Once learnt properly, pelvic floor exercises can produce favourable results that can persist in many women for at least ten years6. At a population level we know little about the status of the pelvic floor or about the number of women who are able or unable to perform pelvic floor contractions or how many do pelvic floor exercises regularly.\n\nIt has been recommended by NICE guidelines that the first line treatment for urinary incontinence should be provided in primary care7. The most appropriate form of PFMT is supervised training given by a physiotherapist or a specialist continence nurse, who will have greater in-depth knowledge and expertise. However, there are insufficient numbers of trained staff to provide this for all women either in primary or secondary care.\n\nWork already completed by a research team at Derriford Hospital, Plymouth has demonstrated that a practice nurse can deliver PFMT with outcomes comparable to those of a specialist nurse8. Practice nurses are in an ideal position to provide this treatment as most undertake cervical smears, which is an appropriate time to assess the pelvic floor and offer PFMT if necessary. We believe that as most practice nurses are more accessible to the woman and her family, it is likely that compliance with training could be better than that given by someone with whom the woman is unfamiliar, for example a continence advisor or a physiotherapist. The results from Waterfield’s study showed good outcomes with practice nurse delivery of PFMT and if this were to be implemented widely would reduce the need for referral to secondary care8.\n\nEvidence suggests that 50% of women with urinary incontinence referred to secondary care have not received PFMT9. Throughout Plymouth Primary Care Trust – known more widely as NHS Plymouth (the body that commissions primary care throughout Plymouth), over 50 referrals were returned to the referring GP during the first six months of 2011 because patients had not received PFMT before referral. If PFMT can be provided in primary care there is evidence to indicate that this can prevent as well as treat urinary incontinence and potentially reduce the need for surgery8.\n\n\nMethods/design\n\nThe study will comprise of two populations – a prevention group and a treatment group (see Figure 1 for a flow diagram of the study design). Women will be allocated to either of these two groups (following gathering of baseline data and assessment of their pelvic floor muscle strength) as follows:\n\n1. Prevention group - Women aged 25–64 years who have had at least one vaginal birth (i.e. parous women) attending their GP surgery for a routine cervical smear. This group will be further subdivided into a primary prevention arm and a secondary prevention arm following the information provided from the completion of the baseline data form at the time of the smear:\n\na) Primary prevention arm - Women with no symptoms of urinary incontinence but a weak Modified Oxford Scale score (≤ 2).\n\nb) Secondary prevention arm - Women symptomatic of mild (i.e. not bothersome) urinary incontinence who have not previously sought medical treatment from their GP for UI irrespective of Modified Oxford Scale score.\n\n2. Treatment group - Women over the age of 18 years who may or may not have experienced a vaginal birth presenting at their GP surgery with urinary incontinence.\n\nWomen (aged over 18) from the primary prevention group who have a score of two or less on the Modified Oxford Scale and all women from the secondary prevention and treatment groups irrespective of their Modified Oxford Scale score will be eligible for the study.\n\nWomen under 18, and those over 18, but unable to speak English or who have special communication needs will be excluded from the study. Patients who have evidence of urinary infection, pelvic pain, blood on urinalysis, visible prolapse, pelvic mass, voiding difficulties or other abnormalities will also be excluded.\n\nNHS Plymouth has 41 GP practices in 5 geographic localities with around 230 fully trained smear takers who are mostly practice nurses. They undertake approximately 16,000 cervical smears every year. In order to implement the delivery of PFMT in primary care, cervical smear takers will need to be trained to undertake pelvic floor assessment and deliver PFMT to women tailored to individual need. Such training will include gaining an understanding of the principles involved in pelvic floor muscle assessment including digital vaginal palpation known as the Modified Oxford Scale score. At the end of this training they will be able to assess pelvic floor muscle condition, be able to advise the patient on an individual programme of exercise and to set targets for that exercise.\n\nTraining will be staggered between localities in order for subsequent evaluation to take place on a rolling basis. Two localities with differing social class demographics will be chosen for initial training roll-out. By doing this, it may be possible to ascertain whether barriers and facilitators relating to the delivery of PFMT in primary care differ by socio-economic status. For the purposes of this evaluation, socio-economic status will be measured by occupation and postcode.\n\nThe evaluation of implementation will be undertaken by the Collaboration for Leadership and Applied Health Research and Care for the South West Peninsula (PenCLAHRC). The study will assess the following outcome measures.\n\nReduce cost by fewer referrals to secondary care. For the treatment group, to assess whether delivering PFMT in primary care results in fewer referrals to secondary care, thereby reducing the number and associated costs of surgical procedures for UI. This is in keeping with the QIPP agenda (a large scale transformational programme for the NHS, involving all staff aimed at improving the quality of care the NHS delivers whilst making up to £20 billion of savings by 2015 to be reinvested in frontline care).\n\nPatient satisfaction. As a marker of service quality, patient satisfaction of practice nurse delivered PFMT will be assessed at the end of PFMT training through the completion of a modified Royal College of General Practitioners (RCGP) Short-Form Patient Satisfaction Questionnaire (PSQ-18) by all women who have been recruited to PFMT training.\n\nCompliance and default rates with PFMT training in the treatment group will be monitored against both sub-groups in the prevention group through attendance at follow-up consultations. Each woman will be offered at least one follow-up consultation three months after the commencement of PFMT training either by telephone or face-to-face depending on preference. Training will be reviewed and further measures for improvement will be suggested if necessary. Follow-up data will be collected using a data collection sheet that will repeat the modified questions from the International Consultation on Incontinence Questionnaire (ICI-Q) completed at baseline (see Questionnaire files). The responses will allow us to ascertain if there have been any changes to the symptoms of UI and indicate levels of compliance with PFMT training offered at baseline. If the woman would like to continue with follow-up after her first follow-up consultation then she will be allowed to do so for a maximum of three further occasions at three monthly intervals.\n\n\n\nClinical outcomes will examine whether patient-reported symptoms of UI are improved after a course of primary care nurse-delivered PFMT, through the completion of a modified ICI-Q Short Form (SF) questionnaire at baseline and at each follow-up (see Questionnaire files). This data will also allow us to establish the prevalence of low grade urinary incontinence and the general pelvic floor muscle condition in the female population in Plymouth.\n\nSemi-structured, in-depth interviews will be carried out by an experienced qualitative researcher with a sample of patients and practice nurses, receiving or delivering PFMT in primary care in order to identify facilitators and barriers to implementation. It is hoped that 60 interviews will be conducted in total, with as far as possible equal numbers from all three groups. Women who refuse PFMT training will also be approached for interview in order to further understand barriers and facilitators to delivering PFMT in primary care.\n\nOnce interview transcripts have been analysed, a matrix will be designed which will enable us to ascertain key themes and create an understanding of the individual and group coding. The interviews will add depth to the analysis of quantitative (baseline and follow-up) data.\n\n\nDiscussion\n\nThe implementation of PFMT in primary care in Plymouth will aim to reduce the number of referrals to secondary care, thereby reducing costs to the NHS. Our evaluation will provide routinely collected data at baseline and at follow-up enabling an assessment of the impact of implementing this type of service change in the NHS. In addition, the qualitative analysis will allow us to ascertain why such a service implementation might be successful and how both practice nurses and patients perceive the benefits and drawbacks of such a change. Our expectation is that the implementation of PFMT in primary care will reduce referrals to secondary care and, through the process of accepting a package of PFMT, empower women to improve their own self-care. It will be interesting to see if these expectations are realised.\n\n\nEthical approval\n\nAfter advice from the National Research Ethics Service South-West (Bristol), UK, the quantitative (baseline and follow-up data) and qualitative evaluations planned for this evaluation of implementation do not require full NHS ethical approval as it is an evaluation of a change in service. However, all interview respondents (both patients and professionals) will be volunteers. Written, informed consent will then be gained using a standardised consent form. Confidentiality will be emphasised at the outset and interview transcripts will be anonymised.",
"appendix": "Author contributions\n\n\n\nSC drafted the manuscript with input from all the authors. RF, CAG, JS, AB and SC designed the study and CAG will supervise the overall study. All authors were involved in the revision of the draft protocol 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 study protocol presents independent research funded by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care (CLAHRC) for the South West Peninsula. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health in England.\n\nThe study has secured funding for a total of £3000 from the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care for the South West Peninsula (PenCLAHRC) for the implementation project to fund the time of the practice nurse trainers to teach practice nurses to understand the principles involved in pelvic floor muscle assessment including observation and digital palpation.\n\n\nAcknowledgements\n\nThe authors acknowledge the preliminary study undertaken by Dr Ann Waterfield as part of her doctoral thesis (2011)8 that showed good outcomes of delivering pelvic floor muscle training with practice nurses.\n\n\nReferences\n\nHunskaar S, Lose G, Sykes D, et al.: The prevalence of urinary incontinence in women in four European countries. BJU Int. 2004; 93(3): 324–30. PubMed Abstract | Publisher Full Text\n\nIrwin DE, Mungapen L, Milsom I, et al.: The economic impact of overactive bladder syndrome in six western countries. BJU Int. 2009; 103(2): 202–209. PubMed Abstract | Publisher Full Text\n\nHay-Smith EJ, Boyle R, Cody JD, et al.: Pelvic floor muscle training for prevention and treatment of urinary and faecal incontinence in antenatal and postnatal women. Cochrane Database Syst Rev. 2012; 10: CD007471. PubMed Abstract | Publisher Full Text\n\nBo K, Talseth T, Holme I: Single, blind randomised controlled trial of pelvic floor exercises, electrical stimulation, vaginal cones, and no treatment in the management of genuine stress incontinence in women. BMJ. 1999; 318: 487–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBump RC, Hurt WG, Fantl JA, et al.: Assessment of Kegel pelvic muscle exercise performance after brief verbal instruction. Am J Obstet Gynecol. 1991; 165(2): 322–327. PubMed Abstract | Publisher Full Text\n\nCammu H, Van Nylen M, Amy JJ: A 10 year follow-up after kegel pelvic floor muscle exercises for genuine stress incontinence. BJU Int. 2000; 85(6): 655–8. PubMed Abstract | Publisher Full Text\n\nNational Institute for Health and Clincial Excellence, Urinary incontinence: The management of urinary incontinence in women. 2009. Reference Source\n\nWaterfield A: A Community Study of Pelvic Floor Muscle Function in Women, in Peninsula College of Medicine and Dentistry. Universities of Exeter and Plymouth 2011. Reference Source\n\nAdekanmi OA, Edwards GJ, Barrington JW: The variation in urodynamic practice in the United Kingdom. J Obstet Gynaecol. 2002; 22(1): 48–50. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "775",
"date": "18 Feb 2013",
"name": "Roger Dmochowski",
"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": "787",
"date": "20 Feb 2013",
"name": "Heinz Koelbl",
"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 think this is an interesting study project, both for the primary aim of the study and for the good study design.It is surely of primary interest to evaluate if the postpartum PFMT, performed as a prevention therapy, can result in fewer referrals to secondary care for urinary incontinence (UI), and then reduce the number and associated costs of surgical procedures for UI.The methodology planned to carry out the study is valid, and in particular the strategy of recruitment seems to be effective.A few suggestions regarding the description of the future results:It would be necessary to stratify the population regarding the time after the last delivery (since the population enrolled is between 25-64 y, it would be interesting to understand if the PFMT performed just after the delivery, will be more effective than the PFMT executed later).More information regarding the PFMT is needed (e.g. for how long, only performed with trained nurse or performed autonomously after training).The results of this trial will help surely to improve the clinical practice and management of UI patients.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-47
|
https://f1000research.com/articles/2-46/v1
|
13 Feb 13
|
{
"type": "Research Article",
"title": "The stretcher spontaneous neurodegenerative mutation models Charcot-Marie-Tooth disease type 4D",
"authors": [
"David Chandler",
"Sash Lopaticki",
"Dexing Huang",
"Michael Hunter",
"Dora Angelicheva",
"Trevor Kilpatrick",
"Rosalind HM King",
"Luba Kalaydjieva",
"Grant Morahan",
"David Chandler",
"Sash Lopaticki",
"Dexing Huang",
"Michael Hunter",
"Dora Angelicheva",
"Trevor Kilpatrick",
"Rosalind HM King",
"Luba Kalaydjieva"
],
"abstract": "Mice affected by a spontaneous mutation which arose within our colony exhibited a neuromuscular phenotype involving tremor and characteristic stretching of the rear limbs. The mutant, named stretcher, was used to breed a backcross cohort for genetic mapping studies. The gene responsible for the mutant phenotype was mapped to a small region on mouse chromosome 15, with a LOD score above 20. Candidate genes within the region included the Ndrg1 gene. Examination of this gene in the mutant mouse strain revealed that exons 10 to 14 had been deleted. Mutations in the human orthologue are known to result in Charcot-Marie-Tooth disease type 4D (CMT4D) a severe early-onset disorder involving Schwann cell dysfunction and extensive demyelination. The stretcher mutant mouse is more severely affected than mice in which the Ndrg1 gene had been knocked out by homologous recombination. Our results demonstrate that the Ndrg1str mutation provides a new model for CMT4D, and demonstrate that exons 10 to 14 of Ndrg1 encode amino acids crucial to the appropriate function of Ndrg1 in the central nervous system.",
"keywords": [
"Over 60 spontaneous mouse mutations that exhibit neurological disorders including movement abnormalities or epilepsy conditions are listed in the Mouse Genome Informatics database. Most of these mutations have been defined at the molecular level. Identifying the genes affected has provided insights into the molecular basis of neurological functions",
"some examples are reviewed in1",
"2. The availability of animal models of disease aids in understanding its molecular basis and is valuable in the search for new treatments. Nevertheless",
"many neurological diseases of humans still lack satisfactory animal models."
],
"content": "Introduction\n\nOver 60 spontaneous mouse mutations that exhibit neurological disorders including movement abnormalities or epilepsy conditions are listed in the Mouse Genome Informatics database. Most of these mutations have been defined at the molecular level. Identifying the genes affected has provided insights into the molecular basis of neurological functions; some examples are reviewed in1,2. The availability of animal models of disease aids in understanding its molecular basis and is valuable in the search for new treatments. Nevertheless, many neurological diseases of humans still lack satisfactory animal models.\n\nPreviously we had mapped a locus, Idd11, which conferred susceptibility to type 1 diabetes in the NOD/LtJ mouse strain3. During the production of congenic mice bearing the C57BL/6J (B6) resistance allele of Idd113,4 on the NOD background in our laboratory, a spontaneous mutation arose. These mutant mice exhibited a neurological defect. This paper describes the phenotypic characterization of these mutant mice, as well as mapping, identification and characterization of the mutant gene.\n\n\nMaterials and methods\n\nMouse work was performed with ethics approval from the Royal Melbourne Hospital Animal Ethics Committee and from the Animal Ethics Committee of The University of Western Australia. All procedures conformed to the Guidelines for the Care and Use of Experimental Animals described by the National Health and Medical Research Council of Australia. BALB/c, C57BL/6J (B6), DBA/2 and NOD/LtJ (NOD) mice were obtained from either the specific-pathogen free colonies of The Walter and Eliza Hall Institute of Medical Research or from the Animal Resources Centre (Murdoch, Western Australia). NOD.Slc9a1b congenic mice4 were maintained in conventional M1 \"shoe box\" mouse cage (335mm Long × 160mm Wide × 130mm High).\n\nEach cage comprised of 1 male and 1 female with litters being weaned from the box at 3 weeks of age. All animals were provided with food and water ad libitum, aspen wood bedding and an environment enrichment consisting of tissue paper for nesting. All animals were cared for by specialist trained staff with experience in clinical observations of ill health, and behaviour irregularities. A vet was on site to provide an opinion to any observations and instigate necropsy if required. Animals that exhibited ill health were euthanased in pre-charged carbon dioxide chambers. The mice displaying the neurological defect, named stretcher (str), were intercrossed with BALB/c mice obtained from The Walter and Eliza Hall Institute of Medical Research. A congenic strain, BALB/c.str was developed after 10 generations of backcrossing to BALB/c, selecting for retention of NOD-derived alleles at markers on chromosome 15. To map the str mutation, we chose to mate NOD.Slc9a1b mutant mice to a third strain, DBA/2 (D2). This was done because the NOD.Slc9a1b mice already had an introduced B6 chromosome region which could potentially complicate mapping.\n\nConventional microsatellite genotyping was performed using MIT markers5 under standard conditions as previously described3. Novel markers were also developed as follows and are listed in Table 1. cDNA sequences of genes previously mapped to the region were BLASTed against GENBANK DNA databases to retrieve genomic sequences. Genomic sequences were also retrieved from the mouse genome sequence6 as reported in the NCBI 37 July 2007 assembly (UCSC Genome Browser). Simple sequence length repeats were selected and primers were designed using the Primer3 program7. Primer sequences are listed in Table 1. These were used to amplify the relevant alleles from NOD and DBA/2 DNA. LOD scores and significance thresholds were calculated as described by Lander and Kruglyak (1995)8.\n\nSimple sequence length repeats were found from inspection of relevant genomic sequences. The location of the nearest known gene, the genomic position (in Mb from the UCSC July 2007 freeze) of the repeat; the primers used to amplify it; the annealing temperature used (Tm), and the sizes of alleles from B6, DBA/2 and NOD mice, are indicated.\n\nWhole kidneys from wild-type BALB/c mice or mutant mice euthanised by exposure to carbon dioxide gas were homogenised in 500µl of Triazol (Gibco) reagent and RNA was extracted according to the manufacturer’s instructions. For cDNA synthesis, 2µg of RNA was reverse transcribed using 1 × reverse transcription buffer (Promega), 1U of RNase inhibitor (Invitrogen), 2mM of dNTPs, 50ng/µl of random hexamers (Promega), and 8U of MMLV-reverse transciptase (Promega) in a total volume of 20µl. Reaction mixes were incubated at 42°C for 60 minutes and the reaction stopped by heat inactivation at 95°C for 10 minutes. The cDNA was used as a template for the amplification of a PCR product spanning exon 6 to 15 of the Ndrg1 gene. The reaction consisted of 1x PCR buffer, 2.5mM MgCl2, 5mM dNTPs, 1.5U Taq polymerase (Kappa), 20ng of each primer (5' GAGGACATGCAGGAGATCAC 3' and 5' CAGAGGCTGTGCGGGACC 3') and water in a total volume of 50µl. PCR cycling conditions consisted of initial denaturation at 95°C followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 45 seconds with a final extension step at 72°C for 7 minutes. The products were cleaned with PCR purification columns (Qiagen) and sequenced using BigDye Terminator chemistry (Life Technologies).\n\nFor northern blotting, 5µg of RNA was electrophoresed on a 1.2% agarose/formamide gel for 2 hours in MOPS buffer. The RNA was transferred to a nitrocellulose membrane via capillary-wick blotting in SSC buffer (Sigma-Aldrich) for 3 hours and the membrane was dried in an oven set at 80ºC for ten minutes. The RNA was fixed onto the membrane by a 4 minute exposure to UV light (312nm) in a UV cabinet. A DNA probe was constructed from a 233bp PCR product spanning exons 2 to 4 of Ndrg1 amplified from mouse kidney cDNA using primers 5' GACCTCGCTGAGGTGAAGCC 3' and 5' GTGATCTCCTGCATGTCCTC 3'. The PCR product was labelled with 32P-CTP using a Random Primed Labelling kit (Roche) according to the manufacturer’s instructions. The membrane was incubated in 3ml of Ultrahyb® hybridization solution (Ambion) for 30 minutes at 42ºC and replaced with 5ml of fresh solution containing the denatured labelled probe (activity of 6.0 × 105 cpm/ml). Hybridization was carried out with rotation at 42ºC for 24 hours. The membrane was then washed twice in 2x SCC, 0.1% SDS buffer pre-warmed to 42ºC for 10 minutes and twice with 0.1xSCC, 0.1% SDS buffer for 15 minutes. The membrane was wrapped in cling film and exposed to Medical X-Ray film for 16 hours at –80ºC. The film was developed on an AGFA CP100 processor.\n\nFor isolation of total protein, sciatic nerves were dissected and homogenised in RIPA buffer (1% Nonidet P-40, 0.1%SDS, 0.5% deoxycholate (Sigma-Aldrich), 150mM NaCl, 50mM Tris pH 8.0, 10µg/ml aprotinin, 1mM PMSF, 1mM benzamidine (Sigma-Aldrich), 0.1mM Na3VO4) and centrifuged at 13,000rpm for 20 minutes at 4ºC. The supernatant was transferred to a new tube and quantitated. 10µg of protein was loaded into single wells of a 12% SDS-PAGE stacking gel (Invitrogen) and electrophoresed at 125V for 30 minutes, and 200V for approximately 1 hr. Proteins were transferred to PVDF membranes (Invitrogen) by western blotting at 30V overnight at 4°C. The membranes were probed first with an affinity-purified polyclonal rabbit antibody raised against the full-length NDRG1 protein (A gift from K. Kokame and T. Miyata). After exposure and subsequent stripping, the membrane was then re-probed with a goat polyclonal IgG directed against the N-terminus of the human NDRG1 protein (Santa Cruz Biotech). Immuno-labelled protein bands were visualised using the ECL+ Chemiluminescence kit (Amersham Biosciences) and exposure to Hyperfilm™ ECL Chemiluminescence film (Amersham Biosciences).\n\nPrimers were derived from the Ndrg1 genomic sequence and used to amplify DNA from B6, NOD/LtJ, and BALB/c.str mice. Sequencing was performed using the Big Dye terminator kit (Life Technologies) followed by capillary electrophoresis on a 3730 DNA analyser (Life Technologies).\n\nHypothetical protein models were constructed from the Ndrg1str cDNA sequence using The HMMSTR/Rosetta Server (available at http://www.bioinfo.rpi.edu/bystrc/hmmstr/server.php) This software implements the HMMSTR (a hidden Markov model for local and secondary structure prediction) and Rosetta (a Monte Carlo Fragment Insertion protein folding program) programs to predict the structure of proteins9. Wild-type and mutant protein sequences were analysed at http://www.predictprotein.org/ to determine whether esterase classification was retained.\n\n\nResults\n\nThe spontaneous mutation was observed in our NOD.Slc9a1b congenic mouse strain4 (referred to as NOD.Idd11B in that paper). The mice showed a characteristic stretching of the rear limbs, especially when they were handled for examination (Figure 1). This feature inspired the mutant strain to be named stretcher (str). The characteristic stretching was accompanied by tremor. Mice also clasped their hind limbs when suspended. The phenotype was most noticeable after 5 weeks of age and progressively worsened, so that after 15 weeks the mice became weak and showed severe tremor of the hind limbs.\n\nThe stretcher mutant is characterized by the stretching and “freezing” of the hind limbs, as illustrated in this photograph. The trait is most apparent when the mice are challenged with some behavioural intervention (e.g. handling for clinical examination).\n\nBecause the strain in which the mutation arose develops type 1 diabetes4, there was a danger of losing the mutant stocks, so we introgressed the str mutation onto the nondiabetic strain, BALB/c. In general, though they are fertile, the male str mice have difficulty in mating. Therefore, the BALB/c.str strain was derived by 10 generations of backcrossing females to BALB/c males (selecting for linked markers that were developed as described below). This strain was maintained by sib mating, taking care to set up brother-sister pairs as soon as they reached breeding age.\n\nAt the same time as the congenic mice were being produced, affected NOD.Slc9a1b mice were also mated with DBA/2 mice in order to map the str locus. The F1 offspring were unaffected, so F2 progeny were produced and observed for the stretcher phenotype. DNA samples from 58 affected F2 mice and 269 unaffected mice were genotyped with markers distributed across the genome. Linkage was observed to markers only on chromosome 15 (Figure 2) with a single-point LOD score = 23.9 at D15Mit63. High resolution genotyping was then performed on both affected and unaffected F2 mice. In this way, it was possible to map the str locus to an interval of approximately 2cM between the markers D15Mit233 and D15Mit144 (Figure 3B). We developed simple sequence length repeat polymorphic markers associated with a number of genes that mapped to the general area, including Kcnq3, Siat4a and Etoile (Table 1). By testing these markers on the panel of F2 mice carrying recombinations between the flanking markers, we excluded Kcnq3 and Etoile as candidates for str, since these mapped either centromeric or telomeric of the critical region, respectively. The D15Mor1 marker defined the new centromeric boundary of the region in which the str locus was mapped. The markers D15Mor3 and –4, defining the Wisp and Siat4a genes respectively, were located within this interval.\n\nAffected F2 progeny (n=49) of (NOD.str x DBA/2) F1 parents were typed with microsatellite markers with an average spacing of 20cM over the 19 autosomes. LOD scores were calculated and the dashed line shows the threshold for significance for an F2 genome-wide scan (Lander & Kruglyak, 1995)8.\n\nA. Affected F2 mice were typed with markers on chromosome 15 and LOD scores calculated as in Figure 1. B. Affected and unaffected mice which had recombinations within 5 cM of the peak of linkage were genotyped with additional markers. “Mit” denotes D15Mit markers, with their given positions in cM; “Mor” denotes novel D15Mor markers developed here (see also Table 1). Filled squares = homozygous for allele derived from the NOD.str strain; d = at least one copy of DBA/2 allele. C. Genomic map of chromosome 15 between the flanking markers D15Mor1 (which is in an intron of Kcnq3) and D15Mit212, from 66.1Mb to 68.5 Mb of the UCSC October 2007 Assembly.\n\nAlthough the critical region covers 3Mb, this interval is relatively gene-poor with only 11 known gene transcripts (Figure 3C). However, several of these genes could be considered candidates for the str mutation. Of these, Ndrg1 was considered as an especially good candidate since mutations in the human orthologue have been shown to be the cause of a demyelinating peripheral neuropathy, Charcot-Marie-Tooth disease type 4D10. This disorder is characterized clinically by distal muscle wasting and atrophy, tendon areflexia, and sensory loss, with onset before ten years of age. Therefore, DNA from str and wild-type NOD mice was amplified using primers designed to amplify Ndrg1 exons from the genomic sequence.\n\nSequences of these amplicons were compared to the available genomic sequences but no polymorphisms which would result in amino acid substitutions were identified. During the course of this work, we were unable to amplify exons 10, 11, 12, 13 or 14 from the str mice. We reasoned the most likely explanation for this finding was that these exons had been deleted. A number of primers flanking exons 9 and 15 were designed and used in various combinations to test this hypothesis. Eventually, we were able to confirm that these exons had in fact been deleted, and to define the exact points between which the deletion had occurred. As shown in Figure 4, over 5kb of DNA encompassing exons 10 to 14 had been deleted. The deletion breakpoints are precise, with no addition of nontemplated nucleotides.\n\nA. Genomic organization of Ndrg1 gene. Exons are represented by filled boxes. The extent of deletion between introns 9 and 14 is indicated; the deleted sequence is indicated by the dotted line and empty boxes. Sequence is shown reversed in comparison to chromosomal orientation. B. Sequence flanking the deletion point. Lower case: sequence from intron 9; upper case: intron 14 sequence; underline: sites for primers to amplify deletion allele.\n\nThe northern blot analysis revealed a shorter mRNA band, present in the str animals at levels similar to the normal product found in WT littermates (Figure 5A). The sequence of Ndrg1str cDNA confirmed that transcripts from the mutant allele were processed with in-frame splicing directly from exons 9 to 15 (Figure 5C). A western blot analysis of protein extracted from sciatic nerve revealed a faint band at ~32 kDa, corresponding to the expected molecular mass of the mutant protein missing the 99 amino acids encoded by the deleted exons (Figure 5B). Bioinformatic analysis of the abnormal protein showed it could remain classified as a member of the esterases/lipases superfamily. The one letter amino acid codes for both the ndrg1 WT and mutant proteins are displayed below:\n\nmsrelhdvdlaevkplvekgesitgllqefdvqeqdietlhgslhvtlcgtpkgnrpviltyhdigmnhktcynplfnsedmqeitqhfavchvdapgqqdgapsfpvgymypsmdqlaemlpgvlhqfglksvigmgtgagayiltrfalnnpemveglvlmnvnpcaegwmdwaaskisgwtqalpdmvvshlfgkeeihnnvevvhtyrqhilndmnpsnlhlfisaynsrrdleierpmpgthtvtlqcpallvvgdnspavdavvecnskldptkttllkmadcgglpqisqpaklaeafkyfvqgmgympsasmtrlmrsrtasgssvtslegtrsrshtsegprsrshtsegsrsrshtsedarlnitpnsgatgnnagpksmevsc.\n\nmsrelhdvdlaevkplvekgesitgllqefdvqeqdietlhgslhvtlcgtpkgnrpviltyhdigmnhktcynplfnsedmqeitqhfavchvdapgqqdgapsfpvgymypsmdqlaemlpgvlhqfglksvigmgtgagayiltrfalnnpemveglvlmnvnpcaegwmdwaaskisgwtqalpdmvvshlfgkpaklaeafkyfvqgmgympsasmtrlmrsrtasgssvtslegtrsrshtsegprsrshtsegsrsrshtsedarlnitpnsgatgnnagpksmevsc.\n\nA. A northern blot of RNA from kidneys of wild-type and str mice. A 233bp probe spanning exons 2–4 detected in the mutant mouse anRNA species shorter than that seen in the wild-type Ndrg1 RNA (left panel). Ethidium bromide-staining of the agarose gel prior to northern transfer showed equal amounts of RNA were loaded (right panel). B. Western blot of sciatic nerve lysates prepared from wild-type and str mutant mice, probed with antibodies raised against the full-length Ndrg1 protein (left) or GAPDH (right). The full length (43 kDa) Ndrg1 protein was absent from the lysate of the str mice but an immunoreactive truncated (32 kDa) protein was present in a lower amount; this size is approximately that predicted for the Ndrg1str mutant protein. C. Chromatograms and translated protein sequences of Ndrg1 cDNA prepared from kidney tissue from 1) wildtype BALB/C and 2) the mutant mouse. The deletion results in the skipping of exons 10–14. Exon 15 is spliced in-frame with exon 9.\n\nFurther biochemical and structural characterization of the effect of the stretcher mutation is described elsewhere11.\n\nSplice sites for exons 9 and 15 were unaffected by the deletion, and sequencing of the transcripts from the truncated gene showed they could be spliced correctly but would encode a smaller protein product than would the wild-type gene. Hypothetical structures for the normal and mutant proteins were generated using the HMMSTR/Rosetta Server9. These models are presented in Figure 6. The predicted structure of the truncated protein has an overall similarity to the wild-type, but also contains conformational changes in compensation for the deleted sequences. The major changes to the first third of the molecule may explain the functional deficit of the mutant Ndrg1 molecule in the str mice. In view of the low amounts detectable by western blot, the mutant protein is likely to be unstable.\n\nCertain residues are indicated for reference. Cyan, amino acids prior to #199; red, residues encoded by exons deleted in the Ndrg1str mutant; blue, residues 298-end of wild-type Ndrg1.\n\n\nDiscussion\n\nHere we report the identification and characterization of the spontaneous mutant stretcher mouse, a new model of Charcot-Marie-Tooth 4D disease, with a spontaneous deletion of exons 10–14 of the Ndrg1 gene. We showed that the Ndrg1str mutation results in low levels of expression of a truncated protein which, compared to the normal protein, is missing 99 amino acids (ie #199 to 297 of the wild-type sequence).\n\nThe absent Ndrg1 fragment is due to the deletion in the Ndrg1str allele. The protein fragment encoded by the deleted exons does not show homology to any particular conserved domain family. The functional importance of the missing domain is highlighted by both the str mutation and the human splicing mutation, 2290787G>A which skipped exon 912. The reading frame was preserved in both mutations, yet the phenotype in each case was severe peripheral neuropathy. The low detectable levels of aberrant protein suggest that it is unstable, leading to the neurological phenotype observed only in homozygote mutant mice.\n\nThe stretcher mutation has been characterized by histology11 and is more severely affected in both molecular and behavioural phenotypes than was reported for the Ndrg1-/- mouse13. Though the comparisons should be made on the same genetic background, the milder phenotype of the Ndrg1-/- mouse is probably due to the knockout strategy which resulted in excision of the promoter and exon 1, but left intact the initiation codon in exon 2 as well as the rest of the coding region. It seems that these mice are able to produce sufficient amounts of full-length protein to avoid the more extreme phenotype displayed by the stretcher mutant mice, and only display the reported milder phenotype of muscle weakness.\n\nWe conclude the Ndrg1str mutant mouse will be a useful resource for investigating the role of Ndrg1 in maintaining the myelin sheath, and for modelling the human disorder, Charcot-Marie-Tooth disease 4D.",
"appendix": "Author contributions\n\n\n\nTK, RHMK, LK and GM designed and analysed experiments. DC, DA, SL, DH and MH performed the research. GM and DC wrote the manuscript. All the authors reviewed and approved this article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by Program Grants 516700 and 37612600 from the National Health and Medical Research Council of Australia to GM; NHMRC grants to LK; by grant DP110102067 from the Australian Research Council; and by L’Association Francaise contre les Myopathies (RHMK and LK). GM is supported by the Diabetes Research Foundation of Western Australia.\n\n\nReferences\n\nWatase K, Zoghbi HY: Modelling brain diseases in mice: the challenges of design and analysis. Nat Rev Genet. 2003; 4(4): 296–307. PubMed Abstract | Publisher Full Text\n\nBaraban SC: Emerging epilepsy models: insights from mice, flies, worms and fish. Curr Opin Neurol. 2007; 20(2): 164–8. PubMed Abstract | Publisher Full Text\n\nMorahan G, McClive P, Huang D, et al.: Genetic and physiological association of diabetes susceptibility with raised Na+/H+ exchange activity. Proc Natl Acad Sci U S A. 1994; 91(13): 5898–902. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrodnicki TC, McClive P, Couper S, et al.: Localization of Idd11 using NOD congenic mouse strains: elimination of Slc9a1 as a candidate gene. Immunogenetics. 2000; 51(1): 37–41. PubMed Abstract | Publisher Full Text\n\nDietrich W, Katz H, Lincoln SE, et al.: A genetic map of the mouse suitable for typing intraspecific crosses. Genetics. 1992; 131(2): 423–47. PubMed Abstract | Free Full Text\n\nWaterston RH, Lindblad-Toh K, Birney E, et al.: Initial sequencing and comparative analysis of the mouse genome. Nature. 2002; 420(6915): 520–62. PubMed Abstract | Publisher Full Text\n\nRozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000; 132: 365–86. PubMed Abstract | Publisher Full Text\n\nLander E, Kruglyak L: Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet. 1995; 11(3): 241–7. PubMed Abstract | Publisher Full Text\n\nBystroff C, Shao Y: Fully automated ab initio protein structure prediction using I-SITES, HMMSTR and ROSETTA. Bioinformatics. 2002; 18(Suppl 1): S54–61. PubMed Abstract | Publisher Full Text\n\nKalaydjieva L, Gresham D, Gooding R, et al.: N-myc downstream-regulated gene 1 is mutated in hereditary motor and sensory neuropathy-Lom. Am J Hum Genet. 2000; 67(1): 47–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKing RH, Chandler D, Lopaticki S, et al.: Ndrg1 in development and maintenance of the myelin sheath. Neurobiol Dis. 2011; 42(3): 368–80. PubMed Abstract | Publisher Full Text\n\nHunter M, Bernard R, Freitas E, et al.: Mutation screening of the N-myc downstream-regulated gene 1 (NDRG1) in patients with Charcot-Marie-Tooth Disease. Hum Mutat. 2003; 22(2): 129–35. PubMed Abstract | Publisher Full Text\n\nOkuda T, Higashi Y, Kokame K, et al.: Ndrg1-deficient mice exhibit a progressive demyelinating disorder of peripheral nerves. Mol Cell Biol. 2004; 24(9): 3949–56. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "899",
"date": "17 Apr 2013",
"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\nThe title and abstract are appropriate for the content of the article. The article is clearly written, and the design, methods and analysis are appropriate. The article shows that the mutation is due to deletion of exons 10-14 of the Ndrg1 gene, resulting in an unstable smaller protein, obtained by translation of exons 1-9 and exons 15 and 16 in frame. The functional deficits in the hind limbs appear clearly at about 5 weeks of age – this represents a relatively early onset which can be compared to the early onset seen in human recessive Charcot-Marie-Tooth type 4D which shows an early onset. The model appears to model the human disease phenotype more faithfully than the previously reported Ndrg1 knockout mouse and therefore represents an additional tool for research.",
"responses": []
},
{
"id": "933",
"date": "07 May 2013",
"name": "Fransesc Palau",
"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 new knowledge on the role of the Ndrg1 gene in the biology of peripheral nerve, and more specifically in the pathophysiology of Charcot-Marie-Tooth disease type 4D (CMT4D) caused by mutations in Ndrg1. Most importantly, the stretcher mouse (Ndrg1str) provides a very useful tool to investigate in depth the myelin biology and defects associated to NDRG1 protein. From the experimental aspects, the authors show a nice and classical genetic approach to isolate and characterize the gene in a spontaneous mutant mouse, which include phenotyping, gene mapping, gene expression and molecular genetics to define the mutation. Further histological phenotypes have been reported elsewhere, as indicated by the authors (Ref. 11- King et al.). It is noteworthy, the finding of a more severe phenotype in the stretcher mouse than in the Ndrg1-/-, which suggests the protein domain encoded by exons 10-14 are very important in protein function (a biological aspect that deserves more attention in future investigations). In this way, the paper remains a description of an animal model of CMT4D, and I want to state the relevance to move forward to the functional role of NDRG1 in the physiology of the Schwann cell and Schwann cell-axon interaction.",
"responses": []
},
{
"id": "959",
"date": "20 May 2013",
"name": "Angelo Schenone",
"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 for the content of the article and the abstract does represent a good summary of the work. Design, methods and analysis are nicely explained and indeed appropriate for the topic of the study. Results are definitely important to the research in the field of hereditary neuropathies, as they give insights on the role of the NDRG1 gene in the development of a rare type of CMT (CMT4D). Having good animal models for the different types of CMT and a detailed description of their phenotype (given in the paper published in Neurobiology of Disease- PMID21303696) is pivotal to unravel disease pathomechanisms and program future therapies. The discussion and conclusions are justified. However, it would have been very helpful to the readers reporting the presence of a canine model of CMT4D which carries a small deletion within the NDRG1 gene. Canine CMT is relatively frequent and, to the best of my knowledge, this is the only model in which a genetic change has been reported.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-46
|
https://f1000research.com/articles/2-45/v1
|
13 Feb 13
|
{
"type": "Research Article",
"title": "UVB irradiation does not directly induce detectable changes of DNA methylation in human keratinocytes",
"authors": [
"Christoph Lahtz",
"Sang-In Kim",
"Steven E Bates",
"Arthur X Li",
"Xiwei Wu",
"Gerd P Pfeifer",
"Christoph Lahtz",
"Sang-In Kim",
"Steven E Bates",
"Arthur X Li",
"Xiwei Wu"
],
"abstract": "Unprotected exposure to UVB radiation from the sun and the resulting DNA damage are thought to be responsible for physiological changes in the skin and for a variety of skin cancers, including basal cell and squamous cell carcinoma and malignant melanoma. Although the mutagenic effects of UVB have been well documented and studied mechanistically, there is only limited information as to whether UV light may also be responsible for inducing epigenetic changes in the genome of exposed cells. DNA methylation is a stable epigenetic modification involved in gene control. To study the effects of UVB radiation on DNA methylation, we repeatedly exposed normal human keratinocytes to a UVB light source. After a recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA) method in combination with high-resolution microarrays. Bioinformatics analysis revealed only a limited number of possible differences between UVB-exposed and control cells. However, these minor apparent changes could not be independently confirmed by bisulfite sequencing-based approaches. This study reveals that UVB irradiation of keratinocytes has no recognizable global effect on DNA methylation patterns and suggests that changes in DNA methylation, as observed in skin cancers, are not immediate consequences of human exposure to solar UVB irradiation.",
"keywords": [
"Solar UV light is divided into three wavelength categories: UVA with a wavelength between 320 nm and 400 nm",
"UVB with a wavelength between 280 nm and 320 nm",
"and far UV light (UVC) with a wavelength between 100 nm and 280 nm. UVC radiation is filtered by the atmosphere and technically does not exist on the earth’s surface. However",
"a fraction of UVB and much of the UVA wavelength radiation reach the surface of the earth and have been implicated in skin cancers and other acute and chronic aberrations of the skin such as sunburn and premature skin aging",
"respectively. Most of the skin cancer-causing effects of sunlight have been ascribed to UVB radiation with a smaller contribution from UVA1",
"2. UVB induces direct DNA damage through the formation of cyclobutane pyrimidine dimers (CPDs) and another dipyrimidine lesion",
"the (6-4) photoproduct3–6. Of these two types of lesions",
"the CPD is thought to be responsible for the majority of mutations induced by UVB or sunlight irradiation7",
"8. These mutations are characterized by a preponderance of C to T transition mutations at dipyrimidine sites containing cytosine",
"for example 5′TC and 5′CC. Very often",
"5-methylcytosines (mC)",
"when part of a dipyrimidine sequence",
"are seen as preferential sites of CPD formation and also as preferential mutational target sites in mammalian cells9–11. These types of mutations",
"i.e. C or mC to T mutations at 5′TC",
"5′CC",
"5′TmC",
"and 5′CmC",
"are recognized as the major mutational events in human skin cancers",
"both in specific genes4 and in large-scale genomic sequencing studies analyzing thousands of different genes simultaneously12–14."
],
"content": "Introduction\n\nSolar UV light is divided into three wavelength categories: UVA with a wavelength between 320 nm and 400 nm, UVB with a wavelength between 280 nm and 320 nm, and far UV light (UVC) with a wavelength between 100 nm and 280 nm. UVC radiation is filtered by the atmosphere and technically does not exist on the earth’s surface. However, a fraction of UVB and much of the UVA wavelength radiation reach the surface of the earth and have been implicated in skin cancers and other acute and chronic aberrations of the skin such as sunburn and premature skin aging, respectively. Most of the skin cancer-causing effects of sunlight have been ascribed to UVB radiation with a smaller contribution from UVA1,2. UVB induces direct DNA damage through the formation of cyclobutane pyrimidine dimers (CPDs) and another dipyrimidine lesion, the (6-4) photoproduct3–6. Of these two types of lesions, the CPD is thought to be responsible for the majority of mutations induced by UVB or sunlight irradiation7,8. These mutations are characterized by a preponderance of C to T transition mutations at dipyrimidine sites containing cytosine, for example 5′TC and 5′CC. Very often, 5-methylcytosines (mC), when part of a dipyrimidine sequence, are seen as preferential sites of CPD formation and also as preferential mutational target sites in mammalian cells9–11. These types of mutations, i.e. C or mC to T mutations at 5′TC, 5′CC, 5′TmC, and 5′CmC, are recognized as the major mutational events in human skin cancers, both in specific genes4 and in large-scale genomic sequencing studies analyzing thousands of different genes simultaneously12–14.\n\nBesides mutations, the other frequent change observed at the DNA level of skin cancer genomes is the aberration of DNA cytosine methylation patterns. Like most cancer types, both nonmelanoma and melanoma skin cancers are characterized by substantially aberrant DNA methylation15–21. DNA hypermethylation is widespread and affects many CpG islands, which are defined as CpG dinucleotide-rich genomic sequences, often found around promoters of genes. This DNA hypermethylation can affect hundreds of genes in individual tumors, sometimes producing a cancer-driving event, for example if genes involved in growth control and/or DNA repair are involved22,23. Although the methylation changes were first described many years ago, the mechanisms of cancer-associated DNA hypermethylation or hypomethylation have remained obscure. One model proposes that environmental influences, in the form of exposure of humans to either chemicals or radiation, may produce these aberrant DNA methylation events24. For example, one could conceive a scenario in which this exposure induces a signaling cascade and transcriptional changes inside cells that would affect DNA methylation patterns, for example by modulating the DNA methylation machinery or the chromatin state at genes that become susceptible to methylation. Such UV-induced heritable DNA methylation changes could lead to an altered phenotype and could provide a selective advantage to cells, perhaps when combined with UVB-induced mutations, and could thus be viewed as a tumor-driving event. In this study, we examined this hypothesis by exposing human keratinocytes chronically to UVB radiation and by assessing DNA methylation patterns on a genomic scale following UV exposure of cells and a recovery period.\n\n\nMaterials and methods\n\nNormal human keratinocytes (Clonetics; San Diego) were grown in EpiLife Medium (Invitrogen; Carlsbad, CA). The restriction enzyme used for combined bisulfite restriction analysis (COBRA), TaqαI (5′-TCGA-3′), was obtained from New England Biolabs (Ipswich, MA).\n\nThe UVB source we used consisted of three fluorescent light tubes (Philips TL 20 W/12R) filtered through a cellulose acetate sheet, which eliminates wavelengths below 295 nm. The source has a peak spectral emission at 312 nm. The keratinocytes were grown in 150 mm cell culture dishes. The cells were irradiated with doses of 260 J/m2 (high dose) and 130 J/m2 (low dose) of UVB after the medium had been removed and cells had been washed three times with phosphate buffered saline. After UVB exposure, the cells were grown in new culture medium for three days. They were then irradiated again, and then nine more times with a two or three day recovery time between each irradiation cycle. After the final irradiation dose was delivered, the cells were grown for a recovery period of eight days or 18 days.\n\nThe cells were trypsinized and collected by centrifugation. After a proteinase K treatment, DNA was isolated with a standard phenol/chloroform extraction method followed by ethanol precipitation25.\n\nTo detect potential genome-wide changes in DNA methylation patterns after the UVB treatments, the methylated-CpG island recovery assay (MIRA) combined with microarray analysis was used as described previously26,27. Nimblegen’s Signalmap program was used to visualize the DNA methylation data and for generation of profiling snapshots.\n\nLoess normalization was applied to the raw intensity files of each array to correct intensity-dependent dye bias and obtain log2 ratios between MIRA and input samples. Then the log2 ratios across all the samples were quantile-normalized. Probes were considered positive if their normalized log2 ratios were above 2-fold. Peaks in each sample were called if a minimum of four consecutive positive probes were present with either one gap or no gaps. To identify hypermethylated and hypomethylated targets in UVB-treated samples vs. untreated control samples, the average probe log2 ratio signals within the peaks identified in each UVB-treated sample were compared to the untreated samples. Only the peaks with an average log2 ratio signal difference of more than log2(3) were considered hypermethylated or hypomethylated peaks. These differential peaks were annotated to the Refseq transcript database downloaded from the UCSC genome database. The microarray data have been deposited into the GEO database (accession number GSE42943).\n\nCandidate loci were investigated by combined bisulfite restriction analysis (COBRA)28. PCR was performed with primers and conditions listed in Supplementary Table S1 and Supplementary Table S2. Briefly, COBRA-PCR was performed with bisulfite-converted DNA-specific primers using 50 ng of bisulfite- modified genomic DNA as template for 60 cycles after a 15 min incubation at 95°C, then 30 s at the TA (see Table S2) and 30 s at 72°C in a 25 µl volume containing 5 nmol dNTPs, 20 pmol primers, and 1.25 units of Hot start Taq DNA polymerase (Qiagen, Valencia, CA). Five microliters of the PCR product was analyzed on a 2% agarose gel. Equal amounts of PCR product were digested with the restriction enzyme TaqαI (5′-TCGA-3′).\n\nCOBRA PCR products were created as described above. The PCR products were ligated into a cloning vector (TOPO® Cloning Kit; Invitrogen, Grand Island, NY, or the pGEM®-T-Easy Kit, Promega, Madison, WI) and transformed into competent cells. Different clones were picked randomly and the plasmids were isolated and sequenced. For the analysis of methylated and unmethylated cytosines, the free software program Bioedit was used.\n\n\nResults\n\nWe irradiated human keratinocytes with two different doses of UVB, 130 J/m2 and 260 J/m2. These doses were well tolerated by the cells and did not produce overt losses in cell viability. Cells were irradiated chronically (11 times total) with these doses with a 2–3-day recovery period between each irradiation cycle. Controls included cells that were not irradiated and cells irradiated once with 130 or 260 J/m2 of UVB, but then harvested immediately after the irradiation.\n\nAfter the irradiation and final recovery times of eight or eighteen days, DNA was isolated from the cells. DNA was sonicated and the methylated fraction of the genome was enriched by use of the methylated CpG island recovery assay (MIRA) technique29. The methylated fraction was hybridized relative to input DNA onto NimbleGen CpG island plus promoter arrays. These arrays cover all ~28,000 CpG islands of the human genome and all Refseq gene promoters from -2.4 kb to +0.6 kb relative to the transcription start site. Analysis of methylation patterns using NimbleGen’s Signalmap display software indicated the excellent reproducibility of the data (Figure 1). The patterns of methylation peaks were remarkably similar between all four controls and all three UVB-irradiated samples. Bioinformatics analysis was used to identify potential differences between the control and treatment groups. Methylation peaks were identified as described in Materials and Methods. Peaks with an average log2 ratio signal difference of more than log2(3) were considered as hypermethylated or hypomethylated peaks, respectively. Table 1 summarizes the number of differences identified between UVB and control treatment groups. Most comparisons revealed only a handful of differences and the numbers of differential peaks were generally below 100 for each comparison. In fact, comparison between two controls, non-irradiated cells grown for 18 days or 8 days (N18con vs. N8con), respectively, showed a greater number of differences than any comparison between a UVB-irradiated and a control sample (Table 1). Nonetheless, a small number of differential peaks could clearly be detected on SignalMap profiles (Figure 2). We show examples for the genes CXXC5, PPP3CB, IL17C, CCDC40 and C21orf29, where either hypermethylation or hypomethylation in a UVB-irradiated sample was observed (Figure 2).\n\nA. A random segment of chromosome 1 is shown to indicate the reliability of the method and to show the uniform peaks between control and UVB-irradiated samples. The positions of transcripts and CpG islands are indicated. The samples labeled ‘low UVB control’ and ‘high UVB control’ represent DNA immediately harvested following a one-time irradiation of cells with either 130 J/m2 (low) or 260 J/m2 (high) of UVB. Samples labeled ‘8 days control’ or ’18 days control’ were grown for the same time periods as the irradiated cells but were never irradiated. Samples labeled ‘18 days low UVB’ or ‘18 days high UVB’ were chronically irradiated with 130 J/m2 (low) or 260 J/m2 (high) of UVB followed by an 18 day recovery period. The sample labeled ‘8 days high UVB’ was chronically irradiated with 260 J/m2 of UVB followed by an 8 day recovery period. B. Methylation peaks are shown for randomly selected genomic regions on chromosomes 1, 3, 5, 7, 11, 17 and X. The positions of transcripts and CpG islands are indicated. The chromosomal coordinates are shown above each snapshot.\n\naTreatments:\n\nLcon: 130 J/m2 UVB once, cells harvested immediately after irradiation\n\nHcon: 260 J/m2 UVB once, cells harvested immediately after irradiation\n\nN8con: no UVB, cells harvested after 8 days\n\nN18con: no UVB, cells harvested after 18 days\n\nH8d: 260 J/m2 UVB, 11 times, cells harvested 8 days following final dose\n\nL18d: 130 J/m2 UVB, 11 times, cells harvested 18 days following final dose\n\nH18d: 260 J/m2 UVB, 11 times, cells harvested 18 days following final dose\n\nbHyper- and hypomethylated peaks were defined as described in Materials and Methods\n\nMethylation peaks are shown for the genes CXXC5, PPP3CB, IL17C, CCDC40, and C21orf29. The positions of transcripts and CpG islands are indicated. The chromosomal coordinates are shown above each snapshot. The signals framed by the red rectangles are significantly (*) differentially methylated pairs of a control sample and a UVB-treated sample, as determined by bioinformatics analysis. The description of the samples is shown in Figure 1.\n\nTo verify the apparent methylation differences observed by microarray analysis, we performed sodium bisulfite conversion-based DNA methylation assays. COBRA analysis is shown for the genes CXXC5, PPP3CB, IL17C, CCDC40 and C21orf29 in Figure 3. Cleaved molecules in these assays indicate methylated restriction sites that are resistant to bisulfite conversion and remain cleavable by the CpG-targeting restriction enzyme after PCR. Uncut molecules represent unmethylated DNA fragments. The COBRA assays indicated that the methylation patterns were the same or very similar between DNA isolated from control cells and DNA from UVB-irradiated cells.\n\nCOBRA analysis of restriction enzyme sites indicates no substantial difference in DNA methylation in UVB-irradiated cells versus controls. Samples labeled ‘8 d high UVB’ and ‘18 d high UVB’ were chronically irradiated with 260 J/m2 of UVB followed by an 8 day or 18 day recovery period, respectively. Samples labeled ‘8 d control’ or ‘18 d control’ were grown for the same time periods as the irradiated cells but were never irradiated. The samples labeled ‘high UVB control’ represent DNA immediately harvested following a one-time irradiation of cells with 260 J/m2 of UVB. The genes CXXC5, PPP3CB, IL17C, CCDC40 and C21orf29 were analyzed by TaqαI digestion. The PCR products were cleaved with the enzyme (E) or were left uncleaved (-). The methylated and unmethylated control samples at the right side of the gel panels were fully CpG-methylated and unmethylated control DNAs.\n\nCOBRA assays, although generally indicative of the methylation status of a genomic target, can score only a limited number of CpG sites. Therefore, we performed sodium bisulfite sequencing to provide the methylation status of all CpG within the amplified target fragments. These assays also indicated no substantial difference between control and UV-irradiated cells (Figure 4). Similar methylation patterns were observed for the five different gene targets, regardless of whether the cells were UVB-irradiated or not. A somewhat lower frequency of methylated CpG sites was observed for the IL17C gene in UVB-irradiated cells (18.4% versus 29%). However, these results may be biased by the few molecules in the population that had almost every CpG methylated. Taken together, our results suggest that the rather small number of differential peaks observed by bioinformatics analysis were false positives. Such small numbers of false positive differences can be expected when comparisons are made for over 28,000 CpG islands and about 20,000 Refseq promoters.\n\nBisulfite sequencing data are shown for the genes CXXC5 (A), PPP3CB (B), IL17C (C), CCDC40 (D), and C21orf29 (E). The chronic UVB dose was 260 J/m2. Open circles represent unmethylated CpG sites and black circles indicate methylated CpG sites. The percentage of methylated sites is indicated below the sequence data for individual cloned molecules.\n\n\n\n\nDiscussion\n\nThe field of environmental epigenetics has recently received much attention24. Environmental genotoxic or non-genotoxic carcinogens, theoretically at least, could alter the epigenome at the levels of DNA methylation or histone posttranslational modifications. Such carcinogen-induced epigenetic effects may be heritable and may contribute to the etiology of human cancer and other diseases. Effects of environmental exposure on the epigenome may be direct or indirect. For example, indirect effects could be produced by carcinogen-induced gene mutations in a gene target encoding an epigenetic modifier protein thus leading to an epigenetic defect. Mutation of histone modifying enzymes or DNA modification enzymes would fall into this category. Such mutations are indeed observed in several types of human cancer30 including melanoma13. A more immediately acting but still indirect effect of an environmental exposure on epigenetic marks may occur via signaling cascades, e.g. through a DNA damage response pathway, impinging on the expression levels of epigenetic modifiers or on local chromatin structure at a susceptible gene locus, thereby modulating DNA methylation patterns.\n\nOn the other hand, exposure of target cells to an environmental agent may produce a more direct effect on the epigenome if the effect is mediated by DNA damage. In the case of UV irradiation, an interesting photochemical deamination and demethylation pathway of 5-methylcytosine has been described31 but its biological relevance has remained unknown. Furthermore, it has been shown several decades ago that UVB irradiation can inhibit DNA methyltransferases in vitro32. UVB-induced pyrimidine dimers may also alter nucleosome association with DNA33, thereby potentially changing DNA methylation patterns at a susceptible gene locus. Repair of the pyrimidine dimer damage may remove methylated cytosines during the excision repair step thus contributing potentially to altered DNA methylation.\n\nHowever, our data are inconsistent with proposals that UVB can change DNA methylation patterns heritably as a direct consequence of chronic exposure. Rather, our sensitive and specific genome-scale analysis of DNA methylation patterns in UVB-exposed keratinocytes has not uncovered any substantial changes in DNA methylation patterns, either 8 days or 18 days following chronic exposure of the cells to UVB. The few differences observed on the microarray could not be confirmed by bisulfite-based sequence analysis thus suggesting that these differences were false positives. While we cannot exclude the possibility that more drastic UV doses or a longer waiting time may produce some changes, the data presented here make it questionable that the many DNA methylation differences observed in human skin cancers are a direct consequence of exposure of skin cells to UVB radiation from the sun. The numerous skin cancer-associated methylation changes must therefore be events occurring later during the cell transformation process. As discussed earlier, the vast majority of the methylation differences seen in cancer are likely passenger events and do not appear to be selected22. These methylation events could be secondary to specific genetic events, as discussed above, or they could represent random or locus-targeted methylation gains or losses occurring over the timeline of enhanced cell proliferation. Alternatively, DNA methylation differences found in skin tumors could be induced by other processes such as inflammation.\n\nFuture studies will determine if methylation changes can be induced directly by UVA, a longer wavelength component of sunlight that also has a strong component of oxidative DNA damage4,34. UVA, however, produces less severe overall levels of DNA damage than UVB and therefore may be even less likely to induce direct DNA damage-dependent changes of methylation patterns. Other skin cell types, in particular melanocytes, will also need to be analyzed to better understand the role of UV irradiation in melanoma pathogenesis.",
"appendix": "Author contributions\n\n\n\nGPP devised and planned the experiments. CL, SIK and SEB carried out the experiments. CL, AXL and XW analyzed the data. CL and GPP interpreted the results and CL and GPP wrote the paper. All authors read and agreed to the final content of the paper\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed\n\n\nGrant information\n\nThis study was supported by NIH grant ES06070 to GPP.\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nDe Fabo EC, Noonan FP, Fears T, et al.: Ultraviolet B but not ultraviolet A radiation initiates melanoma. Cancer Res. 2004; 64(18): 6372–6376. PubMed Abstract | Publisher Full Text\n\nde Gruijl FR: Photocarcinogenesis: UVA vs. UVB radiation. Skin Pharmacol Appl Skin Physiol. 2002; 15(5): 316–320. PubMed Abstract | Publisher Full Text\n\nPfeifer GP: Formation and processing of UV photoproducts: effects of DNA sequence and chromatin environment. Photochem Photobiol. 1997; 65(2): 270–283. PubMed Abstract | Publisher Full Text\n\nPfeifer GP, You YH, Besaratinia A: Mutations induced by ultraviolet light. Mutat Res. 2005; 571(1–2): 19–31. PubMed Abstract | Publisher Full Text\n\nCadet J, Sage E, Douki T: Ultraviolet radiation-mediated damage to cellular DNA. Mutat Res. 2005; 571(1–2): 3–17. PubMed Abstract | Publisher Full Text\n\nMitchell DL, Fernandez AA: Different types of DNA damage play different roles in the etiology of sunlight-induced melanoma. Pigment Cell Melanoma Res. 2011; 24(1): 119–124. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoon JH, Lee CS, O’Connor TR, et al.: The DNA damage spectrum produced by simulated sunlight. J Mol Biol. 2000; 299(3): 681–693. PubMed Abstract | Publisher Full Text\n\nYou YH, Lee DH, Yoon JH, et al.: Cyclobutane pyrimidine dimers are responsible for the vast majority of mutations induced by UVB irradiation in mammalian cells. J Biol Chem. 2001; 276(48): 44688–44694. PubMed Abstract | Publisher Full Text\n\nTommasi S, Denissenko MF, Pfeifer GP: Sunlight induces pyrimidine dimers preferentially at 5-methylcytosine bases. Cancer Res. 1997; 57(21): 4727–4730. PubMed Abstract\n\nYou YH, Pfeifer GP: Similarities in sunlight-induced mutational spectra of CpG-methylated transgenes and the p53 gene in skin cancer point to an important role of 5-methylcytosine residues in solar UV mutagenesis. J Mol Biol. 2001; 305(3): 389–399. PubMed Abstract | Publisher Full Text\n\nIkehata H, Ono T: Significance of CpG methylation for solar UV-induced mutagenesis and carcinogenesis in skin. Photochem Photobiol. 2007; 83(1): 196–204. PubMed Abstract | Publisher Full Text\n\nPfeifer GP: Environmental exposures and mutational patterns of cancer genomes. Genome Med. 2010; 2(8): 54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHodis E, Watson IR, Kryukov GV, et al.: A landscape of driver mutations in melanoma. Cell. 2012; 150(2): 251–263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPleasance ED, Cheetham RK, Stephens PJ, et al.: A comprehensive catalogue of somatic mutations from a human cancer genome. Nature. 2010; 463(7278): 191–196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSathyanarayana UG, Moore AY, Li L, et al.: Sun exposure related methylation in malignant and non-malignant skin lesions. Cancer Lett. 2007; 245(1–2): 112–120. PubMed Abstract | Publisher Full Text\n\nKoga Y, Pelizzola M, Cheng E, et al.: Genome-wide screen of promoter methylation identifies novel markers in melanoma. Genome Res. 2009; 19(8): 1462–1470. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Doorn R, Gruis NA, Willemze R, et al.: Aberrant DNA methylation in cutaneous malignancies. Semin Oncol. 2005; 32(5): 479–487. PubMed Abstract | Publisher Full Text\n\nFuruta J, Nobeyama Y, Umebayashi Y, et al.: Silencing of Peroxiredoxin 2 and aberrant methylation of 33 CpG islands in putative promoter regions in human malignant melanomas. Cancer Res. 2006; 66(12): 6080–6086. PubMed Abstract | Publisher Full Text\n\nSigalotti L, Fratta E, Bidoli E, et al.: Methylation levels of the “long interspersed nucleotide element-1” repetitive sequences predict survival of melanoma patients. J Transl Med. 2011; 9: 78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTellez CS, Shen L, Estecio MR, et al.: CpG island methylation profiling in human melanoma cell lines. Melanoma Res. 2009; 19(3): 146–155. PubMed Abstract | Publisher Full Text\n\nLahtz C, Stranzenbach R, Fiedler E, et al.: Methylation of PTEN as a prognostic factor in malignant melanoma of the skin. J Invest Dermatol. 2010; 130(2): 620–622. PubMed Abstract | Publisher Full Text\n\nKalari S, Pfeifer GP: Identification of driver and passenger DNA methylation in cancer by epigenomic analysis. Adv Genet. 2010; 70: 277–308. PubMed Abstract | Publisher Full Text\n\nLahtz C, Pfeifer GP: Epigenetic changes of DNA repair genes in cancer. J Mol Cell Biol. 2011; 3(1): 51–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCortessis VK, Thomas DC, Levine AJ, et al.: Environmental epigenetics: prospects for studying epigenetic mediation of exposure-response relationships. Hum Genet. 2012; 131(10): 1565–1589. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPfeifer GP, Chen HH, Komura J, et al.: Chromatin structure analysis by ligation-mediated and terminal transferase-mediated polymerase chain reaction. Methods Enzymol. 1999; 304: 548–571. PubMed Abstract | Publisher Full Text\n\nRauch TA, Pfeifer GP: The MIRA method for DNA methylation analysis. Methods Mol Biol. 2009; 507: 65–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRauch TA, Pfeifer GP: DNA methylation profiling using the methylated-CpG island recovery assay (MIRA). Methods. 2010; 52(3): 213–217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXiong Z, Laird PW: COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997; 25(12): 2532–2534. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRauch T, Li H, Wu X, et al.: MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells. Cancer Res. 2006; 66(16): 7939–7947. PubMed Abstract | Publisher Full Text\n\nBaylin SB, Jones PA: A decade of exploring the cancer epigenome - biological and translational implications. Nat Rev Cancer. 2011; 11(10): 726–734. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrivat E, Sowers LC: Photochemical deamination and demethylation of 5-methylcytosine. Chem Res Toxicol. 1996; 9(4): 745–750. PubMed Abstract | Publisher Full Text\n\nBecker FF, Holton P, Ruchirawat M, et al.: Perturbation of maintenance and de novo DNA methylation in vitro by UVB (280-340 nm)-induced pyrimidine photodimers. Proc Natl Acad Sci U S A. 1985; 82(18): 6055–6059. PubMed Abstract | Free Full Text\n\nDuan MR, Smerdon MJ: UV damage in DNA promotes nucleosome unwrapping. J Biol Chem. 2010; 285(34): 26295–26303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSage E, Girard PM, Francesconi S: Unravelling UVA-induced mutagenesis. Photochem Photobiol Sci. 2012; 11(1): 74–80. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "819",
"date": "08 Mar 2013",
"name": "Tom Tullius",
"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, and a good example of how apparently negative results can be important to a field. The authors establish, by three different experiments, that the DNA methylation status of human keratinocytes is not affected by UV irradiation. The results are clear from the data presented in the figures, and the authors are careful to provide bioinformatics analysis to back up visual analysis of the experimental data. This work shows that for the case of UV irradiation, the tantalizing possibility that environmental insult leads to heritable epigenetic changes is not operative. This paper represents a good benchmark for future work that investigates the same hypothesis for other environmental insults.",
"responses": []
},
{
"id": "843",
"date": "18 Mar 2013",
"name": "Shisheng Li",
"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 scientifically sound and clearly written article. The major conclusion, that UVB irradiation of keratinocytes has no recognizable global effect on DNA methylation, is supported by carefully designed and well executed experiments. The authors made appropriate speculations about the causes and timing for DNA methylation changes observed in human skin cancers. In view of the nature of somatic cancer development, namely stochastic transformation followed by clonal selection, it is also possible that UVB exposures cause DNA methylation change in only a small fraction of the cells, which cannot be detected with techniques that can only measure collective overall changes but not those in individual cells.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-45
|
https://f1000research.com/articles/2-43/v1
|
12 Feb 13
|
{
"type": "Research Article",
"title": "The stabilizing effects of genetic diversity on predator-prey dynamics",
"authors": [
"Christopher F Steiner",
"Jordan Masse",
"Jordan Masse"
],
"abstract": "Heterogeneity among prey in their susceptibility to predation is a potentially important stabilizer of predator-prey interactions, reducing the magnitude of population oscillations and enhancing total prey population abundance. When microevolutionary responses of prey populations occur at time scales comparable to population dynamics, adaptive responses in prey defense can, in theory, stabilize predator-prey dynamics and reduce top-down effects on prey abundance. While experiments have tested these predictions, less explored are the consequences of the evolution of prey phenotypes that can persist in both vulnerable and invulnerable classes. We tested this experimentally using a laboratory aquatic system composed of the rotifer Brachionus calyciflorus as a predator and the prey Synura petersenii, a colony-forming alga that exhibits genetic variation in its propensity to form colonies and colony size (larger colonies are a defense against predators). Prey populations of either low initial genetic diversity and low adaptive capacity or high initial genetic diversity and high adaptive capacity were crossed with predator presence and absence. Dynamics measured over the last 127 days of the 167-day experiment revealed no effects of initial prey genetic diversity on the average abundance or temporal variability of predator populations. However, genetic diversity and predator presence/absence interactively affected prey population abundance and stability; diversity of prey had no effects in the absence of predators but stabilized dynamics and increased total prey abundance in the presence of predators. The size structure of the genetically diverse prey populations diverged from single strain populations in the presence of predators, showing increases in colony size and in the relative abundance of cells found in colonies. Our work sheds light on the adaptive value of colony formation and supports the general view that genetic diversity and intraspecific trait variation of prey can play a vital role in the short-term dynamics and stability of planktonic predator-prey systems.",
"keywords": [
"A large body of theoretical and empirical work has shown that the presence of variation among prey in their susceptibility to predation can have profound impacts on the structure and dynamics of predator-prey communities1–9. Prey heterogeneity can mediate trophic-level responses to enrichment and weaken top-down limitation of prey communities by facilitating numerical dominance by defended prey2",
"4",
"7",
"10–12. Such shifts in dominance can have significant dynamic consequences",
"potentially reducing the propensity and severity of predator-prey oscillations and stabilizing community dynamics1",
"3",
"5",
"9. While empirical research has largely focused on the consequences of interspecific variation in prey edibility",
"a growing body of experiments has begun to highlight the dynamic consequences of intraspecific trait variation. When prey populations exhibit phenotypic variation in their susceptibility to predators and show rapid adaptive responses to predation pressure",
"they can fundamentally alter the strength and dynamic consequences of their interactions with their consumers13–18."
],
"content": "Introduction\n\nA large body of theoretical and empirical work has shown that the presence of variation among prey in their susceptibility to predation can have profound impacts on the structure and dynamics of predator-prey communities1–9. Prey heterogeneity can mediate trophic-level responses to enrichment and weaken top-down limitation of prey communities by facilitating numerical dominance by defended prey2,4,7,10–12. Such shifts in dominance can have significant dynamic consequences, potentially reducing the propensity and severity of predator-prey oscillations and stabilizing community dynamics1,3,5,9. While empirical research has largely focused on the consequences of interspecific variation in prey edibility, a growing body of experiments has begun to highlight the dynamic consequences of intraspecific trait variation. When prey populations exhibit phenotypic variation in their susceptibility to predators and show rapid adaptive responses to predation pressure, they can fundamentally alter the strength and dynamic consequences of their interactions with their consumers13–18.\n\nThe effects of prey heterogeneity on predator-prey dynamics has been extensively explored in the context of endogenously driven population cycles. Cycles are a common feature of simple, two-species predator-prey models that incorporate nonlinear functional responses19–22. Such oscillatory dynamics become more probable and increase in amplitude with increasing prey carrying capacity20,21. Inclusion of prey heterogeneity in the form of species that are defended from predation can in theory stabilize predator-prey cycles. This readily occurs when prey species trade off their ability to compete for shared resources with their capacity to resist predation. In such instances, predators can facilitate the invasion and persistence of defended prey which, in turn, siphon resources from more edible species, reducing their carrying capacity. This can, in some cases, decrease the amplitude of predator-edible prey cycles or move systems from periodic to point attractors3,5,23. Predator-mediated increases in the relative abundance of defended prey may also weaken top-down limitation of trophic-level abundance causing an increase in total prey abundance relative to prey community’s lacking trait heterogeneity2,4,7,10–12.\n\nWhile the abovementioned models were developed with the intent of understanding the dynamic consequences of heterogeneity among prey species, their general predictions may, under certain conditions, apply to heterogeneity that occurs within species. For instance, models of trophic structure that incorporate prey evolutionary dynamics show that heritable variation in defense against predators may allow prey populations to adaptively respond to predation pressure, shifting regulation of prey populations from top-down control to stronger bottom-up control13,24. Several models have also explored how adaptive responses in prey defense may impact predator-prey dynamics and stability14–16,18,24–26. These demonstrate the capacity for prey evolution to dramatically alter predator-prey dynamics but stabilization is not a generalizable prediction. While adaptive responses in prey defense may stabilize predator or prey populations under certain conditions14,15,24, evolution can also give rise to destabilization of predator-prey cycles and enhanced extinction probability14,16,17,25,26.\n\nOnly a handful of studies have attempted to address the dynamic consequences of intraspecific variability in prey edibility using direct manipulations in which prey evolution was either suppressed xor promoted16,17,27. Moreover, previous experiments have only considered phenotypes or species with fixed traits. For many organisms, ecological strategies may involve transitions between dynamic classes that vary in their susceptibility to predators, with variation among phenotypes consisting of variation in state transition rates or degree of invulnerability. This could apply to organisms that change behavioral/physiological states or who move in and out of spatial refuges. It could also occur with colony-forming organisms in which colonies of increasing size are more resistant to predators. Prior work has shown that such prey strategies can stabilize predator-prey cycles1. However, this has not been examined within an evolutionary context.\n\nWe tested this experimentally using a laboratory-based aquatic system composed of the zooplankton-predator Brachionus calyciflorus and the algal-prey Synura petersenii, in which the potential for prey evolution was either enhanced or reduced through direct manipulations of initial prey genetic diversity. Our study differed from prior work in its use of an algal-prey species that can transition between a vulnerable and predator-resistant class. Synura petersenii is a common freshwater flagellate that may transition between two states: either free-living cells (which are more susceptible to zooplankton predators) or as swimming colonies (which are less susceptible to zooplankton feeding due to their larger size). Reproduction can occur in either the free-living state or in the colony state through binary fission. The strains of S. petersenii used in our study exhibit heritable variation in their propensity to form colonies and their degree of vulnerability when in the colony state due to variation in colony size (Figure 1). Cellular aggregation and colony formation are viewed as key steps in the evolution of multicellularity28–30. Thus, our work also permits exploration of the selective forces that may favor colonial strategies. We show that the initial presence of trait heterogeneity among prey can reduce top-down limitation of prey and alter predator-prey dynamics by reducing temporal variation in total prey abundance. This stabilization is associated with an increase in the size of colonies and the relative abundance of cells found in colonies.\n\nSource cultures were maintained under common garden conditions using the same environmental conditions as the main experiment. Individuals of each Synura strain were isolated from one week old cultures and used to establish three replicate monocultures at an initial total cell density of 1500 cells/mL. Monocultures experienced the same environmental conditions (light, temperature and nutrient replacement) and sampling procedures as the main experiment. (A) Mean cells per colony of each strain estimated from samples taken on day 14 and day 42 of the assay (shown are means and 95% confidence intervals). (B) Mean relative abundance of cells found in colonies estimated from samples taken on day 14 and day 42 of the assay (shown are means and 95% confidence intervals). (C) Density of free-living cells over time for each strain. Shown are means (+/- S.E.); original units in numbers of individuals per mL. (D) Density of colonies over time for each strain. Shown are means (+/- S.E.); original units in numbers of colonies per mL.\n\n\nMethods\n\nOur experimental system consisted of a single species of zooplankton as a predator, the rotifer Brachionus calyciflorus, and a single species of phytoplankton as the prey, the colony-forming flagellate Synura petersenii. Hereafter we refer to both species by genus. Brachionus cultures were obtained from Florida Aqua Farms (Dade City, FL, USA). Five strains of Synura were used in the experiment, four of which (LB239, LB2403, LB2405, LB2406) were obtained from UTEX (Austin, TX, USA) and one (CBS) from Carolina Biological Supply (Burlington, NC, USA). Synura stock cultures were initiated with a single cell isolated from serial dilutions in sterile medium to ensure that all stocks were initially isogenic. All five Synura strains produced populations composed of a mix of single cells and colonies when under semi-continuous culture conditions. However, short-term trait assays revealed significant genetic variation among the strains in population densities, colony size (number of cells per colony), and the relative abundance of total cells found in colony form (Figure 1). Brachionus used in the experiment were isolated from a clonal culture grown on the CBS strain of Synura. This culture was initially stocked with a single Brachionus clone, minimizing genetic variation within our Brachionus populations and reducing the potential for coevolution between predator and prey. All stock cultures were maintained using the same environmental conditions as in the experiment.\n\nAll experimental materials were autoclave-sterilized prior to use. Experimental containers consisted of 500mL flasks filled with 400mL of COMBO medium31, capped with aluminum foil and housed in a single environmental chamber at 20°C under 24 hour light. The containers were randomly ordered and rotated in the chamber following each sampling event. We used a factorial design in which predator presence/absence was crossed with a manipulation of initial genetic diversity (a low genetic diversity treatment composed of each Synura strain in monoculture or a high genetic diversity treatment composed of all five strains together). The treatments with Brachionus present were replicated four times; treatments without Brachionus were replicated three times. At the initiation of the experiment, flasks were first inoculated with their respective Synura strains from stock cultures. The high genetic diversity treatment received 20 cells/mL of each of the five strains while the low diversity treatment received a single strain at 100 cells/mL. Thus, total Synura density added was kept constant at 100 cells/mL across treatments. While the five strains were added at lower initial densities in the high genetic diversity treatment, each flask received a total of 8000 cells of each strain. Hence, the probability of losing a strain through demographic stochasticity and genetic drift at the initiation of the experiment was low. Synura populations were allowed to grow in the absence of predators for seven days at which time 10 Brachionus adults were added to each plus predator treatment. We refer to this as day 0 of the experiment. Brachionus populations were allowed to grow for 12 days (with periodic medium replacement) at which time sampling was initiated.\n\nSampling occurred every 2–3 days up to day 167, the final day of the experiment. To sample the experiment, flasks were first gently swirled to homogenize their contents and 12.5% of the volume (50mL) was poured into a sample bottle, which served as both a zooplankton and phytoplankton sample. Removed medium was replaced with sterile COMBO medium. Thus, the experiment was maintained under semi-continuous culture conditions. Brachionus was enumerated using a stereomicroscope while Synura was enumerated using a CASY particle counter (Innovatis AG, Germany). Maximum cell diameter of Synura in our cultures ranged between 6–10µm (mean=7.9µm); we found no significant differences among our strains in mean maximum diameter. Consequently, we programmed the CASY to perform total counts and counts of particles below 10.5µm mean diameter as an estimate of the density of free living cells. Colony densities were then calculated by subtracting single cell counts from totals. To measure the density of cells in colonies, we multiplied the colony counts by estimates of the mean number of cells per colony for each treatment replicate. Estimates of cells per colony were performed at four time points during the experiment: at the initiation of the experiment, on day 30, day 97 and at the end of the experiment. To obtain estimates, subsamples of phytoplankton from each replicate were preserved in acid Lugols solution and counts performed using a compound microscope. We counted cells/colony for up to 25 haphazardly chosen colonies per sample and averaged the values. Because we did not have mean colony size estimates for all dates, we time-averaged the estimates of cells/colony over days 30 to 167 to create conversion constants for each replicate prior to multiplying by the colony counts. This should have provided conservative estimates of treatments effects since treatments had not completely diverged by the day 30 sample. Total cell density was calculated by summing free-living cell densities with estimates of the density of cells found in colonies.\n\nBrachionus populations in the low diversity treatment rapidly went extinct in all replicates of the LB239, LB2403, LB2405, and LB2406 strains. We consequently discontinued sampling of these monocultures and focused on the CBS strain for the low genetic diversity treatment. Brachionus populations exhibited an initial exponential growth phase followed by a population decline between days 0 and 40 before settling into more regular population oscillations. To remove the influence of initial transitory dynamics we analyzed data over days 51 to 167. Significant linear trends over time in Synura and Brachionus densities were evident in several replicates (see Results). To remove the influence of temporal trends on measures of temporal variability, we first performed linear regressions of log transformed densities versus time for each replicate for both species. Mean absolute values of the regression residuals were then used as measures of temporal variability (an inverse measure of stability). Analyses of mean Brachionus and Synura densities were performed on log10 transformed values averaged over days 51 to 167. To analyze changes in the degree of colony formation, we examined colony densities and the relative abundance of cells found in colonies (equal to the density of cells in colonies divided by total cell density). Response variables were analyzed using ANOVA. Because replication was not equal among replicates, we used type III sums of squares and max t-tests for post hoc comparisons, which are robust to unbalanced designs and non-normal data32. All response variables met assumptions of normality using Lilliefor’s test. Statistics were performed in R version 2.1533. Code for running max t-tests can be found in32.\n\n\nResults\n\nBrachionus populations persisted in all high genetic diversity treatments. However, populations of the zooplankton failed to establish in all low diversity replicates that were composed of the LB239, LB2403, LB2405, and LB2406 genotypes (sampling of these monocultures was discontinued). Brachionus also went extinct mid-experiment in one low genetic diversity replicate containing the CBS strain; we have removed this replicate from all analyses. Figure 2 displays Brachionus dynamics for all replicates in the presence of either low initial genetic diversity (Figure 2A) or high initial genetic diversity (Figure 2B). When examining dynamics from day 51 to day 167, linear regressions revealed a significant positive trend in log Brachionus densities over time in one low genetic diversity replicate (p=0.02). Three of four high genetic diversity replicates exhibited significant negative trends in log Brachionus densities over time (p<0.05, linear regression). Examining regression residuals, Synura genetic diversity had no effect on detrended temporal variability of Brachionus (Figure 3A; p=0.62, ANOVA). Time-averaged Brachionus densities also showed no responses to initial Synura genetic diversity (Figure 3B; p=0.93, ANOVA).\n\nBrachionus dynamics in the presence of (A) low initial prey genetic diversity (prey populations started with a single strain of Synura) or (B) high initial prey genetic diversity (prey population started with five strains of Synura). Dynamics for each replicate are shown separately (different symbols). Original units in numbers of individuals per mL.\n\nEffects of initial Synura genetic diversity (low versus high) on (A) detrended temporal variability of Brachionus populations (an inverse measure of stability), measured as mean residuals from linear regressions of log transformed Brachionus density versus time, and (B) Brachionus densities averaged over the course of the experiment. Both measures (A and B) were based on dynamics over days 51 to 167. Shown are means (+/- S.E.). Original units in numbers of individuals per mL.\n\nSynura dynamics for all treatments and replicates are shown in Figure 4. Examining dynamics from day 51 to day 167, log Synura densities in the low genetic diversity treatment and in the presence of Brachionus showed no significant trends with time across all three replicates (Figure 4A; all p<0.08, linear regressions). By contrast, in the absence of Brachionus, Synura densities in the low genetic diversity treatments showed significant positive trends with time in all replicates (Figure 4A; all p<0.001, linear regressions). Turning to Synura dynamics in the high genetic diversity treatments, all replicates exhibited significant positive increases over time in both the presence and absence of Brachionus (Figure 4B; all p<0.019, linear regressions).\n\nSynura dynamics in presence of Brachionus (closed symbols) or absence of Brachionus (open symbols) for (A) populations with low initial Synura genetic diversity or (B) populations with high initial Synura genetic diversity. Dynamics for each replicate are shown separately (different symbols). Original units in numbers of individuals per mL.\n\nExamining temporal variability of Synura total cell densities, a significant interaction between Brachionus presence/absence and initial genetic diversity was detected (Figure 5A; F1,9=26.9, p<0.001, ANOVA). Effects of genetic diversity on Synura stability were only strongly evident in the presence of Brachionus, significantly decreasing temporal variability in the high genetic diversity treatment relative to low genetic diversity (Figure 5A; p=0.02, max t-test). In contrast, no effects of initial genetic diversity were detected in the absence of Brachionus (Figure 5A; p=0.14, max t-test). We performed additional analyses of temporal variability of free-living cells and colony cell abundances to examine how these components contributed to variability in total cell density. Measures were detrended using linear regressions in the same manner as total cell densities. Temporal variability of free-living cells showed trends that mirrored variability of total cell abundances (Figure 6A). However, effects of genetic diversity were not strong (main effect: p=0.65, ANOVA; interaction: p=0.10, ANOVA) whereas Brachionus significantly increased temporal variability of free-living cells (Figure 6A; F1,9=11.2, p=0.01, ANOVA). Genetic diversity and Brachionus presence/absence interactively affected temporal variability of colony cell abundances (Figure 6B; F1,9=39.1, p<0.001, ANOVA). Genetic diversity reduced temporal variability in the presence (p<0.001, max t-test) and absence of Brachionus (p=0.053, max t-test), but effects were stronger when Brachionus was present (Figure 6B).\n\nEffects of initial Synura genetic diversity (low versus high) and Brachionus presence/absence on (A) detrended temporal variability of Synura populations (based on total cell densities), measured as mean residuals from linear regressions of log transformed Synura density versus time, and (B) Synura total cell densities averaged over the course of the experiment.Both measures (A and B) were based on dynamics over days 51 to 167. Shown are means (+/- S.E.). Original units in numbers of individuals per mL.\n\nEffects of initial Synura genetic diversity (low versus high) and Brachionus presence/absence (A) detrended temporal variability of free-living cells of Synura, measured as mean residuals from linear regressions of log transformed free cell densities versus time, and (B) detrended temporal variability of colony cells of Synura, measured as mean residuals from linear regressions of log transformed colony cell densities versus time. Shown are means (+/- S.E.).\n\nEffects of Brachionus presence/absence on total Synura cell densities also appeared to be dependent on initial genetic diversity (Figure 4A versus 4B). Analyzing time-averaged densities, a significant interaction was detected (Figure 5B; F1,9=5.5, p<0.043, ANOVA). While Brachionus presence/absence had no effects on mean cell densities in the high genetic diversity treatment (Figure 5B; p=0.99, max t-test), cell densities were significantly depressed in the presence of Brachionus in the low genetic diversity treatment (Figure 5B; p=0.03, max t-test).\n\nPresence of Brachionus reduced Synura colony density in both genetic diversity treatments (Figure 7A). When examining time-averaged colony densities, a Brachionus effect was present (Figure 7B; F1,9=49.7, p<0.0001, ANOVA) but no main effect of genetic diversity or interaction were detected (p<0.37, ANOVA). While the number of colonies was unaffected by initial genetic diversity, colony size (number of cells per colony) varied greatly between diversity treatments (Figure 8). When analyzing log transformed time-averages, a significant interaction between genetic diversity and Brachionus presence/absence on colony size was detected (Figure 8; F1,9=68.9, p<0.0001, ANOVA). Colony size in the high genetic diversity treatment and in the presence of Brachionus was greater compared to all other treatment combinations (all p<0.001, max t-test). Colony size in the high genetic diversity treatment without Brachionus was also significantly greater than both low genetic diversity treatments (p=0.03, max t-test). In the low genetic diversity treatment, no effect of Brachionus presence/absence was detected (p=0.99, max t-test). Effects on colony size were also apparent when examining the relative number of cells in colonies, which increased greatly in the presence of Brachionus and high genetic diversity (Figure 9A). Analyzing time-averaged relative colony cell density produced a significant interaction between Brachionus presence/absence and initial Synura genetic diversity (Figure 9B; F1,9=436.1, p<0.0001, ANOVA). There was no difference between low and high genetic diversity treatments when Brachionus was not present (p=0.49, max t-test) but a strong positive effect of Synura genetic diversity in the presence of Brachionus (Figure 9B; p<0.001, max t-test).\n\nEffects of initial Synura genetic diversity (low versus high) and Brachionus presence/absence on (A) Synura colony densities over time (shown are means across replicates, +/-S.E.), and (B) Synura colony densities averaged over days 51 to 167 (means, +/-S.E.).Original units in numbers of colonies per mL.\n\nThe left panel displays dynamics over time and the right panel time-averaged values (averaged over days 30 to 167). Shown are means (+/- S.E.).\n\nEffects of initial Synura genetic diversity (low versus high) and Brachionus presence/absence on (A) the relative abundance of Synura cells found in colonies over time (shown are means across replicates, +/-S.E.), and (B) the relative abundance of Synura cells found in colonies averaged over days 51 to 167 (means, +/-S.E.). Relative colony cell density was calculated by dividing the number of cells found in colonies (per mL) by the total cell density per mL (colony cells plus free-living cells).\n\n\nDiscussion\n\nWe found clear evidence that the presence of intraspecific trait heterogeneity can positively impact the stability and abundance of prey populations when in the presence of predators. These effects were linked to increases in the degree of aggregation of prey individuals into colonies; diverse prey populations produced larger colonies and a greater relative abundance of cells found in colony form when predators were present. Our results bear many similarities to prior studies that have examined the dynamic consequences of interspecific variation in prey defense against predators. As outlined in our Introduction, several models have shown that the presence of weakly interacting prey can stabilize dynamics by shunting resources away from more edible forms and subsequently reducing the amplitude of predator-edible prey oscillations3,5,23 – a prediction that has garnered some empirical support from studies that have manipulated prey species composition6,34,35 and clonal composition15. Our work further demonstrates that intraspecific variation can stabilize dynamics when prey persist in different dynamic classes that vary in predator resistance. The mechanisms that can give rise to vulnerable-invulnerable class structures within prey populations are diverse and include refuge use, inter-individual variation in behavioral or physiological states, and spatial variation in predation pressure1. Thus, our general findings may have broader applicability beyond planktonic systems of colony-forming prey.\n\nWhile stabilization is one potential outcome of enhanced prey diversity, several models have shown that the evolution of prey defense may also destabilize predator-prey dynamics, depending on assumptions of prey and predator traits15,17,25. For example, Hairston, Ellner and colleagues have examined the dynamic consequences of evolutionary responses among prey using models and an experimental system composed of Brachionus calyciflorus and unicellular green algae as prey14–17. They have shown both theoretically and experimentally that rapid prey adaption in prey edibility can have significant effects on predator-prey dynamics, though effects are not always stabilizing and dependent on the degree of prey phenotypic variation present and the strength of trade-offs among phenotypes in anti-predator strategies and competitive ability for shared resources. When predator-resistance is effective but costly to defended phenotypes, rapid prey adaptation can induce large predator-prey oscillations and destabilization14,16,18. In contrast, when defense against predators is effective but costs in competitive ability are low, prey adaptation can result in cryptic prey cycles in which predators continue to oscillate and total prey abundance is stabilized as edible and predator-resistant phenotypes oscillate out of phase of each other15,18. The production of cryptic cycles via rapid prey adaptation shows a passing similarity to our results, in which high genetic diversity had no effects on predator stability but led to stabilization of total Synura abundance in the presence of Brachionus. Because our sampling intervals were uneven, spanning 2 or 3 days, we could not examine covariation between free-living cells and colony cell abundances using traditional cross-correlation analysis. However, visual examination of detrended abundances of free-living Synura cells and colony cells over time revealed no strong support for asynchronous oscillations between the two groups (Figure 10). Dynamics were instead highly synchronous; significant positive correlations were detected for all replicates (Figure 10).\n\nThe absence of cryptic cycles in our experiment is perhaps not surprising as the life history of our prey species is quite different from that assumed in prior models in which different genotypes have fixed traits. As described above, Synura can persist in two phenotypic states: susceptible free-living cells or a more predator-resistant colonial stage. Cells can transition between states via cellular aggregation and colony disassembly or subsets of the population can remain within states since both free living cells and colonies can reproduce – the latter through binary fission36. The prey life history in our experimental system is similar to the prey strategy presented in model 1 of1, in which a single prey species transitions between a vulnerable and invulnerable class, both of which may reproduce. Their model, in addition to several variants, shows that class transitions can reduce top-down control of prey and stabilize dynamics by moving predator-prey cycles to point attractors. However1, only focused on a two species system in the absence of prey evolution. Exploration of the eco-evolutionary dynamics of multi-class prey systems may prove fruitful as it seems plausible that many natural prey populations could persist in multiple dynamic classes that vary in their ecological traits.\n\nDetrended dynamics of Synura free-living cells and colony cell abundances in the presence of Brachionus (left panels) for all three replicates of the low genetic diversity treatment (A–C) and all four replicates of the high genetic diversity treatment (D–G). Shown for each replicate in the right panel is the relationship between free-living cell and colony cell abundances and Pearson correlation statistics (the line is the linear regression fit). Detrended abundances are the residuals from linear regressions of either log free-living cell densities or log colony cell densities versus time.\n\nOur work also produced results that were similar to prior investigations of the consequences of prey heterogeneity on top-down versus bottom-up control of trophic-level production2,4,7,10–12,37. These studies have all provided strong empirical evidence that predators can facilitate numerical dominance of defended prey species or phenotypes, reducing predator limitation of total prey production. Similarly, we found that presence of predators selected for dominance by large colony phenotypes with reduced susceptibility to predation pressure. This resulted in a significant increase in mean prey abundance when compared to populations that were initially composed of a single, susceptible phenotype. Hence, prey evolution has the capacity to alter trophic structure and the partitioning of production among trophic levels.\n\nAs with any experiment, there are caveats and limitations in our study that deserve mention. First, it is important to note that our contrast between low and high prey genetic diversity was performed with a single genotype (CBS) for the low diversity treatment. This is an approach that has been used in prior experiments in which dynamics in the presence of a highly edible prey phenotype are contrasted with dynamics in which the edible prey has been supplemented with additional phenotypes that vary in their susceptibility to predators2,7,9,16. In the present case, this was done out of necessity since we observed that the CBS Synura strain was the only one capable of maintaining Brachionus populations at densities that were not prone to rapid extinction. This was likely due to the CBS strain’s combination of high cell density, small colony size and low relative abundance of cells in colonies compared to the other strains (Figure 1). The only exception to this was strain LB239, which also had small colonies and a low relative abundance of cells in colony form. However, this strain also exhibited the lowest cell densities among the prey genotypes (Figure 1), which likely contributed to predator extinctions. If using extinction probability as a measure of predator stability, our results bolster our argument that prey genetic diversity enhances stability; in monocultures of Synura, Brachionus extinction probability was high for four out of five strains but low in the prey polyculture. Another limitation of our study is that we did not measure competitive abilities among our Synura strains. A central assumption of many of the abovementioned models is that prey exhibit trade-offs in their ability to resist predation and their competitive ability for shared resources3,11,13,14,23,26,38,39. Hence, investment in defensive traits - whether behavioral, chemical or morphological – comes at a cost to the organism in terms of its ability to acquire or convert limiting resources to growth and reproduction. While we did not measure competition between our Synura strains, it is conceivable that colony aggregation and increasing colony size could incur a fitness cost on individual cells by reducing exposed cell surface area and impairing the ability of individuals to acquire nutrient resources. Assuming a single limiting resource, models predict that phenotypes with high competitive ability should exclude less competitive, defended phenotypes at equilibrium and in the absence of predators. Results from our experiment were consistent with this prediction. In the absence of Brachionus, mean colony size and the relative abundance of Synura cells in colonies fell to low levels that were similar in magnitude to the low diversity treatment. This is indicative of a shift in dominance to phenotypes with lower investment in defense.\n\nCellular aggregation is viewed as an evolutionary stepping stone between unicellular and multicellular life forms28,30,40. A hurdle in achieving this initial transition is the fitness costs associated with colony formation such as reductions in competitive ability, reproductive rates or buoyancy. Our work supports the general idea that colony formation and large colony size provide a selective advantage in the presence of predators and a disadvantage when predators are absent and resource competition is strong. This finding complements previous studies that have shown similar effects of predators on colony formation in unicellular algae17,41 and supports the general hypothesis that a driver in the early evolution of multicellularity was the emergence of heterotrophic life forms and phagotrophy – a transition point where the disadvantages of cellular aggregation began to be outweighed by the advantages of increasing size and predator escape29. Our study also adds to a growing body of experiments demonstrating the effects of rapid evolutionary change on the dynamics of consumer-resource interactions2,16,17,27,42 and the strength of top-down control of prey abundance2,37. How such effects translate to natural communities remains largely unresolved; the majority of prior experiments, including ours, have utilized highly simplified laboratory settings with a minimal number of interacting species. Moreover, the consequences of coevolutionary responses between predator and prey populations on ecological dynamics have not been deeply explored. Natural prey populations are of course exposed to multiple species of predators and embedded in prey communities with a large degree of interspecific variation in competitive ability and defense against predators. Understanding the dynamic consequences of evolution and coevolution within the context of complex communities remains a daunting challenge in ecology but an exciting avenue for future exploration.",
"appendix": "Author contributions\n\n\n\nCFS conceived the study, designed the experiment, analyzed the data and wrote the manuscript. CFS and JM carried out the experiment.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by National Science Foundation grant DEB-0951495 to CFS.\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 Drew Stockwell, Laith Shaman, Monica Tadros, Michelle Nawal, and Mariela N-Tyler for lab assistance and Emily Grman for comments on the manuscript.\n\n\nReferences\n\nAbrams PA, Walters CJ: Invulnerable prey and the paradox of enrichment. Ecology. 1996; 77(4): 1125–1133. Publisher Full Text\n\nBohannan JM, Lenski RE: Effect of prey heterogeneity on the response of a model food chain to resource enrichment. Am Nat. 1999; 153(1): 73–82. 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PubMed Abstract | Publisher Full Text\n\nFussmann GF, Ellner SP, Shertzer KW, et al.: Crossing the Hopf bifurcation in a live predator-prey system. Science. 2000; 290(5495): 1358–60. PubMed Abstract | Publisher Full Text\n\nGilpin ME, Rosenzweig ML: Enriched predator-prey systems: theoretical stability. Science. 1972; 177(4052): 902–4. PubMed Abstract | Publisher Full Text\n\nRosenzweig ML: Paradox of enrichment: destabilization of exploitation ecosystems in ecological time. Science. 1971; 171(3969): 385–7. PubMed Abstract | Publisher Full Text\n\nMay RM: Limit cycles in predator-prey communities. Science. 1972; 177(4052): 900–2. PubMed Abstract | Publisher Full Text\n\nGrover JP: Competition, herbivory, and enrichment: nutrient-based models for edible and inedible plants. Am Nat. 1995; 145(5): 746–774. Publisher Full Text\n\nHochberg ME, Holt RD: Refuge evolution and the population dynamics of coupled host-parasitoid associations. Evol Ecol. 1995; 9(6): 633–661. Publisher Full Text\n\nAbrams PA, Matsuda H: Prey adaptation as a cause of predator-prey cycles. Evolution. 1997; 51(6): 1742–1750. Reference Source\n\nJones LE, Becks L, Ellner SP, et al.: Rapid contemporary evolution and clonal food web dynamics. Philos Trans R Soc Lond B Biol Sci. 2009; 364(1523): 1579–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPimentel D, Nagel WP, Madden JL: Space-time structure of the environment and the survival of parasite-host systems. Am Nat. 1963; 97(894): 141–167. Publisher Full Text\n\nMichod RE: Evolution of individuality during the transition from unicellular to multicellular life. Proc Natl Acad Sci U S A. 2007; 104(Suppl 1): 8613–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStanley SM: An ecological theory for the sudden origin of multicellular life in the late precambrian. Proc Natl Acad Sci U S A. 1973; 70(5): 1486–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrosberg RK, Strathmann RR: The evolution of multicellularity: A minor major transition? Annu Rev Ecol Evol Syst. 2007; 38: 621–654. Publisher Full Text\n\nKilham SS, Kreeger DA, Lynn SG, et al.: COMBO: a defined freshwater culture medium for algae and zooplankton. Hydrobiologia. 1998; 377(1–3): 147–159. Publisher Full Text\n\nHerberich E, Sikorski J, Hothorn T: A robust procedure for comparing multiple means under heteroscedasticity in unbalanced designs. Plos One. 2010; 5(3): e9788. PubMed Abstract | Publisher Full Text | Free Full Text\n\nR Development Core Team R: A language and environment for statistical computing. (R Foundation for Statistical Computing, ISBN 3–900051–502 07–0, Vienna, Austria). 2011. Reference Source\n\nPersson A, Hansson LA, Bronmark C, et al.: Effects of enrichment on simple aquatic food webs. Am Nat. 2001; 157(6): 654–69. PubMed Abstract | Publisher Full Text\n\nJiang L, Joshi H, Patel SN: Predation alters relationships between biodiversity and temporal stability. Am Nat. 2009; 173(3): 389–99. PubMed Abstract | Publisher Full Text\n\nSilver PA: Synurophyte Algae. Freshwater Algae of North America: Ecology and Classification eds Wehr JD & Sheath RG (Academic Press, New York), 2003; 523–557. Reference Source\n\nBohannan BJM, Lenski RE: The relative importance of competition and predation varies with productivity in a model community. Am Nat. 2000; 156(4): 329–340. Publisher Full Text\n\nLeibold MA: A graphical model of keystone predators in food webs: Trophic regulation of abundance, incidence, and diversity patterns in communities. Am Nat. 1996; 147(5): 784–812. Publisher Full Text\n\nHolt RD, Grover J, Tilman D: Simple rules for interspecific dominance in systems with exploitative and apparent competition. Am Nat. 1994; 144(5): 741–771. Publisher Full Text\n\nCarroll SB: Chance and necessity: the evolution of morphological complexity and diversity. Nature. 2001; 409(6823): 1102–9. PubMed Abstract | Publisher Full Text\n\nBoraas ME, Seale DB, Boxhorn JE: Phagotrophy by a flagellate selects for colonial prey: A possible origin of multicellularity. Evol Ecol. 1998; 12(2): 153–164. Publisher Full Text\n\nDuffy MA, Sivars-Becker L: Rapid evolution and ecological host-parasite dynamics. Ecol Lett. 2007; 10(1): 44–53. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "767",
"date": "13 Feb 2013",
"name": "Eva Kisdi",
"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 demonstrates that prey heterogeneity stabilizes prey population dynamics in an experimental predator-prey system. It should, however, be noted that if this happens via the better-defended prey shunting away resources from the edible prey, then the density of the predator should be lower in the high-prey-diversity treatment. But, according to Fig 3B, this is not the case.",
"responses": []
},
{
"id": "2471",
"date": "18 Nov 2013",
"name": "Gregory Crutsinger",
"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 of this study performed a laboratory mesocosm experiment that sought to address the role of intraspecific variation in predator-prey dynamics. While the underlying questions presented an interesting and relevant avenue of research, four of the five clonal lines went extinct in their single-genotype treatment. As a result, their contrasts between low and high prey genetic diversity were performed with a single genotype (CBS strain). Consequently, it is unclear to me whether conclusions could be drawn about the consequences of genetic diversity per se, as opposed to the identity effects of this individual genotype. I agree with the authors that it is a major limitation in interpreting this experiment.",
"responses": []
},
{
"id": "4251",
"date": "09 Apr 2014",
"name": "Jordi Moya-Laraño",
"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 shows how increasing genetic variation in prey can stabilize the dynamics, especially since predator can barely establish in communities without variability in prey. This last finding is by far the strongest and I think that it adds substantially to a current important topic. A second relevant finding is that, despite having only a single strain with low variability, the high genetic variability replicates had also more stable dynamics than the one single strain.The finding that colony formation was linked to predator presence and that there was a trend in which colony formation increased through time also provides a hint on how multicellularity evolved.However, unfortunately I do not think that this article provides convincing evidence of an evolutionary response of defense through colony formation as the authors claim. First, in their preliminary assays the authors did not combine the different strains to see how they form colonies when different strains are present in the same habitat. We do not know if they are able to combine and to cooperate with other strains (at least with the information provided) and if there are emergent patterns of colony formation when more than one strain is combined.The findings that defense (aggregative behaviour) increase when predators are present could be just the result of complex patterns of (plastic) cooperation among strains which change through time, for instance as a response to persistent predation risk. Assays at the end of the experiment to test if after removing predators, colonies were still maintained at substantially high rates (e.g. as in strain LB2405) would have been a convincing evidence for adaptive evolution ocurring during the dynamics.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-43
|
https://f1000research.com/articles/2-42/v1
|
12 Feb 13
|
{
"type": "Commentary",
"title": "Rack ‘em, pack 'em and stack ‘em: challenges and opportunities in teaching large classes in higher education",
"authors": [
"Saravana Kumar"
],
"abstract": "The higher education sector is undergoing tremendous change, driven by complex driving forces including financial, administrative, and organisational and stakeholder expectations. It is in this challenging environment, educators are required to maintain and improve the quality of teaching and learning outcomes while contending with increasing class sizes. Despite mixed evidence on the effectiveness of large classes on student outcomes, large classes continue to play an important part in higher education. While large classes pose numerous challenges, they also provide opportunities for innovative solutions. This paper provides an overview of these challenges and highlights opportunities for innovative solutions.",
"keywords": [
"There continues to be a number of significant changes to the higher education sector around the world. In Australia",
"these changes have been in the form of funding opportunities",
"student enrolments",
"structure and delivery of courses",
"and these were highlighted in the 2008 Review of Australian Higher Education1. Coupled with the global financial crisis",
"which resulted in increasing financial pressures on higher education providers",
"and their funding sources",
"many higher education providers have taken a critical view of how courses are planned",
"structured and delivered. One common strategy that has been implemented is the move towards common (or foundation) courses within particular disciplines (or programs) and degrees. It is in this challenging context",
"educators are also required to maintain and improve the quality of teaching and learning outcomes while the student-staff ratio continues to increase1."
],
"content": "Commentary\n\nThere continues to be a number of significant changes to the higher education sector around the world. In Australia, these changes have been in the form of funding opportunities, student enrolments, structure and delivery of courses, and these were highlighted in the 2008 Review of Australian Higher Education1. Coupled with the global financial crisis, which resulted in increasing financial pressures on higher education providers, and their funding sources, many higher education providers have taken a critical view of how courses are planned, structured and delivered. One common strategy that has been implemented is the move towards common (or foundation) courses within particular disciplines (or programs) and degrees. It is in this challenging context, educators are also required to maintain and improve the quality of teaching and learning outcomes while the student-staff ratio continues to increase1.\n\nFoundation courses are common in the first year of study at university and often encompass a number programs and degrees. As such, these courses tend to have a large cohort of students, ranging from 100 to 1500 students. These large classes pose unique challenges, including the transition from school to university and learning in a university context. While the advantages of large classes include decreased costs, improved efficiency in terms of educators’ time and resources and standardisation of learning experiences, the disadvantages include a limited range of teaching methods, limited opportunities for interaction between educators and students, and a negative perception of educators who teach large classes2. While the driver for large classes may be pragmatic (such as promotion of inter-disciplinary learning) and resource-centric (such as financial), the evidence base on the effectiveness of large classes on student outcomes is mixed3.\n\nCuseo4 undertook a narrative literature synthesis of research evidence on the effect of class size on teaching, learning and retention of first year students. The findings from this literature review indicated that increasing class size had a deleterious effect on educational outcomes for students overall, and first year students in particular. Cuseo4 evaluated the effect of the class size across eight different constructs including reliance on lecture method of instruction, students’ active involvement in the learning process, interaction between educators and students and feedback to students, depth of thinking inside the classroom, breadth and depth of course objectives, assignments and learning outside the classroom, students’ academic achievement and performance, students’ perspective of course satisfaction, and students’ evaluation of course instruction.\n\nThe findings from Cuseo4 are supported by research undertaken by Monks and Schmidt5. Drawing on data from published studies and from administrative records and student course evaluations at a private, highly selective university on the east coast of the USA, they found that both class size and student load negatively impact student assessments of courses and instructors. Large classes and heavy student loads appear to prompt faculty to alter their courses in ways deleterious to students. Other studies by2,6–8 also highlight the potential problems associated with large classes. While there is a large body of evidence on the negative impacts of large classes, some research advice caution in interpreting these results. For example, a review of the literature by Toth and Montagna9, call for future research on this topic which adequately controlled a number of variables which may influence teaching and learning outcomes. Methodological issues, such as lack of controlling of variables, definitional ambiguity on what can be considered to be important learning outcomes, the varying impact of different types of teaching methods, all contribute to the heterogeneity of the evidence base on the impact of teaching large classes.\n\nDespite the challenges posed by large classes, due to financial and organisational drivers, large classes are going to be part of teaching and learning for most educators in the short and long term. These challenges, however, also provide opportunities for innovative thinking and strategies which may be utilised to improve the quality of teaching and learning outcomes. Burnett and Krause10 provide a number of useful strategies when teaching large classes. From an organisational perspective, simple strategies such as ensuring timely and regular communication with students, providing alternate means of communication with educators (other than the traditional lecture-style didactic presentation), recognising and establishing helpful strategies on key issues students might face (such as time management, access to learning materials), and providing online and other support mechanisms might be beneficial. From a pedagogical perspective, organising and presenting effective and interesting lectures which engage the students, using multiple strategies as part of teaching (such as visual and multi-media aids, handouts, problem-based activities), encourages interaction and involvement during the teaching and learning process. This build on active learning principles, ensuring students can relate the content to real world applications and using technology to enhance learning activities.\n\nAssessments can potentially be an important source of frustration and stress for students and educators in large classes. This can be dealt with by ensuring clear linking of course content with assessment processes, using early assessment strategies to identify potential issues, teaching students about how best to approach assessments and using automated assessments so that students can use that as a learning process.\n\nFrom an affective perspective, building interest and rapport with students, taking interest in strong and weak students, ensuring there are clear pathways for communication between educators and students are important strategies to consider. Furthermore, recognising that different students learn differently using diverse means (such as visual, multimedia, and online tools) to enhance student learning opportunities and providing access to support services (such as access to teaching and learning units for international students and students from diverse backgrounds) are important as they can enhance overall student experiences.\n\nFrom a management perspective, supporting and managing other educators (such as tutors) who may undertake teaching as part of the course is important to ensure consistency and standardisation in the teaching and learning process. Regular meetings among teaching staff, supporting teaching staff by providing timely access to learning materials and ensuring all the teaching staff are presented as a cohesive team are all important.\n\nWith large classes becoming entrenched in higher education, it doesn’t have to be a case of rack 'em', pack 'em' and stack ‘em’, as with challenges come opportunities. Stakeholders in education need to collaborate and partner with each other in identifying, implementing and evaluating innovative solutions. From a personal perspective, as an educator and an academic, I am tasked with coordinating a large foundation course which encompasses a number of health programs including physiotherapy, occupational therapy, medical radiations, podiatry, human movement and health sciences. Given the diversity of these programs and their respective student cohorts, my fellow educators and I face a number of challenges while delivering this course. Recognising these challenges, my fellow educators and I put in place a number of enabling strategies to address these challenges.\n\nThey include:\n\nProvision of clear and explicit instructions, and repeated at numerous instances, if necessary, to ensure students were fully informed about the objectives, delivery and outcomes of the course. Provision of clear and explicit instructions also extended to all educators who taught within this course to ensure consistency and uniformity in the teaching process.\n\nUsing a range of teaching and learning approaches including lectures, tutorials, workshops, small group work, which were underpinned by active learning principles.\n\nA self-imposed benchmark of 24 hours response time, during working hours, to all communications from students was maintained throughout the duration of the course. This was to ensure the students’ issues were acknowledged and support provided in a timely manner.\n\nRegular online and face to face feedback was also provided to ensure students concerns, issues regarding the delivery and content of the course and general issues relating to the course were dealt with in a timely and efficient manner.\n\nThe outcomes of this course were also discussed at lengths with students to ensure students were aware what this course would provide to them as future health care practitioners and how the course objectives linked with the assessment items.\n\nAll learning materials (such as lecture and tutorial notes) were made accessible online along with podcast of lectures.\n\nStudents were actively engaged to provide feedback about their learning experiences and this feedback was immediately implemented to ensure students could see how their feedback was utilised to inform improvements in the content and delivery of the course.\n\nWorking in close association with the information technology staff, a number of online resources, such as discussion boards, multimedia, were created to ensure ongoing access to learning and assessment materials. The online resource was also re-constructed to make the resource user-friendly and easily accessible.\n\nStudents who required additional assistance, such as those from non-English speaking background, international students, were provided with the opportunity to seek additional help from the educators, who, depending on the needs of the individual students, offered targeted assistance (such as referral to teaching and learning units).\n\nWhile there is a growing evidence base on teaching large classes in higher education, ongoing further research is required. Future research in this area should focus on establishing the effectiveness of various models of teaching and learning and should also incorporate diverse measures of learning outcomes. With improved research evidence base, which can then inform teaching and learning practices, stakeholders in education can confidently move towards implementing evidence-informed strategies which will assist in the improvement in the quality of teaching and learning outcomes in higher education.",
"appendix": "Competing interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nKeirle P, Morgan R: Teething Problems in the Academy: negotiating the transition to large-class teaching in the discipline of history. J Univ Teach Learn Pract. 2011; 8(3). Reference Source\n\nCarpenter J: Effective teaching methods for large classes. J Fam Consu Sci Educ. 2006; 24(2): 13–23. Reference Source\n\nLearning and teaching. Guide to inclusive teaching - Inclusive spaces (homepage on the internet). 2012.\n\nCuseo J: The empirical case against large class size: adverse effects on the teaching, learning, and retention of first-year students. J Fac Dev. 2007; 21(1): 5–21. Reference Source\n\nMonks J, Schmidt R: The impact of class size and number of students on outcomes in higher education. [Electronic version]. Retrieved 18th Cornell University, School of Industrial and Labor Relations site. 2010. Reference Source\n\nBedard K, Kuhn P: Where class size really matters: class size and student ratings of instructor effectiveness. Econ Educ Rev. 2008; 27(3): 253–265. Publisher Full Text\n\nDillon M, Kokkelenberg EC, Christy SM: The effects of class size on student achievement in higher education: applying an earnings function. (CHERI Working Paper #28). Cornell Higher Education Research Institute (CHERI). 2002; 29. Reference Source\n\nWesterlund J: Class size and student evaluations in Sweden. Educ Econ. 2008; 16(1): 19–28. Reference Source\n\nToth LS, Montagna LG: Class Size and Achievement in Higher Education: A Summary of Current Research. Coll Student J. 2002; 36(2): 253–61. Reference Source\n\nBurnett J, Krause KL: Teaching Large Classes: Challenges and Strategies.2012. Reference Source"
}
|
[
{
"id": "779",
"date": "18 Feb 2013",
"name": "Quinette Louw",
"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": "783",
"date": "19 Feb 2013",
"name": "Janet Wale",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI agree with the author. A formal study of some of the 'interventions' used would be very interesting.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-42
|
https://f1000research.com/articles/2-41/v1
|
12 Feb 13
|
{
"type": "Research Article",
"title": "Has incentive payment improved venous thrombo-embolism risk assessment and treatment of hospital in-patients?",
"authors": [
"Sue Child",
"Rod Sheaff",
"Olga Boiko",
"Alice Bateman",
"Christian A Gericke",
"Rod Sheaff",
"Olga Boiko",
"Alice Bateman",
"Christian A Gericke"
],
"abstract": "This paper focuses on financial incentives rewarding successful implementation of guidelines in the UK National Health Service (NHS). In particular, it assesses the implementation of National Institute for Health and Clinical Excellence (NICE) venous thrombo-embolism (VTE) guidance in 2010 on the risk assessment and secondary prevention of VTE in hospital in-patients and the financial incentives driving successful implementation introduced by the Commissioning for Quality and Innovation for Payment Framework (CQUIN) for 2010-2011. We systematically compared the implementation of evidence-based national guidance on VTE prevention across two specialities (general medicine and orthopaedics) in four hospital sites in the greater South West of England by auditing and evaluating VTE prevention activity for 2009 (i.e. before the 2010 NICE guideline) and late 2010 (almost a year after the guideline was published). Analysis of VTE prevention activity reported in 816 randomly selected orthopaedic and general medical in-patient medical records was complemented by a qualitative study into the practical responses to revised national guidance. This paper’s contribution to knowledge is to suggest that by financially rewarding the implementation of national guidance on VTE prevention, paradoxes and contradictions have become apparent between the ‘payment by volume system’ of Healthcare Resource Groups and the ‘payment by results’ system of CQUIN.",
"keywords": [
"Payment by Results (PbR)",
"Healthcare Resource Groups (HRG)",
"CQUIN",
"NICE",
"Venous Thrombo-embolism (VTE)",
"NHS",
"England"
],
"content": "Background to incentive payments in the NHS\n\nDiagnostic Related Groups (DRGs) offered health organisations the ability to understand provider activity in terms of how many patients they cared for and their case-mix. They allowed activity comparison within and between different organisations as well as providing a means of making longitudinal analyses of trends in treatment and service provision. Local adaptations or variants of diagnosis related groups (DRGs) have been adopted as the unit of payments to hospitals in many health systems1. This is a system that classifies hospital cases into identifiable groups in order to identify ‘products’ that a hospital provides. The system was originally developed in the USA in order to replace ‘cost-based’ reimbursement. DRGs are based on the International Classification of Diseases (ICD) and the presence of complications or co-morbidity. This model of classification has spread to other countries including Australia, Denmark, France, Germany, Austria, the Netherlands and Russia; followed by a less rapid spread from acute care into long-term care and psychiatry2.\n\nIn England, the NHS Plan of July 2000 introduced the government’s intention to link the allocation of hospital funds to the activity they undertake – a system of payment by results (PbR)3. PbR underpinned the NHS reform agenda and was formally implemented by The Department of Health in 2002 as a mechanism to reimburse hospitals in England for levels of activity undertaken. Under this new system of PbR hospitals were reimbursed for activities undertaken using a fixed price tariff that reflected national average prices for hospital procedures4. Such prices are standardised across the NHS with adjustments for higher labour costs in London. The main aim of PbR was to improve efficiency and increase value for money in the NHS by enhancing service quality, facilitating patient choice, enabling service innovation, improving quality of service and reducing waiting times by rewarding healthcare providers for the volumes of work completed.\n\nPbR has continued to be implemented in the NHS (albeit incrementally) both in terms of scope and financial impact. The first NHS foundation trust (FT) applicants moved to the full PbR system in 2005–06 and other NHS trusts in 2006–074. In 2006–2007 PbR was extended to include non-elective, outpatient and emergency admissions for all trusts5. In 2009–2010, the national tariff for admitted patient care and outpatient activity was for the first time defined in terms of Healthcare Resource Groups (HRGs), an English variant of DRGs. The Department of Health in the UK defines HRGs as groupings of treatment episodes that are similar in resource use and clinical response. They are a standard method of analysing clinical procedures and thereby classification of hospital activity6 HRGs are reviewed every four years to ensure they accurately reflect clinical practice.\n\nAlthough the imposition of payment by results was intended as a fair and consistent basis for hospital funding it also rewards hospitals by volume of work completed. Its overarching aim was to allow the commissioning of activity volumes that were required to deliver service priorities from a plurality of providers on the basis of a standard, national pricing tariff. By standardising prices, it was intended to promote non-price competition, i.e. provider differentiation on the basis of service quality4.\n\nNHS commissioners therefore faced a very challenging agenda. They needed to secure high quality services which remained safe for patients, whilst at the same time trying to achieve best value for the budgets available.\n\n\nThe CQUIN (Commissioning for Quality and Innovation) framework\n\nFurther policy aims were in play however. Commissioning specifically to improve clinical quality and safety required additional incentive frameworks through which improvements could be monitored and additional drivers for change generated. Alongside the more traditional payment by results (volume), there was apparently a need to develop more effective patient experience metrics and use these to improve quality and safety. Lord Darzi’s review of the NHS: \"High Quality Care for All\"7 included a commitment to make providers’ income linked to quality of care performance. This commitment led to the creation of the Commissioning for Quality and Innovation (CQUIN) payment framework, introduced nationally in 2009 to help local commissioners identify and agree quality improvement schemes. It enabled commissioners to reward excellence by linking a proportion of healthcare providers’ income to the achievement of local quality improvement goals8. CQUIN targets introduced for VTE in 2010–11 included a financial incentive for reaching a 90% threshold for venous thrombo-embolism (VTE) risk assessment of all adult hospital in-patients. Compliance was to be measured through a monthly audit of patients who received appropriate thrombo-prophylaxis after risk assessment.\n\nIt has been over 23 years since the first evidence-based guidelines9 recommended routine use of thromboprophylaxis for most hospitalised patients but there continue to be large gaps in the provision of this key patient safety intervention. Venous thromboembolism continues to be a major and often unrecognised cause of morbidity and mortality in hospitalised patients10. Over one-third of hospital in-patients are at risk of developing venous thrombo-embolism (VTE)11. This is a condition where a blood clot forms in a vein and travels through the bloodstream to the lungs, where it often proves fatal.\n\nMore recently, national efforts to reduce avoidable deaths from VTE recommenced in 2005, when the House of Commons Health Select Committee estimated that over 25,000 patients admitted to hospital died each year from preventable VTE11. The Committee cited evidence that many deaths might had been avoided if patients had been properly assessed for risk of VTE on admission to hospital and received appropriate prophylaxis12. They concluded that a nationwide system was needed in the NHS to identify patients at risk of VTE on admission to hospital as well as taking steps to ensure that appropriate thrombo-prophylaxis was prescribed unless contra-indicated.\n\nAs a result a financial incentive for hospitals to focus on VTE risk assessment and prevention was introduced through the Commissioning for Quality and Innovation for Payment Framework (CQUIN) for 2010–201113. A percentage of CQUIN payments paid to or withheld from acute providers became conditional on risk assessing for VTE at least 90% of patients admitted to hospital as in-patients. As well as acting as a monitor for ensuring delivery of risk assessment procedures, this measure also supported VTE prevention by providing a pan-NHS picture of compliance.\n\nThere are also national requirements for all acute hospitals to undertake and report a monthly audit of the percentage of their patients who have received appropriate thrombo-prophylaxis after risk assessment and undertake a root cause analysis of confirmed cases of hospital acquired VTE. Further audit requirements include the reassessment of risk after 24 hours and the provision of patient information on VTE prevention. Compliance with all these indicators is measured from a nationally mandated monthly Unify2 data collection for all providers of NHS acute services. Failure to report in line with such contractual obligations allows commissioners to withhold a percentage of contract value.\n\n\nParadoxes and contradictions within the CQUIN payment framework\n\nWhat is interesting about the CQUIN payment framework is that it enables commissioners to reward excellence by linking a proportion of English healthcare providers’ income to the achievement of local quality improvement goals. Unlike incentive systems that reward individuals, CQUIN rewards are made available to providers through contracts with primary care trusts (PCTs). CQUIN funding is up to 1.5% of contract outturn value, conditional upon achievement of quality outcomes specified in the contract. The contract outturn value is a locally agreed package of quality improvement goals and indicators, which, if achieved enables the provider to earn its full CQUIN payment (calculated as 1.5% of the actual outturn value of the provider contract). The financial value of a CQUIN scheme is specified nationally as a % of actual outturn value of the provider’s contract. At the start of the year, the expected value of the scheme should be calculated as a % of the estimated total outturn contract value for commissioned patient services. A year-end reconciliation is then required against actual outturn value13.\n\nTherefore, it might be questioned what motivates individuals working for PCTs to successfully implement local quality improvement goals when they have no personal benefit in terms of monetary reward. The following section discusses different types of motivation and how each may act as either a barrier or facilitator to the successful implementation of such ‘payment by results’ schemes in the wider NHS.\n\nIn virtually every developed country the health service is the largest single employer, and Britain is no exception. The desire to increase capacity within the NHS means that it has probably never been more important to understand what attracts, retains and motivates staff14. The ninth annual survey to collect the views of NHS staff across England was published in 2011. It indicated that 90% of staff felt their role ultimately made a difference to patients, and 87% were satisfied with the quality of care they personally gave15.\n\nYet, despite the individual (subjective) needs of each patient it may be argued that standardisation of effective clinical practice is becoming more commonplace. Risk assessing for VTE is one example. It would prove extremely difficult for compliance to be rewarded unless a standardised, objective system of directly comparable output existed. Revised NICE guidance in January 2010 formalised differing hospital guidelines for attributing VTE risk, administering appropriate prophylaxis and dealing with contraindications. This meant that compliance against a national target was now able to be monitored for every acute provider. As a result, directly measurable financial incentives for acute providers to focus on VTE risk assessment and prevention through the CQUIN payments framework were able to be introduced on a nationwide basis.\n\nIt would appear to be a rational assumption that offering any form of financial incentivisation should drive up employee compliance with new or revised working practices. An employee’s motivation to perform effectively is said to be determined by two variables16. The first is contained in the effort-reward probability. This is determined by two further probabilities:\n\n1. The probability that effort will result in performance.\n\n2. The probability that performance will result in reward.\n\nYet, this would appear to be paradoxical to the aims of CQUIN. Here we see quite clearly that an increased effort by individuals to properly risk assess and treat VTE in hospital in-patients is not financially rewarding to themselves, but to the provider for whom they work.\n\nWhat the CQUIN framework appears to have tried to do is to bridge the gap between individual and organisational reward by creating and rewarding a culture of continuous quality improvement with stretching goals and measured outcomes agreed in contracts with PCTs on an annual basis. Despite there being no individual reward through CQUIN to comply with increasing national guidance (including VTE risk assessment and treatment) for most healthcare professionals, different, less extrinsic motivational factors must influence uptake and compliance. These are discussed below:\n\n1. In the case of Trust chief executives their pay and continuity in their post are often tied to the trust achieving certain targets. Since 2001, public sector hospitals have been given ‘star ratings’ according to their performance on targets and other indicators17. These public service targets in health have commonly become known as \"P45\" targets - if you do not achieve set targets then your job may be at risk17. The total reward package for Trust chief executives commonly includes basic pay, a spot rate salary determined by the role and an organisational weighting factor alongside an annual performance bonus scheme18. CQUIN may be said to be one of those targets on which performance rewarded pay for chief executives is based. As a crude form of motivation it may be promulgated that if hospitals do not achieve their targets then the employment of the Trust chief executive may be at risk. Clearly this will have an impact on performance.\n\n2. However, we may posit that for clinicians who do not receive any form of performance bonus like Trust chief executives, their motivational reasons for complying with NICE guidance may be more complex. Intrinsically, a concern for the welfare and treatment of patients has always been an over-riding factor in driving up standards in healthcare delivery. The NHS staff survey data for 2011 indicated that nine out of ten staff felt that their role made a difference to patients, with 69% saying they were able to deliver the quality of patient care to which they aspired15. What is interesting from the perspective of this paper is the reduction in the number of staff in the same survey indicating their satisfaction with the level of freedom they now have to chose their own working methods (61%, down from 63% in 201015). Here we may see the shift towards a greater standardisation of working practice against what were locally developed working practices for risk assessment and treatment of VTE19.\n\n3. As discussed earlier, CQUIN rewards are made available to providers through contracts with PCTs. Despite not rewarding the majority of individuals through personal rewards, the achievement of local quality improvement goals will increase overall hospital income. Because CQUIN funding is currently up to 1.5% of contract outturn value, conditional upon the achievement of quality outcomes specified in the contract20, by achieving CQUIN goals healthcare professionals may be incentivised to adopt revised working practices in order to ‘earn’ extra monies at a departmental level in order to both improve their working practices and increase patient satisfaction. These may have taken the form of the employment of a lead specialist VTE nurse or improved staff and patient education.\n\n4. A further motivating factor for the improvement of VTE risk assessment and treatment might be through the increased surveillance of clinical practice that the nationwide standardisation of VTE assessment now permits21. Not only does this allow for ongoing intra-organisational surveillance through mandatory audit activity, but inter-organisational, public comparison of how well different hospitals risk assess and treat VTE also becomes possible. Measurements against national targets such as VTE risk assessment and treatment are now reported in annual hospital reviews of performance. As a result of increasing transparency in the outcomes of hospital performance, many healthcare professionals may have become increasingly aware of the need to drive up and maintain professional status by meeting local quality improvement goals in their hospitals.\n\nThis paper examines the organisational and workplace conditions that favour the implementation of national revised VTE guidance in 2010 in four hospitals in the greater South West of England. In particular, it focuses on the attitudes of staff towards changes to VTE risk assessment policies upon which CQUIN targets were based.\n\nThe implementation of evidence-based national guidance on VTE prevention across two specialities (general medicine and orthopaedics) were systematically compared in four hospitals in 2009 and re-assessed for 2010. The review used 816 randomly selected longitudinal case studies of the hospitals’ VTE prevention activities (in each of the four hospital sites 51 records for orthopaedics and 51 records for general medicine were audited for 2009 and 2010), complemented by a qualitative study into the practical responses by healthcare professionals to the revised national guidance and the imposition of payment by results (PbR). This paper suggests that in instances where there were unachievable clauses for achieving CQUIN rewards for VTE, there was less evidence of an improvement in VTE risk assessment and appropriate prescribing of venous thromboembolism prophylaxis.\n\n\nResearch questions\n\nFocussing on four hospital sites in NHS South West, our first research question was:\n\n1. To what extent did the recording of VTE risk assessment and the prescribing of appropriate thrombo-prophylaxis increase after the imposition of revised NICE guidance in January 2010?\n\nIn so far as that did happen, we further ask:\n\n2. In what respects was this increase a response to the financial incentives now available for acute providers through the CQUIN payment framework? Specifically:\n\na) What motivated staff to improve VTE risk assessment and prevention when reward was given at the level of the organisation rather than the individual?\n\nb) Does potential public exposure of how well a hospital does in terms of meeting CQUIN targets for VTE risk assessment and prevention motivate staff to meet new NICE guidance, or:\n\nc) Is concern for patient welfare at the forefront of staff motivation so any improvement in the recording of VTE risk assessment and prevention would have occurred even if incentivised payments were not in place?\n\n\nMethods\n\nIn order to compare the differences in the implementation of evidence-based national guidance on VTE prevention across general medicine and orthopaedics in four hospital study sites, a multi-method approach was adopted. To examine staff motivation in implementing (or not) the NICE guidance on VTE, qualitative methods were necessary. We content-analysed interviews and local documents expressing the attitudes, motives and rationales of the staff most responsible for implementing revised NICE guidance and meeting CQUIN targets. To examine how far staff in the study sites had in fact implemented the NICE guidance and responded to the CQUIN incentives, we extracted data from medical records.\n\nAs study sites, we sampled four hospitals with initially divergent models of VTE prevention activity. For example, Hospital A favoured universal prophylaxis with an ‘opt-out’ strategy where patients received prophylaxis unless contra-indicated. In contrast, Hospital C undertook early and ongoing risk assessment and only prescribed prophylaxis to patients with a clear, clinical need. Our sampling frame was a regional census undertaken in 2009 (reference not cited to protect anonymity of the sites) which reported organisational context and VTE prevention activity for each hospital.\n\nIt was decided to compare patients within general medicine and orthopaedics as it was hypothesised that a speciality with a relatively high proportion of planned admissions (orthopaedics) would find it easier to implement the revised guidance than a speciality with a relatively high proportion of unplanned admissions (general medicine). As we wanted to determine the extent to which the imposition of national guidance on VTE risk assessment and treatment had influenced the development and risk assessment intervention in each hospital we decided to sample records over two years. To minimise the effects of both the seasonal inflows of tourists and the rotation of junior doctors our sample was drawn from a list of all adult admissions for stays over 24 hours for the same three-week period in October 2009 and 2010.\n\nWe sampled staff using a snowballing method. We started by interviewing the medical and orthopaedics consultants leading VTE implementation and from their responses a care pathway for VTE risk assessment, prevention and treatment in each hospital site was able to be mapped. From this we were able to identify staff who made key decisions about VTE prevention and treatment during a hospital episode. These included: junior medical staff, VTE nurses, orthopaedic nurses, pre-operative assessment staff, pharmacists and senior hospital managers.\n\nEach hospital approached our request to devise a sampling frame in different ways. Hospital A gave free access to admission data for the three week period in October from which we sampled the target population. Hospitals B, C and D would not release admission data but did provide a sample of patient files for audit. Subsequent statistical analysis confirmed that in those hospitals where we were unable to take our own sample, a random cross section of files appeared to have been made available without having been ‘cherry-picked’ by hospital records departments in order to show 100% compliance with national VTE risk assessment in orthopaedics and general medicine. An unexpected, later opportunity to compare our statistical analysis of VTE risk assessment in one of the hospital sites with an internal audit undertaken by some ward pharmacists during 2010 appeared to substantiate that the ‘cherry-picking’ of files to indicate stronger compliance was not the case.\n\nTo examine how the NICE and CQUIN norms were actually implemented we extracted from medical records the data shown on the data collection instrument (see data collection file). This was designed for mainly ‘tick-box’ entry of standard data entry fields. Power calculations indicated that a sample of 408 patients across all four hospitals for each year would be sufficient to detect an increase from 50% compliance in 2009 to 60% in 2010 with 80% power at the 5% level of significance. Data from 2009 allowed a retrospective sample predating NICE guidance 2010, whilst 2010 data was a prospective sample following NICE guidance implementation. In all 816 medical records were sampled.\n\n\n\nCase study data were collected by interviews with key informants. We started collecting data in 2009 by interviewing either the medical or orthopaedic consultant responsible for leading VTE implementation in each hospital. A care pathway for VTE prevention and treatment was then mapped for both orthopaedics and general medicine in each hospital site. From this we were able to identify key healthcare professionals who appeared integral to VTE risk assessment and treatment throughout a hospital episode. We were then able to snowball to interview these professionals whose roles included: VTE nurses, pharmacists, orthopaedic nurses, pre-operative assessment staff and senior hospital managers. Forty four interviews were completed in the four hospitals over an 18 month period between March 2010 and July 2011. These were broken down hospital by hospital as follows: 11 each for Hospitals A and B; 10 for Hospital C and 12 for Hospital D. At certain times in Hospital A, time constraints in a busy Acute Assessment Unit meant that some interviews were undertaken with two members of staff at the same time. Fifty professionals were interviewed in total across the four hospitals. A semi-structured interview schedule was used (see interview schedule file). The interviews described the pre-NICE 2009 systems and reported how each hospital had responded to the 2010 NICE guidance and what factors appeared to facilitate or hinder compliance. All the interviews with key informants lasted approximately one hour and were conducted on a face-to-face basis in the four hospitals by SC or OB.\n\nEach interview was transcribed and checked for accuracy by the respondent. We used framework analysis by collating our interview data into tables for each hospital whose rows were categories that reflected our research questions. This approach enabled us to immediately ascertain whether there were any unforeseen emergent patterns in the data that necessitated us revising our initial assumptions. To check for accuracy, anonymised empirical findings were presented to the thrombosis committee in three out of the four sites – A, C and D.\n\nA hospital can be awarded exemplar status as a ‘kite-mark’ of quality and good practice in VTE care. As part of the award they are expected to share examples of good practice, educational and audit material as well as providing advice on VTE care. A further expectation is that they collaborate on clinical research into VTE with other exemplar centres and develop innovate VTE prevention practices. In June 2010, there were sixteen VTE exemplar centres spanning the UK20, including two of our hospitals (A and D).\n\nHospital A is a teaching hospital with over 900 beds. More than 48,000 patients pass through its doors every week. It provides a full range of acute and general hospital services for nearly half a million people. Speciality services are available to a greater population of over two million people. It employs approximately 5400 full-time staff. After becoming a Department of Health VTE exemplar site in 2009 it created a post of VTE Clinical Nurse Specialist to ensure it continued to comply with VTE risk assessment and disseminate best practice across the Trust.\n\nHospital B has over 750 beds spread between three geographically dispersed and semi-remote, rural sites (with the main site undertaking over 90% of the Trusts activity). It serves a population of 450,000 people (often doubled by holidaymakers at the busiest times of the year) and employs approximately 5200 staff. The recent opening of a new medical teaching facility had further enhanced its strong reputation for education and training. It was in the process of applying for Foundation Trust status at the time of this investigation.\n\nHospital C was one of the UK’s first foundation trust hospitals and provides acute hospital services to around 350,000 people as well as offering specialist services to a much larger population of approximately two million. Across all of its numerous sites it has over 30 wards, 20 operating theatres, 850 inpatient beds and 80 day-case beds. It employs approximately 6700 staff. There are over 310,000 consultant-led outpatient attendances per year and 120,000 day case admissions per year. It also has teaching hospital status.\n\nHospital D has foundation trust status and is much smaller in scale than the other three hospitals. It is not a teaching hospital and serves a population of approximately 225,000 spread across three neighbouring counties. Specialist services such as burns, plastic surgery, genetics and rehabilitation extend to more than three million people within a greater geographical area. It employs around 3900 full and part-time staff and had been awarded Department of Health exemplar status for VTE in 2009 (like Hospital A). It continued to be at the forefront nationally on VTE prevention and hosted a national VTE conference in 2010.\n\n\nEthical considerations\n\nEthical approval was not needed for this study as the local NHS Research and Development Service deemed it to be service evaluation and audit. Honorary contracts for the two researchers (SC and OB) were necessary in three of the four hospitals (B, C and D). The fourth hospital (A) only required a letter of access.\n\nAll four hospitals were assured that both they and individual informants would not be identifiable in both data analysis and publications. Researcher safety was addressed by ensuring that someone within the team had knowledge of the date, time and place of interviews.\n\n\nFindings\n\n1. Increase in the recording of VTE risk assessment and prescribing of appropriate thrombo-prophylaxis.\n\nAll four sites show an improvement in the percentage of patients risk assessed for VTE between 2009 and 2010. Table 1 shows the overall pattern of convergence with NICE requirements for VTE risk assessment and the amount of CQUIN monies available for each hospital if local quality improvement goals were met. It also indicates the percentage of CQUIN monies allocated to further improving VTE risk assessment by paying for a Lead VTE Nurse or improving staff education in VTE.\n\nThe biggest overall percentage change in risk assessment was in Hospital A. This was not unexpected. Despite its low figure of 43.1% for risk assessment in 2009, it had been awarded Department of Health VTE exemplar site status in December 2008 due to its policy of prescribing universal pharmaceutical prophylaxis unless contra-indicated. However, poor documentation of risk assessment in patient files meant that we were unable to count that a VTE risk assessment had taken place for many patients for our audit purposes in 2009. This might begin to explain the low 43.1% statistic. Over the 12 months of our audit, it rose from last to first place for VTE risk assessment in the four hospitals in our study as auditable risk assessment rose to 90.2% in 2010. In contrast, Hospital D, also a Department of Health exemplar site for VTE pre-2010 NICE guidance was the leading hospital in 2009 by risk assessing 71.6% of inpatients for VTE. Only a small improvement was able to be statistically shown for 2010 as auditable risk assessments rose to 74.5%. Again, we were only able to count patient files where there was written evidence of an actual risk assessment being undertaken. This statistically non-significant shift in risk assessment might be explained by inaccurate recording of paperwork or poor filing of risk assessment documentation. However, Hospital D was the only hospital to not be eligible for CQUIN incentives and this may have affected the motivation for implementing the required national changes in completing VTE risk assessment documentation.\n\n2. How far was the improvement in VTE risk assessment a response to the financial incentives paid to acute providers through CQUIN?\n\nAll hospitals had local VTE prevention policies in place before national guidance was introduced in 2010. Three of the hospitals (B, C and D) had developed policies that ultimately matched the national standards subsequently set by NICE in 2010. These policies were built on slightly differentiated risk assessments that were often individual for each specialism. For example in Hospital D, Obstetrics and Gynaecology used a VTE risk assessment they had developed from the Royal College of Obstetricians and Gynaecologists risk assessment tool rolled out in 200922. When questioned about the use of differing risk assessment forms across the hospital during interview, the chair of the thrombosis committee at Hospital D described these differentiated risk assessments thus:\n\n\"…(the) end output of the algorithm may vary by speciality but not the input.\"\n\nThis suggests that risk factors across specialisms might vary, but the overarching aim to prevent VTE during a hospital episode remained consistent throughout the hospital. Prevention of venous thromboembolism was therefore based on the cumulative risk factors of assessment and treatment.\n\nIn contrast to Hospital D, Hospital A had developed an ‘opt-out’ policy where all patients (irrespective of admittance speciality) were given a standardised dose of pharmaceutical prophylaxis unless it has been contra-indicated. This ‘opt-out’ policy had originally been devised by the thrombosis committee following a national campaign around the preventable risks of VTE. However, such a blanket policy meant individual risk assessment for VTE was not always officially recorded in hospital notes nor, therefore, reflected in our compliance scores (as shown in Table 1) for 2009 (which appeared to be low in comparison to Hospitals B, C and D). As new drug charts were introduced in Hospital A during 2010 which included VTE risk assessment proformas that needed to be completed before any drugs were prescribed, our audit was able to document a large increase in evidence of risk assessment from 43.1% in 2009 to 90.2% in 2010.\n\nOur analysis appears to support the suggestion that the imposition of national guidelines for VTE risk assessment in 2010 acted as the driving force behind the significant improvements in the recording of risk assessment in Hospital A between 2009 and 2010 as outlined above. The recording of risk and prescription of appropriate VTE prophylaxis appeared to be further underpinned across all the hospitals by the introduction of the CQUIN goal on VTE risk assessment.\n\nHospital A achieved all of its CQUIN targets for VTE risk assessment, resulting in the reward of £500,000 to the hospital operating budget. A member of its VTE committee suggested that as the hospital had performed so well with meeting current CQUIN standards, the PCT would be very unlikely to include as many targeted criteria in following years. The hospital would probably achieve them all thereby making financial incentives too expensive to finance. When asked in early 2012 if any of the £500,000 CQUIN monies had been directly received by the thrombosis committee to improve VTE risk assessment or treatment the committee member replied:\n\n\"The VTE prevention initiative has received nothing from the Trust so far but talks are in progress.\"\n\nHospital B also received payment for achieving CQUIN targets for VTE. The 2010–2011 VTE figure was approximately £400,000. However, as was the case in Hospital A, the Chief Haematologist at Hospital B said:\n\n\"…not a penny has come to our department [haematology] as I have been told there is no linkage between the monies and its use in the specified area.\"\n\nIn Hospital C the value of the Trust’s VTE CQUIN reward for VTE was in the region of £300,000. In line with our findings in Hospitals A and B, the VTE CQUIN monies earned to date in Hospital C had not been used to explicitly invest in VTE assessment. However, an electronic system for capturing VTE risk assessment was in the course of development but the hospital was unable to confirm if research and development monies for this initiative had come directly from CQUIN payments.\n\nHowever, what was very interesting from the point of view of our research was that Hospital D was found to differ significantly from the previous hospitals discussed above as they were said to have experienced a ‘difficult relationship’ with their PCT over a number of years and:\n\n\"…therefore one of the consequences of that is an unachievable clause for achieving VTE CQUIN money has been set into the contract which is outside our control and that is ambulance response times on patients who require thrombolysis.\" (Lead Clinician for Haematology, Hospital D).\n\nThis resulted in no CQUIN monies being made available to the hospital whether they achieved CQUIN targets or not.\n\nDespite the improvements in VTE risk assessment and prevention discussed above, it remains difficult to ascertain with certainty if monetary incentives were the driving force behind the significant changes in the percentages of patients risk assessed between 2009 and 2010 in each hospital. We used interview analysis to try and determine if a more intrinsic type of motivation in driving up patient safety might explain the improvements in risk assessment. A Lead Anti-Coagulant Nurse from Hospital B suggested that CQUIN incentives were actually a crude form of payment by results. His concern appeared to focus on whether any monies obtained by CQUIN would be used to improve VTE risk assessment by employing more staff (as evidenced by Hospital A using the funding to employ a Lead VTE Nurse) or simply swallowed into general operating budgets:\n\n\"I think in the current financial climate you need to be realistic that money will be absorbed but at the same time… it’s a fairly significant amount and you would not need every penny of that money to deliver an effective VTE role. I think what you need is the hospital to be able to say we are prepared to spend what is needed to achieve it and whatever is left over we will absorb that as a hospital. That’s acceptable.\"\n\nCertainly in the three hospitals eligible for VTE CQUIN funding all monies had indeed been swallowed into the general operating budget so it was difficult to ascertain whether extrinsic motivation in the form of payment by results drove increased compliance to NICE guidance.\n\nWe suggest that hospitals were not using CQUIN rewards to improve the patient experience by allocating CQUIN payments to specific departments. Indeed interview data from a Pre-Operative Assessment Nurse in Hospital B suggest that even when CQUIN targets were met the amount of time being allowed for VTE risk assessment in all sites had since been reduced to allow for a greater volume of patient activity, whilst at the same time staff were being expected to increase their recording and frequency of VTE risk assessment in order to comply with national guidelines on which CQUIN rewards were based:\n\n\"…our processes have changed quite considerably in the past three or four years to actually increase the throughput of patients that we see… historically… the doctors that were in clinic would always do a risk assessment [for VTE] so you would always see the bottom of the prescription chart you know – high risk – and then we would measure for the TED stockings but we don’t now.\" (Sister at Pre-Operative Assessment Clinic, Hospital B).\n\nThis finding appears to fit with the NHS Staff Survey (2010), which suggests that just under half of all staff (46%, up from 45% in 2010) felt they do not have enough time to carry out their work and 42% indicated they could not meet all the conflicting demands on their time at work. Yet, paradoxically, 87% of staff indicated they were satisfied with the quality of care provided to patients15.\n\n\nConclusions\n\nThe research–practice gap is widely documented. It can be extremely difficult to implement even those strategies (such as risk assessing for VTE) that are known to carry large health benefits into the healthcare system. The general goal of VTE risk assessment for all patients appears to have been transformed into a metric-specific aim statement with the process measure defined as 90% risk assessment of all in-patients. Despite this, recent figures from the Department of Health show that since national measures were introduced in 2010, patients are now twice as likely to be risk assessed for VTE. More than 14.3 million patients have been risk assessed since the measure was put in place, and an estimated 230,000 patients are being screened per week. So our findings appear to match events in the NHS more widely.\n\nAs our data show, the introduction of CQUIN targets has focussed the attention of hospital management towards improving the recording of risk assessment and the prescribing of appropriate VTE prophylaxis. The role of CQUIN has been re-emphasised as being a key mechanism for delivering the improvements identified as markers of quality by NICE quality standards. However, there appears to remain a contradiction between the more traditional, extrinsically motivated payment by results, to reduce waiting times and increase the volume of work completed in a given period, and the development of a more intrinsically motivated effective patient experience by driving up standards. The improvements in VTE risk assessment between 2009 and 2010 in each hospital suggests that the implementation of national clinical guidelines for VTE risk assessment and appropriate prescribing of prophylaxis appears to have significantly changed the attitudes and behaviour of healthcare professionals towards VTE risk assessment. In turn, this has increased compliance with national guidelines. However, it is very difficult to separate whether improvements in VTE risk assessment and the prescribing of appropriate thrombo-prophylaxis has increased due to risk assessment for VTE becoming a national performance indicator underpinned by the introduction of a national CQUIN goal on VTE risk assessment (motivated by the potential of extrinsic financial reward) or through the continuous improvement in the risk management of VTE in each hospital (intrinsic motivation). The increased measurement and risk assessment and the increased transparency of clinical practice appear to have created what proponents of Foucault (a 20th century philosopher who focussed on the methods by which power is conveyed in society), call a ‘disciplinary’ motive for compliance with NICE and CQUIN norms23. Such motives are certainly intrinsic but not in the positive way that the original theorists of intrinsic motivation seem to have had in mind.\n\nCertainly increased levels of risk assessment and prophylaxis for VTE are evident from our study, but once again, it is difficult to establish with certainty whether clinical activity has actually changed, or simply the recording of it. Whether a year is long enough for the full impact of guideline implementation to be apparent is difficult to say, although our audit of medical records does show substantial changes in the numbers of patients being risk assessed and appropriately treated. Our study appears to mirror that of Tharvarajah and Wetherill who found that by attaching a VTE risk assessment tool to drug charts significant improvements in the recording of risk assessment can be observed24.\n\nWhat would be interesting for future investigation would be whether the focus of hospital management to comply with national policy guidance on VTE risk assessment and prophylaxis remains after the financial ‘carrot’ of CQUIN monies has been withdrawn. To what extent are we able to determine whether the culture of VTE risk assessment will have become so entrenched in the working practices of healthcare professionals that we will continue to see compliance with quality guidelines that may no longer be rewarded financially?",
"appendix": "Author contributions\n\n\n\nSC drafted the manuscript with input from all the authors. SC, RS and OB designed the study and CAG supervised the overall study. All authors were involved in the revision of the draft manuscripts and have agreed 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\nSC, RS and AB are currently employed by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care for the South West Peninsula (PenCLAHRC). Support from the NIHR in England for SC’s, RS’s and AB’s contribution is gratefully acknowledged. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health in England.\n\n\nReferences\n\nBusse R, Schreyogg J, Smith PC: Hospital case payment systems in Europe. Health Care Manag Sci. 2006; 9(3): 211–213. PubMed Abstract\n\nSchreyogg J, Stargardt T, Tiemann O, et al.: Methods to determine reimbursement rates for diagnosis related groups (DRG): A comparison of nine European countries. Health Care Manag Sci. 2006; 9(3): 215–223. PubMed Abstract | Publisher Full Text\n\nDepartment of Health: The NHS Plan. A Plan for Reinvestment. A Plan for Reform. 2000. Reference Source\n\nDepartment of Health Payment by Results team: A simple guide to payment by results. 2011. Reference Source\n\nMaybin J: Payment by Results. 2007.\n\nCSP: Payment By Results. The New funding system for the NHS in England. Practical Support for Allied Health Professionals. 2005. Reference Source\n\nDepartment of Health: High Quality Care For All. NHS Next Stage Review Final Report Summary. 2008.\n\nManchester City Council CQUIN Payments - (Commissioning for Quality and Innovation (CQUIN) payment framework). 2011.\n\nGeerts W: Prevention of venous thromboembolism: a key patient safety priority. J Thromb Haemost. 2009; 7(Suppl 1): 1–8. PubMed Abstract | Publisher Full Text\n\nDouketis JD, Moinuddin I: Prophylaxis against venous thromboembolism in hospitalized medical patients: an evidence-based and practical approach. Pol Arch Med Wewn. 2008; 118(4): 209–15. PubMed Abstract\n\nHouse of Commons Health Committee: The Prevention of Venous Thromboembolism in Hospitalised Patients. HMSO: London 2005. Reference Source\n\nLindblad B, Sternby NH, Bergqvist D: Incidence of venous thromboembolism verified by necropsy over 30 years. BMJ. 1991; 302(6778): 709–711. PubMed Abstract | Free Full Text\n\nDepartment of Health: Using the Commissioning for Quality and Innovation (CQUIN) payment framework - A summary guide. 2010. Reference Source\n\nElliott B: Labour markets in the NHS an agenda for research. Health Econ. 2003; 12(10): 797–801. PubMed Abstract | Publisher Full Text\n\nThe Department of Health: NHS Staff Ninth Annual Survey. 2011. Reference Source\n\nEdward E, Lawler I: Job Design and Employee Motivation. Pers Psychol. 1969; 22(4): 426–435. Publisher Full Text\n\nHood C: Gaming in Targetworld: The Targets Approach to Managing British Public Services. Public Adm Rev. 2006; 66(4): 515–521. Publisher Full Text\n\nDepartment of Health: Pay Framework for Very Senior Managers in Strategic and Special Health Authorities, Primary Care Trusts and Ambulance Trusts. 2012. Reference Source\n\nSheaff R: A Synthetic Discipline; preventing venous thrombo-embolism in hospital in-patients. 2012 (Forthcoming).\n\nKing's Thrombosis Centre: Venous Thromboembolism Prevention. A Guide for Delivering the CQUIN goal, R. Arya and B. Hunt, Editors. 2010. Reference Source\n\nTimmermans S, Berg M: The Gold Standard: The Challenge of Evidence-Based Medicine and Standardization in Health Care. Philadelphia: Temple University 2003. Reference Source\n\nRoyal College of Obstetricans and Gynaecologists: Reducing the risk of thrombosis and embolism during pregnancy and the puerperium. 2009. Reference Source\n\nFerlie E, Mcgivern G, FitzGerald L: A New Mode of Organizing in Health Care? Governmentality and Managed Networks in Cancer Services in England. Soc Sci Med. 2012; 74(3): 340–347. PubMed Abstract | Publisher Full Text\n\nThavarajah D, Wetherill M: Implementing NICE guidelines on risk assessment for venous thromboembolism. Failure, success and controversy. Int J Health Care Qual Assur. 2012; 25(7): 618–624. PubMed Abstract | Publisher Full Text"
}
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[
{
"id": "786",
"date": "19 Feb 2013",
"name": "Oweikumo Eradiri",
"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\nSummary – The article is a robust presentation of current challenges in implementing strategies for effective VTE prophylaxis. I consider it an important contribution to the debate on incentivising managers of NHS Trusts to support clinicians in their bid to innovate for patient safety, or comply with national quality improvement guidance. Methodology – I have not reviewed the power calculations, but the overall approach is sound.Recommendation – The article could perhaps have benefited from additional answers to research questions 2b and 2c.",
"responses": []
},
{
"id": "802",
"date": "28 Feb 2013",
"name": "Lynda Bonner",
"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 mixed methods study. It provides a comprehensive background to incentive payments (payment by volume and payment by results) in the NHS. The authors mention that compliance with VTE CQUIN targets 2010-11 was measured through a monthly audit of patients who received appropriate thrombo-prophylaxis after risk assessment, however, it is worth adding that the financial incentive for reaching a 90% threshold for VTE risk assessment was based on census data collection and that Unify2 contains data on the VTE risk assessment indicator only. The section on motivation in the NHS is particularly relevant to those who are in a position to use CQUIN as a driver to lead the implementation of effective VTE prevention practice in their organisation. The power of CQUIN relies on its meaningful translation of its implications for staff within the healthcare organisation. The authors give examples of how CQUIN can act as a motivator to engage different staff groups that have a role in VTE prevention and suggest that there was less evidence of improvement where unachievable clauses for achieving CQUIN rewards existed. This highlights the importance of commissioners and providers agreeing realistic targets to activate the motivational potential of CQUIN. The study methodology appears justified and sound. Any limitations in methodology were acknowledged and where possible action was taken to minimise their potential impact. Ethical issues were considered and authorisation for the study was granted by the appropriate authorities. Findings are discussed within the wider context of practice nationally and debate created around the intrinsic/extrinsic motivations that may have been triggered by payment by results. It might be worth reflecting on the authors positive view of CQUIN as a financial 'carrot' and whether this may have influenced their interpretation of findings related to motivation. (This is in contrast to the more negative view held by some that CQUIN is a financial 'stick' whereby monies allocated for an organisation will be withheld if they are unable to meet their targets). The snowball sampling technique could have limited the richness of data that might have been gathered from untapped sources. This study adds to the current knowledge base and its findings support a positive correlation between a statistically significant improvement in the percentage of patients risk assessed for VTE and the achievement of CQUIN payments amongst the study sample. It also offers an insight into the perceptions of CQUIN as a motivator and acknowledges that further research in the area is needed. It should be of genuine interest to those in the field of VTE prevention or to those who are aiming to achieve CQUIN payments for their organisation.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-41
|
https://f1000research.com/articles/2-40/v1
|
12 Feb 13
|
{
"type": "Systematic Review",
"title": "Mobile phones affect multiple sperm quality traits: a meta-analysis",
"authors": [
"Madhukar Shivajirao Dama",
"M Narayana Bhat",
"M Narayana Bhat"
],
"abstract": "As mobile phone usage is growing rapidly, there is a need for a comprehensive analysis of the literature to inform scientific debates about the adverse effects of mobile phone radiation on sperm quality traits. Therefore, we conducted a meta-analysis of the eligible published research studies on human males of reproductive age. Eleven studies were eligible for this analysis. Based on the meta-analysis, mobile phone use was significantly associated with deterioration in semen quality (Hedges’s g = -0.547; 95% CI: -0.713, -0.382; p < 0.001). The traits particularly affected adversely were sperm concentration, sperm morphology, sperm motility, proportion of non-progressive motile sperm (%), proportion of slow progressive motile sperm (%), and sperm viability. Direct exposure of spermatozoa to mobile phone radiation with in vitro study designs also significantly deteriorated the sperm quality (Hedges’s g = -2.233; 95% CI: -2.758, -1.708; p < 0.001), by reducing straight line velocity, fast progressive motility, Hypo-osmotic swelling (HOS) test score, major axis (µm), minor axis (µm), total sperm motility, perimeter (µm), area (µm2), average path velocity, curvilinear velocity, motile spermatozoa, and acrosome reacted spermatozoa (%). The strength of evidence for the different outcomes varied from very low to very high. The analysis shows that mobile phone use is possibly associated with a number of deleterious effects on the spermatozoa.",
"keywords": [
"Almost 10% of men of reproductive age are estimated to be subfertile1. Owing to its complexity",
"even after identification of a plethora of underlying factors",
"etiology in almost half of the infertile subjects tested at fertility clinics remains obscure2. Hence",
"the list of the causes of male infertility is growing by the day with recent advances in fertility research3. Though advances in assisted reproduction technologies (ARTs)",
"especially in the form of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI)",
"have helped subfertile couples conceive offspring",
"it is feared that ARTs only bypasses the problem of subfertility and contributes towards hiding the underlying causes which have at times led to serious health problems in offspring4",
"5. Hence",
"identification of unknown aetiologies would help in prescription of specific preventive measures that will ultimately decrease the incidence of male infertility."
],
"content": "Introduction\n\nAlmost 10% of men of reproductive age are estimated to be subfertile1. Owing to its complexity, even after identification of a plethora of underlying factors, etiology in almost half of the infertile subjects tested at fertility clinics remains obscure2. Hence, the list of the causes of male infertility is growing by the day with recent advances in fertility research3. Though advances in assisted reproduction technologies (ARTs), especially in the form of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), have helped subfertile couples conceive offspring, it is feared that ARTs only bypasses the problem of subfertility and contributes towards hiding the underlying causes which have at times led to serious health problems in offspring4,5. Hence, identification of unknown aetiologies would help in prescription of specific preventive measures that will ultimately decrease the incidence of male infertility.\n\nMost nations, especially developing countries, are witnessing an increase in the use of various radiation-emitting domestic-purpose devices that could cause mild to serious health problems based on the duration and intensity of usage6, and reduced fertility is now recognised as one such problem7. Wireless mobile phones are one of the most accepted devices with a tremendous increase in usage across the world in recent times8. Research into the impact of ionizing radiation on the development of various types of health disorders, especially cancers, has been well established9. Similarly, several studies have found an increase in the risk of developing some types of tumors after long-term exposure to non-ionizing radiation from mobile phones10. Research into the effects of mobile phone radiation on male fertility, though growing, is limited and inconclusive11,12. Recently, several case-control studies have reported results from a general population setting alongside a few studies from subfertile populations7,13–20. Like ionizing radiation, non-ionizing radiation is also expected to affect spermatozoa, though in subtle ways21. The aim of this meta-analysis was, therefore, to investigate the impact of mobile phone radiation on semen parameters in vitro as well as in vivo settings in men of reproductive age from both general and subfertile populations.\n\n\nMaterial and methods\n\nA systematic search of an electronic database was conducted to retrieve published studies on the impact of mobile phone radiation on semen parameters in adult men. The results have been reported according to the standards of the guidelines for meta-analysis of observational studies in epidemiology22. All English language research studies published up until January 2012 in scientific journals indexed in the searched databases were included for analysis.\n\nInclusion/exclusion criteria and outcomes of interest: The studies on human males of reproductive age reporting the effect of mobile phone radiation on any or all measures of semen volume, total sperm count, sperm concentration, sperm motility or sperm morphology were included. All the studies that did not satisfy the inclusion criteria were excluded.\n\nSearch strategy, data extraction and meta-analysis: Google Scholar and NLM’s PubMed database were searched for articles by using different combinations of 4 mobile phone related keywords [‘mobile phone’, ‘cellular phone’, ‘radiofrequency electromagnetic waves (RF-EMW)’, ‘radiation’] with 5 sperm quality related keywords (‘spermatozoa’, ‘semen’, ‘sperm concentration’, ‘sperm motility’, ‘sperm morphology’) Data from 11 eligible studies were extracted and separated into in vitro and in vivo categories.\n\nEffect sizes were expressed as Hedges’s g23, separately for in vivo & in vitro studies using individual semen parameters as units of analysis (Supplementary Table 1). A random model was used to test and quantify effect size using ‘Comprehensive Meta-Analysis (v.2)’ trial version24. A random effect model was preferred over a fixed effect model in order to account for differences in both effect size and sampling error25.\n\n\nResults\n\nOur analysis shows that overall, mobile phone users had significant deterioration in semen quality (Hedges’s g = -0.547; 95% CI: -0.713, -0.382; p < 0.001). There was significant heterogeneity among effect sizes (Q = 475.985, p < 0.001), which suggest that some of the semen parameters may not be affected by mobile phone exposure. Hence, combined effect-size for each of the semen parameters were calculated separately (Table 1), and it was found that sperm concentration, sperm morphology, sperm motility, proportion of non-progressive motile sperm (%), proportion of slow progressive motile sperm (%), and sperm viability were deteriorated in individuals exposed to mobile phone radiation. By contrast, semen volume, liquefaction time, semen pH, proportion of rapid progressive motile sperm (%), and semen viscosity were not affected by mobile phone usage.\n\nPublication bias could potentially change the results of meta-analysis but analysis of funnel plot of precision by Hedges’s g using Dual and Tweedie’s trim-and-fill test26 did not change the overall effect size, suggesting little bias. Moreover, Rosenthal’s fail-safe N test27 revealed that 3964 missing studies with a mean Hedges’s g of 0 are required for the combined 2-tailed p-value to exceed 0.050. In other words, there need to be 99.1 missing studies for every observed study for the effect to be nullified.\n\nExperimental exposure of spermatozoa isolated from healthy men of reproductive age to mobile phone radiation significantly affected sperm quality (Hedges’s g = -2.233; 95% CI: -2.758, -1.708; p < 0.001). There was significant heterogeneity among effect sizes (Q = 639.294, p<0.001), suggesting that similar to in vivo exposure, in vitro exposure may also not affect all the parameters of spermatozoa. Hence, combined effect-size for spermatozoa parameters were calculated separately (Table 1), and it was found that exposure to mobile phones significantly reduced straight line velocity, fast progressive motility, Hypo-osmotic swelling (HOS) test score, major axis (µm), minor axis (µm), total sperm motility, perimeter (µm), area (µm2), average path velocity, curvilinear velocity, motile spermatozoa, and acrosome reacted spermatozoa (%). By contrast, DNA fragmentation levels, non-progressive motility, total antioxidant capacity (TAC), progressive motility, reactive oxygen species (ROS) generation, slow progressive motility, sperm concentration, and sperm zona binding was not affected by mobile phone radiation.\n\nA Funnel plot of precision by Hedges’s g using Dual and Tweedie’s trim-and-fill test did not change the overall effect size, suggesting little publication bias. Rosenthal’s fail-safe N test revealed that 3813 missing studies with a mean Hedges’s g of 0 are required for the combined 2-tailed p-value to exceed 0.050. In other words, there need to be 100.3 missing studies for every observed study for the effect to be nullified.\n\n\nDiscussion\n\nThis study was aimed to analyse the data assessing the risk of mobile phone radiation on male fertility. Our results suggest that mobile phone radiation has a tendency to significantly affect sperm quality. Based on the design of the analysed records, we divided studies into in vivo studies and in vitro studies. The effect size was significant in both the categories, suggesting that mobile phone radiation could severely compromise male fertility. This conclusion is robust, as a fail-safe test suggested that the results are not likely to be mediated by publication bias.\n\nThe number of worldwide mobile subscriptions grew from less than 1 billion in 2000 to over 6 billion in 20128, with more than half of these subscribers estimated to be children and young adults. Hence, it is very likely that in the coming decades, we could witness an increase in the incidence of male infertility due to mobile phone radiation exposure, similar to growing concerns over other hazards. Although the mechanism of cell phone radiation-mediated health defects is still obscure, it is proposed that their ability to produce heat, disrupt cell membranes, affect endothelial function, alter the blood-brain barrier, and modulate neuronal excitability have the potential to affect multiple physiological functions simultaneously28–30.\n\nTo our knowledge, this is the first meta-analysis of the effects of mobile phone radiations on various sperm quality parameters. Cellular phones have become integral part of everyday life, and newer versions of these are developed very rapidly these days. Hence, it is necessary to educate the users about the hazards of cell phones as well as test the newer versions like smartphones for health hazards.",
"appendix": "Competing interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nde Kretser DM: Male infertility. Lancet. 1997; 349(9054): 787–90. PubMed Abstract | Publisher Full Text\n\nMatzuk MM, Lamb DJ: The biology of infertility: research advances and clinical challenges. Nat Med. 2008; 14(11): 1197–213. PubMed Abstract | Publisher Full Text\n\nKilgallon SJ, Simmons LW: Image content influences men’s semen quality. Biol Lett. 2005; 1(3): 253–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavies MJ, Moore VM, Willson KJ, et al.: Reproductive technologies and the risk of birth defects. N Engl J Med. 2012; 366(19): 1803–13. PubMed Abstract | Publisher Full Text\n\nWen J, Jiang J, Ding C, et al.: Birth defects in children conceived by in vitro fertilization and intracytoplasmic sperm injection: a meta-analysis. Fertil Steril. 2012; 97(6): 1331–7.e1–4. PubMed Abstract | Publisher Full Text\n\nSage C, Carpenter D: BioInitiative Working G: BioInitiative report : a rationale for a biologically-based public exposure standard for electromagnetic fields (ELF and RF). [United States]: BioInitiative Working Group, 2007. Reference Source\n\nFejes I, Závaczki Z, Szöllosi J, et al.: Is there a relationship between cell phone use and semen quality? Arch Androl. 2005; 51(5): 385–93. PubMed Abstract | Publisher Full Text\n\nWorld Bank. Information and Communication T, infoDev, World B. Maximizing mobile : 2012 information and communications for development. In. Washington, D.C.: World Bank: InfoDev. Reference Source\n\nAhlbom A, Green A, Kheifets L, et al.: Epidemiology of health effects of radiofrequency exposure. Environ Health Perspect. 2004; 112(17): 1741–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMyung SK, Ju W, McDonnell DD, et al.: Mobile phone use and risk of tumors: a meta-analysis. J Clin Oncol. 2009; 27(33): 5565–72. PubMed Abstract | Publisher Full Text\n\nAgarwal A, Singh A, Hamada A, et al.: Cell phones and male infertility: a review of recent innovations in technology and consequences. Int Braz J Urol. 2011; 37(4): 432–54. PubMed Abstract\n\nMerhi ZO: Challenging cell phone impact on reproduction: a review. J Assist Reprod Genet. 2012; 29(4): 293–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWdowiak A, Wdowiak L, Wiktor H: Evaluation of the effect of using mobile phones on male fertility. Ann Agric Environ Med. 2007; 14(1): 169–72. PubMed Abstract\n\nAgarwal A, Deepinder F, Sharma RK, et al.: Effect of cell phone usage on semen analysis in men attending infertility clinic: an observational study. Fertil Steril. 2008; 89(1): 124–8. PubMed Abstract | Publisher Full Text\n\nSajeda S, Al-Wattar YT: Effect of mobile phone usage on semen analysis in infertile men. Tikrit J Pharm Sci. 2011; 7(1): 77–82.\n\nErogul O, Oztas E, Yildirim I, et al.: Effects of electromagnetic radiation from a cellular phone on human sperm motility: an in vitro study. Arch Med Res. 2006; 37(7): 840–3. PubMed Abstract | Publisher Full Text\n\nFalzone N, Huyser C, Fourie F, et al.: In vitro effect of pulsed 900 MHz GSM radiation on mitochondrial membrane potential and motility of human spermatozoa. Bioelectromagnetics. 2008; 29(4): 268–76. PubMed Abstract | Publisher Full Text\n\nAhmad L, Baig NM: Mobile Phone RF-EMW Exposure to Human Spermatozoa: An in vitro Study. Pakistan J Zool. 2011; 43(6): 1147–54. Reference Source\n\nDkhil MA, Danfour MA, Al-Quraishy S: Sperm function is affected by the electromagnetic radiation emitted by mobile phone. Afr J Microbiol Res. 2011; 5(27): 4896–900. Publisher Full Text\n\nAgarwal A, Desai NR, Makker K, et al.: Effects of radiofrequency electromagnetic waves (RF-EMW) from cellular phones on human ejaculated semen: an in vitro pilot study. Fertil Steril. 2009; 92(4): 1318–25. PubMed Abstract | Publisher Full Text\n\nDasdag S, Ketani MA, Akdag Z, et al.: Whole-body microwave exposure emitted by cellular phones and testicular function of rats. Urol Res. 1999; 27(3): 219–23. PubMed Abstract\n\nStroup DF, Berlin JA, Morton SC, et al.: Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group. JAMA. 2000; 283(15): 2008–12. PubMed Abstract | Publisher Full Text\n\nHedges LV, Olkin I: Statistical methods for meta-analysis. Orlando: Academic Press, 1985. Reference Source\n\nBorenstein M, Hedges L, Higgins J, et al.: Comprehensive meta-analysis (Version 2) [Computer software]. Englewood, NJ BioStat, 2005.\n\nSutton AJ: Methods for meta-analysis in medical research. Chichester; New York: J. Wiley, 2000. Reference Source\n\nDuval S, Tweedie R: A Nonparametric \"Trim and Fill\" Method of Accounting for Publication Bias in Meta-Analysis. J Am Statistical Assoc. 2000; 95(449): 89–98. Publisher Full Text\n\nOrwin RG: A Fail-Safe N for Effect Size in Meta-Analysis. J Educational Statistics. 1983; 8(2): 157–59. Publisher Full Text\n\nStraume A, Oftedal G, Johnsson A: Skin temperature increase caused by a mobile phone: a methodological infrared camera study. Bioelectromagnetics. 2005; 26(6): 510–9. PubMed Abstract | Publisher Full Text\n\nBehari J: Biological responses of mobile phone frequency exposure. Indian J Exp Biol. 2010; 48(10): 959–81. PubMed Abstract\n\nJuutilainen J, de Seze R: Biological effects of amplitude-modulated radiofrequency radiation. Scand J Work Environ Health. 1998; 24(4): 245–54. PubMed Abstract | Publisher Full Text\n\nDe Iuliis GN, Newey RJ, King BV, et al.: Mobile phone radiation induces reactive oxygen species production and DNA damage in human spermatozoa in vitro. PloS one. 2009; 4(7): e6446. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFalzone N, Huyser C, Becker P, et al.: The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa. Int J Androl. 2011; 34(1): 20–26. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "780",
"date": "19 Feb 2013",
"name": "Nelson Bennett",
"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": "808",
"date": "14 Mar 2013",
"name": "Gary Klinefelter",
"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\nOn the surface, the results seem quite striking with virtually any sperm endpoint one can imagine being significantly altered in the collective analysis of mobile phone studies compiled. However, upon looking at the data in the supplementary table, it is obvious that the relatively few studies compiled varied widely both in respect to endpoints measured and the sample size. As shown in Table 1, motility is the endpoint representing the greatest combined sample size for both in vivo and in vitro studies. Motility was measured in 4 out of 4 in vivo studies and 5 out of 7 in vitro studies. So motility ‘might’ be an endpoint that is repeatedly altered by cell phone exposure. The reason for ‘might’ is the lack of any reported exposure data in this study. In summary, the small sample size and lack of exposure data significantly weaken the conclusions of this study.",
"responses": [
{
"c_id": "431",
"date": "02 Apr 2013",
"name": "Madhukar Dama",
"role": "Author Response",
"response": "We have studied the reviewer comments and would like to justify our results. Our analysis is showing that mobile phone radiations could affect many sperm parameters. This could be due to interdependence of sperm parameters (Acta Eur Fertil. 1982;13(2):49-54). We also agree with the point that the number of studies is few and total sample size in in vitro studies is smaller. However, it must be noted that the sample size is weighted during meta-analysis, which nullifies the problems posed by smaller sample size studies. Apart from motility, other parameters like morphology, concentration, and viability are also significantly affected by in vivo exposure. Hence we have provided all the effect sizes individually along with p values and sample size. We hope that our points justify the reviewer comments."
}
]
},
{
"id": "861",
"date": "25 Mar 2013",
"name": "Essam- Eldeen M Mohamed",
"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/2-40
|
https://f1000research.com/articles/2-39/v1
|
11 Feb 13
|
{
"type": "Short Research Article",
"title": "Detection of Giardia lamblia stool samples: a comparison of two enzyme-linked immunosorbent assays",
"authors": [
"Tomas Jelinek",
"Stefan Neifer",
"Stefan Neifer"
],
"abstract": "Two commercially produced enzyme-immunosorbent assays (EIAs) to detect antigens of Giardia lamblia in stool specimens, Ridascreen Giardia and Serazym Giardia, were evaluated. A total of 88 stool specimens were collected from patients who presented for various medical complaints in our outpatient clinic. Every specimen was examined by a conventional microscopic examination (CME), PCR and tested by both EIA kits. When microscopy was used as the reference standard, the Ridascreen Giardia showed a sensitivity of 72.9% and a specificity of 100%. Serazym Giardia had a sensitivity of 93.8% and a specificity of 100%. Antigen detection by EIA has been established as a valuable tool to make parasite stool diagnostics more effective.",
"keywords": [
"Giardia lamblia diagnosis",
"antigen detection"
],
"content": "Introduction\n\nIntestinal parasites have a world-wide distribution. One of the most common intestinal protozoan parasites is Giardia lamblia1. Since it has a fecal-oral transmission cycle and is contracted by ingestion of contaminated water or food or by person-to-person contact, the highest disease burden is found in areas where sanitary conditions are poor. The highest rates of infection are therefore encountered in developing countries (10–30% in young children), while in developed countries, infections occur mostly in persons living in closed communities, homosexual men, immigrants and, of increasing importance, travellers returning from highly endemic countries (2–5% of symptomatic patients)1.\n\nThe protozoan flagellate Giardia lamblia has a reported global prevalence of approximately 30% in cases of acute or persistent diarrhea2,3. Acute giardiasis has been repeatedly described in travellers returning from highly endemic areas4,5. The infection may be asymptomatic or present with various symptoms. The main symptom is watery, foul smelling diarrhoea, often accompanied by nausea, abdominal cramps or gurgling, bloating and weight loss1. Symptoms of variable severity may persist for weeks. Giardia lamblia does not invade the tissue6. Its life cycle consists of two different stages: the trophozoite and the infectious form (the cyst).\n\nThe diagnosis of giardiasis is frequently based on microscopical detection of the organisms in stool samples. Yet the method is time and labour intensive and depends on the skill of an experienced microscopist7. Diagnosis via microscopical examination of a single stool specimen has a low sensitivity and may therefore miss up to 50% of Giardia infections7–9. Because of the intermittent shedding of the parasites, microscopic examination of three consecutive stool specimens is required to reach a sensitivity of over 90%1–7. Given these difficulties the development of sensitive, cost-effective and rapid diagnostic methods is of utmost importance. Enzyme immuno-assays (EIAs) for detection of the specific antigens in stools have developed into an efficient diagnostic technique in the detection of Giardia lamblia4,7–9.\n\nIn this study we tested two commercially available EIA kits that detect antigens of Giardia lamblia in stool samples (Ridascreen Giardia; manufactured by r-biopharm, and Serazym Giardia; manufactured by Seramun Diagnostica). The results were compared with those of a conventional microscopic examination (CME), immunofluorescence microscopy (IFM) and with polymerase chain reaction (PCR).\n\n\nMaterials and methods\n\nInstitutional ethical approval was obtained before the study. After written informed consent was obtained, stool specimens were collected from patients who presented for diarrhoea plus various other complaints. All patients were German nationals returning from vacations abroad. All stool samples were investigated for ova and parasites by direct microscopy (iron-hematoxylin stain) and the SAF (sodium-acetate-acetic acid-formaline)/ethyl acetate-concentration technique10. Every slide was read for at least 10 min by two experienced microscopists (conventional microscopy examination, CME) before being considered negative if no ova or parasites could be detected within this time. A part of every fresh stool sample was stored immediately at –20°C and tested later by EIA, according to the relevant manufacturer´s instructions, by one lab technician who was not aware of the results of the microscopy or polymerase chain reaction (PCR) findings. PCR was performed at the Bernhard Nocht Institute for Tropical Medicine, Hamburg (Professor Egbert Tannich) by investigators who were blinded to the results of the EIA and microscopy tests. If different results between the EIA, PCR or microscopy (CME) were obtained, immunofluorescence microscopy was used for additional confirmation of true positive results. EIA results were compared with those obtained by conventional microscopic examination (CME), immunofluorescence microscopy (IFM) and by PCR. The samples that had a positive result in CME and/or immunoflorescence microscopy were considered true positives. The sensitivity, specificity and the positive predictive value (PPV) of both EIAs were calculated. These values were also calculated for PCR against microscopy, and also for both EIAs against PCR.\n\n\nResults\n\nThe 88 specimens were examined by CME, IFM, PCR and two EIAs. Giardia lamblia was detected in 44 (54.5%) of stool samples by both, CME and IFM. Comparing the Ridascreen Giardia EIA against microscopy, 35 stool samples were positive and 40 were negative with both methods (Table 1). Sensitivity of the test against CME and IFM was 72.9%, whilst the specificity was 100%. With the Serazym Giardia test, sensitivity was calculated at 93.8% (45 samples positive), with a specificity of 100%. PCR produced several false negative results against CME and IFM, which are considered the gold standard tests. The sensitivity of PCR was 85.4% (41 samples correctly positive), whilst the specificity was 100% (Table 2). With PCR used as a background for the EIAs, results became more divergent (Table 3): Ridascreen Giardia performed with a sensitivity of 73.2% and a specificity of 83.4%, while Serazym Giardia showed a sensitivity of 95.1%, and a specificity of 87.2%.\n\nSensitivity Ridascreen Giardia 72.9%.\n\nSpecificity Ridascreen Giardia 100%.\n\nPositive predictive value Ridascreen Giardia 100%.\n\nNegative predictive value Ridascreen Giardia 75.5%.\n\nSensitivity Serazym Giardia 93.8%.\n\nSpecificity Serazym Giardia 100%.\n\nPositive predictive value Serazym Giardia 100%.\n\nNegative predictive value Serazym Giardia 93%.\n\nSensitivity PCR 85.4%.\n\nSpecificity PCR 100%.\n\nPositive predictive value PCR 100%.\n\nNegative predictive value PCR 85.1%.\n\nSensitivity Ridascreen Giardia 73.2%.\n\nSpecificity Ridascreen Giardia 83.4%.\n\nPositive predictive value Ridascreen Giardia 85.7%.\n\nNegative predictive value Ridascreen Giardia 79.2%.\n\nSensitivity Serazym Giardia 95.1%.\n\nSpecificity Serazym Giardia 87.2%.\n\nPositive predictive value Serazym Giardia 86.7%.\n\nNegative predictive value Serazym Giardia 95.4%.\n\n\nDiscussion\n\nIn order to find simple, inexpensive and reliable diagnostic techniques for detecting intestinal infections with Giardia lamblia, several EIA test kits have been developed and tested in various studies4,7–9,11–17. In this study, we evaluated the performance of two commercially available EIA kits for detecting Giardia lamblia antigens. We tested 88 specimens and compared the results of EIA to the results of stool microscopy (CME and IFM) of the same sample. When microscopy was used as the reference standard, the EIA kits both showed a specificity of 100%, while sensitivity varied considerably (Table 1). Ridascreen Giardia showed 72.9%, whilst Serazym Giardia performed considerably better with a sensitivity of 93.8%. PCR detection of Giardia DNA was also compared to microscopy (Table 2). Interestingly, PCR showed a lower sensitivity than one of the EIAs (85.4% vs. 93.8% Serazym Giardia). This may be explained by the high concentration of DNase in stool samples that traditionally hamper PCR diagnostics from this material18. It is important to notice though that the use of PCR as gold standard for the evaluation of EIAs in this setting would have produced false low specificity results for both tests (Table 3: 83.4% for Ridascreen, and 87.2% for Serazym, as opposed to the actual 100% specificities in both tests against CME and IFM).\n\nAs mentioned earlier, microscopic examination of one single stool specimen has a low sensitivity7,9. It has been demonstrated that antigens of Giardia lamblia can be detected by EIA even in the absence of intact organisms (cysts or trophozoites). This may result in a greater sensitivity of EIA-tests compared with microscopy9 and therefore provide low specificity results when only one CME is used as a reference standard. In a study conducted by Aldeen et al.16, nine different immunoassay kits for the detection of Giardia lamblia were evaluated: the sensitivity ranged from 93% to 100% and the specificity in all EIAs was above 99%. The authors suggest that the high specificity results are due to the examination of up to seven individual slide preparations on an initially negative microscopic finding. In conclusion, we found that the EIAs evaluated are highly sensitive and specific for detection of Giardia lamblia. In our setting, one of the two EIAs proved to be more reliable than PCR. Antigen detection by EIA methods certainly has developed into a powerful additional method for increasing the effectiveness of stool diagnostics. Results of this study also demonstrate that considerable differences in sensitivity might occur between commercial tests. Thus, careful selection of appropriate test kits seems mandatory. For screening purposes, the antigen tests remain additional tools: there is currently no replacement for microscopical examination of stool specimens, since other potential pathogens could otherwise escape detection.",
"appendix": "Author contributions\n\nTJ designed the study, collected part of the samples, did part of the testing, and wrote part of the paper. SN collected part of the samples, did part of the testing and wrote part of the paper. Both authors approved the final manuscript for publication.\n\n\nCompeting interests\n\nNo competing interests have been disclosed.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nSekisui Virotech GmbH provided the Serazym Giardia assays as well as financial support for purchase of the Ridascreen Giardia assays and for PCR.\n\n\nReferences\n\nAli SA, Hill DR: Giardia intestinalis. Curr Opin Infect Dis. 2003; 16: 453–60.\n\nPawlowski SW, Warren CA, Guerrant R: Diagnosis and treatment of acute or persistent diarrhea. Gastroenterology. 2009; 136: 1874–1886.\n\nHuang DB, White AC: An updated review on Cryptosporidium and Giardia. Gastroenterol Clin North Am. 2006; 35: 291–314.\n\nJelinek T, Peyerl G, Löscher T, et al:Giardiasis in travellers: evaluation of an antigen-capture ELISA for the detection of Giardia lamblia-antigen in stool. Z Gastroenterol. 1996; 34: 237–240.\n\nWahnschaffe U, Ignatius R, Loddenkemper C, et al:Diagnostic value of endoscopy for the diagnosis of giardiasis and other intestinal diseases in patients with persistent diarrhea from tropical or subtropical areas. Scand J Gastroenterol. 2007; 42: 391–396.\n\nTroeger H, Epple HJ, Schneider T, et al:Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum. Gut. 2007; 56: 328–335.\n\nWeitzel T, Dittrich S, Möhl I, et al:Evaluation of seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples. Clin Microbiol Infect. 2006; 12: 656–659.\n\nSchunk M, Jelinek T, Wetzel K, et al:Detection of Giardia lamblia and Entamoeba histolytica in stool samples by two enzyme immunoassays. Eur J Clin Microbiol Infect Dis. 2001; 20: 389–391.\n\nAddis DG, Mathews HM, Stewart JM, et al:Evaluation of a commercially available enzyme-linked immunosorbent assay for Giardia lamblia antigen in stool. J Clini Microbiol. 1991; 29: 1137–1142.\n\nAnonymous. Instruction Sheet DYS004. Parasitology. Fixatives, Reagents and Stains. http://www.diasys.com/PDF/DYS004–StainsReagentsFix.pdf (last accessed 21 JAN 2013).\n\nElkadi IA, Smith DH, Hommel M: Early diagnosis of giardiasis by feacal antigen detection using capture EIA in a cohort of children in the United Arab Emirates. Trans R Soc Trop Med Hyg. 1992; 86: 520–521.\n\nRosenblatt JE, Sloan LM, Schneider SK: Evaluation of an enzyme-linked immunosorbent assay for the detection of Giardia lamblia in stool specimens. Diagn Microbiol Infect Dis. 1993; 16: 337–341.\n\nPillai DR, Keystone JS, Sheppard DC, et al:Entamoea histolytica and the diagnostic methods in a setting of nonendemicity. Clin Infect Dis. 1999; 29: 1315–1318.\n\nRosoff JD, Stibbs HH: Physical and chemical characterisation of a Giardia lamblia-specific antigen useful in the coprodiagnostic of giardiasis. J Clini Microbiol. 1986; 24: 1079–83.\n\nGoldin AJ, Hall A, Sarker RN, et al:Diagnosis of Giardia lamblia infection in Bangladeshi infants: feacal antigen capture EIA. Trans R Soc Trop Med Hyg. 1993; 87: 428–432.\n\nAldeen WE, Carroll K, Robison A, et al:Comparison of nine commercially available enzyme-linked immunosorbent assays for detection of Giardia lamblia in fecal specimens. J Clini Microbiol. 1998; 36: 1338–1340.\n\nBenzeguir AK, Aust KA: Evaluation of an enzyme-immunoassay test kit for diagnosing infections with Entamoeba histolytica. Arch Med Res. 1997; 28: 276–278.\n\nNantavisai K, Mungthin M, Tan-ariya P, et al:Evaluation of the sensitivities of DNA extraction and PCR methods for detection of Giardia duodenalis in stool specimens. J Clin Microbiol. 2007; 45: 581–583."
}
|
[
{
"id": "771",
"date": "14 Feb 2013",
"name": "Rogelio López-Vélez",
"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": "999",
"date": "12 Jun 2013",
"name": "Selwyn Lowe",
"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\nAbstract: The PCR is mentioned, but not the results compared to the standard.Article content:Materials and methods: \"Every slide was read for at least 10 min by two experienced microscopists …\" Was this done in parallel for 2 different slides or sequentially for 1 slide? If sequentially, microscopy was done for 20 min and this could influence the results. If different results between the EIA, PCR or microscopy (CME) were obtained, was immunofluorescence microscopy used for additional confirmation of true positive results? This is not in line with the Results: The 88 specimens were examined by CME, IFM, PCR and two EIA’s.Please clarify the difference between what is stated in the Materials and Methods section and in the Results section. Where all 5 assays performed on all 88 stool samples?Results: It is not quite clear how the different assays compare to each other. Maybe a 5 by 5 table with only the positive results would give a better insight?",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-39
|
https://f1000research.com/articles/2-38/v1
|
11 Feb 13
|
{
"type": "Case Report",
"title": "Presentation of an umbilical cord cyst with a surprising jet: a case report of a patent urachus",
"authors": [
"John Svigos",
"Sanjeev Khurana",
"Christopher Munt",
"Sanjay Sinhal",
"Julie Bernardo",
"Sanjeev Khurana",
"Christopher Munt",
"Sanjay Sinhal",
"Julie Bernardo"
],
"abstract": "We report a baby with an unusual true umbilical cord cyst detected at 12 weeks gestation which as the pregnancy progressed became increasingly difficult to distinguish from a pseudocyst of the umbilical cord. Concern of the possibility of cord compression/cord accident led to an elective caesarean section being performed at 35+ week’s gestation with delivery of a healthy female infant weighing 2170g. At birth the cyst ruptured and the resultant thickened elongated cord was clamped accordingly. After the cord clamp fell off at 5 days post delivery an elongated umbilical stump was left behind from which a stream of urine surprisingly jetted out from the umbilicus each time the baby cried. A patent urachus was confirmed on ultrasound and the umbilical jet of urine resolved at 4 weeks post delivery after treatment of an Escherichia coli urinary tract infection. At 11 weeks post delivery a laparoscopic excision of the urachus was successfully performed. The baby, now 18 months of age, continues to thrive without incident.",
"keywords": [
"A persistent urachus is a result of failure of involution at 10–12 weeks gestation of the allantois which communicates from the dome of the bladder to the umbilicus. Embryologically",
"the allantois is an endodermal diverticulum which becomes the urogenital sinus with the cranial portion developing as the bladder. Persistence of the urachus may be partial resulting in an urachal cyst",
"diverticulum or sinus",
"or it may be completely patent allowing communication with the bladder1. Just over 100 cases in the neonatal period have been documented after the first report in the 16th Century2."
],
"content": "Introduction\n\nA persistent urachus is a result of failure of involution at 10–12 weeks gestation of the allantois which communicates from the dome of the bladder to the umbilicus. Embryologically, the allantois is an endodermal diverticulum which becomes the urogenital sinus with the cranial portion developing as the bladder. Persistence of the urachus may be partial resulting in an urachal cyst, diverticulum or sinus, or it may be completely patent allowing communication with the bladder1. Just over 100 cases in the neonatal period have been documented after the first report in the 16th Century2.\n\n\nCase report\n\nA 36 year old Caucasian woman with a 4 year history of infertility presented at 9 weeks gestation for antenatal care following a successful single embryo transfer after intra-cytoplasmic sperm injection (ICSI). First Trimester screening at 12 weeks gestation demonstrated a trisomy 21 risk of 1:2760 and a trisomy 18 risk of 1:4060. However, detailed ultrasonic examination of the fetus revealed a hypoechoic area on the anterior abdominal wall which was thought possibly to be fluid distending the urethra or an extra-abdominal mass such as an omphalocoele. At 16 weeks gestation a repeat ultrasound examination identified an umbilical cyst, of dimensions 1.7 cm x 1.6 cm x 1.8 cm with vessels coursing around it with no gut present within and a normal anterior abdominal and bladder wall. The uncertainty of the type of cyst and possible reduction in the fetal dimensions required trisomy 18 to be excluded and an amniocentesis revealed a normal XX karyotype. The umbilical cyst increased progressively with advancing gestation, increasing in dimension, to 4.9 x 4.5 x 4.7 cm at 35 weeks gestation (See Figure 1).\n\nOn the ultrasound, the cord developed homogeneous echogenicity, which was thought to be due to increased Wharton’s jelly, seen more commonly with pseudocysts of the cord; or due to oedema which was a more sinister sign of possible cord compression/cord accident. In view of the latter, after a course of preoperative maternal cortico-steroids to enhance fetal lung maturity, the patient underwent an elective caesarean section under a combined epidural/spinal anaesthetic block, with delivery of a healthy female infant. Unfortunately, during the process of the delivery the cord cyst ruptured making accurate diagnosis impossible. However the cord was thickened with Wharton’s jelly and was elongated with an umbilical stump with overlying skin up to 2 cm in length. The cord was divided in the standard fashion and a normal placenta was removed.\n\nOn the 5th day post-delivery, the cord clamp fell off and each time the baby cried a stream of urine jetted out from the umbilicus (see Figure 2). This phenomenon finally ceased 4 weeks after delivery, following successful treatment of a urinary tract infection. A patent urachus was confirmed on the ultrasound. The very prominent umbilical stump created the appearance of a ‘pseudophallus’ due to bulging from increased intra-abdominal and bladder pressure after each normal micturition.\n\nA successful laparoscopic excision of the urachus was performed 11 weeks after delivery. At surgery, the entire dome of the bladder was seen to be in continuation with the umbilicus rather than the more commonly seen urachal tract. The ureteral orifices in the bladder were not affected by the closure, whilst a micturating cysto-urethrogram performed post-operatively failed to show any evidence of uretero-vesical reflux. The baby was discharged home 36 hours after surgery. The baby had no further urinary tract infections post-operatively and follow-up blood pressure, growth and development were normal at 18 months of age.\n\n\nDiscussion\n\nThe prevalence of umbilical cord cysts detected by ultrasound in the first trimester is of the order of 0.4–3.4%, with a patent urachus being a rare anomaly with an incidence of 1–2.5 per 100,0003. Whilst umbilical cord cysts are classified as true cysts or as pseudocysts differentiation from each other is often difficult antenatally and a detailed morphology scan and karyotype is recommended as both types of umbilical cysts may be associated with omphalocoele which has an increased incidence of aneuploidy and other fetal anomalies (25–85%)4. True cysts are less common, ranging in size between 4 and 60 mm, are located towards the anterior abdominal wall of the fetus, split the umbilical vessels, contain fluid and if present in the second and third trimesters, are associated with an omphalocoele or patent urachus4,5.\n\nPseudo cysts are more common, are usually smaller, and may be located anywhere along the length of the umbilical cord. The umbilical vessels are pushed to one side as a result of local oedema and liquefaction of Wharton’s jelly and may be associated with omphalocoele, hydrops and aneuploidy (trisomy 18)4,5. Post-delivery the differential diagnosis of an umbilical cord cyst is between an umbilical pseudo-cyst and an allantoic cyst with a patent urachus which can be distinguished by ultrasound. Ultrasound shows the channel communicating from the umbilicus to the bladder. Bladder outlet obstruction can be excluded by a micturating cysto-urethrogram preoperatively or by an intra-operative cystoscopy. The contemporary treatment of a patent urachus is by laparoscopic surgical excision6 whilst other urinary tract abnormalities (including urachal cysts, fistulae and diverticulae) are investigated and treated appropriately7. Post-operatively these babies are given antibiotic prophylaxis until a normal ultrasonogram of the urinary tract is demonstrated.\n\n\nConclusion\n\nAntenatal ultrasound detection of an umbilical cyst, particularly if located close to the anterior abdominal wall of the fetus should stimulate a search for a patent urachus post-natally. Laparoscopic surgical correction is the treatment of choice for a baby with a patent urachus. Reduced post operative morbidity and length of hospital stay are important advantages in comparison to the open surgical approach with both methods being equally successful in closing a patent urachus.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the mother of the neonate.",
"appendix": "Author contributions\n\n\n\nJS provided the obstetric care for the patient and has written the paper with input from the other authors. SK performed the laparoscopic surgery for the neonate. CM provided the initial neonatal care at Ashford Hospital where the baby was delivered. SS and JB provided neonatal care post surgery.\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\nGupta N, Corbett H, Ismail R, et al.: Allantoic cyst – an unusual umbilical cord swelling. J Surg Case Rep. 2011; 4: 5.Publisher Full Text | Free Full Text\n\nNix JT, Menville JG, Albert M, et al.: Congenital patent urachus. J Urol. 1958; 79(2): 264–273.PubMed Abstract\n\nBunch PT, Kline-Fath BM, Imhoff SC, et al.: Allantoic cyst: a prenatal clue due to patent urachus. Pediatr Radiol. 2006; 36(10): 1090–1095.PubMed Abstract | Publisher Full Text\n\nZangen R, Boldes R, Yaffe H, et al.: Umbilical cord cysts in the second and third trimesters: significance and prenatal approach. Ultrasound Obstet Gynecol. 2010; 36(3): 296–301.PubMed Abstract | Publisher Full Text\n\nSepulveda W: Opinion: Beware of the umbilical cord ‘cyst’. Ultrasound Obstet Gynecol. 2003; 21(3): 213–4.PubMed Abstract | Publisher Full Text\n\nStone NN, Garden RJ, Weber H: Laparoscopic excision of a urachal cyst. Urology. 1995; 45(1): 161–164.PubMed Abstract\n\nCutting CW, Hindley RG, Poulsen J: Laparoscopic management of complicated urachal remnants. BJU Int. 2005; 96(9): 1417–1421.PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "800",
"date": "27 Feb 2013",
"name": "George Condous",
"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 think this case presentation is well written and appropriately referenced. I have one question for the authors:In retrospect, do the authors believe that early delivery at 35+ weeks gestation by LSCS would have been justifiable if they had have made the diagnosis antenatally? And further to this, is there an increase in perinatal mortality if these babies with a patent urachus are allowed to delivery at term? I think the Conclusion should also be changed to read: 'Antenatal ultrasound detection of an umbilical cyst, particularly if located close to the anterior abdominal wall of the fetus should alert the clinician to the possibility of an underlying patent urachus. Once diagnosed post delivery, the laparoscopic approach is the preferred surgical route.'",
"responses": []
},
{
"id": "918",
"date": "02 May 2013",
"name": "Henry Murray",
"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 case report with appropriate referencing and balanced discussion. I would have liked to see in the conclusion a comment about the lack of evidence on antenatal scanning of the patent urachus. For the reader, this would further highlight the need for the postnatal assessment of a baby with the antenatal diagnosis of cord/umbilical cyst(s), even if the antenatal scan suggested normal bladder anatomy.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-38
|
https://f1000research.com/articles/2-37/v1
|
08 Feb 13
|
{
"type": "Research Article",
"title": "Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca ",
"authors": [
"Teresa Docimo",
"Gregor W Schmidt",
"Katrin Luck",
"Sven K Delaney",
"John C D'Auria",
"Teresa Docimo",
"Gregor W Schmidt",
"Katrin Luck",
"Sven K Delaney"
],
"abstract": "Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis.Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca (Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.",
"keywords": [
"qRT-PCR",
"qPCR",
"E. coca",
"Reference genes",
"Normalization"
],
"content": "Introduction\n\nErythroxylum coca has been cultivated by humans for more than 8000 years and has been selected for high-level production of cocaine, a pharmacologically active tropane alkaloid. Cocaine and other tropane alkaloids such as atropine and scopolamine act on the nervous system, and their activity is largely due to their common chemical backbone (the tropane nucleus)1. Despite the socioeconomic importance of cocaine and other tropane alkaloids, the molecular basis for the biosynthesis of the tropane nucleus remains unknown. E. coca is emerging as a model for the investigation of tropane alkaloid synthesis2–4, and shows high-level, localized tropane alkaloid production and storage in its leaf tissue3,4.\n\nWe have performed metabolic and enzymatic studies to identify the molecular and biochemical basis of tropane alkaloid biosynthesis in E. coca, and have developed a number of genomic tools such as expressed sequence tag (EST) libraries and 454 sequence databases2–4. Quantitative real-time reverse-transcription PCR (qRT-PCR) would be a further source of information on candidate tropane alkaloid biosynthesis genes in the different tissues of the coca plant.\n\nqRT-PCR is widely used to quantify and compare levels of gene transcription5. Variables such as RNA quality and the efficiencies of reverse transcription and PCR may compromise the accuracy and reliability of qRT-PCR, and so results are typically ‘normalized’ by comparison with one or more internal reference genes6. The internal reference genes must be stably expressed, and the most stable reference genes vary widely in different species, tissues and sets of experimental conditions. Therefore, the identification of stable reference genes is a crucial step in the design of qRT-PCR experiments.\n\nTraditionally, ‘Housekeeping’ genes such as actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ubiquitin were used for data normalization7,8. These genes were widely assumed to have a uniform level of expression due to their involvement in fundamental cellular processes. However, evaluation of the expression stability of classical housekeeping genes in many species including Arabidopsis thaliana, Oryza sativa, Zea mays and Linum usitatissimus9–12 has revealed unstable expression of these genes under a range of experimental conditions. In addition, several novel reference genes have been shown to be more stably expressed than classical housekeeping genes13. Hence there is a need for systematic validation of internal reference genes in each organism and experiment9,14.\n\nThe stability of candidate internal reference genes may be assessed using a number of models, including geNorm15, NormFinder16 and BestKeeper17. These models differ significantly in their assumptions, and so candidate genes are often assessed with several of these algorithms18. geNorm iteratively calculates an expression stability value (M) for each candidate gene. This is based on the mean pairwise variation between the gene and the other candidate genes across all samples. Genes with lower M values are more stably expressed, and less stable genes (with higher M) are progressively excluded from the analysis. The optimal number of reference genes for qRT-PCR normalization may also be determined by identifying the smallest number of genes needed to minimize mean variation. By contrast, NormFinder estimates the standard deviation for each gene relative to the global expression of all genes included in the analysis, and genes with lower standard deviations are considered better reference genes. BestKeeper uses a third approach involving the calculation of a stability index (the ‘BestKeeper index’ or BKI), which is assumed to represent the highest level of stability because it includes all genes across all samples. The stability of each reference gene is assessed by its correlation with the BKI, with a high correlation indicating a more stable reference gene15–17.\n\nIn this study, we evaluate the stability of nine candidate reference genes (Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4 and Pp2aa3) in a variety of E. coca tissues (four developmental leaf stages, stems, roots and flowers). We then identify the most stable internal reference genes using the geNorm, NormFinder and BestKeeper algorithms and present guidelines for transcript analysis in different tissues of E. coca by qRT-PCR.\n\n\nMaterials and methods\n\nErythroxylum coca was obtained from the Bonn Botanical Garden. Plants were grown at 22°C under a photoperiod of 12 h light/12 h dark with relative humidities of 65% and 70% for light and dark conditions respectively (and fertilized once a week with Ferty 3 (15-10-15) and Wuxal Top N (Planta, Regenstauf, Germany).\n\nThe organs used for RNA extraction and qRT-PCR analysis were obtained from four-month old E. coca plants grown from rooted cuttings. Leaves in four developmental stages, roots, stems and flowers were analysed. The leaf developmental stages were: leaf buds; young expanding leaves in a rolled state (Stage 1); young expanded (unrolled) leaves (Stage 2); and fully mature leaves (Stage 3) (see Figure 1).\n\nLeaf Stage I (L1) young rolled leaves, Leaf Stage II (L2) young expanded leaves, Leaf Stage III (L3) fully mature leaves.\n\nTotal RNA was extracted from 100 mg of fresh plant tissue using a total RNA extraction kit (Invitek, Berlin, Germany). Genomic DNA was removed by treatment with RNAse-free DNAse I (Qiagen, Hilden, Germany). RNA quality was assessed on an Agilent Bioanalyzer 2100 using a RNA 6000 Nano Kit (Agilent, Böblingen, Germany). RNA concentration was determined using a NanoDrop 2000 c spectrophotometer (NanoDrop Technologies, Wilmington, USA). cDNA was synthesized using a Super Script III First Strand Kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. In brief, random hexamer primers and deoxyribonucleoside5’-triphosphates (dNTPs) were added to 5 µg total RNA and the mixture was incubated at 65°C for 5 min before brief chilling on ice. The first strand was then reverse transcribed by adding First Strand Buffer, 20 mM dithiothreitol and Super Script III reverse transcriptase to a final volume of 20 µl and incubating the mixture at 42°C for 1h. The resulting cDNA was diluted 1:20 (vol:vol) with deionized water and stored at -20°C.\n\nCandidate reference genes were selected from an E. coca 454 sequence library2 based on their homology to previously reported reference genes in A. thaliana9. Nine candidate reference genes with an E-value higher than 2e-72 were identified by BlastN comparison as orthologues to Arabidopsis genes: Expressed protein (Ec6409), Expressed protein (Ec10131), Clathrin adaptor complex subunit (Ec11142), Actin (ACT), Adenine phosphoribosyl transferase 2 (APT2), Elongation factor 1 alpha (EF1α), Protein tyrosine phosphatase 1B (TPB1), Peroxin 4 (Pex4) and Pp2aa3-like protein. Primers for qRT-PCR were designed using Primer Express 3.0 (Applied Biosystems) and their sequences are shown in Table 1.\n\nGenBank accession numbers are given for each gene used in this study. The orthologous locus in A. thaliana is referred to by its AGI (Arabidopsis Genome Intitiative) designation. Similarity values are represented by E-values for the pairwise comparison of the coca gene with its Arabidopsis ortholog. PCR amplification efficiencies and the regression coefficients for their standard curves are reported for each primer pair.\n\nAll primer pairs were validated prior to their use in gene expression analysis. PCR reactions were performed with each primer pair and the products were visualised by gel electrophoresis to confirm the presence of a single PCR product of the expected size. The sequence specificity of the PCR products was also verified by sequencing.\n\nAll PCR reactions were performed on a Stratagene Mx3000P (La Jolla, USA). Each reaction contained 12.5 µl Brilliant Sybr Green (Agilent/Böblingen, Germany), 0.375 µl Rox, 0.4 µM primers and 1 µl cDNA in a final volume of 20 µl. All samples were run in triplicate. The thermocycling conditions were denaturation at 95°C for 10 min; followed by 40 cycles of denaturation (95°C, 15 s) and annealing/extension (60°C, 1 min). A melting curve analysis protocol was performed after completion of the PCR reaction to confirm the absence of multiple amplicons and/or primer dimers. A no template control (NTC) was included to ensure the absence of contamination. In addition, the presence of genomic DNA contamination was excluded by performing reactions without reverse transcriptase. PCR efficiency was determined using a standard curve based on between five and seven different four-fold dilutions of a cDNA cloned amplicon.\n\nCycle threshold (Ct) values were exported from the MxPro software (Stratagene) to Microsoft Excel using the qBASE v1.3.5 macro19. PCR efficiencies and regression coefficients were calculated in qBASE and are reported in Table 1. The expression stability of the nine reference genes in E. coca tissues was evaluated with geNorm v3.515, NormFinder16 and Bestkeeeper v117. Relative expression quantities were exported from qBASE and analyzed in Microsoft Excel using the geNorm v3.5 and NormFinder macros. For analysis using the BestKeeper macro, Ct values from the MxPro Software and PCR efficiencies calculated by qBASE were utilized.\n\n\nResults\n\nA similarity search (BlastN) between previously identified reference genes from A. thaliana9 and an E. coca 454 sequence library2 was conducted to identify orthologous sequences. Nine E. coca genes with high similarity to A. thaliana were selected and PCR primers targeting these sequences were developed (see Table 1). To confirm the specificity of the primers and identity of the amplicons, RT-PCR was performed on cDNA from four developmental leaf stages, stems, roots and flowers. Primer specificity was investigated by electrophoresis and a single amplicon of the expected size was obtained for each primer pair (Supplementary Figure 1). Sequence analysis of ten cloned amplicons revealed that the amplified fragments were identical to the targeted sequences in the 454 sequence database. All primer pairs achieved amplification in fewer than 35 cycles in all samples, demonstrating that all of the candidate reference genes are expressed at experimentally useful levels. The ΔCt between samples and no template controls (NTCs) was always greater than five cycles, showing that contamination during the setup of the experiment was negligible20. All RNA samples were tested for contamination with genomic DNA by performing qPCR analysis on negative control reverse transcriptase reactions in which the reverse transcriptase was omitted. No amplification product could be detected in these control reactions.\n\nThe gene-specific amplification efficiency was calculated by linear regression analysis of the standard curve and ranged between 79% (Ec10131) and 97% (Actin). The coefficient of correlation (r2) of the linear regression analysis was always greater than 0.986 as shown in Table 1, indicating a linear relationship between Ct values and log-transformed transcript quantities in the range of the standard curve.\n\nTo ensure that the primer pairs are specific for the desired sequence in all samples and do not target homologous transcripts in some sample subsets, a melting curve analysis of each sample was performed after PCR amplification (Supplementary Figure 2). A single peak in the melting curve specific for each primer pair was obtained for all samples, and no peak could be observed in the melting curves of the control reactions (NTC and negative control reverse transcription reactions).\n\nThe expression stability of the candidate genes were evaluated with the geNorm, NormFinder and BestKeeper algorithms (Table 2). Ct values were transformed to relative quantities using qBASE prior to analysis with geNorm and NormFinder, while Ct values and PCR efficiencies were used in BestKeeper. The cDNA samples were considered as either a single, diverse set derived from all organ samples; or as two subsets derived from leaf buds and leaves (leaf buds, Stage 1, Stage 2 and Stage 3 leaves) or mature organs (Stage 3 leaves, flowers, roots and stems).\n\n*M indicates stability values listed from most stable to least stable.\n\ngeNorm calculates the average expression stability value (M) for each candidate gene on the basis of the average pair-wise variation between all genes analyzed. geNorm analysis indicated that Ec10131 and Ec6409 are the most stable candidate reference genes across all of the E. coca tissues tested (Table 2). In the leaf bud/leaf sample subset, Ec10131, TPB1 and Ec6409 were ranked as the three most stable genes (in that order) (Supplementary Table 1), while in the mature organ subset Ec10131 and Ec6409 were again ranked as the most stable. In contrast, Pex4 and APT2 were consistently ranked as the least stable in all sample subsets (Table 2 and Supplementary Table 1 and Supplementary Table 2). The ‘housekeeping’ genes Actin and EF1α were relatively unstable and were ranked at positions six and seven (respectively) in all sample sets.\n\nThe optimal number of reference genes required for accurate normalization in the respective sample sets (all samples, leaf bud/leaf and mature tissues) was determined by calculating the mean variation in each normalisation factor (V) and then observing the effect of iterative addition of the next most stable reference gene (Vn/Vn+1) (as detailed in Vandesompele et al. 200215). In each case, the two most stable reference genes were sufficient for accurate normalization, since inclusion of a third gene had little impact on the calculation of the normalization factor (Vn/Vn+1 below 0.15).\n\nBestKeeper ranks gene stability by calculating the correlation coefficient (r) between the expression of each candidate gene and the BestKeeper index (BKI; calculated using all genes across all samples). Across all of the samples tested, BestKeeper indicated that Actin (r = 0.784) and Ec6409 (r = 0.768) were the most stable, while Ec10131 was ranked as the least stable (r = 0.638). In the leaf bud/leaf sample subset, Actin (r = 0.869) and APT2 (r = 0.868) had the highest correlation with the BKI, and Ec10131 again showed the lowest correlation (r = 0.385). In the mature organs sample subset, Pex4 and APT2 were strongly correlated with the BestKeeper index (r = 0.767 and r = 0.724, respectively), whereas Ec10131 showed low correlation (r = 0.309) (Supplementary Table 1 and Supplementary Table 2).\n\nTo provide a further ranking of gene stability, the results were also evaluated with NormFinder, in which candidate reference genes are ranked according the variance of their expression relative to the expression variance within a defined group of samples16. Pp2aa3 was the most stably expressed gene with the lowest expression variance (stability value of 0.291), followed by Ec6409 and Ec11142, when all samples were included in the calculation. When the leaf bud/leaf and mature organ subsets of samples were considered, the rankings varied considerably (Supplementary Table 1 and Supplementary Table 2). Actin, APT2 and Pex4 were always ranked as the seventh, eighth and ninth most stable reference genes (respectively), but there was no consistent order of ranking for the other reference genes. The NormFinder rankings were also distinct from the geNorm rankings, although both algorithms identified Actin, APT2 and Pex4 as having the least stable expression profiles.\n\n\nDiscussion\n\nReal time RT-PCR has become a central technique for the evaluation of quantitative changes in gene expression21–24. Reliable and accurate expression data can only be obtained by normalization with stably expressed reference genes. Normalization is an essential prerequisite for the correct measurement of gene expression changes in different plant tissues, organs, developmental stages or treatments of a given plant species and is highly influenced by the choice of reference genes. Traditional reference genes (e.g. actin and ubiquitin) are useful as stable reference genes in some experiments9,25, but their expression is often highly variable26–28, and is often inferior to the stability of less-commonly used genes8. Therefore it is important to assess the expression stability of several candidate reference genes before gene expression studies are performed. Several models including geNorm, NormFinder and BestKeeper have been developed to rank candidate reference genes on the basis of their expression stability. These methods often vary in their stability rankings18,25 and so expression data is commonly analysed using several approaches.\n\nIn this study, we report the identification and validation of nine candidate reference genes in E. coca (Ec6409, Ec10131, Ec11142 Actin, APT2, EF1α, TPB1, Pex4 and Pp2aa3). These genes were identified by analysing a 454 E. coca sequence library for sequences with homology to the top 100 reference genes of Arabidopsis9, on the assumption that homologous genes are likely to have similar expression patterns. Primer pairs specifically targeting the E. coca transcripts were successfully developed and evaluated: all primer pairs produced only the expected amplicon and were highly efficient (Table 1 and Supplementary Figure 1). The relative stabilities of the candidate reference genes were then assessed using geNorm, BestKeeper and NormFinder (Table 2 and Supplementary Table 1 and Supplementary Table 2).\n\ngeNorm produced similar results in all sample sets. Ec6409 and Ec10131 were always identified as two of the three most stably expressed reference genes (although Ec10131 and TPB1 were most stable in the leaf bud/leaf sample subset), and Actin, APT2 and Pex4 were always identified as the least stable. geNorm may identify co-regulated genes as stable reference genes16. However, exclusion of either Ec10131 or Ec6409 did not change the gene rankings (not shown), suggesting that their high ranking is not attributable to co-regulation.\n\nBestKeeper yielded very different rankings to geNorm, and these varied according to the sample subset. The inconsistent results with BestKeeper may be explained by several features of the BestKeeper algorithm. Calculation of the BestKeeper index excludes genes with a standard deviation of more than one Ct value, which results in the exclusion of different genes in different sample sets17. Extensive variation in Ct values is to be expected in a non-normalized data set, and so the algorithm may not be able to effectively distinguish between stable and unstable reference genes. In our experiments, the candidate E. coca reference genes showed very similar correlations with the BestKeeper index, suggesting that the algorithm could not distinguish between the genes to produce useful stability rankings. NormFinder produced a third ranking of gene stability that differed from both BestKeeper and geNorm. Pp2aa3 and Ec6409 were ranked as the most stably expressed genes when all samples were considered (Table 2). geNorm also identified Ec6409 as one of the most stable genes in the entire sample set. However, only Pp2aa3 was consistently ranked by Normfinder, geNorm and BestKeeper as one of the most stable genes in the leaf bud/leaf and mature organs sample sets, and there was no consistency between the algorithms in the order of ranking for the most stable genes (Table 2). The ranking of the least stable genes was more consistent: NormFinder identified Actin, APT2 and Pex4 as the least stable genes in all of the sample sets, and geNorm ranked these genes in the same order.\n\nThe NormFinder, BestKeeper and geNorm models have been shown to produce conflicting stability rankings in many studies18,29. The rankings produced by one or more of the models may be combined to produce a hybrid ranking18, but this complicates the analysis by merging models with very different underlying assumptions. Hence, we favour using a single model when possible.\n\ngeNorm produced a consistent gene ranking across all of our samples, and provides a clear rationale for determining the minimum number of genes required for accurate normalization. We therefore recommend the use of Ec10131 and Ec6409 as internal reference genes for most E. coca sample sets. If leaves and leaf buds are the primary organs of interest, then we recommend the use of Ec10131 and TPB1. These results provide a foundation for qRT-PCR studies in E. coca, and will further its development as a model of tropane alkaloid biosynthesis.",
"appendix": "Author contributions\n\n\n\nTD, GWS and JCD designed the research; TD, GWS, KL, and JCD performed the research; TD, GWS, and JCD analyzed the data; TD, GWS, SKD and JCD wrote the paper. All authors have approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Max Planck Society and an Alexander von Humboldt Foundation postdoctoral fellowship to JCD.\n\n\nSupplementary figures\n\nThe lane marked M represents a 1 Kb ladder (Invitrogen, California) used for size comparisons.\n\nNTC indicates: no template control.\n\n\nReferences\n\nLounasmaa M, Tamminen T: The tropane alkaloids. The Alkaloids, ed Cordell GA (Academic, New York) 1993; 44: 1–114.\n\nDocimo T, Reichelt M, Schneider B, et al.: The first step in the biosynthesis of cocaine in Erythroxylum coca: the characterization of arginine and ornithine decarboxylases. Plant Mol Biol. 2012; 78(6): 599–615. PubMed Abstract | Publisher Full Text\n\nJirschitzka J, Schmidt GW, Reichelt M, et al.: Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae. Proc Natl Acad Sci U S A. 2012; 109(26): 10304–10309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTorre JC, Schmidt GW, Paetz C, et al.: The biosynthesis of hydroxycinnamoyl quinate esters and their role in the storage of cocaine in Erythroxylum coca. Phytochemistry. 2013; 91: 177–186. PubMed Abstract | Publisher Full Text\n\nXu Y, Zhu X, Gong Y, et al.: Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR. Biochem Biophys Res Commun. 2012; 424(3): 398–403. PubMed Abstract | Publisher Full Text\n\nWillems E, Leyns L, Vandesompele J: Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem. 2008; 379(1): 127–129. PubMed Abstract | Publisher Full Text\n\nGuénin S, Mauriat M, Pelloux J, et al.: Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot. 2009; 60(2): 487–493. PubMed Abstract | Publisher Full Text\n\nGutierrez L, Mauriat M, Guénin S, et al.: The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J. 2008; 6(6): 609–618. PubMed Abstract | Publisher Full Text\n\nCzechowski T, Stitt M, Altmann T, et al.: Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 2005; 139(1): 5–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang L, Xie W, Chen Y, et al.: A dynamic gene expression atlas covering the entire life cycle of rice. Plant J. 2010; 61(5): 752–766. PubMed Abstract | Publisher Full Text\n\nSekhon RS, Lin H, Childs KL, et al.: Genome-wide atlas of transcription during maize development. Plant J. 2011; 66(4): 553–563. PubMed Abstract | Publisher Full Text\n\nHuis R, Hawkins S, Neutelings G: Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol. 2010; 10: 71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMafra V, Kubo KS, Alves-Ferreira M, et al.: Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One. 2012; 7(2): e31263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRemans T, Smeets K, Opdenakker K, et al.: Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta. 2008; 227(6): 1343–1349. PubMed Abstract | Publisher Full Text\n\nVandesompele J, De Paepe A, Speleman F: Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real time RT-PCR. Anal Biochem. 2002; 303(1): 95–98. PubMed Abstract | Publisher Full Text\n\nAndersen CL, Jensen JL, Orntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15): 5245–5250. PubMed Abstract | Publisher Full Text\n\nPfaffl MW, Tichopad A, Prgomet C, et al.: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004; 26(6): 509–515. PubMed Abstract | Publisher Full Text\n\nExpósito-Rodríguez M, Borges AA, Borges-Pérez A, et al.: Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol. 2008; 8: 131. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHellemans J, Mortier G, De Paepe A, et al.: qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007; 8(2): R19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNolan T, Hands RE, Bustin SA: Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006; 1(3): 1559–1582. PubMed Abstract | Publisher Full Text\n\nGiulietti A, Overbergh L, Valckx D, et al.: An overview of real-time quantitative PCR applications to quantify cytokine gene expression. Methods. 2001; 25(4): 386–401. PubMed Abstract | Publisher Full Text\n\nGachon C, Mingam A, Charrier B: Real-time PCR: what relevance to plant studies? J Exp Bot. 2004; 55(402): 1445–1454. PubMed Abstract | Publisher Full Text\n\nVanGuilder HD, Vrana KE, Freeman WM: Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques. 2008; 44(5): 619–26. PubMed Abstract | Publisher Full Text\n\nCruz F, Kalaoun S, Nobile P, et al.: Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR. Mol Breed. 2009; 23(4): 607–616. Publisher Full Text\n\nSchmidt GW, Delaney SK: Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Mol Genet Genomics. 2010; 283(3): 233–41. PubMed Abstract | Publisher Full Text\n\nBarsalobres-Cavallari CF, Severino FE, Maluf MP, et al.: Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions. BMC Mol Biol. 2009; 10: 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRadonić A, Thulke S, Mackay IM, et al.: Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004; 313(4): 856–62. PubMed Abstract | Publisher Full Text\n\nTong Z, Gao Z, Wang F, et al.: Selection of reliable reference genes for gene expression studies in peach using realtime PCR. BMC Mol Biol. 2009; 10: 71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArtico S, Nardeli SM, Brilhante O, et al.: Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC Plant Biol. 2010; 10: 49. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "763",
"date": "11 Feb 2013",
"name": "Sheila McCormick",
"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": "773",
"date": "15 Feb 2013",
"name": "Sarah O'Connor",
"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/2-37
|
https://f1000research.com/articles/2-36/v1
|
08 Feb 13
|
{
"type": "Research Article",
"title": "Modulation of proinflammatory activity by the engineered cationic antimicrobial peptide WLBU-2",
"authors": [
"Shruti M Paranjape",
"Thomas W Lauer",
"Ronald C Montelaro",
"Timothy A Mietzner",
"Neeraj Vij",
"Thomas W Lauer",
"Ronald C Montelaro",
"Timothy A Mietzner",
"Neeraj Vij"
],
"abstract": "Background: Host-derived (LL-37) and synthetic (WLBU-2) cationic antimicrobial peptides (CAPs) are known for their membrane-active bactericidal properties. LL-37 is an important mediator for immunomodulation, while the mechanism of action of WLBU-2 remains unclear.Objective: To determine if WLBU-2 induces an early proinflammatory response that facilitates bacterial clearance in cystic fibrosis (CF).Methods: C57BL6 mice were given intranasal or intraperitoneal 1×106 cfu/mL Pseudomonas aeruginosa (PA) and observed for 2h, followed by instillation of LL-37 or WLBU-2 (2-4mg/kg) with subsequent tissue collection at 24h for determination of bacterial colony counts and quantitative RT-PCR measurement of cytokine transcripts. CF airway epithelial cells (IB3-1, ΔF508/W1282X) were cultured in appropriate media with supplements. WLBU-2 (25μM) was added to the media with RT-PCR measurement of TNF-α and IL-1β transcripts after 20, 30, and 60min. Flow cytometry was used to determine if WLBU-2 assists in cellular uptake of Alexa 488-labeled LPS.Results: In murine lung exposed to intranasal or intraperitoneal WLBU-2, there was a reduction in the number of surviving PA colonies compared to controls. Murine lung exposed to intraperitoneal WLBU-2 showed fewer PA colonies compared to LL-37. After 24h WLBU-2 exposure, PA-induced IL-1β transcripts from lungs showed a twofold decrease (p<0.05), while TNF-α levels were unchanged. LL-37 did not significantly change transcript levels. In IB3-1 cells, WLBU-2 exposure resulted in increased TNF-α and IL-1β transcripts that decreased by 60min. WLBU-2 treatment of IB3-1 cells displayed increased LPS uptake, suggesting a potential role for CAPs in inducing protective proinflammatory responses. Taken together, the cytokine response, LPS uptake, and established antimicrobial activity of WLBU-2 demonstrate its ability to modulate proinflammatory signaling as a protective mechanism to clear infection.Conclusions: The immunomodulatory properties of WLBU-2 reveal a potential mechanism of its broad-spectrum antibacterial activity and warrant further preclinical evaluation to study bacterial clearance and rescue of chronic inflammation.",
"keywords": [
"Cationic antimicrobial peptides (CAPs) are one effector of the innate immune response",
"the “first line of defense” against a pathogenic insult. They are ancient",
"structurally diverse elements of the immune responses of all living species. These molecules typically have broad-spectrum antimicrobial activity with conserved recognition patterns to molecules such as lipopolysaccharide (LPS) and lipoteichoic acid. They are rapidly induced on the order of minutes to hours. Their amphipathic structures facilitate their antimicrobial killing activity at the level of the bacterial membrane. It has become increasingly apparent that CAPs also play key roles in inflammatory responses and in orchestrating the mechanisms of innate immunity1."
],
"content": "Introduction\n\nCationic antimicrobial peptides (CAPs) are one effector of the innate immune response, the “first line of defense” against a pathogenic insult. They are ancient, structurally diverse elements of the immune responses of all living species. These molecules typically have broad-spectrum antimicrobial activity with conserved recognition patterns to molecules such as lipopolysaccharide (LPS) and lipoteichoic acid. They are rapidly induced on the order of minutes to hours. Their amphipathic structures facilitate their antimicrobial killing activity at the level of the bacterial membrane. It has become increasingly apparent that CAPs also play key roles in inflammatory responses and in orchestrating the mechanisms of innate immunity1.\n\nLL-37, or human cathelicidin, is a CAP that has been localized to airway epithelium. In its mature form, it is an α-helical peptide made up of 37 residues that has been shown to possess broad spectrum antibacterial activity as well as other host defense functions such as chemotaxis, LPS neutralization, angiogenesis, and wound healing. First cloned from a bone marrow library, the expression of this peptide has been detected in many epithelial tissues, including the testes, epidermis, and the gastrointestinal and respiratory tracts2. The antibacterial and immunomodulatory roles of the cathelicidins are currently under intense investigation and appropriate models of infection are required for understanding their contribution to host defense.\n\nLL-37 is also an important modulator of the human immune response. This host-derived CAP is an antiseptic agent with the ability to inhibit macrophage stimulation by bacterial components such as LPS, lipoteichoic acid, and noncapped lipoarabinomannan3. Using gene expression profiling to identify potential LL-37-modulated macrophage functions, LL-37 directly upregulated 29 genes and downregulated another 20 genes. Among the genes predicted to be upregulated by LL-37 were those encoding chemokines and chemokine receptors. Consistent with this, LL-37 upregulated the expression of chemokines in macrophages and the mouse lung (monocyte chemoattractant protein 1), human A549 epithelial cells (IL-8), and whole human blood (monocyte chemoattractant protein-1 and IL-8), without stimulating the proinflammatory cytokine TNF-α. LL-37 also upregulated the chemokine receptors CXCR-4, CCR2, and IL-8RB. It appears that LL-37 contributes to the immune response by limiting the damage caused by bacterial products and recruiting immune cells to the site of infection4.\n\nWLBU-2, by comparison, is a completely synthetic α-helical, engineered CAP (eCAP) made up of a repeating sequence of Arg, Lys, and Trp residues5–7. Previous work has demonstrated this compound’s broad spectrum antibacterial activity against bacterial pathogens in both in vivo and in vitro systems5,6,8. Among the many antimicrobial peptides currently described in the literature, eCAPs are most chemically and structurally homologous to the magainins9 and LL-37. They are peptides of approximately 30 residues that, when modeled as an α-helix, demonstrate amphipathic character with defined cationic and hydrophobic faces. The selectivity of the eCAPs for bacterial membranes, like other host-derived CAPs, presumably results from their affinity for negatively charged lipids found on the bacterial surface. The high-energy potential of the bacterial membrane facilitates self-promoted CAP uptake, thus compromising the integrity of the bacterial cell by disrupting the lipid bilayer10 and suggests that these α-helical peptides11 may be suitable agents for treating bacterial airway infections.\n\nDespite extensive characterization of the antimicrobial activity of the eCAPs, little is known about their immunomodulatory properties5,6. The purpose of this study was to determine if WLBU-2 modulates an early proinflammatory response to facilitate Pseudomonas aeruginosa (PA) clearance using both in vivo and in vitro models. The in vivo model partially replicates some of the phenotypic lung disease of CF in mice that resemble wild-type animals in size and survival. Development of a murine model of lung inflammation is highly desirable to accelerate pre-clinical testing of novel anti-inflammatory therapeutics. These studies demonstrate the immunomodulatory properties of an engineered, synthetic compound not only for the purpose of its potential development as a novel antibacterial agent, but also for its contribution to study the role of α-helical peptides in the processes of host defense.\n\n\nMethods\n\nFor the in vitro studies, CF airway epithelial cells (IB3-1, ΔF508/W1282X, American Type Culture Collection (ATCC), Manassas, VA) were cultured and maintained in LHC-8 medium containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. WLBU-2 (25μM) was added to the media with qRT-PCR measurement of TNF-α and IL-1β transcripts after 20, 30, and 60min. IB3-1 cells were used for flow cytometry12 and measurement of proinflammatory cytokine activity. Transfected cells containing a wild-type or NFκB mutant-IL-8 promoter gene13 were treated with WLBU-2 followed by measurement of IL-8 reporter activity.\n\nThese studies used CF IB3-1 (ΔF508/W1282X) cells, transiently transfected with α 5' firefly luciferase gene (Clontech Laboratories, Inc, Mountain View, CA) flanking a wild type- or NFκB mutant-IL-8 promoter, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 24h as previously described13,14. The CAP LL-37 (25μM) or the eCAP WLBU-2 (25μM) was added to the media for 30min-4h. The Dual-Luciferase® Reporter (DLRTM) Assay System (Clontech Laboratories, Inc, Mountain View, CA) was used to measure IL-8 reporter activity in IB3-1 cells. Renilla luciferase (Clontech Laboratories, Inc, Mountain View, CA) was used as an internal control to normalize changes in IL-8 promoter-driven firefly luciferase activity across the samples.\n\nIB3-1 cells were incubated overnight at 37°C with LPS-Alexa Fluor 488 (Invitrogen, Carlsbad, CA) and Lipofectamine 2000. PBS or WLBU-2 (25µM) was added to the flask for 24h. After the addition of Cell Dissociation Buffer (Invitrogen, Carlsbad, CA), cells were collected, centrifuged, and rinsed with PBS. Finally, cells were treated with FIX & PERM (Invitrogen, Carlsbad, CA) per the manufacturer’s directions. Flow cytometry was done on a BD FACS can flow cytometer and data were analyzed using CellQuest.\n\nThis protocol was approved by the institutional Animal Care and Use Committee (ACUC). Animals used in this study were followed for 1–5 days (4 animals per cage) in approved satellite housing to allow close observation from the beginning to end of each experiment. Feeding practices, light cycle, and temperature and humidity, and cage and room cleaning procedures were identical to those of this institution’s central animal facility in accordance with ACUC recommendations. Animals were studied in four groups of four animals per experiment. The groups consisted of a) control C57/BL6 wild-type mice; b) C57/BL6 wild-type mice receiving bacteria (positive control); c) the eCAP WLBU-2 or the CAP LL-37 (test agents); or d) the combination of bacteria and WLBU-2 or LL-37. Animals in the experimental groups received the test agents, while control animals received sterile phosphate-buffered saline via the trachea using the posterior pharyngeal approach. These studies were carried out in the presence or absence of a proinflammatory stimulus (bacterial exposure).\n\nMice were anesthetized using a vaporizer set to deliver a mixture of 3% isoflurane and oxygen in preparation for the instillation of control or test agents into the respiratory tree. Complete anesthesia was determined by visual confirmation of a slowed respiration rate and the animal’s response to other clinical tests such as leg withdrawal and tail pinch. Animals were weighed prior to the procedure. The method is based on a published study15. Once anesthetized, the tongue was gently extruded using padded forceps and the control and/or test agents (volume 50µL) were pipetted into the posterior section of the oral cavity. The positive control was 1) P. aeruginosa (ATCC type strain, 1×106 cfu/mL); the negative control was PBS. The test agents were the host-derived CAP LL-37 or the engineered CAP WLBU-2 (dose 1mg/kg). Following aspiration of the test agent, 100% O2 was given until the animal awakened. Animals were then placed into a 37°C chamber until completely recovered.\n\nIntraperitoneal PA infection in age (8 weeks) and sex-matched C57BL/6 mice was established16 followed by WLBU-2 (4mg/kg), LL-37 (4mg/kg), or PBS treatment5, followed by collection at 24h of bronchoalveolar lavage (BAL) samples and lung tissue for bacterial culture on trypticase soy agar plates, quantitative RT-PCR, ELISA, and microscopy12. Mice were anesthetized with 3% isoflurane before and during treatment. After 24h, mice were sacrificed for sample collection.\n\nAt the endpoint of the experiments mice were anesthetized using a vaporizer set to deliver a mixture of 3% isoflurane and oxygen and weighed. Once completely anesthetized, animals were euthanized by cervical dislocation followed by collection of BAL samples from within an exposed thorax using sterile phosphate-buffered saline (PBS) through a cannula inserted in the trachea. BAL samples were used for protein analysis as well as leukocyte differentiation. Lungs were excised and then stored in buffers or preservatives for protein analysis or histologic examination.\n\nMouse lungs were homogenized in TRIzol (Invitrogen, Carlsbad, CA) and processed for isolation of total RNA according to the manufacturer's instructions. The Super Script III First-Strand Synthesis System (Invitrogen, Carlsbad, CA) was used to catalyze the reverse transcription reaction from 1µg of total RNA. Quantitative RT-PCR (qRT-PCR) was performed using TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) for GAPDH (control reagent), IL-1β, and TNF-α (4352932, Mm01336189_m1, Mm99999068_m1, respectively). The qRT-PCR reactions were amplified using an ABI PRISM 7700 Sequence Detection System. Relative gene expression was determined using ∆∆Ct calculations.\n\nELISA kits for IL-1β and IL-6 were obtained from R&D Systems (Minneapolis, MN). The manufacturer’s protocol was followed for the detection of each cytokine in 10µL of BAL fluid as well as in standardized samples provided in the kit. SoftMax Pro (Molecular Devices, Sunnyvale, CA) was used to read the 96-well plates in a Molecular Devices VersaMax microplate reader. A plot of the standardized samples was used to calculate cytokine expression in the BAL samples. For histologic examination, lungs were fixed overnight in 4% paraformaldehyde at 4°C and subsequently embedded in paraffin. Tissue sections (5µm thickness) were deparaffinized and rehydrated with xylenes and a series of decreasing ethanol concentrations, respectively. Hematoxylin and eosin were used to stain the sections prior to microscopic examination.\n\n\nResults\n\nIB3-1 cells were used to examine proinflammatory cytokine effects after WLBU-2 exposure. IL-8 promoter activity was significantly increased compared to control in both WLBU-2-exposed IB3-1 cells after 4h (mean±SD 1.14±0.0004 vs. 0.67±0.0002, p<0.005, Figure 1) and LL-37-exposed cells (mean±SD 1.04±0.0006 vs. 0.67±0.0002, Figure 1). WLBU-2-exposed cells showed significantly less IL-8 promoter activity compared to LL-37-exposed cells (p<0.005). IB3-1 cells with an NFκB mutant IL-8 promoter exposed to either peptide showed minimal reporter activity at 4h (Figure 1), suggesting that both WLBU-2 and LL-37 influence IL-8 secretion through NFκB. Exposure to either WLBU-2 (25μM) or LL-37 (25μM) for 30min (Figure 2) resulted in increased transcript levels of IL-1β (mean±SD fold-change WLBU-2 1.59±0.12; LL-37 1.47±0.12, p<0.005) and TNF-α (mean±SD fold-change WLBU-2 5.74±1.83; LL-37 4.11±1.07, p<0.005) compared to control and decreased by 60min toward baseline (see raw data file).\n\nCF IB3-1 (ΔF508/W1282X) cells transiently transfected with a 5' firefly luciferase gene flanking a 200bp wild type- (solid lines) or NFκB mutant-IL-8 promoter (dashed lines) were treated with 25µM WLBU-2 (solid circles) or 25µM LL-37 (solid squares) followed by measurement of IL-8 promoter activity at time points ranging from 30min-4h by Dual-Luciferase Reporter assay. There was a significant increase in IL-8 promoter activity in cells exposed to either peptide compared to control (asterisks, p<0.005). In cells lacking the NFκB site (dashed lines), there was little IL-8 promoter activity, suggesting that both WLBU-2 (open circles) and LL-37 (open squares) may influence IL-8 secretion through the NFκB pathway.\n\nCF IB3-1 cells were cultured in LHC-8 media with supplements, followed by treatment with PBS (black bars), LL-37 (25µM, light grey bars) or WLBU-2 (25µM, dark grey bars) and measurement of the levels of the proinflammatory cytokines IL-1β and TNF-α by quantitative RT-PCR at time points varying from 0–60min. Data represent fold-change in relative transcript levels of proinflammatory cytokines obtained 30min after peptide exposure; both IL-1β and TNF-α showed significant increases in relative transcript levels that decreased by 60min (see raw data file). CAPs, as effector molecules of the innate immune response, may exert an initial protective effect at the level of the epithelial surface against a potential pathogenic or inflammatory challenge.\n\nUsing equimolar (25μM) peptide concentrations in LPS-stimulated cells, WLBU-2 showed less LPS-induced IL-8 reporter activity compared to LL-37 after 4h (mean±SD 1.82±0.0034 vs. 2.93±0.0009, p<0.005, Figure 3). By flow cytometry, WLBU-2-exposed cells showed 91% uptake of fluorescently labeled LPS after 24h compared to 75% of control cells. These results suggest that WLBU-2 induces a protective inflammatory response through interaction with LPS (T. Mietzner, unpublished observation) and exhibits an initial protective effect against epithelial inflammatory challenges.\n\nCF IB3-1 (ΔF508/W1282X) cells were transiently transfected with a 5' firefly luciferase gene flanking a 200bp wild type-IL-8 promoter. After application of Pseudomonas aeruginosa LPS, WLBU-2 (25µM, circles) or LL-37 (25µM, squares) was added to the plate followed by measurement of IL-8 promoter activity at time points ranging from 30min-4h by Dual-Luciferase Reporter assay. Promoter activity is plotted for both peptides with an LPS-stimulated control. Both peptides showed a significant decrease in LPS-induced IL-8 promoter activity (asterisks, p<0.005); the eCAP WLBU-2 showed a sustained decrease over 4h while LL-37 showed an increasing trend, suggesting that eCAPs may be beneficial as immunomodulators as well as antibacterial agents.\n\n\n\nWLBU-2 treatment of animals (n=5/group) with intraperitoneal PA infection showed more IL-1β suppression compared to LL-37 (mean±SD fold-change 37.6±9.7 vs. 105.9±30.9, p<0.005, Figure 4). There was no effect on TNF-α (data not shown). Consistent with prior efficacy studies comparing the bactericidal activities of WLBU-2 and LL-375,6, bacterial cultures of lung homogenates from WLBU-2-treated animals showed no growth compared to those given LL-37 after 24h (0 cfu/mL vs. 5800 cfu/mL).\n\nWild-type C57BL/6 animals (n=5/group) received intraperitoneal injections of PBS or PA (1×106 cfu/mL), followed by intraperitoneal injections after 2h of PBS (black bars), LL-37 (4mg/kg, light grey bars), or WLBU-2 (4mg/kg, dark grey bars), with subsequent measurement by quantitative RT-PCR of IL-1β transcripts (y axis, fold change in relative transcript level) from lung tissue harvested 24h post exposure. Data represent measurements performed in triplicate. In groups not receiving bacteria, LL-37 and WLBU-2-exposed animals showed an increase in IL-1β transcripts compared to PBS controls. In groups receiving PA, WLBU-2-exposed animals showed significant suppression of IL-1β (p<0.005) compared to LL-37. These data suggest that the eCAP WLBU-2 may modulate proinflammatory cytokine release in the setting of acute infection.\n\n\n\n\nDiscussion\n\nPrevious CAP studies have demonstrated a dose-dependent decrease in LPS-activated neutrophilic proinflammatory cytokine release by LL-3717. Neutrophils stimulated by heat-inactivated bacteria showed a decrease in TNF-α after LL-37 exposure, whereas those from cathelicidin-deficient mice showed less antimicrobial activity and increased proinflammatory cytokine release, suggesting that endogenous cathelicidin modulates the neutrophilic innate immune response. Since Toll-like receptor signaling effects in macrophages differ depending on exogenous or endogenous peptide origin and cellular activation state, exogenous cathelicidin application resulted in blunted activation of p38 and ERK MAPKs and decreased TNF-α release in macrophages exposed to LPS and reversed diminished MAPK activation associated with LPS tolerance. Endogenous cathelicidin release from macrophages in cathelicidin-deficient animals neither inhibited LPS MAPK and cytokine activation nor rendered animals more susceptible to lethal LPS challenges18. Other studies demonstrated that LL-37 and LPS interactions alter endotoxin aggregation, which may explain the observed inhibition of proinflammatory activity19–21.\n\neCAPs represent a novel class of effective antimicrobial peptides6,22–25 that demonstrate broad-spectrum activity against highly resistant bacterial strains22. The antibacterial efficacy of WLBU-2 using murine models of intraperitoneal infection and bacteremia has been described5,6. Intravenous WLBU-2 effectively treated systemic PA infection and was protective when administered 1h prior to establishing bacteremia. Animals treated with subtherapeutic doses of WLBU-2 showed lower IL-1β and TNF-α levels after 3–5h compared to animals exposed to heat killed bacteria (T. Mietzner, unpublished observation). Despite demonstration of in vivo efficacy, further studies are needed to define the immunomodulatory properties of WLBU-2 in the setting of respiratory infection.\n\nThis paper advances previous work on WLBU-2 by defining its antibacterial and immunomodulatory activities in vivo and in vitro. The in vitro studies demonstrate constitutive changes in proinflammatory signaling in the absence of bacteria. The increased LPS uptake in CF epithelial cells indicates the possibility of an LPS interaction as an immunomodulatory function but may have been limited by cellular toxicity resulting from prolonged peptide exposure8. Because peptide inactivation through interaction with LPS may affect cellular uptake26, the optimized hydrophobicity of WLBU-2 could contribute to more effective LPS neutralization27.\n\nThe cytokine response, LPS uptake, and established antimicrobial activity of WLBU-2 demonstrate modulation of proinflammatory signaling as a protective mechanism to clear infection. Because in vivo studies of WLBU-2 have demonstrated effective bacterial killing activity and proinflammatory cytokine suppression, further work examining CAPs in innate immune responses will assist in defining the roles of CAPs in host defense and of eCAPs in the development of novel antibacterial and immunomodulatory therapies.",
"appendix": "Author contributions\n\n\n\nAll authors contributed to the study design and preparation of the manuscript and figures and their revisions. All authors agreed 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 American Lung Association (RG-9247-N, SMP).\n\n\nReferences\n\nZasloff M: Antimicrobial peptides of multicellular organisms. Nature. 2002; 415(6870): 389–395. PubMed Abstract | Publisher Full Text\n\nHuttner KM, Bevins CL: Antimicrobial peptides as mediators of epithelial host defense. Pediatr Res. 1999; 45(6): 785–794. PubMed Abstract | Publisher Full Text\n\nScott MG, Vreugdenhil AC, Buurman WA, et al.: Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein. J Immunol. 2000; 164(2): 549–553. PubMed Abstract\n\nScott MG, Davidson DJ, Gold MR, et al.: The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses. J Immunol. 2002; 169(7): 3883–3891. PubMed Abstract\n\nDeslouches B, Gonzalez IA, DeAlmeida D, et al.: De novo-derived cationic antimicrobial peptide activity in a murine model of Pseudomonas aeruginosa bacteraemia. J Antimicrob Chemother. 2007; 60(3): 669–672. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeslouches B, Islam K, Craigo JK, et al.: Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications. Antimicrob Agents Chemother. 2005; 49(8): 3208–3216. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeslouches B, Phadke SM, Lazarevic V, et al.: De novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity. Antimicrob Agents Chemother. 2005; 49(1): 316–322. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPhadke SM, Islam K, Deslouches B, et al.: Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model. Peptides. 2003; 24(8): 1099–1107. PubMed Abstract | Publisher Full Text\n\nTencza SB, Douglass JP, Creighton DJ Jr, et al.: Novel antimicrobial peptides derived from human immunodeficiency virus type 1 and other lentivirus transmembrane proteins. Antimicrob Agents Chemother. 1997; 41(11): 2394–2398. PubMed Abstract | Free Full Text\n\nHancock RE: Peptide antibiotics. Lancet. 1997; 349(9049): 418–422. PubMed Abstract | Publisher Full Text\n\nSchwab U, Gilligan P, Jaynes J, et al.: In vitro activities of designed antimicrobial peptides against multidrug-resistant cystic fibrosis pathogens. Antimicrob Agents Chemother. 1999; 43(6): 1435–1440. PubMed Abstract | Free Full Text\n\nBodas M, Min T, Mazur S, et al.: Critical modifier role of membrane-cystic fibrosis transmembrane conductance regulator-dependent ceramide signaling in lung injury and emphysema. J Immunol. 2011; 186(1): 602–613. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVij N, Amoako MO, Mazur S, et al.: CHOP transcription factor mediates IL-8 signaling in cystic fibrosis bronchial epithelial cells. Am J Respir Cell Mol Biol. 2008; 38(2): 176–184. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVij N, Mazur S, Zeitlin PL: CFTR is a negative regulator of NFkappaB mediated innate immune response. PLoS One. 2009; 4(2): e4664. PubMed Abstract | Publisher Full Text | Free Full Text\n\nViscardi RM, Kaplan J, Lovchik JC, et al.: Characterization of a murine model of Ureaplasma urealyticum pneumonia. Infect Immun. 2002; 70(10): 5721–5729. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVij N, Min T, Marasigan R, et al.: Development of PEGylated PLGA nanoparticle for controlled and sustained drug delivery in cystic fibrosis. J Nanobiotechnology. 2010; 8: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlalwani SM, Sierigk J, Herr C, et al.: The antimicrobial peptide LL-37 modulates the inflammatory and host defense response of human neutrophils. Eur J Immunol. 2010; 40(4): 1118–1126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPinheiro da Silva F, Gallo RL, Nizet V: Differing effects of exogenous or endogenous cathelicidin on macrophage toll-like receptor signaling. Immunol Cell Biol. 2009; 87(6): 496–500. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBucki R, Janmey PA: Interaction of the gelsolin-derived antibacterial PBP 10 peptide with lipid bilayers and cell membranes. Antimicrob Agents Chemother. 2006; 50(9): 2932–2940. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNagaoka I, Tamura H, Hirata M: An antimicrobial cathelicidin peptide, human CAP18/LL-37, suppresses neutrophil apoptosis via the activation of formyl-peptide receptor-like 1 and P2X7. J Immunol. 2006; 176(5): 3044–3052. PubMed Abstract\n\nRosenfeld Y, Papo N, Shai Y: Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action. J Biol Chem. 2006; 281(3): 1636–1643. PubMed Abstract | Publisher Full Text\n\nTencza SB, Creighton DJ, Yuan T, et al.: Lentivirus-derived antimicrobial peptides: increased potency by sequence engineering and dimerization. J Antimicrob Chemother. 1999; 44(1): 33–41. PubMed Abstract | Publisher Full Text\n\nTencza SB, Mietzner TA, Montelaro RC: Calmodulin-binding function of LLP segments from the HIV type 1 transmembrane protein is conserved among natural sequence variants. AIDS Res Hum Retroviruses. 1997; 13(3): 263–269. PubMed Abstract | Publisher Full Text\n\nTencza SB, Miller MA, Islam K, et al.: Effect of amino acid substitutions on calmodulin binding and cytolytic properties of the LLP-1 peptide segment of human immunodeficiency virus type 1 transmembrane protein. J Virol. 1995; 69(8): 5199–5202. PubMed Abstract | Free Full Text\n\nPhadke SM, Lazarevic V, Bahr CC, et al.: Lentivirus Lytic Peptide 1 perturbs both outer and inner membranes of Serratia marcescens. Antimicrob Agents Chemother. 2002; 46(6): 2041–2045. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBucki R, Byfield FJ, Janmey PA: Release of the antimicrobial peptide LL-37 from DNA/F-actin bundles in cystic fibrosis sputum. Eur Respir J. 2007; 29(4): 624–632. PubMed Abstract | Publisher Full Text\n\nRosenfeld Y, Lev N, Shai Y: Effect of the hydrophobicity to net positive charge ratio on antibacterial and anti-endotoxin activities of structurally similar antimicrobial peptides. Biochemistry. 2010; 49(5): 853–861. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "1957",
"date": "07 Oct 2013",
"name": "Anastasia Nijnik",
"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 presents novel and convincing data for immunomodulatory activity of synthetic cationic peptide WLBU-2. Specifically, the authors demonstrate that WLBU-2 induces IL-8 promoter activity and stimulates TNFa and IL1b gene expression in the IB3-1 cystic fibrosis (CF) airway epithelial cells. Several questions remain to be addressed. In particular, given that TNFa and IL1b production is regulated at post-transcriptional level, it will be important to address whether WLBU-2 stimulates the production and secretion of TNFa, IL1b and/or IL8 proteins. Also, given the relatively high concentration of peptide used in the in vitro studies, it is important to address whether the induction of inflammatory cytokines could be linked to non-specific cytotoxic effects of the peptide on the epithelial cells.The IB3-1 cystic fibrosis (CF) airway epithelial cells were chosen for the study because immunomodulatory peptides are being explored as a potential therapeutic against bacterial colonization and inflammatory tissue damage in CF. At present it is unclear whether there are any differences between wild type and IB3-1 epithelial cells in their responses to WLBU-2. Possible immunomodulatory activities of WLBU-2 on other cell types (e.g. leukocytes) also remain to be addressed. These are possible interesting directions for future studies.The paper is clearly written. Given the many previous studies exploring anti-endotoxic properties of cathelicidins, and activities of cationic peptides in CF models, some sections of the paper could benefit from additional citations.",
"responses": []
},
{
"id": "1956",
"date": "25 Oct 2013",
"name": "Paul Savage",
"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 makes a correlation between the antibacterial and immunomodulatory effects of LL-37 and the synthetic antimicrobial peptide WLBU-2. The authors find that WLBU-2 stimulates cytokine (IL-8) production, which is likely to impact bacterial clearance. This observation implies that the WLBU-2 is interacting comparably with receptors that respond to the presence of LL-37. Overall, the manuscript is well written, the data are well presented, and though the body of work is modest, it should be of interest to a relatively large group of researchers.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-36
|
https://f1000research.com/articles/2-35/v1
|
07 Feb 13
|
{
"type": "Data Article",
"title": "Longitudinal RNA sequencing of the deep transcriptome during neurogenesis of cortical glutamatergic neurons from murine ESCs",
"authors": [
"Kyle S Hubbard",
"Ian M Gut",
"Megan E Lyman",
"Patrick M McNutt",
"Kyle S Hubbard",
"Ian M Gut",
"Megan E Lyman"
],
"abstract": "Using paired-end RNA sequencing, we have quantified the deep transcriptional changes that occur during differentiation of murine embryonic stem cells into a highly enriched population of glutamatergic cortical neurons. These data provide a detailed and nuanced account of longitudinal changes in the transcriptome during neurogenesis and neuronal maturation, starting from mouse embryonic stem cells and progressing through neuroepithelial stem cell induction, radial glial cell formation, neurogenesis, neuronal maturation and cortical patterning. Understanding the transcriptional mechanisms underlying the differentiation of stem cells into mature, glutamatergic neurons of cortical identity has myriad applications, including the elucidation of mechanisms of cortical patterning; identification of neurogenic processes; modeling of disease states; detailing of the host cell response to neurotoxic stimuli; and determination of potential therapeutic targets. In future work we anticipate correlating changes in longitudinal gene expression to other cell parameters, including neuronal function as well as characterizations of the proteome and metabolome. In this data article, we describe the methods used to produce the data and present the raw sequence read data in FASTQ files, sequencing run statistics and a summary flatfile of raw counts for 22,164 genes across 31 samples, representing 3-5 biological replicates at each timepoint. We propose that this data will be a valuable contribution to diverse research efforts in bioinformatics, stem cell research and developmental neuroscience studies.",
"keywords": [
"Transcriptional profiling by RNA sequencing (RNAseq) enables the sensitive and accurate characterization of the transcriptome1–4. Following enumeration of reads",
"various methods are available to normalize counts",
"estimate probability distributions and identify differential gene expression between biological conditions3",
"5",
"6. RNAseq has the added advantages of being extremely high-throughput and relatively inexpensive",
"with a high signal-to-noise ratio and a dynamic range encompassing 4–5 orders of magnitude. This combination of throughput and sensitivity enables the detection of rare transcripts from nanograms of RNA."
],
"content": "Objectives\n\nTranscriptional profiling by RNA sequencing (RNAseq) enables the sensitive and accurate characterization of the transcriptome1–4. Following enumeration of reads, various methods are available to normalize counts, estimate probability distributions and identify differential gene expression between biological conditions3,5,6. RNAseq has the added advantages of being extremely high-throughput and relatively inexpensive, with a high signal-to-noise ratio and a dynamic range encompassing 4–5 orders of magnitude. This combination of throughput and sensitivity enables the detection of rare transcripts from nanograms of RNA.\n\nWe have described a method to produce large quantities of highly enriched, electrically active glutamatergic neurons (ESNs) from suspension-adapted mouse embryonic stem cells (ESCs)7,8. This technique is a modification of the 4/4 method, and is based on the spatiotemporal changes in morphogens that occur during neural induction and patterning9–11. In this method, neuroepithelial stem cells (NESCs) are derived from ESCs by the withdrawal of leukemia inhibitory factor (LIF), then induced to undergo neurogenesis and neural patterning by supplementation with all-trans retinoic acid (RA) in the presence of fetal bovine serum12. We have modified the 4/4 method to include feeder cell-free, suspension culture of ESNs; differentiation under rotary conditions to normalize the intra-aggregate environment; and neuronal induction and neural induction and patterning using 6 µM RA. These refinements have resulted in a facile and economical method to generate large quantities of highly enriched glutamatergic neurons13. Using immunocytochemistry, we have shown that ESNs are composed mostly of glutamatergic neurons (~95%), with about 5% GABAergic neurons, and no evidence of dopaminergic, serotonergic, cholinergic or glycinergic subtypes7. Expression profiling using RNA sequencing has confirmed that derived cultures are primarily glutamatergic, and identified the abundant expression of a wide range of cortical markers, including reelin, Pax6, Otx1, Ctip2 and Cux1/213–19.\n\nAlthough aspects of corticogenesis have been replicated in vitro by the directed differentiation of pluripotent stem cells, spatiotemporal changes in gene expression responsible for regionalization and apical-to-basal patterning of the cerebral cortex are not well understood20–22. The embryological origin of the cerebral cortex is the telencephalon, which is the most anterior structure of the mammalian neural tube23. Cortical glutamatergic neurons are generated by proliferation and laminar patterning of the dorsal telencephalon, whereas inhibitory GABAergic neurons derive from the ventral telencephalon and migrate tangentially into cortical layers24,25. Our ability to derive predominantly glutamatergic neurons that express markers of the telencephalon and all six cortical layers is consistent with an in vitro model of the developing telencephalon and cortical layer formation23. We propose to apply this model to identify the temporal changes in gene and isoform expression associated with neural patterning and corticogenesis. If successful, we intend to use ESNs to functionally interrogate the roles of individual genes in executing the transcriptional programs underlying the formation of the cerebral cortex.\n\nTo evaluate the transcriptional changes associated with differentiation of cortical glutamatergic neurons, we conducted a longitudinal expression profile of the deep transcriptome during neurogenesis from days in vitro (DIV) -8 to 28, where DIV 0 corresponds to the end of differentiation. High quality RNA was isolated from ESCs (DIV -8; n=4); neuroepithelial stem cells (DIV -4; n=3); radial glia (DIV 0; n=3); developmental stage (DS) I/II neurons (DIV 1; n=4); DS III/IV neurons (DIV 7; n=5); and maturing DS IV/V neurons at DIV 16 (n=4); DIV 21 (n=4); and DIV 28 (n=4). The summary data, including quality scores, are presented in Data File 1, and the raw transcript read counts for each biological replicate are presented in Data File 2. The FASTQ files generated for each biological replicate are available at the Sequence Read Archive (SRA), a freely accessible database provided by NCBI (http://www.ncbi.nlm.nih.gov/sra/), under the accession number PRJNA185305.\n\nIn addition to providing the basis for a study to characterize transcriptional processes involved in corticogenesis and neuronal maturation, we can foresee several other applications for this data. First, the identification of genes and isoforms that exhibit differential expression in synchronization with known markers is expected to provide novel insight into molecular mechanisms of neurogenesis and neural patterning. Second, a few algorithms are available for statistical determination of differential gene expression within longitudinal data sets. The depth and quality of these data suggests it may be well-suited for the development and validation of such methods. Third, we envision this dataset facilitating inter-specific comparisons of transcription during neurogenesis and corticogenesis, providing insight into transcriptional mechanisms common to and unique in the evolution of the mammalian cerebral cortex. For these reasons, we are making this data publically available for research efforts in bioinformatics, stem cell research and developmental neuroscience studies.\n\n\nMaterials and methods\n\nESCs were adapted to feeder cell-free, suspension culture and maintained as previously described7,13. In brief, aliquots of R1 ESCs (ATCC, Manassas, VA) were thawed and maintained at 37°C at 5% CO2 in 90% relative humidity in 10 cm bacterial plates in ESM (Knockout DMEM supplemented with 100 µM β-mercaptoethanol, 0.1mM nonessential amino acids, 2.0 mM L-glutamine, 5000 units/mL penicillin/streptomycin, 1000 units/mL recombinant mouse LIF [all Life Technologies, Carlsbad, CA] and 15% ES qualified fetal calf serum [ATCC, Manassas, VA])26. Cells were passaged once aggregates first became clearly visible to the naked eye (4–8 days) and maintained for at least 5 passages prior to differentiation. For passaging, aggregates were allowed to settle by gravity, washed once with 0.5 mL PBS and dissociated for 3 min at 37°C with 0.5 ml of TrypLE Express (Life Technologies). Dissociation was terminated by addition of 0.5 mL ESM followed by gently trituration with a P1000 pipette to achieve a single-cell suspension. ESNs were counted manually using a hemocytometer and ~1.5×106 mESCs were transferred to 10 mL ESM in a fresh 10 cm bacterial dish.\n\nESCs were differentiated into neurons between 5–30 passages after adaptation to suspension culture. A modified 4/4 protocol was used for neuron differentiation7,27. Following routine sub-passaging, 3.5×106 mouse ESCs were transferred to 30 mL differentiation medium (ESM modified to contain 10% ESC-qualified fetal calf serum and without LIF) in a 10 cm ultra-low attachment suspension culture dish (Corning, Lowell, MA). This was designated as DIV -8. Differentiating aggregates were maintained on a rotary shaker at 45 rpm at 37°C, 5% CO2 and 90% relative humidity. Complete media changes were conducted every 48 h, and media was supplemented with 6 µM retinoic acid (Sigma-Aldrich) at DIV -4 and DIV -2.\n\nOn DIV 0, aggregates were dissociated with TrypLE Express for 5 min at 37°C. Trypsinization was halted with 5 mL of 1% soybean trypsin inhibitor (Life Technologies), the aggregates were gently dissociated by trituration with a 10 mL pipet, and the cell suspension was filtered through a 40 µm cell strainer (Thermo Scientific, Waltham, MA). Cells were pelleted for 5 min at 300 x g, washed in N2 medium (Neurobasal-A medium with 1 x N2 vitamins, 2 mM glutamine and antibiotics [Life Technologies]) and counted manually using a hemocytometer. Neuronal progenitors were plated at 1.5×106 cells/cm2 in poly-D coated dishes. Complete washes with N2 medium were conducted at 4 h and 24 h to remove residual serum and non-adherent cells. At DIV 2, N2 was replaced with B27 medium (Neurobasal-A supplemented with antibiotics, 2 mM glutamine and 1 x B27 vitamins [Life Technologies]). Subsequently, ESNs underwent full medium changes with B27 on DIV 4, 8 and 12. On DIV 8 the media was supplemented with 30 µM 5-fluoro-2’-deoxyuridine and 70 µM Uridine (Sigma-Aldrich) to select against any remaining glia. Following DIV 12, ESNs were left undisturbed until RNA harvest. Cultures remained healthy and viable until at least DIV 28 under these conditions.\n\nNeurons were harvested from 6 cm dishes at DIV -8, -4, 0, 1, 7, 16, 21 and 28 (n=3 to 5) and RNA was isolated by QIAcube (Qiagen, Valencia, CA) using the RNeasy mini kit protocol (Qiagen) and submitted to Expression Analysis, Inc. (Durham, NC) for library preparation and sequencing. Typical recovery was ~3 µg per 6 cm dish, with an RNA integrity number exceeding 8 and a 260:280 of 1.8 – 2.2. PolyA+ RNA was purified from 500 ng of total RNA using polyA selection, chemically fragmented and reverse transcribed using random hexamers. This was followed by second strand synthesis and end repair. Libraries were prepared for paired-end sequencing using the TruSeq™ RNA sample prep kits (Illumina, San Diego, CA) per manufacturer’s instructions, and library size and integrity were determined using the Agilent Bioanalyzer 2100 (Santa Clara, CA). Libraries were bound to flow cell surfaces using the Standard Cluster Generation Kit v5 (Illumina). Flow cells were transferred to the Illumina HiSeq 2000 and run using TruSeq SBS Kits (Illumina). Paired-end sequencing data were generated over 2x50 sequencing cycles. Sequence information and quality scores are available at SRA in FASTQ format, with the forward and reverse reads appended with an \"F\" or \"R\" (e.g., DIV7.1F and DIV7.1R). Raw sequence reads were aligned using the University of California, Santa Cruz’s mouse knownGENE track, and transcriptome abundance estimation was performed on completed alignments using RSEM (RNA-Seq by Expectation Maximization)28. A summary flatfile containing the total raw reads for each gene in each biological replicate is presented in Data File 2. Genes were excluded if no reads occurred in any biological sample.\n\n\n\n",
"appendix": "Author contributions\n\n\n\nKSH, IMG and PMM conceived the study. IMG, PMM and MEL conducted ESC culture, produced neurons and extracted the RNA. KSH, IMG and PMM organized the data sets and prepared the manuscript. All authors approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the National Institutes of Health National Institute of Allergy and Infectious Diseases (IAA number AOD12058-0001-0000) and the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division (grant number CBM.THRTOX.01.10.RC.021). This research was performed while IMG held a Defense Threat Reduction Agency-National Research Council Research Fellowship Award and KSH held a National Research Council Research Associateship Award.\n\n\nAcknowledgements\n\nWe thank Angela Adkins, Cindy Kronman and Marian Nelson (USAMRICD, MD) for administrative, logistical and technical assistance. We also thank Expression Analysis, Inc. (Durham, NC) for assistance with RNA sequence generation. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.\n\n\nReferences\n\nBullard JH, Purdom E, Hansen KD, et al.: Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics. 2010; 11: 94.PubMed Abstract | Publisher Full Text | Free Full Text\n\nGriffith M, Griffith OL, Mwenifumbo J, et al.: Alternative expression analysis by RNA sequencing. Nat Methods. 2010; 7(10): 843–847.PubMed Abstract | Publisher Full Text\n\nMarioni JC, Mason CE, Mane SM, et al.: RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 2008; 18(9): 1509–1517.PubMed Abstract | Publisher Full Text | Free Full Text\n\nHebenstreit D, Fang M, Gu M, et al.: RNA sequencing reveals two major classes of gene expression levels in metazoan cells. Mol Syst Biol. 2011; 7: 497.PubMed Abstract | Publisher Full Text | Free Full Text\n\nSultan M, Schulz MH, Richard H, et al.: A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science. 2008; 321(5891): 956–960.PubMed Abstract | Publisher Full Text\n\nRobinson MD, Smyth GK: Moderated statistical tests for assessing differences in tag abundance. Bioinformatics. 2007; 23(21): 2881–2887.PubMed Abstract | Publisher Full Text\n\nMcNutt P, Celver J, Hamilton T, et al.: Embryonic stem cell-derived neurons are a novel, highly sensitive tissue culture platform for botulinum research. Biochem Biophys Res Commun. 2011; 405(1): 85–90.PubMed Abstract | Publisher Full Text\n\nHubbard KS, Gut IM, Lyman ME, et al.: High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research. BMC Neurosci. 2012; 13: 127.PubMed Abstract | Publisher Full Text | Free Full Text\n\nBain G, Kitchens D, Yao M, et al.: Embryonic stem cells express neuronal properties in vitro. Dev Biol. 1995; 168(2): 342–357.PubMed Abstract | Publisher Full Text\n\nBibel M, Richter J, Lacroix E: Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nat Protoc. 2007; 2(5): 1034–1043.PubMed Abstract | Publisher Full Text\n\nGlaser T, Brustle O: Retinoic acid induction of ES-cell-derived neurons: the radial glia connection. Trends Neurosci. 2005; 28(8): 397–400.PubMed Abstract | Publisher Full Text\n\nLevine AJ, Brivanlou AH: Proposal of a model of mammalian neural induction. Dev Biol. 2007; 308(2): 247–256.PubMed Abstract | Publisher Full Text | Free Full Text\n\nHubbard KS, Gut IM, Lyman ME, et al.: High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research. BMC Neurosci. 2012; 13: 127.PubMed Abstract | Publisher Full Text | Free Full Text\n\nGermain N, Banda E, Grabel L: Embryonic stem cell neurogenesis and neural specification. J Cell Biochem. 2010; 111(3): 535–542.PubMed Abstract | Publisher Full Text\n\nWeimann JM, Zhang YA, Levin ME, et al.: Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron. 1999; 24(4): 819–831.PubMed Abstract | Publisher Full Text\n\nChen B, Schaevitz LR, McConnell SK: Fezl regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc Natl Acad Sci U S A. 2005; 102(47): 17184–17189.PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeyer G, Goffinet AM: Prenatal development of reelin-immunoreactive neurons in the human neocortex. J Comp Neurol. 1998; 397(1): 29–40.PubMed Abstract | Publisher Full Text\n\nGeorgala PA, Manuel M, Price DJ: The generation of superficial cortical layers is regulated by levels of the transcription factor Pax6. Cereb Cortex. 2011; 21(1): 81–94.PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi N, Zhao CT, Wang Y, et al.: The transcription factor Cux1 regulates dendritic morphology of cortical pyramidal neurons. PLoS One. 2010; 5(5): e10596.PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaspard N, Bouschet T, Hourez R, et al.: An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature. 2008; 455(7211): 351–357.PubMed Abstract | Publisher Full Text\n\nHansen DV, Rubenstein JL, Kriegstein AR: Deriving excitatory neurons of the neocortex from pluripotent stem cells. Neuron. 2011; 70(4): 645–660.PubMed Abstract | Publisher Full Text | Free Full Text\n\nJing Y, Machon O, Hampl A, et al.: In vitro differentiation of mouse embryonic stem cells into neurons of the dorsal forebrain. Cell Mol Neurobiol. 2011; 31(5): 715–727.PubMed Abstract | Publisher Full Text | Free Full Text\n\nSansom SN, Livesey FJ: Gradients in the brain: the control of the development of form and function in the cerebral cortex. Cold Spring Harb Perspect Biol. 2009; 1(2): a002519.PubMed Abstract | Publisher Full Text | Free Full Text\n\nButler AB: The evolution of the dorsal pallium in the telencephalon of amniotes: cladistic analysis and a new hypothesis. Brain Res Rev. 1994; 19(1): 66–101.PubMed Abstract | Publisher Full Text\n\nFlames N, Marin O: Developmental mechanisms underlying the generation of cortical interneuron diversity. Neuron. 2005; 46(3): 377–381.PubMed Abstract | Publisher Full Text\n\nNagy A, Rossant J, Nagy R, et al.: Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A. 1993; 90(18): 8424–8428.PubMed Abstract | Publisher Full Text | Free Full Text\n\nBibel M, Richter J, Schrenk K, et al.: Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nat Neurosci. 2004; 7(9): 1003–1009.PubMed Abstract | Publisher Full Text\n\nLi B, Dewey CN: RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12: 323.PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "831",
"date": "12 Mar 2013",
"name": "Cliff Ragsdale",
"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\nThere are a growing number of protocols for differentiating stem cells into particular neural cell types. This paper demonstrates the great potential of RNAseq technologies for assessing the identities of such differentiated cells in culture. The authors’ goal is an in vitro population of 'glutamatergic cortical neurons'. Although many of the genes catalogued show the anticipated profiles across the differentiation process (Otx2 abundance decreases with DIV while Kcnh5 reads increase), the dataset also demonstrates that this culture protocol may not be the best for 'glutamatergic cortical neuron' study as transcripts for the predominant cortical vesicular glutamate transporter gene, Vglut1/Slc17a7, are barely detected in the differentiated cell populations.",
"responses": [
{
"c_id": "395",
"date": "19 Mar 2013",
"name": "Patrick McNutt",
"role": "Author Response",
"response": "Despite the presence of a number of transcripts suggestive of telencephalogenesis, the singular derivation of vglut2+/vglut1- neurons has bothered us as well, and was one of the motivating factors underlying this experiment. While vglut2 expression has been reported in the neocortex, transcripts have generally been described to appear postnatally (e.g., see allen brain institute expression data for slc17a6 in the developing mouse brain) and vglut1 expression is much stronger than vglut2 in cortical glutamatergic neurons. We are still wading our way through the differential gene expression data, but preliminary, analysis suggests that the RNAseq data may be more consistent with a thalamic origin than a neocortical origin. Not only is thalamogenesis consistent with a vglut2+/vglut1- phenotype, but it is also less well described than neocortical development. We are currently working toward a clear differential diagnosis based on gene expression (should one exist) in an attempt to more clearly elucidate the structural origin of neurons derived in our model."
}
]
},
{
"id": "947",
"date": "14 May 2013",
"name": "Joyce van de Leemput",
"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 depth and temporal nature of the dataset presented in this paper will be beneficial to any researcher interested in cortical development in general, and potentially lead to many new insights and avenues to pursue. A point of note, in my experience differences in passage number of the cells used for differentiation can affect gene expression levels throughout. The authors state “ESCs were differentiated into neurons between 5-30 passages after adaptation to suspension culture.”, I wonder if that is why the DIV21 samples cluster in between the DIV16 and DIV28 when performing a PCA analysis on the transcript read counts (obtained from Data File 2)? Related question, how raw are the transcript read counts in Data File 2, as I thought raw counts would have to be integers whereas the counts given have decimal points? Finally, with regard to the previous Ref Report (Ragsdale and Albertin; 12 March 2013), have you considered comparative analysis using the Allen Brain Atlas/ Mouse Brain expression data for the thalamic and cortical areas and see which region your samples resemble most?",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-35
|
https://f1000research.com/articles/2-31/v1
|
04 Feb 13
|
{
"type": "Research Article",
"title": "Prediction of post-operative pain following arthroscopic subacromial decompression surgery: an observational study",
"authors": [
"Anthony Davis",
"David J Chinn",
"Sunil Sharma",
"David J Chinn",
"Sunil Sharma"
],
"abstract": "Background: Arthroscopic shoulder surgery is increasingly performed as a day case procedure. Optimal post-operative pain relief remains a challenge due to considerable variations in the level of pain experienced between individuals. Our aim was to examine whether the preoperative electrical pain threshold was a strong predictor of elevated postoperative pain levels following arthroscopic subacromial decompression (ASD) surgery. Methods: Forty consenting patients with American Society of Anesthesiologists (ASA) grade 1-2 presenting for elective ASD surgery were recruited. Patients’ electrical pain thresholds were measured preoperatively using a PainMatcher® (Cefar Medical AB, Lund, Sweden) device. Following surgery under general anaesthesia, the maximum pain experienced at rest and movement was recorded using a visual analogue scale until the end of postoperative day four. Results: In univariate analyses (t-test), the postoperative pain experienced (Area Under Curve) was significantly greater in patients with a low pain threshold as compared with a high pain threshold at both rest (mean 12.5, S.E. 1.7 v mean 6.5, S.E.1.2. P=0.008) and on movement (mean 18.7, S.E. 1.5 v mean 14.1, S.E.1.4. P=0.031). In multivariate analyses, adjusting for additional extra analgesia, the pain experienced postoperatively was significantly greater in the low pain threshold group both at rest (mean difference 4.9, 95% CI 1.5 to 8.4, P=0.007) and on movement (mean difference 4.1, 95%CI 0.03 to 8.2, P=0.049). Conclusions: Preoperative pain threshold can predict postoperative pain level following ASD of the shoulder. Trial registration: Clinicaltrials.gov identifier: NCT01351363 Level of Evidence: II",
"keywords": [
"Shoulder surgery",
"Subacromial decompression",
"Arthroscopic surgery",
"Pain prediction",
"Pain threshold",
"Postoperative pain"
],
"content": "Introduction\n\nIncreasingly, arthroscopic shoulder surgery is being performed as a day case procedure. In this setting, optimal post-operative pain relief is the goal. This not only improves patient comfort and allows expedient discharge from hospital, but also reduces the risk of developing post-operative chronic pain and may improve surgical outcome1–4.\n\nOptimal post-operative pain control in day-case surgery remains a challenge5. There is considerable variation in the level of post-operative pain experienced between individuals and subsequent analgesia requirements. Previous studies have attempted to predict the level of acute post-operative pain an individual will experience, using a variety of complex pre-operative pain and psychological assessments6,7. Other investigators have focussed on a simpler approach, by testing an individual’s pain threshold to a single pre-operative nociceptive stimulus, e.g. heat or pressure8–11. An increase in pain sensitivity in these pre-operative experimental tests appears to correlate with a higher level of post-operative pain and an increased risk of developing persistent post-operative pain12. Electrocutaneous stimulation has also been shown to be a reliable and safe method for assessment of pain and sensory thresholds6,13–18. The technique has previously been used as a predictive tool in the Obstetric surgery setting using the Pain Matcher® (Cefar Medical AB, Lund, Sweden)19.\n\nThe Pain Matcher is a small hand held device that has been validated against the visual analogue scale (VAS) for reliable pain assessment in patients with a range of acute and chronic pain, as well as for pre-operative pain threshold assessment19–24. When the contact pads on the Pain Matcher are gripped between the thumb and forefinger, the device delivers a small, micro-processor controlled, non-harmful electric current to the individual. Variations between individuals’ skin resistance are compensated for. The electrical current consists of rectangular pulses at 10 Hz with 10mA amplitude, which is gradually increased by 4 μs rises in pulse width, from zero to a maximum of 396 μs. This occurs over a total of 99 steps and ceases when the individual releases their grip on the device. The results are displayed on an LCD screen, on a scale from 1 to 99, which is directly related to the pulse width. Higher Pain Matcher values indicate a higher pain threshold17,18,21.\n\nWe aimed to assess the predictive value of pre-operative pain threshold measurements, made via electrocutaneous stimulation, for the intensity of post-operative pain experienced following arthroscopic subacromial decompression surgery.\n\n\nMaterials & methods\n\nEthical approval for this study (09/S0501/25) was provided by the NHS Fife & Forth Valley Research Ethics Committee, Dundee, UK (Chairperson Mr G Costa) on 9th March 2009.\n\nForty adult patients with American Society of Anesthesiologists (ASA) grade 1–2, presenting for elective day case arthroscopic subacromial decompression surgery at the Victoria Hospital, Kirkcaldy, UK, were recruited into the study between May 2009 and October 2010 at the pre-operative surgical assessment clinic, held one week prior to surgery. Full written consent was obtained from all study participants.\n\nExclusion criteria were: inability to give informed consent; inability to perform a telephone interview; an allergy to anaesthetic and analgesic drugs used in this study; drug or alcohol abuse; a formal diagnosis of chronic pain; a formal diagnosis of a neurological or psychiatric disorder; the use of neuromodulatory drugs, or daily analgesics; documented sensory abnormality (eg. peripheral neuropathy); or a documented rotator cuff tear.\n\nPatients were tested on the day of surgery following a six hour period of fasting and abstinence from any analgesics. We recorded age, Body Mass Index (BMI), ASA classification and telephone numbers.\n\nPre-operative electrical pain thresholds (EPT) were recorded using the Pain Matcher device. Patients were asked to grip the device until the stimulus was first experienced as painful. After an initial \"practice run\" with the Pain Matcher, pre-operative pain thresholds were measured using the patient’s hand contra-lateral to that side undergoing surgery.\n\nAnaesthesia was conducted by a single anaesthetist blinded to the patient’s EPT. Following application of standard monitoring, all patients received target controlled total intravenous anaesthesia (Orchestra, Fresenius-Kabi AG, Germany) using propofol (Marsh protocol; effect site concentration 3.5–7 μg/ml) and remifentanil (Minto protocol; effect site concentration 4–7 ng/ml) for both induction and maintenance of anaesthesia. Intermittent positive pressure ventilation was via a laryngeal mask airway, using oxygen and air (FiO2 50%). In addition, all patients received ondansetron 4mg, glycopyrrolate 200 μg and morphine 0.1mg/kg. All patients received an arthroscopic subacromial decompression, performed by a single orthopaedic surgeon, who also administered a subacromial bursa injection of 30ml 0.5% levobupivacaine (Abbott Laboratories, UK) at the commencement of surgery.\n\nUpon recovery from anaesthesia, patients were discharged from the theatre recovery suite once VAS pain scores (0–10) were less than 4. Intravenous morphine was administered to local protocols until this pain score was achieved. Additional rescue analgesia (oral tramadol 100mg) was available on the day case ward prior to discharge.\n\nPatients were discharged home on the day of surgery. Outpatient analgesia consisted of paracetamol 1g 6 hourly, codeine phosphate 60mg 6 hourly and diclofenac 50mg 8 hourly. Additional analgesia, if required, was available from the hospital or the patient’s General Practitioner.\n\nPatients were introduced to the use of a VAS pre-operatively and asked to record the worst pain experienced at rest and at movement for the first four postoperative days. Telephone numbers were obtained to allow the VAS scores to be obtained via a telephone interview on postoperative day five.\n\nThe highest pain score experienced at rest and on movement in each 24 hour period was recorded. The use of additional analgesics was also recorded.\n\nData were analysed with parametric and non-parametric tests using SPSS. The post-operative daily pain scores were used to determine an Area under the Curve (AUC)23 which was related to the pre-operative EPT scores in univariate and multivariate (linear regression) analyses allowing for potential confounders. A power analysis suggested we needed about 40 subjects for the multiple regression based on a recommendation of 10 patients per variable included20. A p value of less than 0.05 was accepted as being statistically significant and 95% confidence intervals (CI) were generated where relevant.\n\n\nResults\n\nForty patients meeting the inclusion criteria were initially approached and recruited into the study. No eligible patients declined to enter the study. Data were incomplete for 9 patients, who were removed from the study, leaving 31 patients (16 women, 15 men) (Table 1). Body mass index was above 30 kg/m2 in 10 patients (6 females).\n\nSD = Standard deviation.\n\nASA = American Society of Anesthesiologists grading score.\n\nThe median EPT for the total study group (n=31) was 21 (range: 4–99). Median EPT was 30 (range: 4–99) in men and 19 (range: 4–37) in women (P=0.022, Mann-Whitney U test).\n\nNo patients required additional morphine or tramadol post-operatively before discharge from hospital. Six patients requested additional analgesia in the first four post-operative days. The median post-operative pain scores and AUC are shown in Table 2.\n\nThe post-operative pain experienced (AUC) did not differ by gender (P=0.93 at rest and 0.78 on movement) or ASA grade (P=0.27 at rest and 0.31 on movement). Patients using additional analgesics (n=6) experienced significantly higher post-operative pain than those who did not (n=25) at both rest, mean (SE) AUC 17.8 (2.7) vs 7.6 (1.0) P<0.001 and at movement, mean (SE) AUC 20.7 (1.7) vs 15.4 (1.2) P=0.053 (un-matched t-tests).\n\nDue to the significant difference between male and female EPT scores, patients were subdivided into one of two groups depending on whether their EPT was below or above the gender-specific median (men 30 and women 19, respectively). Those below the median were judged to have a lower pain threshold compared to those above. In univariate analyses (t-test), the post-operative pain experienced at rest, as expressed by the AUC, was significantly greater in those with a low pain threshold (low EPT) as compared with a high pain threshold (high EPT) (P=0.008) (Table 3). The level of post-operative pain experienced on movement was similarly greater in the lower pain threshold (low EPT) group (P=0.031).\n\n* Below or equal to the gender-specific median Electrical Pain Threshold (EPT).\n\n** Above the gender-specific median EPT.\n\nAUC: Area under the Curve; SE: standard error; CI: confidence interval; P significance level on unmatched t-test.\n\nIn multivariate analyses, after adjusting for the requirement for extra analgesia, the pain experienced post-operatively (AUC) was significantly greater in the low pain threshold (low EPT) group both at rest (mean difference 4.9, 95% CI 1.5 to 8.4, P=0.007) and on movement (mean difference 4.1, 95% CI 0.03 to 8.2, P=0.049).\n\n\nDiscussion\n\nWe have shown that use of a simple pre-operative assessment of pain threshold can be used to predict those patients likely to experience a higher intensity of post-operative pain during the first 4 days following arthroscopic subacromial decompression surgery. Patients with a low pain threshold (EPT below/equal to the gender specific median) reported significantly more post-operative pain both at rest and on movement than those with a high pain threshold (EPT above the gender specific median). These results are in agreement with previous investigations in obstetric surgery that demonstrated pre-operative EPT testing can be used to gain information on the likely intensity of post-operative pain19. The relationship we have demonstrated appears to be clearer with post-operative pain at rest than with pain on movement. This may be because our study did not specify a standardized movement for pain assessment. In addition, we are also in agreement with previous studies, including those using other painful stimuli, which show that women have a lower pain threshold than men21,24.\n\nOur study population, whilst being generally healthy (ASA grade 1 and 2) was, on average, middle aged (mean 51 years) and “overweight” (mean BMI 28.4 kg/m3) with a third of patients having a BMI above 30, and therefore considered obese. We excluded volunteers suffering from conditions that may have influenced the experience of both preoperative and postoperative pain, i.e. chronic pain, heavy analgesic use or any neurological or psychiatric disorder, and only considered one type of arthroscopic shoulder surgery. It is likely that our results may not translate easily to other patient populations and different shoulder pathologies. Further study on these patient population groups is needed.\n\nThe Pain Matcher provides a non-harmful, rapid and easy to administer bedside test of pre-operative pain threshold. Our study did not test levels of pre-existing pain nor undertake any psychological testing, both of which are likely to have effects on pain perception. Despite the simplicity of the Pain Matcher pre-operative testing, our results are comparable to those produced by more elaborate studies8–11. Although our study contained a relatively small sample, the statistical power was sufficient to detect a difference in pain experienced in relation to pre-operative pain thresholds using simple comparisons and multiple regression analyses.\n\n\nConclusion\n\nPre-operative screening of patients with the Pain Matcher before arthroscopic subacromial decompression surgery may allow prediction of the likely intensity of post-operative pain experienced. It is a rapid and simple bedside test and may become a useful tool for targeting postoperative analgesia regimens. Further investigations are needed to elucidate whether this affects surgical outcome or the incidence of post-operative chronic pain.",
"appendix": "Author contributions\n\n\n\nAD, DC and SS contributed towards the study design. AD coordinated the data collection. DC conducted the statistical analysis. All authors contributed towards writing the paper and agreed to its final submission.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was financially supported by NHS Fife, Victoria Hospital, Fife, UK.\n\n\nAcknowledgements\n\nThe authors would like to thank Joy Neal, Physician’s Assistant, for her help during the course of this study.\n\n\nReferences\n\nKehlet H, Jensen TS, Woolf CJ: Persistent postsurgical pain: Risk factors and prevention. Lancet. 2006; 367(9522): 1618–25. PubMed Abstract | Publisher Full Text\n\nKehlet H, Wilmore DW: Multimodal strategies to improve surgical outcome. Am J Surg. 2002; 183(6): 630–41. PubMed Abstract | Publisher Full Text\n\nKehlet H, Holte K: Effect of postoperative analgesia on surgical outcome. Br J Anaesth. 2001; 87(1): 62–72. PubMed Abstract | Publisher Full Text\n\nPerkins FM, Kehlet H: Chronic Pain as an outcome of surgery. A review of predictive factors. Anesthesiology. 2000; 93(4): 1123–33. PubMed Abstract | Publisher Full Text\n\nWhite PF: Pain management after ambulatory surgery: where is the disconnect? Can J Anaesth. 2008; 55(4): 201–207. PubMed Abstract | Publisher Full Text\n\nBhalang K, Sigurdsson A, Slade GD, et al.: Associations among four modalities of experimental pain in women. J Pain. 2005; 6(9): 604–611. PubMed Abstract | Publisher Full Text\n\nWeissman-Fogel I, Granovsky Y, Crispel Y, et al.: Enhanced presurgical pain temporal summation response predicts post-thoracotomy pain intensity during the acute postoperative phase. J Pain. 2009; 10(6): 628–36. PubMed Abstract | Publisher Full Text\n\nAbrishami A, Chan J, Chung F, et al.: Preoperative pain sensitivity and its correlation with postoperative pain and analgesic consumption: a qualitative systematic review. Anesthesiology. 2011; 114(2): 445–57. PubMed Abstract | Publisher Full Text\n\nGranot M, Lowenstein L, Yarnitsky D, et al.: Postcesarean section pain prediction by preoperative experimental pain assessment. Anesthesiology. 2003; 98(6): 1422–6. PubMed Abstract\n\nHsu YW, Somma J, Hung YC, et al.: Predicting postoperative pain by preoperative pressure pain assessment. Anesthesiology. 2005; 103(3): 613–8. PubMed Abstract | Publisher Full Text\n\nWerner MU, Duun P, Kehlet H: Prediction of postoperative pain by preoperative nociceptive responses to heat stimulation. Anesthesiology. 2004; 100(1): 115–9. PubMed Abstract | Publisher Full Text\n\nGranot M: Can we predict persistent postoperative pain by testing preoperative experimental pain? Curr Opin Anaesthesiol. 2009; 22(3): 425–430. PubMed Abstract | Publisher Full Text\n\nAlstergren P, Förström J: Acute oral pain intensity and pain threshold assessed by intensity matching to pain induced by electrical stimuli. J Orofac Pain. 2003; 17(2): 151–9. PubMed Abstract\n\nGracely RH: Pain measurement. Acta Anaesthesiol Scand. 1999; 43(9): 897–908. PubMed Abstract | Publisher Full Text\n\nGracely RH: Studies of pain in normal man. In: Wall PD, Melzack R, eds. Textbook of pain. 4th ed. London: Churchill Livingstone, 1999; 385–407. Reference Source\n\nLund I, Lundeberg T, Kowalski J, et al.: Evaluation of variations in sensory and pain threshold assessments by electrocutaneous stimulation. Physiother Theory Pract. 2005; 21(xx): 81–92. PubMed Abstract\n\nLundeberg T, Lund I, Dahlin L, et al.: Reliability and responsiveness of three different pain assessments. J Rehabil Med. 2001; 33(xx): 279–283. PubMed Abstract\n\nStener-Victorin E, Kowalski J, Lundeberg T: A new highly reliable instrument for the assessment of pre- and postoperative gynecological pain. Anesth Analg. 2002; 95(xx): 151–7. PubMed Abstract\n\nNielsen PR, Nørgaard L, Rasmussen LS, et al.: Prediction of post-operative pain by an electrical pain stimulus. Acta Anaesthesiol Scand. 2007; 51(xx): 582–586. PubMed Abstract\n\nAltman DG: Practical Statistics for Medical Research. Chapman and Hall, London, 1991. Reference Source\n\nKäll LB, Kowalski J, Stener-Victorin E: Assessing pain perception using the Painmatcher in patients with whiplash-associated disorders. J Rehabil Med. 2008; 40(3): 171–7. PubMed Abstract | Publisher Full Text\n\nLundblad H, Kreicbergs A, Jansson KA: Prediction of persistent pain after total knee replacement for osteoarthritis. J Bone Joint Surg Br. 2008; 90-B(2): 166–171. PubMed Abstract | Publisher Full Text\n\nMatthews JN, Altman DG, Campbell MJ, et al.: Analysis of serial measurements in medical research. BMJ. 1990; 300(6719): 230–235. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiley JL 3rd, Robinson ME, Wise EA, et al.: Sex differences in the perception of noxious experimental stimuli: A meta-analysis. Pain. 1998; 74(2-3): 181–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "756",
"date": "07 Feb 2013",
"name": "Darin Correll",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is both interesting and potentially of great importance to help clinicians in improving postoperative pain management for patients. The issue however is that almost 25% of the patients originally enrolled were not included in the analysis without adequate explanation as to why this was (e.g. were they missing at random or not) nor a discussion of the fact that this may have had a major impact on the outcomes. Even though the 'data were incomplete' for these 9 patients there are methods for dealing with this from a statistical perspective that may allow a (more) complete analysis. The other option would have been to recruit more patients to make up for this loss and still meet (or come closer to) the pre-determined sample size.",
"responses": []
},
{
"id": "997",
"date": "12 Jun 2013",
"name": "Claudia Campbell",
"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, well-written and straight-forward study. The analyses appear to be appropriate to the study aims. Some explanation should be given for the exclusion of nine participants and information provided regarding if they are different from the rest of the sample in some way. Additional demographic information would also be of interest. For example, the duration of shoulder pain and pre-operative pain rating would be of value in placing these results in context.",
"responses": []
},
{
"id": "1010",
"date": "19 Jun 2013",
"name": "Irene Lund",
"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 order to contribute to a better control of postoperative pain, this is a very interesting, well described and important clinical study. There are, however, some issues to discuss:Explanation to why there were incomplete data for 9 patients.Description on how the postoperative pain assessments using VAS were performed.The rationale for using both parametric and non-parametric tests. In the manuscript the pain data (by the use of VAS and Pain Matcher) are shown as median and range, referring to that the dataset has ordinal properties, which are adequate for subjectively based assessments such as pain thresholds where the assumption of the distribution of the data is not necessary. In this case, the non-parametric analysis will give more meaning to the analysis than the use of parametric analysis that is based on data that have linear properties and that require knowledge of the data material distribution.The results are presented separate for women and men in table 1 and 2 which is very interesting since obvious gender differences are reported in threshold assessments. It would have been interesting to see separate results based on gender in table 3.The reported values of pain thresholds represent a wide range from 4-99 in men and 4-37 in women. In other studies using the Pain Matcher, the lowest level of your reported pain threshold is reported in the range of the sensory threshold assessed by Pain Matcher. Could the finding be due to the instruction given to the patients on what they were supposed to feel when reaching the pain threshold? In other words, could the sensation of unpleasantness, present before reaching the pain threshold, be a part of the results and thereby explain some of the great dispersion in the data?",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-31
|
https://f1000research.com/articles/2-27/v1
|
29 Jan 13
|
{
"type": "Research Article",
"title": "Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei",
"authors": [
"Claudia Colasante",
"Frank Voncken",
"Theresa Manful",
"Thomas Ruppert",
"Aloysius G M Tielens",
"Jaap J van Hellemond",
"Christine Clayton",
"Claudia Colasante",
"Frank Voncken",
"Theresa Manful",
"Thomas Ruppert",
"Aloysius G M Tielens",
"Jaap J van Hellemond"
],
"abstract": "In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.",
"keywords": [
"glycosome",
"peroxisome",
"membrane",
"lipid",
"proteome",
"Leishmania",
"Trypanosoma"
],
"content": "1. Introduction\n\nIn kinetoplastid protists, several metabolic pathways, including glycolysis, purine salvage and ether lipid biosynthesis, are located in a microbody, the glycosome1,2, which is evolutionarily related to peroxisomes. All evidence so far indicates that the glycosomal membrane, like the peroxisomal membrane, is impermeable to nucleotides, notably adenosine phosphates and NAD(P)(H)3,4. Its permeability to smaller molecules, however, is subject to debate5. Specific transporters would be required if the membrane were impermeable to molecules of the size of glycolytic intermediates, such as glucose, phosphate, malate, pyruvate, phosphoenolpyruvate and various triosephosphates.\n\nIn 1987, the first protein profile of glycosomal membranes from Trypanosoma brucei was published6. It revealed two abundant proteins of 24 and 26 kDa, which were later shown to be trypanosome homologues of the peroxisome biogenesis protein PEX117–9. Subsequent studies, including two of the glycosomal proteome1,10, revealed several more trypanosome PEX proteins that are predicted to be membrane-bound, such as PEX211,12, PEX1013, PEX1213, PEX1314 and PEX1415. The only transporters known to be associated with the glycosomal membrane are the ABC transporters GAT1, GAT2 and GAT3, which might transport fatty acids16. In addition, a member of the mitochondrial carrier protein family was found: MCP6, which is a candidate for nucleotide transport17. MCP6 is found preferentially in the glycosomal membranes of bloodstream-form trypanosomes, whereas in procyclic forms, it is predominantly targeted to the mitochondria17.\n\nNo analysis has yet yielded evidence for glycosomal transporters of metabolites smaller than about 400 Da. In contrast, lipid bilayers that were reconstituted with glycosomal membrane proteins revealed evidence for the presence of anion- and cation-selective pores5. The identities of these pore-forming proteins are still unknown: they could be dedicated exclusively to metabolite transport, or they might be involved in protein import as well5.\n\nIf proteins other than PEX components were indeed involved in metabolite transport, it ought to be possible to find them by mass spectrometry, using highly purified glycosomal membrane protein preparations. A similar proteomics approach has been previously used for mammalian peroxisomes. Analysis of carbonate-washed rat liver peroxisomes initially yielded only two peroxisomal membrane proteins, PMP70 and PMP2218, whereas a later analysis of whole mouse kidney peroxisomes led to the identification of 12 putative glycosomal membrane proteins. These included one tetratricopeptide domain protein, four different ABC transporters, three members of the PMP22 family, PMP34, Pxmp4/PMP4, and the putative solute carrier PMP4719.\n\nSpecific transporters for glycolytic metabolites might have been missed in previous glycosomal proteomic analyses, since glycosomal membrane proteins are likely to comprise a rather small proportion of the total protein content. We have therefore set out to identify the proteins in a highly enriched glycosomal membrane preparation from Leishmania tarentolae, using 30-times more starting material than used for our previously published T. brucei glycosomal proteome study1. Comprehensive mass spectrometry analysis of these highly purified glycosomal membrane protein fractions did not, however, lead to the identification of any novel glycosomal transporters. We therefore postulate that the recently described porin activity5 in the glycosomal membrane might be provided by known components of the glycosomal protein import machinery (peroxins), as has also been suggested for peroxisomes20. In addition, we compared the phospholipid compositions of the glycosomal membranes from bloodstream- and procyclic-form T. brucei, with the lipid composition of the T. brucei cell membrane to see if this could give us more information regarding glycosomal membrane transport.\n\n\n2. Materials and methods\n\nLeishmania tarentolae promastigotes were cultured at 28°C in 3.7 L hemin-supplemented brain-heart infusion medium to a maximum density of 2 × 108 cells/ml. Procyclic-form Trypanosoma brucei Lister 427 was grown at 30°C in 10% (v/v) foetal calf serum-supplemented MEM-PROS medium to a maximum density of 5 × 106 cells/ml21. Bloodstream-form T. brucei 427 was grown at 37°C in 10% (v/v) foetal calf serum-supplemented HMI-9 medium22 to a maximum density of 2 × 106 cells/ml21,23.\n\nProcyclic-form and bloodstream-form T. brucei (1010 cells each), and promastigote L. tarentolae (1012 cells) were harvested by centrifugation for 10 min at 2,000x g, and were washed once in 50 ml of TEDS (25 mM Tris, 1 mM EDTA, 1 mM DTT, 250 mM sucrose, pH 7.8). After centrifugation, the cell pellet was resuspended in 2 ml homogenization medium (250 mM sucrose, 1 mM EDTA, 0.1% (v/v) ethanol, 5 mM MOPS, pH 7.2) containing protease inhibitor (complete EDTA-free, Roche Applied Science) and was grinded in a pre-chilled mortar with 1 volume of wet-weight silicon carbide (Crysalon: Norton Company: porous <400 mesh). Cells were checked for at least 90% disruption by light microscopy. The cell lysate was centrifuged sequentially for 5 minutes each at 100x g and 3,000x g to remove abrasive, intact cells, cell rests and nuclei. The supernatant was centrifuged for 15 minutes at 17,000x g to yield the glycosome-enriched pellet fraction. This fraction was resuspended in 3 ml of homogenization buffer and loaded on top of a 32 ml linear 20–40% (v/v) Optiprep (iodixanol-sucrose, Sigma Biochemicals) gradient, mounted on a 3.5 ml 50% (v/v) Optiprep cushion (Optiprep Application Sheet S9, Axis-shield). Centrifugation was performed for 1 h at 170,000x g and 4°C using a Beckman VTi-50 Rotor. 1 ml aliquots were collected from the bottom of the tube after puncture, and the protein concentration of each fraction was determined using the BioRad Bradford protein assay. Of each fraction, 100 µl was TCA-precipitated and the resulting pellets resuspended in denaturing Laemmli SDS-PAGE buffer. Proteins were separated on a 12% SDS-PAGE gel and analysed by western blotting.\n\nGlycosomes (corresponding to about 0.5 mg protein) were diluted 1:5 in TEDS (see 2.1), subjected to two freeze-thaw cycles, and centrifuged for 40 minutes at 140,000x g and 4°C. The resulting pellet was washed with 5 M urea for 1 h at 4°C to remove proteins that were not tightly associated with the glycosomal membranes. The glycosomal membranes were pelleted by centrifugation for 40 minutes at 140,000x g and 4°C. This 5 M urea wash-step was repeated once. The glycosomal membrane-enriched fraction was resuspended in denaturing Laemmli SDS-PAGE buffer and separated by SDS PAGE. In-gel trypsin digestion and nanoLC-MS/MS analysis of the obtained protein bands were performed as previously described1. The obtained MS/MS spectra were analysed using MASCOT software and visualised in Scaffold. The comparison shown in Supplementary Table 1 was done using the 2012 version of the shotgun sequence of L. tarentolae (http://tritrypdb.org/)24; only proteins for which at least 2 different peptides could be identified with >95% confidence were included. Leishmania proteins were first scanned for the presence of a PTS1 signal based on a published analysis for L. major and T. brucei2 and by manually examining the C-terminal sequences. Additional PTS1-containing proteins were identified using PTS1 Predictor25,26. Trans-membrane domains were identified using the TritrypDB annotation database. For potential glycosomal proteins with no known function and without an annotated trans-membrane domain, we also scanned for trans-membrane domains using the HMMTOP and SOSUI algorithms27,28.\n\nLipids were extracted in triplicate from bloodstream and procyclic T. brucei samples and from a single batch of isolated glycosomes due to the limited amount of purified material. Lipids were extracted according to the method of Bligh and Dyer (1959)29 with the minor modification that 0.5% (v/v) 6 M HCl was added to the second chloroform wash to increase recovery of acidic phospholipids. The phospholipids and free fatty acids were separated from neutral lipids (cholesterol, cholesterol esters and triacylglycerols) by fractionation on a 1 ml silica column prepared from 0.063–0.200 mm silica 60 (Merck, Darmstadt, Germany). Lipid extracts were dissolved in chloroform and loaded on the silica column, then eluted successively with acetone (4 volumes) and methanol (4 volumes). The last fraction, which contained the purified phospholipids, was dried under nitrogen and stored at -20°C until HPLC-MS analysis.\n\nThe purified phospholipids were dissolved in methanol:acetonitrile:chloroform:water (46:20:17:17). Separation of molecular lipid species was performed on a Synergi 4 µm MAX-RP 18A column (250 × 3 mm; Phenomenex, CA, USA). Elution was performed with a linear gradient of water in methanol/acetonitrile (60/40 (v/v)) decreasing from 12.5% to 0% in 25 min, followed by further isocratic elution for another 25 minutes. The flow rate was kept constant at 0.425 ml•min-1 and 1 µM serine and 2.5 mM ammonium acetate were used in all solvents as additives.\n\nMass spectrometry of lipids was performed using electrospray ionization, on a 4000 QTRAP system (Applied Biosystems, Nieuwerkerk aan de IJssel, The Netherlands). Source temperature was set to 450°C and nitrogen was used as curtain gas. The declustering potential was optimized using lipid standards. The optimal collision energy was dependent on the type of experiment performed and was set to +45V (precursor scanning m/z 184), -45V (precursor scanning m/z -196), +35V (neutral loss 141), -30V (precursor scanning m/z -241), and -40V (neutral loss scanning 87 Da) respectively. For quantification of molecular species, samples were measured in multiple-reaction monitoring mode (MRM), monitoring for 95 head-group specific mass transitions with a total dwell time of 1 s, using the same settings as above. Data analysis was performed with Analyst™ v 1.4.1 software (MDS Sciex, Concord, ON).\n\n\n\n\n3. Results\n\nIn preliminary experiments (not shown), we tested various methods for purification of membranes of iodixanol gradient-enriched T. brucei glycosomes1. The methods tested included methanol/chloroform extraction30; ultracentrifugation of glycosomes that had been subjected to osmotic shock with cold water31,32; and different high salt (0–1 M NaCl or 5 M urea) washes of glycosomal membranes32. Although we were able to enrich glycosomal membrane proteins, as judged by the presence of PEX117, the matrix protein aldolase persisted. In addition, the total amount of membrane protein obtained from 3 × 1010 T. brucei was so low that we doubted that any lower-abundance proteins would be detected by mass spectrometry. We therefore decided to isolate glycosomes from the related kinetoplastid L. tarentolae to increase the sensitivity for the detection of even low-abundant glycosomal membrane proteins. In contrast to T. brucei, L. tarentolae can be grown to far higher cell densities, enabling us to isolate glycosomes from as much as 1012 cells. The different fractions obtained after differential fractionation and subsequent density gradient (Optiprep) centrifugation were analysed by western blotting (Figure 1). The gradient distributions of the two marker proteins glyceraldehyde phosphate dehydrogenase (glycosomes) and HSP60 (mitochondria) are shown in Figure 1A. Comparison of previously published density gradient results from T. brucei1 with those from L. tarentolae (Figure 1A) showed that the mitochondria were enriched at similar gradient densities (fractions 22–25), whereas the glycosomes isolated from L. tarentolae appeared to have a higher buoyant density than those of T. brucei. In addition, both the mitochondria and glycosome-containing fractions were spread out over a wide range of fractions for the L. tarentolae gradient, which could be the result of breakage of the organelles during isolation. Judging from the western blotting results (alternate gradient fractions shown in Figure 1A), fractions 9, 11 and 13 contained about 42% of the total GAPDH measured, but only 2% of the total HSP60. We therefore decided to use fractions 9–13 for further glycosomal membrane purification.\n\nA. Western blot analysis of the different L. tarentolae fractions obtained after density gradient centrifugation. Equal volumes of only the odd-numbered fractions were loaded for analysis. Antibodies used for detection are indicated next to the western blot panels. Mitochondrial19–29 and glycosomal9–19 density gradient fractions are indicated. B. SDS-PAGE gel stained with Coomassie brilliant blue, showing protein bands from intact glycosomes (glycosomal fraction, and from the glycosomal membrane-enriched pellet (urea pellet). Arrows indicate enriched proteins in the urea-treated glycosomal membrane fraction.\n\nTo purify glycosomal membranes, we found that the protocol that gave least matrix protein contamination was one that was successfully employed to isolate the cell membrane of E. coli33. It involved washing the glycosomal pellet with 5 M urea, and resulted in strong depletion of some prominent bands (presumably matrix proteins) and the enrichment of various proteins in the 10–25 kDa range (Figure 1B) - similar to the expected sizes of the PEX11 protein homologues7,9. The entire SDS-PAGE lane containing the enriched glycosomal protein fraction (Figure 1B) was subsequently subjected to mass spectrometry.\n\nBy comparison with the predicted proteome of L. tarentolae24, 464 polypeptides were identified (Supplementary Table S1). The first step that we undertook was to identify homologues of all identified proteins from the T. brucei genome (http://tritrypdb.org/tritrypdb/). This was done to facilitate the retrieval of information because most experimental data is available exclusively for T. brucei. All identified proteins were screened for database annotation, including user comments, and in some cases we also updated annotations from publications. We further screened all proteins for their presence in previously published glycosomal1,10 and mitochondrial34 proteomes. The results are summarised in Supplementary Table S1, Sheet 1. Proteins that were clearly located in compartments other than the glycosome were then excluded, resulting in Supplementary Table S1, Sheet 2. Some candidates predicted to contain at least one trans-membrane domain were tested for their locations, by expression of N-terminally and/or C-terminally tagged versions (none has a PTS1 signal). The proteins encoded by Tb927.3.1840 (putative 3-oxo-5-alpha-steroid 4-dehydrogenase), Tb927.5.1210 (putative short-chain dehydrogenase) and Tb927.10.14020 (unknown function) were all targeted to mitochondria, while Tb927.7.3900 (annotated as a vacuolar transporter chaperone) was in the ER (Supplementary Figure S1). All identified proteins were further searched for the presence of known peroxisomal targeting signals in the L. major or T. brucei homologues35; in addition, the L. tarentolae protein sequences in Supplementary Table S1, Sheet 2 were manually scanned for PTS1 signals.\n\nThe L. tarentolae glycosomal membrane preparations revealed the presence of 40 known or predicted glycosomal matrix proteins, and some novel proteins (Supplementary Table S1, sheet 2). A putative glycosomal pathway scheme, incorporating all available information for L. tarentolae and T. brucei, is shown in Figure 2. Glycolytic enzymes, enzymes involved in the conversion of glycerone phosphate to glycerol, the pentose phosphate pathway, steroid and nucleotide biosynthesis as well as enzymes of the succinic fermentation branch were detected. Similar to results obtained for the T. brucei glycosome1, fumarase (EC 4.2.1.2) is the only enzyme of the glycosomal succinic fermentation branch that was not found in the L. tarentolae glycosomal membrane preparation. It is possible that fumarase was removed in the membrane purification; alternatively, the activity may be supplied by one of the four proteins of unknown function that are conserved in kinetoplastids and have an unambiguous PTS1: LtaP34.3290/Tb927.4.1360, LtaP33.2650/Tb927.11.2620, LtaP18.0870/Tb927.10.13240, or LtaP24.1780/Tb927.8.6640 - although fumarase activity would be surprising since they lack known functional domains. Fumarase catalyses the conversion of malate to fumarate, which is an indispensable step towards the generation of succinate in the glycosomal matrix. If indeed the glycosome lacks fumarase then the glycosomal membrane must harbour a malate-fumarate shuttle. This shuttle would be responsible for the transport of glycosome-derived malate in exchange with mitochondria-derived fumarate. Inside the glycosome fumarate could then be converted to succinate by fumarate reductase (EC 1.3.1.6) to maintain the glycosomal NADH balance.\n\nThe scheme summarizes metabolic pathways identified so far in the glycosomes of Leishmania and T. brucei. EC numbers of enzymes identified only in the Leishmania glycosome are indicated in black boxes with white text, those identified only in T. brucei are indicated in italics, and those found in the glycosome of both species are indicated in bold. Predicted transport processes across the glycosomal membrane are indicated by the circled question marks and dashed arrows. Letters in black circles indicate the different metabolic pathways as follows: A: glycolysis; B: succinic fermentation; C: pentose phosphate pathway; D: superoxide and trypanothione metabolism; E: purine salvage; F: pyrimidine metabolism; G: mannose metabolism; H: glycerolipid biosynthesis; I: β-oxidation of fatty acids; L: phosphoarginine metabolism; M: mevalonate pathway; N: phospholipid degradation. Abbreviations used are: Acyl-GPC, 1-acyl-glycero-phosphocholine; Acyl-GPE, 1-acyl-glycero-phosphoethanolamine; AOX, alternative oxidase; DHA, dehydroascorbate; MDHA, monodehydroxyascorbate; NB, nucleobases; PRPP, 5-phosphoribosyl 1-pyrophosphate.\n\nThe L. tarentolae glycosomal membrane preparation contained several enzymes that were not detected during LC-MS analysis of the T. brucei glycosome. For example, glucosamine-6-phosphate isomerase was found in the glycosomes of Leishmania, but not in trypanosomes1,10. In addition, a PTS1-containing D-lactate dehydrogenase-like protein is found in Leishmania, for which there is no obvious substrate, as well as a PTS1-containing xylulokinase and a glucokinase-like protein. These additional enzymes involved in the metabolism of sugars might indicate a higher metabolic flexibility of L. tarentolae compared to African trypanosomes. In T. brucei, the phosphomannomutase Tb927.10.6440 was found in the glycosome, where it can act as phosphoglucomutase during glycolysis36. T. brucei phospho-N-acetylglucosamine mutase (Tb927.8.980) was also partially glycosomal36. Neither has an obvious PTS1 targeting signal so it is possible that they have either an internal glycosomal targeting signal or that they are co-imported via association with other glycosomal targeting signal-containing proteins37,38. The syntenic L. tarentolae homologues LtaP36.1960 and LtaP07.0850 were not present in our dataset, but the different non-syntenic phosphomannomutase-like protein LtaP34.3710, containing the conserved C-terminal PTS1 signal -SKL, was found. The T. brucei aldose 1-epimerase, Tb927.4.1360, is the homologue of LtaP34.3290, but Leishmanias have an additional isoform, LtaP35.110, containing a conserved PTS1 targeting signal.\n\nThe first three steps of ether-lipid biosynthesis in T. brucei, namely the conversion of glycerone 3-phosphate to 1-alkyl-glycerol-3-phosphate, are associated with the glycosome39. T. brucei glycosome proteomic analysis confirmed the presence of the second enzyme of the biosynthetic pathway, namely alkyl-glycerone-phosphate synthetase (EC 2.5.1.26). The first and the third enzymes, glycerone-phosphate acyl-transferase (EC 2.3.1.42) and 1-acyl-glycerol-3-phosphate oxidoreductase (EC 1.1.1.101) were not identified. The T. brucei glycerone 3-phosphate acetyltransferase/acyltransferase homologue, Tb927.4.3160, has a C-terminal PTS1, SRM. Analysis of the glycosomal membrane proteome of L. tarantolae identified not only alkyl-glycerone 3-phosphate synthetase (EC 2.5.1.26) but also glycerone 3-phosphate acetyltransferase/acyltransferase (EC 2.3.1.42, LtaP34.1280, C-terminal PTS1 -SKM) supporting the idea that the first two steps of the ether-lipid biosynthesis can occur inside the glycosome.\n\nThe β-oxidation of long chain fatty acids (LCFA) is one of the hallmark catabolic pathways attributed to peroxisomes (40 and references therein). Our previous analysis suggested that the glycosome of T. brucei was devoid of LCFA β-oxidation enzymes1. On the other hand, 2-enoyl coenzyme A hydratase (EC 4.2.1.17) and NADP-dependent 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) were reported in glycosomal fractions from procyclic-form T. brucei40. According to the results from this proteome analysis, the L. tarentolae glycosome might contain all enzymes involved in the β-oxidation of LCFA except acyl-CoA dehydrogenase (Figure 2, Supplementary Table S1, sheet 2). We found one protein (LtaP24.1780, annotated as hypothetical), which is clearly a fatty acyl Co-A reductase and contains a C-terminal -SSL; another hypothetical protein (LtaP16.0130) contains -AKL and an acyl CoA dehydrogenase domain; and the enoyl-CoA hydratase/enoyl-CoA isomerase/3-hydroxyacyl-CoA dehydrogenase trifunctional enzyme homologues (EC 4.2.1.17/5.3.3.8/1.1.1.35, LtaP26.1590 and LtaP33.2830) containing putative PTS2 signals35. Other detected enzymes of this pathway without obvious peroxisomal targeting signals were long-chain-fatty-acyl-CoA synthetase (EC 6.2.1.3) and thiolase (EC 2.3.1.16); these might have internal signals or could be contaminants from another compartment.\n\nMevalonate kinase is known to be in the glycosome41, and another enzyme of the mevalonate pathway, isopentenyl-diphosphate delta-isomerase, was also present in our glycosomal preparation; but intermediate enzymes (phosphomevalonate kinase and mevalonate-5-pyrophosphate decarboxylase) were not (Figure 2, Supplementary Table S1, sheet 2). Finally, we found a PTS1-containing phosphoribulokinase/uridine kinase family protein, LtaP14.0950, belonging to the P-loop NTPase superfamily; we speculate that this enzyme might be involved in pyrimidine salvage.\n\nWe next focussed on the identification of putative membrane proteins. Known glycosomal membrane proteins and unassigned proteins that had predicted membrane-spanning domains in both L. tarentolae and T. brucei are listed in Supplementary Table S1, sheet 3. These proteins were manually analysed for conserved functional domains, and their protein sequences were aligned to the complete predicted proteomes of Homo sapiens, Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris on the NCBI Web site using Blastp. This enabled the exclusion of further proteins that were deemed likely to be in other (non-peroxisomal) subcellular compartments.\n\nThe list of known glycosomal membrane proteins that we identified is shown in Table 1. It included three proteins related to PEX11 (PEX11, GIM5A and GIM5B), PEX2 and PEX14. The protein encoded by Tb09.160.4700/Tb927.9.6450 has a very weak match to a conserved PEX16 domain (E-value 9e-3). We have therefore annotated this as a putative PEX16. It could therefore be involved in the incorporation of peroxisomal membrane proteins42, although high-throughput RNAi screening revealed no growth defects for RNAi targeting this locus43.\n\nThis list includes all mass-spectrometry-detected glycosomal membrane proteins.\n\nAbbreviations used: Lta, Leishmania tarentolae; Tb, Trypanosoma brucei.\n\nThe trypanosome homologue of the mammalian peroxisomal membrane protein PMP2444 (also called PXMP4 or PMP4) was also found. Like other PMP24/PMP4 proteins, a conserved TIM27 superfamily domain is present in Tb927.9.1720; the function of this domain (and of PMP4) is still unknown. Although S. cerevisiae that lack it show abnormal peroxisome size and numbers45, no growth defects were seen in the high-throughput RNAi screens in T. brucei43. Of the previously reported T. brucei glycosomal ABC transporters GAT1-316, only GAT1 and GAT3 were found in the L. tarentolae glycosomal proteome. This was not unexpected since the annotated L. tarentolae GAT2 protein sequence is severely truncated. Either L. tarentolae lacks a functional GAT2, or this is a genome assembly error; the former is quite possible since no peptides matching L. major and L. infantum GAT2 were found.\n\nOf the remaining potential membrane proteins, four could be tentatively assigned to the mitochondrion, three to the endoplasmic reticulum, two to the flagellum, and one to the nucleus (Supplementary Table S1, sheet 3). The remaining proteins are listed in Table 2. A protein of the major facilitator family (Tb927.3.4070-110, LtaP29.1650) was the only conserved multi-pass membrane protein that had no clear assignment to another subcellular compartment, but the 3 identified peptides covered only 3% of the protein. Best matches to this sequence are ion transporters. The Tb927.9.4310 protein sequence matches a yeast possible alpha-isopropylmalate carrier, which exports alpha-isopropylmalate from the mitochondrion to the cytoplasm for use in leucine biosynthesis. The remaining candidates have either one or two potential trans-membrane domains, usually predicted by only one algorithm.\n\nThis list includes all detected proteins with predicted trans-membrane (TM) domains, for which there is no evidence for location in any particular compartment.\n\nAbbreviations used: Lta, Leishmania tarentolae; Tb, Trypanosoma brucei; Pep nr, number of identified peptides; Cov (%), percentage of protein coverage; TMs (a), number of annotated trans-membrane domains; TMs (b), number of transmembrane-domains identified by THMM TOP; ER sp, endoplasmic reticulum signal peptide.\n\nOverall, our proteomics analysis revealed no clear candidates for major novel glycosomal metabolite transporters.\n\nThe phosphatidylcholine species composition in membranes from total T. brucei cells and glycosomes were analysed to detect possible differences in membrane composition between glycosomal and the other membranes of T. brucei, and to allow comparison in membrane composition between the two cultivatable replicating life cycle stages, the bloodstream form and procylic form. The phosphatidylcholine species composition of total trypanosome membranes differed to some extent between bloodstream and procyclic stages (Table 3), which is consistent with earlier reports on the phospholipid composition in T. brucei46. Membranes of both stages contain predominantly diacyl-phosphatidylcholine species, comprising common fatty acids consisting of 16 to 22 carbon atoms with 0 to 6 desaturations. In addition, membranes of intact trypanosomes also contained ether phospholipids species, both 1-alkyl-2-alkyl phosphatidylcholine species and 1-alkyl-2-acyl phosphatidylcholine species. The phosphatidylcholine species composition of glycosomal membranes differed only to a minor extent from the composition observed in total membranes of both bloodstream form and procyclic form trypanosomes (Table 3).\n\nThe phosphatidylcholine species description comprises the sn-1 linkage type followed by the radyl chains on the sn-1 and sn-2 position, respectively. \"Total cell\" values are mean of three independent experiments. Most abundant species representing over 5 Mol % are marked in bold. Abbreviations: AlkCho, 1-alkyl, 2-acyl phosphatidylcholine; BSF, bloodstream-form T. brucei; EnylCho, 1-alkyl-1-enyl-2-acyl phosphatidylcholine; nd, not detected; PCF, procyclic-form T. brucei; PtdCho, diacyl phosphatidylcholine.\n\n\n4. Discussion\n\nThe glycosome is a major contributor to kinetoplastid energy metabolism and essential for glycolysis3,4,47. Flux rates through the glycolytic pathway are high in trypanosomes and - judging from the peptide counts and protein coverage in our analysis - the enzymes are also abundant in Leishmania. A model of trypanosome glycolysis that assumes free exchange of glucose between the organelle and the cytosol mirrors the in vivo kinetics3,47. This suggests that the protein responsible for glucose transport should be very active and probably also abundant. Our analysis of the glycosomal membrane proteome, however, failed to identify abundant membrane proteins that might fulfil such a role. Given the large number of proteins that we identified - including multiple membrane proteins from other compartments - it seems unlikely that our failure to detect transporters for such major metabolites could be due solely to lack of sensitivity. Dual subcellular locations could be a possible explanation for some proteins, as previously shown for MCP617. The five additional mitochondrial carrier proteins that we detected are predominantly in the mitochondrion of procyclic T. brucei48, but the presence of a minor amount in glycosomes cannot be ruled out. If so, they could possibly function as putative glycosomal isocitrate/2-ketoglutarate and fumarate/malate shuttles.\n\nWe investigated the lipid composition of glycosomal membranes in T. brucei by analysis of the species composition of phosphatidylcholine, the most abundant phospholipid class in membranes of both procyclic and bloodstream form T. brucei46. The phospholipid composition of peroxisomal membranes has been investigated in peroxisomes isolated from rat liver and from the yeasts Saccharomyces cerevisiae and Pichia pastoris49–51. In rat liver peroxisomes the phospholipid classes and their fatty acid composition were similar to those of homogenates and microsomes, although exposure to the endotoxin lipopolysaccharide (LPS) induced significant changes in phospholipid species composition: in particular, the abundance of both long chain fatty acids (>20 C atoms) and poly-unsaturated fatty acids increased in peroxisomal membranes49. The lipid composition of peroxisomes in yeasts was shown to be rather flexible, predominantly depending on the type and amount of fatty acid supply in the medium50,51. The lipid composition of glycosomes in Trypanosomatidae has not been investigated before and our results showed that the phosphatidylcholine species composition in glycosomal membranes resembled that of other cellular membranes in both bloodstream-form and procyclic-form T. brucei. These results suggest that the lipid composition, and thus the biophysical properties, of the glycosomal membrane is similar to that of the other membranes in trypanosomes. Because of this similarity to other cellular membranes, glycosomal membranes are expected to be also impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins.\n\nThere is accumulating evidence that small solutes enter microbodies through pores5,20,52,53. Evidence from a mammalian Pxmp2 (PMP22) knock-out mouse suggested loss of peroxisomal pores for solutes of under 300 Da53 and this type of function was confirmed when the protein was expressed in insect cells. No Pxmp2 homologue appears to exist in kinetoplastids. Instead, it has been speculated that PEX11 family proteins might contribute to glycosomal porin activity5,8. Our failure to find abundant novel glycosomal transporters is consistent with the hypothesis that the PEX11 family proteins are indeed responsible for the transfer of small solutes in and out of the glycosome.",
"appendix": "Author contributions\n\n\n\nC. Colasante was responsible for the glycosome and glycosomal membrane purification, with supervision from FV and C. Clayton. C. Colasante was responsible for biochemical pathway analysis. JvH and AGMT were responsible for the lipid analysis, and TR for the mass spectrometry. C. Colasante, TM and C. Clayton contributed bioinformatic analysis of the identified proteins. The paper was written by C. Colasante, FV, JvH and C. Clayton.\n\n\nCompeting interests\n\n\n\nThe authors have no competing interests to declare.\n\n\nGrant information\n\nWork by C. Colasante and F. Voncken was supported by the Deutsche Forschungsgemeinschaft (DFG) (Cl112/10).\n\n\nSupplementary figure\n\nN-terminally or C-terminally myc-tagged versions of the proteins were detected (green) by using a commercial Myc antibody (Sigma-Aldrich). Mitochondria were visualized (red) using mitotracker. The endoplasmatic reticulum (ER) was detected using an antibody directed against the ER lumen protein BiP (red). Overlays (Merge) of the green staining and the red staining are shown to visualize the common compartmentalization of the proteins. On the right side, western blots are shown to illustrate the expression of the myc-tagged membrane proteins. (+) and (-) indicate tetracycline-induced and -uninduced cells respectively.\n\n\nReferences\n\nColasante C, Ellis M, Ruppert T, et al.: Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei. Proteomics. 2006; 6(11): 3275–3293. PubMed Abstract | Publisher Full Text\n\nVertommen D, Van Roy J, Szikora JP, et al.: Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei. Mol Biochem Parasitol. 2008; 158(2): 189–201. PubMed Abstract | Publisher Full Text\n\nHaanstra JR, van Tuijl A, van Dam J, et al.: Proliferating bloodstream-form Trypanosoma brucei use a negligible part of consumed glucose for anabolic processes. Int J Parasitol. 2012; 42(7): 667–673. PubMed Abstract | Publisher Full Text\n\nBakker BM, Mensonides FI, Teusink B, et al.: Compartmentation protects trypanosomes from the dangerous design of glycolysis. Proc Natl Acad Sci U S A. 2000; 97(5): 2087–2092. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGualdron-López M, Vapola MH, Miinalainen IJ, et al.: Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei . PLoS One. 2012; 7(4): e34530. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAman RA, Wang CC: Identification of two integral glycosomal membrane proteins in Trypanosoma brucei. Mol Biochem Parasitol. 1987; 25(1): 83–92. PubMed Abstract | Publisher Full Text\n\nLorenz P, Maier AG, Erdmann R, et al.: Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p. EMBO J. 1998; 17(13): 3542–3555. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVoncken F, van Hellemond JJ, Pfisterer I, et al.: Depletion of GIM5 causes cellular fragility, a decreased glycosome number, and reduced levels of ether-linked phospholipids in trypanosomes. J Biol Chem. 2003; 278(37): 35299–35310. PubMed Abstract | Publisher Full Text\n\nMaier A, Lorenz P, Voncken F, et al.: An essential dimeric membrane protein of trypanosome glycosomes. Mol Microbiol. 2001; 39(6): 1443–1451. PubMed Abstract | Publisher Full Text\n\nVertommen D, Van Roy J, Szikora JP, et al.: Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei. Mol Biochem Parasitol. 2008; 158(2): 189–201. PubMed Abstract | Publisher Full Text\n\nFlaspohler JA, Rickoll WL, Beverley SM, et al.: Functional identification of a Leishmania gene related to peroxin 2 reveals common ancestry of glycosomes and peroxisomes. Mol Cell Biol. 1997; 17(3): 1093–1101. PubMed Abstract | Free Full Text\n\nGuerra-Giraldez C, Quijada L, Clayton CE: Compartmentation of enzymes in a microbody, the glycosome, is essential in Trypanosoma brucei. J Cell Sci. 2002; 115(Pt 13): 2651–8. PubMed Abstract\n\nKrazy H, Michels PA: Identification and characterization of three peroxins–PEX6, PEX10 and PEX12–involved in glycosome biogenesis in Trypanosoma brucei. Biochim Biophys Acta. 2006; 1763(1): 6–17. PubMed Abstract | Publisher Full Text\n\nBrennand A, Rigden DJ, Michels PA: Trypanosomes contain two highly different isoforms of peroxin PEX13 involved in glycosome biogenesis. FEBS Lett. 2012; 586(13): 1765–71. PubMed Abstract | Publisher Full Text\n\nMoyersoen J, Choe J, Kumar A, et al.: Characterization of Trypanosoma brucei PEX14 and its role in the import of glycosomal matrix proteins. Eur J Biochem. 2003; 270(9): 2059–67. PubMed Abstract | Publisher Full Text\n\nYernaux C, Fransen M, Brees C, et al.: Trypanosoma brucei glycosomal ABC transporters: identification and membrane targeting. Mol Membr Biol. 2006; 23(2): 157–72. PubMed Abstract | Publisher Full Text\n\nColasante C, Alibu VP, Kirchberger S, et al.: Characterization and developmentally regulated localization of the mitochondrial carrier protein homologue MCP6 from Trypanosoma brucei. Eukaryotic Cell. 2006; 5(8): 1194–1205. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIslinger M, Luers GH, Zischka H, et al.: Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments. Proteomics. 2006; 6(3): 804–816. PubMed Abstract | Publisher Full Text\n\nWiese S, Gronemeyer T, Ofman R, et al.: Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling. Mol Cell Proteomics. 2007; 6(12): 2045–2057. PubMed Abstract | Publisher Full Text\n\nVisser WF, van Roermund CW, Ijlst L, et al.: Metabolite transport across the peroxisomal membrane. Biochem J. 2007; 401(12): 365–375. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlibu VP, Storm L, Haile S, et al.: A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei. Mol Biochem Parasitol. 2005; 139(1): 75–82. PubMed Abstract | Publisher Full Text\n\nHirumi H, Hirumi K: Continuous cultivation of Trypanosoma brucei bloodstream forms in a medium containing a low concentration of serum protein without feeder cell layers. J Parasitol. 1989; 75(6): 985–989. PubMed Abstract\n\nvan Deursen FJ, Shahi SK, Turner CM, et al.: Characterisation of the growth and differentiation in vivo and in vitro of bloodstream-form Trypanosoma brucei strain TREU 927. Mol Biochem Parasitol. 2001; 112(2): 163–171. PubMed Abstract | Publisher Full Text\n\nRaymond F, Boisvert S, Roy G, et al.: Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res. 2012; 40(3): 1131–1147. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeuberger G, Maurer-Stroh S, Eisenhaber B, et al.: Prediction of peroxisomal targeting signal 1 containing proteins from amino acid sequence. J Mol Biol. 2003; 328(3): 581–592. PubMed Abstract | Publisher Full Text\n\nNeuberger G, Maurer-Stroh S, Eisenhaber B, et al.: Motif refinement of the peroxisomal targeting signal 1 and evaluation of taxon-specific differences. J Mol Biol. 2003; 328(3): 567–579. PubMed Abstract | Publisher Full Text\n\nHirokawa T, Boon-Chieng S, Mitaku S: SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics. 1998; 14(4): 378–379. PubMed Abstract | Publisher Full Text\n\nTusnády GE, Simon I: The HMMTOP transmembrane topology prediction server. Bioinformatics. 2001; 17(9): 849–850. PubMed Abstract | Publisher Full Text\n\nBligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959; 37(8): 911–917. PubMed Abstract | Publisher Full Text\n\nFerro M, Seigneurin-Berny D, Rolland N, et al.: Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins. Electrophoresis. 2000; 21(16): 3517–3526. PubMed Abstract | Publisher Full Text\n\nDa Cruz S, Xenarios I, Langridge J, et al.: Proteomic analysis of the mouse liver mitochondrial inner membrane. J Biol Chem. 2003; 278(42): 41566–41571. PubMed Abstract | Publisher Full Text\n\nQuinones W, Urbina JA, Dubourdieu M, et al.: The glycosome membrane of Trypanosoma cruzi epimastigotes: protein and lipid composition. Exp Parasitol. 2004; 106(3–4): 135–149. PubMed Abstract | Publisher Full Text\n\nSoualhine H, Brochu V, Menard F, et al.: A proteomic analysis of penicillin resistance in Streptococcus pneumoniae reveals a novel role for PstS, a subunit of the phosphate ABC transporter. Mol Microbiol. 2005; 58(5): 1430–1440. PubMed Abstract | Publisher Full Text\n\nPanigrahi AK, Ogata Y, Zíková A, et al.: A comprehensive analysis of Trypanosoma brucei mitochondrial proteome. Proteomics. 2008; 9(2): 434–450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOpperdoes FR, Szikora JP: In silico prediction of the glycosomal enzymes of Leishmania major and trypanosomes. Mol Biochem Parasitol. 2006; 147(2): 193–206. PubMed Abstract | Publisher Full Text\n\nBandini G, Marino K, Guther ML, et al.: Phosphoglucomutase is absent in Trypanosoma brucei and redundantly substituted by phosphomannomutase and phospho-N-acetylglucosamine mutase. Mol Microbiol. 2012; 85(3): 513–534. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGalland N, de Walque S, Voncken FG, et al.: An internal sequence targets Trypanosoma brucei triosephosphate isomerase to glycosomes. Mol Biochem Parasitol. 2010; 171(1): 45–49. PubMed Abstract | Publisher Full Text\n\nPenha LL, Sant'Anna CB, Mendonca-Previato L, et al.: Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain. Glycobiology. 2009; 19(12): 1462–1472. PubMed Abstract | Publisher Full Text\n\nZomer AW, Michels PA, Opperdoes FR: Molecular characterisation of Trypanosoma brucei alkyl dihydroxyacetone-phosphate synthase. Mol Biochem Parasitol. 1999; 104(1): 55–66. PubMed Abstract | Publisher Full Text\n\nWiemer EA, IJlst L, van Roy J, et al.: Identification of 2-enoyl coenzyme A hydratase and NADP(+)-dependent 3-hydroxyacyl-CoA dehydrogenase activity in glycosomes of procyclic Trypanosoma brucei. Mol Biochem Parasitol. 1996; 82(1): 107–111. PubMed Abstract | Publisher Full Text\n\nCarrero-Lerida J, Perez-Moreno G, Castillo-Acosta VM, et al.: Intracellular location of the early steps of the isoprenoid biosynthetic pathway in the trypanosomatids Leishmania major and Trypanosoma brucei. Int J Parasitol. 2009; 39(3): 307–314. PubMed Abstract | Publisher Full Text\n\nMatsuzaki T, Fujiki Y: The peroxisomal membrane protein import receptor Pex3p is directly transported to peroxisomes by a novel Pex19p- and Pex16p-dependent pathway. J Cell Biol. 2008; 183(7): 1275–1286. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlsford S, Turner DJ, Obado SO, et al.: High throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res. 2011; 21(6): 915–924. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReguenga C, Oliveira ME, Gouveia AE, et al.: Identification of a 24 kDa intrinsic membrane protein from mammalian peroxisomes. Biochim Biophys Acta. 1999; 1445(3): 337–341. PubMed Abstract | Publisher Full Text\n\nMarshall PA, Krimkevich YI, Lark RH, et al.: Pmp27 promotes peroxisomal proliferation. J Cell Biol. 1995; 129(129): 345–355. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatnaik PK, Field MC, Menon AK, et al.: Molecular species analysis of phospholipids from Trypanosoma brucei bloodstream and procyclic forms. Mol Biochem Parasitol. 1993; 58(1): 97–105. PubMed Abstract | Publisher Full Text\n\nBakker BM, Michels PA, Opperdoes FR, et al.: Glycolysis in bloodstream-form Trypanosoma brucei can be understood in terms of the kinetics of the glycolytic enzymes. J Biol Chem. 1997; 272(6): 3207–3215. PubMed Abstract | Publisher Full Text\n\nColasante C, Peña Diaz P, Clayton C, et al.: Mitochondrial carrier family inventory of Trypanosoma brucei brucei: Identification, expression and subcellular localisation. Mol Biochem Parasitol. 2009; 167(2): 104–17. PubMed Abstract | Publisher Full Text\n\nKhan M, Contreras M, Singh I: Endotoxin-induced alterations of lipid and fatty acid compositions in rat liver peroxisomes. J Endotoxin Res. 2000; 6(1): 41–50. PubMed Abstract | Publisher Full Text\n\nTuller G, Nemec T, Hrastnik C, et al.: Lipid composition of subcellular membranes of an FY1679-derived haploid yeast wild-type strain grown on different carbon sources. Yeast. 1999; 15(14): 1555–1564. PubMed Abstract | Publisher Full Text\n\nWriessnegger T, Gübitz G, Leitner E, et al.: Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources. Biochim Biophys Acta. 2007; 1771(4): 455–461. PubMed Abstract | Publisher Full Text\n\nAntonenkov VD, Hiltunen JK: Peroxisomal membrane permeability and solute transfer. Biochim Biophys Acta. 2006; 1763(12): 1697–1706. PubMed Abstract | Publisher Full Text\n\nRokka A, Antonenkov VD, Soininen R, et al.: Pxmp2 is a channel-forming protein in mammalian peroxisomal membrane. PLoS One. 2009; 4(4): e5090. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "758",
"date": "08 Feb 2013",
"name": "Ralf Erdmann",
"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\nGlycosomes are evolutionarily and functionally related to peroxisomes. To fulfill their metabolic functions, the organelles communicate with the cytosol by an exchange of metabolites/products. Driven by the fact that the knowledge on metabolite transport across the glycosomal membrane and the nature of involved protein in this process is still scarce, the authors performed a proteomic approach with purified organelles. The paper is well written, the experimental design and results are conclusive. Some minor comments should be addressed:- Figure 1A: Please indicate top/bottom fractions and provide some information of the density at least of the peak fractions. Please mention in the legend how much of the total fractions was subjected to the gel.- Figure 1B: Please indicate the load. Are the membranes enriched compared to the glycosomal fraction? Please also provide more details on the experimental approach in the methods section, like volumes used for resuspension, fractions loaded on the gel, etc.",
"responses": []
},
{
"id": "868",
"date": "27 Mar 2013",
"name": "Peter J. Myler",
"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 manuscript, Colasante et al. describe an exhaustive analysis of the membrane proteins and lipids present in glycosomes from the kinetoplastid protozoan parasite, Leishmania tarentolae, and conclude that, in the absence of obvious transporters for the major substrates and intermediates of energy metabolism, these molecules must be transported across the glycosomal membrane via pores formed by known glycosomal membrane proteins, which are orthologues of the peroxisomal membrane proteins PEX11, GIM5A/B, PXMP4, PEX2 and PEX16. The paper is well-written and the technical approach is appropriate. While one must be mindful of the adage “absence of evidence is not evidence of absence”, the comprehensive nature of the proteomic analysis in the present study provides a persuasive argument (albeit, not definitive proof) for this conclusion. As a minor point, it could perhaps have been helpful to have indicated the identity of the proteins enriched in the glycosomal membrane fraction (urea pellet) of Table 1B. It would also be good to indicate what proportions of the total glycosomal fraction are represented by the two lanes in this figure.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-27
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https://f1000research.com/articles/2-26/v1
|
29 Jan 13
|
{
"type": "Case Report",
"title": "A brief grief over bowel relief",
"authors": [
"Kamalpreet S Parmar",
"Malvinder S Parmar",
"Kamalpreet S Parmar"
],
"abstract": "Oral sodium phosphate (OSP) solution is commonly used as bowel purgative before colonoscopy. Its safety has recently been questioned with several reports of acute renal failure and chronic kidney disease following its use. All of the cases reported are following bowel preparation for colonoscopy. I present a case of acute renal failure following OSP solution given to relieve constipation. This report further highlights the dangers of OSP and the importance of caution and careful monitoring when OSP solution is used as a cathartic, or for bowel preparation before colonoscopy.",
"keywords": [
"A 72-year-old woman with essentially unremarkable past medical history fell and sustained back injury and was noted to have a T11 compression fracture without any neurovascular compromise. The patient received Tylenol#3 for pain relief and was sent home. A few days later",
"she returned with ongoing vague lower back and abdominal discomfort and was noted to be constipated. Tylenol#3 was stopped and she was given a laxative - oral sodium phosphate solution (OSP",
"45 ml",
"Pharmascience",
"Montreal",
"Canada) to treat constipation."
],
"content": "Case report\n\nA 72-year-old woman with essentially unremarkable past medical history fell and sustained back injury and was noted to have a T11 compression fracture without any neurovascular compromise. The patient received Tylenol#3 for pain relief and was sent home. A few days later, she returned with ongoing vague lower back and abdominal discomfort and was noted to be constipated. Tylenol#3 was stopped and she was given a laxative - oral sodium phosphate solution (OSP, 45 ml, Pharmascience, Montreal, Canada) to treat constipation.\n\nThree days later, she returned to the local emergency department with feeling of generalized weakness, numbness around her lips, ongoing vague abdominal discomfort and nausea, but denied vomiting or diarrhea. Her intake had been poor since the fall and she noted decreased urine output. There is no history of diabetes or hypertension. Her medication was rabeprazole 20 mg a day and acetaminophen as required.\n\nInvestigations at the local emergency department revealed low hemoglobin of 109 g/L, normal white blood cell count WBC of 4.5, elevated blood urea nitrogen BUN of 9.4 mmol/L with serum creatinine of 345 μmol/L, and serum potassium of 3.4 mmol/L. The old records showed that her BUN was 6.1 mmol/L with serum creatinine of 74 μmol/L in December 2007. She was transferred for further management of acute renal failure.\n\nPhysical examination was remarkable for a woman of stated age with mild decreased skin turgor, blood pressure of 106/60 mmHg without orthostatic changes and regular rate of 72 beats per minute. Lungs were clear and heart sounds were normal. Abdominal examination revealed a soft abdomen with mild diffuse tenderness without rebound. There were no masses, renal angle fullness or tenderness. There was mild tenderness in the lower thoracic area. There was no pedal edema and neurological examination was non-focal. She had a Foley catheter with small amount of concentrated urine in the bag.\n\nInvestigations in our emergency department revealed low hemoglobin of 112 g/L, normal WBC of 4.9, elevated BUN of 9.4 mmol/L with serum creatinine of 419 μmol/L, serum potassium of 3.4 mmol/L, low serum calcium of 1.85 mmol/L (2.02–2.60 mmol/L) with serum albumin of 36 g/L, low ionized calcium of 0.85 mmol/L (1.15–1.29 mmol/L) and elevated phosphate of 3.68 mmol/L (0.87–1.45 mmol/L) and creatine kinase [CK] of 349. A urinalysis showed a concentrated urine with specific gravity of >1.030, 1+ protein and trace of blood with few white and red blood cells with few hyaline casts. Random urine sodium was 64 mmol/L with urine creatinine of 5330 μmol/L. A urine culture was negative. An abdominal ultrasound showed normal size kidneys without obstruction. The hospital course is shown in Table 1.\n\nThis patient presented with acute kidney injury (AKI) and the differential diagnosis included ischemic acute tubular necrosis (poor intake, decreased skin turgor, FENa of 3.78%), rhabdomyolysis (history of fall, elevated phosphate). The likelihood of vasculitic process was low in view of bland urine sediment and negative antinuclear and anti-neutrophilic cytoplasmic antibodies. However, significant hyperphosphatemia and hypocalcemia within 72-hours of standard dose (45 ml) of OSP [21.6 gm of monobasic sodium phosphate monohydrate and 8.1 gm of dibasic sodium phosphate heptahydrate] suggests the high probability of acute phosphate nephropathy (APN)1 that results from deposition of calcium-phosphate crystals in renal tubules and parenchyma (nephrocalcinosis)2. A kidney biopsy confirmed findings of acute phosphate nephropathy with acute tubular necrosis (Figure 1). She required supportive dialysis treatment twice.\n\n\nDiscussion\n\nOSP solution is commonly used as a bowel purgative before colonoscopy3. Its safety has recently been questioned1–5 with several reports of AKI and chronic kidney disease following its use. All of the cases reported occurred after bowel preparation with OSP for colonoscopy but AKI in this woman occurred after its use to relieve constipation. Decreased kidney function, use of renin angiotension aldosterone system (RAAS) blockers and older age and female gender are the most probable risk factors for APN, but other contributing factors are - use of non-steroidal anti-inflammatory agents, diuretics, history of hypertension, diabetes or heart failure3–5. Women, because of their smaller body mass, are more sensitive to fluid loss. Adequate fluid intake4 is important to prevent AKI when OSP is used as a bowel purgative. Poor oral intake since the fall in this woman likely contributed to both ATN and APN after OSP use. Although most patients recover renal function, some may have persistent chronic kidney disease4.\n\nThis report further highlights the need for vigilance when using OSP solutions for bowel preparation or to relieve constipation. Alternative solutions should be considered, especially in elderly and high-risk individuals5.\n\n1. Consider acute phosphate nephropathy in the presence of acute renal dysfunction, hyperphosphatemia and hypocalcemia.\n\n2. Consider alternate agents to oral phosphate solutions for bowel preparation or for relief of constipation, especially in elderly patients and in patients with chronic kidney disease.\n\n3. Ensure adequate hydration, if and when these agents (OSP) are used.",
"appendix": "Author contributions\n\n\n\nMSP was involved in the care of this patient and obtained consent from patient for publication, KSP did literature search and prepared the initial draft and both authors approved the final draft.\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\nI wish to thank Andrew Herzenberg (deceased) and John Rohan of the Department of Pathology, University Health Network, University of Toronto, Ontario, Canada, for providing the photomicrograph of the patient’s kidney biopsy.\n\n\nReferences\n\nRocuts AK, Waikar SS, Alexander MP, et al.: Acute phosphate nephropathy. Kidney Int. 2009; 75(9): 987–91. PubMed Abstract | Publisher Full Text\n\nGonlusen G, Akgun H, Ertan A, et al.: Renal failure and nephrocalcinosis associated with oral sodium phosphate bowel cleansing: clinical patterns and renal biopsy findings. Arch Pathol Lab Med. 2006; 130(1): 101–6. PubMed Abstract\n\nHurst FP, Bohen EM, Osgard EM, et al.: Association of oral sodium phosphate purgative use with acute kidney injury. J Am Soc Nephrol. 2007; 18(12): 3192–8. PubMed Abstract | Publisher Full Text\n\nMarkowitz GS, Stokes MB, Radhakrishnan J, et al.: Acute phosphate nephropathy following oral sodium phosphate bowel purgative: an underrecognized cause of chronic renal failure. J Am Soc Nephrol. 2005; 16(11): 3389–96. PubMed Abstract | Publisher Full Text\n\nLien YH: Is bowel preparation before colonoscopy a risky business for the kidney? Nat Clin Pract Nephrol. 2008; 4(11): 606–14. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "844",
"date": "18 Mar 2013",
"name": "Christina Yuan",
"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 acute kidney injury (requiring dialysis) after use of OSP for constipation in an elderly woman. The patient had a kidney biopsy demonstrating findings consistent with acute phosphate nephropathy. This is a valuable reminder that AKI secondary to OSP may occur independently of the indication for OSP prescription.",
"responses": []
},
{
"id": "2323",
"date": "05 Nov 2013",
"name": "Luan D. Trong",
"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 manuscript entitled “A Brief Grief over Bowel Relief,” by K.S. Parmar and M.S. Parmar, the authors describe a patient with acute renal failure after oral sodium phosphate administration for constipation.As the authors mentioned, this type of renal failure is almost always secondary to administration of sodium phosphate for colonoscopy. This report expands the possible complications of oral sodium phosphate in a new clinical context, i.e. treatment for constipation. It is an important finding both clinically and pathologically.A few minor suggestions:The quality of the renal biopsy illustration can be improved. The picture should be sharper with better contrast and the areas of tubular calcification should be better illustrated.In the Discussion, the dose as well as the drug schedule of oral sodium phosphate for colonoscopy should be reviewed and compared with those in this case. This should highlight better the clinical context in which oral sodium phosphate as a treatment for constipation can cause acute renal failure.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-26
|
https://f1000research.com/articles/2-24/v1
|
25 Jan 13
|
{
"type": "Research Article",
"title": "Human iPSC-derived mesoangioblasts, like their tissue-derived counterparts, suppress T cell proliferation through IDO- and PGE-2-dependent pathways",
"authors": [
"Ou Li",
"Karen English",
"Rossana Tonlorenzi",
"Giulio Cossu",
"Francesco Saverio Tedesco",
"Kathryn J Wood",
"Ou Li",
"Karen English",
"Rossana Tonlorenzi",
"Giulio Cossu",
"Francesco Saverio Tedesco"
],
"abstract": "Human mesoangioblasts are currently in a phase I/II clinical trial for the treatment of patients with Duchenne muscular dystrophy. However, limitations associated with the finite life span of these cells combined with the significant numbers of mesoangioblasts required to treat all of the skeletal muscles in these patients restricts their therapeutic potential. Induced pluripotent stem cell (iPSC)-derived mesoangioblasts may provide the solution to this problem. Although, the idea of using iPSC-derived cell therapies has been proposed for quite some time, our understanding of how the immune system interacts with these cells is inadequate. Herein, we show that iPSC-derived mesoangioblasts (HIDEMs) from healthy donors and, importantly, limb-girdle muscular dystrophy 2D patients exert immunosuppressive effects on T cell proliferation. Interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α) play crucial roles in the initial activation of HIDEMs and importantly indoleamine 2,3 dioxygenase (IDO) and prostaglandin E2 (PGE-2) were identified as key mechanisms involved in HIDEM suppression of T cell proliferation. Together with recent studies confirming the myogenic function and regenerative potential of these cells, we suggest that HIDEMs could provide an unlimited alternative source for mesoangioblast-based therapies.",
"keywords": [
"Preclinical models of muscular dystrophy in both mouse (α-sarcoglycan-null",
"dysferlin-null and mdx)1–4 and dog (golden retriever muscular dystrophy)2 provided evidence that mesoangioblasts have the capacity to functionally ameliorate the dystrophic phenotype. Based on these data",
"human allogeneic HLA-identical mesoangioblasts are in a phase I/II clinical trial for the treatment of paediatric patients affected by Duchenne muscular dystrophy (DMD",
"EudraCT no. 2011-000176-33). However",
"human mesoangioblasts have a finite lifespan in culture and the need for large cell numbers to treat each patient may impact the therapeutic efficacy of this approach",
"especially if this treatment should be extended to adults. To address and overcome these issues",
"the concept of deriving patient-specific induced pluripotent stem cells (iPSCs) that could serve as a platform for the genetic correction of autologous cell therapies in the future is being explored5."
],
"content": "Introduction\n\nPreclinical models of muscular dystrophy in both mouse (α-sarcoglycan-null, dysferlin-null and mdx)1–4 and dog (golden retriever muscular dystrophy)2 provided evidence that mesoangioblasts have the capacity to functionally ameliorate the dystrophic phenotype. Based on these data, human allogeneic HLA-identical mesoangioblasts are in a phase I/II clinical trial for the treatment of paediatric patients affected by Duchenne muscular dystrophy (DMD; EudraCT no. 2011-000176-33). However, human mesoangioblasts have a finite lifespan in culture and the need for large cell numbers to treat each patient may impact the therapeutic efficacy of this approach, especially if this treatment should be extended to adults. To address and overcome these issues, the concept of deriving patient-specific induced pluripotent stem cells (iPSCs) that could serve as a platform for the genetic correction of autologous cell therapies in the future is being explored5.\n\nDevelopments in the generation of iPSCs have advanced significantly in the past 6 years6,7; however, concerns related to potential tumorigenicity, incomplete and inefficient terminal differentiation8, as well as immunogenicity of iPSCs9 and in particular their differentiated progeny remains to be fully elucidated. Immune rejection represents a significant hurdle not just for allogeneic cell therapies, but for all cell therapy approaches, and questions surrounding the potential immunogenicity of iPSCs and their derivatives have been largely overlooked. A recent study provides evidence that syngeneic mouse iPSCs are more immunogenic than their embryonic stem cell (ESC) counterparts9. Immune responses against the integrated viral vectors9 as well as allogeneic10 and mutated mitochondrial genome after long term culture11 of iPSCs have been reported. Moreover, rejection of syngeneic mouse iPSCs following transplantation in vivo into mice9 highlights the fact that even autologous iPSCs can be recognised by the immune system and will be subject to standard rejection mechanisms12,13. Therefore, characterising the interactions between iPSC-derived differentiated cells and immune cells and determining whether or not these cells are recognised and rejected by the immune system is critically important.\n\nTo explore these possibilities, we utilised a novel protocol to derive mesoangioblast-like cells (human iPSC-derived mesoangioblasts: HIDEMs) initially from healthy iPSCs and subsequently from iPSCs reprogrammed from skeletal muscle cells of Limb-Girdle Muscular Dystrophy, Type 2D (LGMD2D) patients14. These cells, as expected, exert similar myogenic potential as normal mesoangioblasts, which makes them candidates for future clinical application. Here, we examined the effect of HIDEMs derived from both healthy donors and LGMD2D patients on immune cells in vitro and compared them with conventionally generated mesoangioblasts from healthy donors. Importantly, we demonstrate that HIDEMs from both sources do not induce, but rather suppress mitogen-driven T cell proliferation in vitro. Moreover, consistent with our findings for adult skeletal muscle-derived human mesoangioblasts15, HIDEMs mediate their immune suppressive effects through indoleamine 2,3 dioxygenase (IDO) and prostaglandin E2 (PGE-2).\n\n\nMaterials & methods\n\nHuman mesoangioblasts were isolated from adult skeletal muscle as previously described16. The HIDEM lines utilized in this study were generated from human iPSCs derived from either healthy donors (HIDEMs #1) or patients affected by limb-girdle muscular dystrophy 2D (LGMD2D Pt.3 HIDEMs) and are described in detail in a recent publication14. Both cell types were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; PAA) containing 10% FBS (Gibco), 2 mM glutamine (PAA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 1% NEAA (PAA), 5 ng/ml human bFGF (Peprotech), 100 IU ml–1 penicillin (PAA), 100 mg/ml–1 streptomycin (PAA), 1% insulin transferrin selenium X (ITSX) supplement (Gibco), 0.5 μM oleic and linoleic acids (Sigma-Aldrich), 1.5 mM Fe++ (Iron10 chloride tetrahydrate, Sigma; or Fer-In-Sol, Mead Johnson), 0.12 mM Fe+++ (Iron(III) nitrate nona- hydrate, Sigma; or Ferlixit, Aventis). Myogenic differentiation of mesoangioblasts and HIDEMs was performed as previously described14,16.\n\nHuman peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats. PBMCs from 4 donors were used in this study. All PBMC cultures were carried out in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS (Gibco), 1% v/v L-glutamine (PAA), 0.05mM β-mercaptoethanol (Sigma-Aldrich) and 100 IU ml–1 penicillin (PAA) and 100 mg/ml–1 streptomycin (PAA).\n\nProliferation of PBMCs was measured using the 5,6 carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay. PBMCs were labelled with 10µM CFSE (Invitrogen) in warm phosphate buffered saline (PBS) at room temperature. After 10 minutes, cells were washed in ice cold PBS. CFSE-abelled PBMCs (2.5 × 105/ml) were then seeded into 96-well round bottom plates with or without human anti-CD3/CD28 beads (Dynal-Life Technologies, UK) (0.5 × 105/ml) in complete RPMI 1640 (Sigma-Aldrich). For suppressor assays, mesoangioblasts/HIDEMs were seeded overnight in 96-well round bottom plates in mesoangioblast/HIDEM cell culture medium. The medium was then replaced with complete RPMI 1640 containing PBMC and anti CD3/CD28 beads where appropriate. Cells were harvested on day 6 and CFSE dilution was analysed by flow cytometry (FACSCantoTM II, BD Bioscience, UK). Actual numbers of CFSE-divided cells were calculated using counting beads (BD Biosciences, UK).\n\nMesoangioblasts and HIDEMs were characterised for the following phenotypic surface markers: HLA-ABC, HLA-DR (BD Biosciences), CD105, CD73, CD49b (eBioscience) and CD146 (Biocytex). In addition, mesoangioblast expression of CD40 (BD Biosciences) and PD-L1 (eBioscience) as well as the HLA molecules were analysed before and after stimulation (24h) with the pro-inflam matory cytokines interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α) or interleukin 1-beta (IL-1β) (Peprotech, UK). The effect of mesoangioblast on the expression of early activation markers on T cells was analysed by flow cytometry. Cells were harvested on day 3, 4, 5 and 6 and stained with anti-CD3, anti-CD25, anti-CD69 and 7AAD (eBioscience).\n\nProliferation assays in the form of CFSE dilution were carried out as described above in the presence or absence of neutralising antibodies to IFN-γ (R&D Systems), TNF-α (eBioscience), isotype control (R&D Systems) or recombinant IL-1 receptor antagonist (IL-1RA) (eBioscience) at concentrations of 0.5, 1.0 and 2.0 ug/ml. Inhibitors of IDO-1-Methyl-L-tryptophan (1MT) (Sigma-Aldrich) (0.5 mM), and of Cox-2: NS-398 (Calbiochem, UK) (1.0 uM) were used in this study.\n\nData were analysed using the statistical software Prism (version 5; GraphPad Software) and are reported as mean +/- standard error (SE). The unpaired Student t test was performed to compare 2 mean values. Otherwise, data were analysed using a two-way ANOVA with Bonferonni’s post-test and p values <0.05 were considered statistically significant.\n\n\nResults\n\nCultured HIDEM lines, HIDEMs #1 and LGMD2D Pt.3 HIDEMs, together with mesoangioblast lines XY24TL and XY27FD were characterised for their surface expression of pericyte makers CD73, CD105, CD49b, CD146, and HLA molecule HLA-ABC and HLA-DR. HIDEM lines and mesoangioblast lines expressed all of the aforementioned pericyte markers as well as HLA-ABC and low level expression of HLA-DR, indicating that these cells share the same phenotype (Figure 1A). HIDEM cells were also characterized for their myogenic potential with differentiation into skeletal myocytes/myotubes. Myogenic differentiation was observed in HIDEM lines, like in mesoangioblasts, by immunostaining for the striated muscle specific marker myosin heavy chain (Figure 1B), with the only difference that HIDEM terminal differentiation needed the expression of the myogenic regulator MyoD, as recently reported14.\n\nHealthy donor HIDEM line #1 and LGMD2D HIDEMs from patient no. 3 (HIDEMs Pt.3) and mesoangioblasts XY24TL and XY27FD were analysed for their surface marker expression of HLA-ABC, HLA-DR, CD105, CD73, CD49b and CD146 (A). Mesoangioblasts were stained with fluorochrome-labelled antibodies against the above markers (shown in red) as well as isotype matched control antibodies (shown in blue) followed by flow cytometric analysis. All four cell lines were also induced for myogenic differentiation followed by immunostaining of myocyte/myotube marker Myosin Heavy Chain (red). Cell nuclei were counter stained with Hoechst (blue). Scale bar = 100 µm (B).\n\nIt has been shown that allogeneic stem cells enjoy some level of immune privilege17–21. However, the general consensus is that differentiated cells tend to be more immunogenic than undifferentiated stem cells as demonstrated by studies using embryonic stem cells18,22. Thus, it was important to compare the immunogenicity of HIDEMs with that of mesoangioblasts derived from conventional adult muscle under basal (see above) or simulated inflammatory conditions. Following stimulation with the pro-inflammatory cytokines IFN-γ, TNF-α or IL-1β or a combination of these soluble mediators, expression of HLA-ABC, HLA-DR, PD-L1 were increased after stimulation with IFN-γ, but not TNF-α or IL-1β (Figure 2A and 2B). Stimulation of HIDEMs and mesoangioblasts with IFN-γ and TNF-α or combinations of these cytokines induced low-level expression of CD40 (Figure 2A and 2B).\n\nHIDEMs and mesoangioblasts were stimulated with IFN-γ, TNF-α or IL-1β (20ng/ml) for 24h. Cells were trypsinized and washed, followed by surface staining for HLA-ABC, HLA-DR, CD40, PD-L1 or fluorochrome matched isotype controls and analysis by flow cytometry. Representative histograms of HIDEMs #1 are presented here (A). Data are represented as mean (+/- SE) median fluorescence intensity of the markers examined over 4 replicates. **p<0.01 (B). CFSE-labelled PBMCs were stimulated with anti CD3/CD28 beads (PBMC+B) as a positive control. Non-stimulated or cytokine stimulated HIDEMs/mesoangioblasts (ratio 1:4) were co-cultured with PBMC for 6 days. CD3+ CFSE labelled 7AAD- cells were enumerated using counting beads (C). Experiments were carried out in duplicates. n=4.\n\nTo examine their immunogenicity, HIDEMs and mesoangioblasts cultured under basal conditions or pre-stimulated with the pro-inflammatory cytokines IFN-γ, TNF-α or IL-1β were co-cultured with allogeneic CFSE-labelled PBMCs. Neither HIDEMs nor mesoangioblasts induced CD3+ T cell proliferation (Figure 2C) in vitro after 6 days of co-culture. Furthermore, exposure of HIDEMs to pro-inflammatory cytokines did not alter this lack of T cell response (Figure 2C). Thus, T cells were unresponsive to allogeneic HIDEMs even after pre-stimulation with pro-inflammatory cytokines.\n\nHuman mesoangioblasts have the capacity to modulate T cell proliferation in vitro15. Here, we sought to determine whether HIDEMs could exert immune suppressive effects. All 4 cell lines potently suppressed CD3+ T cell proliferation in vitro in a dose-dependent manner (Figure 3A and 3B). Interestingly, HIDEMs from both healthy donors and patients showed greater suppressive potency compared to mesoangioblasts at the same cell:PBMC ratio. We therefore hypothesised that HIDEMs may alter T cell activation. The effect of HIDEMs/mesoangioblasts on the expression of early T cell activation markers (CD25 and CD69), was directly examined on PBMCs stimulated with anti-CD3/CD28 beads in the presence or absence of HIDEMs or mesoangioblasts. The percentage of T cells expressing CD25 and CD69 increased significantly between days 3 and 4 after stimulation as expected, and the presence of HIDEMs or mesoangioblasts inhibited the expression of CD69 on day 3 but had no effect on days 4–6 (Figure 3C). Expression of CD25 was not significantly affected by HIDEMs or mesoangioblasts at any time point examined (Figure 3C). Thus, although HIDEMs exhibited more potent activity with respect to inhibition of T cell proliferation, they did not differentially impact T cell activation.\n\nCFSE-labelled PBMCs (5 × 104/well) were stimulated with anti CD3/CD28 beads (1 × 104/well) (P+B) in the presence or absence of HIDEMs/mesoangioblasts at decreasing ratios (HIDEM/mesoangioblast:PBMC). On day 6, cells were harvested and stained with anti-CD3 and 7AAD, and analysed by flow cytometry. CFSE dilution was analysed on gated CD3+7AAD- cells. Representative histograms showing CD3+7AAD- cells with CFSE dilutions after co-culture with HIDEM/mesoangioblast (A). The percentage of CD3+CFSE dividing cells calculated for each group and compared to the positive control (P+B). *p<0.05 (B). Cells were harvested on day 3, 4, 5 or 6 and analysed for CFSE dilution and expression of CD25 and CD69. The number of CD3+7AAD- cells expressing CD25 or CD69 using counting beads and the % of CD25+ and CD69+ cells were calculated from the data (C). Experiments were carried out in duplicates. n=2. Unpaired t tests were used for statistical analysis.\n\nAs IFN-γ, TNF-α and IL-1β have been identified as key cytokines required for the activation of mesoangioblast immunosuppressive function in vitro15, we examined the importance of these pro-inflammatory cytokines in the modulation of T cell proliferation by HIDEMs. Neutralising antibodies against IFN-γ at concentrations of 1.0 and 2.0 ug/ml significantly abolished the inhibitory effect mediated by HIDEMs and mesoangioblasts (Figure 4A). A similar, but weaker effect was observed in the presence of a neutralising antibody against TNF-α (2.0 ug/ml) for both cell types. Notably, a lower concentration of anti-TNF-α (1.0 ug/ml) partially restored T cell proliferation in the presence of HIDEMs, but not mesoangioblasts, suggesting that TNF-α may play a more important role in the suppressive function of HIDEMs compared to mesoangioblasts (Figure 4A). The addition of IL-1 receptor antagonist (IL-1RA) at concentrations of 0.5, 1.0 or 2.0 ug/ml did not restore T cell proliferation in this case.\n\nCFSE-labelled PBMCs were stimulated with anti-CD3/CD28 beads in the presence of HIDEMs/mesoangioblasts (1:4) and neutralising antibodies against IFN-γ and TNF-α or irrelevant isotype control antibody (0.5, 1.0 and 2.0 µg/ml) or recombinant IL-1RA (0.5, 1.0 and 2.0 µg/ml). Cells were harvested on day 6 and stained with anti-CD3 and 7AAD. After gating on CD3+7AAD- the number of CFSE diluting cells were enumerated using counting beads. Data are represented as mean CD3+ CFSE diluted cell number +/- SE. *p<0.05, **p<0.01 (A). HIDEMs/mesoangioblasts were left untreated or were stimulated with IFN-γ, TNF-α or IL-1β (20ng/ml) for 24h before setting up co-cultures with CFSE labelled PBMCs. As a positive control, PBMCs were stimulated with anti CD3/CD28 beads. After 6 days cells were harvested and surface stained for CD3 and 7AAD before analysis of CFSE dilution. CD3+CFSE diluted cell numbers were calculated using counting beads as before (B). Experiments were carried out in duplicates. n=4.\n\nOur previous study showed that pre-stimulation with IFN-γ, TNF-α or IL-1β does not enhance or abrogate mesoangioblast suppression of T cell proliferation in vitro. To test if this holds true for HIDEMs, cells were then pre-treated with IFN-γ, TNF-α, IL-1β, or combinations of these cytokines for 24h before adding them to CFSE dilution assays to examine their immunosuppressive capacity. Pre-stimulation did not promote the suppressor capacity of HIDEMs or mesoangioblasts, compared to non-stimulated controls (Figure 4B). However, there was no significant loss of suppressor capacity by HIDEMs following stimulation with pro-inflammatory cytokines, similar to that observed for mesoangioblasts.\n\nIDO and PGE-2 have been identified as key mechanisms in the modulation of T cell proliferation by mesoangioblasts. To test the hypothesis that IDO and PGE-2 are key pathways used by HIDEMs for modulating T cell proliferation, 1-Methyl-L-tryptophan (1MT) (an inhibitor of IDO) or NS-398 (1.0 µM) (a specific Cox-2 inhibitor) were added to T cell cultures containing HIDEMs or mesoangioblasts. Both inhibitors partially, but significantly, restored proliferation, with 1MT having the most potent effect. Addition of both inhibitors showed an additive effect (Figure 5). Restoration of proliferation mediated by the addition of NS-398 was significant in cultures containing HIDEMs, but not mesoangioblasts (Figure 5).\n\nCFSE-labelled PBMCs were stimulated with anti CD3/CD28 beads as before in the presence of HIDEMs/mesoangioblasts and inhibitors of IDO and Cox-2, (1-Methyl-L-trypyophan (1MT) (0.5 mM) and NS-398 (1.0 uM) respectively, or both. On day 6 cells were harvested and stained with anti-CD3 and 7AAD. Cells were gated on live CD3+ populations and analysed for CFSE dilution and the numbers of cells undergoing CFSE dilution were enumerated using counting beads, *p<0.05, **p<0.01. Experiments were carried out in duplicates. n=4.\n\n\nDiscussion\n\nThe advent and unwavering development of iPSC technology makes autologous cell therapy and personalised gene therapy a realistic possibility for many degenerative and genetic diseases like muscular dystrophies. Nevertheless, a number of reports have raised concerns on the overlooked possibility that iPSCs and their derivatives may be targeted by the immune system for various reasons9,10,23. The recent new development of human iPSC-derived mesoangioblasts may provide an unlimited source for mesoangioblast therapy for DMD, but understanding the immunogenicity of these cells is critically important for their successful application.\n\nIn the current study, we demonstrate that HIDEMs behave in a very similar manner to mesoangioblasts with regard to expression of HLA and co-stimulatory molecules, suppressive potency and mechanisms of action. We showed that HIDEMs are comparable to mesoangioblasts in their surface marker expression and that both cell populations respond to stimulation with pro-inflammatory cytokines consistently with increased expression of HLA-ABC, HLA-DR, PD-L1 and low level expression of CD40. Importantly, HIDEMs suppress T cell proliferation driven by anti-CD3/CD28 stimulation in a similar dose-dependent manner as that of mesoangioblasts, but with increased potency. Comparable to mesoangioblasts, HIDEMs did not alter the expression of early activation markers CD25 or CD69 on CD3+ T cells, a phenomenon also seen with immune-suppressive mesenchymal stromal cells (MSC)24.\n\nAn activation step involving IFN-γ or TNF-α was a prerequisite for HIDEM suppression of T cell proliferation as neutralisation of either cytokine significantly abolished the inhibitory effect. IDO and PGE-2 were identified as key mechanisms of action involved in HIDEM suppression of T cell proliferation. Overall, the immunogenicity and immunosuppressive capacity of HIDEMs was almost indistinguishable from that of their conventionally derived counterpart, mesoangioblasts15. The only minor differences between these two cell populations were the increased immunosuppressive potency and sensitivity of HIDEMs to TNF-α and to the specific Cox-2 inhibitor NS-398. Perhaps these three observations are linked and suggest that HIDEMs are more receptive to TNF-α, leading to the induction of enhanced levels of PGE-2 facilitating a more potent inhibition of T cell proliferation. Importantly, this study demonstrates that iPSC derived cells (HIDEMs) display immunomodulatory functions equivalent to those of their tissue-derived counterpart, mesoangioblasts.\n\nGene therapy is an appealing approach to treat many hereditary diseases such as DMD, and adeno-associated virus-based gene therapy trials showed promising results for LGMD2D patients25. However, problems associated with immunity to dystrophin and subsequent loss of transgene expression might limit the successful application of this approach26,27. The use of retro-/lenti-viral vector-based techniques for the generation of iPSCs and for gene correction of their progeny means that cells derived from iPSCs using these technologies could face the same challenge. Nevertheless, HIDEMs have been derived in a complete viral integration-free setting14 and, although we have not examined their immunomodulatory function in vivo, the proven therapeutic value of iPSC-derived mesoangioblasts, combined with their potential immune suppressive characteristics, means that HIDEMs are well placed to overcome some of the challenges facing iPSC-derived cell therapies.",
"appendix": "Author contributions\n\n\n\nOL contributed to conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the article. KE contributed to conception and design, analysis and interpretation of data, drafting and revising the article. RT contributed to acquisition of data. GC contributed to revising the article. FST contributed to acquisition of data and revising the article. KJW contributed to conception and design, analysis and interpretation of data, drafting and revising the article. All authors contributed to the final approval of the version to be published.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by the European Community grant OptiStem (223098), European Research Council (GA 233263), The Wellcome Trust (082519/Z/07/Z) and Medical Research Council (87216).\n\n\nReferences\n\nSampaolesi M, Torrente Y, Innocenzi A, et al.: Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science. 2003; 301(5632): 487–92. PubMed Abstract | Publisher Full Text\n\nSampaolesi M, Blot S, D'Antona G, et al.: Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature. 2006; 444(7119): 574–9. PubMed Abstract | Publisher Full Text\n\nDiaz-Manera J, Touvier T, Dellavalle A, et al.: Partial dysferlin reconstitution by adult murine mesoangioblasts is sufficient for full functional recovery in a murine model of dysferlinopathy. 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}
|
[
{
"id": "743",
"date": "31 Jan 2013",
"name": "Philippe Saas",
"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 reporting the in vitro effect of human iPSC-derived mesoangioblasts on T cells. This is a prerequisite step in order to use these cells in the treatment of patients suffering from muscular dystrophy.How do human iPSC-derived mesoanglioblasts interact with T cells is an open question. This is of interest since these cells can be used in cell-based therapy approaches. Immunogenicity of infused cells depends on cell type.The study by Li and colleagues is well performed. I have only minor comments in order to better explain how the results on Figure #3AB are shown or calculated. Typo (page 3, paragraph “Results”): “makers” instead of “markers”.I propose to add the number of experiments performed at the end of the legend of the Figure #1.I suggest that the authors improve the Figure #3 panel A and B and its legend. For Figure #3A, I understand well the two upper panels: the panel PBMC shows non proliferating PBMC (all the cells are brightly stained with CSFE) and the panel PBMC + Anti CD3/CD28 shows maximal proliferation with all the cells (gated for CD3) exhibiting decreased CSFE fluorescence due to CSFE dilution occurring during cell division. I propose to modify the PBMC histogram and to increase the scale to better appreciate the peak. According to the legend and the Materials and Methods section, the other panels correspond to the coculture of either mesangioblast or iPSC-derived mesangioblast from a healthy donor or a patient with PBMC (fixed number of 5 x 104/well). Thus, the number of mesangioblasts varies and is decreasing (from 1:4 to 1:32). It is logical that the peaks of proliferating CD3+ cells increase in the lower panels. However, if inhibition of T cell activation occurs, a peak in the non dividing area/region should be observed as in the panel “PBMC”. It seems to be the case for the condition “LMGMDD pt.3”. However, this is difficult to observe with the other conditions. I propose to increase the Y scale. Maybe, as performed in the previous Figure (Figure #2), counting beads were added to assess the absolute number of CD3+ CFSElow 7AADneg cells. This explains why the peaks are flat in the 1:4 ratio conditions. Concerning Figure #3B, the shown data represent the percentage of CD3+ cell proliferation in coculture divided by the percentage of CD3+ cell proliferation in the absence of coculture. According to the upper panel of the Figure #3A, all the cells proliferate. I suggest that the authors provide more explanations on how the data are obtained.",
"responses": []
},
{
"id": "3842",
"date": "24 Feb 2014",
"name": "Mayana Zatz",
"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 Li, English et al. propose that HIDEMs behave in a very similar manner to mesoangioblasts, with regard to expression of HLA and co-stimulatory molecules, suppressive potency and mechanisms of action. HIDEMs are comparable to mesoangioblasts in their surface marker expression and that both cell populations respond to stimulation with pro-inflammatory cytokines consistently, with increased expression of HLA-ABC, HLA-DR, PD-L1 and low level expression of CD40. Importantly, HIDEMs suppress T cell proliferation driven by anti-CD3/CD28 stimulation in a similar dose-dependent manner as that of mesoangioblasts, but with increased potency. Comparable to mesoangioblasts, HIDEMs did not alter the expression of early activation markers CD25 or CD69 on CD3+ T cells, a phenomenon also seen with immune-suppressive mesenchymal stromal cells (MSC). These findings are comparable to other publications by the same group. The activation step involving IFN-γ or TNF-α is an apparent prerequisite for HIDEM suppression of T cell proliferation as neutralization of either cytokine significantly abolished the inhibitory effect. IDO and PGE-2 are identified as key mechanisms of action involved in HIDEM suppression of T cell proliferation. Following this line of thinking we suggest that other potential suppressive mechanisms should be investigated. The involvement of regulatory cytokines like IL-10, TGF-β for example should be addressed, as well as the more detailed phenotype of T cells or even the participation of T regulatory cells should be explored. The detailed study of immunological mechanisms is highly relevant and may contribute to advance our knowledge about the use of HIDEMs or mesangioblasts for the therapy of Duchenne muscular dystrophy.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-24
|
https://f1000research.com/articles/2-23/v1
|
24 Jan 13
|
{
"type": "Case Report",
"title": "Neotendon infilling of a full thickness rotator cuff foot print tear following ultrasound guided liquid platelet rich plasma injection and percutaneous tenotomy: favourable outcome up to one year",
"authors": [
"Arockia Doss"
],
"abstract": "This is a case report on excellent clinical outcome and neotendon infilling at one year follow up in a degenerative rotator cuff full thickness tear following percutaneous tenotomy and platelet rich plasma injection.",
"keywords": [
"Rotator cuff",
"Supraspinatus tear",
"Platelet rich plasma (PRP)"
],
"content": "Introduction\n\nRotator cuff tears are an increasingly common cause of morbidity in the aging population. There are surgical and non surgical management options including open or arthroscopic procedures, percutaneous corticosteroid injections, physiotherapy for rotator cuff tears. It is well accepted that there is a poor outcome in degenerative rotator cuff tears after surgery1. This report is based on the clinical and imaging outcome in a patient who received platelet rich plasma (PRP) and percutaneous tenotomy treatment for a full thickness supraspinatus tear.\n\n\nCase report\n\nA 73 year old right hand dominant active lady complained of bilateral shoulder pain for about two months and did not respond to two ultrasound guided subacromial subdeltoid corticosteroid injections. Her shoulder injury occurred whilst she had been caring for her husband and working on their farm. In the past she had recovered from non-Hodgkin’s lymphoma and currently was on antihypertensive medication (candesartan cilexetil once a day). She had led a physically active country life style prior to the presentation of the problem and had never smoked in her life.\n\nAt presentation there was painful limitation of right sided shoulder abduction to less than 90 degrees. Ultrasound documented a 9mm × 14mm partial width full thickness footprint tear of the anterior to mid right supraspinatus with tendinosis of most of the tendon and enthesopathy at the greater tuberosity of the humeral head (Figure 1). A plain radiograph of the right shoulder showed a down-sloping Type 2 acromion. There was mild wasting of the right supraspinatus muscle. A diagnosis of a recent footprint tear superimposed on degenerative supraspinatus tendon with mild muscle atrophy was made. Following written informed consent from the patient, 8ml of autologous unclotted blood was venesected and centrifuged for 5 minutes at about 3000 rotations per minute in a special tube for PRP preparation (BCT, REGEN Labs, Switzerland). 4 to 5ml of liquid PRP was injected through a 22g 5cm long needle into the tear and its margins with simultaneous percutaneous tenotomy directed into the footprint of the anterior facet of the greater tuberosity under direct ultrasound imaging control (GE Logic 9, 9MHz probe). 5ml 1% lignocaine was injected into the superficial soft tissues, subacromial bursa and the supraspinatus tear for local anaesthetic purposes. The shoulder was placed in a sling with 90 degree elbow flexion for 7 days. Physiotherapy was commenced at 4 to 5 weeks post PRP with a home exercise program. At 8 weeks follow up post PRP the patient verbally reported a marked reduction in pain with improvement in shoulder movement. At the 7 and 10 month follow up there was complete relief from pain with full range of movement of the right shoulder and she was able to lift bags of potting mix in her farm. At the 10 month follow up, ultrasound (GE Logic 9, 9MHz probe) performed by the author showed a near complete echogenic infilling obliterating the tear defect. The lateral margin of the tear merged with this neotendon tissue with mild medial retraction (Figure 2). She was completely pain free at a follow up 1 year after PRP injection.\n\nTear outline is well appreciated from distension of the peritendinous and subdeltoid bursal space during percutaneous treatment with tenotomy and liquid platelet rich plasma.\n\nThe lateral tear margin has retracted with mild interval attrition of the supraspinatus tendon. At the time of this follow up study the patient had a dramatic return to a completely pain free state and had full range of movement with no dysfunction.\n\n\nDiscussion\n\nPRP has gained increasing popularity in orthopaedic and musculoskeletal medicine in the last few years despite the lack of large volume data and high quality studies. Aside from its autologous nature, the rationale for the use of PRP is on the basis of its anti-inflammatory properties2. Application of PRP in treating degenerative rotator cuff lesions is made on the basis of its role in the regulation of matrix gene expression and cell proliferation3. PRP has also shown regenerative effects in an animal model of meniscal fibrocartilage tears4 and this supports the use of PRP at the footprint of the supraspinatus insertion where there is fibrocartilagenous tissue at the bone-tendon interface.\n\nThis report documents imaging evidence of the formation of neotendon tissue in a patient who experienced complete resolution of symptoms from a full thickness partial width supraspinatus footprint tear. Symptom resolution at the time of manuscript preparation has lasted for 12 months post PRP injection. The precise histology of the neotendon infilling is of uncertain nature, although it is possible that this is fibrovascular tissue5. Fibrovascular tissue scars at the bone-tendon interface are prone to failure and it remains to be seen if this patient’s relief persists in the long term. The favourable outcome cannot be entirely attributed to PRP. The possible influences of the percutaneous tenotomy, placebo effect, diet, and physiotherapy have to be taken into account. This report serves as anecdotal evidence that PRP does offer an alternative treatment option in some cases of full thickness rotator cuff tears.\n\nRecent papers have contradicted the positive effect of PRP in rotator cuff tears. Two randomized controlled trials with 79 patients and another with 88 patients comparing PRP fibrin matrix (PRFM) versus control on rotator cuff tendon healing showed no demonstrable differences on tendon healing and clinical rating scales1,6. Bergeson et al. also showed similar results with PRFM in at risk rotator cuff tears7. These studies used a semisolid implant material that had to be delivered through the arthroscope cannula. This implant was left at the bone tendon interface and may have resulted in a space occupying effect in addition to an unfavourable biological milieu with increased inflammatory mediators. PRP delivered as a semisolid plug in the form of PRFM through arthroscopic cannula is a different product dissimilar to liquid PRP preparations that are injectable under ultrasound guidance.\n\nRoutine or repeated corticosteroid injections into the subacromial bursa are controversial given that corticosteroid has a catabolic effect that may be harmful to already degenerative tendon tears. Repeated cortisone injections are questionable given that studies have documented an increased loss of bone mineral density after corticosteroid injections in postmenopausal women8,9 with the potential to increase the risk of fractures in this group of patients. In addition corticosteroid does not have a role in the healing cascade of degenerative tears10. Although there is no doubt that in some instances corticosteroid injection will be needed due to individual circumstances, the widespread use of this approach deserves a rethink on the merits and disadvantages.\n\nClearly the routine use of PRP in rotator cuff tears is not recommended for all patients on the basis of this single case report. Until further high quality studies are available, use of PRP in rotator cuff tears may be reserved for recalcitrant pain in individuals with high compliance to post injection rehabilitation and in those where close clinical follow up and documentation of clinical outcome including any adverse events would be possible.\n\nFurther studies are urgently needed on the ultrasound guided percutaneous use of liquid PRP in degenerative rotator cuff tears. Questions remain on the role of PRP in rotator cuff tears. Is there a role for liquid PRP injection into cuff tears under imaging guidance prior to rotator cuff surgery? Does ultrasound guided PRP injection into the rotator cuff prior to surgery improve the outcome of surgical repair in comparison to subacromial subdeltoid bursal corticosteroid injection? Would PRP alone suffice in some patients and preclude the need for surgery thus reducing healthcare costs?\n\nAs far as the author is aware there in no similar published report. This case report documents that liquid PRP may play a favourable role in the stabilization of a full thickness supraspinatus insertional tear with an excellent clinical outcome and fuels the debate on the evolving role of autologous PRP in rotator cuff tears.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.",
"appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nRodeo SA, Delos D, Williams RJ, et al.: The effect of platelet-rich fibrin matrix on rotator cuff tendon healing: a prospective, randomized clinical study. Am J Sports Med. 2012; 40(6): 1234–41. PubMed Abstract | Publisher Full Text\n\nvan Buul GM, Koevoet WL, Kops N, et al.: Platelet-rich plasma releasate inhibits inflammatory processes in osteoarthritic chondrocytes. Am J Sports Med. 2011; 39(11): 2362–70. PubMed Abstract | Publisher Full Text\n\nJo CH, Kim JE, Yoon KS, et al.: Platelet-rich plasma stimulates cell proliferation and enhances matrix gene expression and synthesis in tenocytes from human rotator cuff tendons with degenerative tears. Am J Sports Med. 2012; 40(5): 1035–1045. PubMed Abstract | Publisher Full Text\n\nIshida K, Kuroda R, Miwa M, et al.: The regenerative effects of platelet-rich plasma on meniscal cells in vitro and its in vivo application with biodegradable gelatin hydrogel. Tissue Eng. 2007; 13(5): 1103–12. PubMed Abstract | Publisher Full Text\n\nGulotta LV, Rodeo SA: Growth factors for rotator cuff repair. Clin Sports Med. 2009; 28(1): 13–23. PubMed Abstract | Publisher Full Text\n\nCastricini R, Longo UG, De Benedetto M, et al.: Platelet-rich plasma augmentation for arthroscopic rotator cuff repair: a randomized controlled trial. Am J Sports Med. 2011; 39(2): 258–265. PubMed Abstract | Publisher Full Text\n\nBergeson AG, Tashjian RZ, Greis PE, et al.: Effects of platelet-rich fibrin matrix on repair integrity of at-risk rotator cuff tears. Am J Sports Med. 2012; 40(2): 286–293. PubMed Abstract | Publisher Full Text\n\nKang SS, Hwang BM, Son H, et al.: Changes in bone mineral density in postmenopausal women treated with epidural steroid injections for lower back pain. Pain Physician. 2012; 15(3): 229–36. PubMed Abstract\n\nAl-Shoha A, Rao DS, Schilling J, et al.: Effect of epidural steroid injection on bone mineral density and markers of bone turnover in postmenopausal women. Spine (Phila Pa 1976). 2012; 37(25): E1567–71. PubMed Abstract | Publisher Full Text\n\nKai-Ming Chan, Sai-Chuen Fu: Sports Medicine, Arthroscopy, Rehabilitation, Therapy & Technology. 2009; 1: 23. Publisher Full Text"
}
|
[
{
"id": "753",
"date": "06 Feb 2013",
"name": "Elizaveta Kon",
"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\nLiterature concerning PRP use in rotator cuff pathology is mainly oriented towards intra-operative use of this biological strategy. However, recently, a double blinded randomized controlled trial on 39 patients has been published (Rha DW et al. (2012) Comparison of the therapeutic effects of ultrasound-guided platelet-rich plasma injection and dry needling in rotator cuff disease: a randomized controlled trial). This article is a case report on the same topic, with all the scientific limitations related to the nature of such kind of article. At the present moment, also considering the controversies arisen on PRP application in tendon pathology, we need well designed high quality trials to assess the efficacy of this treatment option. The article is written in a fair manner without big methodological bias. However method is not only how you did what you did but also what you could have done better. Of course case reports provide poor evidence and it is impossible to rely just on findings from this kind of study. The author of the present study should have used some clinical scores (there are many available for the shoulder) to document outcome over time, MRI pre- and post-treatment should be added to better assess tendon healing and the features of PRP used should be discussed as this is one of the crucial points of current debate on PRP application. These changes could improve the scientific value of this case report, and it is important to be exhaustive when you have a single patient examined.",
"responses": []
},
{
"id": "3348",
"date": "27 Jan 2014",
"name": "Nicola Maffulli",
"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 bears witness to how much we fall in love with novelties, and how much we, as a scientific community, do not know yet about a fashionable autologous blood product.This case report is now one year old, and the situation in this field remains unchanged: randomised controlled trials show in a fairly unequivocable fashion that PRP use is at best dubious, and nevertheless case series report success.This should make us think, and use strict stringent scientific methods to plan and evaluate new technologies.",
"responses": []
},
{
"id": "4112",
"date": "21 Mar 2014",
"name": "Marco Patruno",
"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 my opinion the author of this case report describes the results well, although I do agree with Elizaveta Kon that also including MRIs would have improved the quality of the paper. The real action of PRP is still under debate, and the scientific community asks for stringent methods and careful evaluations, even for a single case study. I suggest the author increases the number of patients and improves the quality of the results in future studies concerning PRP.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-23
|
https://f1000research.com/articles/2-22/v1
|
23 Jan 13
|
{
"type": "Short Research Article",
"title": "Memory enhancement produced by post-training exposure to sucrose-conditioned cues",
"authors": [
"Matthew R Holahan",
"Norman M White",
"Norman M White"
],
"abstract": "A number of aversive and appetitive unconditioned stimuli (such as shock and food) are known to produce memory enhancement when they occur during the post-training period. Post-training exposure to conditioned aversive stimuli has also been shown to enhance memory consolidation processes. The present study shows for the first time that post-training exposure to conditioned stimuli previously paired with consumption of a sucrose solution also enhances memory consolidation. Male Long Evans rats were trained on a one-session conditioned cue preference (CCP) task on a radial arm maze. Immediately or 2 hours after training, rats consumed a sucrose solution or were exposed to cues previously paired with consumption of sucrose or cues previously paired with water. Twenty-four hours later, the rats were tested for a CCP. Immediate, but not delayed, post-training consumption of sucrose enhanced memory for the CCP. Immediate, but not delayed, post-training exposure to cues previously paired with sucrose, but not with water, also enhanced CCP memory. The possibility that rewarding and aversive conditioned stimuli affect memory by a common physiological process is discussed.",
"keywords": [
"consolidation",
"modulation",
"conditioned cue preference",
"sucrose"
],
"content": "Introduction\n\nCertain kinds of events that occur during the period immediately after a new task is learned can modulate memory for the task1–5. Events such as electroconvulsive shock6–8 or anaesthetization9,10 can weaken a memory; events such as injections of epinephrine11–13 or amphetamine14–18 can strengthen a memory. In all such demonstrations, the post-training treatment was given shortly after a learning trial and retention was measured one or more days later. When administration of the same treatments was delayed for one or more hours after training, they were ineffective. This pattern of effects is consistent with the idea that memory consolidation is a time-limited process5,19 during which the neural representation of the memory is labile and subject to change20–22.\n\nThe memory enhancing effects of post-training aversive events such as footshock23–25 and of rewarding events such as electrical self-stimulation of the brain26, and ingestion or injection of sucrose or glucose27–33 are well-documented. Furthermore, Holahan and White34,35 found that post-training exposure to conditioned aversive cues previously paired with shock also enhanced memory consolidation. The present study was designed to determine if post-training exposure to rewarding conditioned cues, previously paired with consumption of sucrose, can also enhance consolidation of the memory for a recently acquired task.\n\n\nMaterials and methods\n\nSubjects were 36 test naïve, male Long-Evans rats (Charles River, St. Constant, Québec, Canada; strain code: 006) that weighed 250–275 g (56–58 days old) at the start of the experiment. They were housed in individual cages with free access to water. The temperature (22°C) and lighting (lights on: 0700 to 1900) of the animal housing unit were controlled. Behavioral testing took place from 1000 to 1400. Care of rats and all procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care and protocols approved by the McGill University Animal Care Committee (protocol number: 1417) as well as the Guide for the Use and Care of Laboratory Animals.\n\nRadial-arm maze. The maze was located in a windowless 2.8 × 3.7 × 2.8 m (w × l × h) room partitioned with a sound attenuating divider (1.2 m long) that created a 2.8 × 2.3 m area for the maze. This area contained a number of distal cues. A video camera hung about 1 meter above the maze.\n\nThe maze was made of wood painted flat gray and consisted of an octagonal center platform 29.3 cm edge to edge with arms 42.8 cm long and 9 cm wide. Each arm was surrounded by a wall 15.8 cm high at the entrance; 10 cm out from the entrance the wall height decreased to 5 cm. The surface of the maze was 54 cm from the floor of the room. Wooden blocks (30 cm high) also painted flat gray were placed in the entrances of unused arms. Similar blocks with wooden panels attached (28 cm wide) were used to confine a rat to a 35 cm2 area at the end of an arm during training. The panels confined a rat’s view of the room to an arc of approximately 180° facing away from the maze.\n\nConditioning boxes. This apparatus consisted of a large box made of wood, except the front wall, which was Plexiglas. The box was divided into two compartments of equal size (45 × 45 × 30 cm) by a wooden partition. One of the compartments was painted grey; the other was painted with vertical black and white stripes. The floors in both compartments were made of wood and covered with wood chips. The apparatus was situated in a room across a hallway from the radial arm maze room.\n\nGroups of 7–8 rats were habituated to handling daily for 5 days. They were placed into a large plastic box (70 × 54 × 33 cm) with wood chips covering the floor for 2 h per day while the experimenter picked up and held each rat for 5 min. During these 5 days, food was removed from the rats’ home cages. When returned to its home cage after handling, each rat was given 10 Froot Loops (Kellogg’s, Mississauga, ON) and approximately 5 g of rat chow. At the end of the handling period, all rats weighed 85% ± 3% of their initial free feeding weights. They were maintained at these weights throughout the remainder of the experiment by adjusting the number of pellets given after testing each day. Froot Loops were not given in the home cage after the 5-day handing period.\n\nOn days 3–5 of the handling period, water was removed from each rat's cage at 17h00 and replaced with a bottle containing 20 ml of a 10% (w/v water) sucrose solution. At 8h00 the next morning the sucrose bottles were removed and replaced with water.\n\n\nExperiment 1: Post-training Sucrose\n\nThis experiment compared the effects of immediate and delayed post-training consumption of sucrose on memory for a conditioned cue preference (CCP)36.\n\nEach rat was randomly assigned to a different pair of food-paired and unpaired arms on the radial maze. Food-paired arms contained 30 Froot Loops; unpaired arms were empty. The two arms assigned to each rat were separated by at least 2 other arms. Rats were confined in their food-paired arms for 5 min and then moved to their unpaired arms for 5 min. This sequence was then repeated once more35. Placement on the two arms was always done in this order so that exposure to the unpaired arm was last. When a rat was moved between arms, the experimenter lifted the rat off the arm and placed it onto the other arm. The number of Froot Loops remaining in the paired arm was counted at the end of training and subtracted from 30 to provide a measure of consumption.\n\nEach rat was returned to its home cage when its maze training trials were complete. Rats in the immediate post-training sucrose condition (n=6) were given a 20 ml bottle of 10% sucrose in their home cages and allowed to drink for 20 min. Rats in the delay condition (n=6) were given access to the same solution 2 hrs after being returned to their cages. After the 20 min access period the sucrose bottles were replaced with water bottles for the rats in both groups.\n\n24 hours after the post-training treatments, each rat was placed on the center platform of the maze with the food-paired and unpaired arms open and no food in either arm. The entrances to the other arms were blocked. Each rat was allowed to move freely for 20 min and observed on a monitor connected to the TV camera above the maze. The times of entry into and exit from each arm were recorded and used to calculate the total time spent in each arm. An entry into or an exit from an arm was scored when a rat’s shoulders crossed a line separating the arm from the central platform.\n\nThe results are shown in Figure 1A. The amounts of time spent in the paired and unpaired arms by the rats in each experimental group were compared using pairwise planned comparisons37, (p. 73) following a two-way ANOVA (group by arm) with one repeated measure. The rats in the immediate post-training sucrose group spent significantly more time in their food-paired than in their unpaired arms (F(1,10) = 5.03, p < 0.05) while the group that drank sucrose 2 hours after maze training spent similar amounts of time in their food-paired and unpaired arms (F(1,10) < 1.0). These results show that immediate, but not delayed; post-training exposure to sucrose had a retroactive enhancing effect on memory consolidation rather than a proactive effect on performance.\n\nA) Experiment 1: Modulation by Unconditioned Sucrose. The group exposed to sucrose immediately after training on the radial arm maze conditioned cue preference (CCP) task spent more time in the food-paired arm (* p < 0.05 vs. unpaired arm) 24 hours after training while the group exposed to sucrose 2 hours after CCP training did not spend more time in the food-paired arm compared to the unpaired arm. B) Experiment 2: Modulation by Sucrose-Conditioned Cue. The group exposed to sucrose-paired cues immediately after CCP training (group ISP – immediate sucrose paired) spent more time in the food-paired arm 24 hours after training (** p < 0.01 vs. unpaired arm) while neither the group exposed to water-paired cues immediately after training (group IWP – immediate water paired) nor the group exposed to sucrose-paired cues 2 hours after training (group DSP – delayed sucrose paired) spent more time in the food-paired arm. Data are expressed as mean time (in seconds) ± SEM.\n\nTable 1 shows the mean number of Froot Loops consumed during preference training on the radial maze and the mean volume of sucrose consumed during the post-training period for each of the treatment groups. These data were analyzed with independent samples t-tests. There was no difference in the number of Froot Loops consumed (t(10) < 1.0) but significantly more sucrose was consumed by the 2-hour delay group than by the immediate sucrose group (t(10) = 2.23, p = 0.05). Since the immediate sucrose group exhibited enhanced retention but the delayed group did not, it is unlikely that the difference in sucrose consumption can account for the effects on retention.\n\n#p < 0.05 vs. immed sucrose group.\n\n*p < 0.05 vs. immed sucrose paired group (ISP).\n\n\nExperiment 2: Post-training Sucrose-Conditioned Cues\n\nThis experiment tested the hypothesis that exposure to sucrose conditioned cues would modulate memory in the same way as consumption of sucrose did in Experiment 1.\n\nAfter the initial handling period, all rats in this experiment were given 3, two-day training trials in the conditioning boxes. On one of the two days of each trial each rat was confined in one of the large compartments for 20 min with the 10% sucrose solution. On the other day, each rat was confined in the other large compartment for 20 min with water. The assignment of sucrose-paired compartments and order of sucrose- and water-pairing were counterbalanced within each experimental group.\n\n24 hours after the end of the conditioning trials the rats were trained on the CCP task as described in Experiment 1.\n\nAfter each rat completed maze training, it was placed in the conditioning box for 20 min. Neither the sucrose solution nor water was available in either compartment. Rats in the immediate sucrose-paired (ISP) condition (n=8) were placed in their sucrose-paired compartments immediately after training; rats in the immediate water-paired condition (IWP; n=8) were placed into their water-paired compartments immediately after training. The rats in the 2-hour delayed sucrose-paired (DSP) condition (n=8) were returned to their home cages for 2 hours with no food or water and were then placed into their sucrose-paired compartments.\n\n24 hours after the post-training treatments all rats were tested on the maze as described in Experiment 1.\n\nThe results are shown in Figure 1B. The rats in the ISP group spent significantly more time in their food-paired than in their unpaired arms (F(1,21) = 9.79, p < 0.01). There was no significant difference in the times spent in the 2 arms by the rats in the IWP group (F(1,21) < 1.0) or for the rats in the DSP group (F(1,21) < 1.0). These results show that immediate, but not delayed, post-training exposure to sucrose-conditioned cues, but not water-conditioned cues, had a retroactive enhancing effect on memory consolidation.\n\nTable 1 shows the mean number of Froot Loops consumed on the maze and the average amount of sucrose consumed over the 3 sucrose days. There was a significant difference in the mean number of Froot Loops consumed during preference training on the maze by the rats in the 3 groups (F(2,21) = 3.68, p < 0.05). Tukey's post-hoc tests revealed that the rats given immediate post-training exposure to their sucrose conditioned cues (ISP) ate more Froot-Loops than the rats in the 2 hr delay group (DSP; p < 0.05). It is therefore possible, although unlikely, that the difference in CCP performance of these groups was due to this difference in Froot-Loop consumption. The mean volume of sucrose consumed over the six-day training period in the conditioning box by the rats in each of the treatment groups was analyzed with two-way, repeated measures ANOVA (group by day). This analysis revealed a main effect of day (F(2,4) = 56.53, p < 0.001) indicating all 3 groups increased their sucrose consumption similarly over training days.\n\n\nDiscussion\n\nExposure to sucrose or sucrose-conditioned cues immediately after training enhanced retention of information required for expression of a CCP in the radial maze. Increasing the delay between maze training and either post-training treatment to 2 hours eliminated this effect, showing that this is a classic time-dependent memory modulation effect. This appears to be the first demonstration that post-training exposure to appetitive conditioned cues enhances memory consolidation.\n\nThe enhanced CCP in the immediate groups was not due to learning about the temporal relationship between exposure to the food-paired of unpaired maze arms and any rewarding or conditioned rewarding effects either of the post-training treatments may have had. This is because the rats were always exposed to their unpaired arms immediately before the treatments. Learning that exposure to an arm led to reward would therefore have increased the time spent in the unpaired arm at the expense of time in the food-paired arm. The fact that the rats in the sucrose and ISP groups spent more time on their food-paired than on their unpaired arms shows that this form of learning did not influence the rats' behavior. Moreover, the absence of modulation in the 2 hour delay groups shows that the enhanced CCP for the food-paired arm was due to a retroactive enhancement of memory.\n\nImmediate post-training exposure to water-paired cues in the IWP group did not have a memory modulation effect. Messier and White27 found that post-training consumption of water failed to modulate memory for a conditioned emotional response in an aversive conditioning situation. If it is true that, unlike consumption of sucrose solutions, consumption of water does not have memory modulating effects, it is not surprising that exposure to conditioned stimuli that have been paired with water also lacks these effects.\n\nA number of other aversive and appetitive unconditioned stimuli (UCS) are known to produce memory modulation when they occur during the post-training period and each of these is also associated with several well-studied unconditioned responses (UCRs). For example, food (the classic UCS) improves retention when consumed during the post-training period38, as does the consumption of sucrose solutions27,28. Both of these UCSs produce UCRs in the form of salivation39 and glucose or insulin release40,41, among other responses. Aversive events such as foot shock also modulate memory23,24,34 and produce UCRs, including increased heart rate42 and release of stress-related hormones29,43. When a rat is exposed to a UCS, memory modulation can be seen as resulting from the action of a UCR, either one of those mentioned here, or some other as yet unidentified UCR. These UCRs are subject to conditioning44,45 so it can be presumed that when they occur as conditioned responses (CRs) after conditioning, they have the same effect as the UCRs.\n\nInterestingly, rewarding and aversive UCSs produce many of the same UCRs and CRs. These include increases in blood glucose29,40,41,43, elevation in catecholamine levels such as norepinephrine and dopamine46–50 and activation of the hypothalamic-pituitary-adrenal (HPA) axis51–53, all of which have been shown to modulate memory (see4,54,55). Thus, memory modulation by unconditioned rewarding or aversive events and by rewarding or aversive conditioned stimuli may result from similar internal responses.",
"appendix": "Author contributions\n\n\n\nMRH and NMW conceived the study. MRH designed the experiments. MRH carried out the research. MRH prepared the first draft of the manuscript. MRH and NMW were involved in revising the manuscript and both have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis research was carried out in the laboratory of NMW and supported by a grant from the National Sciences and Engineering Research Council of Canada, RGPIN 7665, to NMW. MRH was supported by a National Institutes of Health, National Research Service Award F31 MH12369 from the National Institute of Mental Health during the course of this research.\n\n\nAcknowledgements\n\nWe acknowledge the assistance of Lauren Enright in the rodent behavioral training and testing.\n\n\nReferences\n\nGold PE, McGaugh JL: A single-trace, two-process view of memory storage processes. In: Deutsch D, Deutsch JA, editors. Short-Term Memory, New York: Academic Press; 1975, p. 355–78.\n\nCahill L, McGaugh JL: Modulation of memory storage. Curr Opin Neurobiol. 1996; 6(2): 237–42. PubMed Abstract | Publisher Full Text\n\nMcGaugh JL: Memory consolidation and the amygdala: a systems perspective. Trends Neurosci. 2002; 25(9): 456. PubMed Abstract | Publisher Full Text\n\nRoozendaal B, McGaugh JL: Memory modulation. Behav Neurosci. 2011; 125(6): 797–824. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcGaugh JL: Memory–a century of consolidation. Science. 2000; 287(5451): 248–51. PubMed Abstract | Publisher Full Text\n\nDuncan CP: The retroactive effect of electroshock on learning. J Comp Physiol Psychol. 1949; 42(1): 32–44. PubMed Abstract | Publisher Full Text\n\nMiller RR, Matzel LD: Memory involves far more than 'consolidation'. Nat Rev Neurosci. 2000; 1(3): 214–6. PubMed Abstract | Publisher Full Text\n\nAlberini CM, Milekic MH, Tronel S: Mechanisms of memory stabilization and de-stabilization. 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Psychopharmacology. 1984; 82(3): 203–9. PubMed Abstract\n\nMcGaugh JL: Time-dependent processes in memory storage. Science. 1966; 153(3742): 1351–8. PubMed Abstract | Publisher Full Text\n\nMcGaugh JL, Herz MJ: Memory consolidation. San Francisco: Albion; 1972.\n\nNadel L, Hupbach A, Gomez R, et al.: Memory formation, consolidation and transformation. Neurosci Biobehav Rev. 2012; 36(7): 1640–5. PubMed Abstract | Publisher Full Text\n\nMorris RG, Moser EI, Riedel G, et al.: Elements of a neurobiological theory of the hippocampus: the role of activity-dependent synaptic plasticity in memory. Philos Trans R Soc Lond B Biol Sci. 2003; 358(1432): 773–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhite NM, Legree P: Effects of posttraining exposure to an aversive stimulus on retention. Physiol Psychol. 1984; 12(3): 233–6. Reference Source\n\nJodar L, Takahashi M, Kaneto H: FS stress induces long-lasting memory facilitation: involvement of cholinergic pathways. Pharmacol Biochem Behav. 1996; 53(3): 735–40. PubMed Abstract | Publisher Full Text\n\nFlint RW, Metzger MM, Benson DM, et al.: Stress-induced memory enhancement for inhibitory fear conditioning in rats. Psychobiology. 1997; 25(1): 89–94. Reference Source\n\nMajor R, White NM: Memory facilitation by self-stimulation reinforcement mediated by the nigro-striatal bundle. Physiol Behav. 1978; 20(6): 723–33. PubMed Abstract\n\nMessier C, White NM: Contingent and non-contingent actions of sucrose and saccharin reinforcers: effects on taste preference and memory. Physiol Behav. 1984; 32(2): 195–203. PubMed Abstract | Publisher Full Text\n\nMessier C, White NM: Memory improvement by glucose, fructose, and two glucose analogs: a possible effect on peripheral glucose transport. Behav Neural Biol. 1987; 48(1): 104–27. PubMed Abstract\n\nGold PE, Vogt J, Hall JL: Glucose effects on memory: behavioral and pharmacological characteristics. Behav Neural Biol. 1986; 46(2): 145–55. PubMed Abstract\n\nGonder-Frederick L, Hall JL, Vogt J, et al.: Memory enhancement in elderly humans: effects of glucose ingestion. Physiol Behav. 1987; 41(5): 503–4. PubMed Abstract | Publisher Full Text\n\nHall JL, Gonder-Frederick LA, Chewning WW, et al.: Glucose enhancement of performance on memory tests in young and aged humans. Neuropsychologia. 1989; 27(9): 1129–38. PubMed Abstract\n\nGold PE: Role of glucose in regulating the brain and cognition. Am J Clin Nutr. 1995; 61(4 Suppl): 987S–95S. PubMed Abstract\n\nGold PE: Modulation of memory processing: enhancement of memory in rodents and humans. In: Squire LR, Butters N, editors. Neuropsychology of Memory, New York: The Guilford Press; 1992, p. 402–14. Reference Source\n\nHolahan MR, White NM: Conditioned memory modulation, freezing, and avoidance as measures of amygdala-mediated conditioned fear. Neurobiol Learn Mem. 2002; 77(22): 250–75. 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Publisher Full Text\n\nSteffens AB: Plasma insulin content in relation to blood glucose level and meal pattern in the normal and hypothalamic hyperphagic rat. Physiol Behav. 1970; 5(2): 147–51. PubMed Abstract\n\nGold MR, Cohen HD: The discharge characteristics of vagal cardiac neurons during classically conditioned heart rate change. J Neurosci. 1984; 4(12): 2963–71. PubMed Abstract\n\nHall JL, Gold PE: The effects of training, epinephrine, and glucose injections on plasma glucose levels in rats. Behav Neural Biol. 1986; 46(2): 156–67. PubMed Abstract\n\nKorte SM, Bouws GAH, Koolhaas JM, et al.: Neuroendocrine and behavioral responses during conditioned active and passive behavior inthe defensive burying/probe avoidance paradigm: effects of ipsapirone. Physiol Behav. 1992; 52(2): 355–61. PubMed Abstract | Publisher Full Text\n\nKorte SM, Buwalda B, Bouws GA, et al.: Conditioned neuroendocrine and cardiovascular stress responsiveness accompanying behavioral passivity and activity in aged and in young rats. Physiol Behav. 1992; 51(4): 815–22. PubMed Abstract | Publisher Full Text\n\nKelleher RT, Morse WH, Herd JA: Pharmacological studies of behavioral influences on cardiovascular function. Neurosci Biobehav Rev. 1981; 5(3): 325–34. PubMed Abstract | Publisher Full Text\n\nGold LH, Swerdlow NR, Koob GF: The role of mesolimbic dopamine in conditioned locomotion produced by amphetamine. Behav Neurosci. 1988; 102(4): 544–52. PubMed Abstract | Publisher Full Text\n\nFeenstra MG, Teske G, Botterblom MH, et al.: Dopamine and noradrenaline release in the prefrontal cortex of rats during classical aversive and appetitive conditioning to a contextual stimulus: interference by novelty effects. Neurosci Lett. 1999; 272(3): 179–82. PubMed Abstract | Publisher Full Text\n\nMc Quade R, Stanford SC: A microdialysis study of the noradrenergic response in rat frontal cortex and hypothalamus to a conditioned cue for aversive, naturalistic environmental stimuli. Psychopharmacology (Berl). 2000; 148(2): 201–8. PubMed Abstract | Publisher Full Text\n\nFeenstra MG: Dopamine and noradrenaline release in the prefrontal cortex in relation to unconditioned and conditioned stress and reward. Prog Brain Res. 2000; 126: 133–63. PubMed Abstract | Publisher Full Text\n\nSmotherman WP: Glucocorticoid and other hormonal substrates of conditioned taste aversion. Ann N Y Acad Sci. 1985; 443: 126–44. PubMed Abstract | Publisher Full Text\n\nBryan RM Jr, Lehman RA: Cerebral glucose utilization after aversive conditioning and during conditioned fear in the rat. Brain Res. 1988; 444(1): 17–24. PubMed Abstract | Publisher Full Text\n\nTomie A, Tirado AD, Yu L, et al.: Pavlovian autoshaping procedures increase plasma corticosterone and levels of norepinephrine and serotonin in prefrontal cortex in rats. Behav Brain Res. 2004; 153(1): 97–105. PubMed Abstract | Publisher Full Text\n\nPackard MG, Cahill L: Affective modulation of multiple memory systems. Curr Opin Neurobiol. 2001; 11(6): 752–6. PubMed Abstract | Publisher Full Text\n\nMcGaugh JL, Roozendaal B: Drug enhancement of memory consolidation: historical perspective and neurobiological implications. Psychopharmacology (Berl). 2009; 202(1–3): 3–14. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "728",
"date": "25 Jan 2013",
"name": "Kent Berridge",
"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": "733",
"date": "28 Jan 2013",
"name": "Edvard I Moser",
"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/2-22
|
https://f1000research.com/articles/2-19/v1
|
22 Jan 13
|
{
"type": "Opinion Article",
"title": "Moving beyond Type I and Type II neuron types",
"authors": [
"Frances K Skinner"
],
"abstract": "In 1948, Hodgkin delineated different classes of axonal firing. This has been mathematically translated allowing insight and understanding to emerge. As such, the terminology of ‘Type I’ and ‘Type II’ neurons is commonplace in the Neuroscience literature today. Theoretical insights have helped us realize that, for example, network synchronization depends on whether neurons are Type I or Type II. Mathematical models are precise with analyses (considering Type I/II aspects), but experimentally, the distinction can be less clear. On the other hand, experiments are becoming more sophisticated in terms of distinguishing and manipulating particular cell types but are limited in terms of being able to consider network aspects simultaneously.\n\nAlthough there is much work going on mathematically and experimentally, in my opinion it is becoming common that models are either superficially linked with experiment or not described in enough detail to appreciate the biological context. Overall, we all suffer in terms of impeding our understanding of brain networks and applying our understanding to neurological disease. I suggest that more modelers become familiar with experimental details and that more experimentalists appreciate modeling assumptions. In other words, we need to move beyond our comfort zones.",
"keywords": [
"Current research in my group involves developing",
"using and analyzing models of neurons and networks in hippocampus. Two aspects relating to the Society for Neuroscience meeting 2012 in New Orleans provided the final push for me to put my thoughts into writing via this opinion piece. First",
"when being questioned at one of our posters involving fast-spiking inhibitory cell models",
"we were asked",
"not for the first time",
"whether our neuron was Type I or II. Second",
"a symposium write-up in the Journal of Neuroscience1 stated that “…fast-spiking inhibitory cells… have a hard Class 2 threshold…”. On the face of it",
"these are reasonable questions and statements given the theoretical basis of Type I/II neurons2. However",
"on further reflection",
"I think that such questions and statements may be obscuring the hard challenges and unintentionally developing a divide by focusing on the theory rather than the theory together with the biology."
],
"content": "Introduction\n\nCurrent research in my group involves developing, using and analyzing models of neurons and networks in hippocampus. Two aspects relating to the Society for Neuroscience meeting 2012 in New Orleans provided the final push for me to put my thoughts into writing via this opinion piece. First, when being questioned at one of our posters involving fast-spiking inhibitory cell models, we were asked, not for the first time, whether our neuron was Type I or II. Second, a symposium write-up in the Journal of Neuroscience1 stated that “…fast-spiking inhibitory cells… have a hard Class 2 threshold…”. On the face of it, these are reasonable questions and statements given the theoretical basis of Type I/II neurons2. However, on further reflection, I think that such questions and statements may be obscuring the hard challenges and unintentionally developing a divide by focusing on the theory rather than the theory together with the biology.\n\n\nHistory and hope\n\nHodgkin’s (1948)3 classification of repetitive firing of axons into three types is common knowledge in the Neuroscience community. Specifically, the first two ‘classes’ are commonly referred to as Type I and II, where Type I neurons are able to exhibit arbitrarily slow frequencies as injected current levels are reduced, unlike Type II neurons, which cannot. In turn, these Types can be ‘translated’ to dynamical systems terminology as saddle node on an invariant circle and Andronov-Hopf type bifurcations respectively2. Such theoretical interpretations have allowed insight and understanding of the control of axonal firing to be obtained, including the annihilation of firing by appropriately timed inputs4. Additional theoretical aspects using Type I/II neurons have been developed. For example, Ermentrout (1996)5 has shown that a general property of Type I neurons is that they have phase response curves (PRCs) that are strictly positive. This means that depolarizing stimuli given at any time point of the firing (oscillating) neuron will always lead to an earlier start of the next action potential. This has subsequent implications for the ability of Type I or II neurons to synchronize with excitatory or inhibitory connections. In essence, whether a neuron is Type I or II endows it with different neuro-computational properties. Type I neurons, so called integrators can encode input strength, whereas Type II neurons, so-called resonators, cannot. However, Type II neurons can exhibit subthreshold oscillations, whereas Type I neurons cannot. These and many other interesting aspects are detailed in Izhikevich’s book2. He envisions a research program in which one is not only concerned with a neuron’s details (ion channels etc., as could be explored by experimentalists) but also with its neuro-computational features or what kind of bifurcations it expresses (as could be explored by mathematicians). I agree with this view. Indeed, he states that one of the goals of his book (p.20) is to “…bring these two groups of people closer together”. But…\n\n\nProblem and challenge\n\nCellular and synaptic biological details are of course important in the functioning brain, but possibly not all details are critical in all contexts. The expanding field of Connectomics is providing much information about synaptic and architectural details that can be used in computational modeling studies6. For cellular aspects, using Type I and II neuron models and their PRC characteristics has been most helpful. For example, Stiefel and Gutkin (2012)7 have described how different acetylcholine levels could lead to switching of cortical, pyramidal cells between Type I and II due to modulating biophysical characteristics in cellular models. Based on previous theoretical studies, this would have effects on network synchrony. A switching of PRC characteristics (in terms of Type I/II) with carbachol has been shown experimentally for Layer 2/3 pyramidal cells. These experimental studies were done in vitro which represents a different synaptic network environment than in vivo8. Prescott et al. (2008)9 created an in vivo-like environment in the dish and compared model and experimental work to indicate that CA1 hippocampal pyramidal cells switch from integrators (Type I) to resonators (Type II) when moving from in vitro to in vivo. The modeling studies implicated an M-type potassium current underlying the Type I/II switching.\n\nI would like to highlight two issues that these studies bring forth. First, these cellular-based studies assume that Type I/II differences are important in network synchrony based on prior theoretical studies. It is of note that these differences have now been fully examined in excitatory networks10 where network structure, synaptic strength and firing frequencies were examined. The underlying assumption of course is that network synchrony (such as at gamma frequencies) is important in cognitive functioning. Unlike in several invertebrate systems – such as the network controlling the movement of teeth in the stomach of crustaceans11 – the function of the neuronal network can be speculative. Second, whether a mathematical model representing a neuron is Type I or II can typically be unambiguously determined using bifurcation analyses. However, whether a neuron in experimental work can be identified as being Type I or II is a bit trickier as the resolution of the injected current as well as the length of time for which the current is injected would affect the resulting frequency-current curve. Tateno et al (2004)12 clearly showed a difference for fast-spiking inhibitory cells (Type II) and regular spiking pyramidal cells (Type I) in rat somatosensory cortex, which underlies the statement of “… hard Class 2” in1. Whether one should extrapolate to fast-spiking inhibitory interneurons as being Type II in general is potentially not appropriate as recent studies would seem to indicate that fast-spiking inhibitory interneurons in hippocampus could be Type I, see Figure 6 in13. As stated at the beginning of this section, knowing whether a neuron is Type I or II is helpful. However, this may not be the essential aspect when the experimental context and biological specifics are also a focus. For example, Sritharan and Skinner (2012)14 used a previously developed biophysical model of a hippocampal interneuron (with Type I-like characteristics) to examine spike reliability. They found that spike reliability at theta frequencies was favoured by an in vivo-like environment that emphasized large inhibitory, but not excitatory, fluctuations. While this did not specifically depend on Type I-like model neurons, the results were more prevalent with Type I-like model neurons. In an earlier study, Tateno and Robinson (2006)15 used dynamic clamp to examine Type I (regular spiking) and Type II (fast-spiking) neurons under various conditions involving fluctuating synaptic inputs. They found that while Type I and II aspects still gave rise to differences, this was not always the case. For example, in the context of relative timing of excitatory and inhibitory inputs, as could be important in cortical columnar circuitries, spike reliability and jitter were similar for both types.\n\nAs neuroscientists, we want to understand the workings of our brains and nervous systems, and it is clear that mathematical modeling and theoretical concepts are key to advancing our understanding. However, today, it sometimes feels as if we are trying too hard to fit the biology to the theory rather than using the theory to help us understand the biology/experiment. Indeed, theoretical and modeling studies provide much needed guidance, and consideration of Type I/II neurons has, and will continue to provide such guidance. However – to borrow a statement from the Society for Neuroscience meeting 2012 – we need to ‘embrace the messiness’ (as stated by Larry Abbott in his Albert and Ellen Grass Lecture “The Collective Wisdom of Neurons”). This interesting talk emphasized having some readout from the biological circuit from which we could come up with reduced descriptions and models to help us understand the system. He pointed out that although we are making progress in determining readouts, progress is limited in linking the reduced circuit models back to the biology. In my opinion, this limited progress may be partly due to insufficient interactions between theoretical, modeling and experimental studies so that the hard but essential questions are less likely to be asked. This is the problem. In the context of what I have presented here, although the dichotomy of Type I and II neurons has been immensely insightful, we need to move beyond this to also be clear about experimental context and biological specifics wherever possible. This is the challenge.\n\n\nWhat to do?\n\nFor theoretical analyses, mathematical models need to be in hand. In order for them to be in hand, experimental data has to be consulted. Exactly what data, how much, at what level, and how to access immediately come to the fore. Many different models have been employed in examining Type I and Type II neurons, but if the goal is to get to the heart of the different spike-generating mechanisms exhibited by Type I or II neurons, then clearly one needs to get into spatial aspects and biophysical details. For example, see16. However, to understand how and why these detailed differences may be important, a clear context (‘readout’) is needed. Unfortunately, often this is either not possible and/or very difficult to obtain and/or speculative and unclear. As such, cellular-based network models have been used in different ways17. Whether one is focused mainly on readouts or on cellular models, they ultimately need to be linked in some way. How best to go about it is far from clear, but in my opinion (and borrowing a well-known quote), “Resistance is futile”. Thus, what to do?\n\nFirst of all, there should clearly never be any confusion or conflict when comparing modeling or theoretical studies given that model specifics, assumptions and limitations can always be spelled out. Next, I think that we all need to move beyond our comfort zones more often. That is, modeling studies should make more linkages with experimental studies, suggesting specific and particular next steps so as to help ‘get the conversation started’. However, to do this, the practicalities and limitations of experimental studies need to be considered by more modelers (e.g., is there too much variability in the experiment to apply the modeling/theoretical insights? How do model parameters relate to experimental measurements being performed? How are the experiments performed?). Also, experimentalists need to read beyond the model results, questioning and understanding model assumptions and theoretical limitations so that one can assess if and how the model insights might apply and be interpreted in particular experimental and biological situations. There are clearly practical challenges in moving beyond one’s comfort zone, but for the greater good (of tackling the immense challenges of understanding brain workings and neurological disease), I think that this needs to be done on a regular basis. While combined efforts require patience, open-mindedness and respect for the realities of different research environments, we should perhaps also try to create more opportunities for “antedisciplinary” science18.\n\n\nIn closing\n\nRecently, I was excited reading an “experimentally inspired theoretical study”19 in which theoretical insights from Rall20 were used to shed light on biophysical design specifics. Besides my own interest in the details of the work, I was happy to see this work given what we know about the challenges faced by Rall in his day when he combined theoretical and experimental work. While I have focused on Type I/II issues and examples here, my opinion applies more widely, so that I end this opinion piece with a similar statement used before – “Neither ignore the details nor be consumed” by them21. Let’s move beyond our comfort zones together.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nThanks to K. Ferguson and V. Sekulic for helpful comments.\n\n\nReferences\n\nCatterall WA, Raman IM, Robinson HP, et al.: The Hodgkin-Huxley Heritage: From Channels to Circuits. J Neurosci. 2012; 32(41): 14064–14073. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIzhikevich EM: Dynamical Systems in Neuroscience: The Geometry of Excitability and Bursting. 1st edition. The MIT Press; 2007. Reference Source\n\nHodgkin AL: The local electric changes associated with repetitive action in a non-medullated axon. J Physiol. 1948; 107(2): 165–181. PubMed Abstract | Free Full Text\n\nGuttman R, Lewis S, Rinzel J: Control of repetitive firing in squid axon membrane as a model for a neuroneoscillator. J Physiol. 1980; 305: 377–395. PubMed Abstract | Free Full Text\n\nErmentrout B: Type I membranes, phase resetting curves, and synchrony. Neural Comput. 1996; 8(5): 979–1001. PubMed Abstract\n\nLeergaard TB, Hilgetag CC, Sporns O: Mapping the connectome: multi-level analysis of brain connectivity. Front Neuroinform. 2012; 6: 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStiefel KM, Gutkin BS: Cholinergic Neuromodulation Controls PRC Type in Cortical Pyramidal Neurons. In Phase Response Curves in Neuroscience. edited by Schultheiss NW, Prinz AA, Butera RJ Springer New York; 2012; 6. : 279–305. Publisher Full Text\n\nDestexhe A, Rudolph M, Paré D: The high-conductance state of neocortical neurons in vivo. Nat Rev Neurosci. 2003; 4(9): 739–751. PubMed Abstract | Publisher Full Text\n\nPrescott SA, Ratté S, De Koninck Y, et al.: Pyramidal neurons switch from integrators in vitro to resonators under in vivo-like conditions. J Neurophysiol. 2008; 100(6): 3030–3042. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFink CG, Booth V, Zochowski M: Cellularly-Driven Differences in Network Synchronization Propensity Are Differentially Modulated by Firing Frequency. PLoS Comput Biol. 2011; 7(5): e1002062. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarris-Warrick RM, Marder E, Selverston AI, et al.: Dynamic Biological Networks: The Stomatogastric Nervous System. The MIT Press; 1992. Reference Source\n\nTateno T, Harsch A, Robinson HP: Threshold Firing Frequency–Current Relationships of Neurons in Rat Somatosensory Cortex: Type 1 and Type 2 Dynamics. J Neurophysiol. 2004; 92(4): 2283–2294. PubMed Abstract | Publisher Full Text\n\nHo EC, Strüber M, Bartos M, et al.: Inhibitory Networks of Fast-Spiking Interneurons Generate Slow Population Activities due to Excitatory Fluctuations and Network Multistability. J Neurosci. 2012; 32(29): 9931–9946. PubMed Abstract | Publisher Full Text\n\nSritharan D, Skinner FK: Fluctuating inhibitory inputs promote reliable spiking at theta frequencies in hippocampal interneurons. Front Comput Neurosci. 2012; 6: 30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTateno T, Robinson HP: Rate Coding and Spike-Time Variability in Cortical Neurons With Two Types of Threshold Dynamics. J Neurophysiol. 2006; 95(4): 2650–2663. PubMed Abstract | Publisher Full Text\n\nHiggs MH, Spain WJ: Kv1 channels control spike threshold dynamics and spike timing in cortical pyramidal neurones. J Physiol. 2011; 589(Pt 21): 5125–5142. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSkinner FK: Cellular-based modeling of oscillatory dynamics in brain networks. Curr Opin Neurobiol. 2012; 22(4): 660–669. PubMed Abstract | Publisher Full Text\n\nEddy SR: \"Antedisciplinary\" science. PLoS Comput Biol. 2005; 1(1): e6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGidon A, Segev I: Principles Governing the Operation of Synaptic Inhibition in Dendrites. Neuron. 2012; 75(2): 330–341. PubMed Abstract | Publisher Full Text\n\nRall W, Segev I, Rinzel J, et al.: The Theoretical Foundation of Dendritic Function: Selected Papers of Wilfrid Rall With Commentaries. MIT Press; 1995. Reference Source\n\nSkinner FK, Mulloney B: Intersegmental coordination in invertebrates and vertebrates. Curr Opin Neurobiol. 1998; 8(6): 725–732. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "726",
"date": "24 Jan 2013",
"name": "Ernest Barreto",
"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": "739",
"date": "29 Jan 2013",
"name": "Horacio G. Rotstein",
"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 think the paper is timely and interesting, here are my comments:Page 2, column 1: \"In turn, these types can be 'translated' to dynamical systems terminology.\" To say 'translated' is to imply that dynamical systems tools provide an alternative (and equivalent) description of the dynamics. I don't think this is correct. I think dynamical systems analysis provide a mechanistic explanation of the dynamics in the form of a description of what causes the dynamics. Also, the dynamical systems description is correct in 2D. Other dynamics may occur in 3D (e.g., canards that may lead to arbitrarily slow frequencies with an underlying Hopf bifurcation. Finally, 2D saddle-node bifurcation (away from an invariant circle) can generate type II dynamics, at least theoretically. Page 2, column 1: \"Phase response curves that are always positive.\"Although it is probably implicit in the text that follows, it should be noted whether these PRCs correspond to excitation or inhibition. The classification of type I neurons as integrators and type II neurons as resonators is far from clear to me. In fact, I am not sure that the concept of integrators and resonators is well defined with a definition that has a broad consensus. I might be mistaken, but I think it would be better to leave all that as open questions.I'm not sure I agree with the statement in \"Second,...\" (page 2, column 2). I think this is true for 2D systems, but not for higher-dimensional systems. I strongly agree with the sentence that follows and I think this should be highlighted. I think that, with appropriate modifications, the sentence at the end of column 2, page 2 \"As stated...\" should be the first sentence in the paragraph.",
"responses": [
{
"c_id": "374",
"date": "21 Feb 2013",
"name": "Frances Skinner",
"role": "Author Response",
"response": "All points made by the referee are well-taken. Thank you!I respond more specifically below - the numbering is for the 6 bullet points of the referee.1) I completely agree that dynamical systems analyses is about trying to obtain a mechanistic explanation and not just an alternative description. Translated in quotes was used loosely here. 2D was assumed but not explicitly stated as the referee correctly points out.2) Yes, the assumption is that it corresponds to excitation .3) The referee has hit upon an essential aspect that I intended to bring forth in this opinion. That is, while mathematical models can be well-defined, biological aspects can only be well-defined when (enough) details and context are also provided. This is what I find is often the main challenge in combining experimental work and mathematical modeling.4) The underlying assumption here again was 2D. Thanks for bringing this to the fore.5) We're on the same page here!6) While I agree that such modifications could be done, after some thought, I think that it will require quite a bit of readjustment of the flow of the entire 'Problem and Challenge' section. I hope that the referee comments and my response will help readers further hone in to the essential point being made. That is, while Type I and II aspects can be helpful, they shouldn't become the focal point of our attempts to understand the biological system (as it may not be an important difference in a given biological context). Rather, Type I/II aspects should be considered as a potentially helpful stepping stone."
}
]
},
{
"id": "766",
"date": "13 Feb 2013",
"name": "Joel Tabak",
"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/2-19
|
https://f1000research.com/articles/2-18/v1
|
22 Jan 13
|
{
"type": "Short Research Article",
"title": "Use of synthetic polymer hydrogels to prepare scaffoldless 3D tissue constructs",
"authors": [
"Gerson Florez",
"Eric Williams",
"Minn H Saing",
"Jasvir S Khurana",
"Solomon Samuel",
"Gerson Florez",
"Eric Williams",
"Minn H Saing",
"Jasvir S Khurana"
],
"abstract": "The use of 3-dimensional tissue cultures is gaining popularity in many fields including drug discovery, toxicity testing and tissue engineering. Currently most of the techniques used to prepare these 3D tissues are time consuming and cannot be reproduced easily. There is an urgent need to optimize the preparation of these 3D tissue cultures. This study evaluated the use of synthetic hydrogel polymers used to manufacture soft contact lenses to guide cells to form multicellular tissue-like structures. It was found that bovine chondrocytes and porcine dental pulp stem cells were able to form 3D tissue structures when placed inside a soft contact lens. Commercially available microarrays, 96 or 384 well plates manufactured using synthetic hydrogel polymers may help overcome many reproducibility issues and simplify the 3D tissue culture process.",
"keywords": [
"Three dimensional tissue cultures are gaining popularity because they mimic the natural cell environment1",
"2 and are used in many applications such as drug discovery",
"toxicity testing and tissue engineering. Scaffoldless 3D tissue cultures may be superior to 3D tissue cultures prepared by immobilizing cells in extracellular matrices such as alginates",
"agarose or chitosan3. The advantages of scaffoldless tissues include avoiding immune responses to the scaffolds",
"degradation of scaffolds in vivo",
"the effects of non-physiological mechanical properties of the scaffold and avoiding cross reactivity to scaffolds during in vitro testing. There are at least four common methods reported in the literature used to prepare these 3D spherical tissue cultures or multi-cellular tumor spheroids (MCTS) without the need for any scaffolds or matrices. They include (a) the use of centrifugal force to produce cell pellets4",
"(b) the hanging drop technique5",
"6",
"(c) culturing cells at high density to enable self assembly",
"or (d) placing cell suspensions onto hydrophilic surfaces (agarose or alginate based hydrogels) so that electrochemical forces coerce cells to assemble into spherical structures7. However",
"these methods are often time consuming and the results are often poorly reproducible8."
],
"content": "Introduction\n\nThree dimensional tissue cultures are gaining popularity because they mimic the natural cell environment1,2 and are used in many applications such as drug discovery, toxicity testing and tissue engineering. Scaffoldless 3D tissue cultures may be superior to 3D tissue cultures prepared by immobilizing cells in extracellular matrices such as alginates, agarose or chitosan3. The advantages of scaffoldless tissues include avoiding immune responses to the scaffolds, degradation of scaffolds in vivo, the effects of non-physiological mechanical properties of the scaffold and avoiding cross reactivity to scaffolds during in vitro testing. There are at least four common methods reported in the literature used to prepare these 3D spherical tissue cultures or multi-cellular tumor spheroids (MCTS) without the need for any scaffolds or matrices. They include (a) the use of centrifugal force to produce cell pellets4, (b) the hanging drop technique5,6, (c) culturing cells at high density to enable self assembly, or (d) placing cell suspensions onto hydrophilic surfaces (agarose or alginate based hydrogels) so that electrochemical forces coerce cells to assemble into spherical structures7. However, these methods are often time consuming and the results are often poorly reproducible8.\n\nThe use of synthetic polymer hydrogels may overcome the limitations associated with the previously described 3D tissue culture methods and simplify 3D tissue manufacture. For example, synthetic hydrogel polymers can be used to manufacture microarrays, 96 or 384 well plates and can be made available off-the-shelf. Preparing 3D tissue structures using commercially manufactured hydrogel plates can be as simple as pipetting a known quantity of cells into these plates, incubating the plates for a certain time period (usually 24 h) and then pipetting out the formed 3D cell aggregates for further tests. In fact, Le Gac et al (2008) showed that polydimethylsiloxane (PDMS) microwell arrays may overcome some of the limitations (e.g. reproducibility, sterility, and low production yield) associated with conventional 3D tissue manufacture9. Synthetic polymer hydrogel materials have been used for a long time in the large scale manufacture of soft contact lenses. Therefore, they may also be ideal hydrogel candidates for large scale manufacture of these microarrays, 96 or 384 well plates, etc. The first logical step is to evaluate whether the synthetic polymer hydrogels used to prepare soft contact lenses have the properties required to guide cells (e.g. chondrocytes) to form 3D tissue-like structures. This property was evaluated by simply placing bovine chondrocytes or porcine dentine pulp cells in commercially available soft contact lens and observing whether they can guide the cells to form 3D structures.\n\n\nMethods\n\nBovine chondrocytes were harvested from the femoral cartilage of a 2 yr old cow obtained from a local slaughter house and the porcine dental pulp cells were harvested from the dental pulp of a euthanized 3–4 month old female Yorkshire pig. The cells were isolated from their respective tissues within 4 h of tissue harvest. It should be noted that to avoid yeast contamination, the porcine dental pulp tissue was briefly (< 30 sec) washed with 70% isopropyl alcohol. To isolate the cells, the tissues were cut into very small pieces using a scalpel blade and washed twice using regular Dulbecco’s Modified Eagle Medium DMEM, (Invitrogen, Carlsbad, CA) and 5 ml solution of TrypLE™ Express (Invitrogen, Carlsbad, CA) containing 10 mg collagenase type 2 (Worthington, Biochemical Corp., Lakewood, NJ) was added to the washed pieces in a 50 ml centrifuge tube. The centrifuge tube was placed in a shaker water bath for 2 h and maintained at 37°C. The dissociated cells along with the remaining partially dissociated tissue fragments were centrifuged to remove the collagenase and washed twice using regular DMEM media. The washed cells along with the tissue fragments were then placed in T25 cell culture flasks containing high glucose concentration DMEM media supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA), 100 units/ml penicillin, and 100 µg/ml streptomycin (complete medium). The cell culture flasks were maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells were allowed to grow in a monolayer for 15 days. Every time (3–5 day interval) the cells reached confluence, they were trypsinized (TrypLE™ Express, Invitrogen, Carlsbad, CA), passaged at 1:1 dilutions and grown in complete medium.\n\n2% agarose hydrogels (control group) were prepared by mixing 2 g of agarose in 100 ml PBS and boiling the mixture in a microwave oven. The clear agarose solution was then poured into six well plates and was allowed to form a hydrogel. For the contact lens group, (chemical composition: galyfilcon A (Acuvue advance™, Johnson & Johnson Vision Care, Inc, Jacksonville, FL), enfilcon (AVAIRA™, Cooper Vision, NY) or Etafilcon A (Acuvue2™, Johnson & Johnson Vision Care, Inc, Jacksonville, FL)), the contact lenses were removed from their sterile packaging and one placed in each well of a 12 well plate. Once placed in the plates, the contact lenses maintained their concave shape and were able to hold up to 200 µl of media without any shape deformation. 100 µl of media containing 6.0 × 105 – 5 × 106 of the desired cells (counted using the automated cell counter, TC10, Bio-Rad Laboratories, Inc., Hercules, CA) were placed carefully into the contact lens or on top of the 2% agarose hydrogels using a pipette. The cells were allowed to coalesce for 3 hours and then 3 ml of complete medium was added to each well surrounding the contact lens to keep them moist (Figure 1). Within 24 hours, the cells coalesced completely and formed a single 3D tissue like structure. The micromass was removed, placed in fresh 12 well culture plates and the soft contact lens or agarose plates were discarded. The constructs were maintained for 3 weeks and the media was changed regularly every 3–4 days. The experiments were conducted in triplicates. After 3 weeks, the micromass was then frozen sectioned; hematoxylin and eosin (H&E) stained and observed under a microscope.\n\nExperimental set up used to evaluate the formation of 3D tissue structures using synthetic polymer hydrogels (in the form of a contact lens, pictured left) and a photograph of a 3D micromass formed using chondrocytes (5 × 106 cells) after 3 weeks (right).\n\n\nResults\n\nBoth bovine femur chondrocytes and porcine dental pulp cells self-assembled when placed in contact either with the contact lens or 2% agarose hydrogel surfaces. None of the cells seemed to adhere to the soft contact lens or 2% agarose hydrogel surfaces. The micromass formed was firm after 24 h and did not crumble during handling (e.g. pipetting). This shows that the cells were already producing an extracelluar matrix to hold them together. However, these micromasses continue to shrink and by day 10 it reaches a stable size. After 3 weeks, the final diameter of the micromass depended on the initial number of cells used. The micromass (irregular in shape) was at least 1.5 mm in diameter when 6 × 105 cells were used and 2–3 mm in diameter when 1.2 × 106 cells were used. Visual examination of 3D constructs showed that the type of contact lens used did not seem to make a difference in the size of the micromass at either day 5 (Figure 2) or day 21 (Figure 3). However, further studies are needed to evaluate whether there are any differences in the matrix composition between 3D constructs formed in agarose gels versus different soft contact lens hydrogel surfaces. From a handling point of view thicker contact lenses worked better (e.g. enfilcon (AVAIRATM, Cooper Vision, NY)) and tended to maintain their shape during media placement (Figure 4). Both galyfilcon and etafilcon had similar thickness/handling properties (they both tended to fold easily) and were thinner compared to the enfilcon. It should be noted that the ease of handling is based purely on thickness of the lens, rather than the materials themselves. An example of the frozen section of micromass tissue formed with either bovine femur chondrocytes or porcine dental pulp cells is shown in Figure 5a and 5b respectively. Hematoxylin and eosin (H&E) stain was used to visualize the cells and the presence of extracellular matrix produced by the cells.\n\nChondrocytes grown in (top to bottom) hondrocytes grown in (top to bottom) enfilcon (AVAIRA™, Cooper Vision, NY), galyfilcon A (Acuvue advance™, Johnson & Johnson Vision Care, Inc, Jacksonville, FL), and etafilcon A (Acuvue2™, Johnson & Johnson Vision Care, Inc, Jacksonville, FL)) at 5 days.\n\nOn visual inspection of the micromass it was concluded that the type of contact lens used did not seem to make a difference. The micromass (left to right) was formed in galyfilcon A (Acuvue advance™, Johnson & Johnson Vision Care, Inc, Jacksonville, FL), enfilcon (AVAIRATM, Cooper Vision, NY), and etafilcon A (Acuvue2™, Johnson & Johnson Vision Care, Inc, Jacksonville, FL) respectively.\n\nThinner contact lenses (bottom) tended to fold and release the cell contents during initial cell placement.\n\nFrozen H&E section (400X) of 3 week old 3D tissue structure formed using bovine femur chondrocytes (a) and porcine dental pulp cells (b). The microscopy slide shows viable cells embedded in a hyalinized background (hydrogel material). The cells show rounded slightly eccentric nuclei with a clear space around them.\n\n\nDiscussion\n\nHydrogels such as agarose, alginate or PDMS have been used before to guide or coax cells to form 3D multicellular tissues3,9. This study evaluated the use of synthetic polymer hydrogels used to prepare soft contact lenses to do the same. The results showed that both chondrocytes and dentine pulp cells reproducibly formed 3D tissue structures when placed inside these soft contact lenses. Therefore, the study demonstrated that the synthetic polymer hydrogels (e.g. galyfilcon A, enfilcon or etafilcon A or even poly HEMA10) may provide an alternative to agarose, alginate or PDMS based hydrogel surfaces.\n\nIt is well known that soft contact lens can be manufactured in large scale without much variability within batches and stored for a long time in sterile solutions. The same technology can theoritically be used to manufacture and store 3D cell aggregation devices in the form of microarrays, 96 or 384 well plates using these synthetic polymer hydrogels. A commercially available 3D cell aggregation device manufactured using synthetic polymer hydrogels has the potential to simplify and help large scale manufacture of reproducible 3D cell cultures for various applications including in vitro chemical toxicity testing and preparation of small scaffold-less tissue engineered constructs.",
"appendix": "Author contributions\n\nSS conceived the study. SS and GF designed the experiments and carried out the research. EW and MS contributed to the design of experiments and the preparation of manuscript. JK helped with the histology sections. 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\nThis study was supported by Albert Einstein Society, Philadelphia, PA with an internal grant provided to Solomon Samuel.\n\n\nReferences\n\nCaron MM, Emans PJ, Coolsen MM, et al:Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures. Osteoarthritis Cartilage. 2012; 20: 1170–1178.\n\nMandl EW, Van Der Veen SW, Verhaar JA, et al:Multiplication of human chondrocytes with low seeding densities accelerates cell yield without losing redifferentiation capacity. Tissue Eng 2004; 10: 109–118.\n\nBernstein P, Dong M, Corbeil D, et al:Pellet culture elicits superior chondrogenic redifferentiation than alginate-based systems. Biotechnol Prog 2009; 25: 1146–1152.\n\nDetzel CJ, Van Wie BJ: Use of a centrifugal bioreactor for cartilaginous tissue formation from isolated chondrocytes. Biotechnol Prog 2011; 27: 451–459.\n\nTung YC, Hsiao AY, Allen SG, et al:High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst 2011; 136: 473–478.\n\nTimmins NE, Nielsen LK: Generation of multicellular tumor spheroids by the hanging-drop method. Methods Mol Med 2007; 140: 141–51.\n\nHu JC, Athanasiou KA: A self-assembling process in articular cartilage tissue engineering. Tissue Eng 2006; 12: 969–979.\n\nNapolitano AP, Dean DM, Man AJ, et al:Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels. Biotechniques 2007; 43: 494–500.\n\nLe Gac S, Rivron N, Wijnperle D, et al:Cellular microtissues spontaneously formed in a microfabricated device for angiongenesis. In: 12th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 12–16 October 2008, San Diego, CA.\n\nPhung YT, Barbone D, Broaddus VC, et al:Rapid Generation of In Vitro Multicellular Spheroids for the Study of Monoclonal Antibody Therapy. J Cancer 2011; 2: 507–514."
}
|
[
{
"id": "749",
"date": "05 Feb 2013",
"name": "Wael Kafienah",
"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 brief report described a method for aggregating matrix producing cells such as chondrocytes using synthetic hydrogel polymers. The polymers were used merely as non-adherent 'containers' to aggregate the cells. Indeed, many labs coat plastic wells with agarose to prevent cells seeded onto other biomaterials from sticking to the plastic well during the tissue engineering process.\n\nIt is important to note that the proposed study does not prove in any way if using the investigated polymers in this fashion is any better than say centrifuged pellets or using ultra-low adherence plates that can come in all kinds and shapes. The study however proves that the tested commercial polymers are biocompatible and permissive to cell aggregation. Hydrogels are inert material by nature unless they are functionalised or modified chemically. Indeed, the authors demonstrate no difference in the gross appearance of the aggregates between the different polymers used.\n\nFurthermore, it is not clear what outcome is expected using either cell type in the study. What was the expected outcome for culturing chondrocytes or pulp cells using this method? The results do not determine whether the polymers merely supported cell aggregation or driven their differentiation. To this end, staining of specific matrix proteins such as aggrecan or type II collagen in the case of chondrocytes for example is critical to answer this question. Other issues that the authors should address include: 1) show photos for histological analysis of all samples (including control). This applies for all kinds of staining (H&E, type II collagen, aggrecan, markers of dental pulp cells?).2) dental pulp cells are uncharacterised. What are they?! How are they expected to behave on plastic or in other similar culture systems?2) state the number of animals used in each case (n number). Anything less than 3 makes the results questionable.3) higher magnification photos are needed for figures 1 to 4. 4) rehash the discussion which is not structured and minimal. The authors are advised to read this article:http://www.bmj.com/content/318/7193/1224 If the authors fulfill all of the above then this short report can be approved for publication.",
"responses": []
},
{
"id": "1362",
"date": "16 Aug 2013",
"name": "Mikko Lammi",
"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 by Florez et al. describes the use of contact lenses as a material to culture chondrocytes and dental pulp cells. The cells formed scaffold-free cell aggregates of variable sizes, which depended on the original amount of cells seeded. The cultures were apparently biocompatible, although the viability was not checked after 3 weeks of cultivation. This would be easy to confirm with Live/Dead fluorescent markers.Centrifugation is often used to form cell pellets of chondrocytes (or mesenchymal stem cells in chondrogenesis assays), so in a way this study appears to aim to replace such technology. Therefore, it would be advisable to compare the results of this technology to pellets made by centrifuging, since centrifuging as such is practically costless. Also, looking at Figure 5A, the H&E staining locates mainly to cells in bovine chondrocytes, which is rather surprising. I would have expected that staining would be more like the one shown for dental pulp cells in Figure 5B. The stainings would benefit from immunostaining for type II collagen, and maybe from additional staining for GAGs by stains such as toluidine blue or Safranin O (even though hematoxylin should do the same in H&E staining).The irregular shape of the aggregates makes me wonder whether the number of cells differs in parallel samples. Therefore, DNA analysis would be useful to clarify this issue. The irregularity is problematic for tissue engineering purposes (even for small constructs), but as commented in the discussion, might be useful for chemical toxicity testing.As another referee commented, the discussion in its present form is very minimal. In particular, what is required is the reasoning for why this technology is needed, and how it can overcome the present methods.",
"responses": []
},
{
"id": "1702",
"date": "25 Sep 2013",
"name": "Carlos E Semino",
"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 described an interesting technology consisting of seeding cells on an hydrogel made out of synthetic polymer used to manufacture contact lenses. They used bovine chondrocytes and porcine dental pulp cells and placed them on top of soft contact lenses. After a few days they observed the formation of 3D-cellular structures. Then, they cultured the constructs for three weeks. It is important to mention that the use of commercial polymers benefits the experimental setup, in terms of better reproducibility and potential production. The idea of turning this into a 96- and 384-well plate platform looks very attractive since future technologies and platforms are required in order to produce easy 3D-constructs for pharmacology, toxicology, stem cell and cancer research. I believe this is a excellent technical contribution, which is simple and feasible to be adapted to a HTS (high-throughput screening). In my opinion the work needs some more characterization at the cellular/construct level with relatively simple tests:I believe it will be important to monitor, as the previous referee mentioned, some live/dead analyses to see how the cells perform inside the construct over time.In addition, constructs could be stained with phalloidin/DAPI to assess cellular configuration by fluorescent microscopy. Also, a general morphological analysis of the construct could be performed, to see if they also form a necrotic core like the classical spheroids, as this would be important. Finally, since the authors used bovine chondrocytes I agree with the previous referee that it would be interesting to test for GAG (glucosaminoglycan) production by simple staining with Toluidine blue. In the discussion section, the authors should further explore the potential use of this technology, especially if they suggest translating it into 96- and 384-well format for potential HTS.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-18
|
https://f1000research.com/articles/2-17/v1
|
21 Jan 13
|
{
"type": "Correspondence",
"title": "Authors are also reviewers: problems in assigning cause for missing negative studies",
"authors": [
"Stephen Senn"
],
"abstract": "I compare two possible extreme hypotheses regarding submission of papers to journals: the Q hypothesis, whereby the decision to submit is based on quality of research; and the P hypothesis, whereby it is based on probability of acceptance. I give five reasons as to why the P hypothesis is more plausible and suggest that problems of missing data may previously have caused researchers to misinterpret the evidence on editorial bias.",
"keywords": [
"Iain Chalmers and Kay Dickersin have written an interesting commentary1 in this journal on an earlier paper of mine2. I am grateful for the attention they have paid to my article but do not agree with their conclusions for reasons I set out below."
],
"content": "Communication\n\nIain Chalmers and Kay Dickersin have written an interesting commentary1 in this journal on an earlier paper of mine2. I am grateful for the attention they have paid to my article but do not agree with their conclusions for reasons I set out below.\n\nA key characteristic of work from the evidence based medicine (EBM) movement has been its stress on the dangers of bias3 and the acclamation of the randomised clinical trial (or sometimes the meta-analysis of such) as representing the very highest level of evidence4. However, EBM enthusiasts sometimes forget that the same pitfalls that beset observational studies of the effects of treatment are a danger for observational studies of the process of evaluating evidence. The claim I made in my previous paper in this journal2 was that researchers in the field of evidence methodology had failed to appreciate the problems with the research instrument they were using and that, in consequence, this research was fundamentally flawed. Much of the argument presented by Chalmers and Dickersin in their commentary on that paper1 consists of citing the research I had already called into question, for example the 2002 JAMA paper5. I think the research was flawed whereas, presumably, they do not but, whatever their opinion, simply citing such research does not answer my criticisms.\n\nWe can consider two extreme hypotheses: the Q hypothesis and the P hypothesis (Mixtures of these two extreme cases can be envisaged, but to understand the problem it is sufficient to consider the extremes.). The Q hypothesis is necessary if the sort of research that Chalmers and Dickersin1 cite is to be valid. The Q hypothesis supposes that negative and positive studies submitted to journals are comparable in terms of quality. That being so, a difference in acceptance rates for negative and positive studies would be evidence of editorial bias, and the fact that such a difference is not found is reassuring. On the other hand, the P hypothesis supposes that a rational decision to submit to a journal would be based on probability of acceptance, which cannot thus (necessarily) be expected to differ by outcome of study, even when bias is present. Thus, an editorial bias would be shown by difference in quality of accepted negative and positive studies despite equal probabilities of acceptance. Equality in acceptance rates would only be reassuring as regard lack of editorial bias if quality did not differ.\n\nThe P hypothesis involves a sort of reverse causation: perceived probability of a future event is what triggers submission and this determines the quality of what is submitted. If the P hypothesis is correct, then EBM researchers who followed the Q hypothesis, which (implicitly) was the case in the JAMA paper that Chalmers and Dickersin cite5, have got things back to front. This may seem far-fetched, but it would not be the first time that such a mistake has been made. For example, some years ago a study showed that Academy Award (‘Oscar’) winners lived longer than a control group of non-winning actors6. This was interpreted as showing the benefit of esteem in terms of years of life gained. However, a more careful analysis suggested it was long life that increased your chance of winning and not vice versa7. I used to explain the point at issue to my students thus: to discover that those who had ever received telegrams from the (British) Queen were unusually long-lived (you would not be proof of the life-preserving effect of royal telegrams, since you receive one if you live to be one hundred.\n\nAnother example can be given. The TARGET study compared lumiracoxib to naproxen and ibuprofen in more than 18,000 patients suffering from osteoarthritis. Patients were stratified by concomitant low-dose aspirin use8,9. An interesting finding was that aspirin users had a significantly higher rate of cardiovascular events than non-aspirin users. The authors commented that this was ‘as expected’8 (p679). In view of the considerable experimental evidence on the cardiovascular prophylactic efficacy of aspirin10, why did they expect this result and not regard it as paradoxical? The answer is that they took it as obvious that anticipated cardiovascular events would increase the probability of low-dose aspirin usage and allowed for it in the design. This reverse-causation explanation reconciles the experimental and the observational evidence.\n\nIn short, the way in which the data have arisen needs to be considered carefully and failure to do so is a fault of many of the studies that Chalmers and Dickersin or, for that matter, Goldacre11 cite. A further irony is that the whole reason why missing negative studies are of such concern is that their missingness causes a bias in evaluating the effects of treatment. The authors of the paper5 cited by Chalmers and Dickersin failed to notice what they should have been sensitised to spot: the studies’ missingness also causes a bias in evaluating the editorial process. The central issue is, ‘what would happen to the studies authors don’t submit if they did submit them?’ It is naïve to suppose that a simple comparison of studies they do submit can say what would happen to those they don’t. Since my investigation of this issue was inspired by readingBad Pharma11, I can’t resist putting it like this: neither the US Food and Drug Agency (FDA) nor the European Medicines Agency (EMA) accept as a ‘strategy’ for dealing with missing data, ‘just ignore the problem and analyse as usual’12–14.\n\nIn fact, I consider the P hypothesis is more reasonable that the Q hypothesis for five reasons, some theoretical and some empirical. I list them below.\n\n1. If researchers behave rationally they will submit according to perceived probability of acceptance. We can suppose that there is a reward, R(Y), and a cost C(Y) of submission of a study with outcome Y where Y = 0,1 according to whether the study is negative or positive. The expected return of submission is positive if P(q,Y) R(Y) – C(Y) > 0, ∴ P(q,Y) > C(Y)/R(Y), where P(q,Y) is the probability of acceptance seen as an increasing function of the quality, q, of the study and also (possibly, for this is the point to be examined) of the outcome. If C(1)/R(1) = C(0)/R(0), which is to say that if the ratio of cost to reward is independent of outcome, then the threshold probability at which authors will submit to a journal is identical for both negative and positive studies, without any implication that the quality will therefore be the same. If C(1)/R(1) < C(0)/R(0) then the threshold probability of acceptance would actually have to be higher for negative than positive studies. Under neither case would observed equal acceptance rates be a proof of lack of editorial bias.\n\n2. As Chalmers and Dickerson note, there is considerable experimental evidence that reviewers are more likely to reject negative versions of a given study. Under the P hypothesis, this is easily reconciled with the observed equality of rejection rates in observational studies. Under the Q hypothesis, the observational and experimental results are at variance with each other. Thus, just as the reverse-causation hypothesis reconciles experimental and observational data on aspirin, so does the P hypothesis for editorial bias.\n\n3. As Chalmers and Dickersin note, we have evidence that authors are less likely to submit negative studies than positive ones. This makes it improbable that the mixture of studies by quality submitted to journals will be identical, which is what the Q hypothesis requires. However, the P hypothesis does not require quality to be equal between submitted positive and negative studies.\n\n4. In support of this we have observational evidence that the quality of submitted negative studies is higher than positive studies despite acceptance rates being the same15. This is exactly what the P hypothesis predicts, but is not compatible with the Q hypothesis.\n\n5. However, the most important point is one everybody seems to have overlooked. By and large authors and reviewers are (in the long run) the same. I doubt that the experience of Chalmers and Dickerson is much different from my own. I write a lot and I review even more. I have a rule of doing one review (if asked) and no more for journals I have no intention of writing for, but review regularly for those journals I publish in often (for example, Statistics in Medicine). Thus I review (mainly) for what I write in, although as a medical statistician I probably review more papers by physicians than physicians do by me. It is true that in his extensive analysis of editorial boards of journals in information science16, Cabanac found the hardly surprising result that editorial board members had in general some considerable seniority whether measured in years since first published or number of published papers, and one might expect that very junior researchers are more likely to submit papers than review them. However, it is pretty obvious that most researchers do both. In their proposal for improving peer review, Hauser and Fehr took it as being so obvious that referees were authors that they suggested punishing tardy reviews by placing the reviewer’s next paper as author in a ‘sin bin’17. Thus, reviewers are (mainly) just authors on another occasion. The Q theory requires researchers to have Jekyll and Hyde personalities. Vile hypothesis tester Mr Hyde chooses inappropriately that the negative studies he has conducted should not be submitted, while journal reviewer Dr Jekyll justly judges similar studies with the Wisdom of Solomon. Faced with a negative paper the referee asks, ‘would I submit something like this?,’ answers, ‘No!,’ and then recommends publication. I regard this as improbable. This leads to my main point.\n\nMy main point picks up on my fifth reason. The whole business of what gets published and what does not does not lend itself to separation. This is a point I made in my original paper. Chalmers and Dickersin1 dismiss this, but I stand by my original statement. The studies that the EBM movement has carried out fail by the very standards the movement promotes elsewhere. Fairness applies not only to the business of judging the effects of medicines but to the business of judging the business by which they are judged.\n\nHowever, I will permit myself an unfair opinion. Nothing much can be hoped for from the sad and sorry mess that is the medical press. I regard it as irredeemable. It makes no difference what the origin of the problem is: whether medical researchers as authors or medical researchers as reviewers are saints or sinners. If they are not guilty one way, they are guilty the other, but the simplest explanation of the facts is that they are guilty in both. In any case, the problem is not just with what is absent, but with what is present. We need to make it possible to check what is published18 and currently very few medical journals do so.\n\nWe need to find a radically different solution: one which renders meaningless the accolade of publishing in a ‘leading’ journal, one which shows the impact factor for the fraud it is. We need to make such journals irrelevant for disseminating the results of primary research. We have to look elsewhere for that.",
"appendix": "Competing interests\n\n\n\nI consult regularly for the pharmaceutical industry and my career is furthered by publishing. I maintain a full declaration of interest here http://www.senns.demon.co.uk/Declaration_Interest.htm.\n\n\nGrant information\n\n\n\n\nReferences\n\nChalmers I, Dickersin K: Biased under-reporting of research reflects biased under-submission more than biased editorial rejection. F1000 Research. 2013; 2 (1). Reference Source\n\nSenn S: Misunderstanding publication bias: editors are not blameless after all. F1000 Research. 2012; 1 (59). Reference Source\n\nEditorial Comment. Fair tests of treatments in health care. The James Lind Library; 2007. Reference Source\n\nPhillips B, Ball C, Sackett D, et al.: Levels of Evidence (March 2009). Oxford: Centre for evidence based medicine; 2009. Reference Source\n\nOlson CM, Rennie D, Cook D, et al.: Publication bias in editorial decision making. JAMA. 2002; 287 (21): 2825–8. PubMed Abstract | Publisher Full Text\n\nRedelmeier DA, Singh SM: Survival in Academy Award-winning actors and actresses. Ann Intern Med. 2001; 134 (10): 955–62. PubMed Abstract | Publisher Full Text\n\nSylvestre MP, Huszti E, Hanley JA: Do OSCAR winners live longer than less successful peers? A reanalysis of the evidence. Ann Intern Med. 2006; 145 (5): 361–3; discussion 392. PubMed Abstract | Publisher Full Text\n\nFarkouh ME, Kirshner H, Harrington RA, et al.: Comparison of lumiracoxib with naproxen and ibuprofen in the Therapeutic Arthritis Research and Gastrointestinal Event Trial (TARGET), cardiovascular outcomes: randomised controlled trial. Lancet. 2004; 364 (9435): 675–84. PubMed Abstract | Publisher Full Text\n\nSenn S: Lessons from TGN1412 and TARGET: implications for observational studies and meta-analysis. Pharm Stat. 2008; 7: 294–301. PubMed Abstract | Publisher Full Text\n\nHennekens CH: Final report on the aspirin component of the ongoing Physicians Health Study. N Engl J Med. 1989; 321 (3): 129–35. PubMed Abstract | Publisher Full Text\n\nGoldacre B: Bad pharma: how drug companies mislead doctors and harm patients. London: Fourth Estate; 2012. 430 p. Reference Source\n\nInternational Conference on Harmonisation. Statistical principles for clinical trials (ICH E9). Stat Med. 1999; 18: 1905–42. PubMed Abstract\n\nCommittee for Medicinal Products for Human Use (CHMP). Guideline on Missing Data in Confirmatory Clinical Trials London: European Medicine Agency; 2010; p. 1–12. Reference Source\n\nLittle RJ, D’Agostino R, Cohen ML, et al.: The prevention and treatment of missing data in clinical trials. N Engl J Med. 2012; 367 (14): 1355–60. PubMed Abstract | Publisher Full Text\n\nLynch JR, Cunningham MR, Warme WJ, et al.: Commercially funded and United States-based research is more likely to be published; good-quality studies with negative outcomes are not. J Bone Joint Surg Am. 2007; 89 (5): 1010–8. PubMed Abstract | Publisher Full Text\n\nCabanac G: Shaping the landscape of research in information systems from the perspective of editorial boards: A scientometric study of 77 leading journals. J Am Soc Inf Sci Technol. 2012; 63 (5): 977–96. Publisher Full Text\n\nHauser M, Fehr E: An incentive solution to the peer review problem. PLoS Biol. 2007; 5 (4): e107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSenn SJ: Overstating the evidence: double counting in meta-analysis and related problems. BMC Med Res Methodol. 2009; 9: 10. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "720",
"date": "22 Jan 2013",
"name": "Paul Silcocks",
"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 Senn’s arguments both plausible and persuasively expounded. I am surprised that the potential confounding by study quality of the relation between acceptance rates and type of outcome has not been more widely studied, although I, like Chalmers and Dickens, am puzzled by Senn’s statement1 that “it also seems plausible that higher quality studies are more likely to lead to a positive result” – possibly what is intended here is “more likely to lead to a convincing result either way”. The correspondence as a whole would be strengthened by distinguishing truly “negative” results from those that are merely inconclusive.",
"responses": []
},
{
"id": "734",
"date": "28 Jan 2013",
"name": "Steff Lewis",
"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": "735",
"date": "28 Jan 2013",
"name": "James Matcham",
"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": "737",
"date": "29 Jan 2013",
"name": "Sara Hughes",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn his concluding statement, Senn argues for a radically different solution for disseminating the results of primary research. Expanding his conclusions to add suggestions for how this may be addressed would be valuable.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-17
|
https://f1000research.com/articles/2-16/v1
|
16 Jan 13
|
{
"type": "Research Article",
"title": "Introduction of structured physical examination skills to second year undergraduate medical students",
"authors": [
"Rano M Piryani",
"P Ravi Shankar",
"Trilok P Thapa",
"Bal M Karki",
"Rishi K Kafle",
"Mahesh P Khakurel",
"Shital Bhandary",
"P Ravi Shankar",
"Trilok P Thapa",
"Bal M Karki",
"Rishi K Kafle",
"Mahesh P Khakurel",
"Shital Bhandary"
],
"abstract": "Introduction: Effective learning of physical examination skills (PES) requires suitable teaching and learning techniques and assessment methods. The Tribhuvan University (Nepal) curriculum recommends involving the departments of Medicine and Surgery in PES training (PEST) for second year students as a part of early clinical exposure. The project was developed to make teaching/learning of PES structured, involving eight clinical sciences departments and using appropriate methods for teaching and assessment in KIST Medical College, Nepal.Methods: Irby’s three stages of clinical teaching model (Preparation, Teaching, Reflection), was applied for teaching. Skill acquisition was based on Millers’ learning pyramid at “show how level” and Dreyfus’ competency model at “competent level”. Teaching/learning was conducted in small groups. A tutorial, demonstration and practice (TDS) model was developed for teaching/learning techniques based on a simple five-step method for teaching clinical skills. Assessment of effectiveness of training was done at “reaction level” as per Kirkpatrick’s model based on students’ feedback, “shows how level” as per Miller’s pyramid of learning by OSCE and “competent level” as per Dreyfus’ model using retro-pre questionnaire.Results: The analysis of retro-pre questionnaire based on the Dreyfus model found the average skill score (max score 184), before the introduction of the project module as 15.9 (median = 13.5) and after as 116.5 (median = 116). A paired t-test showed the difference to be statistically significant (100.5±23 and 95% CI 95.45 – 105.59). The average overall feedback score for the students on PES training based on seven items on a five point Likert scale was found to be 4.30. The mean total objective structured clinical examination (OSCE) score was 3.77 (SD+/- 0.33) out of 5; 80% of students scored more than 70%.Conclusion: Students learned most of the skills with the implementation of the structured PES module and did well in the OSCE. Students and faculty were satisfied with the training and assessment.",
"keywords": [
"Learning physical examination skills (PES) is an important aspect of undergraduate medical students’ training in the early clinical years1. Effective clinical teaching and learning of PES requires appropriate teaching and learning techniques and assessment methods2. KIST Medical College (KISTMC)",
"Lalitpur",
"Nepal",
"is a newly established medical school in the private sector and admitted its first batch of students in November 2008. It follows the curriculum of Tribhuvan University Institute of Medicine (TU-IOM)",
"Kathmandu",
"Nepal3. The curriculum stresses early clinical exposure with the first year being devoted to the acquisition of history taking and communication skills and the second year to physical examination skills. The curriculum recommends involvement of the departments of Medicine and Surgery in PES training (PEST) for second year students as part of early clinical exposure (ECE). The methods for teaching/learning and assessment are not well defined in the curriculum."
],
"content": "Introduction\n\nLearning physical examination skills (PES) is an important aspect of undergraduate medical students’ training in the early clinical years1. Effective clinical teaching and learning of PES requires appropriate teaching and learning techniques and assessment methods2. KIST Medical College (KISTMC), Lalitpur, Nepal, is a newly established medical school in the private sector and admitted its first batch of students in November 2008. It follows the curriculum of Tribhuvan University Institute of Medicine (TU-IOM), Kathmandu, Nepal3. The curriculum stresses early clinical exposure with the first year being devoted to the acquisition of history taking and communication skills and the second year to physical examination skills. The curriculum recommends involvement of the departments of Medicine and Surgery in PES training (PEST) for second year students as part of early clinical exposure (ECE). The methods for teaching/learning and assessment are not well defined in the curriculum.\n\nThe project was developed to make teaching-learning of PES structured, and involved eight clinical departments using appropriate teaching, learning and assessment methods. Students are provided with an opportunity to learn basic physical examination skills in gynecology and obstetrics, orthopedics, ear, nose and throat (ENT), ophthalmology and pediatrics, as well as general medicine and surgery in their second year Bachelor of Medicine and Bachelor of Surgery degrees (MBSS) so that they get sufficient time to learn reasoning, diagnostic, procedural, therapeutic and counseling skills during their clinical years (third, fourth and final year). The objective of the project was that at the end of the second year, students should be able to initiate and perform a basic physical examination of an adult suffering from medical, surgical, gynecology and obstetric, orthopedic, ENT and eye diseases, as well as a basic physical examination of a child.\n\n\nMethods\n\nFaculty members of departments involved in the project identified basic PES to be learnt by students. A checklist for each selected PES was prepared based on Hutichson’s Clinical Methods (22nd edition)4 and was peer reviewed and finalized by a core project committee.\n\nAll faculty members involved in teaching received teacher training before commencement of the module. They were oriented with regards to the implementation of the project in a mini-workshop. The details regarding grouping of students, the posting schedule of various groups in different departments in rotation, the approach to teaching and learning, the teaching-learning strategy and the assessment modalities were also shared with them.\n\nPhysical examination skills involve psychomotor skills. For teaching physical examination skills, Irby’s three stages of clinical teaching were applied5. These are: preparation (stage I), teaching (stage II) and reflection (stage III).\n\nSkills acquisition was based on Millers’ Learning Pyramid6 at the “show how level” and Dreyfus’ competency model6 at the “competent level”. Miller’s four levels of learning are:\n\n1) Whether the learner has knowledge of the skill;\n\n2) Whether the learner knows how the skill is performed;\n\n3) Whether the learner shows how to perform the skill in a controlled or simulated setting; and\n\n4) Finally, whether the learner actually does the skill in clinical practice.\n\nThe basic principle of the Dreyfus model is that the student progresses through five stages of proficiency in this specific order: novice, advanced beginner, competent, proficient, and expert6.\n\nTeaching-learning was conducted in small groups. The one hundred students were divided into seven groups of 14–15 students; each group was further divided into two subgroups of seven or eight students. Each group was posted for four weeks each in Medicine Units I and II, surgery, pediatrics, and gynecology and obstetrics, and each subgroup for two weeks in family medicine, ENT, ophthalmology and orthopedics in rotation. Students learned PES related to the cardiovascular system (CVS) and respiratory system (RSS) in Medicine Unit I, the peripheral nervous system (PNS) and central nervous system (CNS) in Medicine Unit II, the examination of the abdomen in Surgery, the musculoskeletal system (MS) in orthopedics, general PES in family medicine, obstetrics and gynecology examination in the obstetrics and gynecology department, ear, nose and throat examination in ENT and eye examination in ophthalmology. Structured PEST (S-PEST) sessions were held for four hours every Monday for 28 weeks between February and August 2011.\n\nBased on the method used for teaching clinical skills in the American College of Surgeon’s advanced trauma life support course, a tutorial, demonstration and practice (TDP) model was developed. Each S-PEST session had three sub-sessions: Tutorial (T), Demonstration (D) and Practice (P). The ‘Tutorial’ element covered the overview by the faculty preceptor on skills to be taught; ‘Demonstration’ involved actually demonstrating each of the skills taught with a stepwise description, while ‘Practice’ involved performance/practice of each demonstrated skill by the students using a sequential description to be observed by the preceptor. This model follows five (conceptualization, visualization, verbalization, practice and correction and reinforcement) of the seven psychomotor teaching principles based on the taxonomy of psychomotor domain (the other two being skill mastery and skill autonomy)7.\n\nIn most sessions, demonstration and performance/practice were conducted on real patients either in the ward or outpatient department (OPD). Some sessions were conducted on simulated patients, while in a few sessions the students themselves consented to be simulated patients.\n\nAssessment of PES training effectiveness was conducted at Kirkpatrick’s level 1 - Reaction (see below for details) based on student feedback. Skill performance was assessed at Millers’ level 3 (Show How) by an objective structured clinical examination (OSCE), and perceived competence at Dreyfus’ level 3 (Competence) using the retro-pre questionnaire.\n\nDonald Kirkpatrick developed a four-level model of evaluation:\n\n1) Reactions: measures how participants have reacted to the training.\n\n2) Learning: measures what participants have learned from the training.\n\n3) Behavior: measures whether what was learned is being applied on the job.\n\n4) Results: measures whether the application of training is achieving results8–10.\n\nThe following instruments were used for assessment:\n\n1. The retro-pre questionnaire for assessing learners’ self-reported changes. The retrospective post-then-pre design is a popular way to assess learners’ self-reported changes in knowledge, awareness, skills, confidence, attitudes or behaviors. It takes less time, is less intrusive and for self-reported change, avoids pre-test sensitivity and response shift bias that result from pre-test overestimation or underestimation11.\n\n2. A feedback questionnaire to assess the perception of teaching and learning sessions of S-PEST from students and faculty members.\n\n3. The OSCE was used for the end of the posting assessment. Standardized patients (SP) were used in the OSCE. They were trained to follow students’ commands for various aspects of the physical examination. SP were healthy individuals from our house keeping department who consented to be SP. They were given prior briefing regarding appropriate mannerisms and how to respond to students’ commands during OSCEs. A faculty observer at each station used a checklist to rate each student’s performance.\n\nThe following components were developed by the station authors for each OSCE station:\n\nAn instruction sheet for the examinee.\n\nA checklist for the assessment of the skill being examined at that station.\n\nA detailed patient profile for the standardized patient.\n\nA list of the equipment, instruments etc required at the station.\n\nData was analysed using SPSS version 18.0.\n\nThe institutional Research Committee of KIST Medical College approved the project.\n\n\nResults\n\nForty-six skills, representing various systems in different departments during the training were included in the retro-pre questionnaire. Two to four skills from the ‘must know’ category from each subject/chapter were included in this questionnaire. Each skill was scored out of 4. Individual skill scores were added to get overall scores. A paired t-test was used to evaluate the difference in overall scores before and after the module. Scores were found to follow a normal distribution as confirmed by a Shapiro-Wilk test.\n\nThe average perceived skills score (the maximum score being 184) before the module was 15.9, which increased to 116.5 after the module. Students perceived that their level of skill improved after the module. The result from the paired t-test showed that the difference is highly statistically significant (mean 100.5 with SD+/- 23 and 95% confidence interval 95.45 – 105.59), which means that students did learn most of the skills after the Structured Physical Examination Skills Training (S-PEST) module and it did influence them.\n\n\n\nAround 75% of students filled in the feedback questionnaire which used a five point Likert scale. The questions were on the objectives of the session, facilitator/preceptor role, satisfaction with learning activities in each sub-session, overall rating of session and there were two open ended questions (“Suggestion/s for improvement” and “Any other comment/s”). Respondents gave scores out of 5, the higher the score, the higher the satisfaction.\n\nThe average scores of the different questions and global scores (overall session ratings) were calculated. The average feedback score was found to be 4.30 (maximum score 5) and the overall global score was 8.34 (maximum score 10) (see Figure 1 for scores and average scores). The agreement between global (subjective) and overall items average (objective) scores were found to be 78% (Pearson’s Correlation).\n\nOverall scores refer to the average student feedback scores (out of 5) in response to seven questions for each session. Global scores refer to the average student feedback scores (out of 10) for the importance of each session as a whole. Students agreed that they learned the skills of all the systems in various departments and they strongly agreed learning General Physical Examination skills in the Family Medicine department. Session abbreviations: MED-RRS – Medicine-Respiratory System; MED-CVS – Medicine-Cardiovascular System; SURG-ABD – Surgery-Abdomen; MED-CNS – Medicine-Central Nervous System; MED-PNS – Medicine-Peripheral Nervous; PEDS = Pediatrics; OBGYNE-I – Obstetrics; OBGYNE-II – Gynecology; ORTHO-MS – Orthopedics-Musculoskeletal System; FM-GPE – Family Medicine-General Physical Examination; EYE-EX – Eye Examination; ENT-EX – Ear Nose Throat Examination.\n\nAlmost all respondents recognized the importance of each sub-session (Tutorial, Demonstration and Practice). The most frequent remark obtained from the open-response category question was to “provide more time for practice”.\n\n\n\nThe feedback questions were on areas for improvement, how sessions could be improved, what could have been done differently and the faculty members’ perceptions of the development of reasoning and diagnostic skills early on in the students’ clinical years. Fifty-six feedback forms were received from faculty members. The frequency of each item scores on perception of teaching/learning sessions conducted as per protocol together with students’ clinical reasoning and diagnostic skills developed early in clinical years was calculated. None of the faculty members strongly disagreed, one (1.8%) disagreed, 2 (3.6%) remained neutral, 33 (58.9%) agreed and 20 (35.7%) strongly agreed.\n\nThe most frequent comments obtained from the open response category questions were:\n\nI. Areas for improvement:\n\n1) Students require more time for practice.\n\n2) Decrease group size.\n\n3) Increase number of patients available for teaching-learning.\n\nII. How sessions could be improved:\n\n1) More time required for demonstration on patients.\n\n2) Models may be used for demonstration and practice.\n\nIII. What could be done differently:\n\n1) Using videos of PE.\n\n2) Demonstration on manikins.\n\n3) Teaching on models.\n\nOut of 100 students, 98 attended the OSCE. There were 14 OSCE stations; each representing a different system (CVS, RSS, PNS, CNS, Abdomen I & II, Obstetrics, Gynecology, Pediatrics I & II, MS, General PE, Eye, and ENT). The mean total OSCE score obtained by students in each station was 3.77 with a standard deviation (SD+/-) of 0.33 (the maximum score was 5). Eighty percent of the students scored more than 70% (26 students scored more than 80%, 55 students between 70 and 80%, and 15 students between 60% and 70%). A graph of the OSCE scores is shown in Figure 2.\n\nSession abbreviations: Gyne – Gynecology; Obs – Obstetrics; M-CVS – Medicine-Cardiovascular System; M-RES – Medicine-Respiratory System; Sur I – Surgery-Abdomen-liver; Peds I – Pediatrics I; Surgery II – Abdomen-kidney; Ortho – Orthopedics-Musculoskeletal System; M-PNS – Medicine-Peripheral Nervous System; Peds II – Pediatrics II; M-CNS – Medicine-Central Nervous System; FM-GPE – Family Medicine-General Physical Examination; EYE – Eye Examination; ENT – Ear Nose Throat Examination.\n\n\n\n\nDiscussion\n\nClinical skills acquisition is a major focus of education for health professionals extending from undergraduate to postgraduate and continuing to professional education12.\n\nThe current trend in medical education is to introduce clinical teaching early, within the first two years of the curriculum, to help students understand the relevance of the basic sciences to clinical practice and to provide instruction in basic clinical skills in a standardized fashion13.\n\nFollowing these trends, Tribhuvan University (TU), Kathmandu, included early clinical exposure (ECE) in the curriculum, revised in 2008. The clinical examination, a required course for second year students, concentrates on the teaching of physical examination skills in two departments (Medicine and Surgery) with no defined method of teaching/learning and assessment3.\n\nThe importance of structured clinical education has long been recognized. It provides equal learning opportunities and a suitable environment for everyone to acquire clinical skills and competencies. Modules are especially suitable for outcome-based adult learning programs and maximizing adult learning14,15. With this purpose in mind, a teaching/learning module was developed in this project at the KIST Medical College Nepal KISTMC (affiliated to TU) for teaching basic physical examination skills to second year students as part of early clinical exposure. KISTMC involved 8 clinical science departments and made teaching, learning and assessment structured.\n\nTeaching-learning was conducted in small groups as small group teaching and learning is considered effective in clinical settings for tutorials and demonstrations16.\n\nPhysical examination skills are largely psychomotor skills. For teaching physical examination skills, Irby’s three stages of clinical teaching were applied (Preparation, Teaching and Reflection)5,17. Though faculty members and students reflected on experiences at the end of each session, these reflections could not be recorded5.\n\nAll the faculty members involved in teaching received teacher-training before commencement of the course and were oriented about the implementation of the project18.\n\nStudents were well informed about the project implementation but a limitation was that the students’ stage of competency could not be assessed at the beginning of the project (this is why the retro-pre questionnaire was used).\n\nSkill acquisition was based on Millers’ Learning Pyramid at the ‘Show how level’ and Dreyfus’ competency model at the ‘Competent level’ (i.e. consciously competent)5,6.\n\nBased on the method used for teaching clinical skills in the American College of Surgeon’s advanced trauma life support course, a tutorial, demonstration and practice (TDP) model was developed because of the limited time allocated for demonstration and practice session.\n\nFeedback both from faculty members and students was taken on teaching and learning. All students were satisfied with the S-PEST. Almost all recognized the importance of each sub-session (tutorial, demonstration and practice). Students agreed that they learned the skills of all the systems but suggested more time to be provided for practice. Sir William Osler (1849–1919) gave emphasis to practice. He said:\n\n“Observe, record, tabulate, communicate. Use your five senses… Learn to see, learn to hear, learn to feel, learn to smell, and know that by practice alone you can become expert”19.\n\nFaculty members too were generally satisfied with the S-PEST. They commented that with the implementation of this module, students’ clinical reasoning and diagnostic skills seemed to develop early on in the students’ clinical years. Faculty members too felt that the students required more time for practice. They suggested that models, manikins and videos may be used for demonstration in addition to real and simulated patients.\n\nPatsy Stark and F. Fortune had previously suggested that models may not be appropriate for teaching/learning skills but manikins and videos could be used instead20. They suggested that dedicated and structured clinical skills training is the most important factor, whether it takes place in a skills centre, in the ward or in the community20. A significant improvement in first-year medical student performance on the adult PE occurred after the use of a web-based instructional video at the University of Connecticut, School of Medicine, USA21.\n\nIn this study, assessment of skills training effectiveness was done at level 1 (Reaction) as per Kirkpatrick’s model from students through feedback and skill performance done at level 3 (Show How) as per Miller’s pyramid model of demonstrated learning by OSCE and perceived competence at level 3 (competent) as per Dreyfus competency model of skill performance through the use of the retro-pre questionnaire6,8–10,22.\n\nAnalysis of our retro-pre model in line with the Dreyfus model of skill acquisition suggests that students did learn most of the skills following the implementation of the S-PEST Module. One limitation is that although the retro-pre model may reveal valuable information, it is not a substitute tool for an objective measure or a gold standard, but can be used where a large number of skills are to be assessed at one point in time. Don W. Scott et al. from the University of Chicago Pritzker School of Medicine used retro-pre modelling for assessing teaching skills and they recommended this method for assessment23.\n\nThe OSCE is a proven valid and reliable, formative and summative tool for assessing the clinical skills learned by health sciences students24. Standardized patients (healthy individuals trained to portray all the characteristics of an actual patient and to provide constructive feedback) were used in the OSCE. Students did well in the OSCE but one limitation was that only one skill from each system was assessed out of several skills taught in each system. Students and Faculty members also seemed to be satisfied with the OSCE process.\n\n\nConclusion\n\nStudents that were introduced to S-PEST acquired basic PES used to examine medical, surgical, gynecological and obstetric, orthopedic, ENT, and eye adult patients, as well as pediatric patients. It is expected that S-PEST will enhance medical students' performance during their clinical years. In this study, the students did well in OSCE and both students and Faculty members were satisfied with the training and assessment.\n\n\nLimitations\n\nThe main limitations of this study were:\n\n1. Limited time was allocated for each training session;\n\n2. A pre-test for students’ stage of competency could not be done;\n\n3. Both real and simulated patients were used for demo and practice;\n\n4. Reflection on experiences at the end of each session could not be documented; and\n\n5. In OSCE, only one skill from each system was assessed out of several skills taught.",
"appendix": "Author contributions\n\n\n\nRMP conceptualized the project and developed the project proposal in consultation with PRS. RMP, PRS, TPT, BMK and RKK were all involved in designing and implementing the project. MPK also contributed to the implementation of the study. RMP, PRS and SB contributed to the data analysis and interpretation. RMP wrote the initial draft of the article. RMP, PRS, MPK and SB all reviewed the article. All authors approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by KIST Medical College.\n\n\nAcknowledgements\n\nWe acknowledge the support of management, faculty, students, support staff, patients and simulated patients of KISTMC in the implementation of this project. We recognize the efforts of Ms. Shova Poudel, Office Assistant for her assistance in data entry and Dr Suneel in writing. We are grateful to Prof. Dr Thomas V Chacko, Director PSG FAIMER Institute, Coimbatore, Tamal Nadu, India, FAIMER faculties and FAIMER family for support and inspiration.\n\n\nReferences\n\nElliot DL, Hickam DH: Evaluation of physical examination skills. Reliability of faculty observers and patient instructor. JAMA. 1987; 258(23): 3405–8. PubMed Abstract | Publisher Full Text\n\nAligning Assessment with Intended Learning Outcomes. Downloaded on June 03, 2012. Reference Source\n\nCurriculum for Bachelor of Medicine & Bachelor of Surgery Tribhuvan University Institute of Medicine. Revised in 2008 published by Medical Education Department, Institute of Medicine, Kathmandu, Nepal.\n\nMichael Swash, Michael Glynn: Sir Robert Hutchison In: Hutchison’s Clinical Methods: An integrated approach to clinical practice. 22nd ed. Saunders Elsevier; 2007. Reference Source\n\nNadia JI, Charlene MD: Teaching Physical Exams and Procedural Skills. Downloaded on May 1, 2011. Reference Source\n\nAssociation of American Medical Colleges: Recommendations for clinical skills curricula for undergraduate medical education. 2008. Reference Source\n\nGeorge JH, Doto FX: A simple five-step method for teaching clinical skills. Feature Editor Paul M Paulman. Fam Med. 2001; 33(8): 577–8. PubMed Abstract\n\nBenner P: Using the Dreyfus model of skill acquisition to describe and interpret skill acquisition and clinical judgment in nursing practice and education. Bull Sci Technol Soc. 2004; 24(3): 188. Publisher Full Text\n\nSusan Croes: Kirkpatrick's Four Levels of Evaluation. Downloaded on March 27, 2011. Reference Source\n\nWinfrey EC: Kirkpatrick's Four Levels of Evaluation. In: B. Hoffman (Ed.), Encyclopedia of Educational Technology. Retrieved on April 27, 2011, from 1999. Reference Source\n\nProgram Development and Evaluation: Using the Retrospective Post then-Pre Design, Quick Tips #27. University of Wisconsin-Extension, Madison, WI. 2005.Reference Source\n\nOmer R, Amir AA, Ahmed AM, et al.: An experience in early introduction of clinical teaching in a clinical skills laboratory. Sudanese J Publ Health. 2010; 5(2): 29–31. Reference Source\n\nCoupey SM, McEvoy M, Myers DC, et al.: Preparing Einstein Students to Practice Twenty-first Century Medicine. Einstein J Biol Med. 2004; 20: 71–77. Reference Source\n\nVirginia BR: Techniques: Developing Learning Modules for Adults. J Adult Educ Spr. 1990; 18(2): 1–5. Reference Source\n\nDonnelly R, Fitzmaurice M: Designing modules for learning. Downloaded on June 03, 2011. Reference Source\n\nBath D, Smith C: A tutor’s guide to teaching and learning at University of Queensland Australia. Downloaded on March 27, 2011. Reference Source\n\nIrby DM, Bowen JL: Time-efficient strategies for learning and performance. Clin Teach. 2004; 1(1): 23–28. Publisher Full Text\n\nWeston W: Clinical teaching TIPS - A hand Book of Clinical Teachers. 2008; Downloaded on March 27, 2011.\n\nFaculty Development: Teaching clinical skills. Reference Source\n\nStark P, Fortune F: Teaching clinical skills in developing countries: are clinical skills centres the answer? Educ Health (Abingdon). 2003; 16(3): 298–306. PubMed Abstract | Publisher Full Text\n\nOrientale E Jr, Kosowicz L, Alerte A, et al.: Using Web-based Video to Enhance Physical Examination Skills in Medical Students. Fam Med. 2008; 40(7): 471–6. PubMed Abstract\n\nOttawa: Preliminary report with draft Consensus Statements and Recommendations for the Performance Assessment Theme. 2010; Downloaded on June 03, 2011. Reference Source\n\nScott DW, Podrazik P, Levine S, et al.: Practice Makes Perfect? Assessment of Geriatrics Observed Structured Teaching Evaluation (G-OSTE) for Non-Geriatrics Faculty. Downloaded on October 24, 2012. Reference Source\n\nIqbal M, Khizar B, Zaidi Z, et al.: Revising an objective structured clinical examination in a resource-limited Pakistani medical school. Educ Health (Abingdon). 2009; 22(1): 209. PubMed Abstract"
}
|
[
{
"id": "880",
"date": "08 Apr 2013",
"name": "Ramesh K Adhikari",
"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 reads more like a description of an activity, suggesting that it reflects the article which reports on effectiveness of the TDP model in improving PES.In regards to the content, I would suggest that the authors explain the 'retro- pre-questionnaire' in some detail. When did they administer this questionnaire and if there is any relationship between the responses to the questionnaire and OSCE? The conclusions are sensible and balanced.The data are satisfactory to justify the conclusions.",
"responses": [
{
"c_id": "478",
"date": "08 Jun 2013",
"name": "Rano Mal Piryani",
"role": "Author Response",
"response": "Dear Ramesh Adhikari Thanks for reviewing article and providing suggestions for update. The title of the article was finalized after consultation with all authors. A pre test was not done, so 'retro- pre-questionnaire' was used for assessing learners, i.e. self reported changes in acquiring physical examination skills. This was done at the end of project, before OSCE session. This is mentioned in the article. No correlation was done between response of retro-pre and OSCE score.Rano Mal PiryaniPrinciple Author"
}
]
},
{
"id": "969",
"date": "29 May 2013",
"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\nTitle and Abstract: The title is OK, though I would describe it as a structured physical exam skills educational program (italicized words added by me). The abstract focuses largely on the theoretical models underpinning the approaches to evaluation. I think it would be clearer to focus concretely on concisely describing the evaluation performed. The theoretical rationale for that approach can be described in the paper itself.Article content: Overall, the methods and design are adequately explained, though there are a few important exceptions. The authors go into great detail about the theoretical models underpinning their approach to evaluation. This is fine but is not really critical. The critical components of both the survey and the OSCE are whether these are validated instruments. I assume the authors created both for this project, but it is important for them to state that these tools have not been previously validated (unless of course they have). In addition, I did not find the Figures particularly helpful. They might be more clearly displayed in table format than as graphs.Conclusions: The conclusions are reasonable, though I would add the caveat that because the measurement instruments were not validated it is difficult to be certain of their reliability. Also, the authors should emphasize that the meaning of the OSCE scores in particular is not clear since there is no comparison group and the OSCE has not been validated.Data: The data is limited, but the curriculum design is presented in adequate detail.",
"responses": [
{
"c_id": "477",
"date": "08 Jun 2013",
"name": "Rano Mal Piryani",
"role": "Author Response",
"response": "Dear Deborah Korenstein, Thanks for reviewing article and providing suggestions for update. Title and Abstract: As suggested evaluation performed is added in abstract concisely. Article content: Both the survey and the OSCE were validated. Details of validation could not be included in article because of word limits. Validation of OSCE process was done in steps mentioned below. Step I: OSCE academic team was formed. Preliminary workshop on OSCE was arranged in May 2010 for 3 hours for drafting OSCE stations (development of all three components of OSCE station--tasks, checklists and instruction/direction to simulated patients) utilizing standard book of clinical methods and evidence based literature. For drafting of tasks and checklists The Hutchison's Clinical Methods 22nd edition published in 2007 was chosen as standard book. Faculty members involved in teaching physical examination skills were instructed to draft two OSCE stations for each system within 8 weeks time. Step II: Second workshop of OSCE academic team for two half days was organized in July 2010 to assess and finalize all three components of OSCE stations, develop instructions for examiner/assessor and scoring (marking) criteria. Editing of all three components of OSCE stations was done by experienced senior faculty members. Step III: All the documents developed in second workshop printed in first week of August for conducting OSCE session in the end of August 2010 for first batch of undergraduate students.Step IV: OSCE session conducted in the last week of August 2010 and the written feedback obtained from the examiners/assessors, faculty involved in teaching physical examination skills and students. This session was considered as piloting for OSCE session to be conducted for batch II in 2011 for proposed project. Step V: Feedback taken from the examiners/assessors, faculty involved in teaching physical examination skills and students compiled, analyzed and shared with OSCE academic team in a meeting arranged in September 2010. Also score obtained on each station and total score by students shared in the same meeting. The team fully satisfied with the entire process and made some recommendations for conducting session for batch II in 2011 for proposed project. Step VI OSCE academic team developed in 2010, reactivated in May 2011. Faculty members involved in teaching were assigned to review all three components of OSCE stations i.e. Tasks (stem), checklists and instruction/direction (training information) to simulated patients used in 2010 for assessment of first batch technically and as well as in the light of recommendation of OSCE academic team for conducting next session. OSCE Academic Team in depth reviewed all three components of OSCE stations in July 2011 in meeting cum workshop for two half days, edited & finalized. The documents printed in August 2011 and OSCE session held on September 27, 2011 for second batch (for project). Conclusions: Validated tools were used for assessment. Mean of score obtained by students (98 students) on each OSCE station was calculated. Then overall mean was calculated. This data is included in article. Data: All the available data is included in data files of the article.Rano Mal PiryaniPrinciple author"
}
]
},
{
"id": "989",
"date": "07 Jun 2013",
"name": "Bruce Fisher",
"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\nTitle and Abstract: The title is acceptable and clear.Article content: The authors are to be applauded for their thoroughness and attention to accepted pedagogical models when designing and setting up their curriculum. However, there is little description of the actual content and emphasis of the curricular components, and the methods by which key knowledge and skills were actually assessed in the OSCEs. It is not clear if the OSCEs had been validated, or whether there was any comparison group for analysis of effect. There was no information to determine if the OSCE was used in any novel way. Data: The methods used to introduce the curriculum were presented in some detail, but as stated above there were significant areas not described. The figures were unhelpful and the data could be better portrayed in table form. Conclusions: The conclusions were substantiated by the data presented, but without information about any comparison or control group, it is difficult to gauge the impact of the curricular intervention (over that of secular trend etc). It is also not clear what these conclusions add to the existing literature.",
"responses": [
{
"c_id": "480",
"date": "08 Jun 2013",
"name": "Rano Mal Piryani",
"role": "Author Response",
"response": "Dear Bruce Fisher Thanks for reviewing article and providing suggestions for update. Article content: Due to word limitation criteria, every detail of content cannot be included in the article; however, adequate information is provided in the introduction, methods and result section. Curricular components are mentioned briefly in the introduction. Contents are described briefly in method section under development of module, orientation of faculty members, approach to teaching, approach to learning, teaching/learning strategies and assessment section. OSCE was validated, the details of which are mentioned in the comments in response to Dr. Deborah Korenstein (See comments in response to referee 2). OSCE was conducted in a novel way. The summary of practical arrangements for OSCE at KIST Medical College is as follows: 1. Prior to the OSCE session.Suitable venue was selected.Assessors/examiners were identified.Standardized Patients were selected.Running order of the stations in circuit was developed.List of equipments/instruments required was prepared. Tasks, checklists, feedback questionnaires etc printed 2. The day before the OSCE sessionInspection of OSCE stations doneStations were clearly labeledCondition of required equipments/instruments was checked A pack of the documents for the OSCE examiners, students and patients were made availableSigns were displayed at proper place3. On the day of the OSCE sessionReliable stop watch and loud manual bell were usedSupport staffs were placed to direct the candidates, examiners, and SPsAssessors explained SPs about their role once againStudents were briefedSupervisors observed the sessionFeedback was taken from students, assessors and SPs 4. At the end of the OSCE sessionFeedback questionnaires were collectedChecklists were collectedToken money paid to SPsContributions of everyone was appreciated 5. After the OSCE sessionScore was compiled and result declaredFeedback questionnaires data compiled Data: All the available data is included in data files of the article.Conclusions: The conclusions are sensible, reasonable and rational.Rano Mal Piryani Principle Author"
}
]
}
] | 1
|
https://f1000research.com/articles/2-16
|
https://f1000research.com/articles/2-15/v1
|
16 Jan 13
|
{
"type": "Research Article",
"title": "Implementing a successful journal club in an anesthesiology residency program",
"authors": [
"Nathaniel D Pitner",
"Chris A Fox",
"Matthias L Riess",
"Nathaniel D Pitner",
"Chris A Fox"
],
"abstract": "Journal clubs are an integral element of residency training. We report the successful implementation of a monthly structured journal club in our anesthesia residency program. Based on resident surveys before and one year after its start, the journal club led to a significantly higher confidence in how to critically appraise literature and present a manuscript. The journal club also improved the residents' ability to search the literature and their statistical knowledge, skills that are essential in the practice of evidence-based medicine. We describe key features that may aid other training programs in organizing a stimulating an educational and sustainable journal club.",
"keywords": [
"journal club",
"residency",
"anesthesiology"
],
"content": "Introduction\n\nJournal clubs are an integral element of residency training across many medical specialties1–5. Ideally, they teach research design, statistical methods, and critical appraisal skills. Further goals are keeping up-to-date with the current literature and impacting clinical practice through evidence-based medicine4. However, there appears to be no gold standard and organizing a successful journal club that meets these goals and achieves both longevity and high resident participation can be challenging3,4. Thus, we surveyed the residents in our program to facilitate the creation of a successful journal club and to assess its effectiveness in teaching research methodology and critical appraisal skills.\n\n\nMethods\n\nAfter approval by the Institutional Review Board (PRO9864), a voluntary and anonymous resident survey was conducted initially, and repeated one year after implementation of a structured monthly journal club. Thirty-five (67%) of 52 available residents attended the first journal club meeting. Thirty residents (86%) participated in the initial survey, and 21 residents (95% of the 22 attending) completed the follow-up survey one year later [follow-up data in brackets].\n\nResults are expressed as mean ± SEM (standard error of the mean) or percentages. Residents were asked to judge the usefulness of the journal club for their training on a scale of 1 (not useful) to 5 (extremely useful) as well as their abilities in four separate areas (literature search, critical literature appraisal, manuscript presentation, and statistics) on a scale of 1 (not comfortable) to 5 (extremely comfortable). Kruskal-Wallis and Mann-Whitney tests were utilized for statistical analysis of these rating-scale data. Chi square test was used to analyze categorical data, e.g. preference of mandatory vs. voluntary attendance, frequency, time and location of journal club meetings, and preferred type of articles. Spearman’s rank correlation was employed to determine the correlation between two variables. Bonferroni correction was applied to correct for multiple comparisons. Statistical significance was assumed when P < 0.05 (two-tailed). Significance symbols are * vs. alterative, † vs. initial survey.\n\n\nResults\n\nHaving a regular and structured journal club was continuously rated to be useful at 3.5±0.2 [3.3±0.2]. In total, 70%* [62%] of residents favored monthly, 17% [38%] quarterly, and 13% [0%] weekly journal club meetings. A total of 63%* [43%] preferred voluntary, and 37% [57%] mandatory attendance; level of training correlated positively with a shift to mandatory while perceived usefulness did not. A total of 53% [38%] of residents preferred to meet before work, 40% [29%] after work; 57% [57%] preferred the workplace, 40% [33%] outside the workplace; time (before work) correlated significantly with location (workplace). In total, 30% [24%] preferred the selection of review articles, 23% [5%] primary research studies, and 43% [71%] a mix of both types for discussion; interest in primary studies correlated positively with level of training.\n\n67% of responders in the initial survey reported their expectations: 85% preferred articles that had an impact on their clinical decision-making, while 50% wanted to improve their skills in critical appraisal, 20% literature search, 15% manuscript presentation, and 15% statistical methods. Between 1 (not comfortable) and 5 (extremely comfortable), residents rated their ability to search literature 2.8±0.2 [3.2±0.2], to critically appraise lit erature 2.3±0.2 [3.0±0.1†], to present a manuscript 2.6±0.1 [3.1±0.2†], and their statistical knowledge 1.9±0.2* (vs. search and presentation) [2.3±0.2* (vs. search)] (Figure 1). In the follow-up survey, the majority of residents reported a benefit from the journal club in each of these four areas (Figure 1). Interestingly, perceived statistical knowledge correlated significantly with length of previous statistical training, but in-training exam results in statistics did not.\n\nSelf-assessment scores range from 1 (not comfortable) to 5 (extremely comfortable). Data are presented as mean ± SEM. Percentages state the fraction of residents who reported in the follow-up survey to have profited from journal club in these four areas. P values are given for comparisons between initial and follow-up surveys. Statistical significance was assumed when P < 0.05 (two tailed). Significance symbols are * vs. literature search, ** vs. literature search and manuscript presentation.\n\n33% in the follow-up survey favored a single faculty moderator for the entire year, having a different moderator for each meeting was suggested by 52% and having multiple moderators per meeting by 14%* (vs. different moderators).\n\n\nDiscussion\n\nJournal clubs have long been recognized as an effective tool for teaching evidence-based medicine2. They are often used to teach clinical epidemiology and biostatistics to foster critical appraisal skills, an educational goal that is frequently rated higher than keeping up with the medical literature3,5,6. Residents who are being taught appraisal skills reportedly pay more attention to a study’s methodology and are more critical of its conclusions3.\n\nFactors associated with high attendance and longevity includes mandatory attendance, incentives such as food, and a high educational value attributed by the program director. A trained journal club leader to choose articles and direct the discussion leads to higher educational satisfaction3,4. Interestingly, presentation of original articles only is associated with longevity, but lower attendance rates7. Other important characteristics of successful journal clubs are timely dissemination of reading material, preferably via the internet, and regularly scheduled meetings at predictable intervals and at a time appropriate for all participants4; in this context, monthly intervals have been found optimal because of a potentially diminishing interest if conducted too often.\n\nFollowing these recommendations we have successfully implemented a structured Faculty-led journal club with presentation and critical evaluation by a group of two to three second-year residents. In both surveys this was well received and considered very useful. The journal club is held monthly at the workplace and before work, and attendance is mandatory; free food is available. Based on follow-up data, we have switched from initially one faculty moderator for the entire year to now having a different moderator from a variety of anesthesia subspecialties for each meeting. A mix of reviews and seminal original articles is selected by the moderators, and particular attention to methodology and statistics is emphasized. The journal club led to significantly higher ratings in the residents’ ability to critically appraise literature and to present a manuscript, and improvement in literature search skills and statistical knowledge.\n\nAlthough we have to acknowledge the natural limitations of our single-center study – such as a limited sample size and further to be determined discriminative validity, and inter- and intra-rater reliability – its results show that a journal club can be an excellent teaching tool that complements other theoretical and practical training in anesthesiology, and provides a skill set necessary to understand and practice evidence-based medicine. Our experience may aid and encourage other training programs in organizing a stimulating, educational and sustainable journal club that is well-received by its residents and can accomplish these goals.\n\n\nConsent\n\nThis case report is in accordance with Institutional Review Board guidelines and IRB approval was obtained before beginning of the study (PRO9864).",
"appendix": "Author contributions\n\n\n\nAll authors were involved in the draft and revision of the manuscript and have agreed to its final content. Study planning was done by CAF and MLR, data collection by MLR, and data analysis and presentation by NDP and MLR.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nInstitutional support only (NDP, CAF and MLR). No grants were involved in supporting this particular work. Unrelated research support for MLR through the Department of Veterans Affairs (CARA-026-10F) and NIH.\n\n\nReferences\n\nDoust J, Del Mar CB, Montgomery BD, et al.: EBM journal clubs in general practice. Aust Fam Physician. 2008; 37(1–2): 54–6. PubMed Abstract\n\nLee AG, Boldt HC, Golnik KC, et al.: Structured journal club as a tool to teach and assess resident competence in practice-based learning and improvement. Ophthalmology. 2006; 113(3): 497–500. PubMed Abstract | Publisher Full Text\n\nAlguire PC: A review of journal clubs in postgraduate medical education. J Gen Intern Med. 1998; 13(5): 347–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeenadayalan Y, Grimmer-Somers K, Prior M, et al.: How to run an effective journal club: a systematic review. J Eval Clin Pract. 2005; 14(5): 898–911. PubMed Abstract | Publisher Full Text\n\nMoharari RS, Rahimi E, Najafi A, et al.: Teaching critical appraisal and statistics in anesthesia journal club. QJM. 2009; 102(2): 139–41. PubMed Abstract | Publisher Full Text\n\nEbbert JO, Montori VM, Schultz HJ, et al.: The journal club in postgraduate medical education: a systematic review. Med Teach. 2001; 23(5): 455–61. PubMed Abstract | Publisher Full Text\n\nSidorov J: How are internal medicine residency journal clubs organized, and what makes them successful? Arch Intern Med. 1995; 155(11): 1193–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "710",
"date": "18 Jan 2013",
"name": "Bob Phillips",
"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": "738",
"date": "29 Jan 2013",
"name": "Andrew 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",
"responses": []
},
{
"id": "742",
"date": "30 Jan 2013",
"name": "Patricia Khashayar",
"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/2-15
|
https://f1000research.com/articles/2-14/v1
|
15 Jan 13
|
{
"type": "Research Article",
"title": "Characteristics of exhaled particle production in healthy volunteers: possible implications for infectious disease transmission",
"authors": [
"Fatima Wurie",
"Olivier Le Polain de Waroux",
"Matthew Brande",
"Wesley DeHaan",
"Katherine Holdgate",
"Rishi Mannan",
"Donald Milton",
"Daniel Swerdlow",
"Andrew Hayward",
"Olivier Le Polain de Waroux",
"Matthew Brande",
"Wesley DeHaan",
"Katherine Holdgate",
"Rishi Mannan",
"Donald Milton",
"Daniel Swerdlow",
"Andrew Hayward"
],
"abstract": "The size and concentration of exhaled particles may influence respiratory infection transmission risk. We assessed variation in exhaled particle production between individuals, factors associated with high production and stability over time.We measured exhaled particle production during tidal breathing in a sample of 79 healthy volunteers, using optical particle counter technology. Repeat measurements (several months after baseline) were obtained for 37 of the 79 participants.\n\nMultilevel linear regression models of log transformed particle production measures were used to assess risk factors for high production. Stability between measurements over time was assessed using Lin’s correlation coefficients.Ninety-nine percent of expired particles were <1μm in diameter. Considerable variation in exhaled particle production was observed between individuals and within individuals over time. Distribution of particle production was right skewed. Approximately 90% of individuals produce <150 particles per litre in normal breathing. A few individuals had measurements of over 1000 particles per litre (maximum 1456). Particle production increased with age (p<0.001) and was associated with high tree pollen counts. Particle production levels did not remain stable over time [rho 0.14 (95%CI -0.10, 0.38, p=0.238)].Sub-micron particles conducive to airborne rather than droplet transmission form the great majority of exhaled particles in tidal breathing. There is a high level of variability between subjects but measurements are not stable over time. Production increases with age and may be influenced by airway inflammation caused by environmental irritants. Further research is needed to determine whether the observed variations in exhaled particle production affect transmission of respiratory infection.",
"keywords": [
"Exhaled particles serve as a vehicle of transmission for some pathogens. Respiratory infection transmission can be described as either droplet or airborne. Droplet transmission relates to larger particles that are expelled and rapidly settle to the ground",
"usually within 1 minute of production1. Droplet transmission therefore relies on relatively close proximity to the source case. These larger particles are generated from the upper respiratory tract during coughing or sneezing or during procedures such as suctioning or bronchoscopy2. Larger particles tend to deposit on external mucus membranes or high up in the respiratory tract. Settled droplets can also contribute to fomite transmission. Airborne transmission is caused by smaller expelled particles which can stay suspended in the air for long periods exposing a greater number of contacts at greater distance1–3. They are formed by the re-opening of closed airway passages",
"which de-stabilise the mucous surface layer4. These smaller particles penetrate further into the lower respiratory tract to alveolar level. It is not possible to define a cut-off particle diameter at which aerodynamic behaviour changes",
"however",
"the World Health Organisation use a 5 µm cut-off to distinguish between airborne and droplet transmission5."
],
"content": "Introduction\n\nExhaled particles serve as a vehicle of transmission for some pathogens. Respiratory infection transmission can be described as either droplet or airborne. Droplet transmission relates to larger particles that are expelled and rapidly settle to the ground, usually within 1 minute of production1. Droplet transmission therefore relies on relatively close proximity to the source case. These larger particles are generated from the upper respiratory tract during coughing or sneezing or during procedures such as suctioning or bronchoscopy2. Larger particles tend to deposit on external mucus membranes or high up in the respiratory tract. Settled droplets can also contribute to fomite transmission. Airborne transmission is caused by smaller expelled particles which can stay suspended in the air for long periods exposing a greater number of contacts at greater distance1–3. They are formed by the re-opening of closed airway passages, which de-stabilise the mucous surface layer4. These smaller particles penetrate further into the lower respiratory tract to alveolar level. It is not possible to define a cut-off particle diameter at which aerodynamic behaviour changes, however, the World Health Organisation use a 5 µm cut-off to distinguish between airborne and droplet transmission5.\n\nRecently, the development of optical particle counter (OPC) technology has enabled researchers to measure both the density and the full spectrum of sizes of expired droplets, from the submicron level to larger droplets6,7. Studies using that technology have demonstrated that the majority of particles produced during normal breathing and talking are of submicron size. Although coughing and sneezing can produce 5 times more particles than normal breathing7, the latter accounts for the majority of expired bio-aerosols over the course of a day4,7–9. In addition, recent studies have shown that submicron particles exhaled during normal breathing can contain respiratory viruses6,10,11, suggesting that submicron particles could contribute to infectious disease transmission. The relative contributions of droplet and airborne transmission to the spread of different infections remains controversial but there is increasing recognition that airborne spread may be more important than previously thought for the transmission of respiratory viruses such as influenza12–17. For tuberculosis for example, airborne transmission is regarded as obligatory as mycobacteria need to reach alveolar levels to be taken up by macrophages18.\n\nEarly mathematical models of the spread of infectious diseases have tended to assume that infected individuals were largely homogenous within their age group with respect to transmission19,20. More recent modelling work shows substantial heterogeneity in transmission of SARS, measles, monkey pox and pneumonic plague21 suggesting the occurrence of “super-spreaders” of respiratory infections. Previous small-scale studies of exhaled particle production suggest that two distinct populations of particle producers exist: the majority of individuals are low producers (exhaling an average of less than 500 particles per litre during normal breathing) and a few are high producers (producing more than 500 particles per litre)4,7. It has been hypothesised that high level producers of exhaled particles (so-called “super-producers”) may be “super-spreaders” of respiratory infection. To date published studies of exhaled particle production have included small numbers of individuals, limiting the ability to describe the range of particle production and factors associated with high production and have not examined the long-term stability of exhaled particle production within individuals. For example one study with 16 volunteers22 examined the stability of exhaled particles only over the course of 2 months.\n\nThis study aimed to explore the characteristics of exhaled particle production in healthy individuals, its stability over time, and factors associated with high levels of particle production. Findings from this study may have implications for theories and models of infectious disease transmission through the respiratory route.\n\n\nMethods for data collection\n\nEthical approval for this study was received by University College London Ethics Committee (Reference number 1564/001). We collected data from a convenience sample of workers from 4 departments of University College London (UCL). Measurements were obtained during three different sessions (one baseline session and two follow up sessions which were a few months apart) between November 2008 and June 2009. Three measurement cycles were obtained during each session.\n\nEach participant session consisted of a 15-minute interview followed by a respiratory evaluation conducted by a study researcher. The latter consisted of the measurement of exhaled air using an optical particle counter, Exhalair (model 102580-AK), produced by Pulmatrix Incorporated, which measured aerosol size and concentration by optical particle counting technology coupled with respiratory flow rate and volume measurements. Once written informed consent was obtained, participants were asked to provide information regarding personal demographics, any chronic illnesses, prescribed medications, smoking status and any current symptoms of respiratory illness. Indoor and outdoor temperature and humidity readings were taken at the beginning of each session. The background aerosol count was recorded using a Lighthouse handheld 3013 Particle Counter, which measures the total number of particles greater than 0.3 micrometres in diameter per 0.1 cubic foot of air (also referred to as atmospheric-aerosol particle count).\n\nParticipants breathe with a normal tidal breathing pattern into a disposable mouthpiece whilst wearing a nose clip to prevent nose breathing. Valves direct exhaled breath into the optical particle counter. One-way valves and bacterial/viral High Efficiency Particulate Air (HEPA) filters prevent inhalation of infectious particles, ambient or upstream contaminants or previously exhaled breath. Both the one-way valve and inhalation filter are replaced for each individual. The exhaled breath passes by a laser diode, which counts and sizes the particles in the airstream. Prior to exhaust, the airstream is passed through an additional internal large capacity HEPA filter to remove any contaminating elements.\n\nFollowing initial calibration and a first washout period (which includes 3 deep breaths aimed at clearing any ambient particles from the respiratory tract), the Optical Particle Counter measures average size and concentration of exhaled particles in the range of 0.3 to 20µm in diameter over the course of 15 tidal breaths. A visual display provides feedback to participants allowing them to regulate their breathing within standard tidal breathing limits (the software takes the average tidal wash-out period into account and applies the following additional criteria during the sampling interval for a breath to be considered acceptable: peak inhale between 80–130% of average peak inhale and peak exhale between 80–139% of average peak exhale (with maximum exhale set at 28LPM). Minimum inhalation and exhalation volume = 60% of average inhalation and exhalation volumes respectively. This is due to the large variability in tidal volumes by a person so that they are held to being consistent from the tidal washout to the sampling interval. The process was repeated 3 times each session.\n\nThe dataset included 3 measurements per session for each participant, each representing the average number of particles per litre of exhaled breath over the course of 15 breaths. We plotted the particle count per litre during normal breathing at each attempt and each session for each individual included in the study.\n\nGiven the right skewed distribution of submicron bio-aerosol count/L, we log transformed the data and assessed normality through kernel density plots. We explored whether specific individual or environmental factors were associated with high particle production (i.e. ‘super-producers’), and defined high particle production as any particle count equal to or above the 90th percentile of particle count among study participants. The explanatory variables considered were individual factors such as age, sex, ethnicity, height and weight, medical history and flu-like symptoms on the day measurements were taken, and environmental factors which were thought to affect particle production including season, indoor and outdoor temperature, humidity measurements and pollen count. Given that multiple measurements were obtained for the same individuals and that each individual was included in the study for one or more sessions at different periods in time, crude and adjusted odds ratios (ORs) for high particle production were obtained by multilevel logistic regression analysis. Multilevel analysis was required to take the hierarchical structure of the data into account and the non-independence of observations. Univariable models were initially built, and we considered all variables associated with the outcome at p<0.10 for multivariable analysis. The least significant factor was dropped from each model in a stepwise fashion, until all variables remained significant at p<0.05. A sensitivity analysis was performed to explore how changes in the way super-producers were defined impacted on the associations found, using varying thresholds between the 85th and 95th percentile to define superproducers. All analyses were performed in STATA (STATA 12.0 IC, College Station, Texas, USA).\n\nRespiratory/influenza like symptoms on the day of the measurement were defined as any two of the following symptoms: fever, sore throat, rhinitis or cough.\n\nWe explored the stability of bio-aerosol production for individuals between measurements during each session as well as between each session. We did this for each pair of measurements within a session (e.g. measurement 1 and 2 in session 1) as well as between pairs of summary measurements between sessions (e.g. mean measurement in sessions 1 and 2). We used Lin’s concordance correlation coefficient23, which is similar to a Pearson’s correlation coefficient for continuous variables, to assess the agreement between multiple continuous measurements on the same subject.\n\n\nResults\n\nOverall 79 individuals were included in this study, of which 56 (71%) were females (Table 1). The median age of the study participants was 32 years (range 22–62 years). More than half of them were researchers at UCL, and the rest were physicians, nurses, students, clerks and others (Table 1). Further information on the study participants can be found in Table 1. Thirty-seven individuals (47%) were followed up for a second session a few months later, and 13 (16%) were followed up twice (thus included in three different sessions), resulting in a total of 142 sessions. Of these, 50 (35%) were held during the summer, 12 (8%) were during autumn, 43 (30%) during winter and another 37 (26%) during spring. Each individual completed a series of 3 cycles of measurements for each session attended, which resulted in a total of 426 measurements of breathing cycles (59 of which were excluded due to incomplete data on particle size).\n\nThe median total particle count per litre was 38.3 (range 3.3–1456 particle count/L) with 99.9% of the total bio-aerosol production composed of particle sizes smaller than 1 micron and around 75% below 0.5 microns. Figure 1 shows the distribution of exhaled particle counts across all readings. The median sub-micron particle count was 37.3 counts/L (range 3.2–1456.4, 90th percentile 145.8/L).\n\nTable 2 shows results of the logistic regression analysis of the association between a range of exploratory variables and high particle production (i.e. >90th percentile of particle production). We found an association with age, with proportionally more ‘super-producers’ in older age groups compared to younger ones. This association was not confounded by BMI, height, weight, sex or any other factor. There was no association between particle count and respiratory/influenza-like symptoms at the time of measurement, including fever, dry and productive cough, runny nose, myalgia and headache (Table 2).\n\n* 2-sided Fisher’s exact test.\n\nNote: Due to collinearity, tree pollen count, spring season and outdoor temperature were adjusted for age separately. Age in table is adjusted for pollen count.\n\nWe also found a positive association with high tree pollen counts, which was not confounded by age hence the results in Table 2 are from the univariable analysis. We found no other environmental factor associated with high particle counts. Figure 2 shows the variation in pollen counts over the spring and summer study months.\n\nThe analysis with 85th and 95th centiles as the cut-off for defining super-producers yielded similar results and similar associations though point estimates and standard errors differed. Here we only present the results where the 90th centile was used as a cut-off to define super-producers.\n\nWe found that measurements repeated within a session were relatively stable with good agreement between particle counts (concordance coefficient rho ranging from 0.30 to 0.65, p-values <0.01) for all pairs of measurements within each session. However, we found little evidence that bio-aerosol production was stable over time, when comparing the geometric mean submicron particle counts/litre between each session (session 1 and 2: concordance coefficient rho 0.14 (95%CI -0.10, 0.38, p=0.238), session 1 and 3: rho 0.06 (95%CI -0.55–0.66, p=0.859), session 2 and 3: rho 0.36 (95%CI -0.13–0.85, p=0.148)). Figure 3 shows a scatter plot comparing results from session one and session 2 demonstrating minimal evidence of stability over time.\n\n\n\n\nDiscussion\n\nDuring tidal breathing, 99.9% of the total exhaled particle production consisted of particles measuring less than 1µm in diameter, which has confirmed findings from previous studies4,8,9. In common with other studies, we observed high variability in the levels of exhaled particle production between individuals3,24,27. Unlike previous studies we were also able to assess stability over time and found that measurements taken several months apart were not well correlated. The size of our study enabled us to assess a range of putative predictors of exhaled particle production, including age, gender, height and weight, smoking history, chronic respiratory disease and acute respiratory symptoms. We found that high particle production was associated with older age, but not with any other individual factor, and also observed an ecological association between high particle production and high pollen count.\n\nThe predominance of sub-micron particles in exhaled breath underlines the potential importance of airborne transmission in respiratory infection. The high level of variation in particle production between individuals may account for the observed heterogeneity in transmission of respiratory infection21. The lack of stability of particle counts over time, however, suggests that individuals with high particle counts who may be more infectious during one episode of infection may not be as infectious during subsequent episodes of infection. The association with age suggests an age related deterioration of the respiratory system22,25 through decreased elasticity, lower levels of surfactant, age-associated increases in airways closure26 or increased likelihood of chronic inflammation, which may influence production of exhaled particles. There is no evidence from the literature, however, that older adults are more likely to transmit respiratory infections compared with younger adults. The association with pollen counts also suggests that airway irritation may increase the production of exhaled particles.\n\nThis is the largest study to date of exhaled particle production in healthy volunteers and the first to assess the stability of the population in a subset of participants. We did not attempt to gain a representative sample of the population, rather relying on recruiting colleagues who were more easily accessible. This potentially limits generalizability. No children or adults of post-retirement age were included, limiting the conclusions that can be made about age-related trends. Finally, since the hypothesis of an association with high pollen counts was developed post hoc following observations that particle counts tended to be higher in spring and summer months, this association should be treated with caution. The association is also ecological, and therefore potentially confounded by other variables not captured here. It is important that future studies of variation in production assess this over a wider age range, incorporate measures of stability and assess the impact of potential environmental factors on production.\n\nFinally, this study was conducted among healthy volunteers. Whilst a small proportion of these “healthy” volunteers had mild respiratory symptoms at the time of measurement, the study was not designed to assess the impact of respiratory infections or other acute or chronic respiratory problems on exhaled particle production. It may be that particle production in individuals will change through the course of respiratory infections affecting transmission.\n\nA better understanding of the role of airborne transmission in the spread of infections is critical to informing disease transmission models and control policy. For example in influenza, a high risk from airborne transmission may influence decisions about appropriate levels of social distancing, use of respirators rather than surgical masks and appropriate isolation facilities for patients with newly emergent strains28. Further studies focussing on measurements during the course of acute respiratory infections are needed to investigate infection-induced changes in particle production. In addition, studies are needed to explore whether variations in exhaled particle production are associated with an increased respiratory infection transmission risk. Given the lack of stability of production over time it will be important that such studies measure particle production and transmission risk over the same time period. Such studies are fundamental to our understanding of respiratory infection transmission.",
"appendix": "Author contributions\n\n\n\nFW led project management, data collection, collated and dealt with data management, wrote initial drafts of paper and presented the work at scientific conferences. OLPW developed the statistical strategy and analysis and contributed to the interpretation of results. RM, KH and DS completed the ethics application submission and collected the data. WD and MB provided expert technical support to the project. AH provided academic, strategic and statistical advice throughout the study period. DM and AH were responsible for the inception of the study. FW, OLPW, WD, MB, AH and DM refined the final drafts of the paper.\n\n\nCompeting interests\n\n\n\nWD and MB are current employees of Pulmatrix, Inc. which supplied the Exhalair equipment used in the study. Neither have any non-financial competing interests to declare. The remaining authors have no competing interests to declare.\n\n\nGrant information\n\nThe work was funded by a UCL discretionary account.\n\n\nAcknowledgments\n\nWe thank the study participants from the UCL Department of Infection and Population Health, UCL Department of Primary Care and Population Health, The Bartlett School of Architecture, Building, Environmental Design & Planning, UCL Department of Civil, Environmental and Geomatic Engineering and UCL Department of Epidemiology and Public Health.\n\n\nReferences\n\nGralton J, Tovey E, McLaws ML, et al.: The role of particle size in aerosolised pathogen transmission: a review. J Infect. 2011; 62(1): 1–13. PubMed Abstract | Publisher Full Text\n\nXie X, Li Y, Sun H, et al.: Exhaled droplets due to talking and coughing. J R Soc Interface. 2009; 6(Suppl 6): S703–S714. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuguid JP: The size and the duration of air-carriage of respiratory droplets and droplet-nuclei. J Hyg (Lond). 1946; 44(6): 471–479. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdwards DA, Man JC, Brand P, et al.: Inhaling to mitigate exhaled bioaerosols. Proc Natl Acad Sci U S A. 2004; 101(50): 17383–17388. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaslbeck K, Schawarz K, Hohlfeld JM, et al.: Submicron droplet formation in human lung. J Aerosol Sci. 2010; 41: 429–438. Publisher Full Text\n\nFabian P, McDevitt JJ, DeHaan WH, et al.: Influenza virus in human exhaled breath: an observational study. PLoS One. 2008; 3(7): e2691. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFiegel J, Clarke R, Edwards DA, et al.: Airborne infectious disease and the suppression of pulmonary bioaerosols. Drug Discov Today. 2006; 11(1–2): 51–57. PubMed Abstract | Publisher Full Text\n\nPapineni RS, Rosenthal FS: The size distribution of droplets in the exhaled breath of healthy human subjects. J Aerosol Med. 1997; 10(2): 105–116. PubMed Abstract | Publisher Full Text\n\nFairchild CI, Stampfer JF: Particle concentration in exhaled breath. Am Ind Hyg Assoc J. 1987; 48(11): 948–949. PubMed Abstract | Publisher Full Text\n\nStelzer-Braid S, Olive BG, Blazey AJ, et al.: Exhalation of respiratory viruses by breathing, coughing, and talking. J Med Virol. 2009; 81(9): 1674–1679. PubMed Abstract | Publisher Full Text\n\nHuynh KN, Oliver BG, Stelzer S, et al.: A new method for sampling and detection of exhaled respiratory virus aerosols. Clin Infect Dis. 2008; 46(1): 93–95. PubMed Abstract | Publisher Full Text\n\nBrankston G, Gitterman L, Hirji Z, et al.: Transmission of influenza A in human beings. Lancet Infect Dis. 2007; 7(4): 257–265. PubMed Abstract | Publisher Full Text\n\nTellier R: Review of aerosol transmission of influenza A virus. Emerg Infect Dis. 2006; 12(11): 1657–1662. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNicas M, Nazaroff WW, Hubbard A: Toward understanding the risk of secondary airborne infection: emission of respirable pathogens. J Occup Environ Hyg. 2005; 2(3): 143–154. PubMed Abstract | Publisher Full Text\n\nTellier R: Aerosol transmission of influenza A virus: a review of new studies. J R Soc Interface. 2009; 6(Suppl 6): S783–S790. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWong BC, Lee N, Li Y, et al.: Possible role of aerosol transmission in a hospital outbreak of influenza. Clin Infect Dis. 2010; 51(10): 1176–1183. PubMed Abstract | Publisher Full Text\n\nAtkinson MP, Wein LM: Quantifying the routes of transmission for pandemic influenza. Bull Math Biol. 2008; 70(3): 820–867. PubMed Abstract | Publisher Full Text\n\nRoy CJ, Milton DK: Airborne transmission of communicable infection–the elusive pathway. N Engl J Med. 2004; 350(17): 1710–2. PubMed Abstract | Publisher Full Text\n\nAnderson RM, May RM: Infectious Diseases of Humans: Dynamics and Control. Oxford University Press, Oxford 1991: 757. Reference Source\n\nGarske T, Rhodes CJ: The effect of superspreading on epidemic outbreak size distributions. J Theor Biol. 2008; 253(2): 228–37. PubMed Abstract | Publisher Full Text\n\nLloyd-Smith JO, Schreiber SJ, Kopp PE, et al.: Superspreading and the effect of individual variation on disease emergence. Nature. 2005; 438(7066): 355–359. PubMed Abstract | Publisher Full Text\n\nSchwarz K, Biller H, Windt H, et al.: Characterization of exhaled particles from the healthy human lung–a systematic analysis in relation to pulmonary function variables. J Aerosol Med Pulm Drug Deliv. 2010; 23(6): 371–379. PubMed Abstract | Publisher Full Text\n\nLawrence I-Kuei Lin: A concordance correlation coefficient to evaluate reproducibility. Biometrics. 1989; 45(1): 255–268. PubMed Abstract | Publisher Full Text\n\nLoudon RG, Roberts RM: Droplet expulsion from the respiratory tract. Am Rev Respir Dis. 1967; 95(3): 435–442. PubMed Abstract\n\nHeil M, Hazel AL, Smith JA, et al.: The mechanics of airway closure. Respir Physiol Neurobiol. 2008; 163(1–3): 214–221. PubMed Abstract | Publisher Full Text\n\nAlmstrand AC, Bake B, Ljungstrom E: Effect of airway opening on production of exhaled particles. J Appl Physiol. 2010; 108(3): 584–588. PubMed Abstract | Publisher Full Text\n\nDuguid JP: The numbers and the sites of origin of the droplets expelled during expiratory activities. Edinb Med J. 1945; 52: 385–401. PubMed Abstract\n\nKillingley B, Enstone J, Booy R, et al.: Potential role of human challenge studies for investigation of influenza transmission. Lancet Infect Dis. 2011; 11(11): 879–886. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "736",
"date": "28 Jan 2013",
"name": "Jonathan S. Nguyen-Van-Tam",
"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 paper that is generally of high quality and using appropriate methodology. It is a contribution to the literature in terms of its relative size compared to many studies; and it adds new knowledge on the subject of particle emission in health adults. The data are clearly constrained by a lack of data on children and the elderly; this issue is fully acknowledged but one or two statements might be seen as overly bold given these shortcomings. Some of the raw observations require greater explanation (for example why particle emission would be higher at each end of the range of humidity and indoor temperature (are U-shaped curves already recognised? If not the data are more puzzling). There could be improved clarity and separation of the results of univariate and multivariable analyses. But overall, a nice paper that adds something.",
"responses": []
},
{
"id": "752",
"date": "06 Feb 2013",
"name": "Adam T. Hill",
"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 well written article with interesting findingsI would recommend the authors alter the statement that 'high particle production was associated with older age'- the multivariable analysis showed that it was in the group aged 40-49 that was statistically significant but not in the group aged 50+In the multivariable analysis it specifies that it took account of the medical history- did this take account of the treatments patients had e.g. inhaled corticosteroids etc?The authors state that 'measurements repeated within a session were relatively stable with good agreement between particle counts (concordance coefficient rho ranging from 0.3 to 0.65, p<0.01)'. Although I agree this is statistically significant there is still a lot of intrinsic variability and this would merit further discussion in the Discussion section.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-14
|
https://f1000research.com/articles/2-13/v1
|
15 Jan 13
|
{
"type": "Research Article",
"title": "CO2 elevation improves photosynthetic performance in progressive warming environment in white birch seedlings",
"authors": [
"Shouren Zhang",
"Qing-Lai Dang"
],
"abstract": "White birch (Betula paperifera Mash) seedlings were exposed to progressively warming in greenhouses under ambient and elevated CO2 concentrations for 5 months to explore boreal tree species’ potential capacity to acclimate to global climate warming and CO2 elevation. In situ foliar gas exchange, in vivo carboxylation characteristics and chlorophyll fluorescence were measured at temperatures of 26oC and 37oC. Elevated CO2 significantly increased net photosynthetic rate (Pn) at both measurement temperatures, and Pn at 37oC was higher than that at 26oC under elevated CO2. Stomatal conductance (gs) was lower at 37oC than at 26oC, while transpiration rate (E) was higher at 37oC than that at 26oC. Elevated CO2 significantly increased instantaneous water-use efficiency (WUE) at both 26oC and 37oC, but WUE was markedly enhanced at 37oC under elevated CO2. The effect of temperature on maximal carboxylation rate (Vcmax), PAR-saturated electron transport rate (Jmax) and triose phosphate utilization (TPU) varied with CO2, and the Vcmax and Jmax were significantly higher at 37oC than at 26oC under elevated CO2. However, there were no significant interactive effects of CO2 and temperature on TPU. The actual photochemical efficiency of PSII (DF/ Fm’), total photosynthetic linear electron transport rate through PSII (JT) and the partitioning of JT to carboxylation (Jc) were higher at 37oC than at 26oC under elevated CO2. Elevated CO2 significantly suppressed the partitioning of JT to oxygenation (Jo/JT). The data suggest that the CO2 elevation and progressive warming greatly enhanced photosynthesis in white birch seedlings in an interactive fashion.",
"keywords": [
"Global climate warming and increases in atmospheric CO2 concentration are currently key topics for scientists",
"politicians and the general public alike1. Such changes have been observed in the past 150 years and supported by modeling results for the longer term",
"e.g.",
"warming ocean water",
"shrinking mountain glaciers",
"retreating snow cover",
"and CO2 concentration dynamics in Arctic/Antarctic ice cores2–6. It is projected that the global temperatures will increase by an average of 3°C with a range of 2 to 4.5°C under the scenario of doubling atmospheric CO2 concentration by the end of the century2. Global climate warming will likely have profound and diverse impacts on biological systems2",
"4–13."
],
"content": "Introduction\n\nGlobal climate warming and increases in atmospheric CO2 concentration are currently key topics for scientists, politicians and the general public alike1. Such changes have been observed in the past 150 years and supported by modeling results for the longer term, e.g., warming ocean water, shrinking mountain glaciers, retreating snow cover, and CO2 concentration dynamics in Arctic/Antarctic ice cores2–6. It is projected that the global temperatures will increase by an average of 3°C with a range of 2 to 4.5°C under the scenario of doubling atmospheric CO2 concentration by the end of the century2. Global climate warming will likely have profound and diverse impacts on biological systems2,4–13.\n\nIncreases in CO2 and temperature to a certain extent should have positive impact on photosynthesis and growth, as the current atmospheric CO2 concentration is below the saturation point for RuBisCO (Ribulose-1,5-bisphosphate carboxylase oxygenase)14. Furthermore, higher CO2 concentrations suppress photorespiration and increase the partitioning of photosynthetic electron transport to carboxylation14. However, the situation will become complicated if the temperature goes beyond plants’ ability to acclimate, or when the rate of temperature increase exceeds the pace of acclimation. In such cases, temperature and CO2 will have opposite effects on photosynthesis, i.e., the higher temperature induced increase in photorespiration may exceed the beneficial effect of CO2 elevation, resulting in a decline in net photosynthesis. Consequently, the direction and magnitude of change in net photosynthesis will be determined by the relative magnitudes of the two opposite effects15. Kirschbaum16 has conducted a theoretical analysis on the dependence of photosynthesis on temperature and CO2 concentration for C3 plants and found that at 35°C, photosynthesis at the ambient CO2 concentration reaches only 50% of the rate at saturating CO2 concentration, whereas the corresponding value at 5°C is 77%. Therefore, there is greater potential photosynthetic enhancement by CO2 elevations at higher temperatures. This theory has been supported by the results of a number of studies15,17–19. Long20 has suggested that the increase in atmospheric CO2 will not only increase photosynthetic rate, but also alter the photosynthetic response to temperature. Mooney et al.21 indicate that the photosynthetic acclimation to elevated temperature and CO2 mainly involves changes in the heat stability of the thylakoids and RuBisCO activity. Hence, high temperature and CO2 elevations may have synergistic effect on photosynthesis and CO2 elevations may lead to improved acclimation to high temperatures. However, such interactions may vary with plant species22,23 and other environmental conditions. Variations in acclimation ability can change the interactions within and between species and the composition and functioning of plant communities under future climatic conditions. Furthermore, in most past studies, high temperature treatments are achieved in one step, which is in contrast with the gradual, progressive increases in temperature occurring in global climate changes.\n\nThe boreal forest is an important terrestrial ecosystem with a high carbon sequestration potential24. As the global climate change accelerates, the boreal forest has been experiencing progressive increases in temperatures and CO2. The response of the boreal forests could have great impact on the global carbon balance25. White birch is one of the most widely distributed tree species in the boreal forest. The growth conditions of white birch in northwest Ontario are characterized by a long cold winter and short summer. For example, the annual mean temperature in Thunder Bay region is 2.4°C while the January and July average temperatures are -14.9°C and 17.6°C (based on Environment Canada’s online weather records for the time period of January 1943 to December 2003). Nevertheless, based on our past experience in growing white birch seedlings in greenhouses, it appears that the species is capable of acclimating to continuous warming to more than 40°C in the early afternoon on sunny summer days (see Figure 1). In this current study, we test the hypothesis that CO2 elevation will enhance the photosynthetic performance of white birch seedlings growing in a progressively warming environment.\n\n(A) Postmeridian (hour) pattern between 13:00 and 16:00; (B) Diel (hour) pattern between 0:00 and 24:00.\n\n\nMaterials and methods\n\nWhite birch (Betula papyrifera Mash.) seedlings were grown from seeds in the greenhouses at the Thunder Bay campus of Lakehead University. The growing medium was a mixture of peat moss and vermiculite (1:1 (v/v)).\n\nThe seedlings were subject to a progressive warming in the greenhouses as the season progressed from March to August (Figure 1). The temperatures in all the greenhouses were monitored and recorded using a computerized environment control system (Argus, Vancouver, Canada). The highest recorded temperature in the greenhouse was 44.8°C in the later stages of the experiment (Figure 1). The seedlings were grown under two CO2 concentrations (i.e., the ambient (360 µmol mol-1) and elevated (650 µmol mol-1)). The two CO2 treatments were conducted simultaneously in separate greenhouses with identical design and dimensions. The CO2 elevation was achieved using Argus CO2 generators (Argus, Vancouver, Canada). A photoperiod of 16-hour was maintained (the natural light was supplemented by high-pressure sodium lamps on cloudy days, early mornings and late evenings).\n\nThe moisture content of the growing medium was maintained at around 50%, as measured using a HH2 Moisture Meter (DELTA-T DEVICES, Cambridge, UK). The seedlings were watered up to twice a day during the summer to maintain the soil moisture condition. The seedlings were fertilized weekly with a solution of 100 µmol mol-1 N, 35 µmol mol-1 P and 66 µmol mol-1 K.\n\nThe foliage gas exchange was measured using a PP-Systems CIRAS-1 open gas exchange system (Hitchin, Hertfordshire, UK). The environmental conditions in the broad-leaf chamber were controlled automatically. The environmental conditions for measuring the Pn-Ci (Ci = intercellular CO2 concentration) curve were as follows: 26°C and 37°C air temperature, which were close to the highest temperatures in the early and late period of the experiment, 800 µmol m-2s-1 PAR (PAR = photosynthetically-active radiation) and 50% relative humidity. The in vivo maximal carboxylation rate (Vcmax), PAR-saturated electron transport rate (Jmax), triose phosphate utilization (TPU) and other relevant parameters were calculated from the Pn-Ci curves according to Farquhar et al.26, van Caemmerer and Farquhar27, Sharkey28, Harley and Sharkey29 and Harley et al.30. The Pn-Ci curves were fit using the Photosyn Assistant software (Dundee Scientific, Scotland, UK) to estimate Vcmax, Jmax and TPU. The parameters for the kinetics of RuBiscCO, i.e., Kc, Ko and τ, and their temperature dependencies were adopted from Harley et al.30 and Wullschleger31.\n\nThree seedlings were selected randomly from each treatment combination for the measurement. The measurement was taken on the top 5th mature leaf. All the in situ measurements were made between 9:00 and 11:30 AM with the seedlings in their original positions and conditions of the treatments.\n\nThe chlorophyll fluorescence was measured using a FMS-2 portable pulse-modulated fluorometer (Hansatech Instruments Ltd. Norfolk, UK). The probe was integrated in the leaf chambers of the gas exchange system and the control software for the two systems was also integrated to allow the simultaneous measurement of gas exchange and chlorophyll fluorescence. The following variables were obtained: fluorescence intensity at any time, F; the maximal fluorescence in light, Fm’; the actual photochemical efficiency of PSII in light, (Fm’-F)/Fm’ or ΔF/Fm’, which is the efficiency under the actual degree of reaction centre closure32. Fm’ was obtained by illuminating the foliage with a pulse of strong light (around 14000 µmol photons m-2s-1) for 800 ms. The ΔF/Fm’ was measured simultaneously with each gas exchange measurement. Both gas exchange and chlorophyll fluorescence were measured after 5 months of the treatments.\n\nThe apparent rate of total electron transport (JT) and its partitioning between carboxylation (Jc) and oxygenation (Jo) were calculated based on the methods of Farquhar et al.26, Genty et al.33 and Epron et al34.\n\nAll the data were examined graphically for the normality of distribution (probability plots for residual analysis) and the homogeneity of variance (scatter plots) using the Data Desk (version 6.01, Data Description, Inc. 1996)35 before the Analysis of Variance (ANOVA) was carried out. Some of the data were log-transformed to meet the two assumptions for ANOVA. The data were analyzed using the two-way ANOVA procedure of the Data Desk. When the interaction between temperature and CO2 was significant, Scheffe’s F test for post hoc pairwise comparisons was conducted.\n\n\nResults\n\nThere was a significant (P<0.01) interactive effect of temperature and CO2 on Pn (Figure 2). Pn was higher (P<0.01) at 37°C than at 26°C under elevated CO2(Figure 2), but there was no significant (P>0.05) temperature effect on Pn under ambient CO2. CO2 elevation significantly increased Pn at both temperatures (P<0.05, P<0.001 at 26°C and 37°C, respectively). gs significantly (P<0.05) decreased at 37°C under both ambient and elevated CO2(Figure 2), and there was no significant (P>0.05) CO2 effect on gs. Meanwhile high temperature significantly (P<0.05) stimulated E under both ambient and elevated CO2(Figure 2). Water-use efficiency (WUE) was significantly (P<0.05) higher at 26°C than that at 37°C under both CO2 regimes. CO2 elevation greatly (P<0.001) increased WUE at both temperatures.\n\nThe in situ measurements were taken at 26°C and 37°C under ambient CO2 and elevated CO2. The significance levels (*** = P<0.001, ** = P<0.01, * = P<0.05). If the interaction between measurement temperature and CO2 was significant for a given parameter, Scheffe’s F test for post hoc pairwise comparisons was conducted. Means sharing the same letter or letters are not significantly different.\n\nHigh temperature significantly reduced Ci under both ambient and elevated CO2 (P<0.05, P<0.01, respectively) and, also, elevated CO2 significantly (P<0.001) increased Ci at both temperatures.\n\nVcmax, Jmax and TPU at 37°C were significantly (P<0.001) higher than those at 26°C (Figure 3). The temperature dependencies of Vcmax and Jmax were changed by CO2, and those values at 37°C enhanced much more (P<0.05) under elevated CO2 than under ambient CO2.\n\nVcmax, Jmax and TPU were derived from A-Ci curves, which were measured at 26°C and 37°C under ambient CO2 and elevated CO2. See Figure 2 for other explanations.\n\nThere was a significant (P<0.001) interactive effect of CO2 and temperature on (Fm’-F)/Fm’ and JT(Figure 4). (Fm’-F)/Fm’ and JT greatly increased at 37°C as compared to at 26°C under elevated CO2, and there was no significant temperature effect on (Fm’-F)/Fm’ and JT under ambient CO2.\n\nThe pattern of CO2 and temperature effects on Jc was almost the same as (Fm’-F)/Fm’ and JT(Figure 4), and Jc was greater (P<0.001) at 37°C than that at 26°C under elevated CO2, and there was no significant temperature effect on Jc under ambient CO2. Elevated CO2 greatly suppressed Jo/JT, and there was no significant (P > 0.05) effect of temperature on Jo/JT(Figure 4).\n\nSee Figure 2 for other explanations.\n\n\n\n\n\n\n\n\nDiscussion\n\nOur results suggest that the photosynthetic mechanisms of white birch seedlings have high capacity to acclimate to a progressively warming environment, particularly under elevated CO2. This result is in contrast to the results of most studies with a single step warming treatment. Larcher36 has suggested that plants’ optimal temperature is closely related to the climate in which they grow. The measurement temperatures of 26°C and 37°C used in this study are believed to be the normal (or optimal) and stressful temperature, respectively, for most boreal forest tree species growing at their natural environments. Zhang et al.37 have found that the Pn of mature oak even in warm-temperate zones decline greatly at temperatures over 30°C, as compared to measurements at temperatures of 20–30°C, which occurs naturally north of temperate zones or even warm-temperate zones. However, in this experiment the Pn of white birch didn’t decline at 37°C under ambient CO2, as compared to that at 26°C; furthermore, Pn increased substantially at 37°C under elevated CO2. These results indicate that the photosynthetic mechanisms of white birch acclimated to the progressive warming environment, and this high temperature acclimation was greatly strengthened by elevated CO2. Long20 argued that CO2 elevation could change the photosynthesis dependence of temperature.\n\nThe activity of RuBisCO is highly temperature-dependent. According to Jordan and Ogren38, the Rubisco’s specificity for CO2/O2 decreases as increasing temperatures over the optimal range, but the increase in RuBisCO oxygenation will exceed that of carboxylation because the solubility of CO2 declines faster than that of O2 at even higher temperatures, resulting in a decline in net photosynthetic rate. White birch’s acclimation to warming was also evidenced by Vcmax measured at the two different temperatures and two CO2 regimes. Vcmax at 37°C was much higher than at 26°C under both ambient and elevated CO2, indicating a shift in the temperature dependency of RuBisCO. Furthermore, the partitioning of total electron transport to oxygenation was not significantly different between the two temperatures under either ambient CO2 or elevated CO2, suggesting that the higher temperature did not change the RUBisCO specificity for CO2/O2 which could be a contributing factor for the enhanced acclimation of photosynthesis to the progressive warming. Overdieck et al.15 have also found that both the temperature treatment alone and the combination of elevated CO2 and temperature depressed Vcmax in Scots pine at temperatures below the optimum range, but increased Vcmax when the temperature was above the optimum. Additionally, the magnitude of the change in Vcmax increased as temperature increased.\n\nThe decrease in Ci at the high temperature could be attributable to either enhanced RuBisCO activity or declines in stomatal conductance or both. Not only Vcmax, but Jmax and TPU were also higher at 37°C than at 26°C, suggesting that the CO2 assimilation process, including carboxylation, electron transport for RuBP regeneration, ATP supply and the translocation of the primary photosynthates, all maintained at high levels in the warm environment. In this study, there was no down-regulation of RuBisCO activity in association with the CO2 elevation, to the contrary, CO2 elevation greatly increased Vcmax and Jmax at 37°C, as well as Pn at both 26°C and 37°C.\n\nWhile high temperature enhanced Vcmax under both ambient and elevated CO2, the increases in actual PSII efficiency (ΔF’/Fm’) and Jc associated with the high temperature only occurred under elevated CO2, suggesting that the high temperature did not significantly affect the total electron transport, and its partitioning to carboxylation, t under the ambient CO2. Conversely, the partitioning of total electron flow to oxygenation increased more than 40% in response to the high temperature under elevated CO2. The reduced electron transport partitioning to carboxylation and low Ci might explain why Pn was relatively low at 37°C under the ambient CO2, even though the corresponding Vcmax was quite high, implying that the slow electron transport to carboxylation and CO2 supply at high temperature under ambient CO2 didn’t match the high activity of RuBisCO. This again confirms Kirschbaum’s theoretical analysis that photosynthesis has a higher potential to be stimulated by CO2 elevation at high temperatures than at low temperatures16.",
"appendix": "Author contributions\n\n\n\nQLD and SZ conceived and designed the experiment. SZ conducted the measurements and analyzed the data. SZ and QLD wrote and revised the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by Lakehead University, Canada Foundation for Innovation, Ontario Innovation Trust and NSERC Discovery Grants to Q.L. Dang (Project # 203198-2008).\n\n\nAcknowledgements\n\nThe authors thank Dr. J. Wang at Lakehead University for providing white birch seeds, and Dr. K. Brown for statistical advice.\n\n\nReferences\n\nKerr RA: Climate change. Scientists tell policymakers we're are all warming the world. Science. 2007; 315(5813): 754–757. PubMed Abstract | Publisher Full Text\n\nIPCC: Climate Change 2007: Impacts, Adaptation, and Vulnerability. Cambridge Univ. Press 2007; pp 976. Reference Source\n\nKeeling CD, Whorf TD: Atmospheric CO2 records from sites in the SIO air sampling network. In: Trends: a compendium of data on global change. Oak Ridge National Laboratory, US Department of Energy 2002. Reference Source\n\nParmesan C: Influences of species, latitudes and methodologies on estimates of phenological response to global warming. Glob Change Biol. 2007; (13): 1860–1872. Reference Source\n\nRoot TL, Price JT, Hall KR, et al.: Fingerprints of global warming on wild animals and plants. Nature. 2003; 421(6918): 57–60. PubMed Abstract | Publisher Full Text\n\nRosenzweig C, Karoly D, Vicarelli M, et al.: Attributing physical and biological impacts to anthropogenic climate change. Nature. 2008; 453(7193): 353–357. PubMed Abstract | Publisher Full Text\n\nBoth C, van Asch M, Bijlsma R, et al.: Climate change and unequal phenological changes across four trophic levels: constraints or adaptations? J Anim Ecol. 2009; 78(1): 73–83. PubMed Abstract | Publisher Full Text\n\nBradshaw W, Holzapfel C: Genetic shift in photoperiodic response correlated with global warming. Proc Natl Acad Sci U S A. 2001; 98(25): 14509–14511. PubMed Abstract | Publisher Full Text | Free Full Text\n\nColwell R, Brehm G, Cardelus C, et al.: Global warming, elevational range shifts, and lowland biotic attrition in the wet tropics. Science. 2008; 322(5899): 258–261. PubMed Abstract | Publisher Full Text\n\nParmesan C, Yohe G: A globally coherent fingerprint of climate change impacts across natural systems. Nature. 2003; 421(6918): 37–42. PubMed Abstract | Publisher Full Text\n\nSinervo B, Méndez-de-la-Cruz F, Miles DB, et al.: Erosion of lizard diversity by climate change and altered thermal niches. Science. 2010; 328(5980): 894–899. PubMed Abstract | Publisher Full Text\n\nUmina PA, Weeks AR, Kearney MR, et al.: A rapid shift in a classic clinal pattern in Drosophila reflecting climate change. Science. 2005; 308(5722): 691–693. PubMed Abstract | Publisher Full Text\n\nWalther GR, Post E, Convey P, et al.: Ecological responses to recent climate change. Nature. 2002; 416(6879): 389–395. PubMed Abstract | Publisher Full Text\n\nAmthor JS: Respiration in a future, higher-CO2 world. Plant Cell Environ. 1991; 14(1): 13–20. Publisher Full Text\n\nOverdieck D, Kellomäki S, Wang KY, et al.: Do the effects of temperature and CO2 interact? In: European Forests and Global Change: the likely impacts of rising CO2 and temperature. (Eds. Jarvis PG).X Cambridge University Press, 1998; pp 236–273. Reference Source\n\nKirschbaum MUF: The sensitivity of C3 photosynthesis to increasing CO2 concentration: a theoretical analysis of its dependence on temperature and background CO2 concentration. Plant Cell Environ. 1994; 17(6): 747–754. Publisher Full Text\n\nIdso KE, Idso SB: Plant responses to atmospheric CO2 enrichment in the face of environmental constraints: a review of the past ten years’ research. Agricultural and Forest Meteorology. 1994; 69(3–4): 153–203. Publisher Full Text\n\nIdso SB, Idso KE, Garcia RL, et al.: Effects of atmospheric CO2 enrichment and foliar methanol application on net photosynthesis of sour orange tree (Citrus aurantium; Rutaceae) leaves. Am J Bot. 1995; 82(1): 26–30. Publisher Full Text\n\nTissue DT, Thomas RB, Strain BR, et al.: Growth and photosynthesis of loblolly pine (Pinus taeda) after exposure to elevated CO2 for 19 months in the field. Tree Physiol. 1996; 16(1–2): 49–59. Publisher Full Text\n\nLong SP: Modification of the response of photosynthetic productivity to rising temperature by atmospheric CO2 concentration, has its importance been underestimated? Plant Cell Environ. 1991; 14(8): 729–739. Publisher Full Text\n\nMooney HA, Bjorkman O, Collatz GJ, et al.: Photosynthetic acclimation to temperature in the desert shrub, Larrea divaricata I. Carbon dioxide exchange characteristics of intact leaves. Plant Physiol. 1978; 61(3): 406–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStanghellini C, Bunce JA: Response of photosynthesis and conductance to light, CO2, temperature and humidity in tomato plants acclimated to ambient and elevated CO2. Photosynthetica. 1993; 29: 487–97.\n\nTeskey RO: Combined effects of elevated CO2 and air temperature on carbon assimilation of Pinustaeda trees. Plant Cell Environ. 1997; 20(3): 373–380. Publisher Full Text\n\nApps MJ, Kurz WA, Luxmoor RJ, et al.: Boreal forest and tundra. Water, Air, and Soil Pollution. 1993; 70(1–4): 39–53. Publisher Full Text\n\nPeng CH, Apps MJ: Simulating carbon dynamics along the Boreal Forest Transect Case Study (BFTCS) in central Canada. 2. Sensitivity to climate change. Global Biogeochem Cycles 1998; 12(2): 393–402. Publisher Full Text\n\nFarquhar GD, von Caemmerer S, Berry JA, et al.: A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta. 1980; 149(1): 78–90. Publisher Full Text\n\nvon Caemmerer S, Farquhar GD: Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta. 1981; 153(4): 376–87. Publisher Full Text\n\nSharkey TD: Photosynthesis in intact leaves of C3 plants: physics, physiology and rate limitations. Botanical Review. 1985; 51(1): 53–105. Publisher Full Text\n\nHarley PC, Sharkey TD: An improved model of C3 photosynthesis at high CO2: Reversed O2 sensitivity explained by lack of glycerate reentry into the chloroplast. Photosynth Res. 1991; 27(3): 169–178. Publisher Full Text\n\nHarley PC, Thomas RB, Reynolds JF, et al.: Modelling photosynthesis of cotton grown in elevated CO2. Plant Cell Environ. 1992; 15(3): 271–82. Publisher Full Text\n\nWullschleger SD: Biochemical limitations to carbon assimilation in C3 plants – a retrospective analysis of the A/Ci curves from 109 species. J Exp Bot. 1993; 44(5): 907–920. Publisher Full Text\n\nBilger W, Björkman O: Role of the xanthophyll cycle in photoprotection elucidated by measurements of light induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth Res. 1990; 25(3): 173–185. Publisher Full Text\n\nGenty B, Briantais JM, Baker NR, et al.: The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta. 1989; 990(1): 87–92. Publisher Full Text\n\nEpron D, Godard D, Cornic G, et al.: Limitation of net CO2 assimilation rate by internal resistances to CO2 transfer in the leaves of two tree species (Facus sylavatica L. & Castanea sativa Mill.). Plant Cell Environ. 1995; 18(1): 43–51. Publisher Full Text\n\nData Desk. Version 6.01, Data Description, Inc., Ithaca, New York, USA 1996. Reference Source\n\nLarcher W: Physiological plant ecology: ecophysiology and stress physiology of functional groups. Springer-Verlag, Berlin Heidelberg New York 2003. Reference Source\n\nZhang S, Li Q, Ma K, et al.: Temperature-dependent gas exchange and stomatal/non-stomatal limitation to CO2 assimilation of Quercus liaotungensis under midday high irradiance. Photosynthetica. 2001; 39(3): 383–388. Publisher Full Text\n\nJordan DB, Ogren WL: The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase. Planta. 1984; 161(4): 308–313. Publisher Full Text"
}
|
[
{
"id": "711",
"date": "08 Feb 2013",
"name": "Xianzhong Wang",
"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 certainly a valuable manuscript.",
"responses": []
},
{
"id": "1034",
"date": "01 Jul 2013",
"name": "Christian Koerner",
"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\nHaving stated that the work makes a very sound impression to me and reflects state of the art methodology, I still have concerns about the implied meaning. The authors carefully avoid making any statement with regard to growth or productivity implications. However, why would one make such measurements if not for the implied meaning for growth and overall performance? From all what we know to date, such a link has not been shown outside horticultural conditions. It will be most unlikely that a birch seedling in the wild is carbon limited. Whatever the photosynthetic performance, such a seedling would only incorporate structural carbon to the extent nutrients permit, and these are finite per unit land area (except for N) and are competitively foraged for.",
"responses": [
{
"c_id": "493",
"date": "03 Jul 2013",
"name": "Shouren Zhang",
"role": "Author Response",
"response": "We appreciate the invaluable comments from Professsor Christian Koerner. The primary focus of this study was to investigate the physiological plasticity of white birch in response to gradually warming environmental conditions under the elevated CO2 conditions in the future, not growth. Growth is more closely related to the carbon balance and resource availability than to photosynthesis or any single physiological process. While short term physiological studies such as ours are important for understanding the physiological mechanisms and ability in plant responses to stressful environmental conditions, extrapolating the results to predict the growth performance of trees in the field is risky and not reliable because of the long life of trees and the dynamic nature of natural environmental conditions. Therefore, we have refrained ourselves from making such extrapolations."
}
]
}
] | 1
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https://f1000research.com/articles/2-13
|
https://f1000research.com/articles/2-12/v1
|
14 Jan 13
|
{
"type": "Case Report",
"title": "Case Report: Refractory hypotension during general anesthesia despite preoperative discontinuation of an angiotensin receptor blocker",
"authors": [
"Raha Nabbi",
"Harvey J Woehlck",
"Matthias L Riess",
"Raha Nabbi",
"Harvey J Woehlck"
],
"abstract": "Due to their beneficial reduction in morbidity and mortality angiotensin receptor blockers (ARBs) have become increasingly popular to treat hypertension. However, similar to angiotensin converting enzyme inhibitors, they can lead to severe hypotension in conjunction with general anesthesia and thus have been recommended to be withheld in the morning of surgery. Here, we present a 51 year old female who developed severe refractory hypotension after induction of general anesthesia, although she had discontinued her medication 24 hours preoperatively as instructed. Therefore, halting ARBs for more than 24 hours before surgery may be necessary. Heightened awareness of this potential interaction and recognizing the need to treat with vasopressin is required when ARB-induced hypotension occurs.",
"keywords": [
"angiotensin receptor blocker",
"hypotension",
"anesthesia",
"vasopressin"
],
"content": "Introduction\n\nAngiotensin II receptor blockers (ARBs) represent a newer class of effective and well tolerated antihypertensive agents1. Several clinical studies have indicated the beneficial effects of ARBs in hypertensive patients such as reduction of left ventricular hypertrophy, decrease in ventricular arrhythmias, and improved diastolic function1. Inhibitors of the renin-angiotensin system (RAS), either angiotensin converting enzyme (ACE) inhibitors or ARBs, mediate vasodilation and consequently decrease blood-pressure by different mechanisms1. ARBs specifically inhibit angiotensin II from binding to its receptor, the Angiotensin-1 (AT1) receptor on vascular smooth muscle cells. This blockade results in increased angiotensin II and normal bradykinin plasma levels. ARBs were developed to overcome several deficiencies of ACE inhibitors, which, by comparison, lead to decreased angiotensin II, but increased bradykinin levels. Hence, the key advantage of ARBs over ACE inhibitors is their lack of adverse effects related to bradykinin potentiation. ARBs have been shown to reduce morbidity and mortality associated with hypertension, and therefore, it is not surprising that an increasing number of patients scheduled for surgery are chronically treated with ARBs2. However, RAS blockade increases the risk of severe hypotension during and after anesthetic induction. ACE-inhibitors are well known for inducing severe circulatory side effects during anesthesia, which led to the general recommendation to withhold the drug on the day of surgery3. Similarly, refractory hypotension has been described after induction of general anesthesia in patients chronically treated with ARBs who took their medication in the morning of surgery3. Here, we describe a case of severe hypotension under general anesthesia refractory to conventional treatment, despite preoperative discontinuation of an ARB for 24 hours.\n\n\nCase report\n\nA 51 year old female (60 kg) whose hypertension was well controlled with Diovan-HCT (valsartan/hydrochlorothiazide 160/12.5 mg) was scheduled for a T2-10 posterior thoracic fusion. As instructed, she had last taken Diovan-HCT 24 hours prior to surgery. Her preoperative exam was unremarkable. Non-invasive blood pressure measurement, 5-lead ECG with ST segment analysis, heart rate, and pulseoximetry were continuously monitored. Her baseline vital signs were stable with a blood pressure of 136/94 mmHg, heart rate of 86 bpm and SpO2 99% on room air. After induction of anesthesia with 150 mg propofol, 100 mcg fentanyl, and 60 mg rocuronium and an uneventful intubation, her blood pressure decreased and remained 65–75/35–45 mmHg for 45 min with a HR of 75–85 bpm despite rapid administration of 1500 cc Lactate Ringer, repeated 100 mcg phenylephrine boluses followed by a phenylephrine drip and repeated boluses of vasopressin (cumulative dose of 20 units within 25 min). Sevoflurane for anesthesia maintenance was kept low at 0.8 Vol%. Due to her refractory hypotension, the decision was made to postpone the patient’s elective surgery and awaken her. Upon emergence, her blood pressure recovered to 115/65 mmHg with a heart rate of 90 bpm, and she was extubated successfully after neuromuscular blocker reversal. The patient did not suffer any neurologic sequelae. Her ARB was withheld postoperatively and she was successfully anesthetized with the same drugs and operated on five days later without significant hypotension.\n\n\nDiscussion\n\nValsartan is a potent, highly selective antagonist of the angiotensin II at the AT1 receptor leading to vasodilatation and an anesthetic-induced reduction in pre- and afterload. Vasodilation may also be afforded in part by upregulated angiotensin II activating AT2-receptors which causes vascular relaxation4 and reduces peripheral vascular resistance usually without a rise in heart rate. The efficacy, tolerability and safety of valsartan have been demonstrated in large-scale studies on patients with hypertension, heart failure and post-myocardial infarction5. Valsartan’s mechanism of action is to displace angiotensin II from the AT1 receptor, thereby antagonizing AT1-induced vasoconstriction, aldosterone, catecholamine and arginine-vasopressin release, water intake, and hypertrophic responses. All of this results in a more efficient blockade of angiotensin II’s cardiovascular effects and in fewer side effects than ACE inhibitors. In addition, most ARBs control blood pressure for 24 hrs after a single dose.\n\nARBs are non-peptide compounds, and variations in molecular structure result in different binding affinities to their receptors and different pharmacokinetic profiles1. In comparison to other ARBs, valsartan’s plasma elimination half life is of an intermediate duration (5–10 hrs)6. It is eliminated mainly by non-renal routes. However, protein binding greatly affects its biological half life in the body. Valsartan is highly bound to plasma proteins (94–97%), and these may act as a reservoir or depot from which the drug is slowly released and therefore exhibits a longer lasting effect on the vasculature6. As the unbound drug is metabolized and excreted from the body, some of the bound fraction is released in order to maintain equilibrium. In fact, our case demonstrates impressively that valsartan’s biological half life is far outlived by its physiological effects in the human body and can consequently result in severe hypotension despite its prior discontinuation in cases when RAS activation is needed to maintain hemodynamic stability, as for instance during anesthesia. Indeed, during general anesthesia, maintenance of normotension becomes RAS-dependent7 and a pronounced anesthetic-induced hypotension may be prevented or at least attenuated by angiotensin II-mediated AT1 receptor activation. Conversely, by blocking RAS, systemic blood pressures can decrease markedly during general anesthesia4.\n\nIn addition, chronic AT1 blockade also reduces the vasoconstrictor response to α1 receptors activated by norepinephrine, which explains why ARB-induced hypotension can be so resistant to phenylephrine, ephedrine and norepinephrine2,8 as observed in our patient. The lack of response to repeated phenylephrine boluses and a continuous infusion, fluids and a decrease of the volatile anesthetic urgently required a different approach, and we administered vasopressin in repeated boluses. Clinical studies have shown significant vasoconstrictor effects of vasopressin and increased cardiac filling during echocardiographic measurements2. Vasopressin or its synthetic analogues can restore the sympathetic response and may be useful pressors in cases of refractory hypotension during anaphylaxis9 and septic shock10 as well as in patients on RAS inhibitors, although norepinephrine has been reported to have a more favorable effect on splanchnic perfusion and oxygen delivery11.\n\nWe conclude that, similar to ACE inhibitors3, ARBs also need to be withheld for 24 hours prior to elective surgery, and – as our case illustrates – possibly longer to elude unnecessary morbidity and mortality associated with refractory hypotension following induction of general anesthesia in patients on chronic ARB treatment. Heightened awareness of this perilous interaction and recognizing the need to treat with an adequate dose of vasopressin or norepinephrine is required, when ARB-induced hypotension is encountered.\n\n\nConsent\n\nThis case report is in accordance with institutional IRB-guidelines. We have been unable to obtain explicit written consent from the patient.",
"appendix": "Author contributions\n\n\n\nAll authors were involved in the draft and revision of 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\nInstitutional support only (RN, HJW and MLR). No grants were involved in supporting this particular work. Unrelated research support for MLR through the Department of Veterans Affairs (CARA-026-10F) and NIH.\n\n\nReferences\n\nIsraili ZH: Clinical pharmacokinetics of angiotensin II (AT1) receptor blockers in hypertension. J Hum Hypertens. 2000; 14(Suppl 1): S73–86. PubMed Abstract\n\nBrabant SM, Eyraud D, Bertrand M, et al.: Refractory hypotension after induction of anesthesia in a patient chronically treated with angiotensin receptor antagonists. Anesth Analg. 1999; 89(4): 887–8. PubMed Abstract\n\nBertrand M, Godet G, Meersschaert K, et al.: Should the angiotensin II antagonists be discontinued before surgery? Anesth Analg. 2001; 92(1): 26–30. PubMed Abstract\n\nRyckwaert F, Colson P, Andre E, et al.: Haemodynamic effects of an angiotensin-converting enzyme inhibitor and angiotensin receptor antagonist during hypovolaemia in the anaesthetized pig. Br J Anaesth. 2002; 89(4): 599–604. PubMed Abstract | Publisher Full Text\n\nBlack HR, Bailey J, Zappe D, et al.: Valsartan: more than a decade of experience. Drugs. 2009; 69(17): 2393–414. PubMed Abstract | Publisher Full Text\n\nFlesch G, Muller P, Lloyd P: Absolute bioavailability and pharmacokinetics of valsartan, an angiotensin II receptor antagonist, in man. Eur J Clin Pharmacol. 1997; 52(2): 115–20. PubMed Abstract\n\nColson P, Ryckwaert F, Coriat P: Renin angiotensin system antagonists and anesthesia. Anesth Analg. 1999; 89(5): 1143–55. PubMed Abstract\n\nLicker M, Neidhart P, Lustenberger S, et al.: Long-term angiotensin-converting enzyme inhibitor treatment attenuates adrenergic responsiveness without altering hemodynamic control in patients undergoing cardiac surgery. Anesthesiology. 1996; 84(4): 789–800. PubMed Abstract\n\nHussain AM, Yousuf B, Khan MA, et al.: Vasopressin for the management of catecholamine-resistant anaphylactic shock. Singapore Med J. 2008; 49(9): e225–8. PubMed Abstract\n\nLandry DW, Levin HR, Gallant EM, et al.: Vasopressin deficiency contributes to the vasodilation of septic shock. Circulation. 1997; 95(5): 1122–5. PubMed Abstract | Publisher Full Text\n\nMorelli A, Tritapepe L, Rocco M, et al.: Terlipressin versus norepinephrine to counteract anesthesia-induced hypotension in patients treated with renin-angiotensin system inhibitors: effects on systemic and regional hemodynamics. Anesthesiology. 2005; 102(1): 12–9. PubMed Abstract"
}
|
[
{
"id": "708",
"date": "17 Jan 2013",
"name": "Sumio Hoka",
"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": "717",
"date": "22 Jan 2013",
"name": "Yukio Hayashi",
"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 may give us several suggestions and it may be helpful to consider the anesthetic management of similar patients.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-12
|
https://f1000research.com/articles/2-11/v1
|
14 Jan 13
|
{
"type": "Case Report",
"title": "Maintained consciousness during witnessed asystole after spinal anesthesia for Cesarean section",
"authors": [
"Kanishka Rajput",
"Harvey J Woehlck",
"Matthias L Riess",
"Kanishka Rajput",
"Harvey J Woehlck"
],
"abstract": "Despite its low incidence, cardiac arrest after spinal anesthesia carries a high mortality. Counterintuitively, young and healthy patients with low resting pulse are at increased risk. We report the case of a healthy 24 yr G2P0 at term scheduled for elective Cesarean section whose heart rate decreased to 30 bpm, followed by more than 30 seconds of asystole 3 minutes after spinal anesthesia with a T4 level block. Following atropine and epinephrine administration, the patient had several single heart beats when startled by the anesthesiologist’s loud voice and when touching her chest to prepare for chest compressions. Eventually, regular sinus rhythm returned with a heart rate of up to 160 bpm. The patient was rapidly prepped, and within 5 minutes, the fetus was delivered surgically with Apgar scores of 8 and 9. Most unusually, the patient remained responsive during the entire event and denied having lost consciousness. Supine position and volume loading may have contributed to venous pooling within the cerebral vasculature, so even in the absence of cerebral blood flow during asystole venous blood may still have been present and delayed cerebral hypoxia. Therefore, loss of consciousness in the supine position may occur considerably after the onset of asystole which may reduce the time available for treatment and contribute to its high mortality. Inspiration during the two startle reactions may have decreased vagal tone and permitted enough spontaneous cardiac activity to circulate the resuscitative drugs without CPR.",
"keywords": [
"obstetrics",
"cesarean section",
"spinal anesthesia",
"asystole",
"consciousness"
],
"content": "Introduction\n\nCardiac arrest after spinal anesthesia is rare, recently reported to occur in 1.5/10,000 anesthetics1. Despite its low frequency, it was believed to be associated with a high mortality rate, reported as >40% mortality in the closed claims analysis by Caplan et al.2 We present a unique case of cardiac arrest after spinal anesthesia for Cesarean section. We discuss relevant physiological mechanisms to explain why this patient never lost consciousness during the asystolic episode, which could mislead clinicians into delaying treatment.\n\n\nCase report\n\nA healthy Spanish speaking 24 year old G2P0 female (70 kg, 65\", BMI 26.5) at term was scheduled for an elective Cesarean section for breech presentation. The patient’s preoperative heart rate (HR) was 62 beats per minute (bpm), blood pressure was 126/70 mmHg. After administration of 1000 ml Ringer’s lactate solution, spinal anesthesia was performed with 1.6 cc of 0.75% bupivacaine (12 mg) in a sitting position, resulting in a bilateral T4 sensory tested by pinprick. No sedatives or prophylactic vasopressors were administered. The patient was laid supine immediately after spinal anesthesia with a left lateral tilt to prevent aorto-caval compression. SpO2 remained at 98%. Three min later her HR decreased to 30 bpm, followed by 15 sec of asystole, which was immediately noticed via pulse oximeter and EKG. 1 mg atropine was administered intravenously (IV). The anesthesiologist’s loud voice shouting for resuscitative help appeared to startle the patient who turned towards him. Now, 3 beats at 40 bpm were noted, followed again by asystole for 30 sec. 70 mcg of epinephrine IV was given, the anesthesiologist repeated the call for help, and removed the blanket and gown from her chest to begin chest compressions. The patient again appeared startled by this action based on facial expressions while turning towards the anesthesiologist, and a single spontaneous heart beat reappeared on the monitor. Another beat followed within 2 sec, rapidly accelerating to 160 bpm (sinus tachycardia). The patient was rapidly prepared, and the fetus was delivered within 5 min with Apgar scores of 8 and 9. Afterwards, the events were discussed with her with the help of an interpreter. She had been unaware of the cardiac arrest, and denied having lost consciousness.\n\n\nDiscussion\n\nSpinal anesthesia is associated with an increased risk of hypotension and bradycardia3. ASA 1 status, baseline HR <60 bpm, a prolonged PR-interval, beta-blockade, age <50 years, a sensory blockade above T6 and male gender have been described as independent risk factors2–5. The differential diagnosis for asystole in this setting includes hypoxia due to respiratory depression, high or total spinal, local anesthetic toxicity when an epidural dose is administered IV, myocardial ischemia, vasovagal reaction, and bradycardic reflexes after neuraxial anesthesia. Our patient was unsedated, and oxygenation maintained prior to the arrest. In fact, in contrast to earlier studies that suspected oversedation2, it is difficult to invoke hypoxemia as the primary cause of cardiac arrests during this case or historical reports because most asystolic arrests occurred in the setting of 95–100% oxygen saturations6. Spinal anesthesia itself is associated with bradycardia in 9–13% of patients3. Most of these effects are related to sympathetic blockade during spinal anesthesia. Sympathetic blockade is often two to six levels higher than the sensory level, so that a patient with a T4 sensory block may have completely blocked cardioaccelerator fibers originating from T1 to T47. However, in a prospective study of 952 patients, Carpenter et al.3 found that the traditional risk factor peak block height had the weakest correlation with the severity of bradycardia.\n\nA more important effect of sympathetic inhibition is a significant decrease in venous return to the heart that enhances cardiac vagal tone8. The decrease in venous return could further be accentuated by aorto-caval compression despite the application of a left lateral tilt position, since no particular position has been shown to completely abolish caval compression9. Cardiac vagal tone may be enhanced by three different mechanisms4: 1) Decreased venous return results in decreased stretch of atrial pacemaker cells resulting in bradycardia; 2) Firing of low pressure baroreceptors in the right atrium and vena cava and 3) A Bezold-Jarisch reflex, in which left ventricular mechanoreceptors are stimulated and paradoxically cause bradycardia. Earlier studies suggested that vasodilation caused by sympathetic blockade may be best treated by preemptive fluid administration7,8. However, more recent data suggest that crystalloids alone (without vasopressors) are inconsistent in preventing hypotension10.\n\nA high or total spinal anesthesia was unlikely, since our patient responded appropriately to pinprick sensations at or above the T4 level. Nevertheless, a T4 level block could completely block cardioaccelerator fibers originating from T1 to T4 resulting in severe bradycardia. Caplan et al. in their closed claims analysis noted the median time from local anesthetic administration to cardiac arrest to be 36±18 minutes2; however, most of these cases were non-obstetric. The onset of asystole in our case was 3 min from initiation of spinal anesthesia which could have resulted from a sympathetic block accentuated by at least partial aorto-caval compression by the gravid uterus. Supine hypotensive syndrome of pregnancy has been well described in the literature and can cause a marked bradycardia with a reduction in cardiac output and severe hypotension in a few subjects11.\n\nWhile women are 11 to 50 times less likely to develop bradycardia during neuraxial anesthesia than men12, parturients typically have a higher HR and are therefore even less likely to develop bradycardia or cardiac arrest during neuraxial anesthesia5. Carpenter et al. reported a low baseline heart rate (<60 bpm), ASA 1 status (when compared to ASA 3 and 4) and beta-blockade as the strongest predictors of bradycardia3. With her baseline HR of 62 bpm our patient may have been at higher risk.\n\nCase reports exist in which patients were amnestic for episodes of unconsciousness, as documented with carotid sinus syndrome13. Despite the language barrier, our patient exhibited signs of consciousness during asystole via her startle reactions and turning her head to unexpected shouting and light tactile stimulation. This demonstrates that a patient could remain conscious for up to 30 sec despite being asystolic in isolated cases. This delay in loss of consciousness could substantially delay recognition of a life threatening event, especially if a patient is temporarily disconnected from monitoring. It therefore underscores the importance of continuous monitoring during spinal anesthesia with pulse oximetry and EKG at all times.\n\nThere are several theoretical mechanisms that may be postulated to explain the maintenance of cerebral oxygenation in this scenario. First, supine position and volume loading may have contributed to venous pooling within the cerebral vasculature. Studies have also demonstrated that stroke volume and CVP increase during vagally induced bradycardia14, so that a few heart beats during the asystolic episode may have produced a disproportionally large amount of blood flow in this setting. Whether aortic compression by a gravid uterus enhances cerebral blood flow at the expense of lower extremity blood flow remains unknown. Through their experiments on healthy controls Ide et al. showed that an impaired ability to increase cardiac output during exercise with a large muscle mass appears to limit blood flow distribution not only to active muscle15 but also to such a vital organ as the brain, and that this flow restriction was by way of the sympathetic nervous system16. During central blood volume depletion, the increase in sympathetic nerve activity shifts the cerebral autoregulation curve to the right17 and the vasoconstrictor sympathetic nerve activity over-rides vasodilatation18. Ide et al. also showed that sympathetic blockade at the level of the neck eliminated the limitation to the increase in cerebral blood flow following cardio-selective β-1 adrenergic blockade16. Although a theoretical hypothesis, a T4 level blockade in our patient may have blunted the effect of reduced cardiac output on cerebral vasoconstriction, and may have helped maintain cerebral perfusion prior to the asystolic events.\n\nAn auditory startle stimulus produces a transient tachycardia 2 to 5 sec after the stimulus, consistent with our findings; the reflex is mediated by the vagus nerve and sympathetic ganglia19. The initial startle response was followed by 3 spontaneous heart beats and probably occurred before the atropine had time to circulate through the bloodstream. The second startle reaction perhaps permitted enough cardiac activity to circulate the resuscitative drugs without CPR. A deep inspiration accompanying the startle response may have further reduced vagal tone by a mechanism similar to that seen with respiratory sinus arrhythmia (RSA)20. RSA is a cardiorespiratory phenomenon characterized by HR or R-R interval (RRI) fluctuations in phase with inhalation and exhalation. Typically, HR accelerates during inspiration and slows during expiration, and is prominent during high resting vagal tone; increased sympathetic stimulation attenuates it. Rate and depth of respiration also influence the magnitude of RSA with slow deep breathing producing maximal and rapid shallow breathing greatly attenuating RSA. Unfortunately, no EKG strips are available to confirm the above assumptions as the anesthesiologist was busy resuscitating the patient.\n\n\nConclusion\n\nWe present a unique case of sudden cardiac arrest after spinal anesthesia in an apparently healthy patient. Vagal predominance and sympathetic blockade along with activation of baroreceptor reflexes may contribute to bradycardia during spinal anesthesia. Cerebral perfusion may be maintained by venous pooling and a partial sympathetic blockade blunting the effect of reduced cardiac output on cerebral vasoconstriction. Transient tachycardia could be caused by a startle response and the effect of slow, deep respirations on RSA magnitude, allowing sufficient spontaneous cardiac activity to circulate the resuscitative drugs. Consciousness may be maintained even after asystole has occurred, rendering insufficiently monitored patients at high risk.",
"appendix": "Consent\n\nThis case report is in accordance with institutional IRB-guidelines. Despite several attempts, contacting the patient was impossible so we have been unable to obtain explicit written consent.\n\n\nAuthor contributions\n\nAll authors were involved in the draft and revision of the manuscript and have agreed to its final content.\n\n\nCompeting interests\n\nNone of the authors have a relevant competing interest.\n\n\nGrant funding\n\nInstitutional support only (KR, HJW and MLR). No grants were involved in supporting this particular work.\n\n\nReferences\n\nSprung J, Warner ME, Contreras MG, et al:Predictors of survival following cardiac arrest in patients undergoing noncardiac surgery: A study of 518,294 patients at a tertiary referral center. Anesthesiology. 2003; 99(2): 259–69.\n\nCaplan RA, Ward RJ, Posner K, et al:Unexpected cardiac arrest during spinal anesthesia: A closed claims analysis of predisposing factors. Anesthesiology. 1988; 68(1): 5–11.\n\nCarpenter RL, Caplan RA, Brown DL, et al:Incidence and risk factors for side effects of spinal anesthesia. Anesthesiology. 1992; 76(6): 906–16.\n\nMackey DC, Carpenter RL, Thompson GE, et al:Bradycardia and asystole during spinal anesthesia: A report of three cases without morbidity. Anesthesiology. 1989; 70(5): 866–8.\n\nPollard JB: Cardiac arrest during spinal anesthesia: Common mechanisms and strategies for prevention. Anesth Analg. 2001; 92(1): 252–6.\n\nGeffin B, Shapiro L: Sinus bradycardia and asystole during spinal and epidural anesthesia: A report of 13 cases. J Clin Anesth. 1998; 10(4): 278–85.\n\nCook PR, Malmqvist LA, Bengtsson M, et al:Vagal and sympathetic activity during spinal analgesia. Acta Anaesthesiol Scand. 1990; 34(4): 271–5.\n\nBaron JF, Decaux-Jacolot A, Edouard A, et al:Influence of venous return on baroreflex control of heart rate during lumbar epidural anesthesia in humans. Anesthesiology. 1986; 64(2): 188–93.\n\nCyna AM, Andrew M, Emmett RS, et al:Techniques for preventing hypotension during spinal anaesthesia for caesarean section. Cochrane Database Syst Rev. 2006; 18(4): CD002251.\n\nMorgan PJ, Halpern SH, Tarshis J, et al:The effects of an increase of central blood volume before spinal anesthesia for cesarean delivery: A qualitative systematic review. Anesth Analg. 2001; 92(4): 997–1005.\n\nKinsella SM, Tuckey JP: Perioperative bradycardia and asystole: Relationship to vasovagal syncope and the bezold-jarisch reflex. Br J Anaesth. 2001; 86(6): 859–68.\n\nCuratolo M, Scaramozzino P, Venuti FS, et al:Factors associated with hypotension and bradycardia after epidural blockade. Anesth Analg. 1996; 83(5): 1033–40.\n\nParry SW, Steen IN, Baptist M, et al:Amnesia for loss of consciousness in carotid sinus syndrome: Implications for presentation with falls. J Am Coll Cardiol. 2005; 45(11): 1840–3.\n\nSheriff DD, Luo Z: Capacitive function of the heart: Influence of acute changes in heart volume on mean right atrial pressure. Am J Physiol. 1997; 272(1 Pt 2): H553–8.\n\nPawelczyk JA, Hanel B, Pawelczyk RA, et al:Leg vasoconstriction during dynamic exercise with reduced cardiac output. J Appl Physiol. 1992; 73(5): 1838–46.\n\nIde K, Boushel R, Sorensen HM, et al:Middle cerebral artery blood velocity during exercise with beta-1 adrenergic and unilateral stellate ganglion blockade in humans. Acta Physiol Scand. 2000; 170(1): 33–8.\n\nPaulson OB, Strandgaard S, Edvinsson L, et al:Cerebral autoregulation. Cerebrovasc Brain Metab Rev. 1990; 2(2): 161–92.\n\nLevine BD, Giller CA, Lane LD, et al:Cerebral versus systemic hemodynamics during graded orthostatic stress in humans. Circulation. 1994; 90(1): 298–306.\n\nValls-Solé J, Veciana M, Leon L, et al:Effects of a startle on heart rate in patients with multiple system atrophy. Mov Disord. 2002; 17(3): 546–9.\n\nGrossman P, Taylor EW: Toward understanding respiratory sinus arrhythmia: Relations to cardiac vagal tone, evolution and biobehavioral functions. Biol Psychol. 2007; 74(2): 263–85."
}
|
[
{
"id": "724",
"date": "23 Jan 2013",
"name": "John Pollard",
"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 believe that this report should be Approved with Reservations. One concern is the assumption that this patient did not have relatively high levels of spinal blockade. The authors describe a 'T4 level block' 3 minutes after initiating spinal anesthesia. Since the block would be expected to continue to spread upwardly after the initial 3 minutes, it is very likely that this patient had higher levels of sensory blockade after the 3 minute mark. Since the effective dose is significantly decreased in pregnancy, the authors should remind the reader that 12 mg of bupivacaine was a relatively large dose for a short (5 ft 5 inch tall) woman undergoing cesarean section. A second concern is the speculation the discussion based primarily on physiologic studies that either had no sympathetic blockade or in one case unilateral sympathetic blockade. A spinal block that caused a T4 sensory block in 3 minutes would be expected to cause a near if not total sympathetic block in the ensuing minutes so physiologic studies following complete sympathetic blockade would likely be applicable to this case. Despite these reservations, the authors do highlight a very important point. Physicians and nurses are often falsely reassured by a patient who is still awake or even talking despite severe decreases in heart rate or blood pressure. In such cases, there appears to be enough vasoconstriction from endogenous adrenaline or vasopressin to shunt blood flow to the heart and brain despite the low cardiac output. This case demonstrates that in the supine position, the cardiac output needed to maintain consciousness is far less than what is needed to provide adequate perfusion to all of the other key organs. Treating hemodynamically unstable patients aggressively with fluids, atropine and vasopressors while they still conscious improves the likelihood that resuscitation medications will enter the central circulation more expeditiously and should result in better outcomes as observed in this case.",
"responses": []
},
{
"id": "929",
"date": "07 May 2013",
"name": "Michael Paech",
"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\nAlthough not a scientific article, this is a well written and interesting case report of an exceptionally rare event. Although there is nothing special about management it is of educational interest due to the incompletely explained physiological events and clinical presentation and course. Did the patient have a history of syncope? It would have been very helpful to have captured the ECG sequence and to have reported on the final block height (was this checked again?), as very high spinal block remains a strong possible explanation. The case report does not mention when the inspiratory effort, that is later discussed, was made. The decision to give epinephrine prior to preparing to instituting external cardiac massage, when the second episode of asystole occurred, warrants mention given it is contrary to guidelines. Clearly consciousness can be maintained with minimal or episodic cardiac output, and has been described during CPR (Bihari S, Rajavee V. Neurocrit Care 2008). In the conclusion, reference is made to a transient tachycardia in the patient – but this would be more accurately described as transient myocardial conduction or activity or similar.",
"responses": []
},
{
"id": "985",
"date": "05 Jun 2013",
"name": "Bernard Wittels",
"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 describe one 15-second and one 30-second episode of transient asystole after spinal anesthesia for cesarean delivery in a woman with a baseline heart rate of 62 and an intravenous preload of 1 liter of crystalloid over an undefined period of time. In their manuscript, they note correctly that bradycardia is a common dysrhythmia with spinal anesthesia and that asystole and sudden death are relatively rare. Much of the article centers on the purported physiologic mechanisms involved, yet there are no data in this case to support any specific argument. In their manuscript title, the authors imply that their patient's persistent consciousness throughout two transient episodes of asystole is unusual or unique, but most clinicians would view this as normal in a young, healthy individual; in their manuscript, they describe treatment with IV atropine, IV epinephrine, and shouting at the patient, but this omits airway evaluation, oxygenation, ventilation and ventricular pacing that are BLS and ACLS measures to treat asystole in the non-obstetric patient.The authors mention the Bezold-Jarisch reflex only once in the manuscript as a possible contributing influence; and their clinical responses ignored this physiology. Many authors agree that even two liters of IV crystalloid may be insufficient to prevent hypotension with spinal anesthesia, so the authors should have anticipated both hypotension and hypovolemia-induced changes in cardiac physiology. Resuscitation should have included rapid IV fluid administration and raising the patient’s legs to increase venous return to the heart without increasing the cephalad spread of the spinal anesthetic. As in any resuscitation, communication with the patient (“Annie, Annie, are you OK?”), providing 100% oxygen by face mask, assisting with ventilation if necessary, and frequently checking the blood pressure (the ABC’s) should have been performed. There is no role for shouting or frightening the patient who has severe bradycardia or transient asystole. In fact, sudden frightening has been known to induce a transient myocardial stunning and LV dysfunction in young, healthy individuals. By not explicitly doing maneuvers to increase venous return to the heart, the anesthesiologists delayed the patient’s recovery and her response to IV atropine and epinephrine.Most obstetric anesthesiologists know to prevent these types of episodes by administering IV fluids in amounts of 2 liters or more prior to spinal anesthesia, as well as administering pressor agents (ephedrine or phenylephrine) at the earliest sign of hypotension or bradycardia. Continuous calm communication, especially to distressed obstetric patients is an art all obstetric anesthesiologists should master. Consciousness in unmedicated, young, healthy patients without any peripheral vascular disease has no clearly defined relationship to heart rate alone, so that awareness can only be assessed by direct communication.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-11
|
https://f1000research.com/articles/2-10/v1
|
10 Jan 13
|
{
"type": "Research Article",
"title": "III. Cellular ultrastructures in situ as key to understanding tumor energy metabolism: biological significance of the Warburg effect",
"authors": [
"Halina Witkiewicz",
"Phil Oh",
"Jan E Schnitzer",
"Phil Oh",
"Jan E Schnitzer"
],
"abstract": "Despite the universality of metabolic pathways, malignant cells were found to have their metabolism reprogrammed to generate energy by glycolysis even under normal oxygen concentrations (the Warburg effect). Therefore, the pathway energetically 18 times less efficient than oxidative phosphorylation was implicated to match increased energy requirements of growing tumors. The paradox was explained by an abnormally high rate of glucose uptake, assuming unlimited availability of substrates for tumor growth in vivo. However, ultrastructural analysis of tumor vasculature morphogenesis showed that the growing tissue regions did not have continuous blood supply and intermittently depended on autophagy for survival. Erythrogenic autophagy, and resulting ATP generation by glycolysis, appeared critical to initiating vasculature formation where it was missing. This study focused on ultrastructural features that reflected metabolic switch from aerobic to anaerobic. Morphological differences between and within different types of cells were evident in tissue sections. In cells undergoing nucleo-cytoplasmic conversion into erythrosomes (erythrogenesis), gradual changes led to replacing mitochondria with peroxisomes, through an intermediate form connected to endoplasmic reticulum. Those findings related to the issue of peroxisome biogenesis and to the phenomenon of hemogenic endothelium. Mitochondria were compacted also during mitosis. In vivo, cells that lost and others that retained capability to use oxygen coexisted side-by-side; both types were important for vasculature morphogenesis and tissue growth. Once passable, the new vasculature segment could deliver external oxygen and nutrients. Nutritional and redox status of microenvironment had similar effect on metabolism of malignant and non-malignant cells demonstrating the necessity to maintain structure-energy equivalence in all living cells. The role of glycolysis in initiating vasculature formation, and in progression of cell cycle through mitosis, indicated that Warburg effect had a fundamental biological significance extending to non-malignant tissues. The approach used here could facilitate integration of accumulated cyber knowledge on cancer metabolism into predictive science.",
"keywords": [
"“It isn’t what we don’t know that gives us trouble",
"it’s what we know that ain’t so”."
],
"content": "\n\n“It isn’t what we don’t know that gives us trouble; it’s what we know that ain’t so”.\n\n– Will Rogers\n\n\nIntroduction\n\nIn 1976, in his concluding remarks on Otto Warburg’s controversial theory of respiratory impairment in cancer, announced 50 years earlier, Sidney Weinhouse emphasized the need for basic knowledge of the biology of the cell as a way to elucidate the unresolved issues of “regulatory malfunctions which underlie neoplasia”1. Indeed, during the following years much cancer research focused on isolated tumor cells cultured in vitro, analyzed by flow cytometry, subjected to molecular high-throughput genomics and proteomics analyses, or studied in vivo by light microscopy. The outburst of interest in oncogenes dominated over metabolic studies; however, many genetic mutations affecting signaling pathways turned out to be those involved in regulation of metabolic processes. The modern approaches contributed to currently persisting understanding that alterations in cellular metabolism should be considered one of the crucial hallmarks of cancer2,3. Yet the biological significance of the Warburg effect, the aerobic glycolysis, remained elusive and the dependence of tumor growth on glycolysis instead of oxidative phosphorylation (i.e. on the pathway with net gain of two instead of 36 ATP molecules per one molecule of glucose) remained intriguing3,4. Theoretically, the increased rate of glycolysis could compensate for the energetic imbalance; however, such reasoning is not supported by tumor hypoglycemia commonly observed in vivo2.\n\nHere the old controversy on the respiratory impairment in cancer is addressed from the perspective of tissue biology rather than cell biology, following recent findings regarding cellular mechanisms of tumor vasculature formation5,6. The new perspective emerged from putting in vitro grown mammary tumor spheroids back into the tissue context and increasing the resolution of morphological analysis in situ to the ultrastructural level which enabled evaluation of subtle differences in the physiological status of individual cells. Before implantation, tumor spheroids were getting all the energy they needed from properly buffered culture medium in an optimized atmospheric environment and they formed no vessels. After pseudo-orthotopic implantation, the tumors induced stem cells of homologous tissue graft to form neo-vasculature for them5. The ectopic implantation put tumors in a critical situation because none of the first two options were available; the spheroids had to fend for themselves6. They did so in more than one way: (1) by losing part of its population through erythrosomal autophagy, (2) by establishing paracrine dialog with initially non-responsive, non-homologous, local tissue stem cells (TSCs), (3) by self-organizing, i.e. inducing some of the tumor cells to differentiate into hematopoietic stem cells (HSCs). The third option was most astounding as it meant trans-differentiation to a nonmalignant phenotype. Until new functional vessels formed, the growing regions suffered from malnourishment and hypoxia, manifested by changes in ultrastructural features of the cells. The approach used here allowed monitoring of the earliest stages of vasculature morphogenesis occurring in vivo and contributed a much needed qualitatively new perspective to the importance of sub-populations of cancer cells7,8. Natural interactions among cells, within and between types, could be deduced that way and complex morphogenetic processes reconstructed in retrospect, i.e. not in real time but faithfully. That type of information is critical for the integration of multiple types of data for signaling research9. It could also inspire computer modeling to add quantitative aspects to the analysis.\n\nIn situ ultrastructural analysis is suitable to study cellular metabolism because metabolic pathways have variable structural bases. Ultrastructures are as dynamic as the processes supported by them. The most energetically efficient pathway (oxidative phosphorylation) requires the most complex structure (mitochondrion) providing the enclosed space necessary for the existence of a proton gradient because the movement of protons across the inner mitochondrial membrane is the primary energy-conserving event. The less efficient pathway (glycolysis) occurs in cytoplasm and can be reproduced in vitro. Without specific protein markers, no particular glycolysis-associated structure is visualized by transmission electron microscopy (TEM) although in erythrosomes, known for using glycolysis to generate ATP10, abundance of calmyrites suggested that those complexes might be involved5. When not needed, mitochondria become degraded. Therefore, there is a correlation between active oxidative phosphorylation with presence of mitochondria. Uncoupling of oxidation from phosphorylation correlates with subtle but noticeable morphological changes of mitochondria as seen in brown fat11.\n\nThe concept of compartmentalization of specific metabolic processes is widely accepted. A myriad of different metabolic pathways and processes associates with peroxisomes, i.e. relatively simple cytoplasmic organelles (0.1–1.0 µm in diameter), bound with a single membrane and missing DNA. The enzymatic content of such small organelles serves as a criterion to distinguish them from structurally similar, acid hydrolyses containing, lysosomes12. Despite their structural simplicity, peroxisomes are surrounded by a certain aura of mystery13 because of their resemblance to mitochondria with regard to several functional characteristics14. The two kinds of organelles appear to cooperate. Notably, the initial steps of all very long chain fatty acid (VLCFA) metabolism occur in peroxisomes whereas the subsequent steps occur in mitochondria. The advantage of such cooperation is the protection of mitochondria from the damaging effect of by-products (free radicals) when VLCFA (longer than C-18) are broken down into chains shorter than C-9 to be exported to mitochondria15. Historically, catalase activity served as the criterion identifying peroxisomes even if their 3D shape16 was similar to the mitochondrial tubular network discovered later17–19. The interplay between the two organelles in peroxisomal disorders suggests more than just an overlap of functions14,20. Interestingly, they also share fission machinery; although, in addition to fission of existing organelles, peroxisomes are said to be formed de novo from the endoplasmic reticulum (ER)21–23. Yet, no direct evidence of physical contact in vivo between mitochondria and morphogenesis of peroxisomes is available, except for genetically modified cells cultured in vitro24. The nature of those parallels between the two organelles remains unclear.\n\nThis report shows how in situ ultrastructural analysis proved useful for further substantiating earlier hypothesized structural relationship between peroxisomes, ER, and mitochondria14,23 and for revealing the biological significance of the aerobic glycolysis in metazoan vasculature morphogenesis and tissue growth, i.e. of the Warburg effect.\n\n\nMaterials and methods\n\nThe study was performed according to protocols approved by the Sidney Kimmel Cancer Center’s (SKCC) OLAW-approved Institutional Animal Care and Use Committee (Assurance No A4128-01). The protocol numbers were: 03-16A and 05-11 for Grants CA104898 and CA119378, respectively. No human specimens were involved in any of the experiments outlined here.\n\nA total of five recipient mice were used in the study described here and in the two accompanying articles. The same numbering system was used in all three articles. The experimental design is summarized in Table 1.\n\nHost and graft donor female, athymic, nude mice, 8–9 weeks old, were purchased from Harlan. The donor mouse with GFP-labeled ubiquitin was from The Jackson Laboratory (Stock Number: 004353; Strain Name: C57BL/6-Tg(UBC-GFP)30Scha/J). The mice were housed in the SKCC animal care facility. For surgery, they were anesthetized (7.3 mg ketamine hydrochloride and 2.3 mg xylazine/100 g body weight, inoculated i.p.) and placed on a heating pad. Immediately before tissue harvesting the tumor hosting mice as well as the graft donors were euthanized with pentobarbital overdose (100 mg/kg i.p.).\n\nThe parental murine breast cancer cell line, N202.1A25 was stably transfected to express GFP under histone H2B promoter26. The two cell lines, N202.1A parental and N202.1A+H2B-GFP (obtained from Drs. J. Lustgarten and P. Borgstrom) were used to form tumor spheroids by culturing 2×105 cells per well for 2–3 days prior to implantation.\n\nA week after establishment of mouse dorsal skin chambers, the tumor spheroids were implanted on a pad of homologous tissue, namely, minced breast fat pad from a lactating mouse (pseudo-orthotopically) or without graft (ectopically) as described earlier27. The tumors were incubated in the chambers for one or three weeks. Their final size was about 1–3 mm in diameter.\n\nThe GFP-specific rabbit polyclonal IgG (ab290) was from Abcam; CIB1-specific rabbit polyclonal IgG (11823-1-AP) was from ProteinTech Group, Inc.; CD34-specific rat monoclonal IgG2a (sc-18917) and non-reactive goat polyclonal IgG (sc-34284) were from Santa Cruz.\n\nThe tumors with some surrounding tissues were dissected out and cut in halves perpendicular to the host skin surface while immersed in cold fixative (4% paraformaldehyde in 0.1 M Na cacodylate pH 7.4). The skin region served later as a reference to distinguish between the edges of the tumor facing the skin and those facing the glass window of the chamber. The halves were then separated and processed independently for TEM and immunocytochemistry.\n\nThe tissues were transferred into a stronger fixative (4% paraformaldehyde/2.0% glutaraldehyde in 0.1 M Na cacodylate pH 7.4) to better preserve the ultrastructures before further cutting. They were cut into 1 mm thick slices in planes perpendicular to the plane of the first cut and to the skin surface, finally, into ~ 1 mm3 blocks, transferred into a fresh portion of the fixative in which they were cut and incubated for 2 hrs at 4°C. The fixed tissue blocks were washed with 0.1 M Na cacodylate – HCl buffer pH 7.4 (3 × 15 min.) and post fixed in 1% OsO4 in 0.1 M Na cacodylate buffer, pH 7.0 for 60 min. on ice, washed with water and stained with 1% uranyl acetate at RT for one hour. The blocks were embedded in EMbed-12 (EM Sciences, Cat. No 14120). The resin embedded tissues were cut into 60 nm sections, on a Leica Ultracut UCT ultramicrotome and stained with lead citrate28 or viewed without further contrasting.\n\nDuring cutting into ~ 1 mm3 blocks as described above, the tissues were kept in the mild fixative to protect antigenic epitopes (4% paraformaldehyde in 0.1 M Na cacodylate pH 7.4). The tissue blocks were vitrified by infiltrating the pieces with 50% PVP (polyvinylpyrrolidone) containing 2.3 M sucrose in 0.1 M Na-cacodylate buffer, pH 7.4, for 2 hrs or overnight, mounted on metal pins and frozen in liquid nitrogen, as described by Tokuyasu29. Frozen tissues were cut into 70 nm sections, on a Leica Ultracut UCT ultramicrotome with the cryo-attachment. The sections were picked from the knife with 2.3 M sucrose and floated on 1% ovalbumin (Sigma, Cat No.A5378) in 0.1 M Na-cacodylate buffer for at least one hour before incubation with specific or non-reactive antibody (50 µg/ml), at RT for one hour. Sections were then rinsed eight times with 0.1% ovalbumin in the same buffer and incubated for one hour with 10 nm Au coupled to protein A (from Dr G. Posthuma; Cell Microscopy Center, University Medical Center Utrecht, The Netherlands). The eight rinsing steps were repeated before fixation of the immune complexes with 1% glutaraldehyde. After rinsing three times with water, the immunostained cryosections were contrasted with a mixture of uranyl acetate and methyl cellulose (25 centipoises, Sigma M-6385) in water, at a final concentration of 1.3% each, for 10 min at RT. Excess liquid was removed and the sections were dried at RT.\n\nAll sections were viewed and the images captured at 100 kV using a Morgagni 268 electron microscope equipped with a MegaView III digital camera. Images were transmitted from the microscope camera to an iTEM imaging platform from Olympus Soft Imaging Solutions and archived in a designated database. In some cases, the final images were assembled by multiple image alignment (MIA) to increase the surface area without losing the resolution. We used the graphics editing program, Adobe PhotoShop, to add cell type-specific color-coding shown in the twin sets of images included in the Supplement.\n\n\nResults\n\nFormation of vasculature in the avascular tumor spheroids has been shown to depend in our model on the presence of the homologous tissue graft which results in faster tumor growth5,6. However locally, the phenomena described for the ectopically implanted tumors (Figure 1–3 in6) also occurred in the presence of the graft, i.e. after pseudo-orthotopic implantation (see Table 1 to correlate specific images with particular mice). Those phenomena were the early stages of vasculature formation within tumor nodules and the erythrogenesis in fibroblasts separating the nodules, which we have demonstrated to highlight the variable dynamics of the tumor microenvironment in situ. A typical hematopoietic cell triplet, megakaryocyte, erythroblast and EC (with anoikis between the last two cells30) located inside the tumor nodule, suggests that some tumor cells were evolving into re-differentiated (non-malignant) cells (Figure 1 and Figure S1). The erythroblast with depleted mitochondria and accompanied by an EC and a megakaryocyte coexisted side by side with tumor cells displaying the pattern of mitochondrial profiles typical of the resting phase of the cell cycle (fragmented mitochondria31). Such ultrastructural variability would not be possible if all cells used the same type of metabolism, like yeast in suspension do, depending only on the environmental conditions. Therefore, that image provided an answer to the quest for tumor-specific defects of mitochondria. The loss of mitochondria must have resulted in impairment of respiration in erythroblasts and platelets but not in the cells that maintained them. The phenomenon was not tumor-specific. The fibroblasts squeezed between the tumor nodules were also suffering from local hypoxia, and some of them were also converting into erythrosomes (in a slightly different way).\n\nMouse 3. The erythrosomes (ES) and the fibroblast (FB) oriented “diagonally” separated the tumor nodules. One nodule contained plasma cells (T-PC), as partly shown in the upper right corner. Cells in the lower and left part of the image belonged to the other nodule. Flanked by tumor cells were the vasculature-initiating cells: erythroblast (T-EB), as identified by the disappearing nucleus and mitochondria converting into peroxisomes, and endothelial cell (T-EC). The two cells were locally separated by anoikis (arrows)30. To the left of the T-EC, is a partly shown large nucleus with two nucleoli, likely belonging to a megakaryocyte of tumor origin (T-M); peroxisomes are visible in the cytoplasm above the nucleus.\n\nThe erythroblast located in another tumor nodule represented an earlier stage of the erythrogenic conversion (Figure 2 and Figure S2). The nucleus of that cell was more prominent despite having integrity compromised by the local absence of a nuclear membrane and invasion of the cytoplasm. Mitochondria were morphologically different from the fragmented ones in the surrounding tumor cells and highly pleomorphic (Figure 3 and Figure S3). Their profiles resembled those of the organelles in partly differentiated cells (type II) of the mouse preputial gland, where they contained catalase and were thought of as peroxisomes (Figure 1 in16). Both cell types featured mitochondria undergoing the process of conversion into peroxisomes. The mitochondrial properties were: double outer membrane and internal cristae. The peroxisomal-like properties were degradation of the cristae, increasing electron density, remodeling of the double outer membrane into the single membrane, and internal vesicles like those containing catalase. By extrapolation, the 2D pleomorphic profiles in the erythroblast (meta-chondria and peroxi-chondria) shared the 3D structure with the “peroxisomes” of the type II preputial gland cell16,17 and with the mitochondria17,18,31 .\n\nMouse 4. The erythroblast (T-EB), differentiating endothelial cell (T-EC), possibly megakaryocyte (T-M). Ovals contain peroxisomes forming in tumor cells (T). Arrows point to the anoikis30 area.\n\nMouse 4. Mitochondria at various stages of conversion into peroxisomes (white stars); ER (black stars); autophagosomes (+). The arrows point to electron lucent vesicles that might contain catalase (compare with Figure 7 in16). The enlarged boxed area is rotated left in Figure 4[A] whereas the microenvironment of the erythroblast (T-EB) is shown in Figure 2. Mitochondria (M) of the tumor cell (T) in the upper right corner are characteristic of the quiescent cells but some show signs of hypoxia (m), as do dilated cisternae of ER (black stars).\n\nThe early erythroblast contained a region displaying continuity of the membranes from three kinds of organelles. Those organelles were nucleus, mitochondrion and ER (Figure 4[A] and Figure S4[A]). In the tumor cell with intact nucleus (not committed to erythrogenesis), the nuclear membrane was not involved but the membrane continuity between the ER and the deteriorating mitochondrion was seen (Figure 4[B] and Figure S4[B]). The continuity of the membranes indicated their gradual remodeling rather than complete degradation of the existing system of membranes before synthesizing the new, evolving type(s)5. The irregular mitochondrial shape, gradual disassembly of mitochondrial cristae and condensation of the remaining components into an electron-dense region enclosed by a single membrane indicates a morphogenetic relationship with peroxisomes12. Examples of more advanced stages of peroxisomal morphogenesis are shown in Figure 4[C & D] and Figure S4[C & D]. Calmyrites, the para-crystalline structures detectable with calmyrin-specific antibodies and abundant in erythrosomes5, were occasionally associated with mitochondria and with the intermediate forms (Figure 5 and Figure S5). In addition to tumor cells, they were seen in erythroblasts, megakaryocytes, platelets, granulocytes, smooth muscle cells, plasma cells (Figure 6 and Figure S6) and in the epithelium and endothelium of the control sample from normal rat lung, (Figure 8[G] in5 and Figure 6 and Figure S6 [I], respectively). In megakaryocytes and platelets, they co-localized with peroxisomes (Figure 6[B & C] and Figure S6[B & C]). The histone H2B-specific label detected chromatin in the nucleus and some in one of the coffee bean-resembling, “twin” peroxisomes next to it (Figure 7[A] and Figure S7[A]).\n\nMouse 4. The point of contact for mitochondrial outer membrane (white star), ER (black star) and nuclear membrane (x) is located in the center of the circle [A]. Such membrane continuity between the ER and mitochondrion converting into peroxisome was also seen in a tumor cell with intact nucleus [B]. Examples of more advanced forming peroxisomes are shown in [C & D]. Arrows point to peroxisomes with a single membrane [D].\n\nMouse 5. Electron lucent regions were commonly associated with atypically shaped mitochondria (stars). Calmyrites were detected by immunocytochemical staining with calmyrin-specific antibody.\n\nMouse 5 [A–H] and normal rat lung [I]. Erythroblast [A], megakaryocyte [B], platelet [C], granulocyte [D], plasma cell [E], hemangioblast [F], smooth muscle [G], erythrosome [H], and lung endothelium of control sample from normal rat lung [I].\n\nMouse 4. Gold grains associated with chromatin of the interphase nucleus and a cluster of the gold particles (arrow) co-localized with one of the peroxisomes (stars) in [A]. The label was also found in the mitotic chromosomes and mitochondria of the dividing cell [B]. In the cell undergoing mitosis, the mitochondria were comparatively smaller, more electron-dense and often accompanied by an electron-lucent region, similar to those converting into peroxisomes [C]. Mice 3 [A] & 4 [B&C].\n\nCondensation and an increase in electron density of mitochondria during mitosis suggested a lowered rate of respiration at that phase of the cell cycle (Figure 7[B & C] and Figure S7[B & C] and Figure 2[F] in6). At mitosis, the mitochondria were not just fragmented as during the interphase19 but compacted, electron dense, resembling peroxisomes yet capable of reversing those morphological changes. The structural difference between the mini-chondria seen at mitosis and the fragmented eu-chondria during the resting phase of the cell cycle was obvious at the resolution of TEM. It was missed by fluorescent microscopy.\n\nIf forming vessels did not fuse with the circulatory system before the local environment ran out of oxygen and nutrients, even the differentiated ECs would undergo erythrogenic autophagy (Figure 8 and Figure S8). Within the EC type, individual cells behaved variably; the effect of hypoxia was not synchronous. That way some cells could help others to survive longer, although they perished in the process of nucleo-cytoplasmic conversion. Upon a significant change of the redox status, one could envision the surviving ECs guiding the differentiation of other cells into phenotypes matching their level of complexity5. Thus, erythrogenesis could be induced by factors limiting metabolic processes due to hypoxia and malnutrition resulting from either tissue growth (developmental and malignant) or the opposite, i.e. failure of forming vessels to continue growing until they fused with the circulatory system. Hypoxia and a shortage of nutrients affected all cell types in that microenvironment; cell type-dependent differences were subtle (Figure 1 and Figure S1). Non-malignant, collagen-producing fibroblasts outside the blood oxygen diffusion range converted most, but not all, of their bodies into erythrosomes; remnants of the cytoplasm remained, and sometimes mitochondria were present during the ongoing erythrogenic process (Figure 3 in6). To the contrary, erythroblasts of tumor origin were undergoing a complete erythrogenic conversion whilst also initiating the vasculature formation-related cascade of differentiation within the tumor population. With regard to their ability to produce erythrosomes under stressful conditions, those examples were not different from hemogenic endothelium32.\n\nMouse 4. The cell partly shown in [A] (enlargement of the boxed area in [B]) displayed features typical of definitive erythroblasts undergoing nucleo-cytoplasmic conversion: dark nuclear lobes separated from cytoplasm by multilayered corrugated membranes and peroxisomes in the absence of mitochondria. However, the shape and location of that cell were such that by light microscopy, it would look like an EC [B]. At one region, its plasma membrane appeared fused with the membrane of an underlying EC (arrow in [A]) suggesting that simultaneously with experiencing erythrogenic conversion, the cell was penetrating the endothelium of a vessel that already contained some non-circulating blood (as judged by irregular shapes of the erythrosomes). A smaller, forming vessel in [C] appeared to have converted one of its’ ECs into erythroschizosome, which had then moved into the lumen, leaving a vacant space behind, and had already separated one monomer completely and another one partially. The cell in [D] represented an ambivalent (hemangioblastic) phenotype, similar to those seen five days after pseudo-orthotopic implantation5. However, here it had an increased level of phenotypic complexity, as manifested by the presence of caveolae (arrow), a hallmark of EC, and remodeling of the internal membranes covering the emerging erythrosome (upper left corner), in addition to collagen synthesis (bundle of collagen fibers under the right end of the scale bar).\n\n\nDiscussion\n\nSystems biology research struggles with a paucity of satisfactory methods because local micro-environmental differences, meaningful in vivo, are hard to reproduce in vitro where conditions controlled experimentally affect the analyzed phenomenon indiscriminately. For example, purified DNA molecules can easily be condensed in vitro by dehydration and charge neutralization (adding alcohol and salt, respectively) but the entire length of each molecule is affected simultaneously33. However in vivo, the process is controlled in a more shrewd way (through protein-DNA interactions), assuring local conformational differences within a single molecule34. Similarly, cells isolated from tissues and cultured in vitro lose their original functional characteristics. To study cellular interactions, preserving tissue structure is necessary. The in situ analysis of ultrathin tissue sections enabled the examination of complex cellular interactions that would have been missed by other methods. It also allowed dialectical interpretation of the observed phenomena that appeared contradictory when studied independently. That approach exposed new relationships between intra- and inter-cellular events and implied logical connections between tissue morphogenesis and metabolic pathways. In vivo, cells with different types of metabolism were located side-by-side and cooperated (Figure 1, Figure 2, Figure S1 and Figure S2). Therefore, in addition to environmental factors (hypoxia and hypoglycemia) that triggered the metabolic switch from oxidative phosphorylation to glycolysis in some cells, tissue-intrinsic regulatory mechanisms must have been involved to control types of metabolism for each cell individually, yet cooperatively for all. The environmental factors triggered the response, but tissue-inherent factors acted as modulators that made the response differential and resulted in changes most beneficial for the tissue rather than individual cells. The in situ analysis demonstrated great heterogeneity of cellular phenotypes within relatively small tissue fragments. Characterizing metabolic pathways of those individual cell types without changing their properties would not have been possible by methods requiring destruction of the tissue fabric. Such relational characterization is necessary to unravel metabolic processes occurring in vivo.\n\nThe single piece of information that was most consequential for viewing tumor energy metabolism from a new perspective was the one on the mechanism of erythrogenesis5. It showed how the conclusions regarding “erythrocytic enucleation” in other models35,36 had a long-lasting misleading effect on studies of tumor vasculature morphogenesis. While the presence of non-malignant cells of hematopoietic lineage in tumor samples had been known for years, it was difficult to explain. They were assumed to be “recruited to” or “extravasated at” the tumor site3. However, erythroblasts were not identified in tumors until recently5; even though, the morphology of extramedullar erythroblasts (Figure 1, Figure S1, Figure 2, Figure S2, Figure 6[A] and Figure S6[A]) was no different from those in human bone marrow (medulla ossea) seen earlier12. Previously, the megakaryocyte, erythroblast & EC triplets, as well as fibroblasts, were categorized as stromal cells when seen in tumor nodules. Here, the gradual shrinking and disappearance of mitochondria with a simultaneous proliferation of peroxisomes was correlated with the metabolic switch. That led to a glycolytic pathway in mature erythrosomes through a phase of extensive, peroxisomes-requiring restructuring of the entire cell. That phase lasted long enough to make cells with peroxisomes in the absence of mitochondria commonly encountered. The distinct morphology of the erythroblasts going through the nucleo-cytoplasmic conversion conceptually steered the erythrogenesis process from bone marrow to any location where tissue growth was happening. Practically, it helped in identifying specific locations where vasculature morphogenesis started in vivo (via trans-differentiation of designated cells into HSCs and their interactions with other cells) and in following the process as it unfolded. Clearly, the absence of mitochondria in erythroblasts was a good reason for their respiration to be impaired and replaced by the alternative pathway. That is why using isolated mitochondria to search for causes of the respiratory impairment was not successful, and why metabolic studies on tissue slices were showing both pathways.\n\nThe nucleo-cytoplasmic conversion that we observed into erythrosomes, being the extreme case of cellular remodeling, augmented the role of peroxisomes in erythrogenesis and helped us to demystify their biogenesis considerably. The process depended on fundamental restructuring of the entire cell in a controlled purposeful fashion. It required reduction of all organelles and membranes to their primary components from which to form erythrosomes - the new organelles with their own specialized membrane. Peroxisomes, known for homing catabolic as well anabolic processes, played a critical role in the restructuring. The results were consistent with the three hypothesized sources of lipids and proteins for the creation of peroxisomal membrane namely mitochondria, ER and cytoplasm23. However, in cells committed to the erythrogenic autophagy, the nucleus also contributed to the biogenesis of peroxisomes and ultimately erythrosomes (Figure 4[A] and Figure S4[A]).\n\nThe mitochondrial reticulum was shown to be present in cultured cells only at certain phase of the cell cycle when the energy output was the greatest, just before synthesis of DNA (G1/S)31. It could also be induced by starvation and reactive oxygen species37,38. Here, we present similar mitochondrial forms in situ, in a tumor cell that was not preparing to replicate because its nucleus was partly degraded and an early EC and potential megakaryocyte accompanied that cell. Together, the three cells represented the typical hematopoietic triplet. The erythrosomal conversion of the nucleated cells into the organelles containing no visible internal ultrastructures (erythrosomes) involved degradation of all organelles originally present in the erythroblasts5. The molecular products of such degradation became substrates for the formation of the erythrosomes. The tumor cell in Figure 3 and Figure S3 was in the transition phase and so were its organelles. Mitochondria were converting into peroxisomes, and ultimately the peroxisomes would degrade as well. Hypoxia and starvation could have induced those ultrastructural changes by triggering initiation of erythrosomal authophagy.\n\nThe profiles of mitochondria were unlike those fragmented ones in the surrounding tumor cells (eu-chondria). The term meta-chondria describes them better because they were an equivalent of the peroxi-chondria in the secretory cells of glands like the preputial16, Meibomian39 or those producing gastrointestinal secretions40. One can observe that the filamentous mitochondria generating more ATP than any other form31 appear when entire cell content requires restructuring, either due to proliferation or conversion of the cell into something different, even if only gradually; for example, the erythrosomes or fat-containing vacuoles. A major similarity between the two cell types harboring such events is that both eventually perish in the process of producing sub-cellular elements: erythrosomes or lipid droplets, respectively. The partly differentiated cells of the mouse preputial gland (type II)16 were in the process of differentiating into lipid-producing type III and subsequently would degenerate via the lethal cells (type IV)39.\n\nTo form peroxisomes, certain elements of mitochondria were utilized, for example those supporting the overlapping functions, including fission of the organelle22, but others had to be synthesized; hence, the involvement of rough ER. The conversion to peroxisomes appeared simultaneous with division of the mitochondrion. The division started by mitochondrion could be completed by peroxisomes. The continuity of partially disassembled mitochondrial membranes with ER (Figure 4[A & B] and Figure S4[A & B]) and localization of histone H2B in the peroxisome (Figure 7[A] and Figure S7[A]) corroborated the close relationship between mitochondria and peroxisomes41 and provided evidence for direct structural connection in situ. Chromatin was shown here for the first time in peroxisomes, confirming their derivation from mitochondria. A very small amount of mitochondrial DNA found earlier in the preparation of isolated peroxisomes was dismissed as contamination17,42. Participation of the cytoplasmic components could only be assumed here, except for calmyrites that were usually free in cytoplasm, but in some cells commonly co-localized with peroxisomes and mitochondria (Figure 5, Figure S5, Figure 6 and Figure S6). Substrates catabolized in peroxisomes should be those other than glucose (including amino acids, fatty acids and lactate43–45), because glycolysis does not require structural isolation from cytoplasm except in some protozoa where specialized peroxisomes are equipped with glycolytic enzymes14. Therefore, the significance of co-localization of calmyrites with peroxisomes in platelets remains to be clarified.\n\nMitochondrial conversion into peroxisomes, correlating with hypoxia and cellular differentiation into erythroblasts, reflects concomitant metabolic changes. Erythroblasts and neurons appear to be two extreme examples with regard to their type of metabolism and peroxisomal functions. In the former, peroxisomes are more abundant than mitochondria and eventually replace them, and in the latter, using mainly glucose as a substrate for OXPHOS, peroxisomes are absent15. In white blood cells, peroxisomes are common and unaccompanied by mitochondria (similar to erythroblasts), indicating that fluctuating oxygen pressure in circulating blood is incompatible with OXPHOS. Some ill-defined granules of white blood cells may represent variants of peroxisomes; a hint for such a possibility was provided by mitochondrial crystalline inter-membrane inclusions in gliomas46.\n\nAccording to the concept of “hemogenic endothelium”, blood derives from endothelium32 because the hemogenic embryonal tissue is the tissue from which the aorta develops. The term implies that the endothelium exists before the emergence of blood cells. However, the embryonal “hemogenic endothelium” is not the kind of endothelium present in mature vascular system. As portrayed in gerbil embryo, it morphologically resembled tumor cells rather than the specialized lining of the luminal surface of blood vessels47. The essential ultrastructural feature of the embryo shared with the tumor is side-by-side location of cells with differentially modified mitochondria. By contrast, the hallmarks of ECs are a flattened shape, polarized cell membrane (luminal and abluminal), abundant caveolae (mostly on luminal surface), attachment to collagen or basement membrane (on abluminal surface), collagen synthesis and Weibel-Palade bodies. Those bodies have a functional characteristic overlapping with that of alpha granules in platelets, namely involvement in production, storage and secretion of von Willebrand factor which is essential for blood coagulation48.\n\nAlternatively, ECs could evolve from hemangioblasts and possibly megakaryocytes and collagen producers (at a higher level of complexity). As with erythrogenesis, differentiation of ECs appears to occur in multiple ways5. In our studies, tumor spheroids presented themselves as an analog of undifferentiated embryonic tissue but with a dysfunctional morphogenetic control system, except for vasculature that does make tumors vulnerable to natural selection. Erythrogenesis was a primary event; endothelium evolved next, contrary to the hemogenic endothelium concept, if that concept is taken literally. When comparing the tissues rather than words, one would object not to the “hemogenic” part of the term, but to the word “endothelium”. Yet, under hypoxia, the endothelium could become hemogenic (Figure 8 and Figure S8) just like fibroblasts and tumor cells (Figure 1, Figure S1, Figure 2 and Figure S2). By converting differentiated ECs into erythrosomes, the vessel formation could be disturbed before completion, illustrating how the environment could dominate the intrinsic potential to grow and develop (Figure 8 and Figure S8).\n\nGlycolysis was detected in mammalian embryos49,50 when the size of the embryo exceeded the oxygen diffusion range and blood islet appeared (Figure 6(B) in51), marking the initiation of vasculature development. Activation of glycolysis in non-embryonal proliferating cells has also been known for years, for example in murine splenic lymphocytes in vivo52, in cultured rat thymocytes53–55 or mouse lymphocytes56, and in rapidly proliferating prostate cells57). The energy required for nuclear division was reported to be wholly or partly derived from the anaerobic metabolism of glucose58. The reduced size and the increased electron density of mitochondria at mitosis (mini-chondria) suggests that ATP generation by OXPHOS was down-regulated intermittently, with a frequency determined by the length of the cell cycle (Figure 7[B & C] and Figure S7[B & C]). For that reason, inhibiting glycolysis with the intent to stop vasculature formation could have side effects comparable to chemotherapies targeting cell proliferation.\n\nOtto Warburg deserves credit for discovering mammalian glycolysis in 1923 despite it being a minor pathway at tissue level, even if in tissue slices it was enhanced experimentally by cutting off the circulation and providing excess glucose. Warburg was aware of cellular heterogeneity in the tissue slices and thought that solid tumors had mixed metabolic pathways active because of their impurities, i.e. containing non-tumor cells. Concerned with that “contamination”, after 1950 he studied only ascites tumor cells, believing they represented a homogenous population of cells with impaired respiration59. Sidney Weinhouse also used tissue slices and ascites tumor cells to study the same metabolic processes but his technique, employing isotopicly labeled metabolites, was much better suited for quantitative analysis and more sensitive in detecting the two pathways, glycolysis and oxidative phosphorylation, in comparison to the Warburg’s manometric method. He debated Warburg’s interpretation repeatedly1,60–62. The glycolytic process was likely up-regulated in those cells in cyclical, non-synchronous fashion, due to proliferation63 and variable level of hypoxia, depending on the volume of the ascites in vivo, as reflected by morphology of the mitochondria later visualized in ascites tumor cells by TEM64,65. Here, in situ ultrastructural analysis showed the metabolic differences among neighboring cells, also of the same type. Moreover, it suggested co-existence of the two metabolic pathways even within single cells, based on co-existence of mitochondria and calmyrites either co-localizing with mitochondria or free in the cytoplasm (Figure 5, Figure S5, Figure 6 and Figure S6). The central role of erythrogenesis in vasculature formation deepened our understanding of the relationship between tissue growth and the energy aspect of metabolism by delivering the critical missing element necessary to make the connection between processes occurring at molecular and tissue levels: glycolysis and tissue morphogenesis, including malignancy. Tumors appear to be autistic with regard to interacting with morphogenetic control signals from the system of which they are a part, but capable of basic morphogenetic functions related to vasculature formation; hence their cellular heterogeneity. Otherwise, they would not grow.\n\nThe problem with cancer cells is that in many respects they are not different from non-malignant cells. Glycolysis, the less energetically efficient metabolic pathway, appeared critical as a physiological temporary alternative when oxidative phosphorylation diminished due to either growth-related hypoxia or cytokinesis-related slowdown of anabolic processes at the mitotic phase of the cell cycle. It was not tumor specific. Not even yeast could survive for long time on glycolysis alone59. The two metabolic pathways were not mutually exclusive. If the biological significance of aerobic glycolysis in vivo was to address the needs of growing tissues4, the goal was accomplished indirectly, by inducing vasculature formation and by compensating for cyclically down-regulating mitochondrial activity during mitosis. Regardless of the role that the Warburg effect might play in carcinogenesis59, the phenomenon discovered by him appeared to be of profound significance for tissue morphogenesis in general, not only in malignancy. By proposing a fundamental role of metazoan glycolysis in establishing the circulatory system critical for fueling the metabolism of complex organisms, that conclusion indeed brings Warburg’s “anerobic glycolysis” to a renewed prominence, as anticipated by Weinhouse. The essential issues of “regulatory malfunctions which underlie neoplasia” remain unresolved but the level of our thinking about them has changed1.",
"appendix": "Author contributions\n\n\n\nJES conceived the study and participated in the interpretation of results; PO conducted the tissue culture and animal surgery; HW did the electron microscopy and wrote the manuscript. All authors participated in the design of the study and approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThe study was performed according to protocols approved by Sidney Kimmel Cancer Center’s OLAW-approved Institutional Animal Care and Use Committee (Assurance No A4128-01). The protocol numbers were: 03-16A and 05-11 for Grants CA104898 and CA119378, respectively. No human specimens were involved in any of the experiments outlined here.\n\n\nAcknowledgments\n\nThe authors thank Mrs. Carol Casarjian for proofreading the manuscript, Mr. Fred Long for his expert technical Figure preparation assistance and Mr. Zbigniew Malas who contributed the color versions of the Figures included in the Supplement.\n\n\nSupplementary figures\n\nMouse 3. The erythrosomes (ES, red) and the fibroblast (FB, yellow) oriented “diagonally” separated the tumor nodules. One nodule contained plasma cells (T-PC, brown), as partly shown in the upper right corner. Cells in the lower and left part of the image belonged to the other nodule. Flanked by tumor cells (T, brown) were the vasculature-initiating cells: erythroblasts (T-EB, red), as identified by the disappearing nucleus and mitochondria converting into peroxisomes, and endothelial cell (T-EC, green). The two cells were locally separated by anoikis (arrows)30. To the left of the T-EC, is a partly shown large nucleus with two nucleoli, likely belonging to a megakaryocyte of tumor origin (T-M, purple); peroxisomes are visible in the cytoplasm above the nucleus.\n\nMouse 4. The erythroblast (T-EB, red), differentiating endothelial cell (T-EC, green), possibly megakaryocyte (T-M, purple). Ovals contain peroxisomes (red) forming in tumor cells (T, brown). Arrows point to the anoikis30 area.\n\nMouse 4. Mitochondria at various stages of conversion into peroxisomes (white stars); ER (black stars); autophagosomes (+). The arrows point to electron lucent vesicles that might contain catalase (compare with Figure 7 in16). The enlarged boxed area is rotated left in Figure S4[A] whereas the microenvironment of the erythroblast (T-EB, reddish brown) is shown in Figure S2. Mitochondria (M) of the tumor cell (T) in the upper right corner are characteristic of the quiescent cells but some show signs of hypoxia (m), as do dilated cisternae of ER (black stars).\n\nMouse 4. The point of contact for mitochondrial outer membrane (white star), ER (black star) and nuclear membrane (x) is located in the center of the circle [A]. Such membrane continuity between the ER and mitochondrion converting into peroxisome was also seen in a tumor cell with intact nucleus [B]. Examples of more advanced forming peroxisomes are shown in [C & D]. Arrows point to peroxisomes with a single membrane [D]. Mitochondria and peroxisomes are in different shades of brown in comparison to the rest of each cell.\n\nMouse 5. Electron lucent regions were commonly associated with atypically shaped mitochondria (brown). Calmyrites were detected by immunocytochemical staining with calmyrin-specific antibody. Arrows indicate anoikis.\n\nMouse 5 [A–H] and normal rat lung [I]. Erythroblast [A, red], megakaryocyte (purple) [B], platelet (purple) [C], granulocyte [D], plasma cell [E], hemangioblast [F], smooth muscle [G], erythrosome (red) [H], lung endothelium (green) of control sample from normal rat lung [I].\n\nMouse 4. Gold grains associated with chromatin of the interphase nucleus and a cluster of the gold particles (arrow) co-localized with one of the peroxisomes (stars) in [A]. The label was also found in the mitotic chromosomes and mitochondria of the dividing cell [B]. In the cell undergoing mitosis, the mitochondria were comparatively smaller, more electron-dense and often accompanied by an electron-lucent region, similar to those converting into peroxisomes [C]. Mice 3 [A] & 4 [B & C]. Mitochondria and peroxisomes are in different shades of brown.\n\nMouse 4. The light red cell partly shown in [A] (enlargement of the boxed area in [B]) displayed features typical of definitive erythroblasts undergoing nucleo-cytoplasmic conversion: dark nuclear lobes separated from cytoplasm by multilayered corrugated membranes and peroxisomes in the absence of mitochondria. However, the shape and location of that cell were such that by light microscopy, it would look like an EC [B]. At one region, its plasma membrane appeared fused with the membrane of an underlying green EC (arrow in [A]) suggesting that simultaneously with experiencing erythrogenic conversion, the cell was penetrating the endothelium of a vessel that already contained some non-circulating blood (as judged by irregular shapes of the erythrosomes). A smaller, forming vessel in [C] appeared to have converted one of its EC into erythroschizosome (red), which had then moved into the lumen, leaving a vacant space behind, and had already separated one monomer completely and another one partially. Fibroblasts and tumor cells are shown in yellow and brown, respectively. The cell in [D] represented an ambivalent (hemangioblastic) phenotype, similar to those seen five days after pseudo-orthotopic implantation5. However, here it had an increased level of phenotypic complexity, as manifested by the presence of caveolae (arrow), a hallmark of EC (green), and remodeling of the internal membranes covering the emerging erythrosome (red in upper left corner), in addition to collagen synthesis (yellow bundle of collagen fibers under the right end of the scale bar).\n\n\nReferences\n\nWeinhouse S: The Warburg hypothesis fifty years later. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 1976; 87(2): 115–126. 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}
|
[
{
"id": "732",
"date": "28 Jan 2013",
"name": "Ralph DeBerardinis",
"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 provide an interesting and detailed description of the ultrastructure of breast tumor spheroids ectopically implanted into immunocompromised mice. They describe numerous changes in mitochondrial and peroxisomal structure during the development of tumors in this model. Although the structural descriptions are interesting, there are limitations to the conclusions about metabolism that may be drawn from this type of analysis. For example, changes in mitochondrial appearance do not necessarily force an increase in the glycolytic rate, as suggested. Other conclusions drawn from the microscopy studies, including the presence of hypoxia at the level of single cells, and the origin of individual cells within weeks-old tumor structures, are not well validated.",
"responses": [
{
"c_id": "433",
"date": "10 Apr 2013",
"name": "Halina Witkiewicz",
"role": "Author Response",
"response": "A variety of metabolic pathways can be activated in response to hypoxia. However, glycolysis is the most likely one in cells undergoing erythrogenesis because the erythrosomes use it. The main point though is that the changes in mitochondrial structure do indicate impairment of their function – the oxidative phosphorylation, and therefore imply alternative pathways. The correlation between structural integrity of mitochondria and their functioning has been documented in the literature. One more example is PM:10717001. The ultrastructural analysis in situ is the best way to validate hypoxia at the level of single cells. The origin of individual cells was validated in the accompanying articles by immunocytochemical detection of tumor and graft cells harboring GFP coding sequences under histone H2B or ubiquitin promoter, respectively. Once integrated into the host genome the exogenous sequence was inherited by all progeny cells and expressed under control of the particular promoter. Therefore the time of tumor incubation in the chambers was irrelevant for the detection of the label. In this article the distinction between the two possible sources of HSCs was based on the location where the early stages of vasculature formation were seen - within or outside the encapsulated tumor nodule. While our conclusions based on those interesting images appeared correct to us, the readers may be inspired by the same images to formulate their own conclusions, and perhaps, build on them to develop and further test some alternative hypotheses. For the considerations on the significance of Warburg effect most important was the side by side co-existence of cells with variable metabolism, not their origin."
}
]
},
{
"id": "912",
"date": "29 Apr 2013",
"name": "Janusz Byczkowski",
"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 is part three in a series of three articles, which taken together postulate a novel and intriguing hypothetical mechanism of 'vasculature morphogenesis' in tumors involving formation of blood and vessels from local tissue stem cells. The reviewed manuscript, as written, is in some places difficult to read, as the authors tend to discuss several issues at once and often jump from subject to subject, generating sometimes a little bit of narrative chaos. Also, this manuscript may be difficult to comprehend without the context of the remaining two companion articles and quoted references. For this reason, I would suggest, that all three articles should have been published together. Moreover, another problem with this kind of functional morphology research is that the researchers have to present their observational study describing what they see, interpreting pictures qualitatively and deriving working hypotheses without translating visual fields into numbers and supporting statistics, which would be meaningless in this study, as there are no conflicting hypotheses to test, or control group to compare. Anyway, in this manuscript, the authors could more clearly separate their own results from extended discussions (especially in the “Results” section), and perhaps, could easily cut from their interpretations some embellishments, like for example “...The approach used here could facilitate integration of accumulated cyber knowledge on cancer metabolism into predictive science...” (quoted right from the Abstract). But besides its style, this manuscript is well documented and seems to be methodologically sound. It formulates and substantiates a very innovative hypothetical mechanism of vasculature morphogenesis in situ, including enhanced glycolysis in growing tumor tissue under aerobic conditions, seemingly due to cellular heterogeneity of tumors. The strength of this study lies in the use of in situ methods, involving, after Frost and Borgstrom (2003), the implantation of cultured breast tumor spheroids into the healthy mouse skin fold chamber, incubation for 1 or 3 weeks in vivo, followed by dissecting the tissue and processing it for electron microscopy. The electron microscopy was supported by immunocytochemistry, using antibodies to identify specific molecules in situ. Unfortunately, due to methodological constraints, the redox potential in the tissue microenvironment and the rate of respiration/energy production were only indirectly inferred from the structural changes in the mitochondria, but appropriately interpreted as indicative of their temporary functional disability which correlated with mitosis. On the other hand, to my knowledge, currently there is no ethical method for direct measurement of microenvironmental tissue oxygenation and/or ATP/ADP ratio in non-anesthetized animals in vivo. Thus, verification of the postulated hypothetical mechanism may need to wait until such a method will be developed. While the title seems to be appropriate for the content of the article, the abstract deserves a revision to more clearly describe the biological system investigated, the methods used, the results obtained and the hypothetical mechanism postulated. Even though the article is relatively well constructed, its clarity could be further improved by separating the results observed by the authors from interpretations based on reports by other researchers. In any event, the interpretations and the postulated mechanism are sensible and well documented.",
"responses": []
},
{
"id": "974",
"date": "30 May 2013",
"name": "Karolina Kucharova",
"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: “III. Cellular ultrastructures in situ as key to understanding tumor energy metabolism: biological significance of the Warburg effect” is the appropriate for the content of the article. The abstract seems longer than needed – I suggest focusing on the observed findings of the present study.The methods are well documented, except there is little information about the chamber and how cells were implanted into the chamber. In the results section, I suggest: Showing the mitochondrial profiles typical of the resting phase of the cell cycle (fragmented mitochondria) described in the results sectionAdding an enlarged figure of mitochondria which is converting into peroxisomes in the erythroblasts described in the legend of the figure 1., peroxisomes forming in the tumor cells described or showed in the figures 1, 2.Showing the mini-chondria and eu-chondria described in the results section",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-10
|
https://f1000research.com/articles/2-7/v1
|
10 Jan 13
|
{
"type": "Short Research Article",
"title": "High efficiency generalized transduction in Escherichia coli O157:H7",
"authors": [
"Martin G Marinus",
"Anthony R Poteete",
"Anthony R Poteete"
],
"abstract": "Genetic manipulation in enterohemorrhagic E. coli O157:H7 is currently restricted to recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changes inter alia into the genome. Bacteriophage 933W is a prophage in E. coli O157:H7 strain EDL933, which encodes the genes (stx2AB) for the production of Shiga toxin which is the basis for the potentially fatal Hemolytic Uremic Syndrome in infected humans. We replaced the stx2AB genes with a kanamycin cassette using recombineering. After induction of the prophage by ultra-violet light, we found that bacteriophage lysates were capable of transducing to wildtype, point mutations in the lactose, arabinose and maltose genes. The lysates could also transduce tetracycline resistant cassettes. Bacteriophage 933W is also efficient at transducing markers in E. coli K-12. Co-transduction experiments indicated that the maximal amount of transferred DNA was likely the size of the bacteriophage genome, 61 kB. All tested transductants, in both E. coli K-12 and O157:H7, were kanamycin-sensitive indicating that the transducing particles contained host DNA.",
"keywords": [
"Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is among the leading causes of food- and water-borne illnesses affecting humans in the U.S.",
"Europe",
"and Japan (for review",
"see Donnenberg and Whittam (2001)1",
"Kaper et al. (2004)2 and Spears et al. (2006)3). These bacteria are highly infectious and ingestion of only 100–200 organisms is sufficient to trigger debilitating diarrheal disease. Although most EHEC infections resolve spontaneously after 5–10 days of abdominal cramping and bloody diarrhea",
"approximately 2–7% of cases progress to the potentially fatal hemolytic uremic syndrome due",
"in part",
"to the production of cytotoxic Shiga toxins which are capable of promoting kidney failure (Donnenberg and Whittam (2001)1",
"Kaper et al. (2004)2 and Spears et al. (2006)3). The genes for Shiga toxin (stx2AB) are located on a prophage in the late region between genes Q and S and are transcribed with the late region genes from promoter PR’ (Tyler et al. (2004)4)."
],
"content": "Introduction\n\nEnterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is among the leading causes of food- and water-borne illnesses affecting humans in the U.S., Europe, and Japan (for review, see Donnenberg and Whittam (2001)1, Kaper et al. (2004)2 and Spears et al. (2006)3). These bacteria are highly infectious and ingestion of only 100–200 organisms is sufficient to trigger debilitating diarrheal disease. Although most EHEC infections resolve spontaneously after 5–10 days of abdominal cramping and bloody diarrhea, approximately 2–7% of cases progress to the potentially fatal hemolytic uremic syndrome due, in part, to the production of cytotoxic Shiga toxins which are capable of promoting kidney failure (Donnenberg and Whittam (2001)1, Kaper et al. (2004)2 and Spears et al. (2006)3). The genes for Shiga toxin (stx2AB) are located on a prophage in the late region between genes Q and S and are transcribed with the late region genes from promoter PR’ (Tyler et al. (2004)4).\n\nThe most widely used genetic manipulation in EHEC is recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changes inter alia into the genome (Murphy (1998)5, Court et al. (2002)6). A method for using the generalized transducing bacteriophage P1 in EHEC has been reported previously but it requires the construction of galactose mutant derivatives of the recipient strain (Ho and Waldor (2007)7).\n\nIn EHEC strain EDL933, the stx2AB genes are located on prophage 933W, the genome size of which is 61 kB (Penna et al. (2001)8). The DNA sequence of both the prophage and free phage are known. During sequencing of the free phage genome, it was noticed that the ends of the genome could not be sequenced; the investigators interpreted this and other observations as indicating that the packaged chromosome might be terminally redundant (Plunkett et al. (2001)9). Because terminal redundancy is a shared feature among generalized transducing phages, this finding suggested that bacteriophage 933W might be capable of packaging host DNA and transducing genetic markers. We report here that bacteriophage 933W is capable of transducing genetic markers in unmodified EHEC and E. coli K-12 strains.\n\n\nMethods\n\nThe EHEC strain used principally in this paper is KM80 (Carone et al. unpublished document), a derivative of EDL933 which lacks prophage 933W (obtained from Dr. K.C. Murphy). Derivatives of KM80 unable to utilize lactose (GM9289), arabinose (GM9290) or maltose (GM9291) were obtained after a 20 min exposure of a log phase culture in L broth to 40 µl ethyl methane sulfonate (Sigma-Aldrich) per ml at 37°C followed by a 1/100 dilution into broth. Aliquots of the saturated culture were plated on MacConkey Agar Base (Difco) containing lactose, arabinose or maltose to detect non-fermenting colonies. The E. coli K-12 strains used were AB1157 (thr-1 araC14 leuB6(Am)Δ (gpt-proA)62 lacY1 tsx-33 supE44(AS) galK2(Oc) hisG4(Oc) rfbD1 mgl-51 rpoS396(Am) rpsL31(StrR) kdgK51 xylA5 mtl-1 argE3(Oc) thi-1) (Dewitt and Adelberg (1962)10) (obtained from Dr. E.A. Adelberg), MM294 (endA1 hsdR17 supE44(AS) rfbD1 spoT1 thi-1) (Meselson and Yuan (1968)11) (obtained from Dr. M. Meselson), GM1731 (cysG98::Tn5) (Shaw and Berg (1979)12) and GM1748 (leu::Tn10) (Kleckner et al. (1975)13). (Am=amber, AS=amber suppressor, Oc=ochre). Strains containing the Tn5 and Tn10 mutations were obtained from Drs. C. Berg and N. Kleckner respectively. The composition of L broth and minimal medium has been described previously (Nowosielska and Marinus (2008)14).\n\nReplacement of the stx2AB genes in EDL933 by recombineering as described by Murphy and Campellone (2003)15 was carried out to yield strain GM9251. In a polymerase chain reaction containing Herculase II DNA polymerase Agilent Technologies), supplied buffer, 2 µl GM1731, and primers. ggtctggtgctgattacttcagccaaaaggaacacctgtatatgTATGGACAGCAAGCGAACCG and gattacacttgttacccacataccacgaatcaggttatgcctcaTCAGAAGAACTCGTCAAGAAG (sequence in capital letters is of Tn5), a DNA fragment containing the kanamycin-resistance gene was synthesized. This fragment was electroporated into EDL933 containing pKM208, a thermosensitive plasmid bearing the bacteriophage lambda exo and bet genes (Murphy and Camellone (2003)15) followed by selection for kanamycin-resistant clones. These were tested further by the ability of the cells to produce infective centers on strain AB1157 (Dewitt and Adelberg (1962)10) and one of these was designated GM9251.\n\nProphage induction was accomplished by plating 10 µl of a standing overnight culture on solid medium and irradiating with 25 J/m2 ultraviolet light followed by an overlay of 3.5 ml soft agar (0.3%) containing 300 µl of a standing overnight culture of the desired strain for bacteriophage propagation. Alternatively, the desired strain was mixed with dilutions of bacteriophage lysate at 37°C, incubated for 30 min, and incorporated in a soft agar layer on solid medium. In both induction and bacteriophage propagation, the plates were incubated at 37°C overnight. The next day, the soft agar layer was scraped off, and 4 ml of L broth or TM buffer (10 mM Tris-HCl, pH7.4, 10 mM MgSO4) was added together with 0.15 ml chloroform. The mixture was vortexed and left at room temperature for 10 min followed by centrifugation in a microfuge at room temperature for 2 min at 10,000 rpm. The supernatant was either used immediately or mixed with an equal volume of 4 M ammonium sulfate. The bacteriophage suspension in ammonium sulfate is stable for at least one month at 4°C in the dark. During lysate preparation it is crucial that the temperature remain at or above 20°C. Lysates were titered on AB1157.\n\nFor transduction, the recipient strain was grown standing overnight in liquid medium at 37°C, centrifuged, and resuspended in fresh medium. The recipient strain (100 µl) was mixed with varying amounts of bacteriophage lysate and 400 µl fresh medium. For selection using auxotrophic markers, the mixture was spread on selective minimal medium without further incubation. For drug-resistance markers, the mixture was incubated at 37°C for 60 min before plating on antibiotic medium. The plates were incubated at 37°C.\n\nA 933W lysogenic derivative of E. coli K-12 was constructed as follows. Dilutions of a GM9251 induced lysate were mixed with a standing overnight L broth culture of strain MM294 and incubated at 37ºC for 60 min and portions added to L broth-kanamycin plates and incubating them overnight at 37ºC. Colonies were picked into L broth supplemented with kanamycin and grown overnight at 37ºC. The broth cultures were diluted and 10 µl portions placed onto a soft agar (0.3%) layer containing 200 µl of a standing overnight culture of AB1157 on an L broth plate. After allowing the drops to dry the plate was incubated overnight at 37ºC. A kanamycin-resistant lysogen that formed infective centers by this method was designated GM9255 and 933W phage induced from this lysogen was used in all experiments with E. coli K-12.\n\n\nResults and discussion\n\nInitial experiments with bacteriophage 933W stx2AB::Kan were confounded by its instability. It was found that the bacteriophage was cold-sensitive and would not form plaques on indicator strains at 30°C but could do so at 37°C. The lysates rapidly lost infective titer (>90%) upon incubation at 4°C overnight. The addition of 2 M ammonium sulfate to the lysates stabilized the titer.\n\nThe observation that the bacteriophage 933W chromosome had terminal redundancy suggested that, like bacteriophage P1, it should be capable of transducing genetic markers. Strain KM80, which is derived from EDL933 and is not lysogenic for 933W, was mutagenized with ethyl methane sulfonate and the lactose, arabinose and maltose non-fermenting derivatives were isolated. These derivatives were used as recipients for transduction and, after mixing with bacteriophage from strain GM9251, were plated on minimal medium containing the appropriate carbon source. The results in Table 1 show that colonies were present on the plates with the mixture but not on plates with the recipient alone or the bacteriophage alone. Forty recombinants from each transduction were patched onto solid medium with kanamycin and all were sensitive.\n\nLac- (GM9289), Ara- (GM9290) and Mal- (GM9291) derivatives of KM80 were transduced with a lysate prepared on GM9251. The number of transdcutants on each of three plates is shown followed by the average. There were no colonies on plates seeded with the recipient strain alone or from the GM9251 lysate.\n\nA 933W stx2AB::Kan lysogen (GM9255) of E. coli K-12 strain MM294 was constructed and a lysate prepared on E. coli K-12 strain GM1748 (leu::Tn10). This lysate was used to transduce strain AB1157 (Thr- Ara-) to tetracycline-resistance (due to the Tn10). Of the 40 recombinants tested, 100% were Ara+ and 100% Thr-. Given that the gene order is thr-ara-leu and that the intervals between thr and leu, and ara and leu, are 80 and 14 kB respectively, the result indicates that cotransduction is possible for markers separated by 14 kB, but not 80 kB. This result is consistent with the 933W bacteriophage genome size of 61 kB. None of the 40 tetracycline-resistant recombinants were kanamycin-resistant, consistent with the transducing particles containing only host DNA.\n\nBacteriophage 933W is a member of a family of stx-encoding bacteriophages and these may also be capable of transduction. The annotation of a terminase in the 933W genome should be revised to a pacase to more clearly reflect the action of these enzymes. The ability of bacteriophage 933W to transduce genetic markers in EHEC and E. coli K-12 suggests that other related bacteria such as enteropathogenic E. coli or Citrobacter rhodentium may also be amenable to this type of genetic exchange. Only 933W non-lysogens have been used in the present study as recipients and it is not yet known if 933W lysogens can be transduced.\n\n\nConclusion\n\nBacteriophage 933W is capable of transducing genetic markers in EHEC and E. coli K-12.",
"appendix": "Author contributions\n\n\n\nMGM and AP conceived the study, designed and carried out the research and agreed to the final content, and wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nDonnenberg MS, Whittam TS: Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J Clin Invest. 2001; 107(5): 539–548. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKaper JB, Nataro JP, Mobley HL: Pathogenic Escherichia coli. Nat Rev Microbiol. 2004; 2(2): 123–140. PubMed Abstract | Publisher Full Text\n\nSpears KJ, Roe AJ, Gally DL: A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis. FEMS Microbiol Lett. 2006; 255(2): 187–202. PubMed Abstract | Publisher Full Text\n\nTyler JS, Mills MJ, Friedman DI: The operator and early promoter region of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin expression. J Bacteriol. 2004; 186(22): 7670–7679. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurphy KC: Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol. 1998; 180(8): 2063–2071. PubMed Abstract | Free Full Text\n\nCourt DL, Sawitzke JA, Thomason LC: Genetic engineering using homologous recombination. Annu Rev Genet. 2002; 36: 361–388. PubMed Abstract | Publisher Full Text\n\nHo TD, Waldor MK: Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization. Infect Immun. 2007; 75(4): 1661–1666. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPerna NT, Plunkett G III, Burland V, et al.: Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature. 2001; 409(6819): 529–533. PubMed Abstract | Publisher Full Text\n\nPlunkett G III, Rose DJ, Durfee TJ, et al.: Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product. J Bacteriol. 1999; 181(6): 1767–1778. PubMed Abstract | Free Full Text\n\nDewitt SK, Adelberg EA: The Occurrence of a Genetic Transposition in a Strain of Escherichia Coli. Genetics. 1962; 47(5): 577–585. PubMed Abstract | Free Full Text\n\nMeselson M, Yuan R: DNA restriction enzyme from E. coli. Nature. 1968; 217(5134): 1110–1114. PubMed Abstract\n\nShaw KJ, Berg CM: Escherichia coli K-12 auxotrophs induced by insertion of the transposable element Tn5. Genetics. 1979; 92(3): 741–747. PubMed Abstract | Free Full Text\n\nKleckner N, Chan RK, Tye BK, et al.: Mutagenesis by insertion of a drug-resistance element carrying an inverted repetition. J Mol Biol. 1975; 97(4): 561–575. PubMed Abstract\n\nNowosielska A, Marinus MG: DNA mismatch repair-induced double-strand breaks. DNA Repair. 2008; 7(1): 48–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurphy KC, Campellone KG: Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol. 2003; 4: 11. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "705",
"date": "14 Jan 2013",
"name": "Joe Wade",
"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 describe a new transduction system for E. coli O157:H7, a strain that cannot be transduced using P1. Such a transducing phage would be a useful tool. The authors make a fairly convincing case that they have indeed isolated a transducing phage. However, there are some important, missing controls, and the efficiency of co-transduction of linked genes needs to be discussed in greater detail.Specific comments:1. Given that the impact of the transducing phage is likely to be limited to O157:H7 strains (i.e. not K-12), it is important to demonstrate transduction in O157:H7 more convincingly. Specifically, the authors should identify the specific mutation in the recipient strain and then show that this mutation is lost in the transductants. This will rule out spontaneous mutations as the cause of the phenotype. Alternatively, and perhaps more easily, the authors could attempt to transduce an antibiotic resistance gene, as they did for K-12.2. The authors show that co-transduction occurs in K-12 between genes 14 kb apart. The frequency of co-transduction is 100% for 40 colonies tested. This is higher than I would have expected. Assuming random acquisition of host DNA by the phage, you would expect 8 or 9 colonies, on average, in which the two genes did not co-transduce. The probability of getting 40 out of 40 co-transductants would be 3e-5. This suggests that the phage does not package host DNA randomly. The authors should discuss the significance of this, especially in light of the utility of the phage for generalised transduction.",
"responses": [
{
"c_id": "452",
"date": "07 May 2013",
"name": "Martin Marinus",
"role": "Author Response F1000Research Advisory Board Member",
"response": "1. There were never any colonies on the control plates for the O157:H7 transductions for three different markers so we consider the possibility that the transductants are spontaneous revertants to be very low.2. We agree with the reviewer on this point and it is very possible that the DNA is not packaged randomly."
}
]
},
{
"id": "707",
"date": "16 Jan 2013",
"name": "Donald Court",
"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 methods used for E. coli O157:H7 (EHEC) to prepare phage lysates and to use those lysates for generalized transduction of bacterial markers, which is useful since P1 transduction in this strain requires the presence of a special gal mutant in any EHEC recipient. The general phage methods described are also useful. I have a few minor comments below to improve the paper.- The authors point out that the kanR phage marker does not come along during the transduction event. See the last sentence of the abstract and the last sentence in the second paragraph of results. Both should say something to the effect that this indicates phage DNA is not involved instead of arguing that host DNA is. This is pretty obvious but I think the question is more whether there is some kind of host DNA pick into a phage genome.- It would be informative if the authors can provide a frequency of generalized transductants per phage particle (as well as per 10 microliters). What is the frequency of lysogeniziation by this phage as measured by KanR? How does this transduction compare to a similar phage like P22.- How frequently does the tet marker transduce the EHEC strain?I mark this as approved for publication with some minor revisions as suggested.",
"responses": []
},
{
"id": "716",
"date": "21 Jan 2013",
"name": "Michael Donnenberg",
"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 findings are novel and of potential interest in that generalized transduction, if confirmed in other pathogenic E. coli strains, could facilitate more rapid progress in understanding these infections. Such confirmation is necessary before its full impact can be appreciated.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-7
|
https://f1000research.com/articles/2-6/v1
|
10 Jan 13
|
{
"type": "Research Article",
"title": "The novel Arabidopsis thaliana svt2 suppressor of the ascorbic acid-deficient mutant vtc1-1 exhibits phenotypic and genotypic instability",
"authors": [
"Chase F Kempinski",
"Samuel V Crowell",
"Caleb Smeeth",
"Carina Barth",
"Chase F Kempinski",
"Samuel V Crowell",
"Caleb Smeeth"
],
"abstract": "Ascorbic acid is a potent antioxidant that detoxifies reactive oxygen species when plants are exposed to unfavorable environmental conditions. In addition to its antioxidant properties, ascorbic acid and its biosynthetic precursors fulfill a variety of other physiological and molecular functions. A mutation in the ascorbic acid biosynthesis gene VTC1, which encodes GDP-mannose pyrophosphorylase, results in conditional root growth inhibition in the presence of ammonium. To isolate suppressors of vtc1-1, which is in the Arabidopsis Columbia-0 background, seeds of the mutant were subjected to ethyl methanesulfonate mutagenesis. A suppressor mutant of vtc1-1 2, svt2, with wild-type levels of ascorbic acid and root growth similar to the wild type in the presence of ammonium was isolated. Interestingly, svt2 has Arabidopsis Landsberg erecta features, although svt2 is delayed in flowering and has an enlarged morphology. Moreover, the svt2 genotype shares similarities with Ler polymorphism markers and sequences, despite the fact that the mutant derived from mutagenesis of Col-0 vtc1-1 seed. We provide evidence that svt2 is not an artifact of the experiment, a contamination of Ler seed, or a result of outcrossing of the svt2 mutant with Ler pollen. Instead, our results show that svt2 exhibits transgenerational genotypic and phenotypic instability, which is manifested in a fraction of svt2 progeny, producing revertants that have Col-like phenotypic and genotypic characteristics. Some of those Col-like revertants then revert back to svt2-like plants in the subsequent generation. Our findings have important implications for undiscovered phenomena in transmitting genetic information in addition to the Mendelian laws of inheritance. Our results suggest that stress can trigger a genome restoration mechanism that could be advantageous for plants to survive environmental changes for which the ancestral genes were better adapted.",
"keywords": [
"l-ascorbic acid (AA",
"vitamin C) is an important antioxidant with multiple functions in many species. It serves as a scavenger of reactive oxygen species generated under adverse environmental conditions. However",
"AA also influences flowering time and senescence1–3",
"pathogen disease resistance2",
"4",
"the biosynthesis of various plant hormones5–7",
"and root development8–11. This suggests that AA and some of its intermediates have functions in addition to its antioxidant properties."
],
"content": "Introduction\n\nl-ascorbic acid (AA, vitamin C) is an important antioxidant with multiple functions in many species. It serves as a scavenger of reactive oxygen species generated under adverse environmental conditions. However, AA also influences flowering time and senescence1–3, pathogen disease resistance2,4, the biosynthesis of various plant hormones5–7, and root development8–11. This suggests that AA and some of its intermediates have functions in addition to its antioxidant properties.\n\nAscorbic acid biosynthesis in plants occurs predominantly through the d-mannose/l-galactose pathway12,13. Given the multifaceted functions of AA in plants, there is a need to advance our understanding of how plants regulate the biosynthesis and accumulation of AA. Arabidopsis thaliana mutants deficient in AA have provided important insights into the breadth of molecular and physiological functions of AA. One of the Arabidopsis mutants, vtc1-1, contains a defect in the AA biosynthetic enzyme GDP-mannose pyrophosphorylase. The mutant was originally generated by ethyl methanesulfonate (EMS) mutagenesis of Col-0 wild-type seed14. The vtc1-1 mutant contains a point mutation in amino acid 22 that converts a conserved proline into a serine15. The VTC1 gene has recently been shown to be a determinant of ammonium sensitivity in plants. In the presence of ammonium, vtc1-1 mutants exhibit strongly reduced root growth in comparison to the wild type, a phenomenon that is independent of AA deficiency8–11. To better understand the mechanism through which VTC1 mediates conditional ammonium sensitivity, it is important to identify regulatory partners of VTC1. To accomplish this, we undertook a suppressor mutagenesis approach of vtc1-1 homozygous mutant seed in the hope of identifying vtc1-1 suppressor mutants that could then be isolated and studied.\n\nOne of the suppressor mutants isolated in the M0 generation, svt2 (suppressor of vtc1-1 2), contained wild-type AA levels and developed roots similar to the wild type in the presence of ammonium. However, while characterizing the mutant genotypically, we observed that it lost the original vtc1-1 mutation (i.e., svt2 contained the homozygous wild-type allele). Furthermore, we determined that svt2, although generated through EMS mutagenesis of Col-0 vtc1-1 mutant seed, was phenotypically and genotypically similar to Ler. Intriguingly, a small percentage of svt2 M1 plants produced offspring that have phenotypic and genotypic similarities to Col in the M2 generation. Even more remarkably, a small percentage of Col-like revertants in the M2 generation produced progeny that exhibited phenotypic and genotypic svt2 characteristics again in the M3 generation.\n\nPhenotypic instability of Arabidopsis alleles affecting a disease resistance gene cluster has recently been reported16. In their work, Yi and Richards described that exposure to EMS or through the generation of different F1 hybrids induced phenotypic instability in the bal and cpr1 mutant alleles. The authors later proposed that the high phenotypic instability is caused by a genetic mechanism17.\n\nThe presented study focuses on describing and characterizing the Arabidopsis svt2 suppressor mutant and its phenotypic and genotypic behavior. After illustrating the phenotypic features of svt2, we investigate transgenerational changes in the phenome and genome of svt2 and provide evidence that svt2 is a true mutant and not the result of an experimental artifact or contamination. Finally, we discuss our experimental findings in respect to the vtc1-1 mutant background and other reports that previously described similar phenomena of genome instability and restoration, and we briefly speculate on possible mechanisms of phenome and genome instability in svt2.\n\n\nMaterials and methods\n\nArabidopsis thaliana Col-0 wild type and the previously described vtc1-1 mutant14 (in the Col-0 background) were kindly provided by Patricia Conklin (SUNY Cortland, NY, USA). Ler-0 wild-type seed were obtained from The Arabidopsis Biological Resource Center (http://www.arabidopsis.org). Plants were grown in Metromix 360 potting soil at 23°C at both day and night with a 16-hour photoperiod at 160 μmol photons m-2 s-2 (fluorescent bulbs).\n\nFor assessment of root growth, seed of the wild types and mutant lines were surface-sterilized (see below) and grown on basal full strength 1× Murashige and Skoog (MS) medium without vitamins (Cat.# MSP01, Caisson Laboratories, Inc., North Logan, UT), containing 1% Phytoblend (Cat.# PTP01, Caisson Laboratories) in omni trays (Fisher Scientific, Pittsburgh, PA) as described11. Sucrose was omitted from the tissue culture medium. The pH of the medium was adjusted with KOH to 5.7. Trays were sealed with two layers of 3M micropore tape (Fisher Scientific), put in vertical orientation, and placed in the growth chamber under long days (16 h light, 8 h dark) at 23°C day and night, and 160 µmol photons m-2 s-1 in a growth chamber (Percival Scientific, Inc., Perry, IA). Each plate contained wild-type and mutant seed. Primary root length was measured in seven-day-old seedlings using a ruler.\n\nTo assess AA content in leaf tissue, seeds of wild type and mutants were randomly sown on MetroMix 360 soil (BFG supplies Co., Burton, OH) in the same flat under the growth conditions described above. When plants were three weeks old, whole rosettes were harvested for the AA assay.\n\nSeeds were soaked for 1 min in 50% ethanol, followed by washing the seeds in 50% bleach plus 0.01% sodium dodecyl sulphate for 6 min. Finally, seeds were rinsed six times with sterile water and stored in 0.1% sterile Phytoblend agar for 2 d at 4°C18.\n\nSeeds of homozygous vtc1-1 Arabidopsis thaliana (Col-0 background) were mutagenized with 0.2% ethyl methanesulfonate as described (Figure 1;18). Approximately, 1200 M0 seed were stratified for 4 days at 4°C in 0.1% agar, sown on MetroMix soil and grown as above. Plants were screened for wild-type AA levels using the nitroblue tetrazolium assay19. Additional suppressor mutants were isolated by pooling seeds generated from M1 plants. Putative mutants were isolated and allowed to self-pollinate to obtain seed.\n\nTo isolate vtc1-1 suppressor mutants, homozygous vtc1-1 seed (in the Col-0 genetic background) were exposed to chemical mutagenesis using ethyl methanesulfonate (EMS). Over 1000 mutagenized seed (M0) were planted on soil and screened for wild-type levels of ascorbic acid. The only mutant isolated in the M0 generation containing recovered ascorbic acid levels was svt2. The mutant was allowed to self-fertilize and was characterized phenotypically and genotypically in subsequent generations.\n\nPollen was taken from 4.5-week-old flowering plants of Col-0 and Ler wild type and vtc1-1 and svt2 M2 mutants, mounted in glycerol, and photographed using bright field settings on a Nikon E800 microscope equipped with a CoolSNAP cf CCD camera (Photometrics, Tuscon, AZ, USA).\n\nGenomic DNA was isolated from rosette leaves following a previously described protocol3. In case of genomic DNA isolation from vtc1-1 seeds, a small amount of dried seeds was crushed and the extraction procedure described previously3 was followed. Primers for the VTC1 gene and for the Insertion/Deletion (InDel) polymorphisms were designed using sequence data available on The Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org). Polymerase chain reaction (PCR) was used to amplify fragments of the VTC1 gene for sequencing and to assess InDel polymorphisms. Sequences of primers used for sequencing and InDel analysis are summarized in Table 1. PCR reactions were run on 1.0% agarose gels stained with ethidium bromide.\n\nQuantitative PCR reactions were set up to measure gene copy number using 2.5 pmole gene-specific primers, 300 ng of genomic DNA diluted in DNase/RNase free water, and iQ SYBR Green supermix (Bio-Rad, Hercules, CA, USA) for a total volume of 10 μL. Reactions without template were used as negative controls. Each single copy reaction was set up in triplicate and run in a Bio-Rad iCycler for 40 cycles. Threshold cycles (CT) were calculated using iQ software (Bio-Rad).\n\nPrimer efficiencies (E) were calculated using cDNAs synthesized from RNA isolated from Col-0 plants as previously described11. cDNA samples were serially diluted across three orders of magnitude. Serial dilutions were amplified in triplicate using the same protocol as for the copy number experiment. The CTs of each triplicate were averaged and plotted against the dilution factor. A linear trend was fitted to the data and the slope of this trend was used to calculate E for each primer with the formula: E=10(1/-slope).\n\nCopy number of VTC1 (AT2G39770) was calculated using the formula: Reported Quantity (RQ) = 1/ECT normalized to the RQ of a known single copy gene (PAD4, AT3G52430;20,21). VTC1 RQ was calculated from the average VTC1 RQ of three biological replicates per genotype and was normalized to the average RQ of PAD4 from three replicates of each respective genotype, all run in the same reaction plate.\n\nPCR products were purified using the Qiagen Miniprep Kit. Dye-terminator based DNA sequencing was performed at the Genomics Facility in the Department of Biology at West Virginia University. Sequence alignments were performed using the BioEdit program (http://www.mbio.ncsu.edu/bioedit/bioedit.html).\n\nTo screen mutants, AA levels were analyzed qualitatively in small pieces of two-week-old rosette leaves using the nitroblue tetrazolium assay previously described19. The AA content was determined in whole rosettes of three-week-old plants using the iron reduction assay4.\n\nExperiments were performed at least three times. Figures represent individual experiments. Data were expressed as mean values ± SE. P values were determined by Student’s t test analysis.\n\n\nResults\n\nOur laboratory is interested in understanding how the VTC1 gene, which is essential for the biosynthesis of GDP-mannose and AA, is regulated. This would help deciphering the pleiotropic phenotypes displayed by vtc1-1, including its hypersensitivity to ammonium8–11. We employed a gene suppressor analysis with the goal of identifying novel genes that interact or regulate VTC1. Seed of the vtc1-1 mutant, which is in the Col-0 genetic background14, were subjected to chemical mutagenesis using EMS18. One thousand and one hundred mutagenized vtc1-1 seeds (M0 generation) were planted onto soil and screened for recovered (wild-type) leaf AA content using the qualitative nitroblue tetrazolium test19. One of the mutants exhibited wild-type AA levels in the M0 generation. This mutant was named svt2 (suppressor of vtc1-1 2), isolated, and further characterized. The mutant was allowed to self-fertilize and seeds from the plant were collected (M1 generation) (Figure 1). Note that we isolated additional suppressor mutants by pooling M2 seed and by screening for long roots on 1× Murashige and Skoog (MS) medium containing ammonium. Six suppressor mutants were identified among 2000 plants. M3 seed were collected and screened for long roots again to test for segregation. M4 progeny of one line had all long roots, whereas the other five lines segregated in a ratio of three plants producing long roots, and one plant having short roots. Figure 2 summarizes data of four of these suppressor mutants, with D3–4 homogenously producing long roots, whereas D3–3, D3–7, and D3–15 developed long and short roots in a 3:1 ratio. As is illustrated in Figure 2A, these suppressor mutants developed roots that were significantly longer than those of the Col-0 wild type. Analysis of the total AA content revealed that the suppressor D3–4 had an AA content comparable to the Col-0 wild type, whereas that of vtc1-1 was only approximately 40% of that of the wild type (Figure 2B)14,15. Finally, sequence analysis of these four suppressor mutants demonstrated a lack of the vtc1-1 mutation (Figure 2C). Except for the assessments described above, these suppressor mutants were not yet characterized further.\n\n(A) Root length in seven-day-old seedlings grown on 1× MS. Bars represent means ± SE of 18–73 individuals. Since D3-4 homogenously produced long roots, all individuals were included in the calculations. As D3-3, D3-7, and D3-15 developed long and short roots in an approximate 3:1 ratio, only individual seedlings that produced long roots were included in the calculations. (B) Total ascorbic acid content per gram fresh weight in whole rosettes of three-week-old plants. Bars represent means ± SE of three (Col-0 and vtc1-1) or 24 individual replicates. ***P < 0.001 by Student’s t-test indicates significant differences in comparison to the Col-0 wild type. (C) Sequences of the Col-0 wild type, the vtc1-1 mutant and four suppressor mutants. The arrow points to the vtc1-1 mutation, a conversion of cytosine to a thymine.\n\nThe first observation we made when characterizing svt2 M1 plants was that svt2 exhibited a phenotype reminiscent of the Ler ecotype with the characteristic round leaves and erect morphology when compared to Col (Figure 3A). However, svt2 also had features that were distinct from the Ler phenotype, including overall enlarged vegetative and reproductive morphology (insets of rosettes and flowers in Figure 3A). In addition, svt2 was strongly delayed in flowering compared to the Col-0 and Ler-0 wild types and the vtc1-1 mutant (Figure 3A, 3B). Primary inflorescences in four-week-old plants were 1.4-times significantly longer in the vtc1-1 mutant and approximately twice as long in the Ler-0 wild type compared to the Col-0 wild type. In svt2 mutant plants, however, buds of primary inflorescences only began to emerge when plants were four weeks old (Figure 3A, 3B). The flowering data are consistent with previous reports, with Ler-0 wild type entering the reproductive phase before Col-0 wild type. An early flowering phenotype of vtc1-1 has been reported previously3.\n\n(A) Flowering phenotype of four-week-old Col-0 wild type, the vtc1-1 and svt2 mutants and the Ler-0 wild type. Insets show rosette phenotypes of the four genotypes when plants were three weeks old and the flower phenotype of six-week-old plants, respectively. (B) Primary inflorescence length when plants were four weeks old. Bars represent means ± SE of eight individual replicates. (C) Total ascorbic acid content per gram fresh weight in whole rosettes of three-week-old plants. Bars represent means ± SE of three individual replicates. (D) Root length in seven-day-old seedlings grown on 1× MS. Bars represent means ± SE of 30–90 individuals. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test indicate significant differences in comparison to Col-0 and Ler-0 wild type, respectively.\n\nThe AA content in svt2 was similar to levels quantified in Col-0 and Ler-0 wild types, whereas vtc1-1 contained approximately 30% of the AA content as expected14,15 (Figure 3C). Finally, we investigated whether svt2 also exhibits recovered root development in the presence of ammonium by growing the four genotypes in full strength 1× MS medium. The vtc1-1 mutant is conditionally hypersensitive to ammonium8,9,11. Figure 3D illustrates that root length in svt2 was the same as in Col-0 wild type, whereas root development was strongly inhibited in vtc1-1 as expected.\n\nThe enlarged morphology of svt2 raises the question as to whether svt2 is polyploid. In order to test this, we assessed the size of pollen grains from the Col-0 and Ler-0 wild-types and vtc1-1 and svt2 mutants. As is shown in Figure 4, pollen grains of the four genotypes are similar in size. In addition, using qPCR, we determined the number of VTC1 gene copies in the four genotypes. Our results revealed that VTC1 is present as a single copy gene in both the Col-0 and Ler-0 wild types and in the vtc1-1 and svt2 mutants (Table 2). Although an extensive chromosome analysis has not yet been performed in svt2, our results suggest that the mutant does not contain additional sets of chromosomes.\n\nQuantitative PCR was performed as described in Materials and Methods. The PAD4 gene is a known single copy gene. Therefore, an RQ/RQ ratio of approximately 1 indicates that VTC1 is present in similar quantity as PAD4, and therefore a single-copy gene.\n\nLight images were taken when plants were 4.5 weeks old. Scale bar represents 10 µm.\n\nTaken together, based on the phenotypic observations, our data suggest that svt2 represents a novel vtc1-1 suppressor mutant with recovered AA content and root development. Next, we characterized svt2 genotypically in order to determine whether svt2 represents an intragenic or extragenic suppressor.\n\nTo determine whether svt2 represents an intragenic suppressor, i.e., to test whether the suppressor mutation is present within the VTC1 gene, we designed nine overlapping PCR primer sets spanning the entire VTC1 gene and approximately 500 bp of the promoter region directly upstream of the first base in the 5’ UTR (Table 1, Figure 5A). PCR products were generated from genomic DNA extracted from Col-0 and Ler-0 wild types, and vtc1-1 and svt2 mutants. In eight of the nine primer pairs covering the entire VTC1 gene, the PCR products generated using svt2 genomic DNA had the same electrophoretic mobility as those generated using Col-0 wild-type genomic DNA. However, this was not the case for the first primer set. The G1F/G1R primer set, used to amplify the VTC1 promoter region, generated a larger PCR product in svt2 than in the wild type (Figure 5B, Figure 6). The PCR product in the wild type was 567 bp, whereas that in svt2 had a size of approximately 850 bp, suggesting that svt2 contained an approximately 300 bp insertion in this region. We repeated the PCR analysis of the VTC1 promoter region using the G1F-G1R and the G1F-G2R primer sets that should generate a PCR product of 567 bp and 751 bp, respectively (Figure 5A). The expected size was obtained for the Col-0 wild type and the vtc1-1 mutant. However, approximately 300 bp larger PCR products were detected in the svt2 mutant and the Ler-0 wild type (Figure 5B), suggesting a Ler insertion polymorphism. Thus, these data imply that svt2 shares both phenotypic and genotypic similarities with Ler.\n\n(A) VTC1 Col-0 gene model. Light green box indicates VTC1 gene promoter region, light blue rectangles indicate 5´ and 3´ UTRs, dark blue rectangles indicate exons, and lines indicate introns. Shown is the location of the vtc1-1 mutation within the first exon, primer locations, and polymorphism insertion of 283 bp in Ler-0 VTC1. (B) PCR amplification of the VTC1 promoter region in the Col-0 wild type, vtc1-1 and svt2 mutants and Ler-0 wild type. (-) indicates negative control, no DNA. (C) Partial sequence alignment of the VTC1 promoter region from the TAIR database (Col-0), sequenced Col-0 wild type, vtc1-1 and svt2 mutants, sequenced Ler-0 wild type and the Ler-0 sequence obtained from GenBank. The alignment shows the sequence insertion in the svt2 mutant, the Ler-0 wild type and the GenBank sequence. Arrows indicate single nucleotide polymorphisms between the Ler-0 and Col-0 sequence. (D) Point mutation in vtc1-1, a conversion from a cytosine to a thymine.\n\nAmplification of the VTC1 gene including ~500 bp of the promoter region using a series of nine, overlapping primers (G1F+R through G9F+R) in both Col-0 wild type and svt2 M1 mutant genomic DNA. The last lane in each gel contained a negative control (water instead of DNA). Red arrows indicate the different sized PCR products using the same primer set.\n\nWe therefore assessed five additional Insertion/Deletion (InDel) polymorphisms randomly chosen across the five Arabidopsis chromosomes (Table 1) in svt2 compared to the Col-0 and Ler-0 wild types and sequenced the entire VTC1 gene and the promoter region tested. Our data show that the PCR products generated for those five InDels using svt2 genomic DNA had the same electrophoretic mobility as those produced from Ler-0 genomic DNA (Figure 7). Moreover, sequence analysis of the VTC1 gene and promoter region revealed that svt2 contained a 283 bp insertion in the VTC1 promoter (Figure 5C). The insertion is highlighted in gray in Figure S1. Note additional single nucleotide polymorphisms as indicated by upright arrows in Figure 5C and Figure S1. When we aligned the VTC1 gene sequence obtained from svt2 with that of the vtc1-1 mutant, the VTC1 Col-0 gene sequence deposited in the TAIR database, and the VTC1 Ler GenBank sequence, the VTC1 gene sequence in svt2 shared similarities with Ler (upright arrows in Figure 5C, Figure S1) and Col (arrows pointing down in Figure S1). However, note that there are sequences that are unique to svt2 and are not shared between Col, vtc1-1 or Ler (arrowheads in Figure S1). Finally, note the overlap in sequences between Col, vtc1-1, svt2 and Ler on the 5´ end of the sequence flanking the insertion (at approximately base pair 1990); see left-facing horizontal black arrow in Figure S1 compared to the sequence flanking the 3´ end of the DNA sequence insertion (starting at base pair 2273); see right-facing horizontal black arrow in Figure S1.\n\nPrimers were designed for five randomly selected InDel polymorphisms across the five Arabidopsis chromosomes. The polymorphisms represent insertions in Col-0 and deletions in Ler.\n\nFinally, most intragenic suppressor mutants still contain the original mutation in addition to the suppressor mutation. Therefore, we expected that the vtc1-1 mutation is still present in svt2. However, our sequencing analysis demonstrated that svt2 did not contain the vtc1-1 mutation anymore and that the mutation reverted back to the homozygous wild-type allele (Figures 5D; green shading in Figure S1).\n\nIn summary, our data demonstrate that svt2 shares DNA sequence similarity with Col and Ler, but also contains DNA sequences that are unique to this mutant. This is particularly remarkable because svt2 was generated in the vtc1-1 Col-0 background. Also, svt2 did not contain the original vtc1-1 mutation anymore. Although our data already argue against svt2 being a result of an artifact of the experiment or a contamination with Ler, we analyzed subsequent svt2 generations and discovered additional characteristics that are unique to svt2.\n\nOur initial observations revealed that approximately 10% of svt2 M2 plants displayed a Col-like phenotype. Therefore, we planted svt2 M1, M2, and M3 progeny to check whether this result could be repeated and to determine segregation ratios (Table 3). Additionally, we investigated whether svt2 progeny that were phenotypically Col-like revertants would produce svt2 (Ler-like) offspring in the next generation.\n\nThe table summarizes the number of plants screened in each of three svt2 generations (M1, M2 and M3), screens of revertant progeny from Col-like revertants (A8, G7, K1), and the revertant progeny of a Ler-like line (K1 Col R svt2 R). The percent reversion is shown in the last column. Although the number of progeny plants tested is relatively large, some lines did not give rise to revertant progeny. R denotes revertant. *indicates mutant plants that were also analyzed genotypically (see Table 4).\n\nAs summarized in Table 3, revertants could only be detected when a relatively large population was planted. In the svt2 M1 generation, only 1% of Col-like revertants were detected. In contrast, 8–10% of svt2 M2 plants displayed a Col-like phenotype, whereas no revertants were detected in the svt2 M3 generation. These Col-like revertants were isolated and seeds were collected from individual plants and the phenotype of the progeny in the M3 generation was assessed in some examples. In most cases, reversion appeared to be stable, i.e., once svt2 plants reverted, displaying a Col-like phenotype in the M2 generation, their M3 progeny continued to appear as Col-like plants. This was the case for the M3 progeny of the A8 and G7 plants listed in Table 3. However, out of 63 progeny from the K1 revertant plant, one reverted back to a svt2-like phenotype (Table 3), i.e., the K1 double revertant switched from svt2 phenotype in the M1 generation to a Col-like phenotype in the M2 generation, and then reverted back to a svt2-like phenotype in the M3 generation. Note that only a small number of progeny was planted. In a second experiment, the svt2 Col R1 revertant produced 20 individuals displaying a svt2-like phenotype (Table 3). This represents a larger reversion percentage than in the K1 double revertant (22.7% vs. 1.6%). This may be explained by the genotypic make-up of the Col-like reverted parents and will be presented in the next section. Figure 8 illustrates the phenotypic appearance of three examples of svt2 → Col single revertants (Col R1, Col R2, K1 Col R) and a svt2 → Col → svt2 double revertant (K1 Col R svt2 R).\n\nPlants were three weeks old when photographs were taken. Top row represents controls, Col-0 wild type, vtc1-1 and svt2 mutants, and Ler-0 wild type. Bottom row represents three Col-like revertants, svt2 Col R1 M3, svt2 Col R2 M3, svt2 K1 Col R M3, and a double revertant, svt2 K1 Col R svt2 R M4. R stands for revertant.\n\nNext we tested whether a Col-like revertant phenotype correlated with a Col-like genotype. Likewise, we would expect that a svt2 → Col → svt2 double revertant phenotype corresponds with svt2-like genomic markers. To check this we isolated genomic DNA from Col-0 and Ler-0 wild types, svt2, vtc1-1 and revertant mutants, and PCR-amplified the five randomly selected InDel polymorphisms plus the InDel polymorphism in the VTC1 promoter (Table 1). In all cases but the svt2 M2 Col R1 revertant, the svt2-like revertant plants (labeled svt2 M2 Col revertants 1 through 5) produced PCR products that where of the same electrophoretic mobility as the PCR products generated using Col-0 wild-type genomic DNA. In contrast, svt2 M1 plants and svt2 M2 plants that displayed an svt2 phenotype, gave rise to PCR products that were of the same electrophoretic mobility as those of the Ler wild type (Table 4, Figure 9). In addition, the double revertant plant K1 (labeled svt2 M2 K1 Col R) was genotyped in both its M2 and M3 generations. The K1 plant produced InDel PCR products similar to those of the Col-0 wild type in the M2 generation. However, the M3 generation that displayed svt2-like morphology produced PCR products that were comparable to the InDel PCR products generated using Ler genomic DNA (Table 4). The svt2 M2 Col R1 (highlighted in red in Table 4 is intriguing, because it appears to contain DNA that is similar to both Col and Ler genomic DNA. This suggests the presence of chimeric genome sectors, which may explain the higher percentage of Col-like revertants compared to svt2 M2 K1 Col R. Note that the PCR results are in line with the sequencing analysis of the revertants. That is, Col-like revertants and svt2-like revertants share sequence similarity with Col-0 and Ler wild type, respectively (Figure S2).\n\nWith the exception of svt2 Col R1 M2, where Col and Ler markers and one heterozygous marker were found (highlighted in red), phenotype matched genotype. That is, a Col-like phenotype correlated with the presence of Col polymorphisms, while a Ler-like phenotype correlated with Ler polymorphisms. C, L, and H refer to Col, Ler, or heterozygous, respectively. R denotes revertant. n.d., not detected.\n\nPCR amplification of the Col/Ler VTC1 promoter polymorphism in svt2 plants and svt2 revertant (R) plants, amplified with the VTC1 G1F and G2R primers. (-) indicates negative control, no DNA.\n\nTaken together, these data suggest (i) transgenerational phenotypic and genotypic instability in svt2, and that (ii) svt2 offspring do not segregate in a Mendelian fashion. In an attempt to obtain first insights toward a mechanism that is causing this genotypic instability, we investigated whether transgenerational epigenetic inheritance could play a role.\n\nTo investigate whether genome instability is caused by transgenerational epigenetic inheritance in the svt2 mutant, we performed reciprocal crosses between svt2 mutants and Col-0 wild-type plants. It is possible that through the EMS mutagenesis of vtc1-1 seeds, genes involved in the regulation of epigenetic alterations were altered, whereby their activity was affected. There is increasing evidence in both plants and animals that epigenetic marks are not always cleared between generations. Incomplete erasure at genes associated with a measurable phenotype results in unusual patterns of inheritance from one generation to the next, termed transgenerational epigenetic inheritance22,23. Therefore, analysis of the progeny of the reciprocal crosses is expected to provide some first insights on the possibility of transgenerational epigenetic inheritance that is transmitted maternally. If this were the case, only progeny of crosses with a maternal svt2 donor should have a svt2-like phenotype. To determine the genotypes of the F1 progeny of the reciprocal crosses, we performed another InDel polymorphism assay as described above. In addition, progeny were also screened using the VTC1 InDel promoter polymorphism. Table 5 contains a summary of the InDel screen for progeny from each reciprocal cross. In all but six of the progeny from the reciprocal crosses, PCR products similar to those obtained using Col and Ler genomic DNA, respectively, were generated, suggesting that the F1 of the reciprocal crosses were heterozygous. A similar result was obtained for the VTC1 promoter polymorphism marker in all reciprocal crosses. Note, however, that for some polymorphisms and irrespective of whether svt2 or Col-0 served as female or male donor, respectively, PCR products comparable to those obtained using Ler-0 wild-type DNA were prevalent (highlighted in red in Table 5). This is surprising because heterozygosity was expected at all loci. This suggests that some parts of the genome were not inherited equally from both parents. Taken together, these results suggest that maternal epigenetic inheritance may not be the cause of genome instability in svt2. However, at some loci svt2-like alleles dominate over Col-0.\n\nMolecular analysis of the InDel polymorphism markers showed evidence of cryptic but persistent homozygosity, irrespective of the direction of the sexual cross (highlighted in red). However, heterozygosity was expected at all loci.\n\n\nDiscussion\n\nThe svt2 mutant was initially identified as a putative suppressor of the AA-deficient Arabidopsis mutant vtc1-1, as was evident in wild-type levels of AA (Figure 3C) and recovered root development in the presence of ammonium (Figure 3D). However, svt2 manifests other interesting characteristics, including genotypic and phenotypic instability. These unique features could aid in our understanding of the complex mechanisms controlling genome instability and restoration.\n\nSeveral lines of evidence support our findings that svt2 is a novel mutant. First, svt2 was the only suppressor mutant isolated among over 1000 EMS-mutagenized M0 seeds to show unique phenotypic characteristics. Astonishingly, our genetic analysis revealed that both maternal and paternal alleles were affected in five randomly selected InDel polymporphism loci, the newly discovered InDel polymporphism in the VTC1 promoter, and additional SNPs (Figure 5B–D, Figure 6, Figure S1). These data demonstrate that svt2 has acquired new characteristics, presumably as a result of EMS mutagenesis, and that svt2 is neither Col nor Ler. These data also argue against svt2 being an experimental or PCR artifact.\n\nSecond, a number of data provide strong arguments against seed contamination. (1) With high reproducibility, descendents of the original svt2 mutant produce offspring revertants with Col-like features (Table 3, Table 4; Figure 8, Figure 9). (2) One of the Col-like revertants, svt2 Col R1 M3, exhibited heterozygosity at some of the InDels tested (Table 4). (3) One of those Col-like revertants, svt2 K1 Col R M3, produced progeny that reverted back to svt2-like plants (Table 3, Table 4; Figure 8, Figure 9). (4) We were unable to obtain true F1 heterozygotes in all svt2/Col-0 reciprocal crosses (Table 5). (5) Delayed flowering and enlarged morphology phenotypes argue against the fact that svt2 is a result of a Ler-0 wild-type seed landing on the flat during the initial planting of the vtc1-1 M0 mutagenized population. There is the possibility of a Ler seed contamination of the vtc1-1 seed stock used for EMS mutagenesis. Although we have sequenced the vtc1-1 seed stock used for this experiment and confirmed that it is homozygous for the vtc1-1 mutation, one could argue that sequencing the seed stock may not be a sensitive enough method to rule out contamination with a few Ler seed. We performed many other experiments using this very same seed stock and never observed Ler-like plants among the vtc1 population. However, arguments (1) through (4) above speak most compellingly against seed contamination.\n\nThird, the following experimental evidence argues against the possibility that svt2 was generated by cross pollination of vtc1-1 mutant plants with Ler wild-type plants. (1) If svt2 were generated by Ler cross-pollination, the InDel polymorphism markers tested using svt2 genomic DNA should have indicated heterozygosity. This, however, was not the case (Table 4). (2) While svt2 shares phenotypic and genotypic characteristics with Ler and Col, it also has unique features (Figure 3A, Figure S1). (3) svt2 exhibits phenotypic and genotypic instability, causing the appearance of revertants with persistent reproducibility. (4) Ler plants were not grown in our growth chambers at the time of the mutagenesis experiment. Furthermore, svt2 was isolated by placing Aracons over the mutant plant to allow self-fertilization and seed production.\n\nOur results are indicative of genome instability in svt2. Genome instability may be a result of polyploidy24. Polyploids can arise from genome duplication (autopolyploids) or interspecific hybridization (allopolyploids). Our data suggest that svt2 does not contain multiple sets of chromosomes, because VTC1 occurs as a single copy gene in svt2 and vtc1-1 mutants as well as the Col-0 and Ler-0 wild-type controls (Table 2). Furthermore, extra DNA must be replicated with each cell division. Therefore, enlarged cell size is often associated with polyploids25. The chemical mutagenesis of vtc1-1 seed could have resulted in mutations, which may have led to increased ploidy levels in one, two, or all three meristem layers, L1, L2, and L3. However, only mutations in the L2 layer, which gives rise to the reproductive organs, are inherited. Polyploidy in the L2 layer is reflected in pollen size. While svt2 has an overall enlarged morphology (Figure 3A), its pollen size is comparable to that of the other three genotypes (Figure 4). This suggests that svt2 anthers are not polyploid. Finally, allopolyploids often display a greater degree of heterozygosity25, low fertility, and low embryonic viability26–28. This, however, is not the case in svt2. The fact that svt2 is fertile and that its enlarged morphology is heritable from one generation to the next suggests that svt2 is neither a somatic nor a gametic polyploid. Thus, it is therefore unlikely that polyploidy in svt2 contributes to genome instability. This is supported by Ruffio-Chable and co-workers, who reported that between 5% and 21% of F1 hybrids in Brassica oleracea showed aberrant leaf phenotypes, despite normal ploidy levels29.\n\nInstead, we hypothesize that genome instability of svt2 was further aggravated by exposing the already instable genome of vtc1-1 mutants to EMS. It has recently been shown that plants impaired in certain aspects of protection against reactive oxygen species have a higher incidence of spontaneous double-strand breaks30. The AA-deficient vtc1-1 mutant has a three-fold higher spontaneous homologous recombination frequency and has a higher incidence of double-strand breaks (see below). Similar results were reported for the Arabidopsis thaliana flavonoid-deficient mutants tt4 and tt530. One may speculate that through the high level of stress induced by EMS, a yet unknown mechanism of genome restoration was turned on. In fact, genome alterations in soybean and flax in response to environmental stress have been reported previously31,32. In the process of soybean cell culture, massive specific changes in numerous genome-wide loci were observed31. It was suggested that this genetic variation is a consequence of specific recombinational events. Similarly, in flax a single-copy 5.7 kilobase DNA fragment that was not present in the parent line appeared in genotrophs in response to particular growth conditions32.\n\nThe experimental evidence described in this work raises the question as to what mechanism is responsible for the loss or reintroduction of genomic DNA sequences in the original svt2 mutant and its revertant offspring. Several mechanisms may be considered: activity of transposable elements, random mutations, unequal crossing over, gene conversion, double-strand breaks and recombination, and activity of an RNA cache.\n\nTransposons are DNA elements capable of moving around the genome; movement is often associated with chromosome breaks and formation of unstable mutations, which revert frequently but often give rise to new phenotypes. Movement of transposable elements often occurs during meiosis and mitosis and is accelerated by genome damage33. These represent conditions that are present in svt2. However, transposons have a variety of molecular features that do not apply to svt2. Transposons exist as multiple copies in the genome. A blast search of the VTC1 promoter insertion in svt2 did not return any other hits, indicating that the DNA sequence is not present in its entirety anywhere else in the genome. Additionally, transposon termini represent inverted repeats. This, however, is not the case in svt2 (Figure S1). A short, direct repeat of genomic DNA often flanks the transposon, leaving a “footprint”. Our sequencing analysis of the VTC1 promoter region in svt2 did not reveal any footprints, suggesting that transposon activity is not responsible for the insertion or loss of novel sequences in svt2 (Figure S1).\n\nRandom mutations caused by EMS mutagenesis could have activated an unknown mechanism in vtc1-1 seeds, giving rise to the phenome and genome instability in svt2. This may explain the novel SNPs we detected in svt2 that are distinct from the vtc1-1 mutant and Col-0 and Ler-0 wild types (Figure S1). The disappearance of the vtc1-1 mutation in svt2 (Figure 5D, Figure S1) may also be explained by the introduction of a random mutation. However, it is possible that exposure of vtc1-1 seeds to EMS could have reversed the original vtc1-1 mutation to the wild-type sequence, as vtc1-1 was initially isolated in an EMS screen15. Interestingly, Conklin and co-workers previously isolated two vtc1 alleles, vtc1-1 and vtc1-2, containing the exact same single cytosine to thymine point mutation at amino acid position 64 relative to the start codon, despite the fact that vtc1-1 and vtc1-2 mutants were isolated independently from different EMS-mutagenized pools15. The authors suggested that a limited number of mutations are tolerable in the VTC1 enzyme GDP-d-mannose pyrophosphorylase without causing embryo lethality. This is supported by the fact that several independently isolated cyt mutant alleles containing different amino acid mutations in VTC1 are embryo lethal34. To date, only the vtc1-115 and hsn1 mutations8 have been isolated and reportedly do not cause embryo lethality. This suggests some form of allelic constraint that has been reported in Arabidopsis previously35,36. Furthermore, in the EMS screen in which the svt2 mutant was isolated, several other vtc1-1 suppressor mutants with restored root development in the presence of ammonium were identified. Sequencing analysis revealed that in all of these mutants the vtc1-1 mutation was restored to the wild-type allele, while the suppressor mutants neither exhibited a svt2-like phenotype nor did they produce revertants in the subsequent generation (Kempinski et al., unpublished data).\n\nExposure to EMS or γ–radiation has been reported to induce high frequency phenotypic instability in the Arabidopsis disease resistance genes CPR1 and BAL, which map to the RPP5 locus16. Yi and Richards reported destabilization of phenotypes in both the bal and cpr1 mutants in more than 10% of EMS-treated plants in the M1 generation. They also identified exceptions to simple Mendelian inheritance in the M2 generation. Phenotypic instability was also observed in bal × cpr1 F1 hybrids. The authors suggested that the high degree of phenotypic instability in bal and cpr1 mutants is due to the fact that the RPR5 locus can adopt different metastable genetic or epigenetic states, whose stability is highly susceptible to mutagenesis and pairing of different alleles. Yi and Richards later reported that the phenotypic instability of bal mutants is caused mainly by gene duplication and hypermutation of the SNC1 gene17.\n\nAs observed in the cpr1 and bal mutants, we hypothesize that EMS treatment has destabilized the genome of svt2 by interrupting one or more mechanisms involved in genomic inheritance. A combination of unequal crossing over, gene conversion, double-strand breaks, DNA recombination, and/or the presence of an RNA cache template may explain the loss and reappearance of DNA sequences in svt2. Genome-wide non-Medelian inheritance of extra-genomic information in Arabidopsis was reported in the hothead (hth) Arabidopsis mutant37. Self-fertilization of homozygous mutant plants resulted in approximately 10% hth revertants, which were hth/HTH heterozygous, suggesting that the HTH gene was altered in the progeny. However, the authors also detected rare homozygous revertants HTH/HTH embryos, which must have inherited one of their two wild-type HTH genes from the maternal parent and could not have been a result of outcrossing. Inheritable genome-wide high-frequency gene homozygosity in early generations in rice has also been reported38. Lolle et al. postulated that these genetic restoration events are the result of a template-directed process that utilizes an ancestral RNA-sequence cache37. This hypothesis is supported by observations reported by Xu and co-workers38. Therefore, our genetic and phenotypic svt2 data, in conjunction with the observed higher occurrence of double-strand breaks and spontaneous homologous recombination frequency in vtc1-1, are in support of the RNA cache theory. Additional studies are needed to provide experimental support for this hypothesis.\n\n\nConclusions\n\nWe have isolated a novel Arabidopsis mutant that is capable of restoring genetic information that was not present in the chromosomal genome of its parents. We suggest that this ancestral information is present in some cryptic form that is accessible under extreme stress conditions. Genome restoration could be advantageous to plants that encounter environmental changes for which ancestral genes were better adapted. However, the mechanisms responsible for triggering and executing genome restoration remain to be determined. Double strand breaks, DNA recombination, and/or the activity of an RNA cache may be contributing factors. In the future, svt2 may serve as a model to study non-Mendelian inheritance and could provide insight into the evolution and diversification of Arabidopsis ecotypes.\n\n\nAbbreviations\n\nAA, ascorbic acid; EMS, ethyl methanesulfonate; InDel, Insertion/Deletion; MS, Murashige and Skoog.",
"appendix": "Author contributions\n\n\n\nCB and CFK conceived the study and designed the experiments. CFK, SVC, CS and CB conducted the experiments and analyzed the data. CB and CFK 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 relevant competing interests disclosed.\n\n\nGrant information\n\nThis work was supported by a start-up grant of West Virginia University to CB.\n\n\nAcknowledgements\n\nWe would like to thank Dr. Patricia Conklin for providing vtc1-1 mutant and Col-0 wild-type seed. We also wish to thank Dr. Karen Weiler for allowing us to use her microscope and Dr. Rosana Schafer for providing a plate reader.\n\n\nSupplementary material\n\nFigure S1. Sequence alignment of the VTC1 gene sequence of the Col-0 TAIR database, the vtc1-1, svt2 mutants, and the Ler-0 GenBank database.\n\nHorizontal arrows denote 5´ respectively 3´ flanking regions of the sequence insertion, which is highlighted in grey, in the VTC1 promoter region (between base pairs 1990 and 2273). Upright arrows indicate sequences shared between svt2 and Ler. Arrows pointing down denote sequences shared between svt2 and Col. Arrowheads point to sequences unique to svt2. Highlighted in yellow are the start and stop codons, respectively. Highlighted in green is the vtc1-1 mutation.\n\nFigure S2. Sequence alignment of the VTC1 promoter InDel polymorphism sequence of the Col-0 TAIR database, the Ler-0 Genbank database, the svt2 K1 Col R M3 revertant (Col-like phenotype) and the svt2 K1 Col R svt2 R M4 revertant (svt2-like phenotype).\n\nHorizontal arrows denote 5´ respectively 3´ flanking regions of the sequence insertion, which is highlighted in grey, in the VTC1 promoter region in Ler-0 and svt2 K1 Col R svt2 R M4, which exhibits an svt2-like phenotype. The svt2 K1 Col R M3 mutant has a Col-like phenotype and share sequence similarities with the Col-0 sequence. R denotes revertant.\n\n\nReferences\n\nConklin PL, Barth C: Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant Cell Environ. 2004;27(8): 959–970. Publisher Full Text\n\nPavet V, Olmos E, Kiddle G, et al.: Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis. Plant Physiol. 2005; 139(3): 1291–1303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKotchoni SO, Larrimore KE, Mukherjee M, et al.: Alterations in the endogenous ascorbic acid content affect flowering time in Arabidopsis. Plant Physiol. 2009; 149(2): 803–815. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMukherjee M, Larrimore KE, Ahmed NJ, et al.: Ascorbic Acid Deficiency in Arabidopsis Induces Constitutive Priming That is Dependent on Hydrogen Peroxide, Salicylic Acid, and the NPR1 Gene. Mol Plant Microbe Interact. 2010; 23(3): 340–351. PubMed Abstract | Publisher Full Text\n\nArrigoni O, De Tullio MC: The role of ascorbic acid in cell metabolism: between gene-directed functions and unpredictable chemical reactions. J Plant Physiol. 2000; 157(6): 481–488. Publisher Full Text\n\nArrigoni O, De Tullio MC: Ascorbic acid: much more than just an antioxidant. Biochim Biophys Acta. 2002; 1569(1–3): 1–9. PubMed Abstract | Publisher Full Text\n\nPastori GM, Kiddle G, Antoniw J, et al.: Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling. Plant Cell. 2003; 15(4): 939–951. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQin C, Qian W, Wang W, et al.: GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in Arabidopsis thaliana. Proc Natl Acad Sci USA. 2008; 105(47): 18308–18313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarth C, Gouzd ZA, Steele HP, et al.: A mutation in GDP-mannose pyrophosphorylase causes conditional hypersensitivity to ammonium, resulting in Arabidopsis root growth inhibition, altered ammonium metabolism, and hormone homeostasis. J Exp Bot. 2010; 61(2): 379–394. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi Q, Li BH, Kronzucker HJ, et al.: Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity. Plant Cell Environ. 2010; 33(9): 1529–1542. PubMed Abstract | Publisher Full Text\n\nKempinski CF, Haffar R, Barth C, et al.: Toward the mechanism of NH4+ sensitivity mediated by Arabidopsis GDP-mannose pyrophosphorylase. Plant Cell Environ. 2011; 34(5): 847–858. PubMed Abstract | Publisher Full Text\n\nWheeler GL, Jones MA, Smirnoff N, et al.: The biosynthetic pathway of vitamin C in higher plants. Nature. 1998; 393(6683): 365–369. PubMed Abstract | Publisher Full Text\n\nDowdle J, Ishikawa T, Gatzek S, et al.: Two genes in Arabidopsis thaliana encoding GDP-l-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J. 2007; 52(4): 673–689. PubMed Abstract | Publisher Full Text\n\nConklin PL, Williams EH, Last RL, et al.: Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc Natl Acad Sci U S A. 1996; 93(18): 9970–9974. PubMed Abstract | Free Full Text\n\nConklin PL, Norris SR, Wheeler GL, et al.: Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci U S A. 1999; 96(7): 4198–4203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYi H, Richards EJ: Phenotypic instability of Arabidopsis alleles affecting a disease Resistance gene cluster. BMC Plant Biol. 2008; 8: 36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYi H, Richards EJ: Gene duplication and hypermutation of the pathogen Resistance gene SNC1 in the Arabidopsis bal variant. Genetics. 2009; 183(4): 1227–1234. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeigel D, Glazebrook J: Arabidopsis: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 2002, 354. Reference Source\n\nConklin PL, Saracco SA, Norris SR, et al.: Identification of ascorbic acid-deficient Arabidopsis thaliana mutants. Genetics. 2000; 154(2): 847–856. PubMed Abstract | Free Full Text\n\nDe Preter K, Speleman F, Combaret V, et al.: Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay. Mod Pathol. 2002; 15(2): 159–166. PubMed Abstract | Publisher Full Text\n\nDuarte JM, Wall PK, Edger PP, et al.: Identification of shared single copy nuclear genes in Arabidopsis, Populus, Vitis and Oryza and their phylogenetic utility across various taxonomic levels. BMC Evol Biol. 2010; 10: 61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMolinier J, Ries G, Zipfel C, et al.: Transgeneration memory of stress in plants. Nature. 2006; 442(7106): 1046–1049. PubMed Abstract | Publisher Full Text\n\nJablonka E, Raz G: Transgenerational epigenetic inheritance: prevalence, mechanisms, and implications for the study of heredity and evolution. Q Rev Biol. 2009; 84(2): 131–176. PubMed Abstract | Publisher Full Text\n\nWang Y, Jha AK, Chen R, et al.: Polyploidy-associated genomic instability in Arabidopsis thaliana. Genesis. 2010; 48(4): 254–263. PubMed Abstract | Publisher Full Text\n\nRanney TG: Polyploid: From Evolution to New Plant Development. Combined Proceedings International Plant Propagators’ Society. 2006; 56: 137–142. Reference Source\n\nSoltis DE, Soltis PS: The dynamic nature of polyploid genomes. Proc Natl Acad Sci U S A. 1995; 92(18): 8089–8091. PubMed Abstract | Publisher Full Text | Free Full Text\n\nComai L, Tyagi AP, Winter K, et al.: Phenotypic instability and rapid gene silencing in newly formed Arabidopsis allotetraploids. Plant Cell. 2000; 12(9): 1551–1568. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchranz ME, Osborn TC: Novel flowering time variation in the resynthesized polyploid Brassica napus. J Hered. 2000; 91(3): 242–246. PubMed Abstract | Publisher Full Text\n\nRuffio-Chable V, Chatelet P, Thomas G, et al.: Developmentally “aberrant” plants in F1 hybrids of Brassica oleracea. Acta Hort. 2000; 539: 89–94. Reference Source\n\nFilkowski J, Kovalchuk O, Kovalchuk I, et al.: Genome stability of vtc1, tt4, and tt5 Arabidopsis thaliana mutants impaired in protection against oxidative stress. Plant J. 2004; 38(1): 60–69. PubMed Abstract | Publisher Full Text\n\nRoth EJ, Frazier BL, Apuya NR, et al.: Genetic variation in an inbred plant: variation in tissue cultures of soybean [Glycine max (L.) Merrill]. Genetics. 1989; 121(2): 359–368. PubMed Abstract | Free Full Text\n\nChen Y, Lowenfeld R, Cullis CA, et al.: An environmentally induced adaptive (?) insertion event in flax. Int J Biochem Mol Biol. 2009; 1(3): 038–047. Reference Source\n\nLankenau DH, Volff JN: Transposons and the Dynamic Genome. Berlin, Heidelberg: Springer. 2009, 200. Reference Source\n\nLukowitz W, Nickle TC, Meinke DW, et al.: Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis. Proc Natl Acad Sci U S A. 2001; 98(5): 2262–2267. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi J, Last RL: The Arabidopsis thaliana trp5 mutant has a feedback-resistant anthranilate synthase and elevated soluble tryptophan. Plant Physiol. 1996; 110(1): 51–59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKreps JA, Ponappa T, Dong W, et al.: Molecular basis of alpha-methyltryptophan resistance in amt-1 a mutant of Arabidopsis thaliana with altered tryptophan metabolism. Plant Physiol. 1996; 110(4): 1159–1165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLolle SJ, Victor JL, Young JM, et al.: Genome-wide non-mendelian inheritance of extra-genomic information in Arabidopsis. Nature. 2005; 434(7032): 505–509. PubMed Abstract | Publisher Full Text\n\nXu PZ, Yuan S, Li Y, et al.: Genome-wide high-frequency non-Mendelian loss of heterozygosity in rice. Genome. 2007; 50(3): 297–302. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "730",
"date": "25 Jan 2013",
"name": "Andy Pereira",
"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\nVery interesting experimental evidence of an inheritance phenomenon that is non-Mendelian and supports an RNA cache hypothesis. The data support the conclusions drawn, but some alternative explanations are put forth that can be addressed.The EMS mutagenesis treatment of the vtc1-1 genotype yields a single suppressor svt2 mutant that turns out to be a revertant of the vtc1-1 mutation, and is homozygous. Since this screen was from ~1200 seed, it would be interesting to know if such revertant suppressor mutations are also be present in the original batch of vtc1-1 seed used for mutagenesis. Of course since the screen entails a tedious test of TTB on leaves of individual plants for AA content, it is not a recommended control test that should be done, but mechanistically the question remains if the locus is mutable without mutagenesis. What is curious is that the phenotype of the suppressor plant shows a Ler ‘plant type’ phenotype. Looking back at the history of the vtc1 mutant, the only time when the Ler and Col genomes were together, described in Conklin et al (1996), was when the vtc1 mutant was crossed to Ler for mapping. The description of the vtc1-1 (soz1) mutant stock (in TAIR) is given as result of 2 x backcrosses and an F3, presumably as a result of crossing to Col-0, but it might be useful to confirm that the stock has no Ler background and the seed used was progeny of single plant and not from a bulk seed lot. Since the VTC1 locus has also been characterized by cyt1 and emb101 mutants, it would seem that the mutant alleles might have some disadvantage in being propagated and a ‘residual heterozygosity’ might persist by some mechanism. In addition, reversion to a wild-type phenotype svt2 might be facilitated by a selection of vigorous embryos into maturity.The sequence changes in the vtc1-1 and svt2 suggests an origin of a ‘template’ independent of Ler and Col alleles, and might also be sequences from another related ecotype. A screen of available Arabidopsis ecotype genome sequences should show such an alternate donor.Minor comment: On the PDF, page 10, need to use ‘were’ instead of 'where' in the sentence beginning \"In all cases...\"",
"responses": []
},
{
"id": "744",
"date": "31 Jan 2013",
"name": "Igor Kovalchuk",
"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\nVery unusual story. I am still puzzled how this is all possible. I can assume that original vtc1 line had some Ler-1 background (may be from backcrosses). In this case it is possible that the seeds you started with for mutagenesis are highly heterogeneous and some have Ler genomes still present. Now, such a severe case of rearrangements due to combination of EMS and vtc1 background is unbelievable. I wonder why other plants with even greater instability, such as ddm1 or msh2, have never had anything like this reported. Maybe they have not looked for it hard enough? It would make sense to get the vtc1 mutant into rdr2 or rdr6 background (or both) and see whether this RNA cache plays any role - I would expect much lower chance of getting those revertants, same with reverse transcription mutants. I understand that the event is rare – a single plant was produced – but it would really make the entire story stronger if several different plants were produced.",
"responses": []
},
{
"id": "747",
"date": "31 Jan 2013",
"name": "David Oppenheimer",
"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\nIn this paper, the authors characterize a suppressor of the vtc1-1 mutation, which they named svt2. The authors characterize the phenotype of the vtc1-1 mutants that carry the suppressor mutation and show that the plants have characteristics reminiscent of the Ler accession. Molecular characterization of the suppressed plants show that the suppressor mutation is apparently a reversion of the original vtc1-1 mutation, and surprisingly, have additional genomic signatures of the Ler ecotype as well as additional mutations. There are several problems with the experimental methods used in this manuscript. First, according to Figure 1 of the manuscript, the authors screened the M0 generation (the mutagenized seed) for wt ascorbic acid content. This is a significant problem for the subsequent mutant analysis in this manuscript. When Arabidopsis seed are mutagenized, the individual cells of the meristem on the seed are mutagenized independently. When the seed germinate, the plants are genetic mosaics. In addition, only those mutations in the L2 layer that gives rise to the germ cells will pass on the mutations. Therefore, it is highly unlikely that a seedling with wt ascorbic acid levels would be isolated from the M0, because it would take the accumulation of many independent mutations, each of which would need to lead to suppression of vtc1. It is possible that a large sector of an M0 seedling could contain a suppressor mutation that leads to wt ascorbic acid levels, but this sector would have to include cells in the L2 layer for the mutation to be passed on to the next generation. Also, an Ler-like sector should be obvious on a mostly Col-0 plant. Nonetheless, a sector that included the L2 would lead to segregation of the phenotype in the M1 generation, because the cells in the sector would be heterozygous for the suppressor mutation. Second, the concentration of EMS commonly used for mutagenesis in Arabidopsis (0.2%) is known to cause multiple mutations per genome. When one isolates a mutant of interest from an EMS screen, one should back-cross it at least once to allow these other mutations to segregate away. Otherwise, one may observe unexpected results when analyzing the mutant of interest due to the effects of these other mutations. Third, when analyzing the sequence of the vtc1 gene in the original mutant and in the suppressor, svt2, the authors compared the sequence to the Ler and Col-0 sequences reported in Genbank and TAIR. Instead, the authors should sequence the vtc1 gene from their original vtc1-1 stock and the Ler accession that is present in their lab. This is because it is known that nucleotide polymorphisms arise regularly in lab stocks such that a comparison between a lab stock of Col-0 and the reference sequence can show many differences. Because the authors are reporting unexpected sequencing results, they should show the actual sequence traces (from both strands) for the individual base pair differences highlighted in Figure S1. It would be appropriate to show these sequence traces in the supplemental data. Showing the sequencing traces for the base pair differences would demonstrate that the sequence differences are not due to ambiguous base calling or other sequencing errors. Fourth, the authors refer to the suppressed plants and their revertants has having a Ler-like or Col-like phenotypes. Because the phenotype of vtc1 is lower ascorbic acid levels, and the putative suppressor has wt ascorbic acid levels, the ascorbic acid levels in the revertants should be measured to show that they are revertants, instead of relying on the Ler or Col phenotype. Fifth, because seed and pollen contamination can explain the results, the authors need to explicitly state the degrees to which they tried to eliminate these possible sources of contamination. Were plants of more than 1 genotype grown together? Were seeds of more than 1 genotype collected in the same room? Was soil stored where plants were setting seed? etc. The single, Ler-like seedling found in the M0 population can be explained as an Ler seed that contaminated the Col-0 vtc1-1 seed stock used for the mutagenesis. This can be tested by sowing several thousand Col-0 vtc1-1 seed from that seed stock, and screening them for the presence of any Ler contaminants. The results in Table 4 are the same as one would expect from seed contamination: the svt2 plants (with the Ler phenotype) have all Ler markers, and the revertants (with the Col phenotype) have all Col markers. The svt2 Col R1 M2 plant highlighted in red shows the expected results if the parent of that plant was heterozygous for Col/Ler. Again, the authors should state what extraordinary measures they used to eliminate seed and pollen contamination. Once these comments are addressed, the other unexpected results can be examined in a new light.",
"responses": []
}
] | 1
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https://f1000research.com/articles/2-6
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https://f1000research.com/articles/2-4/v1
|
09 Jan 13
|
{
"type": "Correspondence",
"title": "The prion dilemma confounding science educators",
"authors": [
"Igor V Zaitsev",
"Ling Chen",
"Susie Boydston-White",
"Manita Pavel",
"Svetlana N Shugaeva",
"Alla G Petrova",
"Oksana V Williams",
"Ling Chen",
"Susie Boydston-White",
"Manita Pavel",
"Svetlana N Shugaeva",
"Alla G Petrova",
"Oksana V Williams"
],
"abstract": "In this paper, the issue of the prion hypothesis, a simmering controversy within the scientific community, is addressed. We inquire into the appropriateness of the use of certain augmentations and rhetoric approaches used during scientific debates, as well as the aptness of unequivocal statements in textbooks that indicate “abnormal prions” as a primary cause of Transmissible Spongiform Encephalopathies.",
"keywords": [
"According to some in the field",
"one should refrain from discussions concerning controversial issues in science if one is not actively conducting experimental research1. We must dissent",
"most particularly when the prions controversy is under consideration. One does not have to conduct scientific experiments to recognize not only the flaws of the prion protein (PrP) hypothesis2",
"but the inappropriate vocabulary used during discussions of the issue. As science educators",
"we are still confounded when trying to present the cause of Transmissible Spongiform Encephalopathy (TSE) to our students."
],
"content": "Correspondence\n\nAccording to some in the field, one should refrain from discussions concerning controversial issues in science if one is not actively conducting experimental research1. We must dissent, most particularly when the prions controversy is under consideration. One does not have to conduct scientific experiments to recognize not only the flaws of the prion protein (PrP) hypothesis2, but the inappropriate vocabulary used during discussions of the issue. As science educators, we are still confounded when trying to present the cause of Transmissible Spongiform Encephalopathy (TSE) to our students.\n\nTo start with, for the past twenty years, the majority of biology text books unequivocally identified PrPSc as the causative agent of TSE, and some texts even refer to the “prion hypothesis” as the “prion theory”, please see Table 1. Yet, when introducing the scientific method in high schools and college classes, we establish that in order for a hypothesis to become a scientific theory, it has to be supported many times over through experimentation3 providing a substantial and conclusive body of evidence4. Upon reviewing experimental work on PrP, one notes that initial studies are rarely, if ever, repeated by other scientists. Instead, they move on without giving reconsideration to the assumption upon which they base their work5.\n\nWhen describing the scientific method, it is important that we emphasize the difference between faith and fact. Nevertheless, during discussions of the PrP hypothesis in meetings, conferences and private discussions of scientists, “I think” is too often replaced by “I believe”. Perhaps, this inclination began when the Karolinka neurologist Lars Edison told The Times newspaper upon the announcement of the Prusiner’s Noble Prize: “There are still people who don’t believe that a protein can cause these diseases, but we believe it”6. There should be no place in science for such a subjective declaration. Even recent publications emphasize that the scientific community has been split into PrP “believers” and “nonbelievers”. Laura Manuelidis, one of the main scientists who rejects the PrP hypothesis, has been portrayed as a “prion heretic”7. Upon entering the combination of “prions” and “belief” in a Google search, we generated an astonishing 918,000 hits. Another recent tendency in modern science is marginalizing scientists as the “minority” versus the “majority” as is seen in the PrP controversy7, a partition more suitable for political rather than scientific discussions.\n\nIn covering the PrP hypothesis in classrooms, are we also to employ a vocabulary in which the scientific community is divided into “believers” and “nonbelievers” or “majority” and “minority” as if we were referring to a religious conviction or a political debate rather than a scientific dilemma?",
"appendix": "Author contributions\n\n\n\nIVZ was involved in reviewing the literature and writing the letter. LC, SB-W, MP, SNS, AGP and OVW equally contributed to the emerged discussion and conceptualization of the paper and all approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nAAAS: Live Chat: When you’re in the Scientific Minority. Science. 2011. Reference Source\n\nZaitsev IV: Prions: Introducing a Complex Scientific Controversy to a Biology Classroom. Am Biol Teach. 2009; 71(9): 525–530. Publisher Full Text\n\nCampbell NA, Reece JB: Biology. Pearson Educational, Inc. as Benjamin Cummings, San Francisco 2005. Reference Source\n\nMedley D: Biology: Reviewing the Essentials. AMSCO School Publications, Inc., New York 1998. Reference Source\n\nZaitsev IV: Could Prion Protein Assumptions Engender Misleading Sensational Conclusions? 2010. Reference Source\n\nRhodes R: Deadly Feasts. Simon & Schuster 1997. Reference Source\n\nCouzin-Frankel J: The prion heretic. Science. 2011; 332(6033): 1024–1027. Publisher Full Text"
}
|
[
{
"id": "709",
"date": "17 Jan 2013",
"name": "Jose Valpuesta",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI agree with what I think is the main message of the authors, that the scientific debate should be open and should rest in facts and not in beliefs. The first point is very important and the same 'prion hypothesis' is a good example of this, as it was under attack for a long time until substantial evidence was produced in its favour. I agree with the authors in that in science one should refrain from using statements ('I believe ...') more adequate for religious or political debates, but when dealing with educational matters, simple statements need be used to convey a certain message or information. Time will tell whether these messages are correct, and the text books and scientific journals are full of information that later has been proven to be wrong, but which has been useful to stir the scientific debate.",
"responses": []
},
{
"id": "721",
"date": "25 Jan 2013",
"name": "Kai Zinn",
"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 scientific community has been split in the past into those who believed the prion hypothesis and those who did not. During the 1980s and part of the 1990s, most work on the prion hypothesis was from Stanley Prusiner's group, and those who questioned the prion hypothesis were doing so largely by finding potential errors in the work of one laboratory. However, now we have hundreds of papers on mammalian PrP, including, most importantly, the demonstration that transmissible disease can be caused by a pure recombinant prion protein (Wang et al, (2010)), that are not from Prusiner and whose results are consistent with the prion hypothesis. In addition, work by many groups on yeast prions demonstrated the validity of the generalized prion hypothesis (inheritance mediated by conformational changes in proteins) in a more experimentally tractable system in which controls that were not possible for mammalian PrP could easily be done. So, at this point, I see no problems with the statements made in the textbooks that are listed in the Table. The prion hypothesis is as well-established, at least for mammalian PrP, as the chemiosmotic (Mitchell) hypothesis for ATP synthesis by mitochondria, which was controversial at the time it was proposed in the early 60s, but which is now the only mechanism described in textbooks. It is no longer necessary to even mention the alternative ideas from the 60s, such as chemical coupling.",
"responses": []
},
{
"id": "1656",
"date": "09 Sep 2013",
"name": "Hidehiro Mizusawa",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI agree with the authors on how important open discussion is in science. However, the prion hypothesis has been well and openly discussed for many years. Due to the hypothesis, many achievements have been obtained. Abnormal prion proteins resulting from prion protein gene mutations clearly cause genetic prion diseases.Minor point: “Abnormal prions” should be “prions”, because prions all are abnormal.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-4
|
https://f1000research.com/articles/2-3/v1
|
04 Jan 13
|
{
"type": "Short Research Article",
"title": "Concomitant caries and calculus formation from in situ dentin caries model",
"authors": [
"Frederico B de Sousa",
"Pablo J Mangueira",
"David R Tames",
"Sandra S Vianna",
"Neriede S Santos-Magalhaes",
"Pablo J Mangueira",
"David R Tames",
"Sandra S Vianna",
"Neriede S Santos-Magalhaes"
],
"abstract": "The aim of this study was to test the possibility of the concomitant formation of calculus deposits and caries from in situ dentin caries model for short time periods. Six volunteers wore palatal removal appliances with four polished dentin specimens protected from intra-oral mechanical forces for up to 14 days. Each volunteer applied a 50% sucrose solution (four times a day) on the specimens and performed a daily mouthwash with 0.05% NaF. Samples were removed after 2, 5, 9 and 14 days in situ. Demineralization was analyzed by stereomicroscopy and SEM (secondary electrons and backscattered electrons modes) and calculus was analyzed by energy dispersive spectroscopy and fluorescence spectroscopy. Seventeen samples, at least one sample from each volunteer, presented dental calculus on both carious and non-carious ones, detected in all time intervals. Ca/P ratios of dental calculus ranged from 1.1 to 1.7. Some large calculus deposits on carious surfaces were confirmed by fluorescence. In conclusion, concomitant caries and calculus formation can be found in dentin caries formed in situ. This has important repercussions on the study of surface phenomena on the interface between hard dental tissues and dental plaque.",
"keywords": [
"dental caries",
"dental calculus",
"caries model",
"in situ",
"surface reactions"
],
"content": "Introduction\n\nIn situ caries models are not expected to give rise to calculus formation. However, the formation of loosely mineralized deposits in dental plaque concomitantly with carious dissolution of enamel, in an environment with fluoridated mineralizing solution, has been reported1. In a morphological study with transmission electron microscopy, those deposits were shown not to be identical to calculus as they were never present within intact bacterial cells2. In addition, it was shown by scanning electron microscopy (SEM) that calculus (firmly bound to hard dental tissue) could develop on dentin specimens subjected to an in situ carious attack with a daily application of a 50% sucrose solution in individuals living in a water-fluoridated area for periods of four weeks3. While demineralization is likely in such models, no clear-cut evidence was presented. In this study, some samples with calculus had the cariogenic challenge disturbed by weekly mechanical plaque removal, raising the hypothesis that the concomitant development of caries and calculus may occur during shorter periods under the given conditions.\n\nThe aim of this study is to report concomitant formation of calculus deposits and caries on dentin surfaces submitted to an in situ caries model (using sucrose and fluoride) for short time periods (2–14 days).\n\n\nMaterial and Methods\n\nHuman dentin specimens (3 × 3 mm) from non-erupted third molars, previously autoclavated, had their surfaces consecutively polished with slurries of decreasing grain size until the alumina was 1 mm. Then, they were mounted in customized hand-made resinous removable palatal appliances (thin resinous removable plates placed on the hard palate only and extending from the area close to the central incisors to area close to the first molars), each one containing four specimens distributed in two pairs each in a different side of the appliance, where they were protected from intra-oral mechanical forces by means of plastic perforated tape. A space of up to 2 mm was left between the specimen and the tape. In the mouth, specimens were located near the palatal surfaces of the first permanent molar and the first premolar.\n\nSix volunteers, aged 20–29 years, were selected on the basis of the following criteria: (i) to be otherwise healthy (no medication regimen), (ii) no calculus treatment in the last two years and no clinical calculus deposits at the time of the study, and (iii) to present normal stimulated salivary flow. All volunteers were dental students who lived in non water-fluoridated areas, and were given fluoridated dentifrices (1500 ppm of the same brand) daily. All volunteers provided signed informed consent. Ethical approval was obtained from the Universidade do Vale do Itajaí Human Research Ethics Committee.\n\nIntra-oral periods lasted fourteen days and the volunteers were instructed to use the appliances all day long, except during meals. Two drops of a 50% sucrose solution (the same applied previously)3 was applied onto the specimens only, four times a day to mimic meals, and a 0.05% NaF solution was provided to perform one daily mouthwash for about one minute with the appliance in the mouth (at a different time to the sucrose rinsing). Instructions were given to replace the appliance in the mouth just after the sucrose rinsing and to not brush on the specimens. For each volunteer, specimens were removed after 2, 5, 9 and 14 days of intra-oral period. Six other polished specimens prepared in the same way described above, not exposed to the oral cavity, were also selected for reproducibility during morphological analysis, to make a total of 30 specimens. After the in situ period, specimens were stored in 0.1% timol prior to further analysis.\n\nAll samples were treated with NaOCl 5% for 15 minutes, to remove organic coatings, then critical point dried with CO2-atmosphere of 10-2 Torr- and coated with a 10 nm-thick layer of gold. The secondary electron detector of a scanning electron microscope JEOL 5900HV (voltage of 15 KeV) was used. Each specimen was analyzed under tilting angles of 0° and 40°. EDS analyses were performed in representative areas by a VOYAGER system attached to the microscope using the following parameters: spatial resolution of 1 mm, electron beam area of 10 mm, working distance of 8–9 mm, voltage of 15 KeV and a live time of 60 s. Ca/P (mol/mol) ratios (mean of 5 measurements) were analyzed using a standardless method - intensity of the standard calculated on the basis of the spectral properties of the sample4-with corrections for atomic number, absorption and fluorescence (ZAF correction). SEM analysis of surface changes (demineralization and dental calculus) was performed twice by blinded examiners with a time interval of seven days between the two analyses. A set of 3 images from each sample was chosen. The Kappa test was used to calculate agreement. Only Schroeder’s type A dental calculus mineralization centers (with mineralized bacterial bodies outlines)5 were considered.\n\nAfter SEM, all samples were treated with NaOCl 5% for 15 minutes to remove the gold coating and then hemi-sectioned centrally on the exposed surface. The surfaces created after cutting were gently polished with alumina 10 mm and then analyzed under the stereomicroscope (Leica MZ12, Leica, Switzerland). Image analysis for the automated identification of demineralized areas and lesion depth measurements were performed using Leica QWIN Plus software (Leica, Switzerland). A standard margin of color discrepancy detection in the software was used for all samples when the software was asked to differentiate sound and carious dentin. The only influence of the operator was to choose a 1 mm-wide central area for automated lesion depth measurement.\n\nIn samples that presented thick (> 100 mm in height) and extensive calcified deposits, a scalpel was used to remove the surface by scraping after stereomicroscopy. The scraped material was dissolved in a solution of HCl 27% (~ 0.2 ml), dispensed in a quartz cuvette and submitted to fluorescence spectroscopy in a commercial photon-counting spectrofluorometer (PC1, ISI, USA and Vinci software, ISI, USA) equipped with a xenon arc lamp operating at 10 mA, using 1 mm slits (bandwidth of 8 nm). Excitation wavelength (λexcit) was 416 nm and the emission (λemis) was collected from 425 to 800 nm (mean of 15 readings). Fluorescence of the other two saturated solutions in HCl 27%, one with human dental calculus and another with human dentin, were also analyzed.\n\nNext, all samples had their cut surface prepared (polished, dried and metallic coated as described above) for SEM analysis using a backscattered electron detector (voltage of 15 KeV) in order to confirm the presence of demineralization. During this latter procedure, the demineralized layer of most samples broke apart so that only the inner parts remained for BSE-SEM analysis.\n\nThe differences between in situ periods of all volunteers with regard to lesion depth were evaluated by paired t test (significance limit at 5%).\n\n\nResults and discussion\n\nThe occurrence of demineralization and calcified deposits was depicted from SEM examination (Table 1). Kappa’s coefficients of intra-examiner agreement were 0.88 and 0.83, and 0.78 for inter-examiner agreement of the diagnosis of demineralization with SEM. For dental calculus, it was decided to count only cases with full agreement between examiners. Mean lesion depth values were 0.0, 0.197 (± 0.059), 0.442 (± 0.062) and 1.100 mm (± 0.212) for 2, 5, 9 and 14 in situ periods, respectively (differences were statistically significant, p < 0.05). BSE-SEM analysis confirmed the presence of demineralization (lower gray levels due to lower mean atomic number) detected by stereomicroscopy.\n\n* Samples with 50% or more of their surfaces covered by calculus.\n\nSeventeen samples presented calculus deposits (14 with demineralization; Table 1). Seven of these presented ~ 50% or more of the surface area covered by the deposits. In the other samples, deposits formed isolated or connected islands of 50–500 mm wide. Ca/P ratios resemble the structures of dicalcium phosphate (Ca/P of 1.0), octacalcium phosphate (Ca/P of 1.3), magnesium whitlockite (Ca/P of 1.5) and hydroxyapatite (Ca/P of 1.67–2.3)5–7 (Table 1). Mean Ca/P ratio of dentin (for all samples) was 1.88 mol/mol (± 0.22), compatible with hydroxyapatite. Demineralization surrounding calculus deposits occurred in thirteen samples.\n\nThe crystalline structure of young calculus is reported to be mainly of dicalcium phosphate in the early stages, after which octacalcium phosphate develops and, with further growth, whitlockite and hydroxyapatite are formed5. The driving force for the mineralization of dental plaque is the supersaturation of plaque fluid with respect to calcium phosphate, which is pH- and temperature-dependent. It is known that dicalcium phosphate and octalcacium phosphate can be precipitated in acidic environments whereas hydroxyapatite is dissolved7,8. Magnesium whitlockite or simply whitlockite (a type of calcium phosphate where calcium is partly substituted by magnesium) has a structure that is not easily distinguished from β-tricalcium phosphate or magnesium-containing β-tricalcium phosphate, and this is the reason why they have been referred as synonyms9. However, it is known that they are distinguishable by careful powder diffractometry10, although both present a Ca/P ratio of 1.57. While β-tricalcium phosphate does not form in biological systems, whitlockite is found in many biological mineralizations9. Whitlockite precipitates at low, neutral or high pH, with magnesium stimulating whitlockite precipitation at the expense of hydroxyapatite7. The presence of hydroxyapatite most probably reflects pH fluctuations (with either neutral or high pH events) during the periods of time employed. In this context, it is possible to chemically explain the concomitant calculus and caries formation in our model.\n\nCrystallite morphology and Ca/P ratios resembling hydroxyapatite in 10-day old calculus deposits have been reported previously11.\n\nAs the in situ period proceeded, calculus deposits (resembling the morphology of Schroeder's type A calculus deposits5) with increased height, macroscopic in some cases, were seen on demineralized areas (Figure 1A-C). The shapes of intact bacterial bodies (cocci, filaments and rods) remained within extracellular calcified trabeculae, indicating that the mineralization process was preferably extracellular. Some deposits were identified only after tilting the sample to 40°. No bacterial cell remnants were identified within calculus deposits.\n\nA, calculus deposits (arrow), of macroscopic size, on a 9-day sample with demineralization (Bar = 1 mm). B, detail of the area outlined in “A”, showing opening of dentinal tubules (white arrow) and dental calculus (black arrow) (Bar = 20 mm). C, detail of the area close to the white arrow in “B”, showing mineralized bacterial outlines delimitating spaces of ~ 1 mm in diameter (DC, dental calculus) and the demineralized dentin (DE) (Bar = 1 mm). D, histological aspect of the same sample after hemi-sectioning showing demineralization (black arrow) below the experimental surface (Bar = 1 mm). Opaque outline is demineralization caused by bacterial acid infiltrated around the sample. E, BSE-SEM image (bar = 300 mm) showing, below the fractured surface (asterisk), a dark layer (white arrow) representing the preserved part of the demineralised area indicated by the black arrow in “D”. F, fluorescence spectroscopy data of different samples dissolved in HCl 27% and excited with light at 416 nm: (a), human dentin; (b), human dental calculus; and (c), sample from the surface of the sample shown in “A”, showing fluorescence of dentin (arrowhead) and dental calculus (arrow).\n\nThree samples (from two volunteers; in situ times of 9 and 14 days) that presented “large” deposits of calculus and caries were submitted to fluorescence spectroscopy. All of them presented fluorescence of dental calculus (Figure 1F: the same sample of Figs. 1A-E) and histological demineralization (Figure 1C-E). The dark layer shown in Figure 1E represents demineralization (preserved part after polishing) seen under BSE-SEM.\n\nFluorescence of human dental calculus in acid solution has been shown to present the highest emission with λexcit at 416 nm and λemis peaks at 620 and 660 nm (originated from hematoporphyrin)12. A lower fluorescence emission band below 580 nm has also been reported for human dental calculus13. Human dentin fluoresces with a λemis peak at 440 nm and does not present any emissions in the region of 580–700 nm14, which is in accordance with our fluorescence data for λexcit at 416 nm (Figure 1F). The selected samples with “large” calcified deposits (deposits < 100 µm in height cannot be detected by a spectrofluorometer system with a lamp source as excitation light15) showed a mixture of fluorescence bands of human dental calculus and human dentin (Figure 1F). The double treatment with NaOCl 5% and the absence of bacterial cell remnants during SEM examination exclude the influence of loosely bound organic material of bacteria on the observed fluorescence.\n\nThe concomitant development (based on our time intervals) of caries lesions and calculus deposits using an in situ caries model reported here occurred under conditions known to cause the formation and growth of dental plaque. Combining SEM, fluorescence spectroscopy and stereomicroscopy (Figs 1A-F), we have shown caries and calculus developments in the same dentin sample that was originally non-carious and had not been exposed to the oral cavity before the in situ experiment. Our model was able to promote time-dependent demineralisation (seen from lesion depth data), which means that volunteers followed instructions properly. To our knowledge, this is the first time that this phenomenon has been reported on the basis of the combination of such mutually validating techniques.\n\n\nConclusion\n\nIn conclusion, calculus formation in active cariogenic dental plaque has important repercussions on the study of surface phenomena on the interface between hard dental tissues and dental plaque. It is also important in the development and the evaluation of anti-tartar and caries-preventative agents. Our study shows that more attention should be paid by those studying in situ dentin caries to the identification of calculus on hard dental tissues exposed to in situ caries models.",
"appendix": "Author contributions\n\n\n\nSousa FB and Tames DR designed the study and the intra-oral appliances. Mangueira PJ selected volunteers, prepared intra-oral appliances, and dentin samples. Sousa FB and Mangueira PJ conducted the in situ protocols and followed up with volunteers. Sousa FB, Vianna SS and Santos-Magalhaes NS conducted the fluorescence spectroscopy, SEM and BSE analysis, and statistical analysis. Sousa FB and Tames DR wrote the paper. All 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 first author thanks the Brazilian Ministry of Education (CAPES) for financial support (doctorate scholarship). The other authors are grateful to CNPq Brazilian Federal Agency of Research.\n\n\nConsent\n\nAll volunteers who used the intra-oral appliances signed a written informed consent before participating in this study. No payment was provided to volunteers in return for their participation in this study.\n\n\nReferences\n\nPearce EI: Effect of plaque mineralization on experimental dental caries. Caries Res. 1982; 16(6): 460–471. PubMed Abstract\n\nPearce EI, Wakefield JS, Sissons CH: Therapeutic mineral enrichment of dental plaque visualized by transmission electron microscopy. J Dent Res. 1991; 70(2): 90–94. PubMed Abstract | Publisher Full Text\n\nSousa FB: The effect of weekly mechanical plaque removal on the in situ development of dentin caries (in Portuguese). Master dissertation. Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil 1996.\n\nGoldstein JI, Newbury DE, Echlin P, et al.: Scanning electron microscopy and x-ray microanalysis. 2. ed, Plenum, New York 1992. Reference Source\n\nSchroeder HE: Formation and inhibition of dental calculus. J Periodontol. 1969; 40(11): 643–6. PubMed Abstract | Publisher Full Text\n\nKodaka T, Ohohara Y, Debari K: Scanning electron microscopy and energy-dispersive x-ray microanalysis studies of early dental calculus on resin plates exposed to human oral cavities. Scanning Microsc. 1992; 6(2): 475–485. PubMed Abstract\n\nLegeros RZ: Calcium Phosphates in Oral Biology and Medicine. Monogr Oral Sci. 1991; 15: 1–201. PubMed Abstract\n\nJohnsson MS, Nancollas GH: The Role of Brushite and Octacalcium Phosphate in Apatite Formation. Crit Rev Oral Biol Med. 1992; 3(1–2): 61–82. PubMed Abstract\n\nMathew M, Takagi S: Structure of biological minerals in dental research. J Res Natl Inst Stand Technol. 2001; 106: 1035–1044. Reference Source\n\nGopal R, Calvo C: Strucure relationship of whitlockite and β-Ca3(PO4)2. Nature Phys Sci. 1972; 237: 30–32. Reference Source\n\nKodaka T, Ohohara Y, Debari K: Scanning electron microscopy and energy-dispersive x-ray microanalysis studies of early dental calculus on resin plates exposed to human oral cavities. Scanning Microsc. 1992; 6(2): 475–485. PubMed Abstract\n\nReis MM, Biloti DN, Ferreira MM, et al.: PARAFAC for spectral curve resolution: a case study using total luminescence in human dental tartar. Appl Spectrosc. 2001; 55: 847–851. Reference Source\n\nDolowy WC, Brandes ML, Gouterman M, et al.: Fluorescence of dental calculus from cats, dogs, and humans and of bacteria cultured from dental calculus. J Vet Dent. 1995; 12(3): 105–109. PubMed Abstract\n\nMatsumoto H, Kitamura S, Araki T: Autofluorescnce in human dentine in relation to age, tooth type and temperature measured by nanosecond time-resolved fluorescence microscopy. Arch Oral Biol. 1999; 44(4): 309–318. PubMed Abstract\n\nLakowicz JR: Principles of fluorescence spectroscopy. 2nd ed, Kluwer Academic/Plenum, New York 1999; 725. Reference Source"
}
|
[
{
"id": "715",
"date": "21 Jan 2013",
"name": "Leonard A. Crocombe",
"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": "906",
"date": "23 Apr 2013",
"name": "Neville Gully",
"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 was noted that all dentin samples were exposed to sucrose solutions, fluoride and were protected by tape. Only the unexposed slabs were used as controls and none of these three conditions were controlled. A more complete picture would have been possible with their inclusion.The authors claimed “It is known that dicalcium phosphate and octacalcium phosphate can be precipitated in acidic environments whereas hydroxyapatite is dissolved.” They listed references 7 and 8 to support this statement. However, I was unable to access reference 7 and could not find supporting data in reference 8. While DCP is slightly less soluble than HAP around pH4 I would not consider that to mean that either DCP or OCP readily precipitate at that pH.",
"responses": [
{
"c_id": "462",
"date": "20 May 2013",
"name": "Frederico Sousa",
"role": "Author Response",
"response": "I thank the reviewer for his valuable comments and his opinion to accept the article is greatly acknowledged by us. Regarding the first paragraph of the reviewer’s comment, we understood that the critique was that some control was lacking. We think that the inclusion of samples that were exposed to the oral environment but did not develop caries would no add relevant information for the aim of the study. The in situ caries induction aimed at producing caries lesions with known history and with severity directly proportional to the in situ time. Mean lesion depth increased with in situ time, confirming the prediction. In an environment where caries develops, surface features of the samples were analyzed in order to verify whether calculus deposits could have been formed under the same conditions. Such conditions are those common to in situ caries models. Because the length of the article is short, we focused on the more relevant data (samples with dental calculus). If we had exposed dentin samples that remained sound after the in situ period, the presence of absence of calculus deposits on those samples would have not contributed to prove that calculus deposits can be formed concomitantly with caries lesions in short time intervals in an in situ caries model. In the case of precipitation of dicalcium phosphate (DCP) and octacalcium phosphate (OCP) in acidic environments, Johnsson et al. 1992 describes evidence indicating that formation of both DCP and OCP in vivo, during normal mineralization, is quite different from the conditions operating under pathological mineralization. The authors cite reports of the precipitation of DCP and OCP in slightly acidic conditions (please see last paragraph of the second column on page 78 of Johnsson et al. 1992). The critical pH of dentin is between 6.5 and 7 (Hoppenbrouwers et al. 1987). There is a pH range below the critical pH of dentin that can be considered as slightly acidic, and so it is consistent with the possible precipitation of both DCP and OCP in our samples. Regarding formation of DCP and OCP in acidic conditions in vitro, evidence can be found in the following papers; De Rooij et al. 1984 and Lundager Madsen et al. 1991."
}
]
}
] | 1
|
https://f1000research.com/articles/2-3
|
https://f1000research.com/articles/2-2/v1
|
04 Jan 13
|
{
"type": "Web Tool",
"title": "ProfileGrids solve the large alignment visualization problem: influenza hemagglutinin example",
"authors": [
"Alberto I Roca",
"Aaron C Abajian",
"David J Vigerust",
"Aaron C Abajian",
"David J Vigerust"
],
"abstract": "Large multiple sequence alignments are a challenge for current visualization programs. ProfileGrids are a solution that reduces alignments to a matrix, color-shaded according to the residue frequency at each column position. ProfileGrids are not limited by the number of sequences and so solves this visualization problem. We demonstrate the new metadata searching and grep filtering features of the JProfileGrid version 2.0 software on an alignment of 11,900 hemagglutinin protein sequences. JProfileGrid is free and available from http://www.ProfileGrid.org.",
"keywords": [
"The explosion in biological sequence information has led to the generation of large multiple sequence alignments (MSA). For example",
"the biggest protein family alignment currently in the Pfam database (Wellcome Trust-Sanger Institute) has over 288",
"000 sequences1. A new generation of alignment programs",
"such as Clustal Omega2",
"are available that allow the routine calculation of such large alignments. However",
"a Nature Methods review3 noted the lack of software tools for visualizing the results of large alignment calculations. Specifically",
"there was a call for overcoming the conceptual and technical limitations of large data sets to allow one to navigate visually both an overview and the details of an alignment",
"while having mechanisms to query annotated data. We point out that this conceptual limitation was solved in late 2008 by the introduction of ProfileGrids as a new paradigm for visualizing large multiple sequence alignments4. Here",
"we report that the remaining technical limitations have been overcome with version 2.0 of the JProfileGrid software",
"and that therefore",
"the large alignment visualization problem has now been solved. We use the influenza hemagglutinin protein family as a case study to demonstrate the new features of the software."
],
"content": "Introduction\n\nThe explosion in biological sequence information has led to the generation of large multiple sequence alignments (MSA). For example, the biggest protein family alignment currently in the Pfam database (Wellcome Trust-Sanger Institute) has over 288,000 sequences1. A new generation of alignment programs, such as Clustal Omega2, are available that allow the routine calculation of such large alignments. However, a Nature Methods review3 noted the lack of software tools for visualizing the results of large alignment calculations. Specifically, there was a call for overcoming the conceptual and technical limitations of large data sets to allow one to navigate visually both an overview and the details of an alignment, while having mechanisms to query annotated data. We point out that this conceptual limitation was solved in late 2008 by the introduction of ProfileGrids as a new paradigm for visualizing large multiple sequence alignments4. Here, we report that the remaining technical limitations have been overcome with version 2.0 of the JProfileGrid software, and that therefore, the large alignment visualization problem has now been solved. We use the influenza hemagglutinin protein family as a case study to demonstrate the new features of the software.\n\nPrevious MSA visualization paradigms3,5 do not provide both alignment overviews together with the details of each character’s frequency distribution at each homologous position in the entire alignment. A particular technical limitation of stacked sequence representations is that as the alignment size grows, a printed or digital visualization can run out of convenient space. This compounds the conceptual limitation where the user cannot grasp the overall conservation trends and the observed variation details in a large data set. The ProfileGrid paradigm was introduced as a solution to this conceptual problem by converting a multiple sequence alignment to a color-coded matrix of the residue frequency occurring at every homologous position across the entire length of a MSA. Therefore, all MSA information is represented, both at variable regions and of infrequent residues that may yield clues about biological function.\n\n\nImprovements to JProfileGrid software\n\nComparisons of the stacked sequence and ProfileGrid visualization paradigms highlight the challenges of large MSAs (Fig. 1). We downloaded 11,981 full-length, non-redundant hemagglutinin protein sequences from the NCBI influenza virus resource6, aligned them using MUSCLE7, and visualized the MSA8 with the new JProfileGrid 2.0 software. Both paradigms depict the entire width of the alignment, i.e., 650 columns from the approximately 570 protein residues and the 80 inserted gap characters. However, due to space limitations, the stacked sequence overview (Fig. 1a) is only a sampling of 600 sequences from the whole alignment. In the stacked representation, each row is an individual protein sequence with the amino acids represented as pixels colored according to the Taylor scheme9. Below the stacked sequence overview is a similarly colored magnified view (Fig. 1b) from a much smaller alignment with the amino acid one-letter codes shown. By contrast, the ProfileGrid overview representation (Fig. 1c) divides the alignment width into multiple \"tiers\" of which six are 100 characters wide. Within each tier are 21 rows for the 20 amino acids and a gap character. Each cell in the matrix is a color shaded count of the character occurrence at the corresponding MSA column position from low (white) to high (dark blue) frequency. Thus, all of the sequences from the entire alignment are represented in the ProfileGrid by the frequency color shading of each cell in the matrix. As a reference, the left-most column of each ProfileGrid tier is colored according to the Taylor scheme for each amino acid row. ProfileGrids solve the visualization problem of large alignments since there is no limit to the number of sequences that can be represented. Note that a stacked sequence representation of all 11,981 hemagglutinin sequences in this example would be twenty times larger than the number of rows shown in the overview (Fig. 1a).\n\nThe alignment of hemagglutinin sequences is compared side-by-side using the new JProfileGrid 2.0 \"overview\" feature for both stacked sequence (a) and ProfileGrid (c) visualization methods. An example detailed view of a stacked sequence aligment shows only 12 sequences (b) with the one-letter amino acid codes colored according to the Taylor scheme. While space limitations restrict the stacked sequence overview to only 600 sequences, the ProfileGrid overview represents the entire residue content of all 11,981 sequences. See text for details.\n\nThe first version of the Java JProfileGrid software was designed for alignments with hundreds of sequences such as the ubiquitous RecA protein involved in bacterial recombination4. Upon analyzing virus protein families such as influenza hemagglutinins with thousands of sequences, it became apparent that the larger data sets were taxing certain software technical limits. We completely overhauled the program to improve the code with respect to object oriented design, memory management, calculation efficiency, and speed. We strategically reduced memory usage and introduced parallelized code for computing amino acid frequency counts. For example, we were able to reduce the memory requirements by addressing a technical limitation of the Java programming language. Java uses 2 bytes (16-bits) of memory for every Unicode character (UTF-16) reflecting the need to support thousands of characters from hundreds of languages. However, typical MSAs consist of only ASCII characters. Thus, we were able to reduce the memory use 2-fold by introducing a byte (8-bit) map between the integers 0 to 20 and the twenty common amino acid codes and a gap character. Parallelization was possible due to the nature of the ProfileGrid format. Amino acid counts within each column are independent of each other and so were partitioned into separately running threads thereby taking advantage of multi-core processor environments for enhanced ProfileGrid calculation speed.\n\nNew features in JProfileGrid v2.0 accommodate viewing and analyzing very large MSAs. The software now allows sorting and searching the menu list of sequence names for finding a specific homolog to serve as the reference sequence for the ProfileGrid. The new \"overview\" modes enable the user to visualize the entire MSA within one window. The detailed ProfileGrid window with the character counts has a new second pane that facilitates simultaneous viewing of different parts of the MSA. To easily focus on rare residues, the \"highlight\" functionality now identifies residues that occur greater or less than a user-defined threshold of residue frequency. We introduced data sampling to accelerate similarity plot calculations rather than needlessly including every single sequence from large MSAs. Regular expression functionality has also been implemented to find sequences with particular names or amino acid patterns. Finally, JProfileGrid can import simple sequence annotations from flat file spreadsheet databases10 for metadata filtering to reduce large MSAs to subsets of interest.\n\n\nExample: influenza hemagglutinin\n\nAs a demonstration of the new metadata (Fig. 2a) and regular expression (Fig. 2b) software features, the 11,981 sequence alignment (Fig. 2c) was filtered to just 60 hemagglutinin homologs (Fig. 2d) by using a metadata search for \"human\" hosts and a regular expression search for a \"Mexico\" country location. The ProfileGrid views are positioned at alignment column 333 where there is a potential glycosylation site (asn-thr-thr-cys underlined in red) that is found among this sequence subset. Glycosylation is a key post-translational modification that is vital to the proper folding and trafficking of viral proteins. In addition to the role in protein folding and processing, glycosylation in influenza is also important to virus immune evasion strategy11. The majority of antibody recognition is dedicated to the surface antigen on the globular head although stem-directed antibodies have recently been described12. Glycosylation in the stem region could prevent recognition of these antibodies and allow for virus to escape the immune response. The region visualized by ProfileGrid analysis lies in the stem region of the hemagglutinin molecule. ProfileGrids, therefore, permit observing rare natural sequence variation (Fig. 2d) within the context of an entire multiple sequence alignment (Fig. 2c). ProfileGrids have this unique advantage over other compressed alignment visualization methods such as sequence logos13.\n\nThe metadata (a) and regular expression filters (b) allow the entire sequence alignment (c) to be filtered to a select subset of sequences (d). Note that in panel (d), the amino acids are colored and sorted according to the Taylor classification scheme. A glycosylation site (red underline; MSA column position 333) is located within bioinformatic element variable_5a. See text for details.\n\nIn conclusion, ProfileGrids have solved the problem of visualizing very large alignments. As sequence data sets grow, both for the end user and in central database repositories, we anticipate that ProfileGrids will simplify the dissection and analysis of MSAs. Parenthetically, we note that some bioinformatic studies about influenza proteins have lacked figures depicting the alignment upon which the analyses are based14. This omission was probably due to technical limitations that now no longer exist. Thus, we propose that ProfileGrid representations of alignment datasets be included as part of publication to assist science communication. This may initiate establishing a future standard for MIMSA: Minimum Information about a Multiple Sequence Alignment15. Likewise, database curators can use JProfileGrid and its new PNG image output to include ProfileGrid visualizations with protein family descriptions and search results. JProfileGrid v2.0 is available under a GNU General Public License and can be downloaded from http://www.ProfileGrid.org.",
"appendix": "Author contributions\n\nAIR and DJV conceived the study. AIR designed the software, collected hemagglutinin sequences, performed the bioinformatic analysis and biocuration, and prepared the first draft of the manuscript. ACA wrote Java code and contributed to writing the manuscript. DJV interpreted the bioinformatic observations and contributed to writing the manuscript. AIR and ACA contributed equally to the work. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\nWe have developed the software but have no competing interests.\n\n\nGrant information\n\nAIR was supported by the Erasmo Foundation (grant TSC13702); and DJV was supported by a Veterans Administration Career Development Grant. This material is based upon work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development with resources and the use of facilities at the VA Tennessee Valley Healthcare System.\n\n\nAcknowledgements\n\nWe thank David Talalayevsky and Andy Pitt for programming support; Tamara Munzner and Adam Steinberg for visualization design suggestions; and the ISMB KillerApp Award, VIZBI, and SACNAS Postdoc committees for helpful discussions.\n\n\nReferences\n\nPunta M, Coggill PC, Eberhardt RY, et al:The Pfam protein families database. Nucleic Acids Res. (2012)40, D290–301.\n\nSievers F, Wilm A, Dineen D, et al:Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol, (2011)7, 539.\n\nProcter JB, Thompson J, Letunic I, et al:Visualization of multiple alignments, phylogenies and gene family evolution. Nat Methods, (2010)7, S16–25.\n\nRoca AI, Almada AE, Abajian AC, et al:ProfileGrids as a new visual representation of large multiple sequence alignments: a case study of the RecA protein family. BMC Bioinformatics, (2008)9, 554.\n\nPuntervoll P, Aasland R: In Cesareni, G., Gimona, M., Sudol, M. and Yaffe, M. (eds.), Modular Protein Domains. Wiley-VCH, Weinheim, Germany, (2005), pp. 477–486.\n\nBao Y, Bolotov P, Dernovoy D, et al:The influenza virus resource at the National Center for Biotechnology Information. J Virol. (2008)82, 596–601.\n\nEdgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. (2004)32, 1792–1797.\n\nRoca AI: Multiple sequence alignment of influenza hemagglutinin protein sequences.figshare, (2012).\n\nTaylor WR: Residual colours: a proposal for aminochromography. Protein Eng, (1997)10, 743–746.\n\nRoca AI: Meta data for multiple sequence alignment of influenza hemagglutinin protein sequences.figshare, (2012).\n\nVigerust DJ, Shepherd VL: Virus glycosylation: role in virulence and immune interactions. Trends Microbiol. (2007)15, 211–218.\n\nFleishman SJ, Whitehead TA, Ekiert DC, et al:Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science, (2011)332, 816–821.\n\nSchneider TD, Stephens RM: Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. (1990)18, 6097–6100.\n\nAmaro RE, Swift RV, Votapka L, et al:Mechanism of 150-cavity formation in influenza neuraminidase. Nat Commun. (2011)2, 388.\n\nTaylor CF, Field D, Sansone SA, et al:Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project. Nat Biotechnol. (2008)26, 889–896."
}
|
[
{
"id": "465",
"date": "04 Jan 2013",
"name": "Alex Bateman",
"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 have selected an important problem to address which is how to visualise very large multiple sequence alignments. The manuscript describes a new improved version of their software called JProfileGrid originally published in BMC Bioinformatics in 2008. The software ran on a Stockholm formatted alignment I selected from Pfam and so appears to work largely as advertised.My main critique of the work is the claim that ProfileGrids solve the large alignment visualisation problem as stated in the title. In my opinion the visualisation given does not solve the problem. The problem is recast as a visualisation of a profile. Profiles give the frequency of amino acids at each position calculated from a multiple sequence alignment and so their size is independent of the number of sequences in the alignment. So visualising the profile is one way of gaining an overview of a multiple sequence alignment. But others have already done this such as HMM-logos andSUPERFAMILY profile visualisation. Both of these visualisations are more intuitive that the ProfileGrid because it is easier to map these visualisations to a real sequence. However, none of these are really alignment visualisations. They all lose a lot of information that is implicit in the alignment. For example, the correlations between amino acid positions in subfamilies. The JProfileGrid software does provide a compact stacked alignment representation, but that is also possible in other alignment viewers by choosing a sufficiently small font.Overall I think that this is a well engineered update of an existing software package that has some utility. The authors need to remove the claims that this software solves the large alignment visualisation problem. This is in my opinion is not justified. In particular the current title is not acceptable. The text of the paper also needs to include mention that other profile visualisation software exists.",
"responses": []
},
{
"id": "740",
"date": "29 Jan 2013",
"name": "Jaap Heringa",
"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\nVisualising multiple sequence alignments (MSA) is of great importance, and has only become more essential in the wake of next generation sequencing (NGS) projects. The authors report on a new improved version of their software called JProfileGrid 2.0, originally published in BMC Bioinformatics in 2008. In their new implementation, the authors add the possibility to view very large alignments by means of a profile abstracted from the alignment, such that the number of sequences (typically running into the hundreds or even thousands in the NGS era) is compressed to just 21 rows in a protein MSA, corresponding to the 20 amino acid types and a gap character. However, visualising a profile is an entirely different thing than visualising an alignment. The authors' claim, therefore, that they have 'solved' the alignment visualisation problem is unwarranted. They should adapt their title and main conclusion to reflect this fact.Profiles are an abstraction of an MSA, but losing quite a bit of information: for example, correlations between alignment positions, or specific subgroups discernible in an MSA are not visible in a profile. The authors could have opted for alternative ways to compress MSAs containing many sequences, for instance, by clustering the sequences based on sequence similarity, and selecting a representative sequence for each cluster group. The number of clusters could then be selected by the user, or even set automatically based upon optimality criteria concerning the clustering. Many techniques exist already for visualising profiles, such as the widely used SequenceLogos and related representations. As such, just representing the raw amino acid frequencies using a color coding scheme can hardly be considered novel. The authors could make their profile visualization more useful by using other frequency-derived schemes, such as the aforementioned SequenceLogo (entropy-based), or the log likelihood used in PSSMs calculated by BLAST. On a more general note, the authors should cite related work and software by other researchers in the field.Summarising, the authors have compiled a useful software package to visualise alignment profiles, but have not solved the MSA visualisation problem for large sequence sets. Further, they could enhance the usability of the software by implementing more profile scoring schemes as indicated above.",
"responses": []
},
{
"id": "731",
"date": "29 Jan 2013",
"name": "Christos Ouzounis",
"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\nComputing and visualizing multiple sequence alignments might have been considered a solved issue for bioinformatics a few years ago, until the flood of sequence data we have been experiencing recently. In that respect, efforts to address the issue on another scale altogether are indeed necessary. This work reports on a newer version of ProfileGrid that attempts to solve the visualization of very large alignments and a demonstration of its capabilities with the influenza hemagglutinin as an example.JProfileGrid 2.0 appears to perform well, captures some elements of large alignments and provides a useful interface for the exploration of large homologous sequence data-sets. Offering a number of options to users, e.g. color schemes and frequency diagrams might indeed be helpful, but the claim of 'solving' the visualization issues with these data is a bit of an over-statement. For funding agencies and beginners in the field, that might have implications such as not appreciating the importance of this line of research.Beyond this extraordinary claim, the work is fine and provides sufficient detail for interested readers.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-2
|
https://f1000research.com/articles/2-1/v1
|
02 Jan 13
|
{
"type": "Correspondence",
"title": "Biased under-reporting of research reflects biased under-submission more than biased editorial rejection",
"authors": [
"Iain Chalmers",
"Kay Dickersin",
"Kay Dickersin"
],
"abstract": "Stephen Senn challenges Ben Goldacre’s assertion in ‘Bad Pharma’ that biased editorial acceptance of reports with ‘positive’ findings is not a cause of biased under-reporting of research. We agree with Senn that biased editorial decisions may contribute to reporting bias, but Senn ignores the evidence that biased decisions by researchers to submit reports for possible publication are the main causes of the problem.",
"keywords": [
"peer review"
],
"content": "\n\nStephen Senn challenges Ben Goldacre’s assertion in ‘Bad Pharma’1 that biased editorial acceptance of reports with ‘positive’ findings is not a cause of biased under-reporting of research, and concludes that \"the prospects for disentangling cause and effect when it comes to publication bias are not great\"2. Senn apparently overlooks the studies – including controlled experiments - which have investigated reporting biases. These are summarised in an article3 from which the following is an excerpt:\n\n“Who is responsible for biased reporting of clinical research?\n\nReporting bias can be due to researchers and sponsors failing to submit study findings for publication, or due to journal editors and others rejecting reports for publication. Numerous surveys of investigators have left little doubt that almost all failure to publish is due to the failure of investigators to submit reports for publication4,5, with only a small proportion of studies remaining unpublished because of rejection by journals6, although positive-outcome bias has been demonstrated among peer reviewers7. Qualitative studies of editorial discussion indicate that a study’s scientific rigour is the area of greatest concern8. Researchers report that the reason they do not write up and submit reports of their research for publication is usually because they are \"not interested\" in the results (\"editorial rejection by journals\" is only rarely given as a cause of failure to publish). Even those investigators who have initially published their results as (conference) abstracts are less likely to submit their findings for full publication unless the results are ‘significant’9.\n\nInvestigations of biased reporting of research began with surveys of journal articles, which revealed improbably high proportions of published studies showing statistically significant differences10–14. Subsequent surveys of authors and peer reviewers showed that research that had yielded ‘negative’ results was less likely than other research to be submitted or recommended for publication15–18. These findings have been reinforced by the results of experimental studies, which showed that studies with no reported statistically significant differences were less likely to be accepted for publication7,19–21\".\n\nSenn’s use of the term ‘publication bias’ in his commentary suggests that he is restricting it to editorial bias whereas, as indicated above, the origins of reporting bias are largely due to researchers’ decisions not to submit, not editorial decisions not to accept. The analyses of observational data cited by Ben Goldacre in his book ‘Bad Pharma’1 do not detect editorial bias, but neither do they support a confident conclusion that no editorial bias exists. However, we believe Goldacre is correct to castigate researchers and research sponsors as being more culpable than editors in betraying their responsibility to the patients who have participated in trials.\n\nThe controlled experiments suggest that it is the results of studies, not their quality, that predisposes them to editorial bias. Senn believes that any editorial bias that exists can be ‘very plausibly explained’ by preferential publication of ‘positive’ studies, and that it \"seems plausible that higher quality studies are more likely to lead to a positive result\". Unless he is using the word ‘positive’ to mean something other than ‘a beneficial effect’, however, Senn appears to be overlooking substantial evidence challenging the plausibility of his belief (see, for example, reference22). Given the estimated likelihood of new treatments proving superior to standard treatments23 it surprises us that, \"as a statistician\" Senn would find this evidence \"unpalatable\".",
"appendix": "Author contributions\n\n\n\nThe authors both contributed to the text submitted.\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\nGoldacre B: Bad Pharma. London: 4th Estate 2012. Reference Source\n\nSenn S: Misunderstanding publication bias: editors are not blameless after all [v1; ref status: indexed, http://f1000r.es/YvAwwD]. F1000 Research. 2012; 1(59). Publisher Full Text\n\nDickersin K, Chalmers I: Recognising, investigating and dealing with incomplete and biased reporting of clinical research: from Francis Bacon to the World Health Organisation. JLL Bulletin: Commentaries on the history of treatment evaluation. (www.jameslindlibrary.org). Reference Source\n\nTimmer A, Hilsden RJ, Cole J, et al.: Publication bias in gastroenterological research - a retrospective cohort study based on abstracts submitted to a scientific meeting. BMC Med Res Methodol. 2002; 2: 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGodlee F, Dickersin K: Bias, subjectivity, chance, and conflict of interest in editorial decisions. In: Godlee F, Jefferson T, eds. Peer review in health sciences, 2nd edition. London: BMJ Books 2003. Reference Source\n\nOlson CM, Rennie D, Cook D, et al.: Publication bias in editorial decision making. JAMA. 2002; 287(21): 2825–2828. PubMed Abstract | Publisher Full Text\n\nEmerson GB, Warme WJ, Wolf FM, et al.: Testing for the presence of positive-outcome bias in peer review: a randomized controlled trial. Arch Intern Med. 2010; 170(21): 1934–1939. PubMed Abstract | Publisher Full Text\n\nDickersin K, Ssemanda E, Mansell C, et al.: What do JAMA editors say when they discuss manuscripts that they are considering for publication? Developing a schema for classifying the content of editorial discussion. BMC Med Res Methodol. 2007; 7: 44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nScherer RW, Langenberg P, Von Elm E: Full publication of results initially presented in abstracts. Cochrane Database Syst Rev. 2007; 18(2): MR000005. PubMed Abstract | Publisher Full Text\n\nSterling TD: Publication decisions and their possible effects on inferences drawn from tests of significance - or vice versa. J Am Statistical Assoc. 1959; 54: 30–34. Publisher Full Text\n\nSmart RG: The importance of negative results in psychological research. Can Psychologist. 1964; 5(4): 225–232. Publisher Full Text\n\nChalmers TC, Koff RS, Grady GF, et al.: A note on fatality in serum hepatitis. Gastroenterology. 1965; 49: 22–26. Reference Source\n\nLight RJ, Pillemer DB: Summing up. Cambridge: Harvard University Press 1984. Reference Source\n\nSong F, Parekh S, Hooper L, et al.: Dissemination and publication of research findings: an updated review of related biases. Health Technol Assess. 2010; 14(8): iii, ix-xi, 1–193. PubMed Abstract\n\nGreenwald AG: Consequences of prejudice against the null hypothesis. Psychol Bull. 1975; 82(1): 1–20. Publisher Full Text\n\nCoursol A, Wagner EE: Effect of positive findings on submission and acceptance rates: A note on meta-analysis bias. Prof Psychol: Res Pract. 1986; 17(2): 136–137. Publisher Full Text\n\nDickersin K, Chan S, Chalmers TC, et al.: Publication bias and clinical trials. Control Clin Trials. 1987; 8(4): 343–53. PubMed Abstract | Publisher Full Text\n\nShadish WR, Doherty M, Montgomery LM, et al.: How many studies are in the file drawer? An estimate from the family/marital psychotherapy literature. Clin Psychol Rev. 1989; 9(5): 589–603. Publisher Full Text\n\nMahoney MJ: Publication prejudices: An experimental study of confirmatory bias in the peer review system. Cog Ther Res. 1977; 1(2): 161–175. Publisher Full Text\n\nPeters D, Ceci S: Peer review practice of psychologic journals: The fate of published articles submitted again. Behav Brain Sci. 1982; 5(2): 187–195. Publisher Full Text\n\nEpstein WM: Confirmational response bias among social work journals. Sci Technol Hum Values. 1990; 15(1): 9–38. Publisher Full Text\n\nSavović J, Jones HE, Altman DG, et al.: Influence of reported study design characteristics on intervention effect estimates from randomized controlled trials. Ann Intern Med. 2012; 157(6): 429–438. PubMed Abstract | Publisher Full Text\n\nDjulbegovic B, Kumar A, Glasziou PP, et al.: New treatments compared with established treatments in randomized trials. Cochrane Database Syst Rev. 2012; 10: MR000024. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "600",
"date": "08 Jan 2013",
"name": "Riekie de Vet",
"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 comment on a article by Stephen Senn who questions Ben Goldacre’s assertion in the book “Bad Pharma” that editorial process is not the main cause of publication bias. They present a large amount of evidence from the literature that researchers are the main cause of publication bias by selectively submitting paper for publication. They provide a lot of convincing information in this short reaction. However, some sentences are very difficult to read. Especially for readers who haven’t read the book by Goldacre, the comment by Senn, and some of the other references. I had to reread the first sentence about five times before I understood. The sentence is especially difficult to read because there is a double negation. Splitting the sentence in the statement of Ben Goldacre and the comment of Stephen Senn may help. Also the last sentence of the comment is difficult to understand, especially when the reader is unaware of the conclusion of reference 23. The second part of the citation of Goldacre “the prospects for disentangling cause and effect when it comes to publication bias are not great” is difficult to understand and, as far as I can see, does not come back in the comment. Consider whether that part can be omitted, or refer to it again at the end of the comment. The last section starts with ‘The controlled experiments’. It is not clear to which experiments this refers. To ‘studies – including controlled experiments ‘mentioned in the first section? In conclusion, this is a very important and informative comment. However, the readability should be improved in order to make it better understandable for readers who have not read all previous papers.",
"responses": []
},
{
"id": "601",
"date": "08 Jan 2013",
"name": "Luigi Naldi",
"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": "651",
"date": "09 Jan 2013",
"name": "Steven Julious",
"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 just put one anecdotal observation and that is of second studies that replicate the findings of a study published in a journal. An editor may turn down the second study as 'nothing new' is being said although most would argue replication to be important.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/2-1
|
https://f1000research.com/articles/1-71/v1
|
28 Dec 12
|
{
"type": "Short Research Article",
"title": "New record of intraspecific nest parasitism by the Silky Starling (Sturnus sericeus)",
"authors": [
"Zhong-qiu Li",
"Rong-rong Wang",
"Xue-lei Jiang",
"Zhi-yuan Zhang",
"Rong-rong Wang",
"Xue-lei Jiang",
"Zhi-yuan Zhang"
],
"abstract": "The Silky Starling (Sturnus sericeus) is endemic to East Asia and little is know about its’ breeding ecology. We found intraspecific nest parasitism (INP) by this species in a reproductive study conducted from March to June 2011. We found three nests were parasitized using the obvious morphological differences or partition of egg-laying. One egg appeared 3 days after the 26th female had finished laying eggs. One egg was different in color from the other five eggs in the 27th nest. The third instance was discovered in the 37th nest after the fledglings had fledged. Our findings confirmed INP by the Silky Starling.",
"keywords": [
"Intraspecific nest parasitism (INP) occurs when a female lays her eggs in another female's nest1. The number of bird species known to show INP has grown during the recent decades: Yom-Tov summarized INP from 53 species 30 years ago and up to 236 species in 20011",
"2. Gong and Lu (2003) added a further 14 species reported in China to the list. More and more species have been found to show INP",
"and the major reasons of this rapid increase include more detailed investigations of avian reproductive biology as well as the collection of reproductive data from more species3."
],
"content": "Introduction\n\nIntraspecific nest parasitism (INP) occurs when a female lays her eggs in another female's nest1. The number of bird species known to show INP has grown during the recent decades: Yom-Tov summarized INP from 53 species 30 years ago and up to 236 species in 20011,2. Gong and Lu (2003) added a further 14 species reported in China to the list. More and more species have been found to show INP, and the major reasons of this rapid increase include more detailed investigations of avian reproductive biology as well as the collection of reproductive data from more species3.\n\nINP has been found and studied in four species of starlings: the Grey Starling (Sturnus cineraceus)4, the Spotless Starling (S. unicolor)5, the Rose-coloured Starling (S. roseus)6, and the European Starling (S. vulgaris)7. However, information on the Silky Starling is rare, mainly because they live just in China and the surrounding areas, and few studies have been conducted on its reproductive biology8. We provide the first description of INP by Silky Starlings and discuss possible reasons of INP in this species.\n\n\nMethods\n\nThis study was conducted on the Pukou Campus (32º 10’ N, 118º 41’ E) of Nanjing University, China from March to June 2011. The campus is to the north of the Yangtze River. Elevation ranges from 2 to 50 m above sea level. Average annual temperature is 15.4ºC. Annual precipitation is 1,106 mm, ~ 60% of which occurs from June to September9.\n\nWe placed and numbered 15 artificial nests (16 × 16 × 35 cm) on the Pukou campus. Most nests were open to the east or south with a height range from 3 to 6 m. Distances between boxes were at least 15 m. They were mainly placed in the luxuriant trees just like metasequoia (Metasequoia glyptostroboides), weeping willow (Salix babylonica) and Phoenix Tree (Firmiana platanifolia).\n\nWe used 2 criteria to detect parasitic eggs in a nest4:\n\n1) Partition of egg-laying period. The starling usually lays one egg per day, so if two or more eggs appear a day, or there is an average of >1 egg laid per day during the host egg-laying period, it indicates INP. Additionally, if extra eggs appear outside of the egg-laying period, (e.g. the egg is laid after the host has completed its clutch), it also indicates INP.\n\n2) Morphological differences of eggs. There are individual differences in egg morphological features, so the appearance of eggs which were of a different shape, size and color to other eggs in the clutch would also indicate INP.\n\nThe breeding season of the Silky Starling is from late-April to June. We checked nest boxes every two or three days to record the breeding status, and took notes and photos of any changes in the nests.\n\n\nResults\n\nWe placed 15 nests on the Pukou campus on March 24th. We did not find any nest materials in these boxes until 2 weeks later. The first egg appeared on April 20th and hatched on May 9th. Seven boxes were occupied by Silky Starlings. They were nest numbers 26, 27, 30, 33, 37, 38 and 40. The probability of boxes used by Silky Starlings was 46.7%. We found the 26th, 27th, and 37th nests had parasitic eggs.\n\nIn nest box #26, we found three eggs on April 20th, and six eggs 4 days later. Three days later, we found a 7th egg. Birds normally lay no more than one egg per day. On May 13th, 3 days after the six eggs hatched, the last egg hatched. As the incubation period is consistent within species this suggests that the host had completed its clutch before April 24th, but that on April 27th, another female laid the 7th egg. Unfortunately, the nest was destroyed by an unknown reason (probably predation) with only one dead hatchling left on May 17th. As a result, we confirmed that it was a parasitic egg (Fig. 1A-D).\n\n(A). On April 27th, the seventh egg appeared in the nest, but we could not recognize which one was parasitic as they were all of similar size, shape and color; (B). On May 9th, we found six hatchlings and the last egg unhatched; (C). On May 13th, the last egg hatched (in red circle); (D). On May 17th, we found only a dead hatchling in the nest and the other hatchings had disappeared.\n\nIn nest box #27, we found nest materials on April 28th. On May 6th, we found five eggs. On May 9th, there were six eggs in the nest. We numbered them with a pencil and found that egg # 5 was significantly paler than the others. On May 19th, we found the first hatchling in the nest, and 5 days later, there were five hatchlings. However, egg # 5 was still in the nest (Fig. 2A-B). On May 30th, this egg disappeared. As a consequence, we believe the most likely explanation is that the 5th egg came from another female; predation of the egg is unlikely to be an explanation as we would have observed broken egg fragments if this were the case. During our observation, we found that starlings often clean their nests; they take all excreta away after they have fed the hatchlings each time. This may suggest that the 5th egg was removed by the host when it was found that the 5th egg did not hatch.\n\n(A). On May 9th, we found egg #5 was morphologically different from the others; (B). On May 24th, the hatchlings’ color had become darker and had substantially grown, whilst the extra egg had still not hatched.\n\nIn nest box #37, we found six eggs in the nest on May 6th. We did not find any morphological differences between the six eggs. On May 19th, the eggs hatched. It was not until June 7th that we found another egg in the nest after the nestlings had already fledged (Fig. 3). The egg appeared after the hatching season, so it was likely to be a parasitic egg.\n\nOn 7 June, one parasitized egg was found after the nestlings had fledged.\n\n\nDiscussion\n\nWe had seven nests occupied by Silky Starlings, among them, three were parasitized by other Silky Starling females and so the parasitism rate was 43%. Two of the three parasitic eggs did not hatch, and so the hatching rate of parasitic eggs was 33%. The only hatchling died for unknown reasons. The fledging rate of parasitized eggs was therefore zero.\n\nAccording to the criteria suggested by Yamguch and Saitou, we confirm INP exists in Silky Starlings4. That the extra eggs appeared after the female’s clutch in nests #26 and #37 accords with the first INP criterion, and that the extra egg had a different color to the other eggs in nest #27 accords with the second criterion. With the above limited information, we can at least conclude female starlings can lay their parasitical eggs when they found the hosts had started hatching.\n\nThe parasitism rate seemed high in our study, although our samples were limited. Previous research on Spotless Starlings5 and Grey Starlings4, found intraspecific brood parasitism of Spotless Starlings was 19.1% in colony A and 25.3% in colony B, and parasitism of Grey Starlings occurred in 18.5% of 157 nests in 1992 and in 24.1% of 133 nests in 1993. The main reason behind the high parasitism rate in our study might be attributed to the lack of artificial or natural nests. This campus was built in 1993 and most of the trees were planted during this period. Therefore there are too few natural tree holes as nests for the Silky Starlings, while too many related neighboring species compete for the limited nests, such as Gray Starlings, Collared Owlet (Glaucidium brodiei), and even bumblebees (Vespa manderinia). Our other 8 artificial nests were just occupied by the above three species.\n\nOur findings confirmed INP in Silky Starlings, but more studies should be done. The following questions could be addressed. Why have starlings evolved the strategy of INP? Could the hosts recognize the parasitical egg, or have the hosts evolved an anti-INP strategy? How about the reproductive success of these parasitical eggs? More artificial nests should be placed and longer monitoring should be done in future studies.",
"appendix": "Author contributions\n\nZL was involved in designing the study, collecting and analyzing the data and writing the manuscript. RW and XJ helped in collecting the data and writing the manuscript. ZZ helped in collecting the data. All authors have read and approved the final submitted version of the manuscript.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was financially supported by Natural Science Foundation of China (NSFC No. J1103512 & J0730641).\n\n\nAcknowledgements\n\nWe thank Wei Zheng, Chen Ge, Chaoqun Lin, Changyuan Guo, and Lei Jin for help in placing the artificial nest boxes and checking them regularly. We also thank Jia Chen and Guodong Cai for help in providing a place for us to look after our tools.\n\n\nReferences\n\nYom-Tov Y: Intraspecific nest parasitism in birds. Biol Rev. (1980), 55: 93–108.\n\nYom-Tov Y: An updated list and some comments on the occurrence of intraspecific nest parasitism in birds. Ibis (2001), 143: 133–143.\n\nGong G, Lu X: Intraspecific nest parasitism among birds in China, evidence based mainly on abnormally large clutches. Curr Zool. (2003), 49: 851–853.\n\nYamaguchi Y, Saitou T: Intraspecific nest parasitism in the grey starling (Sturnus cineraceus). Ecol Res. (1997), 12: 211–221.\n\nCalvo JM, Pascual JA, Deceuninck B, et al:Intraspecific nest parasitism in the spotless starling Sturnus unicolor. Bird Study (2000), 47: 285–294.\n\nCramp S: The birds of the Western Palearctic. Oxford: Oxford University Press 1994.\n\nSandell MI, Diemer M: Intraspecific brood parasitism: a strategy for floating females in the European starling. Anim Behav. (1999), 57: 197–202.\n\nJiang X, Wang Rong-rong, LI Zhong-qiu, et al:Breeding behavior of sympatric silky starlings and white-cheeked starlings. Chinese J Ecol. (2012), 31: 2011–2015.\n\nZhao X, Shen Shuang-he, Sun Hu-sheng, et al:An investigation into tourism climate comfortable index in Nanjing. J Nanjing Inst Meteorol. (2008), 31: 250–256."
}
|
[
{
"id": "706",
"date": "23 Jan 2013",
"name": "David Lahti",
"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\n'INP' is not a helpful initialism because the most reasonable alternative to intraspecific nest parasitism is interspecific, which also starts with an 'I'. In the past 'conspecific brood parasitism' or 'conspecific nest parasitism', hence CBP or CNP, has been used. Alternatively, just drop the initialism and use the whole phrase.The writing is substandard in places, with respect to spelling (e.g., ' its' '), colloquialisms (e.g., 'more and more'), grammar (e.g. 'reasons of', or referring to the starling as 'they'); all of these examples are from the introduction.The pattern of nest visits to nest #26, combined with the numbers of eggs found, does not establish that more than one egg was laid in a given day, nor that there was a day gap between two eggs. Seven eggs could have been laid consecutively between April 18th-24th. In this case a nest visit on the 20th after laying would have met with 3 eggs; a nest visit on the 24th before laying would have met with 6 eggs; and a nest visit on April 27th at any time would have met with 7 eggs. This is consistent with the report, so no parasitism can be concluded.There is insufficient evidence of parasitism for nest #27. A paler egg, also failing to hatch, do not constitute conclusive evidence for parasitism. The egg laid in nest #37 a month after one clutch was laid could have been laid by the same or another female. The gap method of determining parasitism is relevant during the laying and incubation period of a female, not a month later.So it turns out that all three of the nests where parasitism is claimed are problematic. There is insufficient evidence here for nest parasitism.",
"responses": []
},
{
"id": "722",
"date": "23 Jan 2013",
"name": "Bruce Lyon",
"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 paper examines the occurrence of intraspecific brood parasitism (INP) in the Silky Starling, whereby females lay eggs in the nests of other species. INP is widespread in birds (over 200 species) but because it is difficult to detect without detailed field or genetic studies, it is likely to be more common than currently believed. Simply documenting the occurrence of INP in new species, along with some estimate of its frequency, is quite useful because it will eventually allow us to gain a solid understanding of the factors that favor the evolution of INP through comparative analysis.The study of Silky Starlings by Zhongqui Li and colleagues uses two standard non-genetic techniques for detecting INP: the occurrence of multiple new eggs in 24 hours and morphological differences in eggs. These techniques have been criticized by some, who advocate that genetic techniques are the only proper way to detect INP. However, in my opinion, non-genetic methods can be very powerful when used carefully and properly. Thus, the power of these approaches depends entirely on how they are used—to yield convincing evidence for INP, nests need to be checked daily when parasitism rates are low (one parasitic egg per host nest), nests need to be checked at the same time of day each day, and it can also be helpful to know the time of day when eggs are normally laid. For example, if there is a 28 hour interval between nest checks, it would be possible to have two eggs laid by the same female, if the time of laying was just after the first check and just before the second nest check. In the present study, I am not convinced that the resolution of the data provide clear evidence for INP. I will discuss each of the cases. Nest box 26. The sequence of events was as follows:3 eggs on the first nest check3 new eggs when the nest was checked 4 days later1 new egg 3 days after thatThese observations do not indicate that more than one egg per day was laid, nor do they necessarily indicate a very long pause between egg six and seven. Depending on the time of days when nests were checked and when Silky Starlings lay eggs, it is possible that there were no days where there were skips in laying (i.e. egg 7 could have been laid 24 hours after egg six) or perhaps there was a skipped day. Even with the later, a skip of one day is not powerful evidence for INP because some birds will occasionally skip a day between laying consecutive eggs. In sum, the evidence for this nest for INP is very weak.Nest box 27. The evidence from this nest was not laying sequence but egg color. From my own study of American coots, I often found that the last laid egg in a clutch was often paler in color. Sometimes the first laid egg can also be different. I think the pattern of last egg being different is quite widespread. I therefore believe that using egg color alone is very weak evidence for brood parasitism, especially because when dealing with the last egg laid in the clutch. Egg size is also not a very accurate indicator of INP because size often varies within clutches. Egg shape can be a pretty good indicator of INP but the validity of this method needs to be assessed for each species.One thing that was not clear was whether the authors numbered the first five eggs before the sixth egg was laid or whether you numbered the eggs after all six were laid. In other words, are you certain that the pale egg was not the last egg? Even if you can show this, I would then want to know if the pale egg was the first egg. Regardless, egg color deviation alone is pretty weak evidence for INP. Box 37. This seems fairly convincing as long as the egg was a new egg and not one that had been buried in the material. Based on the photograph, the egg looks pretty dirty. Are freshly laid eggs often dirty like this? As long as this example involves a new egg then I think this is fairly convincing evidence for INP.To sum up, I do not find most of the evidence for INP very convincing. I think it is too early to be trying to publish this work—bad data are worse than not having data yet for a species. I encourage the authors to not be in such a rush to get these data out and to invest more time carefully collecting data that will be more convincing. Check nests daily, measure the eggs to assess shape, and determine when the birds lay eggs (video cameras can be useful for this). I think it would be worthwhile checking two recent studies that assess some of the methods for detecting INP, one by John Eadie (Journal of avian Biology 41: 163-176; 2010) the other by Hannu Poysa (J. Avian Biol. 40: 453-456; 2009). I think you should check these out. I also think my recent review on INP with John Eadie in Annual Reviews (Annual Reviews in Ecology Evolution Systematics 39: 343-363; 2008) would be a useful source for you to consult.I think the data from this species will eventually be very useful, but the study is not yet at the stage where it can contribute to our understanding of INP.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-71
|
https://f1000research.com/articles/1-70/v1
|
21 Dec 12
|
{
"type": "Short Research Article",
"title": "Rapid doubling of Alzheimer’s amyloid-β40 and 42 levels in brains of mice exposed to a nickel nanoparticle model of air pollution",
"authors": [
"Soong Ho Kim",
"Elysse M Knight",
"Eric L Saunders",
"Azita K Cuevas",
"Marusia Popovech",
"Lung-Chi Chen",
"Sam Gandy",
"Soong Ho Kim",
"Elysse M Knight",
"Eric L Saunders",
"Azita K Cuevas",
"Marusia Popovech",
"Lung-Chi Chen"
],
"abstract": "Background: Over 20 genetic risk factors have been confirmed to associate with elevated risk for Alzheimer’s disease (AD), but the identification of environmental and/or acquired risk factors has been more elusive. At present, recognized acquired risks for AD include traumatic brain injury, hypercholesterolemia, obesity, hypertension, and type 2 diabetes. Methods: Based on reports associating various inhalants with AD pathology, we investigated the possibility that air pollution might contribute to AD risk by exposing wild-type mice to a standard air pollution modeling system employing nickel nanoparticle-enriched atmosphere for 3 hr. Results: Mice exposed to air pollution showed 72-129% increases in brain levels of both amyloid-β peptides Aβ40 and Aβ42, as well as Aβ42/40 (p <0.01). Conclusions: These effects on elevation of brain Aβ exceed those associated with trisomy 21, a known risk for early onset AD pathology, raising the possibility that clinical importance might be attached. Further work is required to establish the molecular and physiological basis for these phenomena. The rapid, dramatic effect, if verified, would suggest that inhalant exposures should be evaluated for their possible roles in contributing to the environmental risk for common forms of AD.",
"keywords": [
"air pollution"
],
"content": "Introduction\n\nOne common neurodegenerative disease, Parkinson’s disease, has been linked to exposure to MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and to inhaled manganese1,2. Similarly, inhaled aluminum dust has been associated with neurotoxic effects and pre-clinical cognitive impairment3. Certain inhalation anesthetics have also been implicated in elevating AD risk, possibly by exacerbating the neurotoxic oligomerization of the amyloid-β (Aβ) peptide4. The early involvement of the olfactory cortex in AD has caused longtime speculation that some inhaled agent might play a role in AD risk5.\n\nRecently, AD pathology was identified in young people living in areas with high levels of air pollution6,7. Furthermore, impaired cognition has been recently attributed to air pollution exposure in some populations8. These converging lines of evidence led us to analyze brain levels of Aβ40 and Aβ42 in mice exposed to an inhaled particulate matter (nickel nanoparticle; Ni NP) model of air pollution.\n\n\nMethods\n\nAll procedures involving animals were conducted in compliance with guidelines for ethical animal research and approved by the New York University School of Medicine Animal Care and Use Committee. Two-month-old male and female FVBN mice (Taconic Farm, Hudson, NY) were randomly assigned to Ni NP inhalation (count median diameter 54 nm, at 1 mg/m3, which is the current Occupational Safety and Health Administration’s Permissible Exposure Limit for nickel hydroxide [http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=standards&p_id=9992]) (n = 16 per group) or control filtered air (n = 5 per group) for 3 hours in a nose-only exposure chamber. This protocol has been established as a model for air pollution toxicity in pulmonary disease9, atherosclerosis10, and insulin resistance11. Twenty-four hours post exposure, mice were given pentobarbital, bled out via the vena cava, and then their brains were harvested, snap frozen and stored at -80ºC until assay. For measurement of endogenous mouse brain Aβ40 and Aβ42, we employed the Schmidt method12 and human/rat Aβ 1–40/1–42 ELISA kits (Wako, Richmond, VA). Statistical analysis was performed via Mann-Whitney test. #8 Ni NP is excluded from the analysis due to being more than 2 SD's away from mean or closest value.\n\n\nResults\n\nBoth endogenous Aβ40 and Aβ42 were elevated in the brains of mice following Ni NP exposure (Figure 1). Aβ40 was increased by 1.72-fold (P = 0.0011, Mann-Whitney test), and Aβ42 was increased by 2.29-fold (P = 0.0005, Mann-Whitney test). Aβ42/40 ratio was also increased in the Ni NP-exposed group compared to the filtered air control group (0.27 ± 0.01 and 0.21 ± 0.007, respectively; P = 0.0093, Mann-Whitney test). Both male and female mice responded similarly to Ni NP exposure (male vs. female for Aβ40 and Aβ42 levels; P > 0.1, Mann-Whitney test).\n\nElevated endogenous mouse brain Aβ40 and Aβ42 in mice exposed to nickel nanoparticles (count median diameter 54 nm, at 1 mg/m3) (n = 16 per group) versus filtered air (n = 5 per group) for 3 hours in a nose-only exposure chamber. Data presented as mean + SEM. **P < 0.01, ***P < 0.001 (Mann-Whitney test).\n\n\nDiscussion\n\nThese data add credence to the proposal4 that one or more inhaled neurotoxin(s) might increase the risk for AD by elevating levels of brain Aβ. We have not identified whether this accumulation occurs at the level(s) of transcription, translation, or post-translational processing. It is tempting to speculate that the well-known links between inhaled toxins and brain inflammation, and other links between brain inflammation and AD established by Griffin and colleagues13 may underlie these phenomena.\n\nThe changes that we observed were dramatic, rapid, and unexpected. Human Aβ is more aggregatable than murine Aβ, making it conceivable that the effect on Aβ levels in human brain could be even greater. While elucidating the genesis and molecular underpinnings will be an important next step, an even more important step will be a rigorous application of environmental toxicology and epidemiology to determine whether the elevated brain Aβ caused in mice by this air pollution model corresponds to any situation of authentic human inhalation exposure that is linked to an increased risk for AD.",
"appendix": "Author contributions\n\n\n\nS.H.K., E.M.K., E.L.S. and A.K.C. designed the experiments with L.C.C. and S.G. S.H.K., E.L.S., A.K.C. and M.P. performed the experiments. E.M.K. analyzed the data. S.G. wrote the manuscript. All authors commented on the manuscript. L.C.C. and S.G. supervised the project.\n\n\nCompeting interests\n\n\n\nS.G. holds research grant support from Amicus Pharmaceuticals and Baxter Pharmaceuticals; he is a consultant to Balance Pharmaceuticals and Diagenic; and he is a member of the Data and Safety Monitoring Board for the Pfizer-Janssen Alzheimer's Immunotherapy Alliance.\n\n\nGrant information\n\nThis work was supported by the Cure Alzheimer's Fund (S.G.), VA MERIT Review Award I01BX000348 (S.G.), and the American Health Assistance Foundation A2012625 (S.H.K.). This work was also supported by National Institutes of Health Grant U01ES020126 and P30ES00260 (L.C.C.).\n\n\nReferences\n\nLangston JW, Ballard P, Tetrud JW, et al.: Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science. 1983; 219(4587): 979–80. PubMed Abstract | Publisher Full Text\n\nCotzias GC, Papavasiliou PS, Ginos J, et al.: Metabolic modification of Parkinson's disease and of chronic manganese poisoning. Annu Rev Med. 1971; 22: 305–326. PubMed Abstract | Publisher Full Text\n\nMeyer-Baron M, Schäper M, Knapp G, et al.: Occupational aluminum exposure: evidence in support of its neurobehavioral impact. Neurotoxicology. 2007; 28(6): 1068–1078. PubMed Abstract | Publisher Full Text\n\nXie Z, Xu Z: General anesthetics and β-amyloid protein. Prog Neuropsychopharmacol Biol Psychiatry. 2012. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPearson R, Esiri M, Hiorns RW, et al.: Anatomical correlates of the distribution of the pathological changes in the neocortex in Alzheimer disease. Proc Natl Acad Sci U S A. 1985; 82(13): 4531–4534. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalderon-Garciduenas L, Reed W, Maronpot RR, et al.: Brain inflammation and Alzheimer's-like pathology in individuals exposed to severe air pollution. Toxicol Pathol. 2004; 32(6): 650–658. PubMed Abstract | Publisher Full Text\n\nCalderón-Garcidueñas L, Kavanaugh M, Block M, et al.: Neuroinflammation, hyperphosphorylated tau, diffuse amyloid plaques, and down-regulation of the cellular prion protein in air pollution exposed children and young adults. J Alzheimers Dis. 2012; 28(1): 93–107. PubMed Abstract | Publisher Full Text\n\nWeuve J, Puett RC, Schwartz J: Exposure to particulate air pollution and cognitive decline in older women. Arch Intern Med. 2012; 172(3): 219–227. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGillespie P, Kang GS, Elder A, et al.: Pulmonary response after exposure to inhaled nickel hydroxide nanoparticles: short and long-term studies in mice. Nanotoxicology. 2010; 4(1): 106–119. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang G, Gillespie PA, Gunnison A, et al.: Long-term inhalation exposure to nickel nanoparticles exacerbated atherosclerosis in a susceptible mouse model. Environ Health Perspect. 2011; 119: 176–181. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu X, Liu C, Xu Z, et al.: Long-term exposure to ambient fine particulate pollution induces insulin resistance and mitochondrial alteration in adipose tissue. Toxicol Sci. 2011; 124(1): 88–98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmidt S, Jiang Y, Nixon R, et al.: Tissue processing prior to protein analysis and amyloid-beta quantitation. Methods Mol Biol. 2005; 299: 267–278. PubMed Abstract | Publisher Full Text\n\nGriffin WS, Sheng JG, Royston MC, et al.: Glial-neuronal interactions in Alzheimer's disease: the potential role of a 'cytokine cycle' in disease progression. Brain Pathol. 1998; 8(1): 65–72. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "714",
"date": "21 Jan 2013",
"name": "W.Sue T Griffin",
"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\nNeurodegenerative consequences are a looming possibility with the current increased burden of air-borne pollutants. Many studies have suggested an association between anesthesia administered to older adults and cognitive decline and development or progression of Alzheimer’s disease (AD). A number of studies have already established links between air-borne pollutants and risk for development of systemic disorders several of which, like AD are inflammatory in nature. Gandy and his colleagues have taken a purposeful step toward more directly connecting air-pollution to increased risk for development of AD in a study of wild type mice exposed to a toxin at the OSHA permissible level for an 8h human exposure. The results are very convincing, showing that a one-time 3h exposure to nickel hydroxide nanoparticles at the “permissible” level doubled brain levels of Aβ40 and Aβ42 within 24h—increases that are similar to levels reported in younger adults with Down’s syndrome. Interestingly, as noted by the authors, and in accord with systemic inflammatory consequences reported following exposure to air-borne pollutants, neuroinflammatory changes that drive Alzheimer neuropathological change may also be elicited by such exposure. This may be particularly important as we necessarily inhale air that appears to be “clean,” but which contains permissible levels of agents that may have adverse effects on the brain, especially in persons with genetic risk factors and or co-morbid conditions that already predispose them for development of AD.",
"responses": []
},
{
"id": "725",
"date": "23 Jan 2013",
"name": "George Perry",
"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\nLinkage of environmental air toxicity from nanoparticles leading to Alzheimer-like changes in the brain of mice opens a new avenue to understanding the development of brain diseases. These findings are consistent with prior work showing particulate air pollution can affect the brains of human as well as dogs living in polluted cities.",
"responses": []
},
{
"id": "718",
"date": "25 Jan 2013",
"name": "Ottavio Arancio",
"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 intriguing observation that might potentially have important relevance to the etiopathogenesis of Alzheimer’s Disease.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-70
|
https://f1000research.com/articles/1-69/v1
|
19 Dec 12
|
{
"type": "Review",
"title": "Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?",
"authors": [
"Miriam Matamales"
],
"abstract": "Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.",
"keywords": [
"Among the hundreds of distinct cell types that make up our bodies",
"neurons are the most morphologically complex",
"and also one of the most dynamic in their responsiveness and adaptability. Neurons contain structural specialisations",
"which allow the rapid processing and transmission of the thousands of synaptic inputs that they simultaneously receive. The vast majority of excitatory synapses involving the neurotransmitter glutamate are made into dendrites",
"which are extensions of the plasma membrane that can span hundreds of microns and cover broad fields in the tissue1. Release of glutamate into the synaptic cleft induces transient postsynaptic electrical and biochemical responses",
"which can eventually promote stable changes in the properties of the neuron2. At an excitatory synapse",
"glutamate acts on both metabotropic (mGluRs) and the ionotropic N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors3",
"4. mGluRs are G-protein coupled receptors (GPCRs) that are linked to heterotrimeric G proteins on the intracellular side of the membrane. Activation of mGluRs by glutamate modulates the activity of various signal transduction pathways through the change in concentration of intracellular second messengers such as intracellular inositol 1",
"4",
"5-trisphosphate (IP3) and cyclic adenosine monophosphate (cAMP)",
"which controls the cAMP-dependent protein kinase (PKA)5. Glutamate binding to AMPA receptors (AMPARs) opens the ion channel and induces fast depolarisation of the postsynaptic membrane",
"mainly through the influx of sodium (Na+) ions. By contrast",
"glutamate-gated channel opening of NMDA receptors enables calcium (Ca2+) influx into the dendritic spine",
"which initiates a cascade of signalling events involving the stimulation of the Ca2+/calmodulin-dependent protein kinase (CaMK) as well as the extracellular signal regulated kinase (ERK). The stimulation of CaMK and ERK triggers the phosphorylation-induced activation of a myriad of cellular targets including ion channels and transmembrane receptors",
"which in turn modifies their conductance properties6",
"7."
],
"content": "Introduction\n\nAmong the hundreds of distinct cell types that make up our bodies, neurons are the most morphologically complex, and also one of the most dynamic in their responsiveness and adaptability. Neurons contain structural specialisations, which allow the rapid processing and transmission of the thousands of synaptic inputs that they simultaneously receive. The vast majority of excitatory synapses involving the neurotransmitter glutamate are made into dendrites, which are extensions of the plasma membrane that can span hundreds of microns and cover broad fields in the tissue1. Release of glutamate into the synaptic cleft induces transient postsynaptic electrical and biochemical responses, which can eventually promote stable changes in the properties of the neuron2. At an excitatory synapse, glutamate acts on both metabotropic (mGluRs) and the ionotropic N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors3,4. mGluRs are G-protein coupled receptors (GPCRs) that are linked to heterotrimeric G proteins on the intracellular side of the membrane. Activation of mGluRs by glutamate modulates the activity of various signal transduction pathways through the change in concentration of intracellular second messengers such as intracellular inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine monophosphate (cAMP), which controls the cAMP-dependent protein kinase (PKA)5. Glutamate binding to AMPA receptors (AMPARs) opens the ion channel and induces fast depolarisation of the postsynaptic membrane, mainly through the influx of sodium (Na+) ions. By contrast, glutamate-gated channel opening of NMDA receptors enables calcium (Ca2+) influx into the dendritic spine, which initiates a cascade of signalling events involving the stimulation of the Ca2+/calmodulin-dependent protein kinase (CaMK) as well as the extracellular signal regulated kinase (ERK). The stimulation of CaMK and ERK triggers the phosphorylation-induced activation of a myriad of cellular targets including ion channels and transmembrane receptors, which in turn modifies their conductance properties6,7.\n\nAdditionally, an important consequence of the activation of these signalling pathways upon excitatory neurotransmission is that they can regulate the activity of nuclear factors thereby triggering changes in gene transcription8. The mechanism by which neuronal activity is conveyed to the nucleus for the induction of activity-dependent gene expression programs is one of the most investigated topics in neuroscience, since it is believed to be necessary for the establishment of memories. Indeed, mRNA synthesis inhibitors such as actinomycin D have been shown to prevent late long-term potentiation in hippocampal slices and to impair retention of new memories in several species and learning paradigms9–13. Interestingly, former studies regarding activity-dependent regulation of transcription showed that induction of genes occurs within a few minutes after excitatory electrical and pharmacological stimulation14–17. These studies demonstrate that, in spite of the highly polarised morphology of neurons, synaptic signals are rapidly conveyed to the nucleus to allow the immediate regulation of gene transcription.\n\nVarious mechanisms have been proposed that might couple synaptic activity to gene transcription18–20. The main difference among these models relies on the nature of the signal that carries the message, i.e. synapto-nuclear messengers, IP3-triggered Ca2+ waves or action potentials. This review will critically discuss the primary experimental findings supporting the current models for communication between synapses and the nucleus and ascertain their potential role in efficiently and timely coupling neuronal activity to gene expression.\n\n\nTranslocation of transcription regulators from synapses to the nucleus\n\nThe restriction of effector proteins to particular subcellular locations is a commonly employed strategy used by most known signal transduction cascades, and allows coordination of signalling events in space and time21–23. In many cases, the activation of signalling proteins by upstream regulators, or their activation of downstream effectors, involves their rapid and regulated translocation to specific subcellular compartments24. Indeed, nuclear translocation of signalling molecules is known to play a role in the timely expression of genes in response to extracellular stimuli25–27. For example, although PKA and ERK are preferentially distributed in the cytoplasm in resting conditions, the rise of intracellular second messengers drives their rapid accumulation in the nucleus, where they phosphorylate multiple nuclear targets28,29. Furthermore, transcription factors can also relocate from the cytoplasm to the nucleus in response to stimuli that induce apoptosis, cell differentiation or proliferation30–32. There are several advantages of moving signalling proteins between neighbouring subcellular compartments upon cellular activity, including the enhancement of the specificity as well as speed of signal transmission22–34. Interestingly, there is strong evidence demonstrating that a variety of stimuli mimicking neuronal activity, such as bath application of neurotransmitters or electrical stimulation, also induce the cyto-nuclear translocation of signalling proteins in primary neuronal cultures and brain slice preparations35,36. Moreover, the robust accumulation of gene transcription regulators in the nucleus is also observed in response to a wide variety of physiological and pharmacological stimuli in vivo. For example, immunohistological analysis of mouse brain sections have shown that the rapid nuclear accumulation of activated ERK in various regions of the brain including the hippocampus, amygdala and projection neurons of the striatum, occurs in animals that have been trained to behavioural paradigms that underlie learning, or following acute administration of addictive and non-addictive drugs37–41.\n\n\nSynapse-to-nucleus protein translocation\n\nAlthough the translocation of proteins has been demonstrated to occur between adjacent subcellular compartments i.e. from the cytoplasm to the plasma membrane or from the cytoplasm to the nucleus, it has been hypothesised that it can also occur between more distant compartments, linking dendritic terminals and axon tips with the nucleus42,43. Indeed, the retrograde transport of signals from distal axons to neuronal cell bodies by motor-dependent transport along microtubules is one of the best known mechanisms for long-distance communication44. So far, these mechanisms have been studied in the context of development, survival and axonal injury45. However, recent studies have proposed that activity at excitatory synapses can also promote the movement of proteins from distant parts of the dendritic arbour to the nucleus (Reviewed in18,20,46). This idea of long distance rather than local signalling in response to synaptic inputs provides a tantalising model in which the transport of transcription regulators from stimulated synapses serves to inform the genome about peripheral neuronal activity (Figure 1).\n\na) In non-stimulated conditions, transcription regulators (TR, purple dots) are localised into distant dendrites as well as at the perinuclear zone. Some TRs are transported from the cytoplasm to the nucleus by importins (orange dots). Importins are also distributed across distant dendrites and at the postsynaptic density (PSD). b, c) Excitatory inputs that stimulate synapses (1) are believed to activate TRs, which are then transported to the soma along the neuron (2) through microtubule-based active transport b) or by passive diffusion c). Excitatory inputs are also proposed to activate importins at the PSD, which then associate to synaptic cargoes and facilitate their synapto-nuclear translocation c). Therefore, this model predicts that synaptic activity triggers the accumulation of TRs from distant synapses in the nucleus, promoting the induction of gene expression programs (3).\n\nFormer experimental evidence supporting this hypothesis comes from early studies of brain cell fractionation that were published by different laboratories in the 1990s43,47,48. Intriguingly, it was reported that the nuclear factor κ light chain enhancer of activated B-cells (NF-κB) can be detected in the synaptosomal fraction of cortical and hippocampal preparations, which contains pre- and post-synaptic elements of neurons. Additional subsequent studies have later shown that other transcription factors are also located at synaptosome preparations, including the cAMP response element-binding protein (CREB)49, CREB250 and CREB-regulated transcription coactivator 1 (CRTC1)51. Additionally, fluorescence microscopy analyses of endogenous proteins or fluorescently tagged chimeric constructs conducted to determine the subcellular localisation of these transcription regulators in neurons indicate that they are indeed not confined to the vicinity of the nucleus, but rather extend throughout the whole cytoplasm, reaching distant compartments like dendritic spines. Provided that the only recognised function of these transcription factors takes place in the nucleus where their known cellular targets belong, the intriguing question of how they can be efficiently and timely shuttled to the nucleus to influence transcription arises naturally.\n\nMartin and colleagues have proposed a mechanism for the shipping of activated transcription regulators between synapses and the nucleus52,53. It involves the participation of members of the karyopherin family of nuclear transport receptors, which are soluble carriers that mediate the regulated translocation of proteins across the nuclear envelope54,55. The active import of proteins from the cytoplasm to the nucleus is mostly driven by a heterodimeric carrier composed of importin-β and its adaptor importin-α. Importins discriminate their cargo from other cellular proteins by recognition of amino acid targeting sequences known as nuclear localisation signals (NLSs), which contain one or two clusters of basic residues. Monopartite NLSs, exemplified by the SV40 large-T antigen56, have a single cluster of 4–5 basic residues (e.g. P KKKRKV), whereas bipartite NLSs, such as that of nucleoplasmin57, have a second basic cluster located 10–12 residues downstream from the first cluster (e.g. KRPAATKKAGOA KKKLD). Interestingly, immunofluorescence studies have detected importins far away from the nucleus, including dendrites and the axon of mouse hippocampal and Aplysia sensory neurons, suggesting a role in long-distance trafficking of proteins towards the nucleus52. Furthermore, strong accumulation of importins in the nucleus is observed in response to stimuli that mimic synaptic activity in neuronal primary cultures and hippocampal slice preparations. These observations led to the hypothesis that importins may direct the translocation of synaptic proteins to the nucleus53. In this line, a study by Holmes and co-workers reported that the cytoplasmic tail of the NR1-1a subunit of the NDMAR contains a putative bipartite NLS which is surrounded by four phosphorylation sites i.e. PDP KKKATFRAITSTLASSF KRRRSSKDT58,59. Based on these results, subsequent co-immunoprecipitation experiments by Jeffrey and colleagues showed that importin-α binds to NR1 in vitro, and that this interaction is disrupted by the activity-dependent phosphorylation of the residues flanking the NLS of NR1-1a60. The authors presented a model in which importins are normally tethered to the NLS of the NMDAR in non-stimulated synapses, placing them in close proximity to synaptically localised transcription regulators. Upon stimulation, phosphorylation of the NR1-1a tail by PKA and protein kinase C (PKC) promotes the release of bound importins that are then free to bind soluble synaptic NLS-containing cargoes such as the transcription factor CREB250. In view of this appealing mechanism for the long-range transport of proteins to the nucleus, a series of proteomic and bioinformatic studies have been conducted in order to identify novel NLS-containing proteins undergoing importin-mediated synapto-nuclear translocation61. Surprisingly, around 166 out of 1100 proteins from postsynaptic density (PSD) purified fractions were predicted to contain putative NLSs62. Among them, there were found several proteins involved in RNA trafficking and splicing, as well as regulators of transcription factor activity61,63.\n\n\nLimitations and challenges to the model\n\nAlthough a number of arguments support the existence of synapto-nuclear protein messengers, there are many remaining concerns that challenge the synapto-nuclear translocation model. One important caveat to be considered regards the identification of potential synapto-nuclear messengers by mass spectrometry. It has been observed that due to the high sensitivity of protein identification by this method, low-level contaminants introduced during the biochemical purification of brain fractions are commonly detected64. Moreover, searching for synapto-nuclear messengers by NLS identification may yield false positive results61, since similar clusters of basic residues are present in kinase and phosphatase docking sites and help to mediate protein-protein interaction65. For example, the consensus phosphorylation site for PKC is R/K1–3, X2–0-S/T whereas for PKA, it is R-R-X-S/T-Φ, where X is any amino acid and Φ is a hydrophobic residue which closely resembles the above mentioned NLS found in the NR1-1a subunit of the NMDA receptor66. Accordingly, a detailed functional dissection of these motifs needs to be performed before concluding that they represent bona fide NLSs. Experiments tracking fluorescently tagged proteins through live-cell imaging techniques as well as the use of nuclear import assays in permeabilised cells should be routinely used in order to confirm the possible nuclear translocation of these proteins67–69.\n\nAnother point that needs to be directly demonstrated is whether transcription regulators distributed at distant parts of the neuron actually shuttle to the nucleus. Recent experiments using cultured hippocampal neurons that express a photoconvertible fluorescent form of the synapto-nuclear transcription factor CRTC1 indeed suggest that activation of distant dendrites by local glutamate uncaging can promote its accumulation to the nucleus51. However, the movement of single synaptic messengers on route to the nucleus has never been directly tracked, and therefore this point remains to be clarified. The combination of super-resolution fluorescence microscopy with live-cell single-molecule-based imaging may provide new insights into the dynamics of the long-range transport of proteins in neurons70.\n\nAn important question regarding the long-range transport of proteins to the nucleus concerns the speed at which these messengers may travel towards the soma, which is crucial to explain their potential involvement in timely coupling of neuronal activation to the robust induction of gene programs71. It has been estimated that a transcription factor involved in the rapid induction of activity-dependent genes should travel at nearly 75 microns per minute towards the nucleus, but NF-κB has been calculated to move at only 2 microns per minute72. It is currently unknown whether such a rapid movement of proteins from synapses to the nucleus may occur by diffusion, facilitated transport, signalling endosomes or whether it requires association with the microtubule network. Moreover, a significant limitation of long-range signalling by the synapto-nuclear shuttling of transcription factors is that the signal is not amplified nor regenerated on its way towards the nucleus. Distantly stimulated messengers are likely to be inactivated or lost in transit, thereby compromising the direct involvement of synaptic activity on the control of nuclear functions. For example, if a transcription factor is activated by phosphorylation at the synapse, protein phosphatase activity will likely cause its inactivation on its way to the nucleus. Finally, a critical issue that needs to be addressed is to determine the advantage, if any, of mobilising transcription regulators from distant synapses over those already located adjacent to the nucleus to control gene expression.\n\nDespite the list of synapto-nuclear messengers is rapidly growing, there are considerable ambiguities in the synapse-to-nucleus translocation model that need to be clarified. Further research is essential to gain a more accurate understanding of the presumed role of these signalling mechanisms on the coupling of excitatory synaptic transmission to gene induction during neuronal plasticity.\n\n\nPropagation of calcium waves from synapses to the nucleus\n\nIt is now well documented that the rise of Ca2+ concentration in the nucleus represents an essential step for the activity-dependent induction of gene expression programs19,73–75. Cytoplasmic Ca2+ concentration can increase via voltage- and ligand-gated channels as well as by release from intracellular Ca2+ stores76. The main internal Ca2+ store is the endoplasmic reticulum (ER), which acts both as a sink for Ca2+ that enters from the extracellular space, and as a source for Ca2+ release into the cytosol. The ER forms a continuous vesiculo-tubular system that constitutes an elaborated network distributed throughout the cytoplasm77. In neurons, this network extends from the outer nuclear envelope in the soma into axonal processes and dendritic arborisations, including spines78,79. Release of Ca2+ from the ER can occur upon increase of intracellular IP3 and adenosine diphosphate ribose (ADP-ribose), which bind to and open IP3 receptors (IP3Rs) and ryanodine-type receptors (RyRs), respectively80. As mentioned above, IP3 can be generated by the stimulation of mGluRs which can be coupled to phospholipase C (PLC), the enzyme that cleaves the membrane phospholipid phosphatidylinositol 4,5-bisphosphate to form IP381,82. Moreover, Ca2+ itself activates the opening of RyRs and enhances the Ca2+-releasing action of IP3, therefore stimulating the efflux of more Ca2+ from the ER lumen83. This process of Ca2+-induced Ca2+ release (CICR) is believed to establish regenerative Ca2+ waves that spread bidirectionally from their initiation site and was first discovered in skeletal muscle84,85. Indeed, CICR is considered to be the physiological mechanism of Ca2+ release in cardiac muscle, playing a major role in cardiac excitation-contraction coupling86.\n\nThe development of fluorescent Ca2+ indicator molecules, photolysis techniques and laser-scanning microscopy has enabled the investigation of the Ca2+ dynamics in neurons at very fine temporal and spatial resolutions87–90. Synaptically activated, IP3-mediated propagation of Ca2+ release has been observed in pyramidal neurons in the rodent hippocampus (CA1 and CA3 regions), medial prefrontal cortex and principal neurons in the amygdala91–95. Depending on the neuronal cell type analysed and the stimulation protocol used, dendritic Ca2+ waves can be highly localised or can spread to a larger extent96. For example, when IP3 is generated by focal synaptic stimulation with metabotropic agonists, the generated response is weak and restrained97–99. By contrast, when IP3 production is stimulated by bath application of such agonists, a higher response can be evoked and a rise in Ca2+ concentration can be observed in the nucleus99,100.\n\nThese observations have led to the hypothesis that excitatory inputs trigger IP3R-dependent CICR waves that are able to spread forward from the distant synapses towards the soma and mediate the release of Ca2+ from the inner membrane of the nuclear envelope into the nucleus (Figure 2)76. Therefore, it has been suggested that the process of CICR may serve to communicate between distant cellular compartments in neurons and serve as a means to mediate the coupling of synaptic activity to gene transcription. While this model has the advantage over the synapto-nuclear translocation model of being a regenerative process in which the synaptic signal does not decay over great distances, evidence for the distant communication by this mechanism is controversial. First, it is not well established whether functional Ca2+ channels are present in the nuclear envelope in neurons, and if they can indeed release Ca2+ into the nucleoplasm101–103. More detailed studies on the spatial distribution of receptors involved in Ca2+ release from the ER in neurons may resolve such discrepancies. Second, it is important to note that whereas Ca2+ alone is sufficient to cause its own release through RyRs104–106, it cannot do so through IP3Rs. In the case of IP3Rs, Ca2+ can cause self-release only in the presence of IP3, thus the availability of IP3 in the cytoplasm is a major factor determining the extent of Ca2+ wave propagation107–109. Because of these findings, CICR is generally considered as an exclusive property of RyRs, but not of IP3Rs, even though IP3R exhibits the apparent CICR behavior in the presence of IP386,110,111.\n\nThe endoplasmic reticulum (ER) is distributed throughout the cytoplasm from the nuclear envelope to dendritic spines. Excitatory synaptic stimulation through glutamate causes membrane depolarisation and entry of Ca2+ (blue dots) from the extracellular space through NMDARs (1). Moreover, glutamate also activates mGluRs coupled to PLC thereby stimulating IP3 production (green dots). Ca2+ and IP3 stimulate receptors present at the ER membrane that open and release more Ca2+ from the ER lumen, establishing a Ca2+-induced Ca2+-release wave (2) that propagates from dendrites towards the soma. In the soma, Ca2+ release from the ER activates cytoplasmic Ca2+-dependent signalling cascades that convey the signal to the nucleus (3). Moreover, Ca2+ can be released to the nucleus from the ER, where it activates nuclear transcription regulators (4).\n\nAs noted above, experiments in different types of neurons suggest that the spread of IP3-mediated Ca2+ waves depends on the availability of IP3 in the preparation96. Although IP3 has a fast diffusion constant, it has a limited spatial range of action as it is rapidly metabolised by 3-kinase and 5'-phosphomonoesterase and it is estimated to have a lifetime of a few seconds112,113. Moreover, during the past few years it has become clear that there are functional compartments existing within dendritic trees that control the diffusion of intracellular messengers, particularly within spines87,114. Hence, the restriction of mobilised IP3 to sites close to activated synapses makes it unlikely that synaptically triggered IP3-dependent CICRs spread to the soma and transport a message from the synapse. However, functional metabotropic receptors coupled to IP3 production are present at the level of the soma in the plasma and the inner nuclear membranes of several neuron types, raising the possibility that local rather than remotely generated IP3-dependent release of Ca2+ from the ER may contribute to the induction of gene transcription115–118.\n\n\nPropagation of electrical signals along the plasma membrane\n\nThe models discussed in the previous sections are built on the assumption that activation of postsynaptic glutamate receptors triggers the transmission of intracellular messengers (i.e. transcription regulators and Ca2+ waves) from distant dendritic sites to the nucleus. However, several lines of evidence have suggested that gene expression can be triggered by NMDAR and mGluR-independent mechanisms119,120. Experiments using dorsal root ganglion cell culture preparations, which are devoid of synapses, demonstrated that phosphorylation of nuclear factors and induction of genes could be induced with electrical stimulation121. Furthermore, electrophysiological studies using hippocampal slice preparations have shown that somatic action potentials can trigger gene transcription without the involvement of synaptic activation and in the presence of NMDAR antagonists119,120,122. However, NMDARs appeared to have a role in the activation of nuclear events through their contribution to action potential generation122. These results led to a third model proposed by Dudek and colleagues, which posits that the robust depolarisation of the somatic plasma membrane is sufficient to induce gene transcription without the involvement of transported biochemical signals from distant synapses71,72,123. According to this model, transmission of synaptic events from distant compartments to the soma is achieved through the propagation of electrical signals over long distances across the plasma membrane. There are several mechanisms of long-range electrical propagation that regulate the degree of depolarisation at the soma. A simplified explanation of the process is briefly provided here, however the reader is referred to the excellent available reports fully addressing this topic124,125. On one hand, excitatory synaptic events lead to changes in the membrane conductance that can be forward-propagated to the soma in the form of dendritic spikes126,127. On the other hand, action potentials initiated at the axon initial segment can be retrogradely propagated into the soma and the dendritic tree, spreading back the electrical signal128. The range of propagation efficacies of dendritic spikes and backpropagated action potentials varies in different cell types and is influenced by multiple passive and active properties of the somatodendritic compartment, including dendritic geometry, voltage-gated channel densities and the spatial and temporal profile of synaptic excitation and inhibition125,129,130.\n\nIt is well established that membrane depolarisation in the soma causes the opening of voltage-gated Ca2+ channels (VGCCs), which allow the influx of Ca2+ from the extracellular space to the cytoplasm, thus coupling synaptic activity to intracellular signalling14,131. Interestingly, it appears that, among the different classes of VGCCs, L-type VGCCs seem to be significantly involved in regulating transcription since activity-dependent induction of genes is supressed by exposure to specific antagonists and increased by (-)BayK-8644, a VGCC agonist14,132,133. The differential ability of L-type VGCCs to preferentially convey signals to the nucleus over the other types of VGCCs may be due to their relatively slow inactivation rate, high single-channel conductance for Ca2+ and relative localisation to Ca2+ intracellular sinks134,135. In addition, Ca2+ entry through L-type VGCCs has been suggested to trigger ER-directed Ca2+ waves that propagate from the plasma membrane of the soma to the nucleus without the involvement of cyto-nuclear translocation of proteins73,76. These findings suggest that L-type VGCCs at the soma are well suited for coupling synaptic excitation to activation of transcriptional events.\n\nIt is important to note that the degree of membrane depolarisation, and thus the opening of L-type VGCCs, is influenced by the immediate electrical history of the neuron. Because postsynaptic events to individual synaptic inputs are usually small and transient, action potential initiation and dendritic spikes require strong synchronous synaptic activation to be generated124. Moreover, inhibitory inputs also affect the way in which excitatory synaptic inputs summate in space and time. Thus, the elaborate integration of excitatory and inhibitory inputs will govern the opening of VGCCs, thereby shaping the activation of Ca2+-dependent nuclear functions125. Taken together, the model upheld by Dudek and colleagues provides a cellular mechanism by which synaptic activity is coupled to gene transcription through VGCCs, which transduce electrical signals into biochemical pathways (Figure 3). Through this mechanism, signalling molecules are only required to translocate between neighbouring subcellular compartments, as observed in many other cell types, conveying information timely and efficiently.\n\nExcitatory synaptic activity (arrowheads) generates local changes in membrane conductance, which causes the opening of voltage gated calcium channels (VGCCs). A rise in intracellular Ca2+ triggers the local activation of Ca2+-dependent signalling pathways thereby, coupling membrane depolarisation to intracellular signalling. a) When neurons receive weak excitatory inputs (small arrowheads), the signal spread is small (thin solid arrows) and the threshold to trigger action potentials is not reached, thus somatic L-VGCCs remain closed. b) When neurons receive strong synaptic inputs (big arrowheads), dendritic spikes can efficiently spread (thick solid arrows) in the forward direction and facilitate the initiation of action potentials at the axonal initial segment. Action potentials are backpropagated to the soma and dendrites (dashed arrows), locally generate intracellular Ca2+-dependent signalling cascades. Ca2+ entry in the soma through L-VGCCs will promote the activation of protein effectors that regulate the transcription of plasticity-related genes. In this diagram, the local signal intensity representing both the electrical activity of the membrane and its coupled intracellular signalling is coded by colour. Drawings are adapted from a reconstructed biocytin-filled layer V neuron in the rat cortex (courtesy of B. Chieng and J. Bertran-Gonzalez).\n\nImportantly, this model predicts that action potentials will fail to induce gene transcription programs unless firing above a critical threshold, suggesting that intracellular mechanisms must exist to assess the degree of synaptic stimulation123. Knowledge of how the nucleus computes action potentials to promote gene transcription while avoiding background stimulation is therefore fundamental for understanding how synaptic inputs regulate activity-dependent gene transcription and neuronal plasticity.\n\n\nConclusion\n\nAlthough it has been known for more than 25 years that gene induction upon excitatory transmission is a required step for neuronal plasticity, it is not yet clear whether a direct link between local synaptic activation and gene transcription exists. An increasing body of literature suggests that there are at least three different pathways by which neurons may inform the nucleus about the events happening at distant dendritic compartments18–20. In addition to the unique feature of neurons of communicating messages over long distances by electrical signals, it has been recently proposed that the long-range physical translocation of proteins from remote dendritic sites towards the soma can convey information to the nucleus20,46. The current evidence for the synapto-nuclear translocation model shows that transcription regulators, which were previously considered to be largely localised in close proximity to the nucleus, may also be present at remote sites from the nucleus, in postsynaptic dendritic compartments. Moreover, different stimuli that promote neuronal excitation trigger the accumulation of these gene transcription regulators in the nucleus, although it is still unclear whether they are the same ones activated at distant parts of the neuron. Several critical questions remain enigmatic, as to how and how fast the synapto-nuclear messengers are shipped to the nucleus, and what their actual roles are on gene induction. Taken together, and given the experimental data now available, it is very unlikely that this mechanism participates significantly in the activity-dependent regulation of gene transcription. Future experimental evidence would help to clarify their involvement in other aspects of neuronal physiology.\n\nThe most plausible scenario for relaying synaptic activity to the nucleus involves the robust propagation of action potentials, which are initiated upon integration of excitatory, inhibitory and modulatory inputs123. Influx of Ca2+ from the extracellular space through ligand-gated receptors (e.g. NMDAR) and VGCCs triggers intracellular signalling cascades that remain spatially confined and act locally to regulate cellular processes136. In the soma, the opening of L-type VGCCs causes a rise in intracellular Ca2+ and thereby activates Ca2+-dependent signalling molecules that shuttle from the cytoplasm to the nucleus such as activated ERK and NF-κB74. Moreover, it may also allow the generation of RyR- or IP3R-dependent Ca2+ waves that propagate along the ER from the plasma membrane to the nucleus inducing the increase of Ca2+ concentration within the nucleus76. In addition, it is anticipated that Ca2+-transduction pathways crosstalk with simultaneously activated signalling cascades governed by other second messengers such as IP3 and cAMP that will promote or restrain the spread of the Ca2+ signal and will have a profound influence on the precise timing and extent of gene induction. Future research will need to determine how these coordinated processes contribute to shape neuronal plasticity.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nM. Matamales is a recipient of an EMBO Long-Term Fellowship (ALTF 1228–2010).\n\n\nAcknowledgements\n\nThe author would like to thank Dr. J. Bertran-Gonzalez and Dr. M. Cavazzini for their valuable comments on the manuscript.\n\n\nReferences\n\nFiala JC, Spacek J, Harris KM, et al.: Dendrite structure.Dendrites. Oxford University Press 2nd edition 2008.\n\nFlavell SW, Greenberg ME: Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous system. Annu Rev Neurosci. 2008; 31: 563–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakanishi S: Metabotropic glutamate receptors: synaptic transmission, modulation, and plasticity. Neuron. 1994; 13: 1031–7. PubMed Abstract | Publisher Full Text\n\nNakanishi S, Nakajima Y, Masu M, et al.: Glutamate receptors: brain function and signal transduction. Brain Res Rev. 1998; 26: 230–235. 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}
|
[
{
"id": "702",
"date": "11 Jan 2013",
"name": "Michael Shiflett",
"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 review on the possible mechanisms linking synaptic activity to gene transcription. The author provides a thoughtful consideration of technical and analytic challenges confronted by researchers addressing this question. The outline of a model of how synaptic activity signals are conveyed to the nucleus is given. This model, in conjunction with local synaptic 'tagging' hypotheses, provides a clearer picture of a complex process underlying long-term changes at synapses.",
"responses": []
},
{
"id": "723",
"date": "23 Jan 2013",
"name": "Anne West",
"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 concise, well-written review on synapse to nucleus signaling that does an exemplary job not only of reviewing what experiments have been done in this field but also laying out what data are required to more fully address each of the models proposed.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-69
|
https://f1000research.com/articles/1-68/v1
|
19 Dec 12
|
{
"type": "Case Report",
"title": "Glenoid Labral Tear: follow up case series on ultrasound guided autologous platelet rich plasma in conjunction with a progressive rehabilitation program",
"authors": [
"Robert Vander Kraats",
"Arockia Doss",
"Arockia Doss"
],
"abstract": "Background: Labral tears commonly occur in both the general and sporting population, often leading to significant pain and dysfunction. Patients often engage in progressive rehabilitative programs, and surgical intervention may be required in severe cases. Autologous platelet rich plasma (PRP) injections have been growing in popularity in musculoskeletal medicine as an alternative to corticosteroid injections. This paper looks at the effectiveness of PRP injections in glenoid labral lesions.Methods: The clinical and radiological findings are presented for two patients who have been treated with autologous PRP into the glenohumeral joint adjacent to the labral tear, in conjunction with a progressive rehabilitative program. Follow up occurred at 18 months and 13 months, respectively.Results: Both subjects tolerated the PRP injection well with no adverse effects, and were compliant with their rehabilitative programs. On initial presentation, pain on the visual analogue scale (VAS) was 7/10 and 6/10 and at follow up it was reported as 0/10. Both subjects have now returned to normal sporting and work activities.Conclusions: The findings from this case series suggest that PRP in conjunction with appropriate rehabilitation can assist in the clinical recovery of glenoid labral tears. Further research is required with greater sample numbers and improved methodological parameters.",
"keywords": [
"Glenoid labral lesions can often lead to significant discomfort and restriction during daily living activities",
"as well as various sporting activities. Prevalence can vary between 6% in the general population1 to 35% in the sporting population2. Typical management can range from exercise therapy to surgical intervention. This case report describes the use of ultrasound-guided autologous PRP into the glenohumeral joint adjacent to the labral lesion in two patients",
"in conjunction with a supervised exercise program."
],
"content": "Introduction\n\nGlenoid labral lesions can often lead to significant discomfort and restriction during daily living activities, as well as various sporting activities. Prevalence can vary between 6% in the general population1 to 35% in the sporting population2. Typical management can range from exercise therapy to surgical intervention. This case report describes the use of ultrasound-guided autologous PRP into the glenohumeral joint adjacent to the labral lesion in two patients, in conjunction with a supervised exercise program.\n\n\nCase I\n\nIn June 2011, an otherwise fit and healthy, right arm dominant 27-year-old male electrician was referred for physiotherapy management to the author. He presented with right intermittent deep shoulder ache occurring one month prior when performing multiple supine bench presses in the gym. The patient reported an immediate onset of deep pain distributed around the shoulder region more so posteriorly with an associated ‘click’. There was no pain or discomfort described below the deltoid or associated neck pain or headaches. Grip strength was maintained and any activity above ninety degrees of flexion or abduction was ceased secondary to pain levels increasing. All upper limb gym activities were ceased and sleeping on the right side was avoided due to pain. There was no other relevant medical history reported. The patient was able to continue with work, though he avoided provocative movements and lifting. He took 4g of paracetamol and 100mg of Voltaren Rapid daily for pain relief for the initial 2 weeks.\n\nInitial observation on standing revealed an increase in right upper trapezius bulk with normal resting position of the scapulae. Active abduction was limited by pain to 110°, and hand behind head manoeuvre was provocative. External rotation at 90° was limited by pain to 45% of range and horizontal flexion was mildly painful. Scapular stabilisation in conjunction with active movement resulted in no change to clinical symptoms. The Obrien’s Test was positive (Sen 90%, Spec 98%3), as was the Crank Test (Sen 80%, Spec 20%4). The Biceps Load Test II was positive (Sen 90%, Spec 97%5). The Empty Can Test was positive (Sen 62%, Spec 54%6), the Hornblower’s Test (Sen 100%, Spec 93%7) and the Speeds Test (Sen 90%, Spec 14%8) were positive and posterior glenohumeral joint palpation resulted in pain being reproduced.\n\nOn sitting, the cervical spine was cleared and there was no glenohumeral sulcus sign present. The Shift and Load Test [Sen 90%, Spec 85%9) was negative and there was adequate thoracic spine mobility. In the supine position, the apprehension test was positive and the acromioclavicular joint was tender. Pectoralis minor was shortened and neural integrity and neurodynamics tests were negative. In the prone position, static scapular setting was adequate, although there was a loss of control with dynamic movements.\n\nThe patient agreed to commence a specific rehabilitation program for six weeks in an attempt to improve function and his pain score. The exercise regime consisted of static and dynamic scapular stabilisation in both the prone and sitting positions. Through range scapulohumeral kinematics were addressed with the aid of Theraband. Rotator cuff strength exercises were commenced with a bias on the supraspinatus and teres minor muscle groups, in conjunction with subscapularis training to assist in improving the glenohumeral mechanics. A pectoralis minor lengthening exercise was given and general advice regarding good posture was discussed. All the exercises were to be done in a pain free fashion. Real time ultrasound (RTUS) was used throughout the six-week rehabilitation program to ensure that the correct muscle activation pattern was occurring.\n\n(a) axial T2 fat saturated, (b) axial T2 gradient echo and (c) coronal T2 fat saturated images show the undisplaced posterior labral tear, (d) axial T2 fat saturated and (e) sagittal T2 Fat saturated images shows denervation oedema of teres minor compatible with a brachial neuritis or post traumatic injury.\n\nAfter the six-week progressive exercise program, the patient was reassessed functionally and clinical tests were repeated. The patient reported a 70% improvement in pain scores on the VAS and an increase functional ability. Overhead activities were still unable to be performed secondary to pain and his Obrien’s Test, Crank Test and Biceps Load Test II were all positive. The patient reported a decrease in symptom reproduction when the Empty Can Test and Hornblower’s Test were performed; however, a positive test was recorded.\n\nThe patient was subsequently referred for Magnetic Resonance Imaging (MRI) which showed a 4 mm × 5 mm delamination split of the supraspinatus tendon and an undisplaced posterior labral avulsion extending craniocaudally for 3.2 cm superior to inferior quadrant. The long head of biceps tendon is enlocated within the interbercular groove and there was post traumatic degeneration of the right acromioclavicular joint.\n\nThe results were discussed with the patient with the assistance of the MRI imaging and a model of the shoulder joint. An orthopaedic consult was suggested, but the patient declined as he did not want to go down the surgical treatment pathway. The option of autologous PRP into the paralabral region and glenohumeral joint was discussed on the basis of autologous product with anti-inflammatory properties that may enable an analgesic effect. The patient was made aware that there had been no published evidence, no high level research into such procedure being performed in the shoulder, possible complications and no guaranteed outcomes. He requested the treatment and gave full informed consent. Under ultrasound guidance using a strict aseptic technique, 5cc of 0.5% bupivacaine was injected into the paraglenoid soft tissues, followed by 8cc of autologous PRP (BCT, REGEN Labs, Switzerland) into the glenohumeral synovial cavity near the posterior inferior quadrant of the torn glenoidlabrum with no pain. 2cc of PRP was injected under ultrasound guidance into the acromioclavicular joint. Post injection, the shoulder was placed in a sling for 10 days with elbow ranging allowed.\n\nTwo weeks post PRP injection the patient returned for physiotherapy review and reported an improvement in function and pain scores equating to an 85% improvement on the VAS. The patient could now engage in more functional overhead activities pain free and reported less apprehension. His initial exercise program was recommenced and progressed accordingly to incorporate outer range shoulder movements and to facilitate a return to weight lifting and Muay Thai. At the four-week post PRP review with the radiologist, the patient reported considerable improvement in pain and function of the shoulder and no further injections were needed.\n\nAt his 18 month follow up appointment with the physiotherapist, the patient described complete resolution of all symptoms, a return to Muay Thai, and engagement in pre-injury gym training. Clinical tests included the Obrien’s, Crank, Biceps Load Test II, Empty Can and the Speeds Test, which were all negative. There were no further clinical symptoms to suggest labral lesion involvement, and repeat imaging was therefore not justified in the clinical setting.\n\n\nCase II\n\nIn October 2011, an otherwise fit and healthy 25-year-old female receptionist was referred for radiological intervention to the author. She presented with a 7-year history of gradual onset of right shoulder pain after playing several seasons of softball. She described the symptoms as a deep ache in the joint, particularly made worse with overhead activities. She denied any specific injury to her shoulder, and believed several seasons as a right arm pitcher was the cause. She currently had stopped playing softball and avoided any provocative activities.\n\nPrior to initial assessment with the authors, the patient reported she had received several corticosteroid injections over the 7-year history into her right subacromial bursa, none of which gave her complete relief of her symptoms. In 2009 she had a motor vehicle accident which resulted in a 7% loss of function to her cervical and lumbar spine. She reported spinal function had since improved greatly to date, though she still gets the occasional frontal headache. Six months prior to presentation, she was advised to focus on strengthening her upper limbs, for which she undertook an aggressive gym program for three months. Subsequently, her shoulder pain flared up significantly, resulting in lack of sleep and reduced function. She stopped all exercise after the flare up.\n\nHer MRI showed a thickened coracoacromial ligament with established subacromial bursitis, a 2 mm × 3 mm partial tear on the mid supraspinatus tendon and an undisplaced Superior Labral Anterior to Posterior (SLAP) tear at the junction with the long head of biceps tendon without a measurable fragment.\n\nAt the first visit for radiological intervention there was marked localising tenderness to the insertion of the trapezius into the spine of the scapula, a positive O’Brien’s Test and pain on provocation of the long head of biceps. The posterior shoulder pain was treated with ultrasound guided percutaneous tenotomy and injection of 5cc of unclotted autologous blood mixed with 3cc of 0.5% bupivacaine into the insertion of the trapezius at the medial margin spine of the scapula. At her 4-week review by the radiologist, pain from the above had remarkably improved. However, she had persisting symptoms that was thought to be originating from the SLAP lesion. She was referred for physiotherapy review for assessment and correction of any abnormal biomechanics, shoulder kinetics and glenohumeral instability.\n\n(a) orange arrow in this T2 fat saturated coronal image shows undisplaced tear at the junction of the long head of biceps and superior labrum, this is also seen in (b) yellow arrow in coronal proton density image. Black arrow in (a) shows a thickened coracoacromial ligament and yellow arrow shows subjacent asymptomatic subacromialsubdeltoid bursitis, (c) orange arrow in this axial T2 fat saturation image shows the SLAP tear at the junction of the long head of biceps and superior labrum.\n\nAt her initial physiotherapy review, observation in standing revealed that there was a kyphotic cervical spine resting position with associated protracted scapulae. There was also posterior cuff wasting with compensatory upper trapezius bulk. Active abduction was limited to 100° by pain and external rotation at 90° could not be performed due to provocation. Facilitated seated scapular stabilisation in conjunction with active shoulder range of movements resulted in only minor improvement in function. The Obrien’s Test, Biceps Load Test II and Crank Test were all positive. The Neers Test was positive as was the Speeds Test and the Empty Can Test. Anterior joint line tenderness was considerable.\n\nOn sitting, the cervical quadrant test was negative and there was no tenderness on the acromioclavicular joint. There was no sulcus sign present and the Shift and Load Test was negative. The thoracic spine was hypomobile. In the supine position, the Shoulder Apprehension Test was positive and neural integrity and neurodynamics tests were negative. Prone scapular setting was poor with minimal ability to control upper limb movements with adequate scapular positioning.\n\nRehabilitation involved spinal posture correction exercises incorporating adequate resting scapulae positions and supraspinatus strengthening exercises with the RTUS. Motor control of subscapulariswas also addressed to ensure correct dynamic movements of the glenohumeral joint complex were achieved.\n\nSix weeks after commencing rehabilitation, the patient reported a 40% improvement in symptoms but was still unable to complete tasks above shoulder height without any pain. The patient declined the offer of an orthopaedic consult.\n\nAt radiological review, her Oxford Shoulder Score was 26, indicating moderate affliction. She consented to a PRP injection with an explanation of the procedure and risks similar to case 1. Using an aseptic technique, 5cc of 1% lignocaine and 6cc of PRP mixture (BCT, REGEN Labs, Switzerland) was injected under direct ultrasound visualisation into the glenohumeral synovial cavity through the posterior glenohumeral recess. Immediately post injection there was complete resolution of shoulder symptoms, indicating a positive anaesthetic response, and confirming that the glenohumeral joint was the source of pain. The shoulder was placed in a sling for 10 days and an elbow ranging exercise was given.\n\nTwo weeks after the PRP injection the patient reported a 65% improvement on the VAS, and could now engage in some overhead activities. She continued with her specific rehabilitation with the assistance of the RTUS to ensure accuracy of muscle activation. Six weeks post PRP injection her Obrien’s and Crank Test were negative, as was the Speeds Test. Visible muscle hypertrophy was evident through the posterior cuff, which was confirmed on RTUS, and overhead activities could be performed with minimal pain.\n\nAt her 13 month follow up, the patient reported that all her symptoms had resolved with adequate functional level to compete in high grade volleyball and to throw a medicine ball above shoulder height at boot camp. There were no further clinical symptoms to suggest labral lesion involvement, and repeat imaging was therefore not justified in the clinical setting.\n\n\nDiscussion\n\nThe glenoidlabrum is a triangular fibrocartilaginous structure that assists in deepening the labrum to provide some mechanical stability for the humeral head. This vulnerable structure can result in labral lesions occurring in both the general and sporting population. There has been extensive research suggesting a link between labral pathology and rotator cuff involvement. Up to 45% of patients with labral lesions were also found to have partial thickness tears of the supraspinatus10 as found in both of the reported cases. Therefore, specific rehabilitation that targets rotator cuff deficits in association with glenohumeral positioning is an important part of the recovery process.\n\nThe authors recognise the mixed viewpoints in the literature regarding the exact aetiology and pathomechanics of glenoid labral lesions11–13. It is therefore paramount that a good subjective history is taken and clinical testing is directly correlated to the symptoms and the imaging findings. The authors also recognise the importance of performing all the clinical tests on the non-symptomatic side to ensure a sound baseline for comparison exists. Although the effectiveness has yet to be validated in shoulder rehabilitation, the use of a RTUS to ensure the correct muscle activation occurs is an extremely useful tool for both patient educational purposes and progression of exercises.\n\nRepair of cartilage and tendon lesions is the holy grail of musculoskeletal medicine. The majority of musculoskeletal disorders are due to degenerative pathologies that result in multifactorial chronic pain. There is chronic imbalance between anabolism and catabolism at a cellular level with an unfavourable microenvironment that results in further destruction of normal structure. The reader is referred to the paper by Chikanza and Fernandes for more information14. Inherent catabolic properties and side effects of corticosteroids have resulted in the search for an alternative injectable product for pain relief in these patients. The ideal option would be a non-surgical minimally invasive technique that delivers a product with minimal or no side effects with the ability to restore a favourable microenvironment that allows regeneration of degenerative tissue.\n\nPRP is an autologous agent with a very high concentration of naturally occurring growth factors. It has been in use for at least two decades in maxillofacial surgery and is rapidly gaining popularity in orthopaedic medicine. PRP carries very minimal risks and is distinctly different to cortisone in that it is not a catabolic, with no risk of destruction of normal tissue. PRP has anti-inflammatory properties that inhibit inflammatory processes in osteoarthritic chondrocytes15. Labral tissue is fibrocartilage and in an animal model meniscal tear, PRP enhanced repair of meniscal fibrocartilage16. PRP has also been shown to play a role in regeneration of degenerative rotator cuff tears by enhancing proliferation and matrix synthesis of tenocytes17. However, the use of PRP has had unfavourable review, as seen by recent studies in rotator cuff tears. Two randomized controlled trials, one with 79 patients and the other with 88 patients comparing PRP fibrin matrix (PRFM) versus control on rotator cuff tendon healing showed no demonstrable differences on tendon healing and clinical rating scales18,19. Bergeson et al. also showed similar results with PRFM in at-risk rotator cuff tears20. This may reflect several factors, including differences in individual products of PRP preparation. These studies used a semisolid implant material that had to be delivered through the arthroscope cannula, which is quite different to injectable liquid PRP preparations. This implant was left at the bone tendon interface and may have resulted in a space occupying effect, in addition to an unfavourable biological milieu with increased inflammatory mediators. In a laboratory model, human PRP has been shown to stimulate migration and chondrogenic differentiation of human subchondral progenitor cells21. Glenohumeral intra-articular injection of PRP with the aim of stabilising fibrocartilage and hyaline cartilage lesions has evolved subsequent to safety and efficacy results of studies with good short-term results in osteoarthritis of the hip and knee joints22,23. In the past few years there has been interest in mesenchymal pluripotent stem cells (MSC) in regeneration of cartilage and tendon disorders. Mokbel et al. showed in a dog study that intra-articular injection of MSC is a viable option for treating partial cartilage defects by proving that injected MSC demonstrated homing and incorporation of labelled MSCs in the neocartilage reparative tissue24. PRP has also been shown to enhance proliferation of MSCs and chondrogenic differentiation25.\n\nIn the authors’ practices, patients are informed of the option of promoting proliferation of bone marrow stem cells by nutritional supplements through the consumption of blueberries, Vitamin D3 and green tea26 as an alternative to harvest and injection of MSCs.\n\nThe authors have used PRP in conjunction with optimal biomechanics pre and post PRP injection to ensure the best chances of a favourable outcome. This paper adds to anecdotal evidence on the use of intra-articular PRP in fibrocartilage lesions with favourable clinically significant results. It does not offer a statistically significant result or evidence for routine practice.\n\n\nConclusion\n\nIn both case studies, the patients had complete resolution of symptoms, and were able to return to normal activities, including impact sports. This is the first published report on the use of ultrasound guided PRP in conjunction with physiotherapy to treat glenoid labral tears with a favourable outcome. This report offers evidence that PRP with appropriate rehabilitation is a viable, low risk alternative to corticosteroid injections or surgical procedures in glenoid labral tears. Further research is required with larger sample numbers and improved methodological parameters to further validate these findings.",
"appendix": "Consent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from both subjects and the radiology company.\n\n\nAuthor Contributions\n\nPhysiotherapy comments were by the primary author, radiological comments by secondary author.\n\n\nGrant Information\n\nThere was no grant funding.\n\n\nCompeting Interests\n\nThere was no competing interests.\n\n\nReferences\n\nHandelberg F, Willems S, Shahabpour M, et al:SLAP lesions: a retrospective multicentre study. Arthroscopy, 1998; 14: 856–862.\n\nFunk L, Snow M: SLAP tears of the glenoidlabrum in contact athletes. Clin J Sports Med, 2007; 17(1): 1–4.\n\nO’Brien SJ, Pagnani MJ, Fealy S, et al:The active compression test: A new and effective test for diagnosing labral tears and acromioclavicular joint abnormality. Am J Sports Med, 1998; 26: 610–613.\n\nSnyder SJ, Karzel RP, Del Pizzo W, et al:SLAP lesions of the shoulder. Arthroscopy, 1990; 6: 274–279.\n\nKim SH, Ha KI, Ahn JH, et al:Biceps Load Test II: A clinical test for SLAP lesions of the shoulder. Arthroscopy, 2001; 17: 160–164.\n\nHoltby R, Razmjou H: Validity of the Supraspinatus Test as a Single Clinical Test in Diagnosing Patient with Rotator Cuff Pathology. J Orthop Sports Phys Ther, 2004; 34: 194–200.\n\nWalch G, Boulahia A, Calderone S, et al:The dropping and hornblower’s sign in evaluation of rotator cuff tears. J Bone Joint Surg. 1998; 80: 624–8.\n\nBennet WF: Specificity of the Speed’s Test: Arthroscopic technique for evaluating the biceps tendon at the level of the bicipital groove. Arthroscopy, 1998; 14: 789–796.\n\nAlison Hoens: Cleland Journal of Orthopedic Clinical Examination: An Evidence-Based Approach for Physical Therapists. Carlstadt, NJ: Icon Learning Systems; 2005.\n\nAndrews JR, Carson WG: The arthroscopic treatment of glenoid labrum tears in the throwing athlete. Orthopaedic Transaction Journal, 1984; 8: 44.\n\nAndrews JR, Carson WG Jr, McLeod WD, et al:Glenoid labrum tears related to the long head of the biceps. Am J Sports Med, 1985; 13(5): 337–41.\n\nBurkhart SS, Morgan CD: The peel-back mechanism: its role in producing and extending posterior type II SLAP lesions and its effect on SLAP repair rehabilitation. Arthroscopy, 1998; 14(6): 637–40.\n\nPagnani MJ, Deng XH, Warren RF, et al:Effect of lesions of the superior portion of the glenoid labrum on glenohumeral translation. J Bone Joint Surg, 1995; 77(7): 1003–10.\n\nChikanza IC, Fernandes L: Novel strategies for the treatment of osteoarthritis. Expert Opin Investig Drugs, 2000; 9(7): 1499–1510.\n\nVan Buul GM, Koevoet WL, Kops N, et al:Platelet-rich plasma releasate inhibits inflammatory processes in osteoarthritic chondrocytes. Am J Sports Med, 2011; 39(11): 2362–2370.\n\nIshida K, Kuroda R, Miwa M, et al:The regenerative effects of platelet-rich plasma on meniscal cells in vitro and its in vivo application with biodegradable gelatin hydrogel. Tissue Eng, 2007; 16(5): 1103–12.\n\nChris HJ, Kim JE, Yoon KS, et al:Platelet-Rich Plasma Stimulates Cell Proliferation and Enhances Matrix Gene Expression and Synthesis in Tenocytes From Human Rotator Cuff Tendons With Degenerative Tears. Am J Sports Med, 2012; 40: 1035–1045.\n\nRodeo SA, Delos D, Williams RJ, et al:The effect of platelet-rich fibrin matrix on rotator cuff tendon healing: a prospective, randomized clinical study. Am J Sports Med, 2012; 40(6): 1234–41.\n\nCastricini R, Longo UG, De Benedetto M, et al:Platelet-rich plasma augmentation for arthroscopic rotator cuff repair: a randomized controlled trial. Am J Sports Med, 2011; 39(2): 258–265.\n\nBergeson AG, Tashjian RZ, Greis PE, et al:Effects of platelet-rich fibrin matrix on repair integrity of at-risk rotator cuff tears. Am J Sports Med, 2012; 40(2): 286–293.\n\nKrüger JP, Hondke S, Endres M, et al:Human platelet-richplasma stimulates migration and chondrogenic differentiation of human subchondral progenitor cells. J Orthop Res, 2012; 30(6): 845–52.\n\nKon E, Mandelbaum B, Buda R, et al:Platelet-rich plasma intra-articular injection versus hyaluronic acid viscosupplementation as treatments for cartilage pathology: from early degeneration to osteoarthritis. Arthroscopy, 2011; 27(11): 1490–501.\n\nSanchez M, Guadilla J, Fizl N, et al:Ultrasound-guided platelet-rich plasma injections for the treatment of osteoarthritis of the hip. Rheumatology, 2012; 51(1): 144–150.\n\nMokbel A, El-Tookhy O, Shamaa AA, et al:Homing and efficacy of intra-articular injection of autologous mesenchymal stem cells in experimental chondral defects in dogs. Clin Exp Rheumatol, 2011; 29(2): 275–84.\n\nMishra A, Tummala P, King A, et al:Buffered platelet-rich plasma enhances mesenchymal stem cell proliferation and chondrogenic differentiation. Tissue Eng, 2009; 15(3): 431–5.\n\nBickford PC, Tan J, Shytle RD, et al:Nutraceuticals synergistically promote proliferation of human stem cells. Stem Cells Dev, 2006; 15(1): 118–23."
}
|
[
{
"id": "602",
"date": "08 Jan 2013",
"name": "Nicola Maffulli",
"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 is a case report (two cases) reporting the application of an autologous blood product (PRP) in Glenoid Labral Tear. The recent evidence suggests that PRP, despite the great hype, is of no benefit in the management of musculoskeletal conditions. I have no doubts that these two patients have done well, but, without a control group, this manuscript only perpetuates a myth. In this respect, the authors have chosen to ignore the meta-analysis published in by Sheth et al. 2012, and to jump on the bandwagon. In this respect, therefore, what they report is at best dubious science.",
"responses": [
{
"c_id": "399",
"date": "20 Mar 2013",
"name": "Robert Vander Kraats",
"role": "Author Response",
"response": "The authors are well aware of the limitation of a case report with respect to no control group. We in fact stated this in the case report and the need for further research. It is however hard to compare our paper with other papers directly as this is the first published paper on PRP into glenoid labral tears. Sheth et al.'s 2012 meta-analysis doesn't discredit PRP in isolation, it rather states due to the lack of standardisation of study protocols, platelet-separation techniques, and outcome measures there is uncertainty. Therefore, in a situation like this, one needs to delve into further research before discrediting ones paper and suggesting it it of \"dubious science\". For example, Peerbooms et al. (2010) in his double-blinded randomised controlled trial with a 1 year follow up found a clinically significant reduction in pain and an improvement in function in patients with lateral epicondylitis when PRP was used compared to corticosteriods. Further, Taylor et al. (2011) in his systematic review concluded that PRP has advantages for tendon and ligament injuries, by way of promoting a faster recovery, a reduction in recurrence, and with no adverse reactions described. Therefore, our case report, in conjunction with the above mentioned research, certainly repudiates that PRP is \"of no benefit in the management of musculoskeletal conditions\" as you suggest."
}
]
},
{
"id": "603",
"date": "08 Jan 2013",
"name": "Xander Van Rijen",
"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 believe the article to be a good contribution to support continued clinical study of PRP in labral tears. As a comment to the author, I would seek clarification on the validity of the combinations of shoulder tests used (according to Netter’s Orthopaedic Clinical Examination, combinations of tests are only moderately helpful in identifying labral tears).",
"responses": [
{
"c_id": "398",
"date": "20 Mar 2013",
"name": "Robert Vander Kraats",
"role": "Author Response",
"response": "Lyndsay Somerville, in her 2012 doctorate, and later published in 2013 in the BMC Musculoskeletal Disorders Journal, makes an interesting point suggesting that the sensitivity of SLAP tests in isolation are poor. For example, Somerville reports that amongst other tests, the sensitivity of Speed's Test is 24.6%, Biceps Load I is 10.3% and Resisted Supination ER is 14.3%. Based on the low scores, Somerville proposes that through clinical reasoning considering several tests is likely to paint a better picture than simply one poorly sensitive test in isolation."
}
]
},
{
"id": "653",
"date": "09 Jan 2013",
"name": "Isabel Andia",
"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\nIsabel Andia, reporting on the PRP aspects of this article,From the area of my expertise in PRP therapies, I have some reservations:One of the main questions that remain unanswered regarding the use of PRP is optimal formulation.Differents types of PRP have been identified in base of platelet enrichment and leukocyte concentration (relative to peripheral blood) and activation procedure. These crucial aspects are not mentioned in this article.Moreover, the authors dilute (8:5) (PRP:bupivacaine sol.) in the first case and (6:5) (PRP:lignocaine sol.) which raises concerns about their final PRP product and the capability to clot. I would suggest that the authors describe the formulation of their product and discuss the inconvenience of diluting the PRP product and mixing PRP with anesthetics (Carofino B. Et al., 2012 Corticosteroids and local anesthetics decrease positive effects of platelet-rich plasma: an in vitro study on human tendon cells. Arthroscopy. 711-9.) Michele Abate, reporting on the rehabilitation aspects of the article,General: The authors should provide the timing of the rehabilitation program before and after PRP injections and have to specify the progression of the exercises.Case I: Why was MRI performed after the failure of the rehabilitation program and not before? This could have influenced the outcome of the rehabilitation program.Which stretching exercises were performed? Was it only on the minor pectoralis?Did the patient perform exercises for impingement syndrome (lowering of the humeral head)?What about the strengthening of biceps and triceps? How did the patient perform exercises? Was it with the aid of only Theraband, or other specific devices (isokinetic)?Which suggestions about posture did the author provide?Case II: In this case, the rehabilitation program was wrong in the first phase. The patients suffered, in addition to SLAP (superior labral anterior to posterior) tear, impingement syndrome with bursitis and partial rotator cuff tear.This was the reason the patient had scanty results from the rehabilitation. During this period more attention had to be provided in the treatment of cervical spine and its dysfunction and of impingement syndrome. Which active exercises did the patient perform?",
"responses": [
{
"c_id": "397",
"date": "20 Mar 2013",
"name": "Robert Vander Kraats",
"role": "Author Response",
"response": "Case I: Referring for the MRI when the patient initially presented compared to at the 6 week mark would have made no difference to the rehab outcome. As reported, the patient just with rehab alone reported a 70% reduction on the VAS and improved functionality at the 6 week mark. The other important aspect here is one should never simply diagnose a musculoskeletal complaint from imaging alone; rather a good physical assessment with sound clinical reasoning by an experienced clinician is essential. Refer to Jenson & Modic et al. (1994) and Weishaupt & Boos et al. (1998). Furthermore, Liu et al. (1996) compared physical examination to MRI in assessment of glenoid labral tears confirming that physical examination yielded a sensitivity of 90% and a specificity of 85%, compared to MRI of 59% and 85% respectively, when compared to arthroscopy. Throughout the intensive rehabilitation program, importance was placed on improving the scapulothoracic and scapulohumeral rhythm kinematics. Along with addressing muscular imbalances in the form of length and strength differences comparing anterior to posterior cuff, importance was placed on functional exercises relevant to the patient. In the author's opinion, it is paramount to not get into the habit of prescribing exercises of no relevance to the patient's work or sport for example. Granted in the very early stage this may be acceptable, but certainly this should not be the focus. Doing a basic biceps curl in isolation, as suggested by the reviewer, without any scapulothoracic/ humeral activation or sport specific relevance is unlikely to add favourably to the clinical outcome. Case II: The patient certainly did not \"suffer\" due to a \"wrong\" rehabilitation program after the subject was referred to the author. Despite the chronic nature of the complaint, after just six weeks of intensive rehab the patient reported a 40% improvement on the VAS. One needs to always consider the mechanics and movement patterns of the cervical spine in a patient with a past history of a significant whiplash injury, particularly when considering scapulohumeral/thoracic dysfunction in a symptomatic labral tear. As such, the cervical rehab focussed extensively on the deep cervical muscles as per Jull et al.'s (2008) comprehensive research. Furthermore, with the assistance of a bio-feedback pressure sensor to maintain deep cervical muscle control, shoulder kinematics were added from inner range to outer range, progressed to sport specific positions as able. The other important factor to note is the impingement and bursitis were both not the primary source of symptoms. The subject had a normal acromion on Xray and the multiple cortisone injections failed to give relief. Both were simply secondary to altered kinematics between the cervical/thoracic/scapulohumeral mechanism, with an underlying symptomatic labral pathology. Therefore, addressing the above, in conjunction with the PRP, resulted in initial positive clinical tests all becoming negative, and a symptom free patient who could return to her chosen sport."
}
]
}
] | 1
|
https://f1000research.com/articles/1-68
|
https://f1000research.com/articles/1-38/v1
|
26 Oct 12
|
{
"type": "Research Article",
"title": "Upregulation of human β-defensin-3 and cathelicidin LL-37 in Kaposi’s sarcoma",
"authors": [
"Hanan Fathy",
"Maha M Amin",
"Abdel-Hady El-Gilany",
"Maha M Amin",
"Abdel-Hady El-Gilany"
],
"abstract": "Background: Kaposi’s sarcoma (KS) is a rare neoplasm of lymphatic endothelial cells. Human herpes virus 8 (HHV-8) is considered to be a necessary, but not sufficient causal agent of KS and additional cofactors remain unknown. In this study we evaluated the expression of human β defensin (HBD)-3 and LL-37 in cutaneous lesions of KS in comparison to the healthy skin of normal subjects.Methods: We performed a quantitative immunohistochemical study of HBD-3 and LL-37 on skin lesions from 18 patients having KS, and on healthy skin from 12 normal controls.Results: HBD-3 and LL-37 were significantly upregulated in epidermal and dermal specimens of all KS patients in comparison to normal skin of healthy controls. The immunostaining score of dermal HBD-3 was significantly higher in nodular lesions (9.6 ± 2.4) versus plaque lesions (4.1 ± 2.2), P = 0.001. Also the immunostaining score of dermal LL-37 was significantly higher in nodular lesions versus plaque lesions (P = 0.001).Conclusions: We have demonstrated for the first time that HBD-3 and LL-37 are significantly upregulated in lesional skin of KS in comparison to the skin of healthy controls. The obtained data suggest a possible involvement of these antimicrobial peptides in the pathogenesis of KS. However, the biological significance of HBD-3 and LL-37 in KS lesions needs further research.",
"keywords": [
"Kaposi’s sarcoma",
"Antimicrobial peptides (AMPs)",
"Human beta defensin ((HBD)-3",
"Cathelicidin LL-37"
],
"content": "Background\n\nKaposi’s sarcoma (KS) is a rare disease of lymphatic endothelial cells frequently evident as multiple vascular cutaneous and mucosal nodules. Lymph node and visceral manifestation is seen in cases of strong immunosuppression or aggressive disease1. The four clinico-epidemiological forms of KS are: classic form typically affecting elderly men of the Mediterranean, the endemic presence in Southern Africa, the epidemic form in patients infected with human immunodeficiency virus (HIV) and the iatrogenic KS complicating iatrogenic immunosuppression2.\n\nKS is strongly associated with human herpes virus 8 (HHV-8), which is implicated in the pathogenesis of all forms of KS3. HHV-8 is present in the vast majority (> 90%) of spindle cells and in the neoangiogenic vessels4–6. It is considered to be a necessary, but not sufficient causal agent of KS. Besides immunosuppression and AIDS, additional cofactors remain unknown7.\n\nEnsoli et al.8 speculated that early stage KS is a reactive inflammatory angiogenic process that may be triggered or enhanced by infection with HHV-8 with many lymphocytes and monocytes infiltrating the lesion. These cells produce inflammatory cytokines including interferon-y (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-2, IL-6 and others9. The inflammatory cytokines induce the recruitment of circulating cells into tissues, induce the production of angiogenic factors that mediate angiogenesis and edema and activate endothelial cells to acquire the phenotype of KS spindle cells10.\n\nAntimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune system that defend against invading bacteria, viruses and fungi through membrane or metabolic disruption11. This diverse group of peptides is separated into several classes. Defensins and cathelicidin (also known as LL-37) are considered the two main groups of AMPs in human skin12. It was confirmed that human β defensin (HBD)-3 and LL-37 demonstrated the ability to inhibit viral infection, affecting both enveloped RNA and DNA viruses and non-enveloped viruses13–21. Moreover, beside their role in protecting the host from invasion of pathogens, HBD-3 and LL-37 have immunomodulatory properties in inflammatory skin diseases as psoriasis22,23 and rosacea24.\n\nBased on the antiviral and immunomodulatory effect of HBD-3 and LL-37, as well as the angiogenic role of LL-3725, this study was carried out to assess the expression of the two main antimicrobial peptides (HBD-3 and LL-37) in skin lesions of KS in comparison to normal skin from healthy subjects.\n\n\nMaterials and methods\n\nThis is a case control study including a convenient sample of 18 Egyptian patients with KS recruited sequentially from the outpatient clinic of the Dermatology Unit of Mansoura University Hospital from 2006 to 2011. The diagnosis was based on classic clinical, histopathological features of KS and immunohistochemical staining using the endothelial marker CD34. The controls were 12 healthy subjects that underwent surgical removal of benign lesions.\n\nThorough general and skin examination of patients was done focussing on morphology, localization and number of skin lesions, as well as oral mucosa and lymph nodes examination. Blood cell count, blood chemistry, ELISA test for HIV serology, chest X-ray and abdominal ultrasound were conducted. As classic KS rarely affects other organs26, gastrointestinal tract endoscopies were done only to patients with widespread skin lesions (four patients) to evaluate the presence of gut lesions. Inclusion criteria were newly diagnosed patients without previous treatment of KS. Only patients with fully developed lesions such as plaques and nodules were included in this study. Patch (macular) stage lesions were not included. Staging of KS was assigned using the classification proposed by Schwartz et al.27 and modified by Schwartz et al.28 as follows:\n\nStage I: Localized nodular KS, with ≤ 15 cutaneous lesions or involvement restricted to one bilateral anatomic site, and few, if any gut nodules.\n\nStage II: Includes both exophytic destructive KS and locally infiltrative cutaneous lesions and locally aggressive KS or nodular KS, or > 15 cutaneous lesions or involvement of more than one bilateral anatomic site, and few or many gut nodules.\n\nStage III: Widespread lymph node involvement, with or without cutaneous KS, but with limited if any visceral involvement.\n\nStage IV: Widespread KS, usually progressing from stage II or III, with involvement of multiple visceral organs with or without cutaneous KS.\n\nLesional skin biopsy (intact lesion without evidence of secondary infection) was obtained from each patient. Biopsy from 12 normal subjects served as controls. They were obtained from normal skin beside benign neoplasms such as melanocytic naevus, infundibular cyst and lipoma of age, sex and localization-related subjects. Informed consent was taken from all participants. The study protocol was approved by the ethical committee of the College of Medicine of Mansoura University.\n\nAll specimens were fixed in formalin 10% and sections from paraffin blocks (3–4 um) were cut on glass slides for routine hematoxylin and eosin as well as immunohistochemical staining using indirect avidin-biotin-peroxidase method. Endogenous peroxidase activity was blocked with 0.6% H2O2. After blocking, sections were incubated at room temperature for 60 minutes with antibodies to HBD-3 using rabbit polyclonal antibody (Catalog number D2444; Sigma Aldrich, St. Louis, USA) at dilution of 1:2 and with human LL-37 monoclonal antibody (Catalog number HM2070; Hycult Biotech, Frontstraat 2a, 5405 PB Uden Netherland) at dilution of 1:500. Diaminobenzidine (DAB) reaction was used for visualization, followed by hematoxylin counterstain. Negative controls for all studies were obtained by omission of the primary antibodies of an adjacent section to assess the degree of non-specific staining. All slides were examined by Olympus light microscope.\n\nImmunostaining results of HBD-3 and LL-37 were evaluated in four layers of the epidermis and the proposed score was modified from Meyer-Hoffer et al.20 as follows: 0, none; 1, stratum corneum only; 2, stratum corneum and stratum granulosum; 3, stratum corneum, stratum granulosum and stratum spinosum; 4, whole epidermis. Next an intensity score was assigned, which represented the average intensity of positive epidermal cells as follows: 0, none; 1, weak; 2, moderate; 3, intense staining. Both scores were then added to obtain a total epidermal score which ranged from 0 to 7. Skin samples of psoriasis that are known to exhibit high expression of HBD-3 and LL-37 in the epidermis were used as positive controls. Immunostaining results of dermal lesions of KS were scored as previously described29. Immunoreactivity of HBD-3 and LL-37 were evaluated in the two basic component of this disease (spindle-shaped cells and endothelial cells of newly formed vessels). The percentage of positive cells was graded from 0 to 4 as follows: 0, zero to 10%; 1, 11 to 33%; 2, 33 to 66%; 3, 67 to 90%; and 4, 91 to 100%. Specimens were considered immunopositive when more than 10% of cells showed clear evidence of immunostaining. The intensity of immunostaining was rated as follows: 0, none; 1, weak; 2, moderate; and 3, intense. Because KS lesions frequently showed significant intraspecimen heterogeneity, a score was calculated in which the percentage positive rating was multiplied by the intensity rating. Each component of the lesion was scored independently and the results were added up. The score was calculated on 6 to 10 representative high–power fields after examination of the totality of lesion present in one section for each case. Sweat glands served as internal positive controls for LL-37. Inflammatory cells (monocytes and macrophages) served as internal positive controls for both studied AMPs. However, specimens from healthy subjects served as negative controls. Slides were scored by two independent and trained researchers (the authors).\n\nData were analyzed using SPSS version 16. Qualitative variables were presented as number and percent. Quantitative variable were presented as mean ± SD (median) and Mann-Whitney test was used for group comparison. Spearman’s correlation coefficient was used to calculate correlation between variables in patients with KS. P ≤ 0.05 was considered statistically significant.\n\n\nResults\n\nThe mean age of the patients (fourteen males and four females) was 63.9 ± 7.4 years (ranged from 55 to 82 years). The age and sex-matched control group were eight males and four females with mean age of 63.2 ± 3.7 years (ranged from 45 to 67 years). All investigated patients had classic KS without clinical evidence of immunodeficiency and no history of immunosuppressive drug intake. They had normal total and differential leukocytic count, normal blood chemistry and negative HIV serology. Fourteen KS patients were classified as stage I (≤ 15 cutaneous lesions) and 4 patients as stage II (> 15 cutaneous lesions) (Table 1). All patients had no mucosal lesions, no lymphadenopathy and no clinical or radiological features suggesting visceral involvement and no gut nodules by endoscopy (only done to stage II KS patients).\n\nKaposi’s Sarcoma (KS), human β defensin (HBD)-3, cathelicidin (LL-37).\n\n* Some patients had many anatomic sites affected.\n\nImmunohistochemical staining of HBD-3 and LL-37 were generally cytoplasmic, and membranous staining were also seen. There was significant upregulation of HBD-3 and LL-37 in both epidermal and dermal specimens of all studied patients in comparison to normal skin of healthy controls. The expressions of HBD-3 and LL-37 were seen in the epidermis as well as in the dermis (neoformed vessels, spindle cells and inflammatory cells) (Figure 1D, 1H) with different scores of immunoreactivity in KS lesions (Figure 1). Also positive immunohistochemical staining for LL-37 was seen in sweat glands of KS lesions (Figure 1C). HBD-3 and LL-37 were absent in the epidermis and dermis of control normal skin (Figure 1G, 1K). Only three sections of control normal skin show weak staining within the stratum corneum for HBD-3 and only two sections for LL-37 within stratum granulosum.\n\nLesions with Hematoxylin and eosin (A), CD34 (B), human β defensin (HBD)-3 (D-G) and cathelicidin LL-37 (C, H-K). Immunohistochemistry for CD34 (B) is strongly positive in KS lesions. HBD-3 and cathelicidin LL-37 immunoreactivity are less intense in plaque (E, I; respectively) than in nodular lesions (F, J; respectively). Positive epidermal staining for HBD-3 (D) and cathelicidin LL-37 (H) can be appreciated. Positive internal control for cathelicidin LL-37 in sweat glands (C) and inflammatory cells (E, I) for HBD-3 and cathelicidin LL-37; respectively are seen. Healthy skin as negative control for HBD-3 and cathelicidin LL-37 are seen in (G, K). Original magnification: (A, B, C, D, E, F, H, I, K) X 200; (G) X 100; (J) X 400.\n\nThe immunostaining scores of HBD-3 and LL-37 are detailed in Table 1 and Table 2. We found that in nodular lesions, the mean scores of epidermal HBD-3 (5.4 ± 1.4) and LL-37 (5.6 ± 1.1) were significantly higher in comparison to mean scores of epidermal HBD-3 (3.3 ± 0.9) and LL-37 (3.1 ± 1.2) in plaque lesions (Table 2). The mean scores of dermal HBD-3 (9.6 ± 2.4) and LL-37 (8.9 ± 1.7) in nodular lesions were significantly higher compared to mean scores of HBD-3 (4.1 ± 2.2) and LL-37 (4.1 ± 2.2) in plaque lesions. Furthermore, the mean scores of epidermal and dermal HBD-3 and LL-37 immunoreactivity were significantly higher in KS patients with stage II disease in comparison to patients with stage I (Table 2). There was a strong positive significant correlation between immunostaining scores of epidermal and dermal HBD-3 in KS lesions (r = 0.95 and P ≤ 0.001). Also a strong positive correlation between immunostaining scores of epidermal and dermal LL-37 in KS lesions was found (r = 0.7 and P = 0.001) (Table 3).\n\n\nDiscussion\n\nRecently many reports about the antiviral effect of defensins and cathelicidin have been published. The antiviral activity of HBD-3 has been reported against herpes simplex virus (HSV)13, vaccinia virus14, and HIV15. Similarly LL-37 is found to inhibit replication of vaccinia virus16, kill HSV17, is effective against adenovirus18 and inhibits HIV-1 replication in peripheral blood mononuclear cells19. Furthermore, HBD-3 expression was shown to be upregulated in human papillomavirus induced lesions20,21.\n\nIn our study we investigated for the first time the expression of HBD-3 and LL-37 in KS. They were significantly upregulated in epidermal and dermal (neoangiogenic vessels, spindle cells and inflammatory cells) regions of all studied KS lesions in comparison to normal skin of healthy subjects. These findings suggest a potential role of HBD-3 and LL-37 in pathogenesis of KS. Furthermore, we found that the expressions of these AMPs were increased with progression of KS lesions from plaque stage to nodules. The stage-related differences that we found in HBD-3 and LL-37 expression add more support of the possible role of these AMPs in progression of KS.\n\nThe induction of HBD-3 and LL-37 in KS may be in response to infection with HHV-8 that is implicated in pathogenesis of all forms of KS3. HBD-3 and LL-37 might exhibit antiviral activity against HHV-8 as shown for the related HSV13,17.\n\nIn addition to functioning as direct antimicrobial compounds, AMPs can function as chemokines30. It was found that LL-37 increases natural killer cell proliferation by activating the Toll like receptor 9. LL-37 increases proinflammatory cytokines at the dendritic cell level, promoting CD4+ TH1 cell responses31. LL-37 can synergize with IL-1β to increase the production of cytokines, such as IL-6, IL-8 and IL-10, and chemokines, such as cc-chemokine ligand 232. Similarly to cathelicidin, HBD-3 has chemoattractant properties on different cell types such as T lymphocytes and dendritic cells33. Furthermore, HBD-3 and cathelicidin induce the production of diverse chemokines and cytokines such as monocyte chemotactic protein-1, macrophage inflammatory protein-3, interferon-inducible protein-10, IL-1, IL-6, IL-8, IL-10 and TNF-α mainly in keratinocytes34,35.\n\nTaken together, it is possible that HBD-3 and LL-37 can function in KS by promoting Th1 cell response or by increasing the production of cytokines such as IL-1, IL-6, IL-8 and TNF-α. Several cytokines have been shown to support the growth of cultured KS spindle cells: these include IL-1β, IL-6, the soluble IL-6 receptor 2 and TNF-α36.\n\nHHV-8-specific cytotoxic T-lymphocyte and T helper responses are found in KS patients, and CD4 and CD8+ T cells were present in KS lesions. Also, monocytes-macrophages and dendritic cells were present in lesions8. Furthermore, Sirianni and coworkers37 had shown that NK cell function is important for the control of latent HHV-8 infection and abrogation of this important immune response can lead to a more aggressive KS disease. Moreover, besides LL 37’s function as chemoattractant to T-cells and monocytes, it can stimulate angiogenesis through an increase in endothelial cell proliferation and vessel formation25. AMPs can indirectly sustain angiogenic signals by production of TNF-α and IL-1. These cytokines are powerful inducers of vascular endothelial growth factors38. So the implicated role of HBD-3 and LL-37 in KS may be inhibition of HHV-8 replication, production of several inflammatory cytokines and stimulation of angiogenesis. Ensoli et al.8 speculated that KS is a multistep process including not only HHV-8 infection, but also genetic and angiogenic factors, as well as the production of several inflammatory cytokines.\n\nIt remains unclear whether KS itself is a true malignancy, a reactive proliferation or both28. Whatever the nature of KS, HBD-3 and IL-37 may serve as a growth factor for KS. This is supported by the finding that the expression of these AMPs were increased with progression of KS lesions from plaque stage to nodules in our study. Recent evidence suggests cathelicidin LL-37 to be a putative growth factor for various human cancers39–42. Similarly, HBD-3 may play an important role in the development and progression of oral cancer. HBD-3 stimulated the expression of tumor-promoting cytokines, including IL-1α, Il-6, Il-8 and TNF-α in macrophages43. These cytokines are also important in the progression of KS36. Further study to elucidate the hypothesis that HBD-3 and LL-37 serve as progression factor for KS is therefore recommended.\n\nIt is not known whether upregulation of HBD-3 and LL-37 in KS lesions is signaled indirectly by locally produced proinflammatory cytokines or directly by HHV8 molecules. HBD-3 is upregulated by proinflammatory cytokines, TNF-α, IFN-γ and IL-1β22. However IL-6 is a potent inducer of LL-3744. Interestingly, these inflammatory cytokines are abundant in KS lesions9. Furthermore, the possibility of induction of these AMPs by HHV-8 protein can be supported by the finding of increased expression of HBD-3 and LL-37 with progression of KS lesions from plaque into nodular stage in our study. Similarly, increased load of HHV-8 has been found with progression of KS lesions5. Further study is recommended to verify this point.\n\n\nConclusions\n\nHBD-3 and LL-37 are for the first time shown to be significantly upregulated in KS skin lesions as compared with skin of healthy controls. The obtained data suggest a possible contribution of HBD-3 and LL-37 in the innate and adaptive immune response target against HHV-8. Another possibility is the potential involvement of these antimicrobial peptides in the pathogenesis of KS. However, the biological significance of HBD-3 and LL-37 in KS lesion needs further research.\n\n\nConsent\n\nWA written informed consent for publication of clinical details were obtained from the patients.",
"appendix": "Author contributions\n\n\n\nHF: participated in study design, collecting data, clinical evaluation, participated in pathological evaluation, interpretation of data, writing the manuscript, and i deciding to submit the manuscript for publication. MMA: participated in collecting data, reviewed the pathologic material, photographed the slides, analysis, revision and approval of final and revised manuscript draft, and in deciding to submit the manuscript for publication. AHG: analysis and interpretation of data. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nJakob L, Metzler G, Chen KM, et al.: Non-AIDS associated Kaposi sarcoma: clinical features and treatment outcome. 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}
|
[
{
"id": "337",
"date": "31 Oct 2012",
"name": "Mohamed Badawy Hassan Tawfik Abdel-Naser",
"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\nAlthough I approve this paper, I have a few comments:- The authors stated that “we performed a quantitative immunohistochemistry” however, it seems that they have done a qualitative assessment as quantitative immunohistochemistry is done by image analysis program which is reproducible, avoids interpersonal variations and potential bias. Furthermore, it provides quantitative data. If the authors wished to conduct quantitative immunohistochemistry then they would have to revise the statistical tests used.- It seems that all authors evaluating the immunohistochemistry results were aware of the study design. This may be a potential source of bias.- The authors used specimens of psoriasis as a positive control. Psoriasis is an inflammatory but not infective dermatosis which raises an argument against the impact of the expression of AMPs in KS.",
"responses": [
{
"c_id": "72",
"date": "12 Nov 2012",
"name": "Hanan Fathy",
"role": "Author Response",
"response": "Firstly I thank Professor Naser for his valued comments. Below, I have some answers to the points he has made:1. We performed a quantitative immunohistochemical analysis because the scoring of epidermal and dermal immunostain of the studied AMPs is a quantitative in nature and not a qualitative measure. We do agree that the image analysis program could be more accurate, but unfortunately this was not available to us. Furthermore, false positive results by positive staining of sweat glands (which is not a component of the tumor) with LL-37 can be obtained by the image analysis program used, so there is no need for reanalysis the data.2. The specimens prepared hematoxylin, eosin and the immunohistochemical analysis were coded numerically. So the first two authors evaluating the immunohistochemistry results were unaware about the clinical data of the patients at the time of pathological evaluation. Even so, the stage of the disease (plaques versus nodules) can be determined by hematoxylin and eosin stained sections; Fernández-Figueras et al. (Reference 29 in our manuscript).3. Up until now the pathogenesis of KS has not been clear. HHV8 is considered to be a necessary, but not sufficient causal agent of KS. Apart from immunosuppression and AIDS, additional cofactors still remain unknown (Reference number 7 in our manuscript). This means that the etiology of KS is not just a viral infection. LL-37 was found to be highly expressed in malignant melanoma and squamous cell carcinoma (Kim et al. reference number 42 in our manuscript). Similarly, HBD-3 was found to be highly expressed in oral squamous cell carcinoma (Kesting et al, Cancer Invest 2009, 27: 575-581). These AMPs have been found to be expressed in some inflammatory, infective and malignant diseases, so further research is needed to elucidate the biological function of these AMPs in KS."
}
]
},
{
"id": "338",
"date": "02 Nov 2012",
"name": "Maxwell Fung",
"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": "339",
"date": "02 Nov 2012",
"name": "Frank J Jenkins",
"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": "340",
"date": "19 Nov 2012",
"name": "Barbara Ensoli",
"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 study the authors have evaluated for the first time the expression of human β defensin (HBD)-3 and LL-37 in cutaneous lesions of KS and showed that these antimicrobial peptides (AMPs) are significantly up-regulated in KS skin lesions as compared with skin of healthy controls. The results are of potential interest to the understanding of KS pathogenesis, however there are several points that should be addressed which can improve the work.1. The authors should explain why they excluded from the immunohistochemical evaluation macular lesions considering that these lesions are the one with the highest degree of inflammatory infiltrate, and given the role of the peptides in inflammatory skin diseases.2. It is not clear how the scoring for the immunohistochemical analysis was calculated (see Material and Methods section). In particular it seems that a total epidermal score (ranging from 0 to 7) was obtained by the addition of a score representing the extension of the expression detected in the four epidermal layers with an intensity score, which represented the average intensity of positive epidermal cells. Then the percentage of positive cells is calculated, and a grading from 0 to 4 was assigned. Finally, the authors stated that, because KS lesions frequently showed significant intraspecimen heterogeneity, a score was calculated in which the percentage positive rating was multiplied by the intensity rating. Each component of the lesion was scored independently and the results were added up, so how were all these scorings were combined? Which are the scores indicated in Table 1-3? Finally, it seem that the scoring was done in the open and this may represent a bias.3. In the Table summarizing the “Demographic, clinical and immunohistochemical scoring of HBD-3 and LL-37 of studied population” it would be important to indicate also the patient clinical stage.4. Figure 1: arrows should be included in panels to point out positive cells, particularly in panels E and I. In addition, in order to correctly compare the expression of LL37 in plaque and nodules, panel I and J should have the same magnification.5. The authors stated in Discussion that the expression of AMPs was increased with progression of KS lesions from plaque stage to nodules. However, to support of the possible role of these AMPs in progression of KS lesions from stage III and IV patients should be studied.6. The manuscript does not cite a paper reporting that human beta defensin-2 (HBD-2) is expressed by endothelial cells present in Kaposi’s sarcoma lesions (but not by endothelial cells present in normal tissues), and that inflammatory mediators induce HBD-2 expression by normal endothelial cells Kawsar et al. (2010). This citation should be included in the manuscript and results discussed.",
"responses": [
{
"c_id": "71",
"date": "17 Dec 2012",
"name": "Hanan Fathy",
"role": "Author Response",
"response": "Firstly I thank Professor Barbara Ensoli for her valued comments. Below, I have some answers to the points she has made:1. Patch lesions were not included in this study because it is very difficult to discriminate between tumor cells and activated endothelial cells in this stage of KS (Reference number 29 in our manuscript).2. In this study we used two different independent (i.e not added to each other) scores, one epidermal score and the other is dermal score as detailed in material and method section.3.Tumor stage is presented in table 1. It is based on clinical examination and radiological study as mentioned in material and method section.4. The panel i is replaced by another one with higher magnification (x400) and arrows are included.5. Unfortunately none of the studied patients had stage III or IV.6. We have included the findings of Kawsar et al. 2010 in the discussion.Many Thanks."
}
]
}
] | 1
|
https://f1000research.com/articles/1-38
|
https://f1000research.com/articles/1-66/v1
|
14 Dec 12
|
{
"type": "Short Research Article",
"title": "Introducing the global medical community to the information presented at local scientific conferences through nephrology blogs",
"authors": [
"Tejas Desai",
"Xiangming Fang",
"Maria Ferris",
"Xiangming Fang",
"Maria Ferris"
],
"abstract": "An increasing number of healthcare providers author medical blogs (bloggers) to educate the public and fellow physicians. Traditionally, many bloggers have assumed that readers are most interested in information presented at prestigious and popular scientific meetings. As a result, the readers and bloggers often ignore blogs of local scientific meetings. We hypothesize that blog readers will utilize blogs about local scientific meetings less than those about national meetings.We examined nephrology-pertinent blogs from 2010-2012. Blogs were categorized as \"local/regional\" or \"national/international\" based on the majority of the audience that attended the live scientific meeting. We tracked the number of pageviews, reading time, and location of use per blog for the first 90-days after its first availability on the website. Wilcoxon testing was performed on all data.There were 9 local/regional and 11 national/international scientific meetings for which blogs were available. The mean number of page views was significantly lower in blogs from local/regional than national/international conferences (84.7 versus 160.3, respectively; p < 0.01). However, the mean difference in total reading time between both categories of blogs was not significant (p = 0.25).Data from this investigation do not fully support the hypothesis that readers utilized local/regional blogs less than national/international blogs. Although local/regional blogs attracted fewer readers (lower pageviews), the content in these blogs was compelling enough to keep the reader equally engaged as with national/international blogs.",
"keywords": [
"An increasing number of healthcare providers author medical blogs (bloggers) to educate the public and fellow physicians1–3. Bloggers use this medium to report the events",
"discussions",
"and controversies that occur at scientific conferences. As a result",
"the blog is a valuable tool for the reader who may otherwise not have access to this information. Traditionally",
"many bloggers have assumed that readers are most interested in information presented at prestigious and popular scientific meetings4. Thus",
"they have focused their blogging efforts on large national and international conferences and have ignored smaller",
"local meetings5. Historically",
"local scientific meetings attract a smaller live audience",
"have a geographically restricted educational impact",
"and do not present much novel medical information. Nevertheless",
"the value of blogs that pertain to local conferences has not been studied. Given these limitations",
"we hypothesize that blog readers will utilize blogs about local scientific meetings less than those about national meetings."
],
"content": "Introduction\n\nAn increasing number of healthcare providers author medical blogs (bloggers) to educate the public and fellow physicians1–3. Bloggers use this medium to report the events, discussions, and controversies that occur at scientific conferences. As a result, the blog is a valuable tool for the reader who may otherwise not have access to this information. Traditionally, many bloggers have assumed that readers are most interested in information presented at prestigious and popular scientific meetings4. Thus, they have focused their blogging efforts on large national and international conferences and have ignored smaller, local meetings5. Historically, local scientific meetings attract a smaller live audience, have a geographically restricted educational impact, and do not present much novel medical information. Nevertheless, the value of blogs that pertain to local conferences has not been studied. Given these limitations, we hypothesize that blog readers will utilize blogs about local scientific meetings less than those about national meetings.\n\n\nMethods\n\nWe examined nephrology-pertinent blogs authored by the editors or administrators of Nephrology On-Demand (http://www.mynod.org). These blogs were text-based narrative reports of scientific meetings that occurred between 2010–2012. Blogs were categorized as “local/regional” or “national/international” based on the majority of the audience that primarily attended the live scientific meeting. All of the meetings were based in the United States. The only blogs analyzed were firsthand accounts written by individuals who attended live conferences and not those created from second- or third-party sources. Blogs were posted on Nephrology On-Demand and were freely available to all users at http://goo.gl/28zza. We used Google Analytics to track the number of pageviews, reading time, and location of use per blog for the first 90-days after its availability on the website. Wilcoxon tests were used to compare pageviews and reading time for each blog from different continents. JMP Pro 10 and Microsoft Excel 2007 were used for all statistical analyses.\n\n\nResults\n\nThere were 9 local/regional and 11 national/international scientific meetings for which a blog was available on Nephrology On-Demand (Table 1). The most popular blogs in each category were “Guest Lecture Series: The Cardiorenal Syndrome” (local/regional; 143 pageviews) and “American Society of Nephrology Renal Week” (national/international; 365 pageviews). Overall, the mean number of pageviews was significantly lower in blogs from local/regional than national/international conferences (84.7 versus 160.3, respectively; p < 0.01) (Figure 1). For both groups of blogs, the greatest number of pageviews came from the Americas, but there was a significantly lower number of views in local/regional blogs than national/international blogs across all regions (Table 2).\n\n* URL denotes the prefix: http://blog.ecu.edu/sites/nephrologyondemand/?p.\n\nStandard box plot of pageviews of local/regional and national/international nephrology blog posts by readers from all regions (red) and the Americas only (blue) with lines representing minimum value, 25th percentile, median, 75th percentile, and maximum value within each data set. Where present, inner lines represent 10th and 90th percentile values.\n\nTable 2 also indicates the total time spent reading local/regional and national/international blogs. Readers spent a cumulative total of 2.5 times more hours reading national/international than local/regional blogs. However, the mean difference in total reading time between both categories of blogs was not significant (p = 0.25) (Figure 2). Readers from the Americas spent the greatest amount of total time reading the blogs than from any other region, but there was no statistical difference in the time spent reading either category (p = 0.25).\n\nStandard box plot of reading time of local/regional and national/international nephrology blog posts by readers from all regions (red) and the Americas only (blue) with lines representing minimum value, 25th percentile, median, 75th percentile, and maximum value within each data set. Where present, inner lines represent 10th and 90th percentile values.\n\n\n\n\nDiscussion and conclusions\n\nData from this investigation do not fully support the hypothesis that readers utilized local/regional blogs less than national/international blogs. Although local/regional blogs attracted fewer readers (lower pageviews), the content in these blogs was compelling enough to keep the reader equally engaged as with national/international blogs (as there were statistically similar reading times). The latter finding is surprising because it suggests that information presented at local conferences can keep the attention of the reader as effectively as national conferences. Blogs open local conferences to the global community6. In addition, local conferences are conducted at a greater frequency and held at a wider variety of institutions than national/international conferences. The information presented through blogs would be more frequent and present a greater diversity of ideas than blogs of just national/international meetings7.\n\nFurther investigations are needed to determine what features local/regional blogs need to have in order to be viewed by a similar number of readers as the national/international blogs. Such features, if identified and incorporated, would greatly increase the value of local/regional scientific conferences. This exploratory investigation suggests that once these readers view a blog, the content within that blog will keep them engaged, no matter where it was presented.",
"appendix": "Author contributions\n\n\n\nTD devised the experiment, collected the data, and wrote the manuscript. XF assisted in all statistical analyses. MF provided guidance in the experiment and manuscript composition. All authors agreed to the final manuscript.\n\n\nCompeting interests\n\n\n\nTejas Desai is the creator of Nephrology On-Demand (http://www.nephrologyondemand.org) though receives no funding from the website. Xiangming Fang and Maria Ferris have 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\nAcknowledgements\n\nI would like to thank Pooja Desai for her critical review of the manuscript, and the faculty and fellows of the Division of Nephrology at East Carolina University for their authorship of the blogs. A portion of the data in this manuscript was presented at the American Society of Nephrology Kidney Week 2011 meetings in Philadelphia, PA, USA.\n\n\nReferences\n\nSparks MA, O’Seaghdha CM, Sethi SK, et al.: Embracing the Internet as a means of enhancing medical education in nephrology. Am J Kidney Dis. 2011; 58(4): 512–518. PubMed Abstract | Publisher Full Text\n\neAJKDNephrology Blogs. Am J Kidney Dis. 2011. Reference Source\n\nKovic I, Lulic I, Brumini G, et al.: Examining the medical blogosphere: an online survey of medical bloggers. J Med Internet Res. 2008; 10(3): e28. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarris P: Journal clubs tweeting to success. BioMed Central. 2012. Reference Source\n\nLinzer M: The journal club and medical education: over one hundred years of unrecorded history. Postgrad Med J. 1987; 63(740): 475–478. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBentwood J: Distributed Influence: Quantifying the Impact of Social Media [White paper]. 2008. Reference Source\n\nA Blog Around the Clock: Journal Clubs – Think of the Future. Sci Blogs. 2007. Reference Source"
}
|
[
{
"id": "400",
"date": "20 Dec 2012",
"name": "Tushar Vachharajani",
"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 very important and relevant analysis of the use of modern day technology in education. The education tools utilized in medicine in general has to adapt to the needs and interests of the students in the millennium era. I would suggest that the discussion needs to include a few sentences on how best the authors feel the “millennium multi-tasking student” can be kept engaged and focused so the “take home message” is delivered. Will keeping the blogs short and concise help with delivering the right message?",
"responses": []
},
{
"id": "550",
"date": "08 Jan 2013",
"name": "Manish Ponda",
"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/1-66
|
https://f1000research.com/articles/1-65/v1
|
13 Dec 12
|
{
"type": "Research Article",
"title": "Student feedback about the use of role plays in Sparshanam, a medical humanities module",
"authors": [
"P Ravi Shankar",
"Rano M Piryani",
"Kundan K Singh",
"Bal Man S Karki",
"Rano M Piryani",
"Kundan K Singh",
"Bal Man S Karki"
],
"abstract": "Background: At KIST Medical College, Lalitpur, Nepal, a Medical Humanities module for first year medical students has been conducted. Role plays are used to explore social, medical and sexual issues in the Nepalese context. The present study obtained student feedback about the role plays used in the module, the difficulties faced, and obtained suggestions for further improvement. Method: The module was conducted from January to August 2011 using a total of 15 role plays. Student feedback was obtained using a semi-structured questionnaire. Informal discussions were held and a questionnaire was circulated among the first year students who had participated in the module. Results: Ninety-eight of the 100 students in the module participated in the study. The overall opinion regarding the role plays was positive. Students stated role plays helped to make module objectives concrete and interesting, made students identify with the problem being investigated and improved communication skills. Role plays were designed to address important health issues in Nepal and prepare students for addressing these issues in future practice. A lack of sufficient time for preparing the role plays and initial problems with group dynamics were mentioned by the respondents during the study. Conclusions: Student feedback about the use of role plays during the module was positive. Role plays helped in making module objectives more concrete and interesting, improved communication skills and addressed important health issues in Nepal. Role plays are not resource intensive and can be considered for use in medical schools in developing nations.",
"keywords": [
"Developing nations",
"medical humanities",
"medical students",
"Nepal",
"role plays"
],
"content": "Background\n\nMedical humanities (MH) use subjects traditionally known as the humanities to explore issues in medical education. Literature, painting, sculpture, music, anthropology, philosophy and related subjects are used in MH modules throughout the world. MH supports the exploration of the human side of medicine and is the intersection of the arts and medicine1. MH programs are uncommon in South Asia. A voluntary MH module was conducted at Manipal College of Medical College (MCOMS), Pokhara, Nepal2. For the past three years we have also been conducting a MH module at KIST Medical College (KISTMC), Lalitpur, Nepal, first for faculty and medical/dental officers and later for first year medical students3. The module uses small group, activity-based sessions to explore various aspects of the humanities. Case scenarios, role plays, debates and interpretation of paintings are among the different learning modalities used.\n\nRole plays have been shown to promote active learning of students and are an experiential learning technique with learners acting out roles in case scenarios to provide targeted practice and feedback to train skills4. In Nepal, the use of role plays in educating medical students is limited. However they have been used in certain institutions. At MCOMS, Pokhara role plays were/are used to teach students to communicate non-drug and drug measures with respect to common conditions/diseases using simulated patients by the department of Pharmacology5. At KISTMC, the department of Clinical Pharmacology uses role plays for the same purpose and to optimize the time spent with medical representatives6. At MCOMS, role plays were used to explore issues of human sexuality during the voluntary MH module and during a session on social issues in the use of medicines7. In New Delhi, Indian medical students had used drama to explore the emotional pressures on the humane dimension of a medical student’s life8.\n\nWhen role plays are used in an unplanned and ad hoc manner, students often report dissatisfaction as active learning is impaired4. However, role plays have many potential uses in medical education. Of special relevance to MH are role plays enabling students to place themselves in situations they have not experienced before, to help them empathize and understand other people’s problems and motivations9.\n\nRole plays have been used to teach students the skill of breaking bad news10. The most effective educational interventions present basic steps to delivering bad news and provide opportunities to learners to discuss their concerns, to enable them to practice, and to receive feedback on their skills11. In Germany, video-taped role plays and subsequent analyses are used to teach students the skill of breaking bad news12. At the University of Heidelberg, Germany, introducing role plays enhanced the realism of technical training and improved doctor-patient communication13. In a Malaysian medical school, role plays have been used to teach communication skills in primary care medicine14 and to teach students to obtain a sexual history and discuss sexual health issues15. At MCOMS role plays were used to teach students to critically evaluate drug promotion16.\n\nIn Nepal, students enter medical school after twelve years of schooling with the subjects of physics, chemistry, biology and English being compulsory during the last two years. Students do not have much exposure to life situations and are emotionally immature. In our institution, early clinical exposure for four hours every week starts right from day one. The scenarios used in the MH module and their interpretation by students using role plays exposed them to situations that they are unlikely to have encountered in their life. MH introduces students to problematic life situations, teaches students to communicate better, and can stimulate creativity and the imagination17. Role plays enable students to place themselves in the situation of another person and may help to develop empathy. Role plays early in the course can expose students to different situations they are likely to face in their future career. Students become aware of social issues and other problems of the country so that they can be more active citizens. In Nepal, other medical schools are slowly adopting role plays for specific purposes in medical education. The Patan Academy of Health Sciences (PAHS) has the mission to train doctors for rural Nepal and community health sciences are an important part of the curriculum18. The institution encourages reflection and is planning to introduce MH in its curriculum.\n\nThe module for the third batch of students concentrated on five important areas. The first session was on empathy and the second one on ‘What it means to be sick in Nepal’. The other sessions were on the doctor, the patient and the doctor-patient relationship. There was also a concluding session. The module was held on alternate thursdays for around two hours for a group of 50 students. The students were divided into five small groups and facilitator presentations, case scenarios, role plays, paintings, and student presentations and activities (such as identifying learning issues from case scenarios and interpreting them using role plays, interpreting paintings and photographs among others) were among the different teaching-learning modalities used. Student feedback on the paintings used during the module in 2012 has been published19. Use of more paintings from Nepal and South Asia was suggested by the respondents in the study19. Following this study, we are now using paintings by our medical students which were exhibited during an art exhibition in the institution.\n\nThe content of role plays was often designed around social and political situations in contemporary Nepal. Nepal is recovering from a decade-long conflict and the scars are still visible. Many parts of the country were mined by the two sides and while the problem is not as extensive as in many other nations, land mines can be a danger to innocent civilians. Trafficking of young girls continues to be a problem though many organizations are now active in preventing trafficking and rescuing young girls20. Leprosy and HIV/AIDS continue to be diseases with significant social stigma and the status of women continues to be poor.\n\nIn the United States of America (USA), ‘The art of doctoring’, was introduced as an elective module to third and fourth year students21. Role plays were used along with other learning modalities. Role plays have also been used to teach appropriate interaction with pharmaceutical company representatives22.\n\nDetailed participant feedback on the role plays used in the MH module had not previously been obtained. Hence the present study was carried out with the following objectives:\n\na) To obtain participant feedback about role plays used in Sparshanam.\n\nb) To understand problems and difficulties in interpreting and enacting the role plays.\n\nc) To obtain suggestions for further improving the use of role plays during future modules.\n\n\nMethods\n\nThe topics covered during the MH module were empathy, what it means to be sick in Nepal, the doctor, the patient and the doctor-patient relationship. Each topic was completed in two sessions called ‘bytes’. The module and the role plays were conducted using resources available in the institution. The role plays were video recorded with the consent of the participants.\n\nThe third intake of undergraduate medical (MBBS) students joined the course in November-December 2010 and Sparshanam, the MH module, was conducted from January to early August 2011. The intake of 100 students was subdivided into two batches and sessions were held for each batch on alternate thursdays. The large groups of 50 students were then divided into five small groups of 10 students each.\n\nThe case scenarios were provided to the small groups, who analysed the scenario, identified the learning issues and tried to interpret the various issues using a role play. Students were given 10 minutes for preparation but they often needed more time (up to 15 minutes) which was provided. A group member introduced the role play and the actors. Participants were debriefed regarding their feelings and emotions while playing a particular role after certain role plays. We used 15 role plays during the module. Table 1 shows the scenarios used during the module. These scenarios were agreed upon by the authors to address common and important problems in the country.\n\nThe scenarios and the transcripts of certain role plays mentioned has been recently published in the International Journal of User-driven Healthcare23.\n\nStudents worked in small groups. These groups were kept constant throughout the module. As facilitators, we gave students freedom to explore the role plays according to group opinion and consensus and did not impose our opinions on the group. The scenarios were distributed to the group who then discussed which issues to explore and how to present the role play. Facilitators were present in the background to offer support when requested.\n\nThis study was conducted at the end of July and early August 2011. A semi-structured questionnaire was administered to students. Written informed consent was obtained from all participants. The study was approved by the Institutional Review Board (IRB). The questionnaire was tested for readability and ease of understanding among four faculty members and four second year students. The questionnaire used in the study is shown below.\n\nThe gender and method of financing of medical education was noted. Self-financing students have to pay high tuition fees and tend to be from a higher socioeconomic group compared to scholarship students. Ninety students are self-financing and 10 seats are offered on a full tuition fee scholarship to candidates selected by the Ministry of Education through an entrance exam. Overall comments about use of role plays in Sparshanam, whether students had been exposed to role plays before, and how role plays helped in realizing the objectives of the module were studied. A brief description of all role plays conducted during the module was shown to students on power point slides while filling the questionnaire.\n\nThe participant responses were collected and grouped together and the number of respondents stating each response was noted. Responses were either quoted verbatim by the authors or paraphrased in the findings. The responses quoted verbatim have been put in single inverted commas in the results. For the two ratings the mean score was calculated and compared among subgroups using independent samples t-test. A p value <0.05 was taken as statistically significant.\n\n\nResults\n\nNinety-eight of the 100 students (98%) participated in the study; 53 (54.1%) were male and 43 (43.9%) were female. Two respondents did not mention their gender. Eight students (8.2%) were scholarship students while 90 were self-financing.\n\nTable 1 shows the role plays used during the medical humanities module. Table 2 shows the paraphrased common overall comments about the module. Respondents felt the module was successful in providing knowledge about health and social issues in Nepal. Twenty-seven students (27.5%) were exposed to role plays for educational objectives in school. Seven of these 27 students had done role plays to raise public awareness: among the issues they had covered were drug abuse, education of girls, and trafficking of women. Sixty-eight students had not been exposed to role plays before.\n\nAccording to respondents, role plays helped in realizing the module objectives in many ways. Among the strengths of role plays mentioned were ‘live interaction in real situations’ [n = 27 (27.5%)], ‘made objectives more interesting’ [n = 16 (16.3%)], ‘we understood different aspects of the problem’ [n = 9 (9.2%)] and ‘sessions improved verbal and non-verbal communication’ (n = 6). Fifteen respondents (15.3%) were aware of the use of role plays elsewhere while 72 were not aware of this. In Nepal, the medical schools mentioned as having role plays by the students as a teaching-learning methodology were Patan Academy of Health Sciences (PAHS) (n = 5), and one each mentioned Manipal College of Medical Sciences, Pokhara, and Nepal Medical College. Fifty-two respondents (53.1%) rated their enjoyment of role plays in the module as 4/5 while 39 (39.8%) rated it as 5/5. The mean ± SD enjoyment score was 4.26 ± 0.94. There was no significant difference in the mean score according to gender or method of financing of medical education.\n\nEighty-seven respondents (88.8%) felt the use of role plays was appropriate. Among the reasons provided were role plays covered different health and social issues in the country (n = 39), the issues covered will be experienced in future practice (n = 33), and they dealt with issues of importance to the doctor-patient relationship in Nepal (n = 6). Table 3 shows the paraphrased suggestions of respondents to make role plays more useful. Table 4 mentions the role plays with which respondents identified the most.\n\nParticipants had problems in identifying with the problem of the gay couple (n = 20). Among the reasons cited were that participants felt the problem is less important because it is less prevalent in the country and is not commonly discussed in society. This role play was interpreted differently by different persons with a substantial number also identifying with it. The other role plays that students found difficulty in identifying with were surgery for Aryan features, the problem of leprosy and of mental illness.\n\nAmong the difficulties which students had while planning and enacting the role plays were lack of time (n = 18), problems in working together as a team during the initial days when participants did not know each other well (n = 6), occasionally uncooperative group members (n = 6) and problems in deciding who will act in the role plays (n = 5). These were overcome by group discussion and arriving at a consensus in the group (n = 15), dividing work among group members (n = 9), using a lottery system to select actors (n = 7) and respondents who were shy transformed themselves with the help of others (n = 5). Most small groups were able to resolve their initial problems and work together as a team. During each session, each team democratically elected a team leader, a time keeper, a scribe and a presenter. These roles were rotated during subsequent sessions. There were occasional difficulties in playing roles and students were sometimes reluctant to play roles with a sexual and reproductive component. The lottery system was followed by many groups to select actors and was accepted as fair. Despite a certain degree of reluctance and apprehension in playing and tackling sexual issues, respondents agreed these should be addressed during the MH module.\n\nNinety-two respondents (94%) felt sexual and reproductive issues should be addressed in learning sessions using role plays. Among reasons provided were as doctors they will face these problems in future practice (n = 14), these issues are an important and integral part of medicine (n = 11), and these ‘hidden’ issues are responsible for many problems (n = 8). Fifty respondents opined that these issues sometimes created problems in enacting the role plays compared with 41 respondents who felt that no problems were created. Among the problems mentioned were in acting, people felt awkward due to their conservative nature, and it was hard to create dialogs. These problems were overcome by seeking help from and involving all group members, overcoming initial apprehension in communicating with the opposite gender, using the lottery system to select actors and looking at the issues in a professional manner.\n\nNinety-six respondents (98%) wanted to undertake role plays in future activities. Among the stated advantages were that students will find it easier to deal with patients in future (n = 29), role plays helped in realizing module objectives (n = 22), students were familiarized with health and social problems of Nepal (n = 19), role plays were a refreshing break from routine (n = 18), role plays will improve communication skills (n = 18), and issues in the doctor-patient relationship were introduced (n = 14).\n\nNo significant difference in scores according to demographic characteristics was noted. Among free text comments about the module, one respondent stated: ‘It is appreciable and needs to be continued with more and more innovative ideas. Many other persons from other departments can be included as facilitators’. Another stated: ‘It was a great fun in medical humanities session. I had never had such interesting learning sessions before. It was much more effective and I hope to have such sessions in future also’. Other comments were: ‘Special thanks to the facilitators for organizing such a wonderful event for us. We were among the lucky few to have it’, and: ‘We should open a humanities site of our college where we can post videos of our role plays’.\n\n\nDiscussion\n\nRole plays as mentioned previously have been used for a variety of purposes in medical education. In the United Kingdom (UK), a mixed team of nursing and medical students were involved in an inter-professional pilot learning project on breaking bad news using role plays24. Sufficient trust for learning between medical and nursing students ensued despite the briefness of the program. Our MH module was restricted to only medical students. Last year the college also admitted undergraduate dental students who had however joined the course late. An inter-professional MH module can be considered from the next academic session.\n\nThe high enjoyment scores are a matter of satisfaction. MH was widely regarded by this group as an enjoyable activity which provides a zone of comfort and relaxation, which further supports previous findings25. We feel this aspect of MH is important to maintain student interest and participation and we are therefore satisfied with their responses regarding the enjoyment of the sessions. The MH module is not a formal part of our curriculum and is not assessed. However, the module remains popular with students; attendance at sessions is over 80% during the year the module was conducted and students feel it serves as a welcome break from their routine.\n\nRole plays have also been used as a teaching strategy in Pakistan in community medicine26. A number of benefits were noted by the participants. Role plays were mentioned as the most effective method of teaching; it improved their knowledge of the subject and said it would improve their clinical performance. Role plays would improve their communication skills, were regarded as a feasible method of andragogy and provoked critical thinking about the subject. Role plays have also been used in specific disciplines and amongst third and fourth year students. At MCOMS5 and KISTMC6 role plays were used in pharmacology to teach students to communicate with simulated patients and provide drug and non-drug information. In the present module, role plays were used to explore different issues and to familiarize students with the perspective of different characters involved. Students could experience first-hand what it meant to have a seriously ill child, the problems of the mentally ill, and the status of homosexuals in society among others. We debriefed the student participants occasionally and concentrated on how students felt playing different characters in the role plays. Improving communication skills of students was not a primary objective though it may have been one of the indirect benefits.\n\nIn the University of Chicago, a senior seminar is offered as a four week course in the fourth year to develop fluency in handling conflict and negotiation27. This senior seminar also helps in understanding the elements of persuasive communication. In our module many of the case scenarios did address conflict and negotiation but no special training was provided to the students in these areas.\n\nStudents, despite a certain amount of reluctance, accepted that sexual and reproductive issues were important and should be addressed during the module. This was in contrast to the observation during a previous module conducted for faculty members. Faculty members were uncomfortable with sexual issues which they felt were ‘embarrassing’28. We are happy that first year students realized the important role of these issues in health and that keeping them ‘hidden’ may lead to more problems. The role plays also taught students to interact in a mature and cooperative manner with the opposite gender.\n\nTraining in simulated situations under the guidance and support of facilitators is becoming important in medicine. Role plays provide a safe and low risk learning environment for communication skills4 and can serve to contribute life and a feeling of immediacy and involvement to academic situations. Role plays are rich in cognitive material, understanding and enacting the role play requires more information than what is provided, the problem unfolds and becomes richer over time, there is no single right way to tackle the problem, decisions have to be made in the absence of definitive knowledge and many solutions may exist29. Debriefing participants and providing feedback on performance has been recommended4. Due to time constraints we were not able to do debriefing for all role plays. Time continues to be a problem and we could consider increasing the duration to two hours in future. However, this will reduce time available for early clinical exposure. Another problem faced was that not many faculty members were interested in acting as facilitators. MH is still regarded as something ‘extra’ and not falling within strict subject boundaries. It may also be regarded as extra work which is not rewarded.\n\nThe study had limitations. Feedback was obtained using a semi-structured questionnaire. The data obtained was not triangulated with other sources. The questionnaire was not validated. Certain respondents may have had difficulty in understanding specific questions though facilitators were present to provide support. Participants could have had difficulties recalling role plays performed over a six month period.\n\n\nConclusion\n\nThe study provides valuable feedback about the use of role plays in MH in a medical school in a developing country. Student feedback was positive and they wanted role plays to be used in future modules. Problems in small groups were resolved democratically and role plays introduced students to social and health issues in Nepal and prepared them for future practice. Role plays improved communication skills and explored sexual and reproductive issues. Role plays are not resource intensive and can be easily conducted in medical schools in developing nations with existing resources. Role plays can also be used in communication skills training, breaking bad news, clinical pharmacology and addressing community health issues among others. Role plays have an important role in educating future doctors and should be more widely used in medical schools especially in South Asia and other developing countries.",
"appendix": "Author contributions\n\n\n\nPRS was involved in conceptualizing the study, preparing the questionnaire, collecting the data, reviewing the literature, analyzing the data and writing the manuscript. RMP helped in conceptualizing the study, collecting and analyzing the data and writing the manuscript. KKS helped in study conceptualization, collection and analysis of data and writing the manuscript. BMSK helped in study conception, analysis of the data, review of literature and writing the manuscript. All authors have read and approved the final submitted version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests have been 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 the support of Ms. Shova in organizing the sessions. We acknowledge the help of Ms. Omi Bajracharya and Dr. Suneel Piryani who facilitated a few sessions. We thank Mr. Nabin Ban for his help with logistics and audiovisual equipment. We also thank Ms. Renu Mahat for logistical support to the study. We thank the Principal, Prof TP Thapa for his constant support and encouragement and all students who participated in the module and provided feedback.\n\n\nReferences\n\nHooker C: The medical humanities a brief introduction. Aust Fam Physician. 2008; 37(5): 369–370. PubMed Abstract\n\nShankar PR: A Voluntary Medical Humanities Module in a Medical College in Western Nepal: Participant feedback. Teach Learn Med. 2009; 21(3): 248–253. PubMed Abstract | Publisher Full Text\n\nShankar R, Piryani RM: Three years of Medical Humanities at a Nepalese medical school. Educ Health. 2011; 24(1): 535. PubMed Abstract\n\nJoyner B, Young L: Teaching medical students using role play: twelve tips for successful role plays. Med Teach. 2006; 28(3): 225–229. PubMed Abstract | Publisher Full Text\n\nShankar PR, Dubey AK, Mishra M, et al.: Student attitudes towards communication skills learning in a medical college in western Nepal. Educ Health. 2006; 19(1): 71–84. PubMed Abstract\n\nShankar PR, Jha N, Bajracharya O, et al.: Teaching Pharmacology at a Nepalese Medical School: The Student Perspective. Australas Med J. 2010; 1: 14–22.\n\nShankar PR: Using case scenarios and role plays to explore issues of human sexuality. Educ Health. 2008; 21(3): 108. PubMed Abstract\n\nGupta S, Singh S: Confluence: understanding medical humanities through street theatre. Med Humanit. 2011; 37(2): 127–8. PubMed Abstract | Publisher Full Text\n\nFertleman C, Gibbs J, Eisen S: Video improved role play for teaching communication skills. Med Educ. 2005; 39(11): 1155–1156. PubMed Abstract | Publisher Full Text\n\nVan Ments M: The Effective Use of role play. London: Kogan Page 1999. Reference Source\n\nRosenbaum ME, Ferguson KJ, Lobas JG: Teaching medical students and residents skills for delivering bad news: a review of strategies. Acad Med. 2004; 79(2): 107–117. PubMed Abstract\n\nKopecky-Wenzel M, Maier EM, Muntau AC, et al.: Breaking bad news--a video-based training unit for medical students. Z Kinder Jugendpsychiatr Psychother. 2009; 37(2): 139–44. PubMed Abstract | Publisher Full Text\n\nNikendei C, Kraus B, Schrauth M, et al.: Integration of role playing into technical skills training: a randomized controlled trial. Med Teach. 2007; 29(9): 956–960. PubMed Abstract | Publisher Full Text\n\nSherina HN, Chia YC: Communication skills teaching in primary care medicine. Med J Malaysia. 2002; 57(Suppl E): 74–77. PubMed Abstract\n\nNg CJ, McCarthy SA: Teaching medical students how to take a sexual history and discuss sexual health issues. Med J Malaysia. 2002; 57(Suppl E): 44–51. PubMed Abstract\n\nShankar PR, Dubey AK, Subish P: Critical evaluation of drug promotion using role plays. Med Educ. 2006; 40(5): 472. PubMed Abstract | Publisher Full Text\n\nDownie RS: Literature and Medicine. J Med Ethics. 1991; 17(2): 93–6, 98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarki A, Courneya CA, Woollard R, et al.: Training of physicians for improving rural health care in Nepal: Building bridges to address the urban-rural gap.2009.\n\nShankar PR, Piryani RM, Upadhyay-Dhungel K: Student feedback on the use of paintings in Sparshanam, the Medical Humanities module at KIST Medical College, Nepal. BMC Med Educ. 2011; 11: 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuda S: Sex trafficking in South Asia. Int J Gynecol Obstet. 2006; 94(3): 374–81. PubMed Abstract | Publisher Full Text\n\nShapiro J, Rucker L, Robitshek D: Teaching the art of doctoring: an innovative medical student elective. Med Teach. 2006; 28(1): 30–35. PubMed Abstract | Publisher Full Text\n\nWofford JL, Ohl CA: Teaching appropriate interactions with pharmaceutical company representatives: the impact of an innovative workshop on student attitudes. BMC Med Educ. 2005; 5(1): 5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShankar PR, Singh KK, Dhakal A, et al.: Transcripts of a medical education in humanities module: selection of role plays. International Journal of User Driven Healthcare. 2012; 2(3): 63–76. Reference Source\n\nHolsbrink-Engels GA: Using a computer learning environment for initial training in dealing with social-communicative problems. Br J Educ Technol. 2001; 32(1): 53–67. Reference Source\n\nShankar PR: Medical Humanities In R Biswas. & CM Martin (Eds.) User-driven healthcare and narrative medicine: utilizing collaborative social networks and technologies. (pp. 210–227). Hershey, PA: Medical Information Science Reference 2011. Publisher Full Text\n\nManzoor I, Mukhtar F, Hashmi NR: Medical students’ perspective about role plays as a teaching strategy in community medicine. J Coll Physicians Surg Pak. 2012; 22(4): 222–5. PubMed Abstract\n\nAng M: Advanced communication skills: conflict management and persuasion. Acad Med. 2002; 77(11): 1166. PubMed Abstract\n\nShankar PR: Design the shoe according to the foot! Clin Teach. 2009; 6(2): 67–8. Publisher Full Text\n\nWakefield A, Cocksedge S, Boggis C: Breaking bad news: qualitative evaluation of an interprofessional learning opportunity. Med Teach. 2006; 28(1): 53–58. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "402",
"date": "14 Dec 2012",
"name": "Supten Sarbadhikari",
"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 commendable work on evaluating the student feedback for role plays to identify and interpret the learning objectives for empathy, what it means to be sick in Nepal, the doctor, the patient and the doctor-patient relationship.The authors make a strong case towards the use of role plays for educating future doctors and rightly recommend that they should be more widely used in medical schools especially in South Asia and other developing countries.",
"responses": []
},
{
"id": "655",
"date": "09 Jan 2013",
"name": "Ramalingam Sankran",
"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 have read this submission and think that it is a good piece of work done in Nepal. It is kind of pioneering medical education in South Asia.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-65
|
https://f1000research.com/articles/1-40/v1
|
31 Oct 12
|
{
"type": "Research Article",
"title": "Local indigenous knowledge about medicinal plants in and around Kakamega forest in western Kenya",
"authors": [
"Nickson Erick Otieno",
"Caleb Analo",
"Caleb Analo"
],
"abstract": "Kakamega forest is Kenya’s only rainforest and is distinguishably rich in biodiversity but threatened by agricultural encroachment and other forms of human activity. It is also one of Kenya’s Important Bird Areas and a significant source of natural products to neighboring rural communities, such as medicinal plants, food, wood and other fibers. By using structured questionnaires for direct interviews, local indigenous knowledge was tapped through involvement of a focal group of elderly key informants in three blocks of the forest. Forty key species of medicinal plants used by local people were identified and recorded. Fifty-five percent of these were shrubs, thirty-two percent trees, seven-and-a-half percent lower plants such as herbs or forbs while five percent were climbers. About seventy percent of the medicinal plants occurred inside the forest itself and thirty percent around the edge and the immediate surroundings outside the forest. Thirty-eight (95%) of the plants were indigenous to Kenya and two (5%) exotic. Such extensive indigenous knowledge of the medicinal uses of the plants, including their distribution trends in the forest, may be tapped for decision support in rural health service planning, policy formulation for conserving the forest, tracking and mitigation of climate change impacts.",
"keywords": [
"Please note that the refereeing status of this version was changed from “indexed” to “[v1",
"ref status: approved with reservations 2]”."
],
"content": "Introduction\n\nAlthough community development goals are not always consistent with biodiversity conservation objectives1 there are often many opportunities for mitigating negative effects by tapping into local indigenous knowledge with reference to certain aspects of environmental use and conservation2. Indeed, application of knowledge and values of communities that are resident within or around key biodiversity areas has been gaining increasing global popularity as significant elements in enriching and improving strategies for conserving biodiversity3. This is because integration of such indigenous knowledge into conservation programs facilitates cross-borrowing of ideas, promotes constructive engagement, and instills a sense of common ownership and responsibility towards achievement of a synergy of goals2. This echoes the concept of social capital3 that, apart from amassing local support and goodwill, adoption of local indigenous knowledge in conservation may also promote and provide sustainable insurance against conflicts of purposes. This results in increased chances of achieving the dual goal of biodiversity conservation stewardship as well as community development. For instance, Studies have shown that rainforest ethno-botanical checklists prepared by communities living in or near them tend to be more exhaustive because they are based on practical day-to-day uses that are firmly ingrained in local cultural norms and values4.\n\nLike in many parts of the developing world, there is a growing upsurge in demand for herbal and other traditional remedies for various ailments among communities in Kenya. This is due either to increasing cost of conventional modern medicine or, or inadequacies in public health service delivery. However, the bulk of “technical” information on traditional cures is still disparate and privately held, with limited accessibility to the public or in peer-review domain5.\n\nThis study sought to set in motion a process for comprehensive and systematic documentation of plants of medicinal value for Kakamega forest, with a view to consolidating indigenous knowledge about them and making these available to the wider community around the forest itself as well as other stakeholders, in the process of underscoring highlighting ecosystem and other socio-economic services offered by Kakamega forest to society. The study also sought to highlight any plant species in the forest that may have medicinal value that are also of conservation concern, either as endangered or as a problem species.\n\n\nMaterials and methods\n\nStudy area: Kakamega forest lies in western Kenya between 00°08′30.5′′ – 00°23′12.5′′ N and 34°18′ 08′′ – 34°57′26.5′′ E from 1520–1680 m above sea level6,7. The mean annual rainfall is 2000 mm, with long rains in April/May and short rains in September/October6 while mean annual temperature is 20°C. Due to anthropogenically-driven fragmentation over many decased, its main closed-canopy area now occupies only 60% (85 km2) of its original area (Figure 1). The forest is Kenya’s only true tropical rainforest (BirdLife International, 2004) and constitutes one of Kenya’s 61 Important Bird Areas (IBAs) due to many endemic birds species found in it (BirdLife International, 2004). Apart from birds, it also has remarkable biodiversity richness, hosting several species of mammals, reptiles, amphibians, invertebrates and plants8,9. It is currently under increasing threat of loss to agriculture and settlement by the increasing local human population. The neighbourhood of the forest, where the western Kenya Bantu ethnic community called Luhya reside, is densely populated with an average density of 250 persons per kilometer10,11.\n\nThe study was carried out within the three main blocks of Isecheno-Yala-Ikuywa group of fragments (south), Buyangu-Salaza-Kisere blocks (north) and the detached Kaimosi fragment (see Figure 1). The sections were covered in two field seasons of 11 days each, first during April–May while the effects of the long rains were still evident and many plants bore fruit (wet season) and then late July when full fruiting is reduced and some leaves are shed off (dry season). This was to control for any rainy-season effects.\n\nSampling strategy: A key informant was identified from each block/area of the forest during each sampling week, to be interviewed about the medicinal plants as outlined in Kothari12. The key informant was selected on the following criteria: (1) seniority of age in the community (not less that 50 years old); (2) local residency for a period not less than 20 years; (3) appreciable knowledge of forest plants in the local dialect and well versed with their use(s). Current or previous experience as herbalist was preferable but not essential. Such selection was based on prior consulation with the local community leaders.\n\nData was collected through field excursions using interviews that employed structured questionnaires guided by a mix of closed- and open-ended questions for the key informants. This was combined with free-style discussions, actual field excursions and visits with the respondents. For data consistency, the same informants were involved each sampling season in each area. In addition, eventually there was a joint focused group discussion12 with all the key informants to synergize the information gathered. Information captured and recorded included include: 1) Local name of plant in question; 2) Disease/condition cured by plant; 3) Plant part(s) used for the cure; 4) Preparation method; 5) Common (English) name of plant. These were determined from standard field guides; 6) Scientific name.\n\n\nData analysis\n\nA checklist of all observed plants of medicinal values was compiled, including their indigenous, common and scientific names; condition(s) cured; methods of preparing and administrating them to a patient; as well as the age and gender of the target patients (see link to data file below). All the lists generated by the different key informants were scrutinized and synchronized into a final one at the joint focused group discussion13. With help from the informants/respondents, each plant was observed in its natural habitat and a digital image taken using a digital camera. Further, for each medicinal plant, a small part (preferably with leaves) was collected while fresh and digitally photographed for identification and pressed for herbarium. Plants whose common (English) and scientific names were not immediately identifiable in the field were taken for specialized identification at the East African Herbarium in Nairobi.\n\n\nResults\n\nA total of 40 key species of medicinal plants used by the people around Kakamega forest were identified and recorded (see Appendix). The plants fall into 24 positively identified families while the family of the remaining one species Bequartiodendron oblanceolata was not clearly discerned (Table 1). The most dominant families were Asteraceae, Fabaceae and Lamiaceae, each representing 10.3% each of all plants.\n\nOf the 40 plants, 22 were shrubs, 13 trees, 3 lower plants such as herbs or forbs, and 2 climbers (Figure 2). Twenty-six of the medicinal plants occurred inside the forest itself and 14 occurred outside. One of the medicinal plants (Prunus africana) is also listed in the IUCN Red List as Vulnerable to extinction. The majority of the medicinal plants identified (95%) were indigenous and only 5% exotic.\n\nThe diseases reported to be cured by the medicinal plants identified in the study varied widely but were grouped into 14 categories including use in treatment of a number of livestock diseases (Figure 3). Ninety percent of the diseases cured are those that affect humans and about ten percent for livestock diseases. Most of the human diseases cured using the plants, fell into the categories of digestive or peptic; respiratory, vector-borne; and reproductive ailments (Figure 3). Furthermore, these ‘cures’ are applicable for both genders and almost all age groups except in 17% and 7% of the cases where the ‘cures’ are applicable to adults and elderly people only, respectively.\n\nIn preparing the ‘cures’ from the plants, the local people mainly use leaves, roots and barks, but in a few plants, the ‘cures’ are derived from flowers, fruits and young shoots (Figure 4). Additionally, since many of the plants are used for curing digestive or peptic, respiratory or vector-borne ailments, the majority of them are administered orally.\n\n\nDiscussion\n\nThe results of the study demonstrate that apart from Kakamega forest’s already well known position as a significant Kenyan rainforest in terms of the rich biodiversity, eco-system service provider and as a remarkable tourist site, it is also important to the local community as a repository for ethno-pharmacological resources that play a crucial role in supplementing the government’s effort in providing healthcare at the grass-root level. This also includes remedies for the treatment of livestock diseases. Unfortunately, much of the indigenous knowledge about these plant-based remedies, however, is still restricted to only a minority among the local population, particularly the elderly. Furthermore, these elderly knowledge holders are only those who have ancestry to a select number of families with long histories of the practice of traditional medicine. Traditionally, such indigenous knowledge, which is often regarded as spiritual, is closely guarded by such families, and is only passed on down the generation line to members of the family who use the knowledge and skills as a form of livelihood when they serve society as traditional medical consultants. In the process, such families wield immense respect in the society.\n\nIn-depth discussions with the key informants and a cross-section of some respondents among the local residents further revealed that even when the consultants prescribe treatment to their patients, only the already-prepared form of the cure is provided by the “medicine man” rather than revelation about the plant from which it is obtained, or how the concoctions are prepared. Nevertheless, this system is slowly changing and in recent years, some flexibility appears to be emerging, with the “medicine men”, including the ones interviewed in this study, are quite willing to provide information about the traditional cures in exchange for financial inducement or compensation. The wider society is also getting increasingly skillful in identification, preparation and administration of plant-based remedies at the local level4.\n\nWith the increasing cost of healthcare from modern facilities occasioned by global economic challenges which make medication expensive and out of reach to most rural dwellers in developing countries14, there is an increasing need to identify more affordable alternatives for the treatment of common ailments that affect rural human populations. For this reason, promotion of the use of natural remedies derived from various locally based resources such as medicinal plants, should form an important priority of governments’ strategies to make healthcare accessible to the rural populations in a more affordable way.\n\nWider availability of such knowledge, including from such research projects as these when published and distributed, would go a long way in improving access to basic healthcare. In addition, to protect the local community from exploitation of their indigenous medicinal knowledge by “external” prospectors and their agents for commercial purposes, a modality for a locally-based medicinal plant enterprise including charges for demonstrations, medicinal plant checklists and herbal medicine preparations sold to willing buyers, could be established and proceeds shared with or amongst the local stakeholders.\n\nFor instance, already underway is a project known as the Kakamega Forest Integrated Conservation Project, which involves a section of the local people in collaboration with the International Centre for Insect Physiology and Ecology (ICIPE). Part of this project involves commercial cultivation of two of the medicinal plants (see link to data file) Mondia whytei and Ocimum kilimandscharicum on farms. The income generated benefits the farmers and helps to supplement their subsistence needs. From ICIPE’s science park in Nairobi, an extract from Ocimum kilimandscharicum is used to make a commercial product called Naturub, which helps to relieve nasal congestion, colds, flu, insect bites, aches and pains15.\n\nSimilarly, there is a commercial medicinal product extracted from the roots of one of the plants in the forest. The product is named Mondia Tonic and is produced from extracts of the roots of Mondia whytei (see link to data file) and this is used as an appetizer, a flavoring agent, a stimulant or for mineral supplementation15. Such initiatives, if structured to incorporate indigenous knowledge of the local community, would be a further boost to economic empowerment of the community by using part of the returns from tha market to compensate them for such knowledge.\n\n\nConclusion\n\nIn conclusion, there is sufficient indigenous knowledge among the community around Kakamega forest about medicinal plants, to contribute not only to sustainable provision of grass-root health care but also a potential to share this knowledge beyond western Kenya. This knowledge also has a potential for boosting economic empowerment of the people around Kakamega forest. A tertiary potential benefit is incorporation of the knowledge into policies to guide conservation action for the rainforest and its biodiversity. This crucial benefit has further significant implications for mitigation of climate change impacts that would otherwise result in destruction or loss of this important water catchment for many rivers in western Kenya.\n\n\nRecommendations\n\nMore extensive excursions into the Kakamega forest and its immediate surroundings to reveal more medicinal plant species, particularly through involvement of a larger number key informants.\n\nCollation of the results of this study together with existing but unpublished results of all other studies of medicinal plants of Kakamega forest, anecdotal and otherwise.\n\nEstablishment of a comprehensive working database of the indigenous knowledge of the local community about the medicinal plants and other such resources in Kakamega forest.",
"appendix": "Author contributions\n\n\n\nNO conceived the study and designed the experiments, NO prepared the first draft of the manuscript while both authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nThere are no competing interest with regard to the project, the research, manuscript production and submission, be it financial, in kind or institutional.\n\n\nGrant information\n\nFunds for the project were kindly provided by The Conservation Foundation in UK under its Young Scientist for Rainforests award scheme. The grant was awarded to Nickson Otieno. The grant had no grant number.\n\n\nAcknowledgements\n\nWe wish to thank all our key respondents/informants Mr. John Shunza, Joel Mbogani and Femina Futa as well as our interpreters Laban Shiundu, Patrick and Alphonse Ligaro for assistance with location and identification of the medicinal plants and their uses, Kenya Wildlife Service (Buyangu, Kakamega) and Kenya Forestry Service (Isecheno Kakamega) for allowing us access to the forest to carry out the study as well as the local farmers on whose property some of the plants were identified. The East African Herbarium staff for further assistance with plant identification while Helida Oyieke and Samuel Muchai kindly reviewed the initial project proposal.\n\n\nReferences\n\nBerkes F: Rethinking community-based conservation. Cons Biol. 2004; 18(3): 621–630. Publisher Full Text\n\nBiswas S: Indigenous traditional knowledge integration for forest biodiversity conservation: Needs and priorities. Paper submitted at the VII World Forestry Congress, 2003 Quebec Canada 2003. Reference Source\n\nSmith D, Pretty J: Social capital in biodiversity conservation and management. Cons Biol. 2004; 18(3): 631–638. Publisher Full Text\n\nEllen R: Anthropological approaches to understanding the ethno-botanical knowledge of rainforest populations. In: 'Tropical rainforest research: current issues', Edwards, D. S., Booth, W. E. and Choy, S. C (eds). Kluwer: Dordrecht 1996; 457–465. Reference Source\n\nBiodiversity Monitoring Transect Analysis (BIOTA) East Africa: Biodiversity in conversion: The influence of fragmentation and disturbance on the biodiversity of East African highland rain forests Final Report Phase I (2001–2004). 2005. Reference Source\n\nFashing PJ, Gathua JM: Spatial variability in the vegetation structure and composition of an East African rain forest. Afr J Ecol. 2004; 42(3): 189–197. Publisher Full Text\n\nOtieno NE, Gichuki N, Farwig N, et al.: The role of farm structure on bird assemblages around a Kenyan tropical rainforest. Afr J Ecol. 2011; 49(4): 410–417. Publisher Full Text\n\nBennun AL, Njoroge P: Important Birds Areas in Kenya. East Africa Natural History Society, Nairobi, Kenya. 1999. Reference Source\n\nKokwaro JO: Conservation status of the Kakamega Forest in Kenya: the easternmost relict of the equatorial rain forests of Africa. Mon Syst Bot Miss Bot Gard. 1988; 25: 471–489. Reference Source\n\nAkotsi EFN, Situma CA, Nzioka B, et al.: Mapping land- use/land cover changes in Kakamega forest (1975–2005). International Centre of Insect Physiology and Ecology and the Department of Resource Surveys and Remote Sensing. Nairobi, Kenya. 2006. Reference Source\n\nRietdorf U, Kappel R, Kaikai WO: Linking Local Resources to SME Development. A Pathway Out Off Poverty. Unpublished project Report of BIOTA East Africa. Hamburg, Germany. 2006. Reference Source\n\nKothari CR: Research Methodology: Methods and Techniques. 2nd ed., New Age International (P) Ltd. New Delhi, India. 2000. Reference Source\n\nHughes D, DuMont K: Using focus groups to facilitate culturally anchored research. Am J Community Psychol. 1993; 21(4): 775–806. Publisher Full Text\n\nVander Plaetse B, Hlatiwayo G, Ven Eygen L, et al.: Cost and revenue of health care in a rural Zimbabwe district. Health Policy Plan. 2005; 20(4): 243–251. PubMed Abstract | Publisher Full Text\n\nICIPE: The Kakamega Forest Integrated Conservation Project of the International Center of Insect Physiology and Ecology (ICIPE), Kenya: a project recommendation document. Nairobi 2003. Reference Source"
}
|
[
{
"id": "356",
"date": "06 Nov 2012",
"name": "Martin Potgieter",
"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 potentially a very interesting article, and I think it could ultimately make a positive contribution to the evidence base. However, this paper has a number of serious flaws.More information and detail required:- The abstract and results are superfluous and do not report on some of the major findings.- More can be made of the data in figure 4. Why, for example, are leaves so much used, when in the rest of Africa roots are being predominantly used?- The information from figure 3 is not reported in the results or discussion. For example it would be interesting to know why digestive was so much treated.Discussion, conclusion and recommendations:- The discussion focuses most on generalities and not specifics as is found in the results.- Data on methods of preparation, administration, age and gender are not reflected in the results and discussion. This is particularly important for gender as the level of knowledge of local/rural African communities vary. Women are generally more knowledgeable in local households, but men are more knowledgeable when they are traditional healers.- References are seriously lacking in the discussion – thus no scientific authority is applied to most of the statements presented here. Thus this discussion is basically just an opinion.- Some parts of the discussion need rearranging to either the results (end of 2nd paragraph) or the conclusion (3rd paragraph).- Significant tracts of the discussion do not appear to be relevant to the study at hand, particularly the last two paragraphs of the discussion; the authors should consider removing these.- The current conclusion does not address the core data of this manuscript.- The authors should provide reasons for the points made in the ‘Recommendations’ section.Inconsistencies:- Questionnaires: In the abstract and main text it is implied that multiple questionnaires are used but only one is provided.- It says in the sampling strategy that experience as a herbalist was not essential, yet in the discussion it states that indigenous medicinal knowledge is a closely guarded only passed to family members. It would have been worthwhile to know the ratio of interviewed traditional healers/practitioners vs. lay people – there is a significant difference in their level of knowledge.- In the discussion the authors state that indigenous knowledge is confined to mainly the elderly but this study targeted only people above 50. In Africa that constitutes the elderly. Thus we have no data on the knowledge level of people younger than 50. Therefore we have no data to backup this statement.Language:- Some attention to the accuracy of language used is required; some words need removing, the authors should define what they mean by ‘key species’ (abstract) and appreciable knowledge (page 2) and replace the term ‘cured’ with ‘treated’.Notes on plant family classification:- Bequartiodendron oblanceolata is from the Sapotaceae family.- Malvaceae is the correct family name for Hibiscus spp; Malvoideae is a subfamily.",
"responses": [
{
"c_id": "74",
"date": "14 Dec 2012",
"name": "Nickson Otieno",
"role": "Author Response",
"response": "Dear Dr. Potgeiter, Thank you for your review. We have made the following changes in light of your comments.Title: We have added the word “some” in the title to preclude the presumption that we sought to list all medicinal plants from Kakamega forest in the one survey.Abstract: We have restructured and re-written the abstract to reflect suggested changes. We have also clarified that we used one structured questionnaire and not many types of questionnaire.Introduction: We have cited two references as suggested to support our assertion about other studies having been conducted on the subject.Materials and methods: We have corrected the indicated errors and have also specified how respondents were selected, including the proportions of practitioner to lay respondents, and key to random respondents.Data analysis: Suggested errors now corrected.Results: Bequartioden¬dron oblanceolata is now assigned to the family Sapotaceae as has been helpfully noted by the reviewer. The reference for IUCN is now provided. The word “cure” is now replaced by “treatment”. We have also provided a clarification on methods of administering medicinal plants other than orally.Discussion: We have now merged the Results with the Discussion under the new heading “Results and discussion” to make a more lucid connectivity between the two. In table 1, the Malvoidae subfamily is now corrected to Malvaceae family, as informed by the reviewer. The original Figure 2 depicting proportions of medicinal plant forms is now removed to avoid repeating results in text. As a result, Figure 3 becomes Figure 2 and Figure 4 becomes Figure 3. Figure 2 (new) is now reported in the text of results and discussion, together with an expoundment on the predominance of digestive-related diseases treated using the medicinal species. The new Figure3 now bears, in text, discussion as to the predominance of the use of leaves for treatments, viz-a-viz other plant parts. Table 2 is corrected as suggested; parts of the discussion suggested as not strongly related to the core data and results by reviewer, have been removed. The link between access to information on medicinal cures by local and improvement of basic healthcare is now more clearly explained. The conclusion is now more closely tied to the results of the study. Recommendations are now better justified.References: Corrections on the original reference number 12 is now effected; Due to additional references (also reflected in the body text) the reference section has now been reorganized accordingly."
}
]
},
{
"id": "359",
"date": "17 Nov 2012",
"name": "Hugo Asselin",
"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 piece of work, although very incomplete. I actually hesitated between recommending acceptation or rejection, but decided to settle on the former because any new contribution on traditional knowledge related to medicinal plants should be welcomed. However, I do have several important concerns to raise about this article.First, it should be made very clear that the study is based on the knowledge of only 3 respondents. Each of them might know a lot, but they still are just 3 respondents. Completely different results altogether could have been obtained by interviewing 3 other respondents.Second, it is hard to evaluate the actual contribution of the study to the scientific knowledge, as no other study on traditional knowledge related to medicinal plants in Kenya or tropical Africa is cited. A quick search in Web of Science shows that 87 papers were published in the last 15 years for Kenya alone.Third, more details should have been provided about the forests in which sampling took place. Calling them tropical is not enough. Information on species richness, for example, would have been needed to appreciate if the 40 species recorded as medicinal plants form a significant or trivial proportion of the complete species set. In addition, dominant species and forest dynamics should have been provided to facilitate comparison with other studies. Also, the choice of the forest blocks where sampling took place should have been justified.Fourth, information should have been provided about how ethical issues were addressed. Traditional knowledge is a sensible topic (even the more so when it relates to medicinal plants) and a precautionary approach should be taken to ensure protection of intellectual property rights.Fifth, the choice of the 3 respondents should have been explained in more details. Why only 3? Why these 3? Were they men or women? Knowledge is not shared equally between genders. Etc.Other comments:The abstract is too general and does not provide all the relevant results.The English should be checked by a native speaker. Some sentences are awkward, some words are missing, and some words are uselessly repeated.Figure 1 does not show the effect of forest fragmentation, so the first paragraph of the “Study area” section should not imply that.Why wasn’t BirdLife International (2004) added to the reference list and cited properly?Population density should be given as a number of people per SQUARED kilometers.“Salaza” is not shown on Fig. 1.Please make clearer the distinction between “block”, “fragment”, “section”, etc.Herbarium voucher numbers should be provided, or, at least, the name of the herbarium where samples are kept should be given.A total of 40 medicinal plant species seems low. How does it compare with other studies in African tropical forests?Why use quotation marks when writing “cure”? This uselessly sheds a doubt on the efficacy of medicinal plants.I am surprised that none of the species (especially herbs) was used entirely (instead of just leaves, or fruits, or other parts).How were the disease classes chosen? The “vector-borne” class is not a type of disease, but rather a way of transmission. It can include digestive, psychic, or other types. Furthermore, several common ailment categories were not reported to be treated with medicinal plants. Explanations should have been provided as to why. Authors should have followed, for example, Cook’s (1995) classification : Cook FEM (1995) Economic Botany Data Collection Standard. Kew: Royal Botanic Gardens.",
"responses": [
{
"c_id": "73",
"date": "14 Dec 2012",
"name": "Nickson Otieno",
"role": "Author Response",
"response": "Dear Dr. Asselin,Thank you for your review. We have made the following changes in light of your comments.Choice of respondents: We have now made it clear that there were 3 main focal respondents but that there were 2 other opportunistic random respondents that provided additional information for the study in each forest block, making a total of 9 respondents. Such selection was based on prior consultation with local community leaders and additional guidance by field assistants. Since this study was not meant for gauging opinions, we did not set out to interview as many respondents as possible. That is why we state that we had a key focal group of respondents chosen for their knowledge about the same, and of a minimum age that is generally recognized globally to posses the greatest of such knowledge. One of our key respondents was a practicing healer with long experience in the practice.Consultation of existing literature on the subject, in Kenya: We have incorporated more references to the literature in the revised version.Ethical issues in data use: Prior consent was obtained from each informant before information was obtained including information that the data would be shared widely. All respondents were duly acknowledged in the manuscript and are publicly acknowledged in the final publication. A condition for publication of the manuscript was to provide the detailed data so it was not optional not to disclose the full dataset of all the medicinal plants in detailed form.Abstract too general: We have now provided more details about the results in the abstract.Grammatical errors etc: More careful revision has been made in this regard including in-house pre-review by experienced authors.Figure 1 not related to fragmentation: The reference to Figure 1 is now placed in a more explicitly relevant part of the paragraph.BirdLife International reference: A more recent reference has now been included.Population density unit: This is now provided (in per square kilometers).Block/fragment/section: This is now clarified as referring to forest blocks.Herbarium vouchers: The plant specimens that were collected were not part of herbaria specimens and so did not have voucher numbers. They are yet to be curated and catalogued as the EA Herbarium in Nairobi is rather short of space for replicate specimens.Total of 40 medicinal plants: This number of species identified has now been put in perspective by comparing with other studies elsewhere in Kenya.Quotation marks on “cure”: The term “cure” has been replaced with “treatment” which we feel is more appropriate.I am surprised that none of the species (especially herbs) was used entirely (instead of just leaves, or fruits, or other parts: In Table 1, a number of plants for which more than one part is used for treatment is provided.Choice of classification of disease: The diseases were classified mainly on the basis of the parts of the body that are affected. Obviously this does not apply for “vector-borne” and “livestock” but the idea about including the former was to highlight such vector transmitted diseases, which are common in the area, such as malaria, which would otherwise easily be subsumed by the other classes since malaria presents with a multitude of symptoms. For “livestock” diseases, again this was to highlight them as non-human and compare them with the non-human ones.More details about the forest: We have now added more details in describing Kakamega forest where the study was carried out, including floral and other species status, and the overall vegetation structure.Selection of blocks: We have described the rationale for the choice of forest blocks."
}
]
}
] | 1
|
https://f1000research.com/articles/1-40
|
https://f1000research.com/articles/1-63/v1
|
11 Dec 12
|
{
"type": "Case Report",
"title": "A rare case of monophasic pleuropulmonary synovial sarcoma",
"authors": [
"Rateesh Sareen",
"Pandey Chandralekha",
"Mohit Sareen",
"Akanksha Dutt",
"Pandey Chandralekha",
"Mohit Sareen",
"Akanksha Dutt"
],
"abstract": "Pleuropulmonary synovial sarcoma is a diagnostic challenge. Synovial sarcoma is a soft tissue tumor of joints and extremities and are rarely seen in the mediastinum. We report this tumor at a very unusual location – the mediastinum in a 40-year-old female. The histopathology picture along with the location suggested a differential diagnosis of solitary fibrous tumor or mesothelioma, but immunohistochemistry helped in reaching the diagnosis of synovial sarcoma.",
"keywords": [
"Primary synovial sarcoma arising in the lung is very rarely seen in clinical practice1",
"2. Before 2005",
"only 60 cases of synovial sarcoma in the pleuropulmonary region were reported in the English-language scientific literature3. It is normally a tumor found in adolescents and young adults between 15 and 40 years of age. Males are affected more than females1. The use of molecular techniques like immunohistochemistry (IHC)",
"polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) is required for confirmation of diagnosis. They include detection of translocation T(x",
"18) (p11.2",
"q11.2) is a highly specific gene mutation for synovial sarcoma4."
],
"content": "Introduction\n\nPrimary synovial sarcoma arising in the lung is very rarely seen in clinical practice1,2. Before 2005, only 60 cases of synovial sarcoma in the pleuropulmonary region were reported in the English-language scientific literature3. It is normally a tumor found in adolescents and young adults between 15 and 40 years of age. Males are affected more than females1. The use of molecular techniques like immunohistochemistry (IHC), polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) is required for confirmation of diagnosis. They include detection of translocation T(x; 18) (p11.2; q11.2) is a highly specific gene mutation for synovial sarcoma4.\n\nClassically, synovial sarcoma has a biphasic pattern and is composed of sheets of spindle cells and sharply segregated epithelial cells forming gland-like areas5. Diagnosis of monophasic synovial sarcoma, particularly at an unusual location like the mediastinum, is a challenge as it is often misdiagnosed as primary pulmonary spindle cell sarcoma/carcinoma or metastatic carcinoma6,7. Further work-up including clinical, histopathological and immunohistochemical findings are core when the diagnosis is uncertain and molecular testing is necessary8. This is particularly important for cases like ours from developing countries where financial and infrastructural constraints are major drawbacks.\n\n\nCase\n\nA 40-year-old female presented with complaints of cough, chest pain and shortness of breath with progressive weight loss for the past year. The past medical history was unremarkable. The radiological study x-ray (PA view, Figure 1) showed a large round opacity in the lower zone of the right lung with small nodules in the middle and lower zones of the left lung. Bronchoscopy revealed a right-sided growth involving the middle and lower pulmonary lobes. The computed tomography (CT) of the thorax (Figure 2) showed a well-defined mass measuring 8×7×6 cm on the right lung, infiltrating the diaphragm with small nodules of 1–2 cm in the left upper and lower lung lobes. The origin of mass from lung supstance or pleura could not be identified with certainty.\n\nCT-guided fine-needle aspiration cytology and bronchoalveolar lavage was inconclusive. Finally, a right side partial lobectomy was performed. On histopathology, we received three soft tissue pieces, the largest one measuring 19cm × 15cm × 10cm and consisting of part of the lung tissue with the tumor. A well circumscribed tumor measuring 19cm × 9cm × 9cm was seen. Figure 3 The cut surface of the tumor was fleshy, homogenous gray-white with areas of calcification. The other two pieces comprised of lung tissue measuring 7cm × 6cm × 2cm and tumor measuring 6cm × 6cm × 4cm.\n\nMicroscopy examination showed a spindle cell tumor with a varied pattern of cellularity, with areas of high cellularity mixed with low cellularity areas (Figure 4 and Figure 5). The tumor cells were arranged in random, fasciculate and at places storiform pattern. Cytologically the cells had varying degrees of anaplasia with fusiform to plump cells having hyperchromatic nuclei and mild to moderate pink eosinophilic cytoplasm. Necrosis was minimal, there were occasional mitotic figures seen and metastatic calcification was also noted. It was therefore interpreted as a low-grade spindle-cell neoplasm of uncertain histogenesis. The tumor on immunohistochemical analysis was Epithelial membrane antigen cytokeratin5/6, mic-2, bci-2 and calponin positive. TTF-1, calretinin, WT-1 and CD-34 were negative. The tumor was therefore diagnosed as a synovial sarcoma.\n\n\nDiscussion\n\nThe mediastinum contains various structures and pluripotent cells that are responsible for the origin of a variety of tumors at this anatomical site. As far as primary sarcoma is concerned, mediastinum sarcomas account for 1.4% of all soft tissue sarcomas. Among sarcomas of the mediastinum, the most common one is a malignant peripheral nerve sheath tumor (26%) followed by spindle cell sarcoma (15%), leiomyosarcoma (9%) and liposarcoma (9%). Synovial sarcoma accounts for 2% of all sarcomas of mediastinum9.\n\nThere are four morphological types of synovial sarcoma – biphasic tumor, monophasic tumor, monophasic epithelial tumor and poorly differentiated (round) tumor. The biphasic type is easily diagnosed due to the presence of epithelial and spindle cell components with sharply segregated epithelial cells forming gland-like areas2,5. Primary pulmonary monophasic synovial sarcoma is mainly of monophasic suptype and is difficult to diagnose due to its uniform spindle cell pattern. The differential diagnosis includes malignant peripheral nerve sheath tumor, fibrosarcoma, leiomyosarcoma, sarcomatoid variant of mesothelioma, Ewing’s sarcoma, hemagiopericytoma, spindle cell lymphoma, desmoplastic small round cell tumor and metastatic carcinoma6. Based on morphology, our first differential diagnosis was solitary fibrous tumor followed by mesothelioma. Ancillary immunohistochemical techniques helped to reach the final diagnosis. The immunohistochemical features of pleuropulmonary sarcoma are similar to those of soft tissue synovial sarcoma10. EMA, cytokeratin, and vimentin positivity in combination with CD-34 negativity are the most useful and sensitive biomarkers for diagnosis of monophasic synovial sarcoma. In our case, immunostaining was strongly positive for EMA, vimentin, bcl-2 and focally for cytokeratin. It was negative for thyroid transcription factor (TTF)-1, calretinin, WT-1, CD34, S-100 and CD-99. Theses markers ruled out the possibility of malignant peripheral nerve sheath tumor (S 100 Positive), primitive nerve sheath tumor (CD99 Positive), germ cell tumor (PLAP, Alpha AFP, HCG Positive) and leiomyosarcoma (SMA Positive). P63 and CD34 were negative, which further ruled out thymoma and solitary fibrous tumor. Bcl-2 was positive, but it was not specific, as it could be seen in solitary fibrous tumor and monophasic synovial sarcoma. The diagnosis of pleuropulmonary synovial sarcoma was based on an absence of tumor at any other primary site. Brain metastasis was not seen in our case as it is uncommon in soft tissue sarcoma with only a single reported brain metastasis in the literature11.\n\nP-Positive, N-Negative, V-Variable, MPNST-Malignant Peripheral Nerve sheath tumor.\n\nThe overall prognosis is poor in pleuropulmonary synovial sarcoma12. Tumor size more than 5 cm, male gender, advanced age >20 years, high mitotic activity >10 mitosis/10 HPF, presence of tumor necrosis and SYT-SSX1 gene variant are the main poor prognostic factors13.\n\nTo conclude, pleuropulmonary synovial sarcoma is a rare tumor. The diagnosis of this tumor requires meticulous clinical and pathological correlation, immunohistochemistry and molecular techniques. Familiarity with this entity is essential as it carries poor prognosis.",
"appendix": "Author contributions\n\nRateesh Sareen wrote the article and contributed to the conception and design. Chandra lekha Pandey aided in the design and final approval of the manuscript. Akanksha Dutt contributed in manuscript writing. Mohit Sareen helped in gathering images and literature search.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nReferences\n\nKumar R, Menon S, Desai SB, et al:Primary endobronchial synovial sarcoma confirmed by SYT-SSX1 fusion gene transcript by reverse transcriptase polymerase chain reaction. Indian J Pathol Microbiol 2009; 52: 520–3.\n\nRong R, Doxtader EE, Tull J, et al:Metastatic poorly differentiated monophasic synovial sarcoma to lung with unknown primary: A molecular genetic analysis. Int J Clin Exp Pathol 2010; 3: 217–21.\n\nHosono T, Hironaka M, Kobayashi A, et al:Primary pulmonary synovial sarcoma confirmed by molecular detection of SYT-SSX1 fusion gene transcripts: A case report and review of the literature. Jpn J Clin Oncol 2005; 35: 274–9.\n\nTrupiano JK, Rice TW, Herzog K, et al:Mediastinal synovial sarcoma: Report of two cases with molecular genetic analysis. Ann Thorac Surg 2002; 73: 628–30.\n\nEwing CA, Zakowski MF, Lin O, et al:Monophasic Synovial Sarcoma: A cytolotic spectrum. Diagn Cytopathol 2004; 30: 19–23.\n\nKeel SB, Bacha E, Mark EJ, et al:Primary pulmonary sarcoma: A clinicopathologic study of 26 cases. Mod Pathol 1999; 12: 1124–31.\n\nHartel PH, Fanburg-Smith JC, Frazier AA, et al:Primary pulmonary and mediastinal synovial sarcoma: Aclinicopathologic study of 60 cases and comparison with five prior series. Mod Pathol 2007; 20: 760–9.\n\nCoindre JM, Pelmus M, Hostein I, et al:Should molecular testing be required for diagnosing synovial sarcoma? A prospective study of 204 cases. Cancer 2003; 98: 2700–7.\n\nBurt M, Ihde JK, Hajdu SI, et al:Primary sarcomas of the mediastinum: Results of therapy. J Thorac Cardiovasc Surg 1998; 115: 671–80.\n\nKottu R, Prayaga AK: Synovial sarcoma with relevant immunocytochemistry and special emphasis on the monophasic fibrous variant. J Cytol 2010; 27: 47–50.\n\nSiegel HJ, Dunham WH, Lopez-Ben R, et al:Intracranial metastasis from synovial sarcoma. Orthopedics 2008; 31: 405–7.\n\nMikami Y, Nakajima M, Hashimoto H, et al:Primary poorly differentiated monophasic synovial sarcoma of the lung. A case report with immunohistochemical and genetic studies. Pathol Res Pract 2003; 199: 827–33.\n\nTrassard M, Le Doussal V, Hacene K, et al:Prognostic factors in localized primary synovial sarcoma: A multicenter study of 128 adult patients. J Clin Oncol 2001; 19: 525–34."
}
|
[
{
"id": "399",
"date": "18 Dec 2012",
"name": "Florence Duffaud",
"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 case report does not add any interesting information on monophasic pleural synovialosarcoma. Furthermore to establish a definitive diagnosis of synovialosarcoma we need to have the presence of the translocation t(X; 18).In this case report the presence of the translocation is not mentioned. The global treatment of the patient is not reported.",
"responses": []
},
{
"id": "401",
"date": "19 Dec 2012",
"name": "Matteo Giaj Levra",
"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 spoke about a diagnosis of synovial sarcoma but there is no evidence of the research of the t(x; 18)(p11.2; q11.2) translocation for this case.I’m sorry, but to establish a diagnosis of synovial sarcoma the positivity of the translocation is needed. Without the presence of this test I couldn’t judge the rest of the paper.If the authors forgot to write in the paper the execution of the test, they have to write it; otherwise I do not approve the paper.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-63
|
https://f1000research.com/articles/1-61/v1
|
07 Dec 12
|
{
"type": "Research Article",
"title": "Kyasanur forest disease virus: viremia and challenge studies in monkeys with evidence of cross-protection by Langat virus infection",
"authors": [
"Keerti V Shah",
"Chandu N Dandawate",
"Pravin N Bhatt",
"Chandu N Dandawate",
"Pravin N Bhatt"
],
"abstract": "Kyasanur Forest Disease Virus (KFDV), discovered in 1957, is a member of the tick-borne encephalitis virus (TBEV) complex. Diseases caused by members of the TBEV complex occur in many parts of the world. KFDV produces a hemorrhagic fever in humans in South India and fatal illnesses in both species of monkeys in the area, the black faced langur (Presbytis entellus) and the bonnet macaque (Macaca radiata). Experimental infection of the langur and the bonnet macaque with early mouse passage KFDV strain P9605 resulted in a viremia of up to 11 days duration, peak viremia titers as high as 109, and death in 82 = 100% of the animals. Prolonged passage of the KFDV strain P9605 in monkey kidney tissue culture resulted in a markedly reduced virulence of the virus for both species; peak viremia titers in monkeys decreased by 2.5 to 4.0 log LD 50 (p= 0.001), and the mortality decreased to 10% (p= 0.001). In challenge experiments, monkeys previously infected with tissue-culture-adapted KFDV, or with the related Langat virus from Malaysia, were fully protected against virulent KFDV. These studies in non-human primates lend support to the idea that a live virus vaccine from a member of the TBEV complex may be broadly protective against infections by other members of the TBEV complex.",
"keywords": [
"The studies and results presented in this article were conducted between 1958 and 1970 when ethical standards for animal protect-ion and welfare",
"and acceptable practice with regard to animal research",
"were very different from those standards considered normal today."
],
"content": "Introduction\n\nFlaviviruses of the tick-borne encephalitis virus (TBEV) complex are widely and focally distributed in nature. New viruses of the complex and illnesses associated with them are frequently being described from different parts of the world. Human illnesses associated with infections with these viruses include mild or severe encephalitis and hemorrhagic fevers1. Kyasanur Forest Disease Virus (KFDV), a member of the hemorrhagic fever group, was discovered in 1957 in the Shimoga district of Karnataka State, India, where it produced a febrile illness and deaths in humans, and deaths in both species of monkeys, the black-faced langur (Presbytis entellus) and the red-faced bonnet macaque (Macaca radiata), that inhabit the endemic area2. Most of the human infections with KFDV occur in the drier months of the year, with peaks in March and April, and the number of cases is variable, but may reach over 500 in some years. Alkhurma Hemorrhagic Fever Virus (AHFV), described in 1997, a cause of severe hemorrhagic fever in Saudi Arabia and other regions of the Middle East3,4 and Nanjianyin Virus recovered from a febrile patient in Yunnan, China5, are closely related to KFDV. Omsk Hemorrhagic Fever Virus (OHFV), which is endemic in western Siberia, Russia, and recognized as a tick-borne flavivirus in 1947, is distantly related to KFDV1. In addition to the hemorrhagic fevers noted above, viruses of the TBEV complex are also responsible for over 10,000 cases of encephalitis, annually, in Europe and Asia, and for localized illnesses in different parts of the world1.\n\nIn 1958–59, a formalin-inactivated mouse-brain virus vaccine prepared with the heterologous Russian spring-summer encephalitis virus (RSSE) was administered to over 10,000 residents in the KFDV-endemic area. Although this vaccine was protective against KFDV challenge in the mouse model6, it was completely ineffective in preventing human KFDV infections in the endemic area7. It also failed to prevent KFDV infections in laboratory personnel8,9. Serologically, individuals immunized with the RSSE vaccine showed a barely detectable immune response to KFDV antigens10. In contrast to the lack of effectiveness of an inactivated RSSE vaccine, an inactivated KFDV vaccine reduced the incidence of KFDV in the affected area11. Thus, KFDV infections in humans were preventable by an inactivated homologous vaccine, but not by an inactivated heterologous vaccine.\n\nIn the late 1950s and 1960s, investigators from the Virus Research Centre (VRC) [now the National Institute of Virology (NIV)] in Pune, India, conducted infection and challenge experiments in non-human primates using virulent and tissue-culture-adapted (TC-adapted) strains of KFDV as well as Langat virus (LGTV), a related virus of the TBEV complex isolated from ticks in Malaysia12. The results of these studies were described only in the annual reports of the VRC and in a doctoral thesis published in 19636, so they are not readily available. Because data obtained from experimental infection and challenge of non-human primates with viruses of the TBEV complex are valuable and may help efforts aimed at developing prophylactic vaccines, we have combined the information from the doctoral thesis and from an unpublished manuscript (Bhatt and Dandawate, unpublished observations, 1971) for this report.\n\nWe show here that 1) KFDV produces a high-titered viremia and death in both species of monkeys that inhabit the endemic area, 2) a TC-adapted KFDV has markedly reduced virulence for both monkey species, and 3) monkeys previously infected with TC-adapted KFDV or with heterologous LGTV completely resist challenge with virulent KFDV. Together, these data lend support to the idea that in contrast to the inactivated vaccines that do not cross-protect, an attenuated live virus vaccine derived from a member of the TBEV complex may be broadly protective against the different viruses of the complex.\n\n\nMaterials and methods\n\nStudies described here were conducted at the VRC between 1958 and 1970.\n\nThe animals were trapped by professional catchers in the forested areas of Shimoga district, Karnataka State in south India, outside the known area of KFD infection, and were transported to the laboratory in Pune by train or by truck. The animals were held for observation for several weeks prior to inoculation. At the commencement of the experiments, the weight range (and mean weight) of the animals was 2.0–7.2 kg (4.6 kg) for langurs, and 1.8–5.6 kg (3.3 kg) for bonnets. Animals were first classed by weight and then individuals in each weight class were allocated randomly to the different groups in the experiment.\n\nPre-inoculation serum specimens from the animals were screened for antibodies to KFDV, LGTV, West Nile and dengue 2 by hemagglutination inhibition (HI) tests13. Sera that had HI antibodies to any of the viruses (less than 10% of the sera) were screened for neutralizing antibodies to KFDV10 and the rare animal that had KFDV neutralizing antibodies was not included in the experiment. The animals were inoculated with KFDV by the subcutaneous (SC) or the intravenous (IV) route and with LGTV by the IV or intracerebral (IC) route. The inoculated animals were monitored daily for viremia and general state of health. Rectal temperature was taken daily but these data were found to be uninformative and are not described in this report. Tissues of some of the animals that died during the observation period were tested for virus. Pathological studies of tissues were not routinely performed.\n\nData are reported from a total of 72 animals: 42 langurs and 30 bonnets. Three animals that did not develop either viremia or antibodies following inoculation are excluded from analyses.\n\nThe virus preparations used and their designations are listed below.\n\na) Virulent KFDV. Early passage KFDV is designated as virulent KFDV. Two strains of virulent KFDV were used. Most of the experiments were performed with strain P9605 derived from serum of a febrile individual in the endemic area and isolated and maintained by infant mouse brain passage in Swiss albino strain of mice. Passages 4–12 in infant mouse brain were employed with passage 9 used for a majority of the animals. These preparations are referred to as virulent KFDV (mouse brain) in this report. A second strain, KFDV 603260–2, was isolated and maintained in the bonnet macaque after inoculation from a mixture of two KFDV-positive tick pools. Monkey serum from the second passage of this strain was used as inoculum for one bonnet and four langurs. This strain is referred to as virulent KFDV (monkey serum).\n\nb) Tissue-culture-adapted KFDV. The virulent KFDV P9605 was passaged in primary rhesus or bonnet monkey kidney tissue culture (MKTC) prepared from healthy wild-caught animals which were non-immune to KFD, and was shown to be attenuated for mice as judged by the decrease in mortality in these animals following intraperitoneal inoculation14. The four preparations used in the primate studies were all derived from P9605, which had been adapted to MKTC following one infant mouse brain and two chick embryo tissue culture passages. The 137th and 138th monkey kidney tissue culture passage virus was used in the studies described in the doctoral thesis6. This preparation is designated ‘TC-adapted KFDV MK 137–138’. Preparations employed in the experiments in the unpublished observations of Bhatt and Dandawate (1971) were i) 177th monkey kidney tissue culture passage (designated TC-adapted KFDV MK 177); ii) virus plaque purified by three passages in monkey kidney cell monolayers after 144 passages in monkey kidney tissue culture and then passaged twice again in monkey kidney tissue culture (designated TC-adapted KFDV, plaque); and iii) the plaque-purified virus passaged 41 times in chick embryo tissue culture (designated TC-adapted KFDV, CE 41).\n\nc) Langat virus LGTV. The TP 21 isolate of LGTV derived from a tick pool was used in its 8th and 9th infant mouse brain passage (LGTV, M8 or M9), or as the 14th chick embryo tissue culture passage following nine infant mouse brain passages (LGTV, CE14).\n\nHeparinized blood or serum specimens were collected daily beginning with day 1 or day 2 post-inoculation (pi) until day 10 or 11 pi. They were titrated on the day of the bleeding and, if end points were not reached, an aliquot stored at -70°C was re-titrated at a later date. Titrations for KFDV were performed in adult mice intracerebrally, except in one experiment where they were determined by inoculation of chick embryo tissue culture tubes (which have a sensitivity similar to that of adult mice). Titrations for Langat virus were performed in infant mice intracerebrally. Viremia values are reported as the positive logarithms of the LD50 titer per 0.03 ml (when titrated in adult mice or tissue culture) or 0.02 ml (when titrated in infant mice for Langat virus) of the inoculum. If the undiluted serum killed three of six mice, the estimate of the LD50 titer was 100.0. If it killed less than one half of the mice the titer was reported as “trace virus (T)”.\n\nA lowess smoother was used to generate the estimated regression lines in Figure 1 and Figure 2. For continuous variables, non-parametric Wilcoxon test for differences between medians was used as a test of statistical significance. All graphs and statistical analyses were performed using Stata (version 9.0).\n\n\nResults\n\nVirulent KFDV in the black-faced langur. Thirteen langurs were inoculated with virulent KFDV by the SC or the IV route (Table 1). The amount of virus in the inoculum ranged from 2.8–5.1 log LD50. Viremia was detected continuously from day 2 pi to the day of death or the day before death for 12 of the 13 animals. Peak titers in individual monkeys were reached between the 3rd and 7th day and they ranged from 104.5–108.5 with a median value (one value for each animal) of 106.6.\n\n* Positive logarithms of adult mouse LD50 titers/0.03 ml.\n\nPeak titer is underlined.\n\nT = Trace; N = Negative; X = Died; IV = Intravenous; SC = Subcutaneous.\n\nAll 13 animals died between day 5 and day 9 pi, with an average survival time of 6.5 days. With one exception, animals died during the viremic phase and the majority died at or near the peak of viremia. The exception was animal #84 that died on day 9, but for whom viremia was not estimated on the day of or day before death. Tissues of two animals collected post mortem (#84 and #85) were titrated for KFDV; all tested organs (blood, brain, lung, liver, spleen, kidney) were positive with titers ranging from 101.9–106.5.\n\nTC-adapted KFDV in the black-faced langur. Twenty langurs were inoculated with TC-adapted KFDV by the IV or SC route. The amount of virus in the inoculum ranged from 101.9 to 107.9 (Table 2). There was considerable variation in the duration and peak titers of viremia between animals given different passages of the TC-adapted KFDV. For example, four of the five animals given MKTC 138 had viremia of 5–6 days duration, and their peak titers did not exceed 103.6. In contrast, viremia of 10 days duration, with peak titers of 105.0 or greater, were seen in animals that received CE 41. The highest titers in the 20 individual animals (one value for each animal) ranged from 100.6 to 105.7 with a median value of 104.5.\n\n* Positive logarithms of adult mouse LD50 titers/0.03 ml.\n\nPeak titer is underlined.\n\nT = Trace; N = Negative; X = Died; IV = Intravenous; SC = Subcutaneous.\n\nThere were two deaths among the 20 animals, one (#225) on day 6 at the peak of viremia and the other on day 10 (#85) in a monkey which was viremic from days 1–10. In tests of brain, lung, spleen, liver, kidney and heart tissues of animal #85, KFDV was isolated only from the lung tissue. The lung isolate was identified as the TC-adapted strain on the basis of its low pathogenicity for subadult mice14.\n\nVirulent KFDV in the bonnet macaque. Eleven bonnet macaques were inoculated with virulent KFDV by the IV or SC route. The amount of virus in the inoculum ranged from 104.0 to 108.1 LD50 (Table 3). The duration of viremia after inoculation of the mouse brain virus was from 5 to 10+ days. Peak titers of between 104.2 and 109.0 were reached on the second, third or fourth day. In the single animal given the monkey serum virus, viremia was observed from days 2 to 11 with high titers ranging from 107.2 to 109.0 from days 5 to 11. The median value of the highest titers in the 11 bonnets was 106.5.\n\n* Positive logarithms of adult mouse LD50 titers/0.03 ml.\n\nPeak titer is underlined.\n\nT = Trace; N = Negative; X = Died; **#281 died on day 21, #302 on day 20; IV = Intravenous; SC = Subcutaneous.\n\nNine of the 11 animals inoculated with the virulent virus died. Deaths occurred either at the end of the viremic phase (4 deaths on day 9 and one on day 10), or at the height of viremia (one death on day 11 with the monkey serum virus), or in the post-viremic phase (one death each on day 14, 20, and 21). Tissues of five of these animals were tested for KFDV. Three of these animals (#276, #279, #174) died early in the illness during the viremic phase; virus was recovered from all of their tissues (blood, liver, lung, spleen, kidney) that were examined. The amount of virus was highest in spleen or blood and lowest in the brain. In the two animals that died in the post-viremic phase at the end of the third week (#281 and #302), virus was present almost exclusively in the brain with titers of 104.2 and 104.9.\n\nTC-adapted KFDV in the bonnet macaque. Ten animals were inoculated either by the IV or the SC route with 105.3–106.3 LD50 of the virus (Table 4). The duration of viremia varied from 6–8 days. The peak titers in individual monkeys ranged from 101.6–105.5 and were obtained between 2 and 5 days pi. The median value of the highest titers in the 10 bonnets was 102.3.\n\n* Positive logarithms of adult mouse LD50 titers/0.03 ml.\n\nPeak titer is underlined.\n\nT = Trace; N = Negative; ** #139 died on day 26; IV = Intravenous; SC = Subcutaneous.\n\nThere was one death during the observation period. On day 26, animal #139 which was viremic for 7 days pi died after a bout of diarrhea. In tests of the tissues of this animal for virus, lung, liver, heart and kidney were negative but less than 10 LD50 of virus were recovered from brain tissue. The isolate was identified as attenuated by its low pathogenicity in adult mice. The isolate when inoculated in four bonnets each by the IC and SC routes produced viremia in all, but no death or overt encephalitis in any animal.\n\nThe viremia data of the virulent and TC-adapted strains of KFDV in the two species of monkeys are summarized in Figure 1 and Figure 2. As compared to the virulent strain, the TC-adapted strain had 2.15 log10 units lower peak titers in the langur and 4.0 log10 units lower peak titers in the bonnet macaque. These differences were highly significant (p < 0.001) (Table 5). The mortality in the two species was reduced from 82–100% for the virulent strain to 10% for the TC-adapted strain (p < 0.001) (Table 5).\n\n* One value for each animal, underlined in Table 1–Table 4.\n\nIn the black-faced langur. Peak titers in individual monkeys inoculated with the ninth infant mouse brain passage of the TP21 strain of Langat virus ranged from 100.5–101.5 and were observed on days 2 or 3 pi. The duration of viremia varied from 2–7 days with a mean duration of 4.2 days. Four animals inoculated IC with the tissue culture virus had a somewhat longer duration of viremia (mean duration 5.8 days), peak titers were observed on days 3 and 4 pi and they ranged from 101.6–102.\n\nIn the bonnet macaque. All nine animals inoculated either IV (5 animals) or IC (4 animals) with Langat TP21 M8-M9 were viremic on the first two days; peak titers ranging from 101.6 to >104.0 (mean of 102.6) were observed. The proportion of monkeys circulating the virus as well as the level of viremia decreased after the second day and viremia was not demonstrable in any monkey after day 6. The average duration of viremia was 3.6 days.\n\nGroups of animals, previously infected with Langat virus or with TC-adapted KFDV, were challenged with virulent KFDV. Data are combined in Table 6.\n\n1Survivors of IV or IC infection.\n\n2Survivors of animals infected with MK137 and MK138 in Table 4.\n\n3Survivors of animals infected with MK177, plaque and CE41 in Table 2.\n\n4Survivors of animals infected with CE41 in Table 4.\n\n5Described in Table 3, top 8 animals.\n\n6Described in Table 1, last 3 animals.\n\nIV = Intravenous; SC = Subcutaneous.\n\nEight bonnets previously infected with Langat virus, and 4 bonnets previously infected with TC-adapted KFDV were challenged IV with 7.6–8.1 log LD50 of virulent KFDV. The interval between the immunizing infection and challenge was 31–38 days. In daily tests for viremia for 10 days, no viremia was detected in the four animals previously infected with TC-adapted KFDV, but ‘trace’ viremia was detected in 3 of the 8 bonnets previously infected with Langat virus, on day 1 or 2 pi. All but one animal survived challenge. One bonnet previously infected with Langat virus died on day 10 after challenge; blood, liver, lung, kidney, spleen and brain of this animal did not yield virus. This death was considered unrelated to the challenge.\n\nIn a second series of challenge experiments in the unpublished observations of Bhatt and Dandawate (1971), 9 langurs and 2 bonnets previously infected with TC-adapted KFDV were challenged with 4.2–5.0 log LD50 of virulent KFDV. The interval between the immunizing and challenge infections was 4–30 months. In daily tests for viremia for 10 days pi, none were shown to be viremic. All of the animals survived the challenge.\n\n\nDiscussion\n\nKenyon et al.15 have stated that “one of the most serious constraints for the study of TBE pathogenesis and for vaccine development has been the absence of realistic animal models that mimic human disease”. Our results of infection of two species of monkeys with virulent and TC-adapted KFDV and with Langat virus, and challenge of survivors with virulent KFDV, address this question.\n\n\nAnimal model for KFDV pathogenesis\n\nBoth the black-faced langur and the red-faced bonnet appear to be good models to study KFDV infection. Both species are naturally infected by KFDV in the endemic forested area in south India and many succumb to the disease16,17. A large majority of the animals found dead in the forest are langurs. The results of our experimental infections showed that the langur was highly susceptible to KFDV, and that the disease was acute, of short duration and always fatal. Viremia titers were high and deaths occurred between days 5–10 pi at or near the peak of viremia. Disease in the bonnet macaque seemed to resemble more closely the biphasic human disease. In some infected humans, the acute viremic phase is followed by a second non-viremic phase in which the patient has neurological symptoms and abnormal cerebrospinal fluid, but the symptoms are not localizing and there are no neurologic sequelae18,19. In the bonnet macaque, some deaths occurred during the viremic phase, but others occurred in the third week, at which time virus was recovered from the brain but not from other organs. In previous investigations, Webb and Burston20 have described neurological changes in the post-viremic period in experimentally infected bonnet macaques. Kenyon et al.15 have reported that the bonnet macaques experimentally infected with KFDV have a 100% mortality and that they show significant hematological and neurological abnormalities similar to those in KFDV-infected humans as well as significant histopathological changes in the small and large intestine, spleen and lymph nodes.\n\nIn contrast to its high pathogenicity in the bonnet macaque, KFDV does not produce illness or death in the rhesus macaque, although the pattern of viremia is similar in the two species2,6,21.\n\nReduced virulence of TC-adapted KFDV: Bhatt and Anderson14 have reported that the passage of the prototype P9605 strain of KFDV in monkey kidney tissue culture (MKTC) resulted in a decrease of the virulence of the virus for adult mice as judged by a decrease in LD50 titer of the virus following intraperitoneal (IP) inoculation. In the current investigation, the lower virulence of the virus was also clearly evident in both species of monkeys. In the langurs, the viremia levels seemed to be lower after infection with MKTC 138 passage virus as compared to titers after inoculation of the other three preparations of TC-adapted KFDV. Nevertheless, combined for all passage levels and for both species, the mortality decreased from 82–100% with the virulent virus to 10% with the TC-adapted virus. The reduced virulence was also reflected in a decrease in peak titers of viremia of 2.5–4.5 log LD50 in the two species of monkeys. Identification of changes in the KFDV genome associated with this decrease in virulence may help us to understand the genetic determinants of KFDV virulence.\n\n\nProtection against homologous and heterologous challenge\n\nViruses of the TBE complex are distributed focally in many parts of the world and they produce diseases of varying severity and in varying numbers. So, a single vaccine which can provide protection against all members of the complex would be beneficial. Among viruses of the TBE complex, Langat virus from Malaysia is the least virulent for mice, and mice infected intraperitoneally with Langat virus survive and they resist IP challenge by all of the viruses of the TBE complex that were tested22. Monkeys that had survived infection with the TC-adapted KFDV or with Langat virus completely resisted challenge with virulent KFDV. There was not a single virus-related death in these animals. In addition, there was no viremia at all in the animals previously infected with attenuated KFDV and only traces of viremia in some of the monkeys previously infected with Langat virus. A similar protection against homologous as well as heterologous challenge has been demonstrated in a report by Rumyantsev et al.23 in which chimeric live virus vaccines which contained sequences of TBEV and dengue 4 (TBEV/den4) or Langat virus and dengue 4 (LGT/den 4) were tested in rhesus monkeys. Animals immunized with either of the chimeric vaccine did not develop viremia when challenged with Langat virus.\n\nLive Langat virus has been used in experimental treatment of human malignant disease in the United Kingdom and also as a live virus vaccine against TBE in Russia. In both cases, the neurotropism of Langat virus for humans became apparent. In the United Kingdom, two of 23 cancer patients infected with Langat virus developed encephalitis 8–11 days after the end of viremia24. Immunization of over 600,000 individuals with live Langat virus was reported to have reduced markedly the incidence of encephalitis in the endemic region in Russia, but the vaccine was associated with a serious adverse effect and was responsible for the occurrence of 35 cases of meningoencephalitis with permanent sequelae1. Attempts have been made to construct Langat virus which retains its immunogenicity but is less neuroinvasive25.\n\nThe TC-adapted strain of KFDV has a markedly decreased virulence for non-human primates and it merits consideration for further development as a live virus vaccine. It may be anticipated that an attenuated KFDV vaccine will provide significantly greater protection against KFD than inactivated KFDV vaccine, and it may also be effective against other viruses of the TBE complex. An attenuated KFDV vaccine may be less likely to produce paralytic sequelae than the Langat live virus vaccine because human KFDV infections in south India, while they cause transient neurological illness in some infected individuals, are not associated with paralytic disease. Components of the attenuated KFDV strain could also be employed to construct a genetically engineered non-infectious recombinant TBEV vaccine23,26 in the search for a safe and broadly effective vaccine strategy against viruses of the TBEV complex.",
"appendix": "Author contributions\n\n\n\nAll three authors contributed to the design and conduct of the studies, and to the preparation of the manuscript.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThe Virus Research Center (renamed National Institute of Virology) was supported by the Indian Council of Medical Research, New Delhi and the Rockefeller Foundation, New York.\n\n\nAcknowledgements\n\nWe thank Dr. Mahboobeh Safaein and Ms. Michelle Silver for statistical help.\n\n\nReferences\n\nGritsun TS, Nuttall PA, Gould EA, et al.: Tick-borne flaviviruses. Adv Virus Res. 2003; 61: 317–371. PubMed Abstract | Publisher Full Text\n\nWork TH: Russian spring-summer virus in India: Kyasanur Forest disease. Perspect Med Virol. 1958; 1: 248–279. PubMed Abstract\n\nZaki AM: Isolation of a flavivirus related to the tick-borne encephalitis complex from human cases in Saudi Arabia. Trans R Soc Trop Med Hyg. 1997; 91(2): 179–181. PubMed Abstract | Publisher Full Text\n\nCharrel RN, deLamballerie X: The Alkhurma virus (family Flaviviridae, genus Flavivirus): an emerging pathogen responsible for hemorrhage fever in the Middle East. Med Trop (Mars). 2003; 63(3): 296–299. PubMed Abstract\n\nWang J, Zhang H, Fu S, et al.: Isolation of Kyasanur Forest Disease Virus from Febrile Patient, Yunnan, China. Emerg Infect Dis. 2009; 15(2): 326–328. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShah KV: Studies towards the development of a vaccine against Kyasanur forest disease. Thesis, Johns Hopkins University School of Hygiene and Public Health. 1963.\n\nShah KV, Aniker SP, Murthy DP, et al.: Evaluation of the field experience with formalin-inactivated mouse brain vaccine of Russian spring-summer encephalitis virus against Kyasanur Forest disease. Indian J Med Res. 1962; 50: 162–174. PubMed Abstract\n\nMorse LJ, Russ SB, Needy CF, et al.: Studies of viruses of the tick-borne encephalitis complex. II. Disease and immune responses in man following accidental infection with Kyasanur Forest disease virus. J Immunol. 1962; 88: 240–8. PubMed Abstract\n\nBanerjee K, Gupta NP, Goverdhan MK, et al.: Viral infections in laboratory personnel. Indian J Med Res. 1979; 69: 363–73. PubMed Abstract\n\nPavri KM, Gokhalet T, Shah KV, et al.: Serological response to Russian spring-summer encephalitis virus vaccine as measured with Kyasanur Forest disease virus. Indian J Med Res. 1962; 50: 153–161. PubMed Abstract\n\nDandawate CN, Desai GB, Achar TR, et al.: Field evaluation of formalin inactivated Kyasanur forest disease virus tissue culture vaccine in three districts of Karnataka state. Indian J Med Res. 1994; 99: 152–158. PubMed Abstract\n\nSmith CEG: A virus resembling Russian spring-summer encephalitis virus from an Ixodid tick in Malaya. Nature. 1956; 178(4533): 581–582. PubMed Abstract | Publisher Full Text\n\nClarke DH, Casals J: Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am J Trop Med Hyg. 1958; 7(5): 561–573. PubMed Abstract\n\nBhatt PN, Anderson CR: Attenuation of a strain of Kyasanur Forest disease virus for mice. Indian J Med Res. 1971; 59(2): 199–205. PubMed Abstract\n\nKenyon RH, Rippy MK, McKee KT Jr, et al.: Infection of Macaca radiata with viruses of the tick-borne encephalitis group. Microb Pathog. 1992; 13(5): 399–409. PubMed Abstract | Publisher Full Text\n\nGoverdhan MK, Rajagopalan PK, Narasimha Murthy DP, et al.: Epizootiology of Kyasanur Forest Disease in wild monkeys of Shimoga district, Mysore State (1957–1964). Indian J Med Res. 1974; 62(4): 497–510. PubMed Abstract\n\nSreenivasan MA, Bhat HR, Rajagopalan PK, et al.: The epizootics of Kyasanur Forest disease in wild monkeys during 1964 to 1973. Trans R Soc Trop Med Hyg. 1986; 80(5): 810–814. PubMed Abstract | Publisher Full Text\n\nWebb HE, Rao RL: Kyasanur forest disease: a general clinical study in which some cases with neurological complications were observed. Trans R Soc Trop Med Hyg. 1961; 55: 284–298. PubMed Abstract | Publisher Full Text\n\nAdhikari PMR, Prabhu MG, Raghuveer CV, et al.: Clinical study of 100 cases of Kyasanur Forest disease with clinicopathological correlation. Indian J Med Sci. 1993; 47(5): 124–130. PubMed Abstract\n\nWebb HE, Burston J: Clinical and pathological observations with special reference to the nervous system in Macaca radiata infected with Kyasanur Forest Disease virus. Trans R Soc Trop Med Hyg. 1966; 60(3): 325–331. PubMed Abstract | Publisher Full Text\n\nIlyenko VI, Pokrovskaya OA: Clinical picture in M. rhesus monkeys infected with various strains of tick-borne encephalitis virus. In: Libikova, H. (Ed), Biology of viruses of the tick-born encephalitis complex. Czechoslovak Acad Sci, Praha. 1962; 266–269.\n\nShah KV, Cole GA, Russ SB, et al.: Relative avirulence in laboratory rodents of the Malayan virus, TP 21. In: Libikova, H. (Ed), Biology of viruses of the tick-born encephalitis complex. Czechoslovak Acad Sci, Praha. 1962; 303–310. Reference Source\n\nRumyantsev AA, Chanock RM, Murphy BR, et al.: Comparison of live and inactivated tick-borne encephalitis virus vaccines for safety, immunogenicity and efficacy in rhesus monkeys. Vaccine. 2006; 24(2): 133–43. PubMed Abstract | Publisher Full Text\n\nBurston J, Webb HE, Gordon Smith CE, et al.: Neuropathological changes following treatment of human malignant disease with Langat virus. J Neuropathol Exp Neurol. 1967; 26(3): 511–517. PubMed Abstract | Publisher Full Text\n\nPletnev AG: Infectious cDNA clone of attenuated Langat tick-borne flavivirus (strain E5) and a 3' deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice. Virology. 2001; 282(2): 288–300. PubMed Abstract | Publisher Full Text\n\nLai CJ, Monath TP: Chimeric flaviviruses: novel vaccines against dengue fever, tick-borne encephalitis, and Japanese encephalitis. Adv Virus Res. 2003; 61: 469–509. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "394",
"date": "17 Dec 2012",
"name": "Dennis A Bente",
"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": "406",
"date": "18 Dec 2012",
"name": "Michael Holbrook",
"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": "396",
"date": "19 Dec 2012",
"name": "Alexander N. Freiberg",
"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
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https://f1000research.com/articles/1-61
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https://f1000research.com/articles/1-12/v1
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22 Aug 12
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{
"type": "Opinion Article",
"title": "Diversion at the ER: How Plasmodium falciparum exports proteins into host erythrocytes",
"authors": [
"Karin Römisch"
],
"abstract": "Malaria is caused by parasites which live in host erythrocytes and remodel these cells to provide optimally for the parasites’ needs by exporting effector proteins into the host cells. Eight years ago the discovery of a host cell targeting sequence present in both soluble and transmembrane P. falciparum exported proteins generated a starting point for investigating the mechanism of parasite protein transport into infected erythrocytes. Since then many confusing facts about this targeting signal have emerged. In this paper, I try to make sense of them.",
"keywords": [
"P. falciparum infects erythrocytes and causes malaria in humans. The parasite resides intracellularly in a parasitophorous vacuole (PV)",
"and exports proteins into the erythrocyte that are important for parasite survival (Figure 1)1",
"2. The identification of a host cell targeting signal in exported P. falciparum proteins was an important first step towards understanding the export mechanism",
"but left cell biologists puzzled: Marti et al. (2004) and Hiller and colleagues (2004) identified a short sequence",
"RxLxE/Q",
"which is present in many proteins in the P. falciparum genome known to be exported from the parasite into the erythrocyte3",
"4. This P. falciparum protein export element (PEXEL) was found by both groups to be necessary and sufficient for protein export into the host cell3",
"4."
],
"content": "The problem\n\nP. falciparum infects erythrocytes and causes malaria in humans. The parasite resides intracellularly in a parasitophorous vacuole (PV), and exports proteins into the erythrocyte that are important for parasite survival (Figure 1)1,2. The identification of a host cell targeting signal in exported P. falciparum proteins was an important first step towards understanding the export mechanism, but left cell biologists puzzled: Marti et al. (2004) and Hiller and colleagues (2004) identified a short sequence, RxLxE/Q, which is present in many proteins in the P. falciparum genome known to be exported from the parasite into the erythrocyte3,4. This P. falciparum protein export element (PEXEL) was found by both groups to be necessary and sufficient for protein export into the host cell3,4.\n\nP. falciparum (blue) resides in red blood cells (pale orange) inside a parasitophorous vacuole (white). In order to survive, P. falciparum needs to export numerous proteins into the red blood cell, which remodel the host cell to suit the purposes of the parasite. The mechanism by which these proteins are exported is still unclear (Figure modified from Römisch, 2005)6.\n\nWhat remains unclear to date is the mechanism by which an export signal present in both soluble and transmembrane proteins can mediate transport of both types of protein into the erythrocyte. This issue was debated hotly, but our ideas at the time were limited, because they were solely based on the classical secretory pathway in mammalian cells5–7.\n\n\nThe fact(or)s\n\nSince then, a lot more data related to the PEXEL sequence have been generated, but rather than clarify they seem to confuse the issue further. Meanwhile it has been shown that:\n\nThe secretory signal peptides of the exported soluble proteins, which are located 20–40 amino acids upstream of the host cell targeting signals, are not cleaved upon endoplasmic reticulum (ER) targeting8,9.\n\nThe host cell targeting signal is cleaved and the new N-terminus is N-acetylated at the ER membrane9–11.\n\nThe protease responsible for the cleavage has been identified9,10,12.\n\nCleavage by this protease is a prerequisite for transport into the erythrocyte9,10; if you generate the cleaved N-terminus by modifying the gene and combine it with a cleavable signal peptide, the resulting protein is secreted into the PV and remains there9.\n\nThe uncleaved targeting signal binds phosphoinositol-3-phosphate (PI3P) at the ER membrane with the same specificity required for protease cleavage and host cell targeting; the cleaved signal no longer binds PI3P13.\n\nA putative ‘translocator’ complex resides in the PV membrane (PVM); it consists of 5 proteins that coprecipitate some of the proteins bearing a host cell targeting signal, but a function of the complex has not been demonstrated in any way, nor has it been investigated whether the association of the complex and the PEXEL proteins is mediated by the PEXEL signal14,15.\n\n\nThe hypothesis\n\nThe authors of the respective papers assume that proteolytic cleavage, N-acetylation, and PI3P binding take place in the ER lumen (Figure 2A)9,10,13. This cannot be right: N-acetylation is a cytosolic modification, the active site of plasmepsin V is almost certainly on the cytoplasmic face of the ER membrane (based on the biochemical & sequence data characterizing the protease), and the only possible location for PI3P at the ER membrane is in the cytosolic leaflet and the only possible location for PI3P at the ER membrane is in the cytosolic leaflet. In detail:\n\nA) The hypothesis. Most proteins targeted for export into the host cell have a signal sequence (yellow) or transmembrane domain, which leads to their SRP-mediated targeting to the protein translocation channel (Sec61) in the ER membrane of the parasite (1). Many but not all of the P. falciparum exported proteins have an N-terminal extension (red zigzag) whose function is unknown. In addition, host cell targeted proteins contain, in a distance of 20–40 amino acids from the signal peptide, a PEXEL sequence (RxLxE), which is also required for binding to phosphoinositol-3-phosphate (PI3P; orange balls) and for cleavage by the ER-membrane associated protease plasmepsin V (PM5). The current hypothesis in the field is that, after signal-peptide mediated translocation into the ER lumen, the PEXEL sequence binds to PI3P in the lumenal face of the ER membrane and is cleaved by PM5 (2). The cleaved protein is released from PM5 (3) and continues through the P. falciparum secretory pathway by vesicular transport (4). Note that this model cannot explain the NatD-mediated N-acetylation of the PM5-cleaved N-terminus, because NatD resides in the cytosplasm. B) The alternative. After SRP-mediated targeting of the protein destined for export to the parasite ER (1) and insertion into the Sec61 channel, the N-terminal extension of the signal peptide (red zigzag) delays signal cleavage, perhaps by preventing reorientation of the signal peptide in the Sec61 channel. This delay in completing translocation allows the RxLxE sequence to bind to PI3P (orange balls) on the cytoplasmic face of the ER membrane, which creates a recognition signal for PM5 and results in proteolytic cleavage (2). Cleavage releases the protein from the translocation machinery and allows N-acetylation by NatD (3). The mature protein is handed over to a PI3P-associated putative transmembrane receptor (R; 4), which may itself be a PEXEL protein.\n\nAll known enzyme complexes mediating N-acetylation including NatD, which is likely responsible for the N-acetylation of proteolytically cleaved PEXEL proteins, are located in the cytosol16. The presence of a secretory signal sequence indeed strongly reduces the likelihood of proteins being N-acetylated, confirming that N-acetylation does not take place inside compartments of the secretory pathway17.\n\nIn the initial characterization of plasmepsin V, Klemba & Goldberg (2005)12 found a hydrophobic region at the N-terminus, which they described as a putative signal sequence. There is no discernible signal peptidase cleavage site C-terminal of this hydrophobic region and indeed Klemba and Goldberg did not observe processing of the N-terminus of plasmepsin V in pulse-chase experiment. Russo and colleagues (2010)10 showed later that this region of plasmepsin V was not able to target the protein to the ER, which demonstrates that it is not a signal sequence. In the same paper Russo and colleagues demonstrated that the C-terminal hydrophobic region of plasmepsin V was required to anchor the protein to the ER membrane. Both Russo and colleagues and Klemba and Goldberg describe this region as a transmembrane domain, but Klemba & Goldberg show that 50% of plasmepsin V can be extracted from the membrane by carbonate, pH 11.012. A standard feature of a transmembrane protein, however, is that it is resistant to carbonate extraction at pH 11.518. Altogether these data suggest that plasmepsin V is a carboxy-terminally membrane-anchored or membrane-associated protein with its entire N-terminal domain including the active site in the cytoplasm.\n\nLocalization of PI3P has been investigated in yeast and mammalian cells where it is found on early and late endosomes and transiently at the plasma membrane19. PI3P is generated by PI3-kinases on the cytoplasmic leaflet of intracellular membranes and regulates membrane trafficking events20. The localization of the kinase determines the localization of the PI3P patch20. There are no PI3 kinases inside the secretory pathway. The only known example of PI3P occurring at the ER is during formation of autophagosomal membrane precursors, the so-called omegasomes21. Even in this case, PI3P is generated by the Vps34 PI3-kinase in the cytoplasmic leaflet of the ER membrane22.\n\n\nThe alternative\n\nAn alternative explanation for most of the available data is that their secretory signal peptides target PEXEL proteins to the P. falciparum ER, but are inefficiently cleaved - perhaps due to their long N-terminal extensions (Figure 2B, (1)). This is similar to the biogenesis of some autotransporters in pathogenic E. coli, where delayed signal peptide cleavage due to N-terminal signal peptide extension allows the passenger domain to remain unfolded in the periplasm while the porin domain assembles in the E. coli outer membrane23. Stuck in the protein translocation channel in the P. falciparum ER membrane, the PEXEL protein in transit is oriented such that the host cell targeting signal can bind PI3P at the cytoplasmic face of the ER membrane (Figure 2B, (2)). Binding creates the cleavage site for the protease plasmepsin V (Figure 2B, (2)). This possibility has also been mentioned, but not been pursued experimentally, in Bhattacharjee et al.13. After cleavage and N-acetylation (Figure 2B, (3)) the new N-terminus is not released, but the protein remains membrane-tethered or associated with a transmembrane protein (Figure 2B, (4)) until it reaches the cell surface from where it is transferred to the erythrocyte, perhaps by vesicular transport (Figure 3).\n\nCleaved N-acetylated PEXEL proteins (red triangles) associated with their receptor (black bar) could be transported to the cell surface in a complex (1) in one of two ways. Either the receptor/soluble protein complex remains associated with the PI3P patch (green) in the ER membrane. The patch and associated proteins are transported by vesicle budding (2A) and fusion through the parasite secretory pathway to the cell surface, where a further budding event (3A) liberates vesicles containing the PI3P patch and the PEXEL proteins. These vesicles then fuse with the PVM (4A). Alternatively, similar to the formation of omegasomes during autophagy, the PI3P patch may trigger the budding of processed PEXEL protein-containing vesicles into the ER (2B). PEXEL proteins would then be transported through the secretory pathway in double membrane vesicles (DMV), and released into the PV by fusion of the outer membrane with the parasite plasma membrane (3B). The released vesicle may then fuse with the PVM (4B) (Figure modified from Römisch, 2005)6.\n\nUnusual biogenesis of a host cell targeted P. falciparum membrane protein has already been shown: the protein PfEMP-1 remains peripherally membrane-associated throughout the P. falciparum secretory pathway and only becomes transmembrane in the erythrocyte24. But conventional transmembrane proteins, i.e. proteins that become membrane-integrated in the parasite ER membrane, with PEXEL signals also exist25. The biogenesis of a soluble PEXEL protein has not been studied in similar detail to date.\n\n\nThe key: PI3P in the ER\n\nRecruitment of PEXEL proteins to specific locations within the ER membrane shows interesting parallels to autophagosomal membrane formation at the ER membrane21,22. As mentioned above, the only known example of PI3P occurring in ER membranes is during the induction of autophagosome formation22. Here the Atg14L protein recruits the Vps34 PI3-kinase to the cytosolic face of ER membranes; during amino acid starvation this leads to the formation of membrane patches or bulges, which seem to be attached to the ER and contain PI3P21,22. These patches recruit proteins with PI3P-binding domains, promoting formation of so-called omegasomes, which are invaginations into the ER that ultimately lead to pinching off of a crescent-shaped membrane structure21,22. The mechanism responsible for omegasome formation is not understood21,22,26. In order to be recruited to the right place, the proteins binding to PI3P at the cytosolic face of the ER membrane have to contain, in addition to their PI3P-binding domains, a not yet characterized ER-targeting signal21.\n\nSo maybe during erythrocyte invasion, which is controlled by IP3 signalling and calcium release from the ER27, a PI3-kinase is recruited to or activated at the P. falciparum ER. This PI3-kinase generates PI3P in patches localized in the cytoplasmic leaflet of the ER membrane in proximity to the protein translocation channel (Sec61 channel) or the SRP receptor (Figure 2B). The signal peptide of soluble PEXEL proteins promotes their targeting to the ER membrane where the signal peptide inserts into the Sec61 channel (Figure 2B). The as yet unexplained long N-terminal extensions of many of the PEXEL protein signal peptides may lead to their inefficient cleavage by signal peptidase in the ER lumen, similar to delayed signal sequence cleavage in bacterial autotransporters with long N-terminal extensions23. Delayed cleavage may be caused by the extension preventing reorientation to an N-cytoplasmic/C-lumenal topology in the protein translocation channel as shown in Figure 2B28. Alternatively, the N-terminal signal peptide extension may interact with a cytoplasmic domain of the Sec61 channel, which in turn may interfere with signal peptidase access to the cleavage site in the ER lumen (topology shown in Figure 2A). At least some of the N-terminal hydrophobic regions of PEXEL proteins also simply do not contain signal peptidase cleavage sites6. Delayed signal cleavage will lead to prolonged residence of the nascent PEXEL protein in the Sec61 channel, which in turn would allow interaction of the still cytosolic PEXEL signal with PI3P in the cytosolic leaflet of the ER membrane (Figure 2B). This interaction keeps the protein from entering the ER and thus the conventional secretory pathway. It may also generate a protein conformation recognized by the cytosolically located active site of plasmepsin V and result in cleavage of the PEXEL signal, which liberates the protein from the Sec61 channel, and aborts translocation into the ER (Figure 2B). Plasmepsin V might itself be a PI3P-binding protein, or interact with the Sec61 channel. After cleavage the cytosolic NatD complex would acetylate the new N-terminus (Figure 2B). Importantly, the interaction of the PEXEL protein with plasmepsin V leads to a handing over of the cleaved protein to a receptor protein (or complex) in the PI3P-patch (Figure 2B).\n\nTransmembrane PEXEL proteins are similarly targeted to the Sec61 channel in the ER membrane, but released laterally into the lipid bilayer which allows their cytosolically exposed PEXEL sequence to bind to PI3P and be recruited into the patch (Figure 2B). In this case the topology of the PEXEL/PI3P interaction may prevent cleavage by plasmepsin V. That transmembrane proteins can be recruited to PI3P in the ER membrane has also been shown during autophagosomal membrane formation at the ER21. One or more of the transmembrane PEXEL proteins may form the receptor in the PI3P patch for soluble plasmepsin V-cleaved PEXEL proteins (Figure 2B).\n\n\nThe way out\n\nOne option is that the PI3P patches and their associated proteins are simply packaged into a specific subset of ER-to-Golgi transport vesicles, and are then transported through the secretory pathway by a series of vesicle budding and fusion events (Figure 3, pathway A). After transport vesicle fusion at the plasma membrane, the plasma membrane then could either bud vesicles outwardly that subsequently fuse with the PVM (Figure 3), or there might be a transient fusion of parasite plasma membrane and PVM to transmit the proteins perhaps by interaction of the PI3P patch with a receptor in the PVM (Figure 3). Release of soluble proteins from the PI3P patch might be triggered by different (ion etc.) conditions in the erythrocyte cytosol. Membrane proteins would be transported by vesicular transport from the PVM to Maurer’s clefts where their release from the PI3P patches could be triggered by a PI3P-phosphatase.\n\nAlternatively, similar to what has been observed during autophagosomal membrane formation at the ER, the recruitment of specific proteins to the PI3P patch may lead to an invagination of the ER membrane, resulting in vesicles inside the ER containing the PEXEL proteins (Figure 3, pathway B). These proteins could then be transported through the secretory pathway as double membrane vesicles (DMVs) whose outer layer would ultimately fuse with the parasite plasma membrane (Figure 3, 3B). DMVs have been detected in electron micrographs of P. falciparum, and fusion with the parasite plasma membrane and vesicle release into the PV have been reported5. The released vesicles might subsequently be able to fuse with the PVM (Figure 3, 4B).\n\nEither of the transport pathways depicted in Figure 3 would satisfy the Brefeldin A sensitivity of (at least some) protein transport into the erythrocyte29,30. Either would explain how soluble and transmembrane proteins can be targeted into the host cell using the same signal3,4. Both scenarios could also explain how proteins without a PEXEL signal or proteins without a signal sequence or transmembrane domain could end up in the erythrocyte: these proteins could be packaged into the PEXEL-protein containing vesicles by interaction with these on the cytoplasmic face of the ER membrane6,31. Both hypotheses could also explain how the PEXEL signal leads to targeting to the erythrocyte even though the signal itself is cleaved already at the parasite ER13.\n\nIf the key decision - entry into the conventional secretory pathway or entry into a distinct export pathway that ultimately leads to arrival in the erythrocyte - is already made during PEXEL protein biogenesis at the parasite ER, this would explain why a protein that has been engineered to contain a conventional signal peptide and an N-terminus equivalent to the cleaved PEXEL signal ends up in the PV, not in the erythrocyte9. The construct with the conventional signal peptide is fully translocated into the secretory pathway, and hence separated from the PI3P patch-associated PEXEL proteins (Figure 2B). It will therefore, like conventional secretory proteins, follow the classical secretory pathway and be secreted into the PV, from which there seems to be no direct access into the erythrocyte.\n\nThat this is true is also confirmed by a carefully done set of experiments by Gehde and colleagues (2008)32. The authors aimed to investigate whether protein folding has an effect on PEXEL protein access to the erythrocyte. They generated fusion proteins that contained the signal peptide and PEXEL region from either the transmembrane protein STEVOR or the soluble protein GBP130 fused to dihydrofolate reductase (DHFR), followed by green fluorescent protein (GFP) and expressed these fusion proteins in P. falciparum. They found that in the absence of folate analogues (which promote DHFR folding) these constructs were targeted to the erythrocyte. In the presence of folate analogues, the constructs were found in the PV. Strikingly, only newly synthesized proteins could be transported into the erythrocyte, i.e. it was impossible to chase pre-existing fusion proteins from the PV into the erythrocyte after washout of the folate analogue. The authors’ interpretation of the data was that proteins must be unfolded in order to be transported across the PVM into the erythrocyte, that the PVM therefore contained a protein-conducting channel with similar requirements for transport as the Sec61 channel in the ER membrane, and that the time window after synthesis during which proteins were transport-competent was limited.\n\nThe scenario depicted in Figure 2B suggests a different interpretation of the data. Immediately after targeting of a PEXEL protein to the Sec61 channel in the ER membrane, there is a competition between full translocation of the fusion protein into the ER lumen and binding of the PEXEL region to PI3P on the cytoplasmic leaflet of the ER membrane, which ultimately leads to an abortion of translocation. At this stage the signal peptide and PEXEL regions of the fusion protein are already synthesized, but the ribosome is still associated with the nascent chain and protein synthesis is still going on (between step 1 and step 2 in Figure 2B, not shown). When the DHFR part of the protein emerges from the ribosome, it is initially located in the ER lumen. In the absence of the folate analogue, the DHFR chain remains sufficiently flexible for the PEXEL signal to interact with PI3P, plasmepsin V cleavage occurs, and translocation is aborted; as a result the fusion protein remains in the cytosol associated with the PI3P patch as in Figure 2B. In the presence of a folate analogue, DHFR will fold tightly in the ER lumen during its synthesis and this will interfere with or override the interaction of PEXEL with PI3P in the cytoplasm. As a result the fusion protein will be fully translocated and end up inside the secretory pathway. That folding accelerates translocation into the ER has been shown33. If protein folding in the ER lumen and PI3P-binding at the cytoplasmic leaflet of the ER membrane interfere with each other this might also explain why some of the PEXEL proteins contain large intrinsically unstructured regions34.\n\nOn the whole the model depicted in Figure 2B and Figure 3 makes sense of the vast majority of the available data on trafficking of proteins from P. falciparum into the host cell and suggests that the decision of where to go is made early during biogenesis of exported proteins at the parasite ER membrane. My hypothesis has a number of easily testable elements that might give the research in this field the appropriate direction for a full understanding of the mechanism of protein export from P. falciparum into the erythrocyte.",
"appendix": "Competing interests\n\n\n\nThe author declares no competing interests related to this article.\n\n\nGrant information\n\nThis work was funded by research support from the Saarland University.\n\n\nAcknowledgements\n\nI would like to thank my colleague Frank Breinig for twisting my arm to contribute to a graduate course on infectious disease biology, which inspired a lecture leading to this paper. I would also like to thank my secretary Juncal Gonzalez for help with the reference list and many hours of babysitting, and Gert-Wieland Kohring, Thomas Tretter and Christina Servas for critically reading the manuscript.\n\n\nReferences\n\nMaier AG, Cooke BM, Cowman AF, et al.: Malaria parasite proteins that remodel the host erythrocyte. Nat Rev Microbiol. 2009; 7(5): 341–354. PubMed Abstract | Publisher Full Text\n\nGoldberg DE, Cowman AF: Moving in and renovating: exporting proteins from Plasmodium into host erythrocytes. Nat Rev Microbiol. 2010; 8(9): 617–621. 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}
|
[
{
"id": "343",
"date": "28 Aug 2012",
"name": "Malcolm McConville",
"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 proposes a new model for how proteins in intraerythrocytic stages of the malarial parasite could be transported from the parasite endoplasmic reticulum to the host cell cytoplasm.The new model is compatible with much of the existing Plasmodium protein export literature and incorporates additional mechanistic insights from other protein trafficking process. In particular, a compelling case is made for key processing and transport steps in Plasmodium protein export occurring on the cytoplasmic leaflet of the ER and transport vesicles based on the known topology of phosphatidylinositol-3-phosphate and protein N-acetylation reactions, both of which have been shown to be involved in plasmodium protein export. Another major feature of this model is that exported soluble and membrane proteins are transported from the parasite plasma membrane to the parasitophorous vacuole via membrane vesicles. The internalization of proteins into transport vesicles draws on precedents observed during autophagy and is plausible, but raises important questions about the role of a parasite-derived multi-protein complex in the PV membrane that is thought to translocate parasite proteins across this membrane.",
"responses": []
},
{
"id": "344",
"date": "28 Aug 2012",
"name": "Norma W. 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 a very interesting and compelling article. In a simple and extremely clear style Karin Romisch does a beautiful job placing protein export by intracellular malaria parasites into a solid cell biological context.She corrects important topological misconceptions (unfortunately common in the parasitology/infectious disease fields), acknowledges other authors with similar ideas, and overall makes a crystal clear and important contribution to our understanding of one of the most fascinating problems in protein transport to emerge in recent years.",
"responses": []
},
{
"id": "345",
"date": "30 Oct 2012",
"name": "Tobias Spielman",
"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 very thought provoking review on protein export in the malaria parasite Plasmodium falciparum. The proposed model is centered on the fact that in other systems, N-terminal acetylation of proteins is found in the cytosol, not the ER lumen.If this is adopted for P. falciparum, it literally turns the current model for protein export inside out: instead of the previously assumed binding of the PEXEL export motif to PI3P and subsequent processing of the PEXEL within the ER, cytosolic acetylation of the processed N-terminus would place these events to the outside of the ER. As these events are considered to be the key requirements for export, this is of great importance to understand protein export in malaria parasites.The review then draws on previously published models for vesicular trafficking in P. falciparum and autophagy-related processes to propose pathways for the de-PEXELed protein on the outer ER leaflet to reach the host cell. For instance this would make possible a previously considered elegant model (REF 7) of serial events of vesicular budding and fusion leading to delivery of the protein into the host cell without direct crossing of any membranes.Overall this is a well written review with a clear hypothesis that adds an important new angle for the field to consider. Although the proposed model fits a lot of the data on protein export, there are also some discrepancies that become obvious when delving into the details. There are a number of points I would like to raise for discussion:- The explanation why the mDHFR fusion constructs (REF 32) do not challenge the hypothesis (page 5, third and fourth paragraph) is difficult to understand. Is it assumed that the mDHFR domain is in the ER lumen while the PEXEL binds to the outside of the ER and that this binding would be able to rescue the unfolded domain but not the folded version out of the ER? In this case it is not entirely clear how this fits into the model for translocation of proteins into the ER. A graphical explanation of the topology of this situation with the mDHFR fusion protein and how it can ensue would be helpful. This is important, as this part of the review is central to discounting the prominently proposed protein translocation at the PVM (REF 14). As an aside on this topic: while it is true that functional evidence for the PVM translocon is lacking, it may be worth noting that there is some earlier evidence for ATP-dependent translocation of an exported protein across the PVM (Ansorge et al., 2006; Biochem J).- Boddey et al. (REF 8) carried out a detailed analysis of 2 proteins with mutated PEXELs. For one of these proteins they found a signal peptidase-cleaved species that was retained in/at the ER. Despite an R->A mutation in the PEXEL (position 1) this protein was N-acetylated. Thus, in this case acetylation seemed independent of PEXEL mediated PI3P binding and PMV cleavage. As the signal peptide is usually cleaved during translocation, this could also indicate N-acetylation after ER entry. For the second protein, the PEXEL cleaved E->A mutation (position 5) was analysed. This protein was found in the PV despite being N-acetylated. This would even more strongly argue that protein entering the ER can still be acetylated. Finally, N-acetylation also occurred with the signal peptidase-cleaved mutated PfEMP3 trafficked to the PV (REF 9), a construct mentioned in this review as an example not entering the proposed pathway for export. These findings seem to be at odds with the proposed model.- The data on the exact localisation of plasmepsin V, while not exactly excluding the possibility suggested in this review (page 2), could as well indicate a localisation within the ER. If I interpret the images from the episomally expressed constructs from REF 10 correctly, then the signal peptide mediates (and is sufficient for) PV targeting and thus also for ER entry. This would indicate a failure of ER retention rather than lack of ER targeting in the mutants lacking the transmembrane domain. The conclusion here that this data ‘demonstrates that it is not a signal sequence’ may therefore not be correct. It is also not clear why PMV would possess an N-terminal hydrophobic region at all if it is targeted by its C-terminal hydrophobic region (P. falciparum contains components for the insertion of tailanchored proteins which do not require a signal peptide).- The explanation for PEXEL negative protein (PNEP) export is a bit weak (page 5, first paragraph). Binding of PNEPs to PEXEL proteins as a means for export could occur in most models for export and is no particular feature of the alternative proposed here. In fact, for this model, I would expect PNEPs not to require a hydrophobic region at all.- As a general consideration. If N-acetylation in Plasmodium parasites is expected to strictly follow the current cell biological concepts from other systems, then this should equally be considered for the vesicle trafficking needed as a result of this (i.e. to make possible the export of a protein on the outer face of the ER). In particular this concerns how proteins get across the PVM. There is no tangible evidence for the presence of the components known to mediate vesicular trafficking (e.g. coats, SNAREs, rabs etc) beyond the parasite plasma membrane (absence of evidence of course is not necessarily evidence for absence, but in contrast many classical components in anterograde and retrograde vesicular trafficking seem to be present within the parasite, see e.g. Deponte et al., MBP 2012). How the vesicle trafficking necessary to suit the model is achieved may therefore be a similarly (or even more) enigmatic cell biological problem than how proteins could be acetylated within the ER.- Although various types of vesicles have been documented in the host cell (on the morphological level, directed trafficking has not been demonstrated), I am unaware of any report convincingly showing vesicles in the PV. The close proximity of the PPM and PVM seem to make vesicle trafficking not the most suitable way for this. Is there any precedent for vesicular trafficking in such a situation?- Although an opinion article, alternatives to cytoplasmic acetylation could perhaps be discussed. Possibly there still is an unknown N-terminal acetylation activity in the ER of Plasmodium parasites (for instance there is one GNAT domain containing protein, PF14_0350, with 2 hydrophobic segments). Or, at least as a formal possibility, can it be entirely excluded that the observed N-acetylation of processed PEXEL proteins is non-physiological and occurs during affinity purification of these proteins from parasite extracts? This would require posttranslational N-terminal acetylation activities which, at least in yeast, seem to exist (Helsens et al., 2011 J Proteome Res).",
"responses": [
{
"c_id": "62",
"date": "20 Nov 2012",
"name": "Karin Romisch",
"role": "Author Response",
"response": "Dear Dr. Spielmann,thank you very much for your careful review of my opinion article. I will answer your questions in the order you raised them:You understood correctly what I was saying about the methotrexate-mediated DHFR folding in the ER lumen competing with PEXEL binding to PI3P and plasmepsin V on the cytoplasmic face of the ER membrane. I had considered adding a drawing to the paper, but in the end I decided against it, because I felt it would distract the readers from the central issues I discuss. The paper you mention (Ansorge et al., Biochem J 1996) describes elegant work from the Lingelbach lab in which they showed ATP-dependent release of GBP from streptolysin O-permeabilized infected erythrocytes. During the permeabilization, the vacuolar membrane stays intact and GBP remains protease-protected. Upon addition of ATP and cytosol GBP is released from the PV and becomes protease-accessible. This could either be ATP-dependent translocation of GBP across the PVM, or it could be ATP-induced fusion of GBP-containing vesicles with the PVM; the data is compatible with either model.The N-acetylation of signal-peptide cleaved proteins is indeed puzzling, and I do not have a physiological explanation for it. As you point out, one possibility is that this is a post-lysis artefact and therefore not relevant for the topology of the targeting of proteins exported from P. falciparum to the host cell. Topology predictions for the transmembrane GNAT-domain protein you mention are contradictory using several programmes, so it is unclear which side of the membrane the active site of the protein would be. N-acetylation within the secretory pathway, however, would also require import of Acetyl-CoA across a membrane (ER, Golgi) and a transporter. Is there such a protein? – It also remains unclear whether N-acetylation is actually part of the export signal for erythrocyte targeted proteins. Given that there is no conservation of the residue which is modified in the cleaved PEXEL (the x before the E/Q), it seems that this modification may not be important which is why I prefer not to expand this part of my review.Thank you very much for pointing out my failure to understand the data characterizing the function of the hydrophobic N-terminal domain of plasmepsin V in Russo et al., Nature 2010. To me the images of the YFP staining and the SP-YFP staining looked fairly similar and I took the text in the paper “the transmembrane domain, but no other portions of plasmepsin V were sufficient to target a reporter to the ER” to mean YFP and SP-YFP were both cytosolic and remained inside the parasite. But I agree if you blow up the images, the hydrophobic region at the N-terminus fused to YFP results in ring-shaped staining around the cytoplasm which could be the PV, or the parasite plasma membrane. I will modify this part of the text in the next version of my review. This does not, however, alter my prediction that the active site of plasmepsin V will be in the cytoplasm. The N-terminal hydrophobic domain does not have a signal peptide cleavage site, so it is likely a transmembrane domain, and both TMHMM and TopPred predict it to insert with the N-terminus in the ER lumen such that the subsequent soluble region containing the active site would be in the cytoplasm. But this is all in silico, and since it is a really important issue, I sincerely hope that somebody will characterize the actual topology of the enzyme in the ER membrane biochemically.Regarding vesicles in the PV and vesicle fusion in the absence of SNAREs etc.: If these vesicles were present in the PV only transiently and given the very narrow lumen of the PV it would be exceedingly difficult to capture images of such vesicles by electron microscopy unless their fusion were inhibited by some means. Perhaps they could be accumulated in SLO-permeabilized infected erythrocytes as in Ansorge et al., 1996. There are examples of vesicle fusion in the absence of the conventional eukaryotic fusion machinery: gram-negative bacteria can shed outer membrane derived vesicles which are subsequently either taken up by or fuse with eukaryotic cells. In many, but not all, cases these vesicles contain pathogenicity factors. It is conceivable that transport vesicles containing host cell-targeted P. falciparum proteins fuse with the PVM (which is derived from the erythrocyte plasma membrane) in a manner similar to these bacterial vesicles. The ATP-dependent vesicle fusion observed by Ansorge et al, 1996, may actually be mediated by the ‘translocation complex’ isolated by Cowman’s lab which contains a protein with homology to a bacterial pore-forming protein.One final argument against the lumen of the ER being involved in the biogenesis of proteins exported into the erythrocyte is the apparent absence of essential elements of the Unfolded Protein Response (UPR) in P. falciparum. The UPR is switched on in all eukaryotes under conditions that require an expansion of protein secretory capacity (e.g. insulin secretion in the pancreas, or maturation of plasma cells for antibody production) and is essential under those circumstances. One would expect P. falciparum to have to expand its secretory capacity when upon invasion of an erythrocyte it suddenly has to secrete hundreds of proteins into the host cell. In yeast, the UPR is mediated by a single transmembrane protein in the ER, Ire1, which upon accumulation of misfolded proteins oligomerizes and splices the mRNA for a transcription factor, Hac1, which then activates transcription of ER chaperones etc. This branch of the UPR is highly conserved in eukaryotes. Mammals have two further UPR sensors in the ER, PERK and ATF6. The last time I looked (ca 2006) in the P. falciparum genome there were no ORFs with significant similarity to the central UPR transcription factor Hac1 or its mammalian orthologue XBP-1, or ATF6, and only proteins with weak similarity to Ire1 and PERK. So either P. falciparum has evolved a novel way to adapt to increase of the load on the secretory pathway, or it does not experience much variation in the flux through the ER, because it does not use the conventional secretory pathway for secretion of host cell targeted proteins."
}
]
}
] | 1
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https://f1000research.com/articles/1-12
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https://f1000research.com/articles/1-60/v1
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05 Dec 12
|
{
"type": "Research Article",
"title": "Constraints, synergies and avenues for scaling up breastfeeding, antibiotics for pneumonia and IMCI interventions in the Cusco region, Peru",
"authors": [
"Giselle Sarganas",
"Robert Scherpbier",
"Christian A Gericke",
"Giselle Sarganas",
"Robert Scherpbier"
],
"abstract": "Objective: The purpose of this qualitative case study was to assess the feasibility of scaling up exclusive breastfeeding for 6 months, antibiotics for pneumonia and integrated management of childhood illness (IMCI) child interventions in three districts of the Cusco region, Peru.Methods: During field visits, constraints, synergies and solutions to the implementation of the selected interventions were collected through observational recording and interviews of mothers, health workers, and health managers/decision makers. Results are presented for each intervention according to the health system level where they occurred: mother/community, health worker, health centre, and political/managerial levels.Findings: This case study demonstrates that it is feasible to scale up exclusive breastfeeding, antibiotics for pneumonia and IMCI interventions in poverty-stricken rural areas of a low-income country. Factors that helped and hindered the implementation were identified for each intervention.Conclusions: The need for a coherent multi-sector approach that includes regulation, implementation and monitoring of health policies and education of all involved stakeholders was apparent. This study also demonstrates that global health interventions need to undergo local adaptation. Identifying local constraints and facilitating factors in a systematic way as proposed in this study is a useful step to increase their effectiveness and reach at the local level and to identify areas for improvement in the original intervention policies.",
"keywords": [
"child health",
"breastfeeding",
"antibiotics for pneumonia",
"IMCI",
"scaling up"
],
"content": "Introduction\n\nThe global mortality rate in children younger than 5 years fell by 28%, from an estimated 90 deaths per 1000 live births in 1990, to 65 deaths per 1000 live births in 20081. Still it was estimated that 7.2 million children under the age of five died in 2011 mostly because of treatable and preventable causes2. Continued success towards achieving the Millennium Development Goal to reduce child mortality by two-thirds of the 1990 rate depends on renewed efforts to prevent and control pneumonia, diarrhoea, malaria and malnutrition1,3. UNICEF, the World Health Organization (WHO), and their technical partners, have developed the Integrated Management of Childhood Illness (IMCI) strategy, consisting of three components: improving the case management skills of health workers; improving the health system support needed for effective management of childhood illness; and promoting key family and community practices through the education of caretakers and other members of the community4.\n\nThe Bellagio Child Survival Study Group and the Lancet Neonatal Survival Team have identified and evaluated 31 newborn and child preventive and treatment interventions5,6. Breastfeeding intervention emerged as the most important single intervention in terms of prevention, showing that if it is implemented with 90% coverage, it could reduce 13% of all under-five deaths. According to the Countdown Report (2010), the median coverage of exclusive breastfeeding in the 68 Countdown countries was only 34%1. Antibiotics for pneumonia was identified as a treatment intervention, that if implemented with 99% coverage could reduce under-five mortality by 6%5,6. According to the Countdown Report (2010) the median coverage of antibiotics for pneumonia in the 68 Countdown countries was 27%1. Although substantial progress has been made in reducing mortality and improving coverage, two major challenges remain: how to improve the quality of health interventions, and how to reach the most disadvantaged children7,8.\n\nThere is limited evidence on the process of implementing effective child health interventions, their cultural appropriateness, cost-effectiveness, and effects on health inequalities, all of which are important considerations for policy-making9. It is not only a matter of how many children are receiving effective interventions; the quality with which these are delivered is also critical10. To encourage the full implementation of health interventions, international recommendations need to be adapted locally, taking into account the political environment and the socio-cultural context. For that to happen, there is a need to evaluate how interventions are implemented, and which factors help and hinder their success. Child health interventions may vary substantially in the degree of effort to implement them, as implementation barriers may differ for different interventions. Every intervention requires for its implementation and sustainability both financial and non-financial resources that vary in terms of quality and quantity, known as intervention complexity11.\n\nThe purpose of this study was to assess the feasibility to scale up exclusive breastfeeding for 6 months, antibiotics for pneumonia and IMCI child interventions in selected communities of the Cusco region, Peru.\n\nIn pursuing this purpose, the constraints, synergies and possible solutions to the implementation of these child interventions have been systematically assessed according to the conceptual framework developed by Gericke et al.12.\n\n\nMethods\n\nThis is a qualitative exploratory case study which involves an empirical investigation of a contemporary phenomenon within its real life context using multiple sources of evidence in order to reach new insights and to assess phenomena in a new light13. The purpose was to find out implementation characteristics of exclusive breastfeeding for 6 months, antibiotics for pneumonia and IMCI child interventions in order to scale them up. This field study was carried out in August 2007 in the Paucartambo, Canas and Calca districts of the Cusco region of Peru. These districts were selected as they have high under-five mortality rate in comparison with other districts in the region, are geographically representative (the regions are located in the North, South and Canchis Espinar areas, respectively) and should have an IMCI program based on their high under 5 mortality rates. One first-level facility per district (Huancarani, Yanaoca and Calca, respectively) was selected according to regional and local health manager consultations. The main characteristics of the selected districts and their health facilities are presented in Table 1.\n\nARI: acute respiratory infections.\n\nDuring the visits, the constraints, synergies, and solutions to the implementation of the selected child interventions were collected through observational recording and interviews with mothers, health workers, and health managers/decision makers.\n\nA check list guide was created based on a previous systematic literature review in order to assist with the observational recording of health centres and their consultation characteristics, including a structured inventory of essential IMCI treatments, vaccines, and equipment. The child’s consultation was observed and recorded prior to the mother’s interview.\n\nStandardized face to face in-depth interviews were performed with 11 mothers, nine health workers (three medical doctors, four nurses and two community health workers), and five health managers and decision makers. To standardize the interviews, an open questionnaire-guide tool in Spanish (see Interview and check list file) was created for each intervention based on the previous systematic literature review, and on the conceptual framework with its four dimensions: a) characteristics of the basic intervention, (basic product design, supplies and equipment) b) characteristics of delivery: facilities, human resources, communication and transport; c) requirements on government capacity: regulation/legislation, management systems and collaborative action; and d) usage characteristics (ease of usage, pre-existing demand and black market risk). Mothers were chosen on a convenience basis i.e. as long as they came with their children for the consultation. The child’s case management was observed prior to an interview with the mother. A local research assistant was appointed by the local collaborator to help with translation for mothers speaking the Quechua language. All interviews were audio-taped and transcribed. Full anonymity to all interviewees was guaranteed. Verbal consent was obtained from all interviewees.\n\nThe constraints, synergies, and solutions from the observational recordings and interviews of the three key informant groups were labelled and classified according to the level where they were found to be present and through the application of the selected conceptual framework. The constraints and synergies were then counted to calculate their frequency (for each intervention the number of constraints or synergies adds up to 100%). Triangulation of information was used as a tool for testing one source of information against the others, considering that its by-products were as useful as its primary purpose in validating information.\n\n\nResults\n\nResults are presented for each intervention according to the level they were found to be present: mother/community, health worker and political/managerial levels. For the IMCI intervention, the same levels were identified and a health centre level was added. The frequency of detected constraints and synergies guided the presentation’s order.\n\n\nExclusive breastfeeding for 6 months intervention\n\nMost barriers (55%) to exclusive breastfeeding (EBF) for 6 months correspond to the mother/community level (Table 2). Even though the majority of the mothers are aware that health workers recommend EBF for 6 months, find it easy to breastfeed, and have support from their partners, they still practice mixed feeding in the first 6 months of the infant’s life. A large proportion of the interviewed mothers had also introduced mate (a traditional sort of tea in the region), either alone, or with other foods before the 6th month of life. Some of their reasons for giving mate included treating their baby’s abdominal spasms, the baby’s preferences, and not having enough maternal milk. The reason for not having enough breast milk also leads to the early introduction of cow’s milk; one mother responded, \"I don’t have enough milk, because it is my 7th child that is why I help myself with cow milk\". Mothers also justify this practice by arguing that cow’s milk is better than their own milk.\n\nEBF practice is also affected by a mother’s educational level and nutritional status; some examples of health worker responses were \"mothers tend to forget what we, health workers, have said\", and \"it’s not difficult to breastfeed, but if I don’t eat I don’t have milk\". Not giving colostrum to the baby or delaying breastfeeding, particularly in home deliveries, have been reported by health workers and managers. Even though they acknowledged that these practices have been partially overcome by the institutionalisation of deliveries, they are still present as highlighted by responses such as, \"the mother is considered more important than the baby and that is why in the first 6–7 hours after delivery they don’t practice breastfeeding\", and \"mothers think that the newborn does not need to be breastfed right away\".\n\nThe solutions proposed by health professionals and managers to overcome these obstacles include mother and community education, addressing knowledge, beliefs, attitudes and practices, and increasing promotion of EBF for 6 months by mainly designating specific local health workers, but also including all stakeholders, to increase awareness. For that to occur, informative printed media and audio-visual kits in waiting rooms of health establishments, or community education programs such as socio-drama (market theatre) is needed.\n\n18% of reported obstacles correspond to the political/managerial level under the label ‘lack of supportive work regulations’. This constraint is found throughout working mothers, including health professionals. Examples of responses provided in this respect include \"we, as health staff, recommend to practice EBF for 6 months, but we don’t practice it because we can’t do that due to our own working conditions\", and \"even though there is a law that states the allowance of one hour per day for working mothers to breastfeed, nobody respects that\".\n\nThe majority of mothers in this region work on farms and have the same workload as men. Health workers reported a low frequency of breastfeeding among these mothers; an example of one such response was that \"mothers think the more the baby sleeps the better it is, so they can work more\". Mothers have the socio-economic pressure to perform at work and the feeling that they should not waste their time. While some of them carry their babies on their back, others leave them at the farm or even at home and breastfeed when they return, as encapsulated by one response \"I take my baby to the farm and breastfeed him while resting or when he cries\".\n\nAccording to the health centres’ observations, breastfeeding is promoted mainly by nurses who are in charge of monitoring the healthy child, while doctors focus more on the illness of the child. Supportive policies are trying to establish a specific place at health centres called ‘Lactarios’ where multidisciplinary teams made up of doctors, nurses, obstetricians and social assistants could educate and correct breastfeeding techniques when necessary. Proposed solutions by health managers include strengthening the implementation and monitoring of health policies, including the implementation of ‘Lactarios’, and the monitoring the compliance of health policies and regulations. Health managers consider themselves, as well as the Ministry of Labour and the Ministry of Women, as responsible for the implementation of these actions.\n\nOther synergies in terms of collaborative actions such as the annual celebration of Breastfeeding Week in August, include support from NGOs who organize breastfeeding community lectures, and the Ministry of Health which supports and prioritises breastfeeding within the model of integral child care.\n\nMost detected synergies (60%) to EBF correspond to the health worker level (Table 2). Health professionals reported that speaking the Quechua language is a facilitating factor when promoting breastfeeding while others believe that it should be a requisite for working in this area. Another facilitating factor to support communication and counselling to mothers is the role of community health workers (CHWs). According to health professionals, CHWs help address community beliefs and practices, stating that \"thanks to the CHWs we can have more information about local practices\", and \"it is important to train CHWs for promoting an early breastfeeding practice\".\n\nHealth worker actions for EBF intervention include promotion and education at health centres and in the community. Of the interviewed mothers, 50% had not received home visits, postnatal breastfeeding counselling, or had participated in community talks/lectures about breastfeeding. Some health workers acknowledged this fact, saying that \"some home visits are performed, but not all\".\n\nHealth workers and managers reported difficulties in knowing about mother’s practices, claiming that \"even though mothers say that they practice EBF, we cannot prove whether it is done exclusively and with an adequate frequency\", and \"mothers say that they practice EBF, but we really don’t know if they give to the babies also mate or water\".\n\nDuring field observations, breastfeeding techniques were not regularly checked, but 70% of all interviewed mothers reported they have had their breastfeeding technique checked at least once. The short time of consultations was also identified as a constraint to proper counselling according to health professionals, with one saying that \"mothers have not so much time, they need to walk long distances and there is a big demand at the health centre, we cannot spend much time with each patient\".\n\nUntil at least 2006, an annual one-day breastfeeding training course was held, and even though none of the interviewed health professionals had attended any of them, they had a good knowledge of EBF importance and duration as they had acquired it as part of other courses. Despite this, a lack of human resources and specific training for EBF was reported by health workers as a constraint.\n\nThe solutions proposed by health workers to overcome these obstacles are training and service provision related, including improving counselling and orientation, training at least one nurse specifically in breastfeeding, promoting and introducing breastfeeding as soon as babies are born, monitoring breastfeeding during home visits, taking the opportunity of crowded days (i.e. during vaccination days) to intensify counselling and breastfeeding promotion at the health centres, to learn how to negotiate with the mother, and to be able to adapt to every situation and learn practices for better counselling.\n\n\nAntibiotics for pneumonia intervention\n\nThe majority of reported obstacles (40%) to antibiotics for pneumonia (ATB-Pn) child intervention correspond to the health worker level, and among this, 23% correspond to no-adherence to guidelines (Table 3). It was found that guidelines and protocols are either partially followed, or not followed at all by health professionals. Some of them disagreed with the current guidelines because of what they had experienced, stating that \"we have seen that if you don’t prescribe an antibiotic against pharyngitis then the child will come again into the health centre with pneumonia. This is because the majority of our patients are malnourished and the weather is cold\", \"sometimes, I prefer to prescribe an antibiotic for preventing a pneumonia episode\", and \"we try to avoid complications, our patients are from long distance communities, they don’t come again to the health centre, that is why we prescribe an antibiotic, so we cut out any infection that could be present\". According to interviewed health managers, even though guidelines need to be updated, those that are available are not used by doctors, stating that \"they resist to act as the protocol says so they prescribe what they want\", and \"nurses are the only ones adhering to protocols\". The proposed solutions by health managers regarding this obstacle include updating guidelines and protocols, conducting a study to prove if cotrimoxazole is still effective, and evaluating local antibiotic resistance.\n\nHealth workers reported that some mothers forget to give the whole treatment to the child or sometimes withhold the antibiotic after 2 or 3 days when the child improves. A key factor to achieving good adherence to treatment as identified by the health workers is to properly explain to mothers how to use the antibiotic and also to speak with them in Quechua so they can understand better. Also in order to facilitate communication with mothers, health workers stressed the fact that they must consider local practices and beliefs which are not directly against the intervention, but need to be addressed for improving adherence to treatment, with one stating that \"if they want to give alcohol or egg to the child we cannot prohibit this because if we are against those practices they don’t listen anymore and don’t accept the antibiotic\". According to health workers and managers, communication skills need to improve in order to improve the mother’s counselling, explaining to them the importance of taking the whole treatment.\n\nThe first dose of antibiotic is usually not given at the health centre, supported by the testament of one respondent, \"the antibiotic is prepared and given only to mothers that don’t understand well how to give it\". According to field observations, the person in charge of delivering the antibiotic at the health centre’s pharmacy reads the prescription and re-explains to the mother how to administer the antibiotic, and an interviewed mother could recall correctly how to prepare the antibiotic, how many doses per day and for how long she must give it to her child.\n\nAccording to health managers, the monitoring and follow up of patients’ treatments should be improved, with comments along the lines of, \"we don’t know if patients are adhering to and following the whole treatment\", and \"we need to improve the monitoring and follow up of each patient’s treatment, we also need to find a place in the health centre for providing the antibiotic\". Health professionals reported that even though they ask mothers to bring the child to the health centre for monitoring after 48 hours, they don’t bring them. Health workers express the need to increase the number of home visits and monitoring in the communities; for that they suggest training CHWs to follow up the fulfilment of the child’s treatment.\n\nAntibiotics for pneumonia are provided free of charge at health centres on presentation of the prescription, and seem to be well accepted by the local population. A big concern among health managers and health workers is that mothers, instead of going to the health centre, go to a private shop or pharmacy to get antibiotics because they don’t trust the health centres. Another associated problem reported by health professionals is that pharmacies and other private shops sell antibiotics without prescriptions: \"the pharmacy sells the antibiotic for a one day treatment and then the child comes to the health centre with pneumonia\". Health workers and managers proposed solutions to this obstacle include regulating and restricting the sale of antibiotics in private shops and pharmacies, and educating mothers to address local beliefs and practices.\n\nHealth managers are noted to have been working on the promotion and education of mothers to improve their knowledge regarding pneumonia’s signs and symptoms. Most of the interviewed mothers recalled quite well when to seek care regarding pneumonia: \"when my baby gets tired or breaths fast, I bring him to the health centre\", and \"cold, fast breathing and heart beats; when that happens I bring my child to the health centre\" were some symptoms provided by interviewed mothers.\n\nEven though during the three visits to health centres, the supply and availability of antibiotics was good, health workers expressed that sometimes they have a shortage of supplies, especially in the cold season.\n\nThe local health insurance which finances the health system requests IMCI guidelines and protocols are followed for prescribing antibiotics, but to justify the cost of the child’s consultation, health professionals feel a need to prescribe. Health managers suggested that these political decisions bias the procedure, saying that, \"health workers do not know what to do, because they are aware of the need of generating income for the health centre and that this is only possible through prescription\", and \"it is not compatible what we say and what the health insurance request we do\".\n\nAnother problem reported by health professionals is that the work of some NGOs are in disagreement with IMCI guidelines, with one respondent reporting that \"some NGOs are delivering antibiotics to the communities in an irrational way, not aligned with IMCI protocols, and this can cause antibiotic resistance\". It was stressed by health professionals the importance to establish a collaborative action enhancing alliances and agreements to overcome this obstacle.\n\n\nIMCI intervention\n\nMost of IMCI detected constraints were at the health worker and health centre level (Table 4). A lack of staff, high workload, and low job satisfaction were found to be obstacles affecting IMCI intervention. Health managers attributed the high workload to the fact that the population is growing, and yet the number of health workers remains the same. Health workers reported that the high number of consultations and the high load of administrative work have led to a short time per consultation, with one person responding that \"on market days, time is scarce not only because of the amount of patients but also because of the filling of health insurance administrative forms which take time\".\n\nThe short time for consultation and the shortage of personnel not only affect the delivery of care at the health centre but also at the community level. Even though health managers report that there are already complete teams of doctors, nurses and obstetricians at the health centres, and that they have the possibility to go to long distant communities, health workers disagree, arguing that, \"because of a shortage of personnel we cannot go more frequently to the communities to perform follow up activities\"; \"due to the high administrative work, we need to make the consultation faster, so there is no time for checking if the mother has understood what we said\", and \"theoretically, we should perform between 6–7 community visits per month but that is difficult to achieve with the low amount of personnel\". Among mothers this obstacle was also found, with one reporting that \"sometimes there are two doctors but one of them goes out to the community so only one stays at the health centre and we have to wait too long for the consultation\".\n\nA lack of staff is also affecting the supervision of activities, and health managers acknowledged a low frequency in those activities. Health professionals reported that they had been supervised for the completion of the clinical registry form but not for the clinical consultation.\n\nThe high turnover of trained staff associated with low job satisfaction caused by a lack of incentives and poor working conditions has also been reported, corroborated by responses such as \"there is a frequent change of personnel, those who are trained go and new people come\"; \"personnel leave because of low salaries, there are no economic nor moral incentives, that is why they are not motivated\", and \"the administrative work as well as the community work which take a lot of time are not counted in the productivity, only the number of children that I attend in the consultation is counted\". Poor commitment of the staff was also reported by health managers, which could be related to the low morale. Health professional and manager-proposed solutions include the regulation of contracted personnel working longer than 3 months to include the administrative work and travel time to communities as paid work, and to increase the number of health professionals according to population growth, especially on market days.\n\nLack of incentives and low morale is also present among CHWs. The role of CHWs has changed; previously they were able to implement health interventions, but nowadays they perform only preventive and promotional work. According to health professionals this has affected their image among the community, with one respondent claiming that \"now CHWs have less recognition and more rejection among the community\". Some mothers support this idea, stating that \"there are CHWs, but they don’t know as much as they knew before and they don’t participate much. We are obliged to have our deliveries here at the health centre\". However, others had a good view of the CHWs, with some saying that \"when something happened to my child, I consult with the CHW and he helps me with the solution\" or \"I am very satisfied with the work of the CHW, he visits me and gives me advice on how to take care of my baby\". As health professionals consider the work of CHWs very important, they propose increasing their training and supervision.\n\nFacilitators to IMCI intervention reported by health professionals are IMCI training, clinical experience, speaking the Quechua language, and team work. IMCI training is being performed twice a year and is given to doctors, nurses and obstetricians, but according to health managers and professionals there is a need to pool funds to increase and update training, including two weeks of field training and neonatal-IMCI.\n\nMedical attention at health centres in this region is free for children under five years old. However, the long waiting times for consultations were reported by most of the interviewed mothers and health workers as an obstacle related to the lack of staff, high workload, and the high administrative work, as mentioned at the previous level. According to health managers, a long waiting time for the consultation could be one of the reasons for not seeking consultation at the health centre and some mothers support this fact, with one stating that, \"it takes too long for the medical assistance and then when you are in, it is very fast\".\n\nEven though all visited health centres are open every day from 8 am to 8 pm and in case of emergencies 24 hours a day, some mothers reported obstacles, labelled as availability of service provision, with responses such as, \"once I came to the health centre and I didn’t receive care because there were too many patients\", and \"last August there was a strike at the health centre, it lasted for a whole week and during that week they didn’t provide medical attention\".\n\nAccessibility has also been reported as an obstacle affecting the mothers’ ability to come to the health centre, with some claiming that, \"it is difficult to come to the health centre because I need to walk and it is far away\", and, \"it is difficult to come to the health centre especially in an emergency at night when I need to walk through the darkness\". Accessibility also affects the ability of health professionals to visit communities, especially those communities not accessible by car, and they therefore they sometimes have to walk for hours. A health professional suggested that motorcycles could help them to access those communities. Health managers are aware of the present geographical constraints, with one stating that \"accessibility has been improved but still there are places where there is a need to establish health centres\". Even though accessibility problems are present, mothers still come to the health centre and while some of them are pleased with the medical attention, stating that, \"they give us good attention, they have a lot of patience and explain everything to us all\", others are not satisfied, claiming that, \"sometimes they tell me that I came only for taking advantage of the health insurance and because I want free medicines, but this disturbs me because it is not true\", \"there are doctors who don’t give good care or good prescriptions\", and \"the health personnel should work more motivated\".\n\nThe availability of drugs and vaccines in all visited health centres was good. Poor infrastructure of health centres was considered by health workers as a limiting factor affecting their performance. Also the lack of well-functioning resources and equipment such as defective scales, having only one stretcher and one nebuliser, no wheelchair, and a lack of clinical registration forms, were reported by health professionals as a constraint. The lack of resources was also reported as an obstacle by the CHWs for the communities. In order to better help, especially in an emergency, they expressed the need to be supported with more resources, saying that, \"we don’t have a cellular phone nor a radio to call to the health centre in case of an emergency, we need to walk to the health centre most of the times at night without having any lantern, waistcoats or rain capes\". Health professionals suggest providing CHWs with cellular phones or radios to improve their communication with the health centre during emergencies.\n\nUnder this level most obstacles point toward the referral system, particularly difficulties at the hospital level, in communication, and with the transportation system. According to health managers and health workers some hospitals reject referrals, stating that \"they say they do not have space and sometimes we think they have\"; \"the people at the hospital are not involved and committed to the work and they don’t know how the referral system should work\", and \"there is an emergency and the hospital says they don’t have any bed, or we call and they don’t answer, all that makes a waste of time\". Health workers also stated that there is a deficient communication system between health centres and the hospital because radios are not working well, with one claiming that, \"sometimes the radio has a bad sound or no sound at all, so we can’t understand each other\". Besides these obstacles, the referral system is affected by the transportation system because there are not enough ambulances or available drivers, with one stating that, \"we have two drivers for the ambulance but sometimes one is on leave and the other is driving health workers to the communities, so we don’t have a driver when there is an emergency\". According to health workers, the contra-referral system also has difficulties, examples being, \"after hospital care, the patient comes back with a prescription directly to his home instead of coming to the health centre for a proper follow up\"; \"the contra-referral fails, they don’t send the patient back or the mother doesn’t bring the child back to the health centre\", and \"sometimes the contra-referral form is not correctly filled in\". Problems have also been reported with coordinated referral, \"when we set a specialist consultation date at the hospital for a patient, the mother doesn’t take the child to it, this could happen because the mother needs the authorization from the father or she needs money to pay for public transportation to the hospital and she doesn’t have it\". To overcome these obstacles, health professionals and managers suggest a need to improve the administration of hospitals (including bed occupancy rates and the reception of referrals), improving management of available resources (including technical personnel), improving the referral and contra-referral system, and improving communication by phone or radio between health workers for better medical care and patient follow up.\n\nUnder collaborative action, facilitating factors such as the presence of governmental programs, NGOs, the health insurance system, local Community Associations for the Administration of Health (CLAS) (where the health centre manages its resources together with community representatives), as well as the help of CHWs were reported. A governmental program that started in 2007 has already had an impact on the monitoring of healthy children, with respondents quoted as saying, \"this program gives an amount of 100 Soles to each family, but the condition is that they should bring the child to the health centre for monitoring and vaccination\", and \"there is an NGO that helps CHWs with teaching methodologies and they also pay for snacks and lunches\".\n\nAccording to some health managers, the collaborating NGOs are in line with the IMCI. Others think that the work should be of a better standard, with some reporting that, \"some NGOs and governmental programs help with food donation, the problem is that mothers only give that type of food to the child, without giving anything else, then the child is not well nurtured and gets tired of eating the same thing\", and \"some families receive double donations while others who are also in need don’t receive any\".\n\nOther obstacles reported by health managers relate to other authorities and ministerial priorities: \"the Ministry of Finance and Economics does not prioritise the same as we do\", and \"authorities responsible for the population have other priorities, they don’t support much health issues\", being two examples provided. The lack of specific budget lines and fund identification, as well as no proper institutionalisation was identified by health managers as obstacles for the implementation of IMCI, with some stating that, \"there is not much of a budget for training and follow ups\", and \"the IMCI health system component is too weak, and we need more support\". On the other hand facilitating factors such as having an annual operational plan and the strength of community-IMCI were reported. The fact that only a few areas were selected for the IMCI implementation was reported as an obstacle, but at the same time as a facilitator, with respondents stating that, \"not every health centre has IMCI, those doctors who have not been trained with IMCI, do not accept it\", and \"the work has been prioritized to those areas with higher risk and mortality\". Regarding health information systems, health managers reported that even though a validated information system for clinical registries exists, there is always something missing or something to correct when the forms come. Health managers’ proposed solutions to overcome these obstacles included performing an updated IMCI situational analysis, to institutionalise the IMCI, to implement IMCI in all health centres, to train all health workers, to unify all nutritional supportive programs with the same cause, and to increase the commitment and involvement of the authorities.\n\nHealth workers think that health interventions are culturally accepted, but that the population acts according to its own beliefs and trusts their natural medicine as a first line treatment. Interviewed mothers have verified that, \"when my child has a fever, I pass urine on him and give him fresh mate to drink and the fever goes\", and \"when my child has diarrhoea, I give her everything fresh and cold, like lemon-mate and Ayrampo\". Other mothers go directly to the pharmacy to buy medicines, one reporting that \"when my child had fever, I bought a small pink pill in the pharmacy and the fever went and since then he has not had any more\". According to health workers and managers, local practices and beliefs and low economic resources are influencing the way mothers seek care, with responses such as, \"because of the low education level and beliefs, they still seek for care but a bit late\";, \"there is some resistance when we need to hospitalise a child or administer an intravenous treatment, mothers think that we are hurting the child and they do not like to stay at the health centre because they have animals and other children to take care of\", and \"even though people receive economic and food support from several institutions, they don’t care about the health of their children, they sell what they get\".\n\nAccording to health workers, community education and promotion have good results, with reports such as \"we have a mobile television with an IMCI video in Quechua that we take to the communities and show it\", and \"we are sensitising the communities and we are getting involved, we are starting to know each other\". Even though some mothers were illiterate, they knew when to come for the next visit. Most interviewed mothers had the vaccination and IMCI card with them.\n\nEven though health managers reported that it is still difficult to effectively teach the mother to recognize alarm symptoms, most of the interviewed mothers had a correct idea of when to come back to the health centre, stating that, \"if my baby doesn’t get better from diarrhoea, or doesn’t breastfeed or cry I have to bring him again to the health centre\". Most of the interviewed mothers could properly recall what health workers have recommended in terms of treatment and nutrition, but there are some foods that they cannot get because of socio-economic reasons such as, \"I should give daily eggs, cheese, milk and chicken, also green vegetables and add a little oil. She usually eats potatoes and soup and sometimes I give her fruits but not much, only when I go on Sunday to the market. When we have some money, we try to buy what they recommend and give it to the child or also when we kill an animal\" or \"they told me to add cheese, milk, eggs and vegetables and to feed my child 6 times a day to have a good weight. I give her what we produce on our farm: potatoes, wheat, barley, cabbage, carrots and chard\". According to health workers and managers’ proposed solutions, it is important to improve counselling of mothers on child nutrition and on the exchange and sale of food products in order to support their establishment in cooperatives, and to increase their income potential.\n\n\nDiscussion\n\nAs far as we know this is the first study that, in order to scale up child interventions, systematically assesses constraints, synergies and possible solutions to the implementation of exclusive breastfeeding for 6 months, antibiotics for pneumonia, and IMCI child interventions in selected communities of the Cusco region, Peru. The inclusion of key informants at different levels of the health system allowed a more comprehensive and well-rounded picture of the implementation of the selected interventions and the feasibility to scale them up. The IMCI strategy includes among its components exclusive breastfeeding and antibiotics for pneumonia child interventions. The reason for assessing them independently was to increase the possibility of detecting constraints, synergies and solutions at all health system levels in order to scale them up.\n\nMothers from this region practice breastfeeding but not exclusively. Even though there are facilitators, beliefs and local practices make mothers choose a mixed feeding practice. In order to scale up EBF it is important to intensify EBF´s educational and promotional components, involving mothers, families, new generations of scholars, teenagers, health workers and authorities. Health workers are a key pillar when trying to improve this intervention. It appears that BF training for health workers is required to improve the counselling and communication skills with mothers. Also time management and organizational skills for health workers are needed in order to better allocate resources for counselling, home visits and field activities. Nankunda et al. showed the importance of individual peer counselling in Uganda in scaling up exclusive breastfeeding17.\n\nThe mother’s socio-economical needs, together with the lack of political support, make the feasibility of practicing exclusive breastfeeding quite difficult for working mothers. Even though there are some written supportive policies, the lack of regulation and monitoring when looking at their implementation was identified as a bottleneck. In order to overcome these obstacles and scale up the intervention we recommend strengthening supportive regulations and legislative policies to improve mother’s working conditions and the monitoring and implementation of Lactarios in health centres. The strengthening of collaborative actions to improve the mother’s economy and nutrition could also help the EBF scale up. This aligns with the findings of Bhandari et al.18 who stated the need for a legal framework with political will, strong advocacy, enabling policies, well-defined short- and long-term programme strategy, sustained financial support, clear definition of roles of multiple stakeholders and emphasis on delivery at the community level for scaling-up exclusive breastfeeding.\n\nEven though there was a good availability of antibiotics for pneumonia at the visited health centres, and these were provided free of charge, we need to take into account that there might be children in the region who need antibiotics for pneumonia and who never reach a health facility. A cross-sectional study of two other regions in Peru showed that poorer households consulted less frequently for pneumonia symptoms and used less antibiotics than less poor households19. According to the Indian National Health Survey 2006, only 12.5% of children received antibiotics for pneumonia when needed20.\n\nIn the studied health centres, we detected room for scaling up antibiotics for pneumonia intervention, in strengthening their rational use by updating guidelines and protocols based on high-quality local studies and strengthening policies for assuring the appropriate use of antibiotics. Training health professionals by reinforcing the proper use of antibiotics and resistance risk, including improving their counselling techniques and communication skills for improving adherence to treatment is also needed. A study for improving prescribing practices in Nepal showed that peer-group discussion and a bottom-up approach of supervision and monitoring, implemented through the district health system, improved prescribing practices21. Another measure is to educate mothers on how to seek appropriate care and to educate them on the importance of following a whole treatment through, taking into account their own beliefs and practices. Other measures that we propose consist of unifying and strengthening the collaboration and coordination between NGOs, governmental programs, private providers and CHWs; strengthening the monitoring and follow-up care of pneumonia patients through home visits in collaboration with CHWs; regulating the sale of antibiotics at retail sector providers; and reducing the out-of-pocket payments in the health insurance system.\n\nIMCI is a very complex intervention with many components, which is accepted globally as one of the key strategies to reduce child mortality22. The findings of this study add to previously identified constraints of IMCI implementation in Peru as well as globally22,23. Based on the present analysis, it is possible to formulate the following recommendation in order to scale up IMCI in the Cusco region: at the political and managerial level we recommend to institutionalise IMCI and implement it in all health centres; to reform health workers’ contracting and procurement procedures; to strengthen the health information system; and to intensify the coherent implementation of collaborative actions. At the health centre and health system level our recommendation is to increase health worker staffing and improve the distribution and management of human resources; to improve working conditions and incentives to retain health workers; to provide material resources including communication technology and vehicles for facilitating community workers access to health centres; to support the work of CHWs with basic resources; and to improve the referral system, in particular the reception of patients and referrals to hospitals. At the health worker level our recommendation is to train and supervise health professionals; to train and supervise CHWs; to improve skills for mothers’ counselling; and to add a mobile support team at the health centre for days with high demand and on other days for performing community and home visits. At the mother and community level tour recommendation is to strengthen collaborative action for poverty alleviation; and to continue general education and promotion of child health.\n\nWhile we successfully interviewed health managers, health workers and mothers from three selected health centres, it is possible that we missed important data, especially from mothers who did not participate as we interviewed only those who came that day with their child for the consultation. There are mothers in the communities who do not consult health centres; it would be interesting to undertake interviews in the community instead of only at health centres.\n\nThe influence of external observers on the behaviour and practices of health personnel is well documented24, and therefore the observed performance might differ from their usual performance. We addressed this issue by combining observational-recording with interviews, in order to obtain more precise information on the usual characteristics of child consultation and management in those health centres.\n\nThis case study focuses on current events and can provide theoretical generalizations and solutions on how these selected interventions could be scaled up but they do not permit statistical generalizations.\n\n\nConclusions\n\nThis case study from the Cusco region in Peru demonstrates that it is feasible to scale up exclusive breastfeeding, antibiotics for pneumonia and IMCI interventions in poverty-stricken rural areas of a low-income country. The need for a coherent multi-sector approach that includes regulation, implementation and monitoring of health policies as well as education of all involved stakeholders was apparent. This study also demonstrates that global health interventions such as EBF, AB-Pn and IMCI need to undergo local adaptation. Identifying local constraints and facilitating factors in a systematic way as proposed in this study is a useful step to increase their effectiveness and reach at the local level and to identify areas for improvement in the original intervention policies. This requires feedback loops to be built-in to get the information back from the local health centres to national Ministries of Health and to the international organisations developing global guidance in the first place, such as WHO and UNICEF.",
"appendix": "Author contributions\n\n\n\nGS, CAG and RS planned the study. GS collected the field data and interpreted it under the supervision of CAG. GS wrote the first draft of the manuscript. All authors contributed to the final draft of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was coordinated and financed by the Department of Child and Adolescent Health and Development of the World Health Organization. Prof Gericke was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care for the English South West Peninsula (PenCLAHRC). The views expressed in this publication are those of the author(s) and not necessarily those of the World Health Organization, the NHS, the NIHR or the Department of Health in England.\n\n\nAcknowledgements\n\nDr Ricardo Bado participated actively in the realization of interviews at health centres as well as further translation from Quechua to Spanish. Dr Miguel Davila and Dr Zonia Rozas coordinated the visits at the districts. Dr Lorena Dini was helpful as a consultant before, during and after the field study. Joel Hernandez supported the logistics. Dr Luis Huicho supported the field planning as well as logistics.\n\n\nReferences\n\nBhutta ZA, Chopra M, Axelson H, et al.: Countdown To 2015 Decade Report (2000–10): Taking Stock Of Maternal, Newborn, And Child Survival. Lancet. 2010; 375(9730): 2032–44. PubMed Abstract | Publisher Full Text\n\nLozano R, Wang H, Foreman KJ, et al.: Progress towards Millennium Development Goals 4 and 5 on maternal and child mortality: an updated systematic analysis. Lancet. 2011; 378(9797): 1139–65. PubMed Abstract | Publisher Full Text\n\nBryce J, Boschi-Pinto C, Shibuya K, et al.: WHO Child Health Epidemiology Reference Group. WHO estimates of the causes of death in children. Lancet. 2005; 365(9465): 1147–52. PubMed Abstract | Publisher Full Text\n\nTulloch J: Integrated approach to child health in developing countries. Lancet. 1999; 354(Suppl 2): SII16–20. PubMed Abstract | Publisher Full Text\n\nDarmstadt GL, Bhutta ZA, Cousens S, et al.: Evidence-based, cost-effective interventions: how many newborn babies can we save? Lancet. 2005; 365(9463): 977–88. PubMed Abstract | Publisher Full Text\n\nJones G, Steketee RW, Black RE, et al.: How many child deaths can we prevent this year? Lancet. 2003; 362(9377): 65–71. PubMed Abstract | Publisher Full Text\n\nVictora CG, Barros FC: Global child survival initiatives and their relevance to the Latin American and Caribbean Region. Rev Panam Salud Publica. 2005; 18(3): 197–205. PubMed Abstract\n\nVictora CG, Barros AJ, Axelson H, et al.: How changes in coverage affect equity in maternal and child health interventions in 35 Countdown to 2015 countries: an analysis of national surveys. Lancet. 2012; 380(9848): 1149–56. PubMed Abstract | Publisher Full Text\n\nHaines A, Kuruvilla S, Borchert M: Bridging the implementation gap between knowledge and action for health. Bull World Health Organ. 2004; 82(10): 724–31. PubMed Abstract | Free Full Text\n\nCountdown to 2015: Tracking Progress in Child Survival: The 2005 Report. 2005. Reference Source\n\nGericke CA, Kurowski C, Ranson MK, et al.: Intervention complexity–a conceptual framework to inform priority-setting in health. Bull World Health Organ. 2005; 83(4): 285–93. PubMed Abstract | Free Full Text\n\nGericke CA, Kurowski C, Ranson MK, et al.: Feasibility of Scaling-up Interventions: The Role of Intervention Design. Disease Control Priorities Project Working Paper 13; 2003. Reference Source\n\nRobson C: Real World Research: a resource for social scientists and practitioner researchers. Blackwell. 1993. Reference Source\n\nASIS: Análisis de Situación de Salud del Cusco. Cusco: Ministerio de Salud del Perú; 2005 Dirección de Estadistica e Informatica DISA Cusco, UNICEF Perú Contract No: Document Number.\n\nFONCODES: Indices Absolutos de Pobreza.2000 [updated 2000; cited].\n\nPNUD: Informe sobre Desarrollo Humano. 2002 PNUD, Peru [updated 2002; cited]. Reference Source\n\nNankunda J, Tylleskar T, Ndeezi G, et al.: Establishing individual peer counselling for exclusive breastfeeding in Uganda: implications for scaling-up. Matern Child Nutr. 2010; 6(1): 53–66. PubMed Abstract | Publisher Full Text\n\nBhandari N, Kabir AK, Salam MA: Mainstreaming nutrition into maternal and child health programmes: scaling up of exclusive breastfeeding. Matern Child Nutr. 2008; 4(Suppl 1): 5–23. PubMed Abstract | Publisher Full Text\n\nKristiansson C, Gotuzzo E, Rodriguez H, et al.: Access to health care in relation to socioeconomic status in the Amazonian area of Peru. Int J Equity Health. 2009; 8: 11. PubMed Abstract | Publisher Full Text\n\nIndia Clinical Epidemiology Network (IndiaCLEN) Task Force on Pneumonia: Rational use of antibiotics for pneumonia. Indian Pediatr. 2010; 47(1): 11–8. PubMed Abstract | Publisher Full Text\n\nKafle KK, Bhuju GB, Karkee SB, et al.: An intervention improving prescribing practices and monitoring drugs availability in a district. Nepal Med Coll J. 2009; 11(4): 217–21. PubMed Abstract\n\nGoga AE, Muhe LM: Global challenges with scale-up of the integrated management of childhood illness strategy: results of a multi-country survey. BMC Public Health. 2011; 11: 503. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuicho L, Davila M, Campos M, et al.: Scaling up Integrated Management of Childhood Illness to the national level: achievements and challenges in Peru. Health Policy Plan. 2005; 20(1): 14–24. PubMed Abstract | Publisher Full Text\n\nFavin M, Naimoli G, Sherburne L, et al.: Improving Health through Behavior Change: A Process Guide on Hygiene Promotion. Washington. Bureau for Global Health2004. Reference Source"
}
|
[
{
"id": "393",
"date": "13 Dec 2012",
"name": "Elizabeth Molyneux",
"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 paper have looked at the difficulties in scaling up breast feeding and pneumonia and the IMCI interventions in a region of Peru.They are to be commended for their ’360 degree look’ at the problems and in trying to find solutions from the workers and carers within the system. The answers are not new but need repeating; many problems could be solved if the recommendations from within the service are listened to and carried out. Other health services could ask similar questions of their health service providers and users and would probably find similar suggestions to act upon.",
"responses": []
},
{
"id": "395",
"date": "17 Dec 2012",
"name": "Gill Walt",
"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 no new insights to the issues it addresses, but it does discuss the difficulties of implementing specific child policies in particular settings in Cusco, Peru, which will have resonance for other parts of the world. There is a useful discussion on recommended actions at the various levels of the health service.",
"responses": []
},
{
"id": "604",
"date": "08 Jan 2013",
"name": "M. Rashad Massoud",
"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
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https://f1000research.com/articles/1-60
|
https://f1000research.com/articles/1-58/v1
|
04 Dec 12
|
{
"type": "Research Article",
"title": "Identification of two telomere-proximal fission yeast DNA replication origins constrained by nearby cis-acting sequences to replicate in late S phase",
"authors": [
"Amna Chaudari",
"Joel A Huberman"
],
"abstract": "Telomeres of the fission yeast, Schizosaccharomyces pombe, are known to replicate in late S phase, but the reasons for this late replication are not fully understood. We have identified two closely-spaced DNA replication origins, 5.5 to 8 kb upstream from the telomere itself. These are the most telomere-proximal of all the replication origins in the fission yeast genome. When located by themselves in circular plasmids, these origins fired in early S phase, but if flanking sequences closer to the telomere were included in the circular plasmid, then replication was restrained to late S phase – except in cells lacking the replication-checkpoint kinase, Cds1. We conclude that checkpoint-dependent late replication of telomere-associated sequences is dependent on nearby cis-acting sequences, not on proximity to the physical end of a linear chromosome.",
"keywords": [
"The genome of the fission yeast",
"Schizosaccharomyces pombe",
"is organized into three chromosomes with a total of six protein-DNA telomere structures. The outermost portions of the telomeres consist of telomerase-encoded simple-sequence repeats rich in G residues as read in the 5´ to 3´ direction toward the end of the chromosome. Fission yeast telomeric simple-sequence repeats appear",
"at first sight",
"to be more complicated than those of other organisms1",
"but analysis of motif frequencies2 and analysis of the sequence of the fission yeast telomerase template RNA3 show that the simple-sequence repeats consist mainly of the heptamer GGTTACA with variable numbers of additional G residues (due to polymerase-template slippage) and variable extents of template-coded spacer sequences."
],
"content": "Introduction\n\nThe genome of the fission yeast, Schizosaccharomyces pombe, is organized into three chromosomes with a total of six protein-DNA telomere structures. The outermost portions of the telomeres consist of telomerase-encoded simple-sequence repeats rich in G residues as read in the 5´ to 3´ direction toward the end of the chromosome. Fission yeast telomeric simple-sequence repeats appear, at first sight, to be more complicated than those of other organisms1, but analysis of motif frequencies2 and analysis of the sequence of the fission yeast telomerase template RNA3 show that the simple-sequence repeats consist mainly of the heptamer GGTTACA with variable numbers of additional G residues (due to polymerase-template slippage) and variable extents of template-coded spacer sequences.\n\nAt the ends of most chromosomes, telomere-associated sequences (TAS) are located internal to the simple-sequence repeats1. In all fission yeast strains, TAS are found at both ends of the two larger chromosomes (1 and 2). Chromosome 3 contains arrays of ribosomal DNA (rDNA) repeats near both ends. In some strains, these rDNA repeats directly abut the simple-sequence repeats1, while in other strains there are TAS between the rDNA and the simple-sequence repeats at one or both chromosome 3 ends1,4,5. The complete TAS array consists of approximately 50 kb, forming large inverted repeats at the ends of chromosomes 1 and 2 (Figure 1A). Most of this sequence is \"unique\" (except for the fact that each cell contains 4–6 copies of TAS), but there are clusters of direct repeats within the first 3 kb and also 4 to 6 kb away from the telomere (Figure 1B, C). Within the reference Schizosaccharomyces pombe genome sequence of October, 2008 (ftp://ftp.sanger.ac.uk//pub2/yeast/pombe/Chromosome_contigs/OLD/20080922), only the right end of chromosome 2 extended sufficiently close to the telomere to include any of these direct repeats. The ends of the other chromosomes were less completely sequenced. Fortunately, the right end of chromosome 2 provides an excellent model for the structure of the other chromosome ends (Figure 1A).\n\n(A) The October, 2008, versions (ftp://ftp.sanger.ac.uk//pub2/yeast/pombe/Chromosome_contigs/OLD/20080922) of the nucleotide sequences from the ends of fission yeast chromosomes 1 and 2 were aligned with each other on the basis of restriction map similarities and Pustell DNA matrix alignments using MacVector software (http://www.macvector.com/). The left ends of chromosomes 1 and 2 were reverse-complemented so that their sequences would have the same polarity as those of the right ends of chromosomes 1 and 2. The green arrows show the locations and orientations of documented genes (dubious and pseudo genes were omitted). The orange arrows show regions of high sequence similarity between the four chromosome ends. The positions of deletions and an insertion within these highly similar regions in the ends of chromosome 1 (compared to the ends of chromosome 2) are shown by additional orange arrowheads (deletions) or a gap in the orange arrow (insertion). Thin black guide lines are also provided, to facilitate the alignments of similar sequences despite the presence of deletions and an insertion. The far right end of chromosome 2 overlaps for about 4 kb (panel C) with the left end of pNSU21, a clone of a 7992-bp telomeric HindIII fragment prepared by Neal Sugawara1. Here we have used the reverse-complement of the pNSU21 nucleotide sequence, as determined partially by Neal Sugawara1 and completely by the Sanger Centre fission yeast sequencing project (ftp://ftp.sanger.ac.uk//pub2/yeast/sequences/pombe/telomeres). We have employed the reverse complement (and we’ve changed the numbering correspondingly) so that (i) the pNSU21 sequence would have the same orientation as the right end of chromosome 2, and (ii) so that the G-rich strand of the simple-sequence repeats would be the top strand, read from 5´ to 3´. In this numbering system, the simple-sequence repeats are at nucleotides 7881–7992. The HindIII site at position 1 of pNSU21 is not shown in the diagram. The only restriction sites shown are BamHI, NsiI, PvuII, SacI, and XhoI. These have been given distinct colors to enhance the reader’s ability to perceive similarities between restriction site patterns. A thin vertical line at the right of the diagram shows the estimated position of the simple-sequence repeats for all four chromosome ends. In other words, this line provides an estimate of how far the nucleotide sequence of each chromosome end would extend if the sequence were established all the way to the telomere. (B) A Pustell DNA matrix diagram was prepared (using MacVector software) of pNSU21 against itself, in order to identify significant internal repeat sequences. The conditions employed were: window size 20, minimum % score 100, hash value 6, jump 1. Two families of direct internal repeats were identified. These are called \"Telomere-Proximal Repeats\" (TPR). The TPRs closest to the telomere are called TPR1, while those further from the telomere are called TPR2. The line of sequence identity is colored blue. (C) A Pustell DNA matrix diagram was prepared of the right end of chromosome 2 against pNSU21, using the same conditions as in (B). The blue line shows the region of (near) sequence identity. Approximately 3840 nucleotides would need to be added to the sequence of the right end of chromosome 2 for it to reach the simple-sequence repeats. The TPR2 family of internal direct repeats is included in the sequence of the right end of chromosome 2.\n\nMoser et al.6,7 have shown that the replication forks emanating from the most telomere-proximal origins in fission yeast reach telomeres asymmetrically, with the leading strand polymerase arriving about 20 minutes ahead of the lagging strand polymerase. The lagging strand delay appears to provide an opportunity for differential processing of the leading and lagging strands at the telomere. What could be the mechanism of this telomere-specific replication fork asymmetry? There may be something special about the replication origins located closest to telomeres, or cis-acting sequences that accelerate leading strand synthesis and/or decelerate lagging strand synthesis (perhaps in collaboration with bound proteins) may be located between telomere-proximal origins and the telomeres themselves. In either case, it would be of great interest to identify and characterize the telomere-proximal replication origins in fission yeast.\n\nKim and Huberman8 previously used two-dimensional (2D) agarose gel electrophoresis to study the origin function and replication timing in fission yeast of the TAS-derived telomere-proximal HindIII restriction fragments (bounded by a HindIII restriction site on one side and a telomere on the other side; 7–8 kb each). They found that these fragments were replicated in late S phase. They also observed that these fragments usually produced only Y-arc signals, indicative of passive replication by forks from a centromere-proximal origin or by forks from an internal origin close to one of their ends. Occasionally, however, bubble-arc signals indicative of active origin firing within the middle portions of the fragments were also observed. These results suggested that the replication origins closest to telomeres in fission yeast may be located within these HindIII fragments. In this paper, we describe our characterization of two replication origins located in the centromere-proximal 30% of the HindIII fragments. These two late-firing origins appear to be the closest origins to the telomeres.\n\nLate telomere replication has also been observed in the budding yeast, Saccharomyces cerevisiae9–12. In budding yeast cells treated with hydroxyurea (HU), lateness of telomere replication is maintained by the Rad53-dependent replication checkpoint13.\n\nIn this paper, we report our investigation of the causes of late telomere replication timing in fission yeast. In agreement with previous studies8,14–16, we found that the replication check-point regulates telomere replication timing in fission yeast as in budding yeast. In addition, we found that late replication appears to be a consequence of cis-acting sequences located between the two telomere-proximal replication origins and the telomere itself.\n\n\nResults\n\nPrevious observations8 suggested that the telomere-proximal HindIII restriction fragments of fission yeast were likely to contain replication origins. These would necessarily be the most telomere-proximal origins in the genome. Sugawara1 cloned five such fragments, all of similar sizes and with similar restriction maps. Sugawara cloned these five fragments between the HindIII and SacI sites in the plasmid, pMLC12. The nucleotide sequences of the inserts were subsequently determined by the fission yeast genome sequencing project and are available from the Sanger Centre (ftp://ftp.sanger.ac.uk//pub2/yeast/sequences/pombe/telomeres). Of these sequences, that of pNSU21 (7992 bp) most closely resembles the sequence of the right end of chromosome 2 in the region of overlap (Figure 1, Figure 2A), so we chose pNSU21 for further study.\n\n(A) Diagram showing overlap between the right end of Chromosome 2 and the THF, which was cloned by Neal Sugawara1 as pNSU21. (B) Relative positions within the THF of the smaller regions (R1–R7) generated by PCR. (C) AT content in sliding windows of 100 bp (dotted line) and 500 bp (solid line) along the THF. P1–P4 are peaks of unusually high AT content. (D) Results of ARS assays for plasmids containing the sequences in panel B or controls. The results are the averages ± standard deviation of 3 experiments, with triplicate plating in each experiment.\n\nThe classic method for identifying potential yeast DNA replication origins is to test the abilities of stretches of the DNA of interest to serve as replication origins in a plasmid context. Stretches of DNA that meet this criterion are called \"Autonomously Replicating Sequences\" or ARS elements17. To identify potential origins within pNSU21, we extracted the ~8-kb telomere HindIII-SacI fragment (hereafter called THF for Telomeric HindIII Fragment) from pMLC12 and re-cloned it into the yeast shuttle vector, pRS30618. We also used PCR to create smaller subfragments of the THF, called R1–R7 (for Region 1 through Region 7), and we cloned them into pRS306 (Figure 2B; Table S1).\n\naThe relative positions of the regions are indicated in Figure 1B.\n\nbUnless otherwise indicated, the reference sequence is the reverse complement of pNSU21, which may be downloaded from the Sanger Institute web site (ftp://ftp.sanger.ac.uk/pub/yeast/sequences/pombe/telomeres/). We used the reverse complement of pNSU21 so that the orientation of pNSU21 would match that of the right end of chromosome 2 and so that the \"top\" strand of the sequence would contain the G-rich portion of the telomere simple-sequence repeats. The reference sequence for the vector, pRS306, is the sequence with GenBank accession number U03438. Those cases in which a primer sequence corresponds to the \"bottom\" strand (in other words, the reverse complement of the top strand) of the reference sequence are indicated by \"RC\" for \"reverse complement\".\n\ncWhere indicated, the following restriction sites with flanking non-coded nucleotides were appended to the 5´ ends of the template-encoded primers. The flanking non-coded nucleotides are indicated by plain type, while the restriction sites themselves are indicated by bold face:\n\nBamHI: CGGGATCCCG.\n\nEcoRI: CGGAATTCCG.\n\nXbaI: GCTCTAGAGC.\n\nAdditional non-coded nucleotides, indicated by violet coloring (CG), were inserted where indicated in the region 1 reverse primer.\n\ndThe region 1 forward primer was located in the pRS306 vector, and the resulting PCR fragment contained a HindIII site at its left end.\n\neThe region 4 reverse and region 7 reverse primers were located in the pRS306 vector, and the resulting PCR fragments contained a SacI site at the right end.\n\nWhen designing the positions of the boundaries of subfragments R1–R7, we took into account information derived from the nucleotide sequence of the THF regarding the likely positions of replication origins. Because the N-terminal portion of fission yeast Orc4 contains nine \"AT-hook\" motifs, fission yeast ORC binds preferentially to AT-rich sequences19–21. Indeed, the locations of functional replication origins in fission yeast can be predicted with surprising accuracy based on AT content alone22,23. Figure 2C displays graphs of the AT content of the THF in sliding windows of 100 bp (thin line) and 500 bp (thick line). In both graphs, four peaks (P1–P4) of high AT content are apparent.\n\nTo test whether any of these AT-rich peaks (or other regions in the THF) might serve as replication origins, we used the transformation frequency assay, which is based on counting the number of colonies formed when yeast cells are transformed by a plasmid containing the sequence to be tested for origin activity. The transformed cells are able to form colonies at high frequency only if the plasmid they have been transformed with contains DNA that can serve as an origin of replication for the plasmid. We used pRS306 without an insert as a negative control (Figure 2D). As positive controls, we employed a weak origin, ars300124,25 and the strong compound origin in the plasmid, pDblet26. The intact THF had weak activity, similar to that of ars3001 (Figure 2D). In contrast, subfragment R1 was more active than pDblet, while R2–R7 were completely inactive (Figure 2D). These results show that one or more plasmid replication origins (ARS elements) are contained within R1 and suggest that there may be cis-acting sequences outside of R1 that can suppress origin activity.\n\nIn order to better localize the ARS element(s) within R1, we used an exonuclease III deletion procedure to reduce the size of R1 by ~500 bp, ~1000 bp, and ~1.5 kbp from both ends (Figure 3B; constructs E1–E6). Each of the resulting constructs contained at least one of the peaks of high AT content (P1 and P2) found within R1 (Figure 3A,B). When we assayed these constructs for ARS activity (Figure 3C) we found that all of them were active, but none was as active as R1. The results in Figure 3 suggest that either AT peak within R1 is sufficient to serve as an origin of replication. Thus there are two closely spaced, but independent, ARS elements close to the centromere-proximal end of the THF.\n\n(A) Intact Region 1 with AT content, expanded from Figure 2. (B) For constructs E1–E6, the thin lines show the portions of R1 that were deleted, and the thick lines show the portions of R1 remaining in each construct. The deletions were constructed using the Promega Erase-A-Base Kit. See Materials and Methods for details. (C) Results of ARS assays for the plasmids containing the deleted versions of Region 1 shown in panel B. Compare with Figure 2D.\n\nTo test the replication timing of the THF and of R1 in their plasmid context, we used the hydroxyurea (HU) block-and-release procedure8,27 to synchronize cells. First we treated the cells with HU for 4 hours. This resulted in depletion of the dNTP pools, causing DNA replication to be blocked in early S phase. Early-firing replication origins can fire in the presence of HU, but the replication forks from these origins stall within a short distance (usually less than 10 kb). When cells are released from the HU block, they resume DNA replication. We took time points after release to follow progress of the cells through S phase. We analyzed the DNA from each time point by two-dimensional (2D) agarose gel electrophoresis to test for the presence of replication intermediates (RIs). We probed our blots with vector sequences, so as not to confuse the plasmid signals with those from the corresponding chromosomal sequences. Under our conditions, the time points with the most intense RI signals corresponded to the most frequent times of plasmid replication within the cell population.\n\nThe 2D gel results for the THF-bearing plasmid (Figure 4, top panels) show that the majority of cells replicated the plasmid at 30 or 45 minutes after release from the HU block. There are RIs visible in all time points, but they are most abundant at 30 minutes and somewhat less abundant at 45 minutes. The corresponding flow cytometry results (Figure 4, topmost panels) show that these times correspond to late S phase, consistent with the observed late replication of the THF in the chromosome8. The 2D gel results for the R1 subfragment indicate that RIs were most abundant at 0 minutes (Figure 4, lower panels), meaning that these RIs formed in the presence of HU and were therefore a consequence of early replication. RIs persisted, though less abundant, at 15 and 30 minutes, and then nearly disappeared at 45 and 60 minutes. Thus the majority of R1 plasmid replication took place in early S phase, while most THF plasmid replication took place in late S phase. This leads us to conclude that there are cis-acting sequences within the THF that force late replication, but these sequences are not located within R1.\n\nTopmost panels: Flow cytometric analysis of the passage through S phase of cells containing plasmids bearing the intact 8-kb THF. The majority of cells arrested with an approximately 1N DNA content after four-hour treatment with hydroxyurea (HU; 0 minutes). The DNA content of these cells gradually increased after removal of HU (15 minutes through 60 minutes). Upper middle panels: Two-dimensional (2D) gel analyses show that replication intermediates from the plasmid bearing the THF were most abundant 30 and 45 minutes after removal of HU (late S phase). Lower middle panels: Similar 2D gel analyses show that plasmids containing only R1 replicated primarily during the HU block (0 minutes; early S phase) and nearly all replication was complete by 45 minutes. Bottommost panels: Flow cytometry showing progression through S phase of cells containing plasmids bearing only Region 1. These cells progressed through S phase with kinetics indistinguishable from those of cells containing plasmids with the intact THF (topmost panels).\n\nNext we examined the effects of deleting various sequences from the THF, while leaving R1 intact. Segments of the THF bordered by the restriction sites indicated in Figure 5A were deleted from the THF. Each of the deletions left the R1 portion of the THF intact. Figure 5B shows the ARS activities of the four deletion-containing fragments compared to the activities of pRS306 (negative control), the intact THF, and the R1 fragment by itself (see also Figure 2D). Each of the deletions significantly increased ARS activity compared to the intact THF, but each also decreased activity compared to the R1 fragment by itself. This means that, although each deletion removed some of the sequences that inhibit firing of the origins within the R1 fragment, none of the deletions removed all of the inhibitory sequences. Inhibitory sequences must be distributed broadly throughout the rightmost 70% of the THF.\n\n(A) Diagram of the THF and the regions deleted from it. The thick horizontal bars show regions deleted. The restriction enzyme sites bordering each deletion are indicated. (B) Results of ARS assays for controls and for plasmids containing the deleted THF sequences shown in panel A. Compare with Figure 2D and Figure 3C.\n\nNext we measured the replication times of these deletion plasmids, using the same methods as in Figure 4. Flow cytometry showed that the rate of passage through S phase was almost identical for each of the four strains containing deleted plasmids (Figure 6). Each deletion (Figure 6) accelerated replication timing compared to the intact THF (Figure 4). Deletion ∆A had the least effect. Each of the other deletions, ∆B, ∆C, and ∆D, accelerated replication timing so much that the strongest RI signal was in early S phase (15-minute time point). The strongest effect was produced by the largest deletion, ∆D. However, none of these deletions (Figure 6) permitted replication to be as early as the R1 subfragment by itself (Figure 4), for which the strongest RI signal is at the 0-minute time point. We conclude that cis-acting sequences promoting late replication are distributed throughout the rightmost 70% of the THF and are especially abundant in the rightmost 45% covered by deletion ∆D.\n\nFlow cytometry and 2D gel results (as in Figure 4) are shown for plasmids containing each of the deletions described in Figure 5. The unusual shapes of the replication-intermediate signals for ∆C and ∆D are a result of using the standard 2D-gel-electrophoresis procedure on large (>6 kb) DNA fragments. For ∆A and ∆B typical Y-arc shapes were generated, because in those cases the large plasmid was digested with two restriction enzymes, and the detected restriction fragment was approximately 4.3 kb.\n\nWe also examined the replication timings of the two individual ARS elements within the R1 fragment (Figure 3). The flow cytometry profiles and 2D results are shown in Figure 7 for deletions E4, E5, and E6 (deletions E1, E2, and E3 produced essentially identical results). Similar to the intact R1 fragment (Figure 4), these deletions maintained high concentrations of RIs at the 0-, 15- and 30-minute time points. In contrast to the R1 fragment, their RI concentrations may have been slightly greater at 15 minutes than at 0 minutes. The fact that deletions E4 and E5 appear to complete replication somewhat earlier than deletion E6 (Figure 7) suggests the possibility that cis-acting sequences promoting early replication may be present in the stretch of about 500 bp that is deleted in E6 but not E4 or E5 (Figure 3). Thus multiple cis-acting sequences within the 8-kb THF regulate its late replication timing, with sequences in the rightmost 45% favoring late replication and sequences in the leftmost portion (R1 fragment) possibly promoting early replication.\n\nFlow cytometry and 2D gel results (as in Figure 4 and Figure 6) are shown for plasmids containing some of the deletions described in Figure 3B. The shapes of the Y arcs are unusual due to the large sizes of the fragments analyzed.\n\nIn the fission yeast replication checkpoint pathway, the Rad3 kinase phosphorylates and contributes to activation of the downstream checkpoint kinase, Cds1 (reviewed in28). The fact that telomeres are shortened in rad3∆ cells suggests that Rad3 is important for telomere structure29. However, deletion of the gene encoding the downstream checkpoint kinase, Cds1, has no detectable effect on telomere structure29. Two laboratories14,15 have demonstrated that loss of either Rad3 or Cds1 permits earlier replication in HU-treated cells of TAS in their natural context near the ends of chromosomes. We wanted to test whether this advanced replication timing in checkpoint-deficient cells would also apply to the 8-kb THF described above (Figure 1, FIgure 2, Figure 4 and Figure 5) in a plasmid context. Figure 8 shows the results of an HU block-and-release experiment on cds1Δ cells bearing this plasmid: the THF-containing plasmid replicated primarily in early S phase, in contrast to its late replication when in wild-type cells (Figure 4). Comparison between wild-type cells (Figure 4) and cds1∆ cells (Figure 8) is somewhat complicated by the fact that, after release from the HU block, passage through the rest of S phase is much slower in cds1∆ cells than in wild-type cells, as previously demonstrated by Kim and Huberman8 (S.M. Kim and J.A. Huberman, unpublished). Despite this complication, the 2D gel results (Figure 8) show maximum RI abundance at 0–30 minutes, corresponding to early S phase. The abundance of RIs decreased as the cells progressed further into S phase (45 and 60 minutes). This indicates a loss of late-replication-timing control for the plasmid containing the 8-kb THF in cds1Δ cells, similar to the loss of late-replication-timing control for TAS in checkpoint-deficient cells in their chromosomal context14,15.\n\nFlow cytometry and 2D gel results (as in Figure 4, Figure 6 and Figure 7) are shown for the plasmid containing the intact THF, replicating within cells lacking the Cds1 checkpoint kinase. Compare these results with those in Figure 4 (top two panels), where the same plasmid was replicating in cells with functional Cds1 kinase.\n\n\nDiscussion\n\nWe have identified two DNA replication origins in the fission yeast telomere-associated sequences, TAS. The origins were found within a cloned, sequenced 8-kb Telomeric HindIII Fragment (THF) originally identified by Sugawara and cloned by him into the plasmid, pMLC12 (Figure 1;1). We recloned the intact THF and eight portions of it, regions R1–R8, into a yeast shuttle plasmid (pRS30618) and then tested these plasmids for origin activity. We found that all of the origin activity was confined to region 1, which in its chromosomal context is 6–8 kb from the simple-sequence repeats at chromosome ends (Figure 2).\n\nIn fission yeast, replication origins tend to be highly AT-rich. Conversely, high AT richness is a good predictor of origin locations and activities22,23. Within region R1 there are two peaks of high (>80%) AT content, P1 and P2 (Figure 2, Figure 3). Measurements of the origin activities of exonuclease-generated fragments of region R1 showed that fragments containing P1 alone or P2 alone were individually capable of functioning as replication origins, although neither P1 nor P2 was as active as region R1, which contained both P1 and P2 (Figure 3). Thus region R1 contains not one, but two, active replication origins, and these two origins are nearly adjacent to each other, separated by about 1.6 kb. The two origins are closer to telomeres than are any other origins within the fission yeast genome (Figure 1, Figure 2).\n\nInterestingly, there are two additional high-AT peaks, P3 and P4, within the THF (Figure 2). Each of regions R2, R5 and R6 contains at least one of peaks P3 and P4. Region R5 contains P2 and P3. Although peak P2 is active by itself (Figure 3), and although P3 and P4 are just as AT-rich as P1 and P2, none of regions R2, R5 or R6 has detectable origin activity (Figure 2). This suggests that regions R2, R5 and R6 must contain cis-acting sequences that inhibit origin function, and region R1 must be relatively free of such sequences. Our preliminary investigation revealed (Figure 5) that these inhibitory sequences may well be located in multiple places within regions R2, R5 and R6, and additional inhibitory sequences also appear to be present in regions R3, R4 and R7.\n\nIn its chromosomal context, the THF replicates in late S phase8 . Interestingly, we found that the intact THF also replicates late in a plasmid context (Figure 4). This suggests that the late replication of telomere-proximal sequences in fission yeast chromosomes may not be a consequence of their proximity to chromosome ends, since the plasmids we constructed were circular, not linear.\n\nInstead, our results suggest that cis-acting sequences within the non-R1 portion of the THF must be responsible for the late replication of the THF. A plasmid bearing only region R1 replicated early in S phase, while a plasmid containing the complete 8-kb THF replicated late (Figure 4). None of four deletions, which together covered all of the non-R1 portion of the THF, was sufficient to permit the remaining fragment to replicate as early as does region R1 by itself (Figure 4, Figure 5, Figure 6). These observations suggest that cis-acting sequences promoting late replication are widely distributed throughout the non-R1 portion of the THF.\n\nWhat could these late-replication-determining cis-acting sequences be? We don’t know the answer, but a non-exhaustive search for sequence motifs present only in the non-R1 portion of the THF revealed multiple possibilities (Figure 9). In addition to, or instead of, contributing to late replication of the THF, the motifs identified in Figure 9 that are located in regions R2, R5 and R6 may also contribute to suppression of origin activity in these regions by the AT-rich peaks P3 and P4 (Figure 2).\n\nThe diagram is an expansion of Figure 2B, with the positions of color-coded sequence motifs added. The motifs were identified using Meme software (see Materials and Methods).\n\nIt is interesting that two of the motifs in Figure 9, GGTGGG and TGGGT, seem related to the cis-acting \"LCS\" motif (GKKGGGGGAW, where K represents G or T and W represents A or T) previously identified by Yompakdee and Huberman as being capable of restraining plasmids containing ars727 or ars3001 to replication in late S phase25. All three of these G- and T-rich motifs are also related to a motif (CNWWGTGGGGG, where N represents any nucleotide and W represents A or T) observed by Hayano et al. to be enriched in sites that bind Rif1, a protein essential for normal replication timing in fission yeast30.\n\nThese similarities suggest that the Rif1 protein may be at least partially responsible for late replication of the THF.\n\nAnother sequence motif that should be considered a candidate for contributing to low origin efficiency and/or to late replication timing is the simple-sequence telomeric repeat motif (GGTTA-CA), 12 copies of which are located between coordinates 7882 and 7981of the pNSU21 sequence shown in Figure 2 and Figure 5. If this motif plays a role in reducing replication efficiency or promoting late replication timing, it is likely that it does so only as a repeat sequence, because a single copy is found in region R1. Since a plasmid containing only R1 replicates efficiently in early S phase, the single copy of GGTTACA within R1 is clearly insufficient to reduce replication efficiency or retard its timing. Even if the 12 repeats of GGTTACA contribute to regulating plasmid replication efficiency or timing, they cannot be the only motif doing so, because plasmids lacking these repeats (∆C and ∆D, Figure 5, Figure 6) nevertheless replicate less efficiently and later than a plasmid containing only region R1 (Figure 4, Figure 5, Figure 6).\n\nWe noted above that a plasmid containing the intact, approximately 8-kb telomeric HindIII fragment (THF) replicates in late S phase, at the same time as the corresponding chromosomal sequences. Late replication of the chromosomal sequences requires the Cds1 checkpoint kinase8. Similarly, in this study we found that late replication of the THF-containing circular plasmid also requires the Cds1 kinase (Figure 8). We conclude that, at least for the approximately 8-kb stretch of telomere-associated sequences (TAS) at the ends of chromosomes 1 and 2, both checkpoint-dependence of late replication and late replication itself are dependent primarily on cis-acting sequences, not on proximity to the physical end of a linear chromosome.\n\n\nMaterials and methods\n\nWe used three Schizosaccharomyces pombe strains, ura4-D18 h- 31, 501 ura4- h-32 and cds1::ura4 ura4-D18 leu1-32 ade6-704 h-33. Cells were cultured in YES medium (5 g/l yeast extract, 30 g/l glucose, 225 mg/l adenine, uracil, histidine, lysine hydrochloride, and leucine;34) at 30°C. Cells were also grown on Edinburgh minimal medium + supplements (ade-nine, histidine, lysine hydrochloride, and leucine) (EMMS-ura;34) at 30°C for 8 days for transformation with plasmids. E. coli (DH5α) cells (New England Biolabs) were used to clone TAS sequences prior to yeast transformation.\n\nura4-D18 h- fission yeast cells31 were transformed by a modified version of the lithium acetate procedure as described35. For each sample, two transformations – one with 500 ng and the other with 1000 ng plasmid DNA – were carried out simultaneously. The results shown are an average of 3 separate experiments, with triplicate plating of 1% of each transformation mixture. The cells were plated on EMMS-ura medium and incubated at 30°C. Colonies were counted on day 8 after transformation.\n\nS. pombe telomere sequences were downloaded from ftp://ftp.sanger.ac.uk//pub2/yeast/sequences/pombe/telomeres. The S. pombe reference genome of October, 2008, was downloaded from ftp://ftp.sanger.ac.uk//pub2/yeast/pombe/Chromosome_contigs/OLD/20080922.\n\nNeal Sugawara cloned and sequenced multiple fission yeast telomeric restriction fragments1. We obtained his 8-kb clone, pNSU21, from the Sanger Center (see http://www.pombase.org/tools/clone-and-mapping-resources; send e-mail to archives@sanger.ac.uk).\n\nThe plasmid, pRS30618, was used as recipient for all clonings, due to its URA3 selectable marker and convenient multiple cloning site. The 8-kb HindIII-SacI fragment (the THF) from pNSU21 was cloned between the HindIII and SacI sites of pRS306.\n\nFragments representing Regions 1–8 were generated by PCR amplification from the 8-kb THF. The primers are listed in Table S1. Due to multiple internal sequence repetitions within the TAS fragment, all of the PCR reactions resulted in multiple bands. The band of interest (identified by its electrophoretic mobility) was gel-purified and cloned into pRS306. Many of the primers had restriction enzyme sequences added for cloning purposes.\n\nAs implied in Table S1, the PCR fragment representing Region R1 was cloned into pRS306 between the HindIII and BamH1 sites. The Promega Erase-a-Base Kit, which employs exonuclease III, was used to make the deletions indicated in Figure 3. For deletions E1–E3, the plasmid containing R1 was first digested with BamHI and KpnI. The KpnI site is protected from exonuclease III digestion, so deletions were made leftward (orientation of Figure 3) from the BamHI site. For deletions E4–E6, the plasmid containing R1 was digested with HindIII and KpnI. In this case, the deletions were made rightward from the HindIII site. After exonuclease III digestions, the plasmids were recircularized. Plasmids containing deletions of the desired size were first selected by gel electrophoretic size characterization and then confirmed by sequencing.\n\nCells were grown in EMMS-ura medium to maintain plasmid selection. On the day of the experiment, they were transferred to YES at a density of 0.7 to 1.2 × 107 cells/ml at 30°C and treated with 25 mM hydroxyurea (Sigma-Aldrich) for 4 hours. The cells were then washed twice with 30°C water by 5-min centrifugations in a Sorvall RC5B or RC6 using the GS3 or SLA-3000 rotors respectively. The cells were then resuspended in 30°C YES, and 200-mL samples were collected at T0 (release) and every 15 minutes thereafter for 1 hour. Sodium azide (Sigma-Aldrich) was added to 0.01% to block further cellular metabolism. The cell samples were pelleted in a Sorvall RC5B GSA rotor and stored at -70°C until needed.\n\nAn aliquot of cells was fixed in 70% ethanol and stored at 4°C. The day before flow analysis, the fixed cells were washed twice in 50 mM sodium citrate, then resuspended in 500 µL of 50 mM sodium citrate with 100 µg/ml RNAse A (Sigma-Aldrich). These cells were incubated overnight at 37°C. The next day, 500 µL of 50 mM sodium citrate supplemented with 2 µM Sytox Green (Molecular Probes) were added to the cells. The cells were sonicated and stored at 4°C until analyzed by flow cytometry on a Becton Dickinson FACScan.\n\n200 mL of cells (7–10 × 106 cells/mL) were used to prepare DNA for neutral-neutral 2D gel electrophoresis by previously described methods from Huberman et al.36 and Brewer and Fangman37. The procedure can be found at our website: http://joelhuberman.net/HubermanLabArchives/2D_Gel_Docs_HTML.html. The BND-cellulose enrichment of replication intermediates was omitted from the procedure. The DNA was digested by the enzymes used in cloning the TAS into pRS306. The final southern blots were hybridized with vector sequences alone (Sac1-linearized pRS306 DNA), so as not to confuse the results for the plasmids with those for chromosomal TAS. The autoradiograms were scanned using a Molecular Dynamics PhosphorImager to produce 16-bit TIFF files. IP Lab 3.5 software (originally from Scanalytics.com, currently updated and sold as iVision-Mac by BioVision Technologies) was used to adjust image intensities so that the intensities of the 1N spots would be uniform for each set of figures.\n\nWe used MEME software (http://meme.sdsc.edu/meme/cgi-bin/meme.cgi) to identify sequence motifs present in regions R2–R7 but not in R1. We used regions R2, R3 and R4 as positive sequences and region R1 as a negative sequence. We looked for zero or one occurrence of each motif among the three positive sequences, with maximum motif width 10 bases and minimum width 5 bases. We set the number of different motifs at 20, the minimum number of sites at 2, and the maximum number of sites at 50. From the many motifs identified, we selected those with the broadest distributions and highest frequencies through R2–R4.",
"appendix": "Author contributions\n\n\n\nAC and JAH conceived the study and designed the experiments. AC carried out the research. AC prepared the first draft of the manuscript, and JAH revised the manuscript. Both authors have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests disclosed.\n\n\nGrant information\n\nThis work was funded by NIH grants CA095908 and GM070566 to JAH. This study also benefited from Roswell Park Cancer Institute’s Flow Cytometry facility, which is supported by Roswell Park Cancer Institute’s Cancer Center support grant P30-CA16056 from the National Cancer Institute.\n\n\nAcknowledgements\n\nWe would like to thank Dr. Neal Sugawara and the Sanger Center for providing us with the TAS clones.\n\n\nReferences\n\nSugawara N: DNA sequences at the telomeres of the fission yeast, Schizosaccharomyces pombe.Ph.D. thesis. Harvard University, 1988. Reference Source\n\nHiraoka Y, Henderson E, Blackburn EH: Not so peculiar: fission yeast telomere repeats. Trends Biochem Sci. 1998; 23(4): 126. PubMed Abstract | Publisher Full Text\n\nLeonardi J, Box JA, Bunch JT, et al.: TER1, the RNA subunit of fission yeast telomerase. Nat Struct Mol Biol. 2007; 15(1): 26–33. PubMed Abstract | Publisher Full Text\n\nNakamura TM, Cooper JP, Cech TR: Two modes of survival of fission yeast without telomerase. Science. 1998; 282(5388): 493–496. PubMed Abstract | Publisher Full Text\n\nBaumann P, Cech TR: Protection of telomeres by the Ku protein in fission yeast. Mol Biol Cell. 2000; 11(10): 3265–3275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoser BA, Subramanian L, Chang YT, et al.: Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres. EMBO J. 2009; 28(7): 810–820. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoser BA, Nakamura TM: Protection and replication of telomeres in fission yeast. Biochem Cell Biol. 2009; 87(5): 747–758. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim SM, Huberman JA: Regulation of replication timing in fission yeast. EMBO J. 2001; 20(21): 6115–6126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcCarroll RM, Fangman WL: Time of replication of yeast centromeres and telomeres. Cell. 1988; 54(4): 505–513. PubMed Abstract | Publisher Full Text\n\nFerguson BM, Brewer BJ, Reynolds AE, et al.: A yeast origin of replication is activated late in S phase. Cell. 1991; 65(3): 507–515. PubMed Abstract | Publisher Full Text\n\nFerguson BM, Fangman WL: A position effect on the time of replication origin activation in yeast. Cell. 1992; 68(2): 333–339. PubMed Abstract | Publisher Full Text\n\nRaghuraman MK, Winzeler EA, Collingwood D, et al.: Replication dynamics of the yeast genome. Science. 2001; 294(5540): 115–121. PubMed Abstract | Publisher Full Text\n\nFeng W, Collingwood D, Boeck ME, et al.: Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication. Nat Cell Biol. 2006; 8(2): 148–155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHayashi M, Katou Y, Itoh T, et al.: Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast. EMBO J. 2007; 26(5): 1327–1339. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMickle KL, Ramanathan S, Rosebrock A, et al.: Checkpoint independence of most DNA replication origins in fission yeast. BMC Mol Biol. 2007; 8: 112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHayashi MT, Takahashi TS, Nakagawa T, et al.: The heterochromatin protein Swi6/HP1 activates replication origins at the pericentromeric region and silent mating-type locus. Nat Cell Biol. 2009; 11(3): 357–362. PubMed Abstract | Publisher Full Text\n\nHuberman JA: Genetic methods for characterizing the cis-acting components of yeast DNA replication origins. Methods. 1999; 18(3): 356–367. PubMed Abstract | Publisher Full Text\n\nSikorski RS, Hieter P: A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989; 122(1): 19–27. PubMed Abstract | Free Full Text\n\nChuang RY, Kelly TJ: The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks. Proc Natl Acad Sci USA. 1999; 96(6): 2656–2661. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKong D, DePamphilis ML: Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4 subunit. Mol Cell Biol. 2001; 21(23): 8095–8103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee JK, Moon KY, Jiang Y, et al.: The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein. Proc Natl Acad Sci USA. 2001; 98(24): 13589–13594. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSegurado M, de Luis A, Antequera F, et al.: Genome-wide distribution of DNA replication origins at A + T-rich islands in Schizosaccharomyces pombe . EMBO Rep. 2003; 4(11): 1048–1053. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDai J, Chuang RY, Kelly TJ, et al.: DNA replication origins in the Schizosaccharomyces pombe genome. Proc Natl Acad Sci USA. 2005; 102(2): 337–342. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim SM, Huberman JA: Multiple orientation-dependent, synergistically interacting, similar do-mains in the ribosomal DNA replication origin of the fission yeast, Schizosaccharomyces pombe. Mol Cell Biol. 1998; 18(12): 7294–7303. PubMed Abstract | Free Full Text\n\nYompakdee C, Huberman JA: Enforcement of late replication origin firing by clusters of short G-rich DNA sequences. J Biol Chem. 2004; 279(40): 42337–42344. PubMed Abstract | Publisher Full Text\n\nBrun C, Dubey DD, Huberman JA, et al.: pDblet, a stable autonomously replicating shuttle vector for Schizosaccharomyces pombe. Gene. 1995; 164(1): 173–177. PubMed Abstract | Publisher Full Text\n\nSantocanale C, Diffley JF: A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature. 1998; 395(6702): 615–618. PubMed Abstract | Publisher Full Text\n\nGarabedian MV, Noguchi C, Ziegler MA, et al.: The Double-Bromodomain Proteins Bdf1 and Bdf2 Modulate Chromatin Structure to Regulate S-Phase Stress Response in Schizosaccharomyces pombe . Genetics. 2012; 190(2): 487–500. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDahlen M, Olsson T, Kanter-Smoler G, et al.: Regulation of telomere length by checkpoint genes in Schizosaccharomyces pombe. Mol Biol Cell. 1998; 9(3): 611–621. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHayano M, Kanoh Y, Matsumoto S, et al.: Rif1 is a global regulator of timing of replication origin firing in fission yeast. Genes Dev. 2012; 26(2): 137–150. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrimm C, Kohli J, Murray J, et al.: Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet. 1988; 215(1): 81–86. PubMed Abstract | Publisher Full Text\n\nMurray JM, Doe CL, Schenk P, et al.: Cloning and characterisation of the S. pombe rad15 gene, a homologue to the S. cerevisiae RAD3 and human ERCC2 genes. Nucleic Acids Res. 1992; 20(11): 2673–2678. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurakami H, Okayama H: A kinase from fission yeast responsible for blocking mitosis in S phase. Nature. 1995; 374(6525): 817–819. PubMed Abstract | Publisher Full Text\n\nMoreno S, Klar A, Nurse P, et al.: Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991; 194: 795–823. PubMed Abstract | Publisher Full Text\n\nGietz D, St Jean A, Woods RA, et al.: Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992; 20(6): 1425. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuberman JA, Spotila LD, Nawotka KA, et al.: The in vivo replication origin of the yeast 2 microns plasmid. Cell. 1987; 51(3): 473–481. PubMed Abstract | Publisher Full Text\n\nBrewer BJ, Fangman WL: The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987; 51(3): 463–471. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "450",
"date": "07 Dec 2012",
"name": "Hisao Masai",
"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 provides useful information regarding the mechanisms of how telomere-proximal origins are suppressed for initiation in early S phase.The authors identified two origins near the telomere and found that they are suppressed by cis-acting elements present in adjacent segments (closer to the telomere). These latter sequences enforce late replication or suppression of replication even on the circular plasmid bearing the ARS fragment containing the above two putative origins. The late replication depend on the Cds1 function, suggesting that checkpoint suppresses the replication of from these origins through the putative cis-acting “late replication regulatory sequences”.The data provided are not complete to dissect the sequences and cellular functions required for late replication of subtelomere segments, but provide useful data that can be explored for further studies.Some of the 2D data are not so clear (Figures 6 and 7) and could be redone under modified conditions that would give better separation of RIs. The analyses of ∆A, ∆B, ∆C and ∆D are a bit confusing. Although it is stated that ∆A had least effect and still is replicated late (Figure 6), ∆A is comparable to ∆B and ∆D and better than ∆C in ARS assays in Figure 5. A potential role of rif1 in the late replication is suggested in Discussion, and it is certainly of interest to see the replication timing of THF in rif1∆ cells.Figure 5 title; “leftmost 2/3″ should be “rightmost 2/3″.",
"responses": [
{
"c_id": "103",
"date": "07 Dec 2012",
"name": "Joel Huberman",
"role": "Author Response",
"response": "We thank Dr. Masai for his useful comments and for pointing out our careless mistake in the title for Figure 5.We agree that some of the 2D data in Figures 6 and 7 are not optimally clear, because the analyzed fragments were large and therefore migrated anomalously. Despite that problem, however, the times at which signal strengths from replication intermediates were maximal are obvious in Figures 6 and 7, so we believe that our conclusions are sound. Unfortunately, our laboratory is now closed, due to my retirement and to Dr. Chaudari’s move to Houston, so we cannot redo the experiments with smaller fragment sizes.We also agree that it would be highly interesting to test the effect of the rif1 deletion on replication extents and timings of the THF and its subfragments. We hope that other laboratories will also be curious about these possibilities and will follow up with appropriate experiments. As implied in our manuscript and as pointed out by Dr. Masai, we think that further investigations of the replication properties of the THF and its subfragments (and also of mutant THFs and subfragments in which various sequence motifs have been modified) in appropriate mutant strains will shed much more light on the mechanisms regulating both extent and timing of replication of these two telomere-proximal origins."
}
]
},
{
"id": "451",
"date": "14 Dec 2012",
"name": "Toru M. Nakamura",
"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 current manuscript identifies new replication origins near telomeres in fission yeast. The information provided will serve as a useful starting point for future investigations.The most interesting finding is the identification of potential cis-acting sequences within subtelomeres that can cause telomeric ARS to be late replicating. As suggested by the authors, it would be very interesting to determine if those cis-acting regulatory sequences might collaborate with Rif1 to enforce late S-phase replication of telomeres in the future.Overall, the manuscript is clearly written and conclusions are supported by experiments shown. On the other hand, 2D gel data, especially Figures 6 & 7, are somewhat hard to understand since the signal for replication intermediates is weak and there appears to be non-specific background signals from hybridization on those gels as well. It may be helpful to provide cartoons of 2D gel hybridization signals to clearly indicate what part of the gel corresponds to replication intermediate signals.",
"responses": [
{
"c_id": "102",
"date": "19 Dec 2012",
"name": "Joel Huberman",
"role": "Author Response",
"response": "We thank Dr. Nakamura for his useful comments. Especially helpful is his suggestion that cartoons of 2D gel hybridization signals might help clarify the unusual gel patterns in Figures 6 and 7. We will create such a cartoon and submit it as a supplementary figure in the appropriately revised version of the manuscript."
}
]
},
{
"id": "452",
"date": "17 Dec 2012",
"name": "Jurg Bahler",
"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/1-58
|
https://f1000research.com/articles/1-59/v1
|
04 Dec 12
|
{
"type": "Opinion Article",
"title": "Misunderstanding publication bias: editors are not blameless after all",
"authors": [
"Stephen Senn"
],
"abstract": "In analysing whether there is an editorial bias in favour of positive studies, researchers have made implicit assumptions that are implausible. In particular, to justify the conclusion that there is no bias because observed editorial acceptance rates do not favour positive studies, the assumption that the decision to submit an article is based solely on quality would be required. If, on the other hand, submission were based on perceived probability of acceptance, negative and positive studies would not differ in terms of acceptance rates, but in terms of quality.It is shown, using a simple graphical model, how similar underlying situations as regards the relationship between quality and probability of acceptance on the one hand and study outcome (positive or negative) and probability of acceptance on the other could produce dramatically different results depending on the behaviour of authors.Furthermore, there is, in fact, some evidence that submitted negative studies are, on average, of higher quality than positive ones. This calls into question the standard interpretation of the studies examining editorial bias. It would appear that despite similar probabilities of acceptance for negative and positive studies, editors could be discriminating against negative studies.",
"keywords": [
"In his recent book Bad Pharma1",
"Ben Goldacre dismisses the possibility that the reason that many negative studies are unpublished is because there is an editorial bias in favour of positive and against negative studies. He cites a number of papers2–5 in support of this",
"writing: \"Here again the journals seem blameless: 745 manuscripts submitted to the Journal of the American Medical Association (JAMA) were followed up",
"and there was no difference in acceptance and non-significant findings. The same thing has been tried with papers submitted to the BMJ",
"The Lancet",
"Annals of Internal Medicine and the Journal of Bone and Joint Surgery. Again and again no effect was found\". (p34)."
],
"content": "Bad JAMA?\n\nIn his recent book Bad Pharma1, Ben Goldacre dismisses the possibility that the reason that many negative studies are unpublished is because there is an editorial bias in favour of positive and against negative studies. He cites a number of papers2–5 in support of this, writing: \"Here again the journals seem blameless: 745 manuscripts submitted to the Journal of the American Medical Association (JAMA) were followed up, and there was no difference in acceptance and non-significant findings. The same thing has been tried with papers submitted to the BMJ, The Lancet, Annals of Internal Medicine and the Journal of Bone and Joint Surgery. Again and again no effect was found\". (p34).\n\nA very thorough meta-analysis carried out by Song et al.6, which provides a useful summary of the studies that Goldacre cites, would appear to justify Goldacre’s conclusion. Song et al. find an overall odds ratio in favour of positive studies of 1.06, with confidence limits of 0.8 to 1.39, writing: \"After the submission of a manuscript for publication, editorial decisions were not clearly associated with study results\" (p10). The wording is more circumspect than Goldacre’s, but the sentiment seems broadly the same.\n\nHowever, as I explain below, these studies are inherently incapable of showing that there is no publication bias by journals and in fact, properly interpreted, they provide evidence that could be compatible with the opposite of what Goldacre and others claim.\n\n\nQuality matters\n\nFigure 1 shows two imaginary curves for a given journal illustrating the probability of acceptance for a manuscript submitted to a journal as a function of the quality (measured on an arbitrary scale from 0 to 100) of that manuscript. In the figure it is assumed that the probability of acceptance for a manuscript of a given quality is always higher for a positive study than for a negative one.\n\nThe acceptance curve for negative studies is shown in solid red and that for positive studies in dashed blue. The vertical dashed line shows a postulated quality threshold for submission and the horizontal dashed lines indicate the acceptance probabilities that would result.\n\nThe curves are not meant to be realistic in detail, but to suffice conceptually to represent the idea that higher quality increases the probability of acceptance (programmed in GenStat: see 'Publish or perish GenStat program'). Of course, the reader could always object that the curves do not represent what he or she considers the relationship between quality and probability of acceptance might be. However, the point is that the details of the precise shape of the curves are irrelevant provided only that two features survive: first that other things being equal higher ‘quality’ leads to higher probability of acceptance and second, other things being equal, that positive studies are more likely to be accepted than negative ones. We can refer to the two curves in Figure 1 as quality acceptance curves. Now, consider two assumptions. First, that authors make a decision to submit to a given journal in their field based on quality and not on whether the study is positive or negative. Call this the equal quality assumption. Second, that the distribution of positive and negative studies by quality is equal. Call this the identical distribution assumption. If these assumptions are true, then a lack of observed difference in probabilities of acceptance is indicative that the two quality acceptance curves are the same and this would imply that studies of a given quality have the same chance of being published whether negative or positive: In other words, that comparing like with like, there was no bias in favour of positive studies.\n\nThe quality assumption is illustrated in Figure 1. Here a vertical quality submission threshold is shown. Papers below the threshold (to the left of the boundary) would not be submitted but those above (to the right) will be. The figures show the probability of acceptance at the threshold for the two types of study, and these are very different, being about 5% for negative studies and 27% for positive ones.\n\n\nProbability matters\n\nHowever, if authors act rationally, setting a quality submission threshold is not, except indirectly, how they will judge whether or not to submit a manuscript to a given journal. Self-interest would require them to take into account the cost of submission (in terms of effort), the reward if published (in terms of kudos and so forth) and the probability of acceptance. Here, an alternative threshold might be supposed. We might imagine, other things being equal, that authors employ a probability submission threshold. We can call the assumption corresponding to this the equal probability assumption. In that case what might apply is the situation shown in Figure 2. Now we have a horizontal probability threshold. Now it is the case that authors will not submit papers unless the probability of acceptance is at least equal to 20%. However, this threshold probability does not differ between negative and positive studies and the fact that it does not differ does not show a lack of bias. It is now the quality that differs at the probability threshold not the probability that differs at the quality threshold. Negative studies will have to be of higher quality to be worth submitting. In fact, the quality at the threshold for positive studies is about 46, whereas for negative studies it is 66. Again, whether this would translate into observed identical probabilities of acceptance depends on whether the mixture of studies submitted is identical.\n\nThis time however, it is supposed that authors make their decision to submit based on probability of acceptance. As before, the solid red curve gives the probability of acceptance for negative studies and the dashed blue curve for positive studies. The horizontal dashed line shows a postulated probability threshold for submission and the vertical dashed lines indicate the quality thresholds that would result.\n\n\nPs or Qs?\n\nTherefore, in judging the evidence, a key issue is which of the two assumptions, the equal quality assumption of Figure 1 or the equal probability assumption of Figure 2 is more relevant (there is still, of course, the issue of the identical distribution assumption, to be discussed below). One might argue that one has no more reason to suppose that the equal probability assumption is true than the equal quality assumption.\n\nHowever, this would be to ignore the evidence of a paper Goldacre cites3. This is by Lynch et al. and has a most revealing title ‘Commercially funded and United States-based research is more likely to be published; good-quality studies with negative outcomes are not’ and the abstract states, ‘Studies with a positive outcome were no more likely to be published than were those with a negative outcome (p=0.410) …studies with a negative outcome were of higher quality (p=0.003)’. In other words this paper provides evidence compatible with Figure 2.\n\n\nIn conclusion: caution is called for\n\nAll too easily we make the unconscious assumption that data we have seen have arisen in an inferentially neutral way so that from the point of study onwards, their message can be read at face value. However, studying papers submitted to journals is not like studying patients who have been randomised to treatment in a clinical trial. In fact, had researchers paused to think about it, they would have realised that the quality assumption was implausible. Goldacre himself points out that many studies are not submitted and it seems that negative studies are less likely to be submitted. That being so, it seems almost impossible to believe that the mixture by quality of submitted positive and negative studies could be identical. It would then follow that even if we could satisfy ourselves as to which of the quality or distributional assumptions were more reasonable, it would be difficult to know how this would pan out in terms of crude acceptance probabilities.\n\nA further difficulty is that it also seems plausible that higher quality studies are more likely to lead to a positive result. Certainly, to believe the contrary, is very unpalatable to a statistician like myself. (Of course, one could imagine a world in which researchers were naturally tending to produce studies that were larger than they needed to be but were persuaded by statisticians to get by with fewer subjects. In that case the difference a statistician would make would be mainly in reducing cost and not in increasing the probability of a positive result, but my personal experience, at least, does not support this).\n\nIn conclusion, I consider that the prospects for disentangling cause and effect when it comes to publication bias are not great. Certainly I do not think that the studies carried out so far can be taken as proof that editors have no bias in favour of positive studies. On the contrary, a very plausible explanation is the authors believe that editors are biased in favour of positive studies and are right.",
"appendix": "Author contributions\n\n\n\nSS did the research, conceived the argument, programmed the figures in GenStat® and wrote the paper.\n\n\nCompeting interests\n\n\n\nI consult regularly for the pharmaceutical industry. My career is furthered by publishing. I maintain a full declaration of all my interests here.\n\nThe views expressed in this paper should not be attributed to any organisation with which I am associated.\n\n\nGrant information\n\nThis work was funded by my employers, CRP Santé, for whose support I am extremely grateful.\n\n\nAcknowledgement and funding\n\nThis work was funded by my employers, CRP Santé, for whose support I am extremely grateful.\n\n\nReferences\n\nGoldacre B: Bad Pharma: How Drug Companies Mislead Doctors and Harm Patients. London: Fourth Estate, 2012. Reference Source\n\nOlson CM, Rennie D, Cook D, et al.: Publication bias in editorial decision making. Jama. 2002; 287(21): 2825–8. PubMed Abstract | Publisher Full Text\n\nLynch JR, Cunningham MR, Warme WJ, et al.: Commercially funded and United States-based research is more likely to be published; good-quality studies with negative outcomes are not. J Bone Joint Surg Am. 2007; 89(5): 1010–8. PubMed Abstract | Publisher Full Text\n\nLee KP, Boyd EA, Holroyd-Leduc JM, et al.: Predictors of publication: characteristics of submitted manuscripts associated with acceptance at major biomedical journals. Med J Aust. 2006; 184(12): 621–6. PubMed Abstract\n\nOkike K, Kocher MS, Mehlman CT, et al.: Publication bias in orthopaedic research: an analysis of scientific factors associated with publication in the Journal of Bone and Joint Surgery (American Volume). J Bone Joint Surg Am. 2008; 90(3): 595–601. PubMed Abstract | Publisher Full Text\n\nSong F, Parekh-Bhurke S, Hooper L, et al.: Extent of publication bias in different categories of research cohorts: a meta-analysis of empirical studies. BMC Med Res Methodol. 2009; 9: 79. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "389",
"date": "07 Dec 2012",
"name": "James Roger",
"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 opinion piece that suggests authors of scientific papers, may through their decision to submit, be inducing a correlation between the quality of paper and whether the results are negative or positive.It also suggests there may be an inherent tendency for better quality research to come up with truly positive results. So there is possibly an association between quality and positiveness of results in papers submitted to journals. If so, then we would expect to see papers selected on quality showing more positive results than those rejected. The lack of such an association suggests there may indeed be something amiss, but hardly anything more than that.A suggestion for the author is to change the title to: ‘Misunderstanding publication bias: editors are perhaps not blameless after all’.",
"responses": []
},
{
"id": "392",
"date": "10 Dec 2012",
"name": "Vance Berger",
"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\nNormally when reviewing I like to add something novel, with some added value. But in this case, I have nothing to add. The paper is complete as it is; the point is a valid one, and it was made very well.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-59
|
https://f1000research.com/articles/1-57/v1
|
28 Nov 12
|
{
"type": "Commentary",
"title": "Does vitamin D deficiency contribute to post-burn bone loss?",
"authors": [
"Gordon L Klein"
],
"abstract": "Burn injury results in the acute loss of bone as well as the development of progressive vitamin D deficiency. Bone loss occurs acutely due to resorption, which is then followed by apoptosis of osteoblasts preventing repair of the bone loss. The acute resorption is due to a combination of the inflammatory response and the stress response to the burn injury. The resultant production of inflammatory cytokines and endogenous glucocorticoids initially stimulate the osteoblasts to produce RANK ligand, which stimulates marrow stem cell differentiation into osteoclasts. As the stress response persists for approximately one year post-burn the glucocorticoids produced by the body will cause osteoblast apoptosis and adynamic bone, impairing the ability of bone to recover its resorptive losses. The vitamin D deficiency is due to the failure to supplement the diet of burn patients with vitamin D on discharge from hospital and to failure of the skin to make normal quantities of vitamin D on sunlight exposure. Because the bone resorption can be prevented by the acute administration of bisphosphonates it is unlikely that vitamin D deficiency is responsible for the early-onset bone loss following burns. However, because a deficit in trabecular bone remains for at least two years post-burn, it is possible that vitamin D deficiency prevents the recovery of trabecular bone density over the long term.",
"keywords": [
"Children who have been severely burned develop a progressive vitamin D deficiency1 as characterized by low circulating levels of 25-hydroxyvitamin D (25(OH)D) between 142 and 24 months post-burn with normal circulating levels of 1",
"25-dihydroxyvitamin D (1",
"25(OH)2D). Unlike rickets there is no compensatory rise in 1",
"25(OH)2D and this may be due to persistent low serum parathyroid hormone (PTH) concentrations2",
"3. The hypoparathyroidism is likely due to a persistent post-burn up-regulation of the parathyroid calcium sensing receptor3",
"4. By 7 years post-burn",
"there is a continued reduction in serum levels of 25(OH)D in all patients but now there is also a reduction in circulating concentrations of 1",
"25(OH)2D1."
],
"content": "Commentary\n\nChildren who have been severely burned develop a progressive vitamin D deficiency1 as characterized by low circulating levels of 25-hydroxyvitamin D (25(OH)D) between 142 and 24 months post-burn with normal circulating levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). Unlike rickets there is no compensatory rise in 1,25(OH)2D and this may be due to persistent low serum parathyroid hormone (PTH) concentrations2,3. The hypoparathyroidism is likely due to a persistent post-burn up-regulation of the parathyroid calcium sensing receptor3,4. By 7 years post-burn, there is a continued reduction in serum levels of 25(OH)D in all patients but now there is also a reduction in circulating concentrations of 1,25(OH)2D1.\n\nThere are at least two reasons for this progressive deficiency. The first explanation is that burn patients are not routinely given vitamin D supplements following hospital discharge1 and even if they are given a standard amount of vitamin D supplementation, i.e. 400 international units per day, for six months post-burn, serum levels of 25(OH)D are found to be in the \"insufficient\" range, approximately 20 ng/ml5. Therefore, the proper dose of vitamin D to administer to these children is unknown. The second explanation is that following burn injury, not only the burn scar tissue but also the adjacent normal-appearing skin cannot convert normal quantities of the precursor 7-dehydrocholesterol (7DHC) to vitamin D3 when subjected to UVB irradiation2. Moreover, the epidermal cells both in burn scar tissue and in adjacent normal-appearing skin contain subnormal quantities of 7DHC suggesting abnormalities in cholesterol biosynthesis in skin following burn injury2.\n\nBone loss following burn injury can be attributed to least two non-specific adaptive responses: the inflammatory response and the stress response. Both occur as a result of the destruction of skin, which is an important barrier to infection. The inflammatory response involves significant elevations of circulating cytokines, especially interleukin (IL)-1β and IL-66. The stress response is characterized by a 3–8-fold rise in endogenous glucocorticoid production as measured by urine free cortisol excretion6,7. Acutely, both processes stimulate osteoblast production of the ligand of the receptor activator of NFκB (RANKL), which then stimulates marrow stem cell differentiation into osteoclasts and results in increased bone resorption. The success of acute intravenous administration of bisphosphonates either as a single or once-repeated dose in preventing the bone loss8 is evidence that this is the case. Because the stress response is sustained for as long as one year post-burn, persistence of the elevated endogenous production of glucocorticoids leads to osteoblast and likely osteocyte apoptosis and reduction of marrow stromal cell differentiation into osteoblasts7. Thus the now adynamic bone cannot recover the deficit caused by the acute resorption. It is only after the stress response has dissipated after about one year post-burn, that remodeling resumes9.\n\nBecause the intravenous administration of bisphosphonates prevents the acute bone loss entirely8 for up to two years9, it is highly unlikely that the documented vitamin D deficiency plays a role in the immediate post-burn bone loss. Moreover, we have also shown that, at least acutely, there are low circulating levels of both vitamin D binding protein and albumin10, with albumin recovering by 6 months post-burn5. Therefore, whether there is true vitamin D deficiency during the first six months post-burn is difficult to determine. Nevertheless, what we do see in these burn patients is that by two years post-burn, cortical bone deficits, as measured by total body bone mineral content, in those not receiving acute bisphosphonate therapy recover to the level of those who did receive bisphosphosphonates9 while the deficit in trabecular bone, as measured by lumbar spine bone density, persisted9,11.\n\nA recent report by Zhou et al.12 has shown that in adult human marrow stromal cell culture, the clinical conditions under which the bone biopsies were obtained influenced the ability of the stromal cells to differentiate into osteoblasts in response to 1,25(OH)2D. In particular it was noted that vitamin D deficiency impaired this differentiation. This finding raises the possibility that progressive vitamin D deficiency as experienced by children post-burn may explain the persistence of their trabecular bone deficit, although why vitamin D deficiency would uniquely affect trabecular bone is not at all clear. Perhaps future studies looking at the differential effects of vitamin D on cortical and trabecular bone might determine whether this hypothesis is viable.",
"appendix": "Competing interests\n\n\n\nThe author has served on the Bone Toxicity Advisory Board for Novartis Pharmaceuticals, August 2012.\n\n\nGrant information\n\n\n\n\nReferences\n\nKlein GL, Langman CB, Herndon DN: Vitamin D depletion following burn injury in children: a possible factor in post-burn osteopenia. J Trauma. 2002; 52(2): 346–50. PubMed Abstract\n\nKlein GL, Chen TC, Holick MF, et al.: Synthesis of vitamin D in skin after burns. Lancet. 2004; 363(9405): 291–2. PubMed Abstract | Publisher Full Text\n\nKlein GL, Nicolai M, Langman CB, et al.: Dysregulation of calcium homeostasis after severe burn injury in children: possible role of magnesium depletion. J Pediatr. 1997; 131(2): 246–51. PubMed Abstract\n\nMurphey ED, Chattopadhyay N, Bai M, et al.: Up-regulation of the parathyroid calcium-sensing receptor after burn injury in sheep: a potential contributory factor to post-burn hypocalcemia. Crit Care Med. 2000; 28(12): 3885–90. PubMed Abstract\n\nKlein GL, Herndon DN, Chen TC, et al.: Standard vitamin D supplementation does not improve vitamin D insufficiency after burns. J Bone Miner Metab. 2009; 27(4): 502–6. PubMed Abstract | Publisher Full Text\n\nKlein GL, Herndon DN, Goodman WG, et al.: Histomorphometric and biochemical characterization of bone following acute severe burns in children. Bone. 1995; 17(5): 455–60. PubMed Abstract\n\nKlein GL, Bi LX, Sherrard DJ, et al.: Evidence supporting a role of glucocorticoids in the short-term bone loss in burned children. Osteoporos Int. 2004; 15(6): 468–74. PubMed Abstract | Publisher Full Text\n\nKlein GL, Wimalawansa SJ, Kulkarni G, et al.: The efficacy of acute administration of pamidronate on the conservation of bone mass following severe burn injury in children: a double-blind, randomized, controlled study. Osteoporos Int. 2005; 16(6): 631–5. PubMed Abstract | Publisher Full Text\n\nPrzkora R, Herndon DN, Sherrard DJ, et al.: Pamidronate preserves bone mass for at least 2 years following acute administration for pediatric burn injury. Bone. 2007; 41(2): 297–302. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKlein GL, Herndon DN, Rutan TC, et al.: Bone disease in burn patients. J Bone Miner Res. 1993; 8(3): 337–45. PubMed Abstract | Publisher Full Text\n\nKlein GL, Herndon DN, Langman CB, et al.: Long-term reduction in bone mass after severe burn injury in children. J Pediatr. 1995; 126(2): 252–6. PubMed Abstract\n\nZhou S, Glowacki J, Kim SW, et al.: Clinical characteristics influence in vitro action of 1,25-dihydroxyvitamin D(3) in human marrow stromal cells. J Bone Miner Res. 2012; 27(9): 1992–2000. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "382",
"date": "28 Nov 2012",
"name": "Dominique Garrel",
"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": "383",
"date": "30 Nov 2012",
"name": "Daniel Bikle",
"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": "384",
"date": "22 Jan 2013",
"name": "Jon Tobias",
"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\nMy reservations are as follows:1. Since children are not routinely supplemented with vitamin D, the lack of vitamin D supplementation in burn victims is not in itself an explanation for their low vitamin D levels.2. The assertion that elevated endogenous glucocorticoids contribute to bone loss in this context, through inhibition of osteoblast survival and generation, is somewhat over-stated. Although adverse effects of glucocorticoids on osteoblast function are well described, the literature mainly applies to pharmacological doses of glucocorticoid. The evidence cited here, concerning possible adverse effects of endogenous glucocorticoids, relates to a small observational study which provides weak circumstantial evidence at best.",
"responses": [
{
"c_id": "82",
"date": "30 Nov 2012",
"name": "Gordon Klein",
"role": "Author Response",
"response": "The comments from Dr Tobias are greatly appreciated. If I did not make it clear in the commentary I listed two contributory factors to post-burn vitamin D deficiency. The failure to supplement is not of itself a causative factor, I agree, but the fact that the skin cannot make vitamin D normally on exposure to UVB radiation coupled with the lack of routine supplementation means that the burn victims have no way to compensate for the skin’s failure to normally synthesize vitamin D3 from its precursor.With regard to the comment on endogenous glucocorticoids clearly Dr Tobias is correct in stating that the literature on glucocorticoid-induced osteoporosis relates to the exogenous administration of glucocorticoids. the studies that I have cited, albeit they are my own, demonstrate that the body produces large quantities of endogenous glucocorticoids in response to stress, coupled with lack of surface osteoblasts and a reduction in markers of osteoblast differentiation in marrow stromal cells taken from the burn victims as compared to unburned controls. The point to be emphasized, I believe, is that currently there is no way to equate the quantity of endogenous glucocorticoids produced in response to stress with the dosage of glucocorticoids given as therapy. In the absence of work corroborating our observations I raise the issue of similar effects of endogenous and exogenous glucocorticoids in order to increase awareness that in the case of burns at least the classic manifestations of glucocorticoid induced osteoporosis also exist in children who did not receive exogenous glucocorticoids, I would certainly agree that more studies in this area and in different conditions in which a patient is subject to stress would help clarify the situation, but I would not dismiss the argument for lack of evidence."
}
]
}
] | 1
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https://f1000research.com/articles/1-57
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https://f1000research.com/articles/1-36/v1
|
25 Oct 12
|
{
"type": "Research Article",
"title": "Termination of mid-trimester pregnancies: misoprostol versus concurrent weighted Foley catheter and misoprostol",
"authors": [
"Ayman Shabana",
"Hesham Salah",
"Mohamed Kandil",
"Emad Soliman",
"Dalia Morsi",
"Ayman Shabana",
"Hesham Salah",
"Emad Soliman",
"Dalia Morsi"
],
"abstract": "Objective: To investigate whether the use of a weighted trans-cervical fluid-filled Foley’s catheter would improve the effectiveness of 400µg vaginal misoprostol regimen in terminating mid-trimester pregnancies.Methods: This study was conducted at the department of Obstetrics and Gynecology, Menofyia University Hospital in Egypt. Fifty eligible primigravidae were allocated into 2 groups. Termination was carried out in group I using vaginal misoprostol while in group II, a weighted fluid-filled intra-uterine Foley’s catheter was inserted and a similar misoprostol regimen was followed as in group I.Results:The combined group showed shorter induction to termination interval (15.6 ± 4.9 versus 21.9 ± 5.4 hours; P<0.05). There was no significant difference in the occurrence of side effects between the groups.Conclusion: A combination of a weighted Foley’s catheter and 400µg of vaginal misoprostol every 4 hours is more effective than misoprostol alone in terminating mid-trimester gestations.",
"keywords": [
"Misoprostol",
"mid-trimester termination",
"Foley catheter"
],
"content": "Introduction\n\nUniversal prenatal screening programs have led to an increase in the diagnosis of congenital malformations with subsequent gradual increase in second trimester termination of pregnancy (TOP)1. This represents 10–15% of total abortions performed worldwide2.\n\nTermination in the second trimester is more risky than during the first trimester3 and, therefore, the pharmacologic management seems to be an appealing alternative to surgical evacuation. Misoprostol has been widely used in different dosages and routes for second-trimester pregnancy terminations. Doses ranging from 200 to 800 µg at intervals ranging from 3 to 12 hours have been described4–8. High doses at short intervals have been linked to a higher percentage of side effects.\n\nSeveral studies have described the Foley’s catheter as an effective method in ripening the cervix. Obed and Adewole9 showed that the Foley’s catheter increased the Bishop’s score in women with unripe cervices. Sciscione et al.10 stated that the Foley’s catheter appears to be of comparable effectiveness as to intravaginal misoprostol for pre-induction cervical ripening. Its use is common in poor countries because it is safe, inexpensive and has a low incidence of contractile abnormalities11.\n\nThis study was designed to investigate whether the insertion of a weighted fluid-filled trans-cervical Foley’s catheter would further improve the effectiveness of 400 µg vaginal misoprostol in terminating mid-trimester pregnancies.\n\n\nMaterial and methods\n\nThis study was carried out at the department of Obstetrics and Gynecology, Menofyia University Hospital, Egypt. Women were recruited and enrolled in the study from July 2011 until the end of April 2012. The institutional review board approved the protocol of the study and an informed consent was obtained from all participants. (see Supplementary Material).\n\nWomen enrolled were primigravidae with a singleton pregnancy at a gestational age ranging between 13 and 23 weeks, with a fetal anomaly that is incompatible with life or a fetal demise within one week. Gestational age was calculated from the date of the first day of last menstrual period and confirmed by a vaginal ultrasound scan. Women with any of the following were excluded from the study: 1) Previous uterine scar; 2) Contraindication to misoprostol; 3) Low-lying placenta; 4) Operation on the cervix; 5) Clinical or laboratory evidence of chorioamnionitis.\n\nInitially we planned to recruit 50 women divided equally between two groups. However, after starting enrollment, we were confronted with the fact that many women preferred to be enrolled in the combined group rather than the misoprostol-only group. When patient number 50 was enrolled, there were 20 women assigned to group I and 30 women to group II.\n\nAll participants were subjected to complete history taking and thorough clinical examination. Induction of abortion was carried out in group I by inserting two tablets; each contains 200 microgram misoprostol (Sigma Pharmaceuticals, Egypt), in the posterior vaginal fornix every four hours. In group II, with the patient in the lithotomy position, the cervix was visualized using a Cusco’s speculum and then sterilized with povidone iodine. The anterior lip of the cervix was grasped with a ring forceps and another ring forceps was then used to push the catheter through the cervix under direct visualization. The balloon was then inflated with 30 ml saline (50 ml for those >20 weeks) and the catheter was pulled back snugly against the internal os and taped to the inner aspect of the thigh. A bag filled with saline was applied to the distal end of the catheter to provide moderate traction. Different filling volumes were used at different gestational ages. We used 250 ml at 13–17 weeks, 300 ml with ages at 18–20 weeks and 500 ml at gestational ages more than 20 weeks. The filled bag was approximately equivalent to 250, 300 and 500 g in weight, respectively. A 400 µg misoprostol vaginal dose “two tablets” was then inserted in the posterior vaginal fornix/4 hours as in group I. A maximum of 4 misoprostol doses were used in both groups. Each patient received 1 g ampicillin/6 hours as a prophylactic antibiotic. As soon as the catheter was expelled, re-assessment of the dilatation and effacement of the cervix was carried out. Oxytocin infusion was started 4 hours after the last dose of misoprostol in both groups according to the standard protocol in our department.\n\nThe patient was assessed every 4 hours for vital signs, cervical dilatation, expulsion of fetus and occurrence of complications such as nausea, vomiting and abdominal pain. All patients received intravenous oxytocin 20 IU after expulsion of the fetus to help placental separation and delivery. Retained placenta was considered if no manifestations for placental separation appeared 30 minutes after fetal expulsion. The patient was anesthetized and manual separation of the placenta was carried out. Surgical evacuation was performed if any remnant of conception such as missed placental lobe or piece of membranes was suspected to be retained inside the uterus.\n\nAfter placental expulsion, women were observed for the presence of uterine atony and post-abortive bleeding. This was assessed by counting the number and change in the weight of the sanitary pads used by the patient. Bleeding was considered average if less than 200 cc and excessive if more than 200 cc.\n\nThe measured primary outcome parameter was the induction to abortion interval. Secondary parameters were: 1) Occurrence of side effects; 2) Need for manual separation of the placenta; 3) The need for surgical evacuation; and 4) Occurrence of post-abortive bleeding.\n\nFor the purpose of statistical analysis, women enrolled were categorized into 3 groups according to gestational age (Table 1). Early mid-trimester group between 13 and 17 weeks, mid mid-trimester group 18–20 weeks, and late mid-trimester group 21–23 weeks.\n\nResults were tabulated and analyzed on an IBM personal computer using Epi Info, version 6, a word-processing database and statistics program. Descriptive statistics were expressed as mean and standard deviation. The Student t test was used to evaluate independent variables that are normally distributed. The Chi square test was used to compare 2 rates unless the number in the contingency table was < 6 where Fisher’s exact test was used. A significant statistical level was considered if P was < 0.05.\n\n\nResults\n\nThe age of the participants was not significantly different between both groups (27.93 ± 6.48 years in group I versus 27.19 ± 5.23 years in group II; P > 0.05) There was no significant difference in gestational age at enrollment (18.0 ± 2.41weeks in group I versus 17.9 ± 2.88 weeks in group II; P > 0.05). Women enrolled in the study were categorized into 3 categories as shown in Table 1.\n\nIn the present study, the combined group showed shorter induction to abortion interval at all gestational ages compared to the misoprostol only group. However; this duration increased with increasing gestational age. The mean cumulative evacuation time was 15.6 ± 4.9 hours in the combined group versus 21.9 ± 5.4 hours in the misoprostol-only group (P < 0.01). The evacuation times for different gestational ages is shown in Table 2. In the combined group, 94% of cases (28/30) showed complete evacuation in less than 24 hours from the start of induction compared to only 55% (11/20) in the misoprostol group. At a gestational age of 21–23 weeks, 80% of women had aborted before 24 hours in the combined group but none in the misoprostol group. At a gestational age between 18 and 21 weeks, expulsion of fetus occurred in 100% and 57.1% in the combined and misoprostol groups, respectively. Rates of fetal expulsion were 100% in both groups at a gestational age between 13 and 17 weeks (Table 2).\n\nTable 3 shows that the secondary outcome parameters are not statistically different between both groups. Table 4 reports the side effects in both groups.\n\n\nDiscussion\n\nThis study showed that the combination of weighted Foley’s catheter and misoprostol resulted in a significantly shorter induction to abortion interval compared to misoprostol alone. Because we used the same misoprostol dose in both groups, this decrease in duration was attributed to the use of weighted Foley’s catheter with moderate traction applied over its distal end. Early studies comparing the Foley catheter with PGE2 tablets found no difference in the termination time between both methods12,13. No traction was applied in either of these studies. Bani-Irshaid et al.14 compared the efficacy and safety of Foley catheter (with and without traction) and prostaglandin E2 vaginal tablets in terminating second- and early third-trimester pregnancies in 258 women. They concluded that the efficacy of the Foley’s catheter can be enhanced with the use of traction to give similar results to prostaglandin E2. No studies have compared the use of prostaglandin E1 vaginal tablets alone versus its combined use with weighted Foley’s catheter in terminating mid-trimester gestations. The catheter exerts its effect by disrupting the integrity of amnion-chorion and myometrium in addition to cervical collagen increasing prostaglandin and cytokine release, rendering the uterus susceptible15–17.\n\nThe induction to delivery interval increased with increasing gestational age in both groups. We also had rates of fetal expulsion of 100% in both groups at a gestational age between 13 and 17 weeks, which decreased with advancement of gestational age. This is in agreement with other investigators’ findings. Gómez et al. in 200918 found that the mean induction-to-abortion interval increases by 4 hours after 20 weeks gestation. Ashok et al. (2003)19 in another study on mid-trimester medical termination of pregnancy concluded that the induction-to-abortion interval was significantly longer in the higher gestational age pregnancies. Similar findings were reported by Dilbaz et al.20 in pregnancies between 13 and 20 weeks. We used a misoprostol dose of 400 µg administered every 4 hours. Doses of 600 and 800 µg have shown comparable successful abortion rates, but were associated with high rates of side effects21,22. Wong and colleagues6 compared the efficacy and side effects of 400 µg misoprostol administered every 3 and every 6 hours. They concluded that the three-hour regime was more effective in terms of a significantly shorter drug administration-to-abortion interval at the expense of more side effects. We had no significant difference in the incidence of side effects between both groups. Side effects are less common with the vaginal route compared to other routes of administration23.\n\nIn the present study, there was no significant difference in the need for post-abortive manual separation of the placenta. Surgical evacuation should only be considered if there is clinical evidence that the abortion is incomplete24. In the current study, an incidence of 20% was reported in the misoprostol group while only 10% required surgical evacuation in the combined group. Ashok et al. 200319 studied mid-trimester medical termination of pregnancy using misoprostol and concluded that surgical evacuation of the uterus under general anesthesia was required to complete the abortion in 8.1% of women. In 2005, a report from the Scottish group estimated the rate of surgical evacuation as low as 2.5%25. The high incidence of surgical evacuation in this study may reflect the inadequate experience of dealing with second-trimester abortions. Cases of induced abortion in our society are rare because they are considered illegal unless performed to save a maternal life. Our findings showed that the majority of cases who required surgical evacuation in both groups were between 13 and 17 weeks5,7. This is in agreement with the results of other investigators26. It is probably wise to inform women undergoing pregnancy termination early in the second trimester of a possible higher chance of incomplete abortion. There was no statistically significant difference in the amount of blood loss between both groups.\n\nTo conclude, the use of a weighted trans-cervical Foley’s catheter filled with 30 ml saline improved the effectiveness of 400 µg vaginal misoprostol in terminating mid-trimester pregnancies, as reflected by a shorter induction-to-delivery interval with no significant increase in the incidence of side effects.",
"appendix": "Author contributions\n\n\n\nAS, conceived the study and designed it, HS Enrolled participants and collected the scientific material, MK shared the design of the study and wrote the manuscript, ES collected scientific material and performed the statistical analysis and DM enrolled participants.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nSupplementary Information – protocol of the study\n\nSecond trimester or mid-trimester is a period ranging from 14th to 28th weeks of gestation. It is subdivided into an early period between 14th and 20th weeks and a late period between 20th and 28th weeks27.\n\nThere is gradual increase in second trimester terminations of pregnancies because of the availability of prenatal screening programs allowing the diagnosis of congenitally malformed fetuses1.\n\nCervical ripening before surgical termination is an effective method for reducing the complications associated with suction curettage. It is a routine before second-trimester dilation and evacuation28. Misoprostol, a prostaglandin E1 analogue, is commonly used for this purpose29. It is also useful for elective medical abortion and evacuation of the uterus in cases of emberyonic or fetal death28. The vaginal route of administration is favored compared to other methods in clinical routine owing to its high clinical efficacy and lack of gastrointestinal side effects30.\n\nSeveral studies have described that the Foley’s catheter as an effective method in ripening the cervix. Obed and Adewole9 showed that it increased the Bishop’s score in women with unripe cervices in a way comparable to that of prostaglandins.\n\nThis study was designed to investigate whether the insertion of a weighted fluid filled trans-cervical Foley’s catheter would improve the effectiveness of 400 µg vaginal misoprostol in terminating mid trimester pregnancies.\n\nThis comparative study will be carried out at the department of Obstetrics and Gynecology Department, Menofyia University Hospitals. Participants will be women with an indication for second trimester termination. Eligible women will be those with BMI ranging between 20–30 with singleton pregnancies at a gestational age between 14–23 weeks. Pregnancies sonographically diagnosed with a fetal anomaly incompatible with life or a fetal demise within one week will be enrolled.\n\nThe procedure will be explained to all women and an informed consent will be obtained from all participants. All women enrolled will have the following routine work up:\n\n1. Complete history taking.\n\n2. Thorough clinical examination with special emphasis on:-\n\nPulse, blood pressure and temperature.\n\nNausea (mild, moderate and severe).\n\nVomiting (mild, moderate and severe).\n\nAbdominal pain (mild: less or equal to menstrual cramps, moderate: slightly stronger than menstrual cramps & severe: double strength of menstrual cramps). Women with mild pain will receive no analgesia while those with moderate pain will receive diclofenac sodium & women with severe pain will receive pethidine.\n\nCervical dilatation and consistency by vaginal digital examination (Firm or Soft).\n\nPresence or absence of vaginal bleeding.\n\nWatery vaginal discharge.\n\n3. Laboratory investigations:\n\nComplete blood picture.\n\nProthrombin time, bleeding time, clotting time and INR.\n\nLiver enzymes.\n\nKidney function.\n\nRandom blood sugar.\n\n4. Pelvic ultrasound to detect:\n\nNumber of gestations, viability and gestational age.\n\nWomen with any of the following will be excluded from the study:\n\n1. Previous uterine surgery.\n\n2. Contra-indication to misoprostol. E.g. bronchial asthma and coronary heart disease.\n\n3. Law lying placenta.\n\n4. Operation on the cervix.\n\n5. Chorioaminitis (diagnosed by leucocytic count >12000, positive CRP & fever >38).\n\nFifty women will be recruited and will be divided into 2 equal groups. In group A, termination will be induced by inserting 400 micrograms vaginal misoprostol (2 misotac tablets-sigma pharmaceuticals) every four hours while in group B. termination will be induced by inserting a 16 F Foley’s catheter trans-cervically then inflating its balloon by (30 ml–50 ml) saline. A bag filled with saline will be applied to the distal end of the catheter to provide moderate traction. Different volumes will be used at different gestational ages; 250 ml. at 13–17 weeks, 300 ml. with ages at 18–20 weeks and 500 ml at gestational ages more than 20 weeks. The filled bag is approximately equivalent to 250, 300 and 500 grams in weight respectively. The same dose of misoprostol as in group A (400 ug/4 hours) will be used as well.\n\nThe outcome parameters that will be measured in this study are:-\n\n1. Induction to expulsion interval.\n\n2. Maternal side effects (nausea, vomiting, abdominal pain, rupture of membranes, sepsis, manual separation of the placenta, surgical evacuation and hemorrhage). The amount of vaginal bleeding will be assessed by counting the number and change in the weight of the pads changed by the patient from the start of induction till complete expulsion has occurred. Hemorrhage will be considered as average if less than 200 cc and excessive if more than 200 cc.\n\nParticipants will be assessed every 4 hours for vital signs, cervical dilatation, expulsion of fetus and occurrence of complications.\n\nThe steps of the procedure in the Foley’s catheter group:\n\n1. Speculum insertion and exposure to the cervix.\n\n2. Steralization with povidine iodine.\n\n3. Holding the anterior lip of the cervix with ring forceps.\n\n4. Foley’s catheter will be inserted till it passed the internal os (about 5 cm of the catheter was passed through the cervical canal).\n\n5. Inflation of the balloon by 30 ml saline for women at 14–18 weeks and by 50 ml for 19–23 weeks.\n\n6. The ring forceps and the speculum will be removed.\n\n7. 400 ug misoprostol wetted with saline will be inserted in the posterior vaginal fornix.\n\nAll patients will receive 20 mIU intravenous oxytocin after expulsion of the fetus. If the placenta does not separate in 30 minutes, the patient will be anaesthetized and undergo manual separation of the placenta or surgical evacuation.\n\nAll women will be observed for the occurrence of side effects during the procedure and for the first 12 hours after termination.\n\nResults will then be tabulated and statistically analyzed. Results will then be compared to those of other investigators.\n\n\nReferences\n\nLalitkumar S, Bygdeman M, Gemzell-Danielsson K: Mid-trimester induced abortion: a review. Hum Reprod Update. 2007; 13(1): 37–52. PubMed Abstract | Publisher Full Text\n\nNewmann SJ, Dalve-Endres A, Diedrich JT, et al.: Cervical preparation for second trimester dilation and evacuation. Cochrane Database Syst Rev. 2010; (8): CD007310. PubMed Abstract | Publisher Full Text\n\nYapar EG, Senöz S, Urkütür M, et al.: Second trimester pregnancy termination including fetal death: comparison of five different methods. Eur J Obstet Gynecol Reprod Biol. 1996; 69(2): 97–102. PubMed Abstract | Publisher Full Text\n\nJain JK, Kuo J, Mishell DR Jr: A comparison of two dosing regimens of intravaginal misoprostol for second-trimester pregnancy termination. Obstet Gynecol. 1999; 93(4): 571–575. PubMed Abstract | Publisher Full Text\n\nNuutila M, Toivonen J, Ylikorkala O, et al.: A comparison between two doses of intravaginal misoprostol and gemeprost for induction of second-trimester abortion. Obstet Gynecol. 1997; 90(6): 896–900. PubMed Abstract | Publisher Full Text\n\nWong KS, Ngai CS, Yeo EL, et al.: A comparison of two regimens of intravaginal misoprostol for termination of second trimester pregnancy: a randomized comparative trial. Hum Reprod. 2000; 15(3): 709–712. PubMed Abstract | Publisher Full Text\n\nBebbington MW, Kent N, Lim K, et al.: A randomized controlled trial comparing two protocols for the use of misoprostol in midtrimester pregnancy termination. Am J Obstet Gynecol. 2002; 187(4): 853–7. PubMed Abstract | Publisher Full Text\n\nDickinson JE, Evans SF: The optimization of intravaginal misoprostol dosing schedules in second-trimester pregnancy termination. Am J Obstet Gynecol. 2002; 186(3): 470–474. PubMed Abstract | Publisher Full Text\n\nObed JY, Adewole IF: The unfavourable cervix: improving the Bishop score with the Foley's catheter. West Afr J Med. 1994; 13(4): 209–12. PubMed Abstract\n\nSciscione A, Larkin M, O'Shea A, et al.: Preinduction cervical ripening with the Foley catheter and the risk of subsequent preterm birth. Am J Obstet Gynecol. 2004; 190(3): 751–4. PubMed Abstract | Publisher Full Text\n\nSherman DJ, Frenkel E, Pansky M, et al.: Balloon cervical ripening with extra-amniotic infusion of saline or prostaglandin E2: a double-blind, randomized controlled study. Obstet Gynecol. 2002; 97(3): 375–80. PubMed Abstract | Publisher Full Text\n\nSlater DM, Zervou S, Thornton S: Prostaglandins and prostanoid receptors in human pregnancy and parturition. J Soc Gynecol Investig. 2002; 9(3): 118–24. PubMed Abstract | Publisher Full Text\n\nCaliskan E, Dilbaz S, Gelisen O, et al.: Unsucessful labour induction in women with unfavourable cervical scores: predictors and management. Aust N Z J Obstet Gynecol. 2004; 44(6); 562–7. PubMed Abstract | Publisher Full Text\n\nBani-Irshaid I, Athamneh TZ, Bani-Khaled D, et al.: Termination of second and early third trimester pregnancy: comparison of 3 methods. East Mediterr Health J. 2006; 12(5): 605–9. PubMed Abstract\n\nThomas IL, Chenoweth JN, Tronc GN, et al.: Preparations for induction of labour of the unfavorable cervix with Foley catheter compared with vaginal prostaglandin. Aust N Z J Obstet Gynaecol. 1986; 26(1): 30–5. PubMed Abstract\n\nSt Onge RD, Conners GT: Preinduction cervical ripening: a comparison of intracervical prostaglandin E2 gel versus the Foley catheter. Am J Obstet Gynecol. 1995; 172(2 Pt 1): 687–90. PubMed Abstract | Publisher Full Text\n\nKierce MJ, Thiery M, Parewijck W, et al.: Chronic stimulation of uterine prostaglandin synthesis during cervical ripening before the onset of labor. Prostaglandins. 1983; 25(5): 671–82. PubMed Abstract | Publisher Full Text\n\nGómez Ponce de León R, Wing DA: Misoprostol for termination of pregnancy with intrauterine fetal demise in the second and third trimester of pregnancy - a systematic review. Contraception. 2009; 79(4): 259–71. PubMed Abstract | Publisher Full Text\n\nAshok PW, Penny GC, Flett GM, et al.: An effective regimen for early medical abortion: a report of 2000 consecutive cases. Hum Reprod. 1998; 13(10): 2962–5. PubMed Abstract | Publisher Full Text\n\nDilbaz S, Caliskan E, Dilbaz B, et al.: Frequent low-dose misoprostol for termination of second-trimester pregnancy. Eur J Contracept Reprod Health Care. 2004; 9(1): 11–5. PubMed Abstract | Publisher Full Text\n\nHerabutya Y, Chanrachakul B, Punyavachira P: Vaginal misoprostol in termination of second trimester pregnancy. J Obstet Gynaecol Res. 2000; 26(2): 121–125. PubMed Abstract | Publisher Full Text\n\nPongsatha S, Tongsong T: Second trimester pregnancy termination with 800 mcg vaginal misoprostol. J Med Assoc Thai. 2001; 84(6): 859–863. PubMed Abstract\n\nWinikoff B: Pregnancy failure and misoprostol-time for a change. N Engl J Med. 2005; 353(8): 834–6. PubMed Abstract | Publisher Full Text\n\nel-Refaey H, Templeton A: Induction of abortion in the second trimester by a combination of misoprostol and mifepristone: a randomized comparison between two misoprostol regimens. Hum Reprod. 1995; 10(2): 475–78. PubMed Abstract\n\nHamoda H, Ashok PW, Flett GM, et al.: A randomized trial of mifepristone in combination with misoprostol administered sublingually or vaginally for medical abortion at 13–20 weeks gestation. Hum Reprod. 2005; 20(8): 2348–2354. PubMed Abstract | Publisher Full Text\n\nLo TK, Lau WL, Lai FK, et al.: The effect of gestational age on the outcome of second-trimester termination of pregnancies for foetal abnormalities. Prenat Diagn. 2008; 28(6): 508–11. PubMed Abstract | Publisher Full Text\n\nChia KV, Ogbo VI: Medical termination of missed abortion. J Obest Gynaecol. 2002; 22(2): 184–6. PubMed Abstract | Publisher Full Text\n\nGoldberg AB, Greenberg MB, Darney PD: Misoprostol and pregnancy. N Engl J Med. 2001; 344(1): 38–47. PubMed Abstract | Publisher Full Text\n\nSong J: Use of misoprostol in obstetrics and gynecology. Obstet Gynecol Surv. 2000; 55(8): 503–10. PubMed Abstract\n\nTang OS, Gemzell-Danielsson K, Ho PC: Misoprostol: pharmacokinetics profiles, effects on the uterus and side-effects. Int J Gynaecol Obstet. 2007; 99(Suppl 2): S160–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "332",
"date": "02 Nov 2012",
"name": "Nathalie Kapp",
"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 statistical analysis used by the authors is inappropriate as they did multiple testing, and the primary outcome needs a time-to-event analysis (like a survival analysis).As women chose their treatment group, the conclusions cannot be stated as one may about results from an RCT, given the potential for bias.",
"responses": []
},
{
"id": "333",
"date": "03 Nov 2012",
"name": "Vikram Talaulikar",
"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\nIt is an interesting study adding to the emerging recent evidence on the efficacy of balloon catheters for cervical ripening and induction of labour. However, the above issues need to be addressed in order for the conclusions to be valid:1. Allocation is by patient choice. Without true randomization the potential for bias exists.2. How were the sample numbers calculated (power)?3. Introduction of mechanical devices such as a balloon catheter through cervix into uterine cavity may lead to increased incidence of infection. Although this was an outcome measure in the study protocol, there was no mention of infection rates (whether they were the same or were higher) in the results section – this would have been important if the results are to be useful for general populations in future. Ampicillin was used prophylactically in the combined group – were the authors worried about sepsis too?4. The age and gestational ages in comparison groups look remarkably similar!5. Why were only primigravid women chosen?6. The authors should avoid multiple statistical testing which makes the conclusions invalid.",
"responses": []
},
{
"id": "334",
"date": "12 Nov 2012",
"name": "Nathan S. Fox",
"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 study examining the additional benefit of transcervical Foley in second trimester termination of pregnancy with misoprostol. Overall, this study is valuable, but I have a few reservations regarding their methodology.1. The authors should explain why this was not randomized. It seems like a relatively simple thing to do and it would improve the methods significantly.2. The authors list more baseline characteristics. Especially considering this was not blinded, how are we to be sure that the groups were equal at baseline?3. The authors should report the actual p-value, not just whether it was less than or greater than 0.05.4. How did the authors determine their sample size of 50? There was no power analysis done. A skeptical reader might conclude that the study was stopped only because statistical significance was reached.5. With so few patients, was the primary outcome normally distributed? If not, the t-test would not be the appropriate statistical test.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-36
|
https://f1000research.com/articles/1-56/v1
|
26 Nov 12
|
{
"type": "Case Report",
"title": "Inhalational sevoflurane in severe bronchial obstruction unresponsive to multipharmacologic therapy: a case report",
"authors": [
"Thomas Weber",
"Christian Schiebenpflug",
"Engelbert Deusch",
"Christian Schiebenpflug",
"Engelbert Deusch"
],
"abstract": "Introduction: Bronchial asthma with respiratory failure is a challenge for the intensivist as mechanical ventilation is often difficult due to bronchoconstriction and air-trapping. We describe a case of severe asthma with respiratory acidosis in a 10-year-old patient unresponsive to multipharmacologic broncholytic therapy. Only the initiation of sevoflurane inhalation resolved severe bronchoconstriction and dynamic hyperinflation, leading to complete recovery.Case presentation: A 10-year-old Caucasian boy was intubated and mechanically ventilated due to an asthmatic attack. Bronchoconstriction and dynamic hyperinflation were severe while multipharmacological broncholytic therapy was unsuccessful. Inhalation with sevoflurane via an anaesthesia machine was the key intervention leading to gradual resolving of severe hypercapnia and respiratory acidosis. Furthermore bilateral pupil dilation occurred during hypercapnia, but no intracranial pathology could be detected. The patient made an uneventful recovery. To our knowledge this is the first case where hypercapnia and respiratory acidosis were so profound and long lasting yet the patient survived without any damage.Conclusions: Inhalational anaesthetics must be considered as an early treatment option in ventilated asthmatic patients with bronchial obstruction unresponsive to conventional therapy even though their administration in intensive care units may be difficult.",
"keywords": [
"A 10 year-old boy weighing 40 kilograms was admitted to the Paediatric Intensive Care Unit after being found unconscious at home",
"and was subsequently intubated by an emergency care team. He had a history of asthma starting at the age of 4. Asthmatic episodes in the past were treated with intermittent salbutamol disc inhaler applications."
],
"content": "Case description\n\nA 10 year-old boy weighing 40 kilograms was admitted to the Paediatric Intensive Care Unit after being found unconscious at home, and was subsequently intubated by an emergency care team. He had a history of asthma starting at the age of 4. Asthmatic episodes in the past were treated with intermittent salbutamol disc inhaler applications.\n\nOn admission the patient had a plethysmographic oxygen saturation of 80% on mechanical ventilation with an inspired oxygen fraction of 100%. Initial blood gas analysis (ABL 700 Radiometer Copenhagen) revealed a paCO2 (partial pressure of arterial carbon dioxide) of >256 mm Hg, which was exceeding the cut-off-level of the analyser, and a pH of 6.69. The initial chest X-ray showed hyperinflated lungs and a discrete subcutaneous emphysema at the neck and the upper mediastinum (Figure 1). The patient was sedated with fentanyl 4 µg/kg/h, midazolam 0.5 mg/kg/h and ketamine 2 mg/kg/h and ventilated with a Draeger Evita 4 respirator (Draeger, Luebeck, Germany) using a pressure controlled mode. Initial ventilator settings were plateau pressure 36 cm H2O, positive endexspiratory pressure (PEEP) 5 cm H2O, respiratory rate 12/min and I/E ratio 1:3 with 100% inspired oxygen (ratio of inspiration to expiration in mechanical ventilation). This achieved sufficient oxygenation, but the flow pattern display on the respirator revealed massive air-trapping.\n\nBroncholytic therapy was started with prednisolone 2 mg/kg, terbutaline 0.02 mg/kg/h i.v., magnesium sulphate 2 g over 20 min i.v. q6h and inhalation with salbutamol and ipratropium bromide q4h. An antibiotic treatment with amoxicillin/clavulanic acid 1.5 g q8h and clarithromycin 300 mg q12h was initiated.\n\nThe ventilation management proved to be difficult in this boy. Ventilator settings had to be increased in a stepwise mode to plateau pressures up to 45 cm H2O and PEEP reduced to 0 cm H2O in the first two hours. However, neither severe hypercapnia nor air-trapping improved. Then, a combined high and low-frequency ventilation was initiated (VDR4 percussion ventilator, Reiner, Germany) with a percussion frequency of 400/min and a conventional frequency of 10/min. With this regime, paCO2 could be reduced to 98 mm Hg and pH raised to 7.03. Unfortunately hypercapnia worsened again and mediastinal emphysema was more prominent. 10 h after admission, the blood gas analysis revealed a paCO2 of >256 mmHg and a pH of 6.79. The respirator was replaced by an anaesthesia machine (Draeger Julian, Luebeck, Germany) and inhalation of 3% sevoflurane was started. Within minutes tidal volume increased from 100 to 320 ml while the plateau pressure could be reduced from 43 to 35 cm H2O. Other respiratory settings were PEEP 0, respiratory rate 6/min, I:E 1:5 and inspired oxygen fraction 100%. From that time blood gases improved continuously and air-trapping decreased with a paCO2 falling below 100 mm Hg after 20 hours and a pH exceeding 7.2 after 24 hours. Oxygen fraction could then be reduced to 50%. No buffering was performed throughout the whole treatment. Because blood pressure levels tended to be low (MAP < 60 mm Hg) despite a positive fluid balance, Dobutamine 4 µg/kg/min and Norepinephrine 0.08 µg/kg/min i.v. were administered. The time course of the patient´s blood gases is shown graphically in Figure 2.\n\nPartial pressure of arterial CO2(paCO2) is shown on the right side of the y-axis. Time course in hours (h) is shown on the x-axis.\n\n15 h after admission a fixed dilation of both pupils was observed. A cerebral CT-scan showed no abnormalities like brain swelling or intracranial bleeding. Since there was a risk of a longer hypoxic period, cooling to a core temperature of 34°C was initiated (Arctic Sun cooling system, Medivance, CO, USA) and intracranial pressure monitoring was performed (Codman® ICP monitoring system) revealing normal values.\n\nAfter 36 h, the situation had significantly improved: sevoflurane, terbutaline and ipratropium bromide could be stopped, magnesium sulphate was reduced and the anaesthesia machine could be replaced by an intensive care respirator. Chest X-rays showed that the subcutaneous and mediastinal emphysema had resolved. Subsequently, magnesium infusions were stopped and prednisolone was tapered. After 48 h, pupil dilation slowly resolved and 2 further days later pupils showed intact light reaction. Catecholamines were stopped and sedation was gradually weaned. On day 5 spontaneous breathing started, intracranial pressure monitoring was terminated and the trachea was extubated after 8 days. The boy was transferred to the regular ward on day 10 without any neurologic impairment and could be discharged in good condition 4 days later.\n\n\nDiscussion\n\nWe report a young patient suffering from a severe asthmatic attack that only resolved after therapy with inhalational anaesthesia using sevoflurane. During the treatment period prolonged severe hypercapnia and respiratory acidosis was observed. Moreover, the patient developed pupil dilatation that persisted for more than 30 hours. However, therapy was successful and the patient recovered completely.\n\nTo our knowledge the duration of hypercapnia with a peak paCO2 >256 mmHg, a paCO2 >100 mmHg for 20h and respiratory acidosis with a pH less than 7.2 for 24 h associated with a complete recovery without any complications has not been reported so far. In a case of an asthmatic patient described by Mazzeo and colleagues, where peak paCO2 level was 293 mmHg and pH 6.771, hypercapnia and respiratory acidosis resolved approximately 12 h after onset.\n\nIn our patient, pharmacological therapy with different topic and intravenous broncholytic agents failed. Ventilation management was complicated whereas adequate oxygenation could be achieved without major problems.\n\nThe major risk of massive bronchospasm with consecutive air-trapping is pulmonary hyperinflation leading to barotrauma and, on the other hand, increased pulmonary vascular resistance resulting in right ventricular failure. Therefore, low tidal volumes, a low respiratory frequency and a low I:E ratio are recommended strategies, whereas application of external PEEP remains a controversial issue2,3. The goal is to achieve sufficient oxygenation and a reduction of hypercapnia. Several animal studies showed that even high levels of paCO2 and respiratory acidosis can be well tolerated4,5 whereas buffering respiratory acidosis was found to worsen lung injury6.\n\nIn our patient a trial with high-frequency ventilation to facilitate CO2 elimination was initially successful, but subsequently resulted in deterioration of ventilation parameters.\n\nA multipharmacologic approach was used combining i.v. corticosteroids (prednisolone), i.v. and inhaled β-adrenergic agents (terbutaline and salbutamol), inhaled anticholingergic agents (ipratropium bromide) and i.v. magnesium and ketamine. All these agents influence bronchial tone by different mechanisms and our goal was to achieve a synergistic effect. However, the key therapeutic intervention for resolving airway obstruction was inhalational anaesthesia with sevoflurane. In the aforementioned case, different broncholytic agents and sevoflurane inhalation were applied but it remained questionable which agent was most effective1. Sevoflurane is known to modulate bronchial tone via voltage-dependent Ca++-channel activity and intracellular cyclic adenosine monophosphate levels7. Among anaesthetists sevoflurane inhalation is common practice in the theatre today in cases of bronchial obstruction after tracheal intubation. However, in the majority of intensive care settings, sevoflurane cannot be easily applied since most intensive care respirators are not designed to administer volatile anaesthetics. Getting an anaesthesia respirator to the ICU and changing the machine is cumbersome and may put the patient at further risk. Recently a new rebreathing device for the application of volatile anaesthetics in the ICU has become available (AnaConDa, Sedana Medical, Sundbyberg, Sweden), that allows wash-in kinetics for sevoflurane comparable to a regular vaporizer8.\n\nExtracorporal CO2 elimination can be considered another treatment option to remove hypercapnia and respiratory acidosis, and a pumpless arterio-venous system has been recently used for treatment in children9.\n\nThe clinical course of our patient was further complicated by bilateral dilated pupils. The findings could be explained by the occurrence of intracranial pathologies. Indeed, there are some case reports in ventilated asthmatic patients where permissive hypercapnia resulted in intracranial hypertension and even subarachnoidal haemorrhage10–13. In our patient however, CT scan was unremarkable. Moreover, contamination with inhaled anticholinergic drugs (like ipratropiumbromide) has also been blamed for unilateral pupillary dilation14, however, in our patient, symmetrical abnormalities were observed, the patient was ventilated before arrival on the ICU and had eye protection while receiving broncholytic therapy. Due to a potentially prolonged hypoxic event, systemic cooling therapy and continuously invasive intracranial pressure monitoring was performed for 48 h, but unfortunately the cause of symmetrical pupil dilation remains unclear.\n\n\nSummary\n\nInhalational anaesthetics should be considered as an early treatment option in ventilated asthmatic patients with unresponsive bronchial obstruction.\n\n\nConsent\n\nWritten informed consent for publication was obtained from the patient`s parents for publication of this case report and accompanying images.",
"appendix": "Author contributions\n\n\n\nTW is the first author and wrote the paper and collected and interpreted the data. TW and CS were the attending physicians responsible for the patient in this case report. ED participated in the clinical care of the patient and assisted in editing the draft. All authors have participated in the concept and design, analysis and interpretation of data, drafting and revising the manuscript, and they have given final approval for the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was funded by institutional resources only.\n\n\nReferences\n\nMazzeo AT, Spada A, Pratico C, et al.: Hypercapnia: what is the limit in pediatric patients? A case of near-fatal asthma successfully treated by multipharmacological approach. Pediatr Anesth. 2004; 14(7): 596–603. PubMed Abstract | Publisher Full Text\n\nBrenner B, Corbridge T, Kazzi A: Intubation and mechanical ventilation of the asthmatic patient in respiratory failure. J Allergy Clin Immunol. 2009; 124(2 Suppl): S19–28. PubMed Abstract | Publisher Full Text\n\nJain S, Hanania NA, Guntupalli K: Ventilation of Patients with asthma and obstructive lung disease. Crit Care Clin. 1998; 14(4): 685–705. PubMed Abstract | Publisher Full Text\n\nVannucci RC, Towfighi J, Heitjan DF, et al.: Carbon dioxide protects the perinatal brain from hypoxic-ischemic damage: an experimental study in the immature rat. Pediatrics. 1995; 95(6): 868–874. PubMed Abstract\n\nLitt L, Gonzalez-Mendez R, Severinghaus JW, et al.: Cerebral intracellular changes during supercarbia: an in vivo 31P nuclear magnetic resonance study in rats. J Cereb Blood Flow Metab. 1985; 5(4): 537–544. PubMed Abstract | Publisher Full Text\n\nNichol AD, O´Cronin DF, Howell K, et al.: Infection-induced lung injury is worsened after renal buffering of hypercapnic acidosis. Crit Care Med. 2009; 37(11): 2953–2561. PubMed Abstract | Publisher Full Text\n\nIwasaki S, Yamakage M, Satoh J, et al.: Different inhibitory effects of sevoflurane on hyperreactive airway smooth muscle contractility in ovalbumin-sensitized and chronic cigarette-smoking guinea pig models. Anesthesiology. 2006; 105(4): 753–763. PubMed Abstract\n\nSturesson LW, Johansson A, Bodelsson M, et al.: Wash-in kinetics for sevoflurane using a disposable delivery system (AnaConDa) in cardiac surgery patients. Br J Anaesth. 2009; 102(4): 470–476. PubMed Abstract | Publisher Full Text\n\nConrad SA, Green R, Scott LK: Near-fatal pediatric asthma managed with pumpless arteriovenous carbon dioxide removal. Crit Care Med. 2007; 35(11): 2624–2629. PubMed Abstract | Publisher Full Text\n\nGaussorgues P, Piperno D, Fouque F, et al.: Hypertension intracranienne au cours de l’etat de mal asthmatique. Article in French. Ann Fr Anesth Reanim. 1987; 6(1): 38–41. Publisher Full Text\n\nDimond JP, Palazzo MG: An unconscious man with asthma and a fixed, dilated pupil. Lancet. 1997; 349(9045): 98. PubMed Abstract | Publisher Full Text\n\nRodrigo R, Rodrigo G: Subarachnoid hemorrhage following permissive hypercapnia in a patient with severe acute asthma. Am J Emerg Med. 1999; 17(7): 697–699. PubMed Abstract | Publisher Full Text\n\nEdmunds SM MD, Harrison R: Subarachnoid hemorrhage in a child with status asthmaticus: Significance of permissive hypercapnia. Pediatr Crit Care Med. 2003; 4(1): 100–103. PubMed Abstract | Publisher Full Text\n\nUdy A: A 10-year-old child with status asthmaticus, hypercapnia and a unilateral dilated pupil. Pediatr Anesth. 2005; 15(12): 1120–1123. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "388",
"date": "26 Nov 2012",
"name": "Cesar Picado",
"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 report. Currently, there is very limited information regarding the treatment of severe asthma exacerbation which is difficult-to treat and doesn’t responding to conventional therapy. The approach described in this case report suggests an alternative treatment that could contribute to saving the lives of asthma patients.",
"responses": []
},
{
"id": "390",
"date": "10 Dec 2012",
"name": "Peter Nagele",
"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": "391",
"date": "18 Dec 2012",
"name": "Robin Steinhorn",
"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 report of a child who presented with profound acidosis due to an asthma exacerbation.While the idea of terminating asthma with inhaled isoflurane or sevoflurane has been reported in the literature, this case report is notable for its severity, use in conjunction with core cooling, and good outcome.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-56
|
https://f1000research.com/articles/1-55/v1
|
22 Nov 12
|
{
"type": "Research Article",
"title": "The relationship between political ideology and mortality in Poland",
"authors": [
"Piotr Romaniuk",
"Priyamvada Paudyal",
"Krzysztof Krajewski-Siuda",
"Roman Topór- Mądry",
"Raglan Maddox",
"Christian A Gericke",
"Piotr Romaniuk",
"Priyamvada Paudyal",
"Krzysztof Krajewski-Siuda",
"Roman Topór- Mądry",
"Raglan Maddox"
],
"abstract": "Introduction: The political preference of voters has been shown to be associated with their health status. We investigated the relationship between political preferences and mortality in Poland around the time of the last three general elections. Methods: We used the electoral data from the general elections held in 2001, 2005 and 2007. Indicators of political ideological preference were constructed based on the percentage of votes gathered by each party. Data on mortality, education and income level were obtained from 2002-2007 from the Polish Central Statistical Office. Pearson correlation was computed between standardised mortality ratios (SMR) and political preference. Finally, the influence of political preference on SMR was examined in a multivariate analysis controlling for socio-economic factors. Results: SMR was positively correlated with liberal political views (0.26; p<0.05) and negatively correlated with both secondary education (-0.49; p<0.05) and monthly income (-0.239; p<0.05). The correlation between SMR and conservative political views was negative, although the result did not reach statistical significance. Education and income explained more of the variation in SMR than political views. In a multivariate regression, the liberal views factor and secondary education were significantly associated with the SMR (p<0.001 for both). Conclusion: Our findings are consistent with earlier studies conducted in western countries showing a positive correlation between liberal political ideology and SMR, but differ in that an inverse relationship was found between conservative political orientation with education and income. The importance of socioeconomic and geographical factors in relation to political affiliation and health inequalities in Poland should be further explored.",
"keywords": [
"mortality",
"political ideology",
"political identity",
"demography",
"social determinants of health"
],
"content": "Introduction\n\nIn recent years, there has been growing interest regarding the relationship between political ideology and health status. Strong associations between voting patterns, social deprivation and mortality were reported in studies conducted in England and Ireland1–3. These studies, in general, observed reduced mortality rates in areas where the majority voted for the conservative party. This effect has been explained in relation to socio-economic status; people with better socio-economic status tend to be more conservative and thus, support a party that will improve their existing privileged condition1. Studies conducted in the USA have also reported similar findings. Kondrichin and Lester4 analysed data from the 1980 election that pitted Ronald Reagan against Jimmy Carter and found an inverse correlation between mortality and the percentage voting for the Republican Party. Studies assessing political affiliation at an individual level also suggest that political ideology correlates with the degree of the importance survey correspondents attach to health care5,6. These studies have indicated that Republicans are healthier and also assign a lower priority to issues related to health care than Democrats. It could also be possible that a reverse association exists whereby individuals with poor health are more likely to be influenced by ‘leftist’ ideology; however, this investigation requires longitudinal data on health and political ideology which is not available7.\n\nContemporary Poland lacks a clear social cleavage that could determine the shape of a stable party system because of its unique history8. The Polish political scene has been somewhat unstable since the collapse of communist rule in 1989. It has been regularly exposed to political shocks, splits and transformations. It has been postulated that as a consequence, the political preferences of voters have not been very stable, with evidence demonstrating significant flows between individual parties and coalitions9. Moreover, since the re-establishment of parliamentary democracy in 1989, none of the governing coalitions have managed to win a second election after being in power until 2011. This instability does not necessarily mean that there is irregularity in the voting preferences of particular social groups. However, there is a paucity of research investigating the voting behaviour of the Polish population in relation to their health. As a result of this lack of empirical evidence, a robust discussion regarding the potential relationship has been largely amiss. This study, therefore, is the first to examine the relationship between political preference and mortality in Poland.\n\n\nMethods\n\nThe findings of this study are based on the results of three general elections held in 2001, 2005 and 2007. A proportional voting system is applied in the case of the lower chamber of the Polish Parliament and a majority rule system (plurality-at-large – multi-seat constituencies; first-past the post since 2011) in the case of the upper chamber of Parliament. Only the elections for the lower chamber of Parliament were considered in this study. Each political party was assigned a political \"programme factor\" varying from 3L (extreme left), through 2L (moderate left), 1L (centre-left), 1R (centre-right), 2R (moderate right) to 3R (extreme right). The economic programmes of the political parties in Poland are not usually coherent and stable. Hence, the assignment of a \"programme factor\" was based on other postulates such as concepts that refer to morals, the state’s world-view on neutrality, and opinions on the church, religion and individualism. The population indicator for political preference (left or right) was constructed based on the percentage of votes gathered by each party in an election. The results were then unified using a weighted mean, where the results of each party had the weight equal to its \"programme factor\". Finally, a higher positive number indicated more conservative views dominating in the examined population, whereas a higher negative number demonstrated more liberal views. It is worth noting that some of the parties have been excluded from the analysis, as their political programmes were impossible to be reconstructed, or were ambiguous in the relevant political spheres. These were generally small parties with an extremely narrow electoral support. The lists of the parties that have been included and excluded in the analysis have been listed in the supplementary material. We then analysed the results of the general elections on the level of the poviat, which represent the second tier of local self-government administration in Poland, equivalent to a county in many countries. In 2010, there were 379 poviats with population sizes varying between less than 30,000 to more than 200,00010.\n\nData on mortality, education and income levels from 2002–2007 was obtained from the Polish Central Statistical Office. Standardised mortality ratios (SMR) were calculated in each poviat using the overall age-specific death rates for Poland for the period under consideration. We analysed the data using SPSS (statistical software, version 14). A correlation matrix was computed between SMR, voting preference, secondary education and income. Finally, a multiple linear regression analysis was conducted to examine the influence of political preference on SMR, controlling for socio-economic factors and election year.\n\n\nResults\n\nThe results of the three general elections are displayed in Table 1. The results presented show that the electoral turnout was relatively low in all three elections, although the proportion of eligible people voting increased significantly between 2005 and 2007.\n\nThe right wing parties dominated in all three elections – both in terms of the electoral support, and the distribution of seats in Parliament. In addition, the results show an increasing trend of electoral support towards right wing parties over the years, with the proportion of votes won by right wing parties increasing from 55% to 85% between 2001 and 2007.\n\nThe correlation matrix between SMR and other socio-political factors is presented in Table 2. SMR showed a modest positive correlation with the liberal political views factor, a medium negative correlation with secondary education and a modest negative correlation with monthly income. The correlation between SMR and the conservative views factor was negative; although the result did not reach statistical significance. The conservative factor showed a medium negative correlation with secondary education and monthly income whereas the relationship was smaller and positive with the liberal factor (p<0.05 for all).\n\nNote: *p<0.05.\n\nThe contribution of the political views factor, together with socioeconomic variables, to the statistical explanation of variance in SMR is summarised in Table 3. Secondary education and income explained more variation in SMR than did the political factor. The conservative factor alone had no influence on SMR and the liberal factor could explain only 6.8% of the variation. In a multivariate regression analysis, the liberal factor and secondary education were significantly associated with the SMR (P<0.001 for both) (Table 4).\n\n\nDiscussion\n\nHere we report the results of the first study of voting behaviour in relation to mortality in Poland. In our study, we found a positive correlation between liberal political views and SMR. Conservative political views were inversely correlated with SMR, although the correlation did not reach statistical significance. An inverse correlation was also found between the conservative views factor and secondary education and income levels, whereas, the relationship with the liberal view factor was positive. In a multivariate regression analysis, liberal political views and secondary education significantly predicted the SMR.\n\nOur results are in alignment with previous studies conducted in the UK and the USA, where conservative political views are negatively correlated with SMR1–2,4. However, the finding of an inverse correlation between conservative political views with education and income levels is opposite to the findings of a US study6. The most likely explanation for this difference is the very different social structure and political party landscape in a post-communist, catholic country. Another explanation is that conservative views are less defined by economic status or social class in Poland, but more by geographical differences. For example, the north-west of Poland usually manifests more liberal political attitudes, while the south-eastern areas have a more conservative orientation. This may again be partly determined by the economic characteristics of the regions with western regions being more industrialised and urbanised compared to the eastern regions which are mostly rural and agricultural.\n\nOne of the limitations of our study is that we were not able to adjust for socioeconomic variables other than secondary education and income which could have influenced the study results. Moreover, the results are based on regional socioeconomic indicators and so are prone to suffer from this ecological fallacy. Thus, the results from this aggregated study could potentially have differing health determinants and characteristics from individuals within the study. However, other recent studies investigating political views at both the individual and regional levels did not show major differences between individual and ecological level political preference data, suggesting that political ideology influences health through several pathways at the individual and contextual level6,11,12.\n\nImportant differences in political traditions, political agendas and political party labelling should be acknowledged when comparing studies on political views and health from different countries13,14. While it is important to consider the evidence base when analysing and interpreting the results of studies regarding political ideology and health, it is critical that policy decision makers and others utilising such evidence tailor their policies and programmes to meet local population needs. Furthermore, the hypothesis that conservative values, ideologies and latent attitudes could potentially prove conducive to health promoting behaviours and lifestyles6,15 has to be treated with caution. Unlike in many western countries, the results from this study indicated that higher levels of education and income were commonly correlated with more liberal political attitudes of examined individuals and groups in Poland.\n\nThere has been a wide-ranging debate on the possibility for political ideology to influence health; however, the reverse association is also possible. Health could potentially influence political ideology. For example, those with poor health may be inclined to liberal values and ideology as liberal parties typically tend to value and support social equality which in turn favours disadvantaged populations15.\n\nOverall, our findings are consistent with earlier studies conducted in western countries in showing a positive correlation between liberal political views and mortality, but differ in that an inverse relationship was observed between conservative political orientation and education and income. The importance of socioeconomic and geographical factors in relation to political ideology and health inequalities in Poland should be further explored in a longitudinal observational study.\n\nPolitical preference of voters has been found to be associated with their health status.\n\nOur findings corroborate the findings of studies conducted in western countries in showing a positive correlation between liberal political views and standardised mortality ratios (SMR).\n\nIn contrast to western countries, conservative political views were inversely related with education and income levels in Poland. This counters the argument that the relationship between political views and health is primarily explained by socioeconomic status and that political preference is only a proxy for the latter.",
"appendix": "Author contributions\n\nPR and KKS designed the study. PR and PP analysed the data with guidance from KKS, RTM and CAG. PR and RM wrote the first draft of the article with revisions by all co-authors.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Medical University of Silesia under the agreement No. KNW-1-207/08. Support for CAG and PP from the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care for the South West Peninsula (PenCLAHRC) is gratefully acknowledged. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health in England.\n\n\nReferences\n\nDorling D, Davey Smith G, Shaw M, et al:Analysis of trends in premature mortality by labour voting in the 1997 general election. BMJ 2001; 322(7298): 1336–1337.\n\nDavey Smith G, Dorling D: \"I’m all right, John\": voting patterns and mortality in England and Wales, 1981–92. BMJ 1996; 313(7072): 1573–1577.\n\nKelleher C, Timoney A, Friel S, et al:Indicators of deprivation, voting patterns, and health status at area level in the Republic of Ireland. J Epidemiol Community Health 2002; 56(1): 36–44.\n\nKondrichin SV, Lester D: Voting conservative and mortality. Percept Mot Skills 1998; 87(2): 466.\n\nBlendon RJ, Altman DE, Benson JM, et al:Voters and Health Reform in the 2008 Presidential Election. N Engl J Med 2008; 359(19): 2050–2061.\n\nSubramanian SV, Perkins JM: Are republicans healthier than democrats? Int J Epidemiol 2010; 39(3): 930–931.\n\nHuijts T, Kraaykamp G: Immigrants’ health in Europe: a cross-classified Multilevel approach to examine origin country, destination country, and community effects. Int Mig Rev 2012; 46(1): 101–137.\n\nDomanski H: A new dimension of social stratification in Poland? Class membership and electoral voting in 1991–2001. Eur Sociol Rev 2008; 24(2): 169–182.\n\nZuba K: Polska scena polityczna. Ciągłość i zmiana. [In Polish: Polish Politica Scene. Continuity and Change]. Wydawnictwo Sejmowe, Warszawa 2012.\n\nSize and structure of population and vital statistics by territorial division in 2010. Central Statistical Office. Warsaw 2011 (Last accessed on 19 Oct 2012).\n\nPage A, Morrell S, Taylor R, et al:Suicide and political regime in New South Wales and Australia during the 20th century. J Epidemiol Community Health 2002; 56(10): 766–772.\n\nHuijts T, Perkins JM, Subramanian SV, et al:Political Regimes, Political Ideology, and Self-Rated Health in Europe: A Multilevel Analysis. PLoS One 2010; 5(7): e11711.\n\nMishra R: Globalization and the Welfare State. Cheltenham: Edward Elgar, 1999.\n\nBorrell C, Espelt A, Rodríguez-Sanz M, et al:Analyzing Differences in the Magnitude of Socioeconomic Inequalities in Self-Perceived Health by Countries of Different Political Tradition in Europe. Int J Health Serv 2009; 39(2): 321–341.\n\nSubramanian SV, Huijts T, Perkins JM, et al:Association between political ideology and health in Europe. Eur J Public Health 2009; 19(5): 455–457."
}
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[
{
"id": "380",
"date": "18 Dec 2012",
"name": "Martin McKee",
"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 is subject to a number of weaknesses, in particular omitted variable bias, whereby political affiliation is almost certainly confounded by other factors – a point conceded by the authors when they note the limited variables they have.A further problem is the lack of a plausible mechanism. Another is the fact that the factors that give rise to different causes of death act over different time periods. As voter preference must act through these risk factors, voter preference at a single point in time will influence deaths at many different times in the future, depending on the cause. For example, starting smoking will kill you in 30 years, getting drunk may kill you today. Hence, although interesting, interpretation of the findings is difficult",
"responses": []
},
{
"id": "464",
"date": "04 Jan 2013",
"name": "Olga Vikhireva",
"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\nAs pointed out by the authors themselves and by the first reviewer, the study by Romaniuk and colleagues has some serious methodological limitations, such as omitted variable bias, residual confounding, ecological fallacy, and reverse causation.In addition, it is not entirely clear whether the data clustering (within povyats over three consecutive elections) was properly adjusted for in the regression models. As the authors state in the Discussion, the geographical areas of the country differ substantially; therefore, it could have been sensible to include a wider geographical area (such as industrialized north-west vs. agricultural south-east) in the analytical models. It might also be advisable to have a closer look at cause-specific mortality – as, for example, tobacco- and alcohol-related mortality could be stronger associated with political affiliations.The authors might also wish to strengthen the conclusive section on research and policy implications and emphasize that their findings, despite methodological issues, are relevant to a wider audiencePersonally, my main take-home messages from the present paper were as follows. First, “conservative” and “liberal” mean different things to different people in different countries over different historical periods – a seemingly simple, but often forgotten point.Second, while the voters’ preference may somehow reflect their health, the actual policy of the political party they voted into power could affect health to a much greater extent. For example, conservative values have been demonstrated to be “pro-health” in the West – but would one wholeheartedly claim that Conservative Party policy in the UK is “pro-health” for everybody who voted for Conservatives? Therefore, in an ideal world, the measures of population health would be an ultimate litmus test for the political party in power.Third, while the world is not ideal, it makes sense to invest in education and its economic pay-offs – which is hardly surprising, but highly relevant.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-55
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https://f1000research.com/articles/1-54/v1
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21 Nov 12
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{
"type": "Short Research Article",
"title": "Impact of iron levels on cognitive functioning among dental students of Udaipur, Rajasthan (India)",
"authors": [
"Mohit Sareen",
"Rateesh Sareen",
"Sarang Khajuria",
"Sayak Roy",
"Rateesh Sareen",
"Sarang Khajuria",
"Sayak Roy"
],
"abstract": "Health and intelligence are two closely related aspects of human well being. Nutrition, including iron levels, affects cognitive function and thereby may influence the occupational performance of an individual. Inadequate nutrition during adulthood may lead to decreased work efficiency, poor exercise tolerance and increased susceptibility to infections. The profession of dentistry requires keen recognizing abilities and decision making skills as well as ample physical stamina, which may be impaired in a state of malnourishment. Keeping this in view, this study was designed to assess the possible impact of iron levels on cognitive function among dental students. In this study 206 dental students (150 females and 56 males) participated and their cognitive functioning was determined by answering a questionnaire. The questionnaire evaluated the confidence level, work pattern and the tendency to be distracted by the physical environment of the study participants. Iron status was determined by estimating the hemoglobin level of the individuals. Each of the three cognitive traits was correlated with hemoglobin levels. The results revealed that that majority of dental students had good levels of confidence, work patterns with a low level of restriction and low levels of distraction by the physical environment. No significant correlations were found between any of the cognitive parameters and hemoglobin levels (p>0.05) in female participants. In male participants significant correlations were found in two out of three cognitive functioning tests, confidence levels and work pattern (p<0.05). The results of this study suggest that these three cognitive functions may not be influenced by hemoglobin levels in females and may be slightly or, due to the small male sample size which may have confounded the results, not influenced by hemoglobin in males.",
"keywords": [
"nutrition",
"brain function"
],
"content": "Introduction\n\nNutrition is a fundamental pillar of human life1. The influence of nutrition on cognitive functioning may in turn influence the occupational performance of an individual1,2. Several studies in this regard have been done among growing children3–11. For instance in a study conducted by Gabr M. Sayed et al to assess the magnitude of malnutrition among pre-school and primary school children and its impact on health, intellectual development and scholastic achievement the results yielded a positive correlation between nutritional status and intellectual development as well as scholastic achievement4.\n\nOne partially accepted theory is that malnutrition is associated with low iron levels and that anaemia, via cerebral hypoxia and other possible mechanisms, has a major negative influence on cognitive function1.\n\nBut nutritional deficiencies and their adverse outcomes are not age limited. Inadequate nutrition during adulthood may lead to decreased work efficiency, poor exercise tolerance and increased susceptibility to infection2.\n\nStudies related to the impact of hemoglobin levels on mental functioning in adult populations are few and the outcomes are inconclusive12,13. Keeping this in view, the present study was designed to assess the possible impact of iron nutritional status on cognitive functioning among dental students between the ages of 18–25 years as the profession of dentistry requires keen recognizing abilities, decision making skills as well as ample physical stamina, which may be impaired by low iron/hemoglobin levels thereby influencing one’s ability to be a successful dentist.\n\n\nMaterials and methodology\n\nA total of 206 students, 150 (73%) females and 56 (27%) males studying dentistry at Udaipur, Rajasthan, India, volunteered for the study. The brain functioning of the individuals was determined by answering a questionnaire.\n\nThe questionnaire was adapted from the Inventory of Barriers to Creative Thought and Innovative Action14. This test identified and measured the degree of inhibitors affecting a person’s ability to create and innovate. The questionnaire consisted of thirty-six items, set up in a sixpoint Likert-scale format. This test identifies and measures barriers in six different categories. Out of these six traits the scores identifying barriers related to ‘need for conformity’, ‘ability to abstract’ and ‘ability to use systematic analysis’ were not considered. Only scores of three traits were included which consisted of barriers related to self confidence, task achievement and ease of distraction by the physical environment. Each trait was graded according to the scores obtained and a grading system was introduced to classify each trait into different categories (Table 1, Table 2 and Table 3).\n\nThe second stage in the study involved estimation of haemoglobin levels of the individuals. The haemoglobin level was determined by Sahli’s method. In this method the fingertip was first sterilized using rectified spirit. A quick prick was made to get a moderately large drop of blood. The blood was aspirated in the haemoglobin pipette up to the 20 cubic millimeter mark. The blood was immediately transferred into the hydrochloric acid taken in the diluting tube. The acid and blood was mixed and kept undisturbed for 10 minutes, to ensure that the haemoglobin was converted to acid haematin. After 10 minutes, the contents were diluted by adding distilled water drop by drop and mixing the contents after each drop with the stirrer, till the colour matches with the colour of the standard. Then the reading was taken both in grams and percentages by noting the lower meniscus.\n\n\nResults\n\nThe mean haemoglobin level of female participants was 10.4gm/dL and that of male participants was found to be 11.6gm/dL. Considering the normal range of haemoglobin is 12–14g/dL for females and 14–16g/dL for males, only 8% females (n=13) had haemoglobin within the normal range and none of the male participants (n=0) had haemoglobin level over 14g/dL.\n\nThe outcome of the cognitive functioning tests revealed that no study participant scored grade 1 in any of the categories, and the greatest proportion of participants scored grade 4 in all three categories indicating good level of confidence, less restricted work patterns and lower amounts of physical distraction respectively. 47% females and 57% males belonged to grade 4 in confidence level, 61% females and 53% males belonged to grade 4 in work pattern and 50% females and 57% males belonged to grade 4 in physical distraction [Table 4, Table 5 and Table 6].\n\nA significant positive correlation between was revealed between haemoglobin levels and both confidence levels and work pattern restrictiveness in males (r=0.20 and 0.25 respectively). However, no significant correlation was found between heamoglobin levels and distraction in males and heamoglobin levels did not significantly correlate with any of these three parameters in females (r=-0.11, 0.10 and 0.10 for confidence levels, work pattern restrictiveness and ease of distraction by the physical environment respectively) [Table 7].\n\nNS-Not significant, S-Significant.\n\n\nDiscussion\n\nThe present study revealed that the correlations of all the cognitive function tests with haemoglobin levels in male participants were found to be positive. This could be explained by the fact that the number of male participants compared with the number of female participants was far less. Out of the 206 study participants 150 (73%) were females and 56 (27%) were males. This could be a reason for getting such correlations in male participants as the smaller number of male participants might have affected the statistical analysis.\n\nFurther the results of the study reflected a few limitations of the present study.\n\nFirst as the evaluation of the cognitive function was determined by using questionnaire, the chances of getting exact results were diminished. The chances of giving fake answers by the study participants, particularly by the male participants who had a chauvinistic approach in answering questions evaluating confidence level and work efficiency could have attributed to such results.\n\nSecondly the assessment of nutritional status was done by evaluating only the haemoglobin level of study participants, which had its own limitations. For the assessment of haemoglobin level Sahli’s method was used. This method is not highly sensitive15,16 and resulted in errors in determining the exact values of haemoglobin levels of individuals in the present study.\n\n\nConclusion\n\nThe results of the study revealed that iron levels appears to have less of an impact on cognitive functioning in adult female participants than it does in adult male participants, though the smaller male sample size may have contributed to a false positive result in males. These results may be due, in part, to limitations with the study. As such we recommend further studies in this regard.",
"appendix": "Author contributions\n\nMohit Sareen wrote the article and carried out research. Rateesh Sareen contributed to the design of study. Sarang Khajuria contributed in conception of study and revised the article for intellectual content. Sayak Roy contributed to the data analysis.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.\n\n\nReferences\n\nDuska Petranovi, Vladimir Taki, Renata Dobrila-Dintinjana, et al:Correlation of Anaemia and Cognitive Functions Measured By The Complex Reactiometer, Drenovac. Coll Antropol 2008; 32: 47–51.\n\nBandhu R, Shankar N, Tandon OP, et al:Effects of Iron Therapy on Cognition in Anaemic School Going Boys. Indian J Physiol Pharmacol 2003; 47(3): 301–310.\n\nIjarotimi OS, Ijadunola KT: Nutritional Status and Intelligence Quotient of Primary School Children in Akure Community of Ondo State, Nigeria. Tanzan Health Res Bull 2007; Vol. 9; No. 2: 69–76.\n\nGabr M Sayed, Mohammed F Kaseem, Yehia M Hassan, et al:Nutritional Status & Its Impact on Health, Intellectual Development and Scholastic Achievement in Pre-School and Primary School Children in Cairo. Bull Nutr Inst Cairo 1990; Egypt; Vol-10, No-1: 43–59.\n\nBuzina-Suboticanec Kornelija, Buzina Ratko, Stavljenic Ana, et al:Effects of Iron Supplementation on Iron Nutrition Status and Cognitive Functions in Children. Food Nutr Bull 1998; 4: 298–306.\n\nIvanovic D, Leiva BP, Pérez HT, et al:Nutritional Status, Brain Development and Scholastic Achievement of Chilean High School Graduates From High and Low Intellectual Quotient and Socio-Economic Status. Br J Nutr 2002; 87(1): 81–92.\n\nSungthong R, Mo-Suwan L, Chongsuvivatwong V, et al:Effects of Haemoglobin and Serum Ferritin on Cognitive Function in School Children. Asia Pac J Clin Nutr 2002; 11(2): 117–122.\n\nSen A, Kanani SJ: Deleterious Functional Impact of Anaemia on Young Adolescent School Girls. J Indian Pediatr 2006; Vol-43: 219–226.\n\nSachdev H, Gera T, Nestel P, et al:Effects of Iron Supplementation on Mental Motor Development in Children. Systematic Review of Randomized Controlled Trails. Public Health Nutr. 2005; 8(2): 117–132.\n\nRajbir Sachdeva, Simran Chug, Jaswinder Sangha, et al:Haemopoitic, Serum Minerals and Intellectual Status of Institutionalized and Non Institutionalized Boys. J Hum Ecol 2006; 19(4): 235–238.\n\nNemati A, Barak M, Dehgan MH, et al:Relation Between Iron Deficiency and Anemia with School Success, Weight and Height in Schoolgirls Aged 12 Year Old in Ardebil Province of Iran. Research Journal of Biological Sciences 2007; 2(3): 263–267.\n\nKretsch MJ, Fong AKH, Green MW, et al:Cognitive Function, Iron Status, and Haemoglobin Concentration in Obese Women. Food Nutr Bull July 1998; Vol. 52; No. 7: 512–518.\n\nPaul B Jacobsen, Linda L Garland, Margaret Booth-Jones, et al:Relationship of Haemoglobin Levels to Fatigue and Cognitive Functioning Among Cancer Patients Receiving Chemotherapy. J Pain Symptom Manage 2004; Vol. 28; No. 1: 7–18.\n\nJossey-Bass/Pfeiffer: Inventory of Barriers to Creative Thought and Innovative Action The Pfeiffer Library (1998), Volume 8, 2nd edition, 46–56.\n\nBalasubramaniam P, Malathi A: Comparative study of hemoglobin estimated by Drabkin's and Sahli's methods. J Postgrad Med 1992; 38: 8–9.\n\nManual of Basic Techniques for a Health Laboratory, [WHO] 2nd edition, 2003, Page–271."
}
|
[
{
"id": "1842",
"date": "17 Sep 2013",
"name": "Gerald Keusch",
"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 title is misleading because iron levels are not measured in this study. The summary does reflect the content of the article. However, there are serious concerns about the validity of even the limited findings related to cognition and iron status in Indian dental students. First of all, dental students as a population reflect a group of academic attainers, who have distinguished themselves sufficiently to be admitted to dental school. This is a skewed and privileged cohort to begin with, who are likely to have come from economically stable families and are less likely to have suffered from significant malnutrition earlier in life. Second, early nutritional insults affecting cognitive development can be overcome with nutritional and educational interventions, and this cohort is likely to have benefitted from these inputs. Third, the hemogloblin levels as reported (means without standard deviations) are mostly in the low normal range, with the lowest reported hemoglobin (8 Gm/DL) in 5 females who would be labelled moderately anemic. The lowest hemoglobin a male was 9.7 Gm/DL, and would not likely be symptomatic except with considerable physical stress. Fifth, the authors themselves do not have confidence in the accuracy of the methodology to assess hemogloblin. Sixth, iron levels are not in fact measured, just hemoglobin. While iron deficiency in menstruating females can explain the lower hemogloblin levels compared to males, there are many other causes of mild-moderate anemia which have not been assessed. Finally, the testing of cognitive function was by questionnaire rather than more rigorous assessment tools. It is for these reasons that I judge the work to be irrelevant to any evaluation of the relationship between iron levels and cognitive function in Indian dental students.",
"responses": []
},
{
"id": "2242",
"date": "02 Dec 2013",
"name": "Victoria Arija",
"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 is appropriate for the content of the article and the abstract gives a suitable summary of the investigation. The design, methods and analysis of the results from the study have been correctly explained and correspond with the topic being studied. The conclusions are in accordance with the results obtained from the study. The authors have provided enough information to be able to replicate the experiment. The format/structure of the article are adequate.",
"responses": []
},
{
"id": "4633",
"date": "02 May 2014",
"name": "Huguette Turgeon",
"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 my opinion, the title is misleading because iron levels are not measured in this study, only hemoglobin levels. The summary does reflect the content of the article. However, the introduction needs to be revised. Firstly, it is mentioned that malnutrition is associated with low iron levels and that anaemia, via cerebral hypoxia and other possible mechanisms, has a major negative influence on cognitive function. Malnutrition is not always associated with low iron levels as it depends on what nutrients are missing in the diet. Also, anemia (low hemoglobin levels) is not always caused by a low iron status. Considering that only hemoglobin levels are measured, I suggest approaching the introduction from this angle. Materials and methodology have been correctly explained and correspond with the topic being studied. However, the precision of the Sahli’s method of measuring hemoglobin compared to the hemiglobincyanide or cyanmethemoglobin method should be mentioned, as well as the statistical software and correlation coefficients used. Concerning the discussion and conclusion, I don’t think that the smaller number of male participants explains why positive results were observed in male participants only. Factors related to women’s physiology such as the moment of the menstrual cycle is an important factor affecting cognitive functioning and this should be brought up here. In my opinion, we can’t explain the absence of significant relationships in female participants saying that male participants reached statistical significances because there were a smaller number of them. The discussion and conclusion need to be revised.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-54
|
https://f1000research.com/articles/1-53/v1
|
21 Nov 12
|
{
"type": "Correspondence",
"title": "Continuation of dual anti-platelet therapy after drug-eluting stents in primary angioplasty beyond 12 months",
"authors": [
"Iman Mohasseb",
"Christian A Gericke",
"Iman Mohasseb"
],
"abstract": "In this correspondence we discuss the results of the meta-analysis by De Luca et al. (2012) in the Archives of Internal Medicine which found that late myocardial reinfarction and stent thrombosis is more common in drug-eluting stents than in bare-metal stents. We discuss the clinical implications of this paper for dual anti-platelet therapy which did not receive sufficient attention in the original publication and the accompanying editorial.",
"keywords": [
"The meta-analysis by De Luca et al.1 showed that the incidence of late (> 2 years) myocardial reinfarction and stent thrombosis is significantly higher in drug-eluting stents (DES) compared to bare-metal stents (BMS) in primary angioplasty despite the significant reduction in long-term target vessel revascularization associated with DES."
],
"content": "Correspondence\n\nThe meta-analysis by De Luca et al.1 showed that the incidence of late (> 2 years) myocardial reinfarction and stent thrombosis is significantly higher in drug-eluting stents (DES) compared to bare-metal stents (BMS) in primary angioplasty despite the significant reduction in long-term target vessel revascularization associated with DES.\n\nWhile the Comment section of the paper briefly mentions the role of more potent and prolonged dual anti-platelet therapy in countering these worrisome findings, the related Commentary2 does not. However, in our view the current practice of discontinuing dual anti-platelet therapy after 12 months in DES in most patients is the most likely explanation for the observed increase in late stent thrombosis and reinfarction incidence, in concordance with pathological evidence that even beyond 40 months, DES do not fully epithelialize3. In the De Luca et al.1 meta-analysis, the DES survival curves for both reinfarction and stent thrombosis start diverging from the BMS curves one year after stent implantation until year 6.\n\nThis also raises the most relevant question for practitioners: should we prolong dual anti-platelet therapy beyond 12 months after DES implantation? The Dual Antiplatelet Therapy Study (DAPT) is expected to give us a definitive answer to this question in 20144. For the time being, it seems that the argument to continue dual anti-platelet therapy beyond 12 months, which is fully in line with the current ACCF/AHA/SCAI recommendation5 to continue dual anti-platelet therapy for at least 12 months after DES implantation, has gained in strength.",
"appendix": "Author contributions\n\n\n\nIM drafted the first version of this correspondence article and IM and CAG have substantially revised it after discussion.\n\n\nCompeting interests\n\n\n\nCAG is a member of the Asia-Pacific Advisory Board for Bayer Pharmaceuticals.\n\n\nGrant information\n\n\n\n\nReferences\n\nDe Luca G, Dirksen MT, Spaulding C, et al.: Drug-Eluting vs Bare-Metal Stents in Primary Angioplasty: A Pooled Patient-Level Meta-analysis of Randomized Trials. Arch Intern Med. 2012; 172(8): 611–21. PubMed Abstract | Publisher Full Text\n\nBrophy JM: Drug-Eluting Stents in ST-Segment Elevation Myocardial Infarction: Comment on “Drug-Eluting vs Bare-Metal Stents in Primary Angioplasty”. Arch Intern Med. 2012; 172(8): 621–2. Publisher Full Text\n\nJoner M, Finn AV, Farb A, et al.: Pathology of drug-eluting stents in humans: delayed healing and late thrombotic risk. J Am Coll Cardiol. 2006; 48(1): 193–202. PubMed Abstract | Publisher Full Text\n\nU.S. National Institutes of Health [cited 8 May 2012]. Reference Source\n\nLevine GN, Bates ER, Blankenship JC, et al.: 2011 ACCF/AHA/SCAI Guideline for Percutaneous Coronary Intervention. A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines and the Society for Cardiovascular Angiography and Interventions. J Am Coll Cardiol. 2011; 58(24): e44–122. Publisher Full Text"
}
|
[
{
"id": "372",
"date": "29 Nov 2012",
"name": "Sripal Bangalore",
"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": "376",
"date": "05 Dec 2012",
"name": "Bruce Biccard",
"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/1-53
|
https://f1000research.com/articles/1-52/v1
|
20 Nov 12
|
{
"type": "Short Research Article",
"title": "Long double stranded RNA is present in scrapie infected cells and tissues",
"authors": [
"Yervand E Karapetyan"
],
"abstract": "Despite decades of research efforts, the nature of the infectious agent causing scrapie and other Transmissible Spongiform Encephalopathies (TSE) remains an enigma. The protein-only prion hypothesis posits that an abnormal conformer of a host protein is the infectious agent. Virus and virino theories include host-independent nucleic acids in the genome of the infectious agent, in addition to the protein component (a host protein in the case of virino, and a viral protein in the case of a virus).Viral or sub-viral nucleic acids have long been sought in scrapie to explain the existence of multiple agent strains. Despite a plethora of different approaches to the search, no scrapie-specific nucleic acid sequences have been found in infected cells or tissues.Most viruses induce synthesis of long double stranded RNA (dsRNA) during their replication in cells, and thus the presence of long dsRNA would be an indication of viral infection in cells. J2 monoclonal antibody against long dsRNA is a useful tool for easy screening of cells and tissues for the presence of suspected viral infection; however, this antibody has not previously been used for testing of scrapie infected tissues.Here, we present evidence for long dsRNA in scrapie infected cells and tissues. Such dsRNA is also found in scrapie free tissue culture cells. We believe this may be the first evidence of viral infection in scrapie susceptible and infected cells.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: not approved 2",
"approved with reservations 1",
"approved 1]”."
],
"content": "Introduction\n\nTransmissible Spongiform Encephalopathies are rare neurodegenerative brain disorders in both humans (e.g. Creutzfeldt-Jakob disease and Kuru) and animals (Scrapie in sheep, Bovine Spongiform Encephalopathy in cows and Chronic Wasting Disease in deer and elk), characterized by a long incubation period after initial infection. Once symptoms become apparent in humans, the disease progresses inevitably to death within weeks or months, and, to date, no treatment or early preclinical diagnostics are available.\n\nThe nature of the infectious agent causing these disorders remains unexplained. The most advertised, but not proven to date, prion protein-only theory simply states that the agent is nothing more than a misfolded host glycoprotein called prion protein1. How this single host protein “encodes” the numerous agent strains that have distinct clinical and pathological features remains to be demonstrated by prion scientists. The infectivity of recombinant prion protein misfolded in a test tube in a mixture with RNA and lipid and later injected into the animal brains was demonstrated a few years ago but was never reproduced independently in a laboratory free of contamination2. The statement that cell free replication of TSE infectivity in a test tube excludes the possibility of the agent being a virus3 ignores the well known fact of human poliovirus replication in a cell free system4.\n\nOn the other hand, virino and virus theories claim that host-independent nucleic acid is the genome of the infectious agent5. Virus theory states that the agent is a virus that has not been discovered yet6, while virino theory postulates that the agent is a chimera composed of a host-independent nucleic acid (the genome of the agent) and a host protein, probably the prion protein that protects the genome7. Obviously, nucleic acid-containing theories explain the existence of many agent strains since nucleic acid sequences are the only molecules known to-date that encode phenotypes of all living organisms including microbes, with the smallest among them being the nucleic acids of viroids and satellite RNAs of plant viruses (only few hundred non protein coding nucleotides)8. Despite decades of research efforts, no TSE-specific nucleic acid sequences have been found yet9, leading to the popular conclusion among many scientists that no such nucleic acid exists.\n\nWhile many different approaches were undertaken to hunt for the elusive viral or subviral nucleic acid, surprisingly, the simplest and easiest of them was not employed. J2 monoclonal antibody recognizes double-stranded RNA (dsRNA) provided that the length of the helix is ≥ 40 bp10. Importantly, dsRNA-recognition is independent of the sequence and nucleotide composition of the antigen. All naturally occurring dsRNA investigated up to now (40–50 species) as well as poly(I)•poly(C) and poly(A)•poly(U) have been recognized by J211. In a systematic study of different viruses, J2 detected dsRNA in cells infected with positive-strand RNA viruses, double-stranded RNA viruses and DNA viruses, but not negative-strand RNA viruses12. This shows that most viruses induce synthesis of long double stranded RNA (dsRNA) during their replication in cells that can be detected by J2. Therefore, the presence of long dsRNA would be an indication of viral infection in cells. J2 antibody has not been used for testing of scrapie infected tissues and the first attempt is made in the present work.\n\n\nMethods/results\n\nJFH1 Huh7 cells (human hepatoma cells harboring hepatitis C replicon) and Huh7.5 cells (human hepatoma cells free of replicon) were used as a positive (JFH1 Huh7) and negative (Huh7.5) control for dsRNA in immunofluorescence detection experiments (Figure 1a) when probing PK1 cells (a clone of mouse neuroblastoma N2A cells) and RML (Rocky Mountain Laboratory strain of mouse scrapie) infected PK1 (RML/PK1) cells with J2 antibody (mouse monoclonal from Englsih and Scientific Consulting) (Figure 1b). A secondary anti-mouse antibody labeled with Alexa 488 fluorophore is used to visualize J2 binding sites. dsRNA was detected in the cytoplasm of both PK1 and RML/PK1 cells (Figure 1b). The signal was abolished after RNase A (Invitrogen) treatment at 50 µg/ml in 50mM NaCl as it is shown for PK1 cells in Figure 1c.\n\nA secondary anti-mouse antibody labeled with Alexa 488 fluorophore is used to visualize J2 binding sites. (a). In contrast to the absence of green signal in Huh7.5 cells, JFH1 Huh7 cells show punctuate staining in some of them which harbor hepatitis C replicon. (b). dsRNA is detected in both scrapie free PK1 cells and RML scrapie infected RML/PK1 cells. (c). dsRNA disappears from PK1 cells after treatment with RNase A at low salt conditions where it destroys both single stranded and double stranded RNA.\n\nJ2 antibody was also used for immunoblotting of dsRNA, as described previously13. Crude RNA extracts from JFH1 Huh7, Huh7.5, PK1 and PK1/RML cells were size separated using non-denaturing TBE-polyacrylamide gel, transferred to positively charged Nylon membrane and immunoblotted with J2. Secondary anti-mouse antibody linked to HRP (horseradish peroxidase) and a substrate for it was used for visualization of the blots. Results showed the presence of replication intermediate-RI (upper part of the gel slot, black arrow) and replicative form-RF (seen as a strong band bellow RI, dark blue arrow) in JFH1 Huh7 that were both absent in Huh7.5 (Figure 2). In PK1 and RML/PK1 in addition to dsRNA in the upper part of the gel slots several bands were seen including a duplet with a molecular weight much lower than that of RF of HCV replicon (Figure 2).\n\nTwo bands of dsRNA were detected in JFH1 Huh7 (RI and RF shown by black and dark blue arrows). In PK1 and PK1/RML cells several bands of dsRNA are detected including top ones in the gel slots corresponding to long dsRNA that did not enter the gel. A much lower duplet band could be seen in both specimens. In addition there are several bands in between the duplet and the upper most band.\n\nJ2 antibody was recently used for successful detection of viral dsRNA in formalin-fixed paraffin-embedded tissues14. Here an attempt was made to detect dsRNA in 22L scrapie infected mouse brains fixed in Carnoy’s solution and embedded in paraffin. Proteinase K treatment was used as described15, followed by inhibition in glycine (2mg/ml in nanopure water) and short post-fixation in formalin to expose dsRNA for detection. IHC detection of dsRNA in brains using J2 was done with a secondary system described in ref.6. Uninfected C57Bl/6 mice brains were used as control and brains of terminally sick C57Bl/6 mice infected with 22L strain of mouse scrapie were used for the experiment. As a result, dsRNA was detected in scrapie-infected brain predominantly in the cytoplasm of large neurons in the cortex (Figure 3a) and brainstem (Figure 3b). Nuclear staining was also detected in some neurons of the infected brain. In uninfected brain, nuclear staining of some Purkinje cells was detected in the cerebellum (Figure 3c). Otherwise the staining in the control brain was largely absent.\n\nUninfected C57Bl/6 mice brains were used as control and brains of terminally sick mice infected with 22L strain of mouse scrapie were used for the experiment. (a). Mostly cytoplasmic and some nuclear staining can be seen in cortical neurons of infected brain. While staining is absent in uninfected control brain. (b). Similar staining pattern is observed in brainstem especially in large neurons. And again staining is absent in parallel uninfected control sections. (c). Some nuclei of Purkinje cells are stained in the cerebellum of both uninfected control and 22L infected brain.\n\n\nDiscussion\n\nData presented here shows dsRNA is detectable by J2 antibody using immunofluorescence in scrapie susceptible and scrapie infected tissue culture cells. In contrast, it seems that only scrapie infected brain has dsRNA in the cytoplasm of some neurons. Immunoblotting shows long as well as short dsRNA bands in scrapie susceptible and scrapie infected tissue culture cells (Figure 2). Long dsRNA is not present in uninfected mammalian cells and can only be present as a result of viral infection. Therefore long dsRNA presence in scrapie susceptible as well as scrapie infected cells is a strong indication of viral infection of these cells. Shorter dsRNA bands also detected in these cells might point to the presence of subviral nucleic acids16. These data provide an experimental basis for speculation that scrapie agent could be a satellite nucleic acid of a silent and persistent virus that infects susceptible host cells. A virusoid (satellite RNA) in order to replicate would need a helper virus to be in every species and cell that is susceptible to infection. Gajdusek proposed such scenario four decades ago: “These viruses could be associated or satellite viruses which serve to activate or are themselves activated by some helper virus latent in the susceptible host”17.\n\nIn analogy to plant satellite RNAs18, scrapie agents’ nucleic acid can function via RNAi to silence host neuronal survival genes (e.g. bcl-2 anti-apoptotic group genes) and cause lethal disease due to its homology with host gene sequences.\n\n\nConclusion\n\nFor the first time experimental evidence is provided for the presence of long dsRNAs in scrapie infected cells and tissues. This is the strongest argument presented so far for the existence of a virus in scrapie infected cells and tissues. These molecules deserve sequencing and characterization of their relationship to scrapie agent and disease.",
"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\nAknowledgments\n\nI thank Dr. Corinne Lasmezas for supervising and supporting my work at Scripps Florida. I am grateful to Dr. Timothy Tellinghuisen, from whom at his lab meeting presentation at Scripps Florida I learnt about the existence of J2 antibody and who sincerely provided human hepatoma cells with and without HCV replicon. I greatly appreciate Franco Sferrazza’s expert help in setting up the immunoblotting experiment.\n\n\nReferences\n\nPrusiner SB: Novel proteinaceous infectious particles cause scrapie. Science. 1982; 216(4542): 136–44. PubMed Abstract | Publisher Full Text\n\nKarapetyan Y: Is Recombinant Prion Protein by Itself Infectious? Reference Source\n\nKlingeborn M, Race B, Meade-White KD, et al.: Lower specific infectivity of protease-resistant prion protein generated in cell-free reactions. Proc Natl Acad Sci USA. 2011; 108(48): E1244–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarapetyan YE: Viruses do replicate in cell-free systems. Proc Natl Acad Sci USA. 2012; 109(8): E461. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDickinson AG, Outram GW: Genetic aspects of unconventional virus infections: the basis of the virino hypothesis. Ciba Found Symp. 1988; 135: 63–83. PubMed Abstract\n\nManuelidis L: A 25 nm virion is the likely cause of transmissible spongiform encephalopathies. J Cell Biochem. 2007; 100(4): 897–915. PubMed Abstract | Publisher Full Text\n\nKimberlin RH: Scrapie agent: prions or virinos? Nature. 1982; 297(5862): 107–8. PubMed Abstract | Publisher Full Text\n\nElena SF, Dopazo J, de la Peña M, et al.: Phylogenetic analysis of viroid and viroid-like satellite RNAs from plants: a reassessment. J Mol Evol. 2001; 53(2): 155–9. PubMed Abstract | Publisher Full Text\n\nSimoneau S, Ruchoux MM, Vignier N, et al.: Small critical RNAs in the scrapie agent. Nature Precedings. 2009. Reference Source\n\nSchönborn J, Oberstrass J, Breyel E, et al.: Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res. 1991; 19(11): 2993–3000. PubMed Abstract | Publisher Full Text | Free Full Text\n\nhttp://www.engscicons.de/monoclonal2005_ger/J2_desc2005.htm.\n\nTargett-Adams P, Boulant S, McLauchlan J: Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication. J Virol. 2008; 82(5): 2182–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVeliceasa D, Enünlü N, Kós PB, et al.: Searching for a new putative cryptic virus in Pinus sylvestris L. Virus Genes. 2006; 32(2): 177–86. PubMed Abstract | Publisher Full Text\n\nRichardson SJ, Willcox A, Hilton DA, et al.: Use of antisera directed against dsRNA to detect viral infections in formalin-fixed paraffin-embedded tissue. J Clin Virol. 2010; 49(3): 180–5. PubMed Abstract | Publisher Full Text\n\nKarapetyan YE, Saá P, Mahal SP, et al.: Prion strain discrimination based on rapid in vivo amplification and analysis by the cell panel assay. PLoS One. 2009; 4(5): e5730. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOwens RA, Diener TO: RNA intermediates in potato spindle tuber viroid replication. Proc Natl Acad Sci USA. 1982; 79(1): 113–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGajdusek DC: Spongiform virus encephalopathies. J Clin Pathol Suppl (R Coll Pathol). 1972; 6: 78–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith NA, Eamens AL, Wang MB: Viral small interfering RNAs target host genes to mediate disease symptoms in plants. PLoS Pathog. 2011; 7(5): e1002022. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "385",
"date": "22 Nov 2012",
"name": "Hubert Laude",
"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 paper is scientifically poor. The author’s claim is based only on the data presented in figure 3, which is far from being convincing. What’s more, the data presented in Figures 1 and 2 (the documentation of the specificity of the J2 antibody) clearly show the presence of long ds RNA in both prion-infected and uninfected cell cultures.",
"responses": [
{
"c_id": "81",
"date": "22 Nov 2012",
"name": "Yervand Karapetyan",
"role": "Author Response",
"response": "I thank Dr Laude for his comments.The claim “Long double stranded RNA is present in scrapie infected CELLS and TISSUES” is based on data shown in all 3 figures. As far as cell cultures are concerned, indeed long dsRNA is present in both scrapie free and scrapie infected cells and this in no way undermines the claim “Long double stranded RNA is present in scrapie infected CELLS…”. The brain tissue experiment is a single experiment and the purpose here was to do a pilot test and that is what the results show."
}
]
},
{
"id": "386",
"date": "27 Nov 2012",
"name": "Robert Somerville",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the use of the J2 monoclonal antibody to detect differences in the amount of double stranded RNA between uninfected and TSE (prion) infected brain and cell cultures.The antibody was used to detect dsRNA by immunofluorescence in control JFH1 cells and cells infected with hepatitis C, and in PK1 cells that were or were not infected with a TSE strain. In this experiment the detection of antigen in the JFH1 cells was poorly demonstrated. However the PK1 cells showed high levels of immune-reactivity whether or not they were infected. Staining was reduced by RNAase A treatment, although in this case the untreated control showed poorer staining.Crude undenatured RNA extracts from these cells were also run on gels and immunoblotted with the J2 antibody. The antibody detected bands in the hepatitis C infected cells which were absent from the uninfected cells. The banding pattern differed between the Huh 7.5 cells and the PK1 cells. Although there are minor differences between uninfected and infected PK1 cell extracts no major difference was detected or commented on by the author.In the third experiment control and TSE infected brain sections were subjected to immunohistochemistry using the J2 antibody. In this experiment some differences are observed between the control and TSE infected samples. However there are some weaknesses in presentation which compromise the assessment of these images. Firstly figure 3A is difficult to interpret because the colour of the sections from control brain are markedly different from those from the infected animal, which could be misleading. The author could consider trying to do some form of quantification of the numbers of positive cells within a defined field using a software package such as image J. Figure 3B is a better panel but again the colour balance between the negative and positive panels differs. Figure 3C is less convincing because the images are again poorly presented. It would have been helpful to present them with a similar and lighter colour balance, and with the same area in the same orientation. The use of the term “some cells” is imprecise and could be improved by conducting some quantification, e.g. by counting positive cells in a defined area (blind). Scale bars should be included throughout.In summary, the results presented in the paper show J2 binding to antigen in cells and brain sections. There are differences between uninfected and hepatitis C infected cells and some indication of differences between control and TSE infected brain. However the results are very preliminary and much work will be required to substantiate these observations and establish their relevance.",
"responses": []
},
{
"id": "387",
"date": "28 Nov 2012",
"name": "Igor Zaitsev",
"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 interesting data on a fact that has been ignored by many researchers working on the transmissible spongiform encephalopathies (TSEs) and that is the presence of virus-like particles in the experimental animal tissue samples.For the first time, the J2 monoclonal antibodies against long dsRNA had been used in the scrapie infected tissue. The accumulations of the antibodies in cortical neurons of the infected brain seem to be present in contrast to the controls. This fact itself deserves attention and suggests further testing of the J2 monoclonal antibodies in TSEs. In the discussion section, the author suggests the satellite nature of the infectious agent without supporting evidence that comes from his experimental data. Nevertheless, it is still a viable reminder that such a possibility should not be ruled out at the present development in the field of TSEs research. The experimental approach that had been used is unique and deserves further experimental trails by other investigators since more has to be done to identify the nature of the infectious agent of TSEs. This work could be a valuable contribution to the discovery of its identity.",
"responses": []
},
{
"id": "772",
"date": "15 Feb 2013",
"name": "Pascal Leblanc",
"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 present study of Karapetyan indicates that dsRNAs are detected in tissues and cells infected with the scrapie agent. For this purpose Karapetyan used the J2 monoclonal antibody that recognizes dsRNAs and tested this antibody in different experimental conditions i.e. Huh7.5 hepatocyte cells in the presence (positive control) or absence (negative control) of HCV infection. Similarly, he tested the presence of dsRNA on the Neuroblastoma N2a PK1 cellular model infected or not by the RML scrapie agent strain, and finally he investigated if dsRNAs can be detected in the brain of scrapie infected animals.As expected, the data indicated a positive signal in HCV infected cells, whereas no signal was observed in non-infected cells. The panel for Huh7.5 non-infected cells is not depicted. Additional immunofluorescence data or an immunoblotting data confirming the HCV replication in the infected cells should be added.Similarly, the N2a PK1 cells like RML infected PK1 cells were found to be positive for dsRNAs, indicating that no difference is detected between scrapie infected and non-infected cells. It should be noted that my group previously found that N2a cells replicate a murine leukemia endogenous retrovirus (Neuroblastoma endogenous retrovirus) that does not modulate scrapie replication and release (Alais et al. 2008). We also found that these cells express high levels of Intra A cisternal Particles (IAP) endogenous retroviruses and that IAP-Gag proteins co-localize and co-fractionate with PrP (Alais et al. 2008). Similar data was also obtained by the group of Laura Manuelidis (Manuelidis et al.1995) . All these endogenous retroviruses produce structured dsRNAs that could be detected by this antibody. So in this context, the detection of dsRNA in these cells are expected. It should be considered that the persistence of a signal in scrapie infected cells after RNAse treatment can be due to the interaction of of PrPSc with these viral RNAs.The last piece of data indicates that a dsRNA signal is detected in brain of infected mouse whereas no signal was observed in non-infected control mouse. This result is interesting and should be extensively investigated. If we think in term of virology, many hypotheses can be proposed for this. My group and others have found that PrPC displays some antiviral properties. We could imagine that the decrease of PrPC could favor the expression and replication of endogenous viruses (especially endogenous retroviruses). On the other hand, the scrapie infection can also modulate the replication of endogenous viruses. Micro RNAs (miRNAs) can also be deregulated during scrapie infection and miRNAs (or pre-miRNAs) could be detected with this J2 antibody. So in this context and through the data presented here, we can not say that \"this is the strongest argument presented so far for the existence of a virus in scrapie infected cells and tissues\". Many other alternatives can be suggested.The results presented here are very preliminary and much work will be required to substantiate these observations and establish their relevance.",
"responses": [
{
"c_id": "647",
"date": "13 Dec 2013",
"name": "Yervand Karapetyan",
"role": "Author Response",
"response": "\"The panel for Huh7.5 non-infected cells is not depicted.\" This panel is shown, and is the one on the right upper corner of Figure 1A. \"Additional immunofluorescence data or an immunoblotting data confirming the HCV replication in the infected cells should be added.\" Immunoblotting data confirming the HCV replication in the infected cells is shown in Figure 2. [Two bands of dsRNA are detected in JFH1 Huh7 (RI and RF shown by black and dark blue arrows) which are absent in Huh7.5 cells]. \"All these endogenous retroviruses produce structured dsRNAs that could be detected by this antibody.\"All these retroviruses produce only partial dsRNAs, where dsRNA parts are not longer than 40bp, therefore they cannot be detected by the J2 antibody. If Dr Leblanc has published or unpublished data proving the contrary, I would sincerely ask him to recommend this work to us. Subsequently his next sentence - \"So in this context, the detection of dsRNA in these cells are expected. - is also false, because the detection of dsRNA in these cells was unexpected and is not due to the presence of retroviruses, but is most likely due to the replication of a virus of non-retroviral origin. \"It should be considered that the persistence of a signal in scrapie infected cells after RNAse treatment can be due to the interaction of of PrPSc with these viral RNAs.\" There is no such data shown in the paper. Instead it is shown in Figure 1C that dsRNA disappears from PK1 uninfected with scrapie cells after treatment with RNase A. \"Micro RNAs (miRNAs) can also be deregulated during scrapie infection and miRNAs (or pre-miRNAs) could be detected with this J2 antibody\". miRNAs can indeed be deregulated during scrapie infection, but they or pre-miRNAs cannot be detected by J2 antibody, since there are no dsRNA stretches longer than 40bp in either of them. miRNAs are no longer than 23-24 nucleotides, and they are not perfectly double stranded, but are only partially double stranded. The same applies to pre-miRNAs that are longer in length but do not have dsRNA stretches of longer than 40 bp in them. \"So in this context and through the data presented here, we cannot say that \"this is the strongest argument presented so far for the existence of a virus in scrapie infected cells and tissues\". Many other alternatives can be suggested.\" There cannot be many other alternatives suggested, since no retroviral sequences can be detected by J2, for the reasons outlined above. And indeed we can say that this is the strongest argument presented so far for the existence of a virus of non-retroviral origin in scrapie infected cells and tissues.We would welcome Dr. Leblanc’s response to our clarifications on his comments."
},
{
"c_id": "654",
"date": "19 Dec 2013",
"name": "PASCAL LEBLANC",
"role": "Reviewer Response",
"response": "Specificity of this antibody should be controlled. If Dr Karapetyan is right, immunofluorescence experiments carried out on cells expressing a MuLV retrovirus (for example 293T cells expressing or not a murine retrovirus like MoMuLV and NIH3T3 infected or not by MoMuLV ) should be negative...You have just to test the specificity of this antibody. No, I have no published or unpublished data proving the contrary.My major concern is just the specificity of this antibody, and the fact that N2a cells express endogenous retroviruses. If immunofluorescences are negative in suggested cells, the message of Karapetyan will be stronger."
},
{
"c_id": "3089",
"date": "12 Oct 2017",
"name": "Yervand Karapetyan",
"role": "Author Response",
"response": "In fact, I found the answer to the question posed by Dr. LeBlanc in the published literature. That is - whether endogenous retroviruses expressed in N2a cells can be responsible for the positive staining with J2 anti-dsRNA antibody observed in this paper. And the answers is no, endogenous infectious Murine Leukemia Virus (MuLV), a retrovirus amplified and produced in mouse Neuro-2a neuroblastoma cells is not producing dsRNA detectable by J2. Indeed all Neuro-2a cells are producing infectious MuLV (https://www.ncbi.nlm.nih.gov/pubmed/16550601) but when they are not infected with another virus of non retroviral origin, they are negative for J2 staining and are stained only when they are infected with a virus of non retroviral origin (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4206540/#SUP1). So, the cells used in our experiments, which are susceptible to scrapie infection, are infected with a virus of non retroviral origin in addition to endogenous MuLV and this is the first evidence for the replication of such a non retroviral virus in scrapie susceptible as well as scrapie infected cells."
}
]
}
] | 1
|
https://f1000research.com/articles/1-52
|
https://f1000research.com/articles/1-51/v1
|
20 Nov 12
|
{
"type": "Case Report",
"title": "Adult onset recurrent seizures as the first presentation of primary hypoparathyroidism",
"authors": [
"Pamela Correia",
"Rajeev Ranjan",
"Chandrashekhar Agrawal",
"Rajeev Ranjan",
"Chandrashekhar Agrawal"
],
"abstract": "Introduction: Hypoparathyroidism leading to hypocalcemia is an important treatable cause of recurrent seizures. Primary hypoparathyroidism presenting for the first time as seizures in adulthood is quite infrequent. Patients may present with hypocalcemic seizures even in the absence of subtle hypocalcemic signs.Case report: A 30 year old male, was presented to the emergency facility in an unconscious condition. He was intubated on the way to the hospital as he had suffered from two episodes of ventricular tachycardia. He had previous history of recurrent seizures for 6 years inspite of multiple anticonvulsants including phenytoin sodium, sodium valproate, and levetiracetam. The seizure frequency increased in the last year and he would have 5-6 episodes/ month. A MRI brain scan and EEG at the onset were both normal, as was the general examination but he had history of bilateral cataracts. There were no signs of tetany. Investigations revealed a normal hemoglobin and glucose level with normal electrolytes and both TLC and DLC levels were also normal. He had a serum calcium level of 3.3 mg% with a serum parathyroid hormone level of 1pg/ml, serum 25(OH) vitamin D levels of 6.6ng/ml and hypomagnesemia. NCCT head scan showed bilateral basal ganglia, and deep white matter calcification.Conclusions: 1) Ironically, increasing reliance on high end investigations such as a MRI brain scan could lead to certain conditions being missed; conditions that could be easily identifiable by the humble CT scan. 2) All treatable metabolic conditions should be excluded at first before commencing with anticonvulsants; this will restrict patients from burdensome polytherapy and related side effects.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: approved with reservations 2]”."
],
"content": "Introduction\n\nHypoparathyroidism leading to hypocalcemia is an important treatable cause of recurrent seizures. Even though it is not an uncommon condition, primary hypoparathyroidism presenting for the first time as seizures in adulthood is quite infrequent. Patients may present with hypocalcemic seizures even in the absence of subtle hypocalcemic signs inclusive of tetany, Chvostek’s sign or carpopedal spasms. As this is an entirely treatable condition, a high index of suspicion for primary hypoparathyroism with hypocalcemic seizures should be maintained even in otherwise asymptomatic adults.\n\n\nCase report\n\nA 30 year old male, was presented to the emergency facility in an unconscious condition. He had been intubated on the way to the hospital as he had suffered from two episodes of ventricular tachycardia in the cardiac ambulance. He was being transported from a local hospital where he had been admitted for profuse diarrhea with dehydration.\n\nHe recovered during the hospital stay and on further inquiry it was discovered that he had a past history of recurrent seizures for the last 6 years inspite of being on multiple antiepileptic medications including phenytoin sodium, sodium valproate and leviteracitam. The seizure frequency had increased considerably in the last year, and he would have at least 5–6 episodes in a month, thereby creating a considerable toll on his personal and professional life. He had been evaluated with an MRI brain scan and an EEG at the onset of symptoms 6 years earlier and both were reported to be normal.\n\nGeneral physical examination was relatively normal, though he had a past history of being operated for bilateral cataracts six months ago. Also fundoscopic examination showed bilateral acute papillodema. There was no carpopedal spasm or any other signs of tetany like Chvostek’s or Trousseau’s sign.\n\nInvestigations revealed normal hemoglobin and glucose level with normal sodium and potassium levels. TLC and DLC levels were also normal. He was found to have a serum calcium level of 3.3 mg% with a serum parathyroid hormone level of 1pg/ml, serum 25(OH) vitamin D levels of 6.6ng/ml and hypomagnesemia. NCCT head scan was done which showed bilateral basal ganglia calcification and deep white matter calcification. A 2D ECHO study was performed, and showed normal results (Figures 1 and 2).\n\nA diagnosis of primary hypoparathyroidism was made. He was treated with anticonvulsants, oral calcium, magnesium and vitamin D supplementation. During his hospital stay he did not have any other seizure events.\n\n\nDiscussion\n\nIntracranial calcifications can be classified mainly into 6 groups based on their etiopathogenesis: age-related and physiologic, congenital, infectious, endocrine and metabolic, vascular, and neoplastic1 (Table 1). The function of the parathyroid hormone is primarily maintaining the plasma calcium levels. Hormonal disturbance of the parathyroid glands including hypoparathyroidism, hyperparathyroidism and pseudohypoparathyroidism may lead to intracranial calcifications. Calcium accumulation is demonstrated primarily in the bilateral basal ganglia, dentate nuclei, and peripheral subcortical white matter sites2.\n\nThe principal function of the parathyroid hormone (PTH) is the maintenance of calcium plasmatic levels, withdrawing the calcium from bone tissue, reabsorbing it from the glomerular filtrate, and indirectly increasing its intestinal absorption by stimulating active vitamin D (calcitriol) production. There are two mechanisms that may alter its function, limiting its control on calcium: 1) insufficient PTH production by the parathyroids (hypoparathyroidism), or 2) a resistance against its action in target tissues (pseudohypoparathyroidism). In both cases, there are significantly reduced levels of plasmatic calcium associated with hyperphosphatemia3.\n\nIn acute and/or severe symptomatic hypocalcemia there is a predominance of neuromuscular, neuropsychiatric, and cardiovascular abnormalities. There is an increase in neuromuscular excitability, latent or evident, with sensory and motor disruption. Perioral or extremity paresthesia, cramps, myalgia, and muscular weakness are mild to moderate symptoms. Neuropsychiatric manifestations include irritability, anxiety, psychosis, hallucinations, dementia, depression, mental confusion, and extrapyramidal abnormalities. Increased intracranial pressure, papilledema, and convulsions can also be present, and must be differentiated from severe tetany muscular spasms4,5. Typical clinical signs of neuromuscular irritability associated with latent tetany include hyperreflexia and Chvostek’s and Trousseau’s signs, respectively. Severe hypocalcemia may result in bradycardia or ventricular arrhythmias, cardiovascular collapse, and hypotension that is non-responsive to fluids and vasopressors3.\n\nA decrease in myocardial contractility occurs, as well as a typical electrocardiographic abnormality, which is the rate-corrected QT interval (QTc) prolongation. Patients with chronic hypocalcemia may or may not have symptoms of discreet neuromuscular irritation, even with markedly low calcium levels. Asymptomatic cases may be detected by chance, by the dosage of calcium in routine exams, during periods of greater calcium demand (i.e. gestation, lactation, menstrual cycle and states of alkalosis), or during the use of hypocalcemic drugs (i.e. bisphosphonates)6.\n\nSignificant cognitive deficits, neuropsychiatric abnormalities, and extrapyramidal symptoms that resemble Parkinson’s disease or chorea are associated with the calcification of basal ganglia, which occurs in all forms of chronic hypocalcemia and may be detected with greater sensibility using computerized tomography7. Other findings of chronic hypocalcemia include sub-capsular cataracts, an increase in bone mineral density (BMD), and greater susceptibility to dystonic reactions induced by phenothiazines4.\n\nDifferential diagnosis of hypocalcemia will depend largely upon PTH and phosphorus levels, evaluated along with other clinical and laboratory data (Table 1). Cases presenting hypophosphatemia should include differential diagnosis of vitamin D, while cases associated with hyperphosphatemia are determined according to PTH levels. Hypoparathyroidism is an abnormality caused by a parathyroid hormone (PTH) secretion deficiency, and encompasses heterogeneous conditions (Table 2), which makes etiological differentiation crucial to the detection of abnormalities associated with some of these diseases beforehand, thereby preventing complications4. Signs and symptoms are caused by hypocalcemia.\n\nLaboratory measurements present hypocalcemia, hyperphosphatemia, and inappropriately low or undetectable PTH. Generally, levels of 1.25(OH) 2D are low and the alkaline phosphatase level is normal. In the majority of cases, hypoparathyroidism is sporadic, but there are familial cases in which transmission may be autosomic recessive, dominant, or X-linked.\n\nManagement of acute or severe symptomatic hypocalcemia must be made with intravenous calcium, with the goal of interrupting symptoms, preventing laryngeal spasm, and maintain total calcium levels above 7.0–7.5 mg/dL (ionized calcium greater than 0.7 mmol/L). Long-term treatment of patients with chronic hypocalcemia is done with 1 to 3 grams of elementary calcium per day in the various forms of salts available3.\n\nAll patients with hypoparathyroidism or pseudohypoparathyroidism who become hypocalcemic must use vitamin D or analogues in addition to calcium. The vast majority of patients obtain control with calcitriol in dosages of 0.25 μg, taken twice daily, up to 0.5 μg four times daily. Hypoparathyroidism causes increased excretion of urinary calcium in relation to serum calcium and predisposes hypercalciuria, nephrolithiasis, and nephrocalcinosis. The product of calcium × phosphate must be kept below 55. Patients must have their kidneys radiologically evaluated regularly in order to rule out nephrocalcinosis4.\n\n\nConclusion\n\n1. Ironically, increasing reliance on high end investigations such as a MRI brain scan could lead to certain conditions being missed; conditions that could be easily identifiable by the humble CT scan.\n\n2. All treatable metabolic conditions should be excluded at first before commencing with anticonvulsants; this will restrict patients from burdensome polytherapy and related side effects.\n\n\nConsent\n\nWritten consent was obtained for publication of the patient’s clinical details and images obtained from the patient.",
"appendix": "Author contributions\n\n\n\nPC compiled the entire dataset and wrote the entire report. RR was a treating neurophysician for the work and overlooked the writing of the report and CSA contributed to the data collection and report writing.\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 the Department of Endocrinology & Radiology for their support in the diagnosis of the patient at Sir Ganga Ram Hospital, New Delhi, India.\n\n\nReferences\n\nKıroğlu Y, Çallı C, Karabulut N, et al.: Intracranial calcifications on CT. Diagn Interv Radiol. 2010; 16(4): 263–9. PubMed Abstract | Publisher Full Text\n\nYou JS, Kim HJ, Chung SP, et al.: Intracranial bilateral symmetric calcification in hypoparathyroidism. Emerg Med J. 2008; 25(3): 162. PubMed Abstract | Publisher Full Text\n\nMaeda SS, Fortes EM, Oliveira UM, et al.: Hypoparathyroidism and Pseudohypoparathyroidism. Arq Bras Endocrinol Metabol. 2006; 50(4): 664–73. PubMed Abstract | Publisher Full Text\n\nLevine M: Hypoparathyroidism and pseudohypoparathyroidism. In: DeGroot LJ, Jameson JL. Endocrinology, 4th ed. Philadelphia: WB Saunders, 2001; 1133–53. Reference Source\n\nWeiss-Guillet EM, Takala J, Jakob SM: Diagnosis and management of electrolyte emergencies. Best Pract Res Clin Endocrinol Metab. 2003; 17(4): 623–51. PubMed Abstract | Publisher Full Text\n\nAbugassa S, Nordenstrom J, Eriksson S, et al.: Bone mineral density in patients with chronic hypoparathyroidism. J Clin Endocrinol Metab. 1993; 76(6): 1617–21. PubMed Abstract | Publisher Full Text\n\nKowdley KV, Coull BM, Orwoll ES: Cognitive impairment and intracranial calcification in chronic hypoparathyroidism. Am J Med Sci. 1999; 317(5): 273–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "367",
"date": "21 Nov 2012",
"name": "Gordon Klein",
"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\nA few comments should be made about this case report. The first is that the authors note low serum levels of 25 hydroxyvitamin D. This is not parathyroid hormone (PTH) dependent and an etiology should be sought, i.e. inadequate sun exposure or failure of adequate vitamin D in the diet or any dietary supplements. Also, the low serum magnesium levels, while not reported, might contribute to peripheral PTH resistance.Another thing to note is that advances in this field should be anticipated. For example, the use of parathyroid hormone replacement therapy and the advent of calcilytics (which are not yet marketed), would interfere with the action of the parathyroid calcium sensing receptor preventing a decrease in the set point for circulating calcium suppression of parathyroid hormone production or release.",
"responses": []
},
{
"id": "370",
"date": "08 Feb 2013",
"name": "Michael A Levine",
"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 do not believe that this case report advances this field as such findings are quite trivial. The tables are incorrect or incomplete and omits GCM2 mutations as a cause of hypoparathyroidism.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-51
|
https://f1000research.com/articles/1-50/v1
|
19 Nov 12
|
{
"type": "Research Article",
"title": "The distribution of circulating microRNA and their relation to coronary disease",
"authors": [
"Jane E Freedman",
"Bahadir Ercan",
"Kristine M Morin",
"Ching-Ti Liu",
"Lulufer Tamer",
"Lokman Ayaz",
"Mehmet Kanadasi",
"Dilek Cicek",
"Ali Ihsan Seyhan",
"Rabia Eker Akilli",
"Celalettin Camci",
"Beyhan Cengiz",
"Serdar Oztuzcu",
"Kahraman Tanriverdi",
"Bahadir Ercan",
"Kristine M Morin",
"Ching-Ti Liu",
"Lulufer Tamer",
"Lokman Ayaz",
"Mehmet Kanadasi",
"Dilek Cicek",
"Ali Ihsan Seyhan",
"Rabia Eker Akilli",
"Celalettin Camci",
"Beyhan Cengiz",
"Serdar Oztuzcu",
"Kahraman Tanriverdi"
],
"abstract": "Background: MicroRNAs (miRNAs) are small RNAs that regulate gene expression by suppressing protein translation and may influence RNA expression. MicroRNAs are detected in extracellular locations such as plasma; however, the extent of miRNA expression in plasma its relation to cardiovascular disease is not clear and many clinical studies have utilized array-based platforms with poor reproducibility. Methods and Results: Initially, to define distribution of miRNA in human blood; whole blood, platelets, mononuclear cells, plasma, and serum from 5 normal individuals were screened for 852 miRNAs using high-throughput micro-fluidic quantitative RT-PCR (qRT-PCR). In total; 609, 448, 658, 147, and 178 miRNAs were found to be expressed in moderate to high levels in whole blood, platelets, mononuclear cells, plasma, and serum, respectively, with some miRNAs uniquely expressed. To determine the cardiovascular relevance of blood miRNA expression, plasma miRNA (n=852) levels were measured in 83 patients presenting for cardiac catheterization. Eight plasma miRNAs were found to have over 2-fold increased expression in patients with significant coronary disease (≥70% stenosis) as compared to those with minimal coronary disease (less than 70% stenosis) or normal coronary arteries. Expression of miR-494, miR-490-3p, and miR-769-3p were found to have significantly different levels of expression. Using a multivariable regression model including cardiovascular risk factors and medications, hsa-miR-769-3p was found to be significantly correlated with the presence of significant coronary atherosclerosis. Conclusions: This study utilized a superior high-throughput qRT-PCR based method and found that miRNAs are found to be widely expressed in human blood with differences expressed between cellular and extracellular fractions. Importantly, specific miRNAs from circulating plasma are associated with the presence of significant coronary disease.",
"keywords": [
"Blood",
"plasma"
],
"content": "Introduction\n\nMicroRNAs (miRNAs) are 20–26-nt single stranded RNAs that participatein the regulation of various biological functions in numerouseukaryotic lineages, including plants, insects, vertebrate, and mammals1–3. More than 850 human miRNAs have been cloned and bioinformatic predictions indicate that mammalian miRNAs can regulate approximately 30% of all protein-coding genes4–6. The expressionof many miRNAs is specific to a tissue or developmentalstage, and the miRNA expression pattern is altered during thedevelopment of several diseases7,8.\n\nStudies measuring small numbers of miRNAs have shown their presence in circulating blood; specifically in platelets9, plasma10, and mononuclear cells11. In studies examining specific miRNAs12,13, differential expression was noted both in hematopoietic cell lines13 and between human and mouse cells12. Interestingly, miRNAs have been detected in cell-free human plasma preparations14. They have been found to be stable and protected from endogenous RNase activity14. In addition, levels of a specific miRNA (miR-141) can distinguish patients with prostate cancer from healthy controls14.\n\nBasic studies have shown that miRNAs regulate cardiac differentiation, angiogenesis, and myocyte growth15,16. Small clinical studies have also shown that levels of specific miRNA have been correlated with myocardial infarction in cardiac tissue from humans17 and animal models18,19. A recent study examined one miRNA (miR-1) from plasma and related it to acute myocardial infarction10. In stable and unstable coronary artery disease patients, 157 miRNAs were measured from peripheral blood mononuclear cells and differential expression was found11.\n\nAs shown by these studies, there are some publications about circulating miRNAs in cardiovascular disease9,20. In addition, the existing studies are restricted by incomplete evaluation of currently known miRNAs. Available arrays are constrained by incomplete miRNA coverage, issues in discriminating between closely related miRNAs, as well as ongoing discovery of new miRNAs (and the inherent lack of flexibility of the array platform). In addition, miRNA arrays have poor reproducibility when across clinical populations or in larger samples sizes21. We have developed a combined method of miScript miRNA assays (Qiagen, Germantown, MD) and Dynamic Arrays (Fluidigm, South San Francisco, CA) that employs quantitative RT-PCR (qRT-PCR) using a high-throughput process that allows us to analyze more samples vs. more miRNAs in a very short time22. Lastly, unlike hybridization-based microarray profiling techniques, qRT-PCR is considered the gold standard for RNA expression and does not require further confirmation analysis. Using this platform, a complete analysis of circulating whole blood, cellular, and cell-free miRNA was performed. The relevance of these findings to coronary disease was determined by measuring plasma miRNA expression in patients presenting for coronary angiography. The findings suggest that the distinct patterns of miRNA expression in components of whole blood may reflect specific patterns of disease.\n\n\nResults\n\nThere is minimal information defining miRNA expression in human blood and a complete screen of all known miRNAs has never been reported due to the limitations of current arrays and the cost of extensive qRT-PCR screening. Using blood samples from normal subjects and high-throughput qRT-PCR, the miRNA expression profile was determined for whole blood, isolated platelets, mononuclear cells, plasma and serum from five healthy subjects. Of the 852 miRNAs measured (Supplemental Table 1), the distribution of circulating miRNA that were most abundantly expressed in plasma and many of the blood derived sources is shown in Table 1. Gene expression is listed as cycle threshold value (Ct) consistent with RT-PCR-based data. Because the Ct values are listed, higher gene expression is reflected by a lower number. A complete list of miRNA expression for all sources is shown in Supplemental Table 2 (mean Ct value) and Supplemental Table 3 (mean delta Ct, accounting for housekeeping gene hsa-RNU1A-1). Unlike mRNA expression, it is currently unclear if miRNA data is more reliable when normalized with a housekeeping gene. This is especially germane for the cell-free plasma samples where a fixed volume was used and gene expression does not need to be normalized for cell count.\n\n\n\n\n\n\n\n\n\nOne-hundred and ninety four miRNAs were not expressed in any of the blood-based samples. Peripheral blood mononuclear cells (PBMCs) contained the highest number of miRNAs (658) followed by whole blood (609), platelets (448), serum (178) and plasma (147). As the abbreviated list shown in Table 1 demonstrates, while there is consistency between the groups, some miRNAs are more highly expressed in select sources. Sixty miRNAs were expressed in all five components. As the precise source of miRNA in plasma is not yet known, there was particular interest in comparing the expression between plasma and cellular miRNA patterns. Based on the significant overlap between the groups, it is difficult to determine the source for many of the specific plasma-derived miRNAs. It is not clear from the current data which miRNAs have a non-blood-based cellular source. Interestingly, miR-1185 and miR-548a-5p were much more abundantly expressed in plasma as compared to PBMCs or platelets, or only expressed in plasma. Although an interesting observation, the source of these two miRNAs cannot be determined from this data. In addition, some genes were expressed in platelets or PBMCs and not whole blood. This is likely due to the greater dilution used with PAXgene tubes and the loss of measurable expression of less abundant miRNAs.\n\nThe initial hypothesis of this study was that patients with significant coronary disease would have altered expression of plasma miRNA. Patients were divided into two groups (Table 2); 1) ≥70% coronary stenosis of any coronary artery or 2) <70 coronary stenosis. There were notable differences in expression of some of the miRNAs between these two groups (Table 3). By direct comparison, several plasma miRNAs were found to have over 2-fold increased expression in patients with significant coronary disease (≥70%) as compared to those with minimal coronary disease or normal coronary arteries (Figure 1). Initial statistical analysis demonstrated that anti-hypertensive therapy, smoking, and lipid lowering therapy have a positive association with coronary artery status (Table 2). Increased expression of miR-494, miR-769-3p and miR-490-3p was associated with ≥70% coronary stenosis. Next, the six variables were fit into a logistic regression analysis. As seen in Table 4, anti-hypertensive therapy, smoking and miR-769-3p were significantly associated with the coronary status.\n\nContinuous measures, mean ± S.D., or number and percent with stated characteristic.\n\nData is expressed as the mean delta Ct (Normalized to the RNU1A) and standard deviation. A lower number indicates increased miRNA expression. Bold valuesones are associated with CAD. More explanation in Table 5.\n\nmiRNAs shown had expression increased >2-fold between the groups; 1) ≥70% coronary stenosis of any coronary artery or 2) < 70 coronary stenosis.\n\nWe initially determined whether presence of significant coronary disease, as defined by ≥70% stenosis is associated with specific miRNA expression. A secondary question is whether presence/absence of coronary disease is associated with miRNA expression. To study this, patients were placed into one of three groups; 1) patients with CAD; at least one of the coronary arteries have ≥70% occlusion; 2) patients with minimal CAD; coronary occlusion 1%–<70%; and 3) coronary atherosclerosis-free, patients with no angiographically documented coronary artery stenosis. Because the numbers are small, a simple (non-statistical) comparison was made to determine trends. As seen in Figure 2 and supplemental table 4, 18 miRNAs had a varied expression pattern between group 1 and group 3. Seventeen miRNAs were upregulated at least 2-fold with only miR-1914 downregulated 0.5-fold. Interestingly, most miRNAs demonstrated a dose response with the greatest expression in patients with the most coronary disease and the least expression in disease-free patients (Supplemental table 4).\n\nmiRNAs shown had expression increased >2-fold or decreased <0.5-fold for the groups; 1) patients with CAD; at least one of the coronary arteries have ≥70% occlusion; 2) patients with atherosclerosis but not clinically significant CAD; coronary occlusion 1%–<70%; and 3) coronary atherosclerosis-free, patients with no angiographically documented coronary artery stenosis.\n\nCurrently, the capacity to measure miRNAs far outpaces our ability to understand their function in a given tissue. However, to better understand the potential significance of our findings, we conducted analyses that predict targets of the miRNAs that were up- or downregulated in coronary disease using two methods; current publications (www.ncbi.nlm.nih.gov/pubmed/) and TARGETSCAN 5.1 (http://www.targetscan.org). Using these methods, results for miRNA targets varied between 3 to 369 targets per miRNA when identified. With the TARGETSCAN search, target genes for individual miRNAs were identified using the context score for specific sites within genes. The context score is the sum of site-type contribution, 3’ pairing contribution, local AU contribution, and position contribution. The lower the context score indicates the most highly predicted targets for each miRNA. By TARGETSCAN search, miRNAs miR-1914 and miR-7-2, had no target gene identified.\n\nDetailed predictions for the miRNAs found to be significant in CAD are shown in Table 5. In broad terms, some miRNAs appear to target transcription factors, growth factors, cytokine regulation, transmembrane proteins, signal transduction pathways, and epigenetic pathways such as histone acetylases. Prediction results for specific miRNAs in TARGETSCAN include the following: miR-129-3p and miR-494 target HMGCS1; miR-150 targets MMP14; miR-150, and 92b target MMP16. Additionally, miR-1207 appears to target energy metabolism (most likely involving glucose metabolism).\n\nIndividual miRNAs target genes identified as significantly different between patients with and without significant coronary disease were selecting using the context score, a sum of; site-type contribution, 3’ pairing contribution, local AU contribution, and position contribution. The lowest scores show highest predictions.\n\nWe also analyzed the miRNAs that were unique to plasma, miR-1185-1 and miR-548a-5p-1. While many potential targets were identified, these miRNAs were predicted to target genes involved in controlling transcription factors, as well as several growth and cell cycle components.\n\n\nDiscussion\n\nMicroRNAs (miRNAs) are short regulatory RNAs that participatein the control of approximately 30% of all protein-coding genes4–6. The expressionof many miRNAs is usually specific to a tissue or developmentalstage, and the miRNA expression pattern is altered during theprogression of several diseases7,8. Most miRNAs are transcribed by RNA polymerase II from individual miRNA genes, from introns of protein coding genes, or from polycistronic transcripts that often encode multiple related miRNAs4,23. Although miRNAscan guide mRNA cleavage, the basic function of miRNA is to mediateinhibition of protein translation1,7,24–27 through miRNA-inducedsilencing complexes (miRISCs). The guiding strand of miRNA ina miRISC interacts with a complementary sequence in the 3’-untranslatedregion (3’-UTR) of its target mRNA by partial sequence complementarities, resulting in translational inhibition1,7.\n\nIn this study, the distribution of miRNA expression in whole blood, platelets, PBMCs plasma and serum showed significant overlap. Of particular interest are the nucleus-lacking platelet and the cell-free plasma expression levels. A primary question is why platelets would have miRNA? Platelets are produced in the bone marrow from megakaryocytes as cytoplasmic fragments without genomic DNA28. Platelets, however, retain a small amount of megakaryocyte-derived messenger RNAs (mRNAs) that have recently been suggested to be of physiological significance. Platelets can respond to physiological stimuli at the levels of protein translation and mRNA splicing29,30. There are few published studies describing platelet miRNAs13. In this study, cells of hematopoietic lineage were described to have a limited number of miRNAs (this study only tested for 19 miRNAs) and functionality was not shown. Interestingly, our data demonstrate that platelets express nearly the same number of miRNAs as PBMCs. The number of miRNAs in PBMCs is slightly less than in whole blood with the reason likely being dilution of low abundant PBMC miRNAs in whole blood.\n\nIn limited numbers, miRNAs have been detected in cell-free human plasma preparations14. They have been found to be stable and protected from endogenous RNase activity14. In addition, levels of a specific miRNA (miR-141) can distinguish patients with prostate cancer from healthy controls. In our analysis, we found moderate to high levels of expression of miRNAs in plasma in both normal subjects and patients with coronary disease, albeit in lower numbers as compared to platelets and PBMCs. What these data do not provide is the specific source of circulating miRNAs. This is a fundamental and fascinating question. They are believed to arise from three potential mechanisms: apoptosis, cellular activation with release of protrusions, and microsome/microvesicle formation. For example, Mitchell et al. found miR-141 differentially expressed in plasma in microsomes of prostate cancer patients14. It is possible that the miRNAs detected by our measurements in plasma or blood could be derived from endothelial cells or the atherosclerotic plaque itself. Further fundamental experiments are needed to answer this question. Recently, it has been shown that HDL particles deliver miRNAs31. Additionally, Wang et al. reported that plasma and whole blood miR-133 and miR-328 levels are increased in AMI patients32.\n\nBy the current data, we cannot assign precise punitive targets; however, specific bioinformatics approaches have been developed to predict miRNAs present in the genome of different organisms. These techniques are based on the observation that transcripts are usually highly conserved between related species and produced from precursor transcripts of similar size and structure. Using these bioinformatics approaches and the limited information available in the literature, we assembled potential target genes for the miRNAs expressed in significantly different amounts between patients with and without ≥70% coronary disease (Table 5). The list included a diverse range of functional and structural genes. This includes leukocyte and platelet recruitment to the atherosclerotic tissues, matrix reorganization, foam cell formation, growth/proliferation, and angiogenesis. However, these predictions do not provide definitive targets and additional basic studies are needed to provide clearer mechanistic information.\n\nAlso unique to our study is the specific method of measurement we used, which allowed for flexibility in adding newly discovered miRNAs, the use of small volumes, and high-throughput methods for qRT-PCR. Currently, there are several miRNA microarray products available that measure fewer miRNAs and some consist of older versions of the Sanger miRBase Sequence Database. Using the universal cDNA reaction feature of miScript provided the ability to profile all miRNAs with one cDNA reaction. Unlike the hybridization-based microarray profiling techniques, by coupling the miScript and Biomark Systems, confirmation analysis was not required for individual miRNAs. However, there are important limitations of our study. Despite the expansive miRNA survey for blood components, we cannot define the specific source for the plasma miRNA nor its eventual destination. Our study of coronary patients was limited by only being able to evaluate plasma miRNA, as other blood components were not available to us. In addition, while the miRNA data provided from these patients are unique, the numbers are still small making further analysis based on any subgroup statistically unfeasible.\n\nIn summary, miRNAs are small RNAs that play an important role in the negative regulation of gene expression by suppressing protein translation and have been detected in cell-free plasma and have been related to select diseases. By examining all measurable miRNAs, we defined the relative expression in blood components and find significant expression in platelets, PBMCs, whole blood, plasma and serum. By comparing plasma miRNA expression in patients with coronary disease, we begin to define specific miRNAs that are altered and provide potential targets that influence atherosclerosis.\n\n\nMaterials and methods\n\nThis study was approved by Mersin University Ethical Committee (06/05/2009, #6/144) and written consent was obtained from the subjects to test the hypothesis of whether coronary occlusion of ≥70% is associated with increased plasma miRNA expression levels. Upon enrollment, a study coordinator identified the presence of the following risk factors: (1) age, (2) male sex, (3) clinical history of diabetes, (4) clinical history of hypertension, (5) cigarette smoking, (6) clinical history of hypercholesterolemias, and (7) family history of coronary disease. Coronary angiograms were analyzed off-line in a blinded fashion with the use of digital calipers to measure stenosis severity, and stenosis was defined as a dichotomous variable: if a stenotic lesion was ≥70%, that vessel was counted as stenosed. The presence or absence of stenotic disease was also noted. Patients were ranked as having 0- to 3-vessel disease (number of coronary arteries with detectable atherosclerotic disease). For each patient, K3EDTA arterial blood (5 mL) was collected just prior to coronary angiography. Blood samples were centrifuged (3,000 g) and 400 µl plasma samples were stored at -80°C until RNA isolation.\n\nIn a separate smaller study, blood was obtained from healthy consented volunteers (n=5; 3 female, 2 male, average age=45) at Boston University School of Medicine as previously described33. The study was approved by Boston University IRB and written consent obtained from the subjects. All subjects were free of medications or supplements, and had no history of hypertension, diabetes, smoking, or hyperlipidemia. Blood was collected into PAXgene RNA tubes (Becton Dickinson, Franklin Lakes, NJ) for whole blood, into CPT tubes (Becton Dickinson) for peripheral blood mononuclear cells (PBMCs), into citrate tubes for platelets and plasma, and empty tubes for serum. Isolated platelets were prepared as previously described.\n\nRNA isolation: Total RNA including miRNAs was isolated from 200 µl plasma samples using miRVana Paris Kit (Ambion, Austin, TX). The RNA samples were stored at -80°C until cDNA conversion.\n\ncDNA conversion: Isolated RNA samples were converted to cDNA using miScript Reverse Transcription Kit (Qiagen, Germantown, MD). The RNA was converted to cDNA using the following conditions: 37°C for 60 min, 95°C for 5 min, and 4°C hold until further processing or storage. cDNA samples were kept at -80°C until PCR analysis.\n\nPre-amplification: Prior to PCR, cDNA samples were pre-amplified using Taqman PreAmp Master Mix (Applied Biosystems, Foster City, CA). PreAmp Master Mix and 0.2x Primers were added to the cDNA samples and pre-amplified as follows: 95°C for 10 min once, 95°C for 15 sec, 55°C for 30 sec, 70°C for 4 min (final three steps repeated for 14 cycles).\n\nqRT-PCR: Quantitative Real-Time PCR reactions (qRT-PCR) were performed using the high-throughput BioMark Real-Time PCR system (Fluidigm, South San Francisco, CA). Pre-amplified cDNA samples were diluted with 0.1mM EDTA in TE Buffer (1:5) and mixed with Power Sybr Green PCR Master Mix (Applied Biosystems), AmpliTaq Gold DNA Polymerase (Applied Biosystems) and Sample Loading Reagent (Fluidigm), then pipetted into sample inlets of Dynamic Array 96.96 chips (Fluidigm). Assay Loading Reagent (Fluidigm) and primers (Qiagen) were mixed and pipetted into assay inlets of Dynamic Array 96.96 chips. The IFC Controller HX (Fluidigm) was used to distribute primers and samples into chip reaction wells for qRT-PCR by microfluidic delivery. Gene expression experiments performed at Mersin and Gaziantep Universities in Turkey. The data were normalized using RNU1A1. Coefficient variations were less than 10% for almost all of the assays. Plasma volumes for all samples were constant (200 µl) and all following steps such as cDNA, PreAmplification and qRT-PCR had the same volumes always for all samples.\n\nWhen examining miRNAs that had larger fold changes between the CAD groups (Figure 1 and Figure 2) and those more highly expressed in the circulation using similar bioinformatics methods (such as targetscan), there were many miRNA targets that are known to control the processes important in the development of atherosclerosis (list not shown).\n\nWe initially summarized our data in different stratifications based on our outcome variables (coronary disease status). Next, we examined the bivariate relationship between the response variable and quantitative covariates using either two-sample t-test or Kruskal-Wallis test, where appropriate. Specifically, the t-test was conducted to test for a mean difference in quantitative demographic variables and miRNA expression level between two categories of coronary disease status. The pooled variance or the Satterthwaite’s method was used to estimate variance based on the equality of variance test. We employed Kruskal-Wallis test to determine whether there was any mean difference among groups in the scenario with three-category outcome variable.\n\nTree-based methods have been increasingly applied to biological research such as microarray data analysis and genome-wide association studies34,35. RandomForest is a flexible nonparametric approach, which consists of many decision trees from bootstrap samples. In this study, we constructed randomForest25 in identifying relevant variables to our outcome variable using the randomForest package in R (2.10.1)36. Furthermore, we also conducted logistic regression and multinomial logistic regression where appropriate37.",
"appendix": "Author contributions\n\n\n\nAll authors contributed to this work. There were no paid authors or writing assistants used in the preparation of the manuscript or analysis of the data. All authors declare that the did not submit related or duplicate manuscripts elsewhere. Jane E. Freedman, MD: design of the study and writing the manuscript. Bahadir Ercan, PhD: conducting the PCR analysis. Kristine M. Morin, MPH: analyzing the data. Ching-Ti Liu, PhD: analyzing the data. Lulufer Tamer, PhD: recruiting patient and collecting the blood samples and conducting the RNA isolations. Lokman Ayaz, MSc: conducting the RNA isolations. Mehmet Kanadasi, MD, Dilek Cicek, MD, Ali Ihsan Seyhan, MD, Rabia Eker Akilli, MD, Celalettin Camci, MD, and Beyhan Cengiz, PhD: recruiting patients and collecting the blood samples. Serdar Oztuzcu, MD: conducting the PCR analysis. Kahraman Tanriverdi, PhD: design of the study, conducting the PCR analysis and writing the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was partially supported by HL087201 (Jane E. Freedman.).\n\n\nReferences\n\nBartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004; 116: 281–297. PubMed Abstract | Publisher Full Text\n\nAmbros V, Chen X: The regulation of genes and genomes by small RNAs. Development. 2007; 134: 1635–1641. PubMed Abstract | Publisher Full Text\n\nWinter J, Jung S, Keller S, et al.: Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol. 2009; 11: 228–234. PubMed Abstract | Publisher Full Text\n\nSuarez Y, Sessa WC: MicroRNAs as novel regulators of angiogenesis. Circ Res. 2009; 104: 442–454. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFilipowicz W, Bhattacharyya SN, Sonenberg N: Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008; 9: 102–114. PubMed Abstract | Publisher Full Text\n\nGriffiths-Jones S, Saini HK, van Dongen S, et al.: miRBase: tools for microRNA genomics. Nucleic Acids Res. 2008; 36: D154–158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang J, Liang Z, Yang B, et al.: Derepression of microRNA-mediated protein translation inhibition by apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members. J Biol Chem. 2007; 282: 33632–33640. PubMed Abstract | Publisher Full Text\n\nRana TM: Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol. 2007; 8: 23–36. PubMed Abstract | Publisher Full Text\n\nLandry P, Plante I, Ouellet DL, et al.: Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol. 2009; 16: 961–966. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAi J, Zhang R, Li Y, et al.: Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction. Biochem Biophys Res Commun. 2010; 391: 73–77. PubMed Abstract | Publisher Full Text\n\nHoekstra M, van der Lans CA, Halvorsen B, et al.: The peripheral blood mononuclear cell microRNA signature of coronary artery disease. Biochem Biophys Res Commun. 2010; 394: 792–797. PubMed Abstract | Publisher Full Text\n\nRamkissoon SH, Mainwaring LA, Ogasawara Y, et al.: Hematopoietic-specific microRNA expression in human cells. Leuk Res. 2006; 30: 643–647. PubMed Abstract | Publisher Full Text\n\nMerkerova M, Belickova M, Bruchova H: Differential expression of microRNAs in hematopoietic cell lineages. Eur J Haematol. 2008; 81: 304–310. PubMed Abstract | Publisher Full Text\n\nMitchell PS, Parkin RK, Kroh EM, et al.: Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA. 2008; 105: 10513–10518. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCare A, Catalucci D, Felicetti F, et al.: MicroRNA-133 controls cardiac hypertrophy. Nat Med. 2007; 13: 613–618. PubMed Abstract | Publisher Full Text\n\nLatronico MV, Catalucci D, Condorelli G: MicroRNA and cardiac pathologies. Physiol Genomics. 2008; 34: 239–242. PubMed Abstract | Publisher Full Text\n\nBostjancic E, Zidar N, Glavac D: MicroRNA microarray expression profiling in human myocardial infarction. Dis Markers. 2009; 27: 255–268. PubMed Abstract | Publisher Full Text\n\nDong S, Cheng Y, Yang J, et al.: MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem. 2009; 284: 29514–29525. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang GK, Zhu JQ, Zhang JT, et al.: Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J. 2010; 31: 659–666. PubMed Abstract | Publisher Full Text\n\nKondkar AA, Bray MS, Leal SM, et al.: VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA. J Thromb Haemost. 2010; 8: 369–378. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSato F, Tsuchiya S, Terasawa K, et al.: Intra-platform repeatability and inter-platform comparability of microRNA microarray technology. PLoS One. 2009; 4: e5540. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpurgeon SL, Jones RC, Ramakrishnan R: High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS One. 2008; 3(2): e1662. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee Y, Kim M, Han J, et al.: MicroRNA genes are transcribed by RNA polymerase II. Embo J. 2004; 23: 4051–4060. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHutvagner G, Zamore PD: A microRNA in a multiple-turnover RNAi enzyme complex. Science. 2002; 297: 2056–2060. PubMed Abstract | Publisher Full Text\n\nMeister G, Landthaler M, Patkaniowska A, et al.: Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell. 2004; 15: 185–197. PubMed Abstract | Publisher Full Text\n\nAmbros V, Lee RC, Lavanway A, et al.: MicroRNAs and other tiny endogenous RNAs in C. elegans. Curr Biol. 2003; 13: 807–818. PubMed Abstract | Publisher Full Text\n\nPillai RS, Bhattacharyya SN, Filipowicz W: Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol. 2007; 17: 118–126. PubMed Abstract | Publisher Full Text\n\nItaliano JE Jr, Bergmeier W, Tiwari S, et al.: Mechanisms and implications of platelet discoid shape. Blood. 2003; 101: 4789–4796. PubMed Abstract | Publisher Full Text\n\nKieffer N, Guichard J, Farcet JP, et al.: Biosynthesis of major platelet proteins in human blood platelets. Eur J Biochem. 1987; 164: 189–195. PubMed Abstract | Publisher Full Text\n\nDenis MM, Tolley ND, Bunting M, et al.: Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets. Cell. 2005; 122: 379–391. PubMed Abstract | Publisher Full Text\n\nVickers KC, Palmisano BT, Shoucri BM, et al.: MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 2011; 13: 423–433. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang R, Li N, Zhang Y, et al.: Circulating MicroRNAs are Promising Novel Biomarkers of Acute Myocardial Infarction. Intern Med. 2011; 50: 1789–1795. PubMed Abstract | Publisher Full Text\n\nFreedman JE, Ting B, Hankin B, et al.: Impaired platelet production of nitric oxide predicts presence of acute coronary syndromes. Circulation. 1998; 98: 1481–1486. PubMed Abstract | Publisher Full Text\n\nZhang H, Yu CY, Singer B: Cell and tumor classification using gene expression data: construction of forests. Proc Natl Acad Sci USA. 2003; 100: 4168–4172. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen X, Liu CT, Zhang M, et al.: A forest-based approach to identifying gene and gene gene interactions. Proc Natl Acad Sci USA. 2007; 104: 19199–19203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBreiman L: Random Forests. Machine Learning. 2001; 45: 5–32. Publisher Full Text\n\nLiaw A, Wiener M: Classification and Regression by random Forest. R News. 2002; 2/3: 18–22. Reference Source"
}
|
[
{
"id": "381",
"date": "26 Nov 2012",
"name": "Peter Sarnow",
"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": "652",
"date": "09 Jan 2013",
"name": "Terry Elton",
"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": "701",
"date": "11 Jan 2013",
"name": "Jennifer 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\nI would like to note that the manuscript could be improved by use of error bars in Figures 1 and 2 to display some measure of variance and that future studies of this type could utilise spike-ins prior to RNA extraction to normalise for variability in RNA extractions between samples.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-50
|
https://f1000research.com/articles/1-49/v1
|
15 Nov 12
|
{
"type": "Research Article",
"title": "Student feedback about the use of literature excerpts in Sparshanam, a Medical Humanities module",
"authors": [
"P Ravi Shankar",
"Kundan K Singh",
"Ajaya Dhakal",
"Arati Shakya",
"Rano M Piryani",
"Kundan K Singh",
"Ajaya Dhakal",
"Arati Shakya",
"Rano M Piryani"
],
"abstract": "Medical humanities (MH) modules have been conducted for first year students at KIST Medical College, Lalitpur, Nepal for the last four years. Literature excerpts are widely used in MH programs in developed nations. In Nepal English language literature excerpts had been used previously in two modules. Problems noted were difficulty in comprehending the excerpts and relating them to the Nepalese scenario. The MH module for the 2011 intake was conducted from December 2011 to March 2012. The present study was conducted in the third week of March to obtain student perceptions about use of literature excerpts and suggestions for further improvement using a questionnaire. Literature excerpts used in the module dealt with Nepal and health-related topics. Sixty-eight of the 80 students (85%) participated in the study. The majority were male, self-financing and from urban areas. Respondents felt the excerpts introduced them to different aspects of the medical profession, prepared them for future practice, and underscored the importance of understanding the patients’ feelings. The literature excerpts with which they could identify the most and the least were noted. There were no differences in median enjoyment and effectiveness scores of the literature excerpts according to subgroups of respondents. The suggested benefits of using literature in medical education were similar to those reported previously. Most respondents were able to appreciate the English language excerpts. They felt that Nepali language excerpts and those by Nepali writers could also be included. The findings would be of interest to educators in other developing nations introducing MH modules.",
"keywords": [
"colleges",
"questionnaires",
"medical education"
],
"content": "Introduction\n\nKIST Medical College (KISTMC) is a private medical school in the Lalitpur district of the Kathmandu valley affiliated to the Institute of Medicine, Tribhuvan University for the undergraduate medical (MBBS) course. The college admitted its first batch of students in November 2008 and four batches of students have been admitted since. A medical humanities (MH) module has been conducted for first year medical students right from the first batch1 using small group, activity-based learning methods. Case scenarios, role plays, facilitator presentations and paintings are among the different modalities used to explore various aspects of MH2,3.\n\nThe first author had used literature excerpts during a voluntary module for medical students and faculty members at the Manipal College of Medical Sciences (MCOMS), Pokhara, Nepal4. Literature excerpts were also used during a module conducted for faculty members and medical and dental officers at KISTMC5. Problems were noted with the use of literature excerpts. The excerpts were in English and were mainly obtained from the book ‘Ten years of the medicine and the arts’6 published by the Association of American Medical Colleges. Participants felt the excerpts did not reflect the Nepalese scenario, the language used was difficult to understand and they could not relate to certain excerpts.\n\nLiterature has been widely used in MH programs especially in developed nations. At the Northwestern Medical School in Chicago, United States (US) literature is taught in courses from the first day of medical school through residency7. Literature has been used in the MH program at the University of Missouri-Kansas City School of Medicine in the US8 and during a family medicine clerkship at the University of California, Irvine College of Medicine9. In Asia literature from both Western and Arabic contexts has been widely used at the Weill Cornell Medical College in Qatar10.\n\nLiterature has a number of advantages in the education of doctors. It develops skills in observation and interpretation, develops clinical imagination, and promotes clarity of expression7. Literature can foster tolerance for uncertainty inherent in clinical practice, and increase empathy towards patients. A review states that five broad goals are met by the study of literature in medicine. Literary accounts can teach medical students concrete and powerful lessons about the lives of sick people11. Physicians are able to better understand and empathize with narrative stories of patients, literature increases physicians’ expertise in narrative ethics and literary theory can offer new perspectives on the work and genres of medicine.\n\nThe MH module at KISTMC is named as ‘Sparshanam’ meaning ‘touch’ in Sanskrit, an ancient South Asian language. The 80 students of the 2011 intake were divided into six groups each consisting of 14 or 15 students. The module was conducted for 90 minutes every Thursday during the early clinical exposure for first year students. Eight sessions were conducted from December 2011 to March 2012. The topics covered during the eight sessions were empathy, what it means to be sick in Nepal, the patient, the doctor, the doctor-patient relationship, the family, the medical student and a wrap up session. Literature excerpts were reintroduced in this module and were used during certain sessions. Keeping in mind previous feedback we used excerpts with relatively simple language and which were directly related to Nepal. The excerpts were by western authors and in the English language. Further information about the excerpts used is provided in the Methods section. The present study was conducted in the third week of March 2012 with the following objectives:\n\na. Obtain feedback on the literature excerpts used and\n\nb. Get suggestions for further improvement.\n\n\nMethods\n\nThe study was conducted among first year undergraduate medical (MBBS) students in the third week of March 2012 on conclusion of the Medical Humanities module. The aims and objectives of the study were explained to the students and they were invited to participate. Written informed consent was obtained from all participants. The study was approved by the Institutional Review Board of KIST Medical College.\n\nLiterature excerpts were used during certain sessions of the module. Among the books used were ‘The snow leopard’ by Peter Matthiessen12, ‘Three men in a boat’ by Jerome K Jerome13, ‘Window on to Annapurna’ by Joe Stephens14, ‘Aama in America’ by Broughton Coburn15 and ‘The tennis partner’ by Abraham Verghese16. ‘The snow leopard’ is a book about the author’s trek in the remote Dolpo district of Nepal in 1973. He was trekking with a naturalist who was studying mountain goats and the snow leopard. The excerpt used was towards the end of the book where the author was returning back and his Sherpa falls sick. He is treated by a Japanese doctor from a mountaineering expedition. ‘Three men in a boat’ is a humorous account of a boat journey undertaken by three friends on the Thames River. The excerpt deals with the author reading a medical textbook and beginning to imagine that he suffers from the different diseases enumerated. ‘Window on to Annapurna’ is a story of a woman and her husband staying for a year in a Magar village outside the town of Baglung in western Nepal and how they become a part of the life of the villagers. The excerpt used dealt with different faith healing practices which the author had witnessed. ‘Aama in America’ is the story of a Peace Corps worker who stays in a remote village and is adopted by an old Gurung lady as her foster son. Later he takes the old lady to America and shows her his country. The excerpt used describes the lady’s fear that she might die in America away from her family and also describes Gurung funeral rites. ‘The tennis partner’ describes a young Australian student who joins the author’s medical school and who is fighting a losing battle against drug abuse. The excerpts deal with doctors from India emigrating to America and about doctors and drug abuse. The student groups were asked to interpret the excerpt in the context of the topic of the day’s session and present their interpretation to the house using flip charts.\n\nSixty-eight of the 80 students (85%) participated in the study. Student responses were collected using a questionnaire. Basic information about the respondents like gender, method of financing of medical education, medium of instruction at school, place of family residence and occupation of parents were noted. The questionnaire used is shown below. Free text comments were collected about participants’ perception regarding the use of literature excerpts in Sparshanam, how literature excerpts helped in realizing the objectives of the module, participants’ opinion regarding the use of excerpts mainly by western writers, which of the different excerpts they could identify with the most and the least. Opinion was also sought on what they would suggest to make the exercise more useful, whether they faced difficulties in putting the excerpts in a Nepalese context and if yes, how they overcame these. Similar responses were noted and the frequency of different responses worked out. Common responses were tabulated.\n\nParticipants were asked to rate on a scale of 1 to 5 their enjoyment of the literature excerpts used and their perception regarding the effectiveness of the excerpts. The mean and median scores were calculated. One sample Kolmogorov-Smirnoff test was used to test the normality of distribution of the scores. Both were found not to be normally distributed and non-parametric tests (Mann Whitney test was used for variables with two subgroups and Kruskal Wallis test for the others) were used to compare the median scores among subgroups of respondents.\n\n\nResults\n\nSixty-eight of the 80 students (85%) participated in the study. Table 1 shows the demographic characteristics of the student respondents. The majority of students were male, self-financing and from urban areas. Only a few students had parents from health related fields.\n\n* The numbers may not add up to 68 and the percentage to 100 in all cases as certain respondents did not complete all demographic details.\n\nTable 2 shows the respondents’ comments about the use of literature excerpts in the module along with the frequency of the comments. Other interesting comments in addition to those mentioned in the table were “excerpts introduced us to different ways of thinking, and gave us ideas about right and wrong”. Ten students (14.7%) stated they had previous experience with use of literature for educational purposes. Among the books mentioned were ‘Diary of Anne Frank’ and ‘Animal farm’. Regarding how the literature excerpts helped in realizing the objectives of the module, 11 respondents (16.2%) stated it helped by preparing doctors for future practice, 10 (14.7%) said it helped them understand patient’s feelings, eight (11.8%) stated it helped them by highlighting the reality of various issues discussed during the module, while five respondents (7.3%) wrote it helped them in visualizing different situations covered during the MH module.\n\nOnly six respondents (8.8%) were aware of the use of literature in medical education elsewhere. They mainly mentioned the examples of medical schools in western nations without giving specific names. Forty-eight respondents (70.6%) felt the use of literature excerpts by western authors in the module were appropriate. A respondent mentioned, “Nepal is also rapidly developing. Being in a poor country does not mean we should be away from the modern drama of medical development. And apart from that, we are influenced predominantly by the western world”. Among the reasons mentioned were the excerpts widened their horizons, they could learn from the western writers, the writers mainly described their experiences in Nepal, they showed cultural aspects of illness, students have been exposed to western literature since childhood, and this type of literature is more easily available. Twelve (17.6%) felt this was not appropriate. Among reasons mentioned were Nepali excerpts were also available, the local context was always better and the excerpts dealt with a different set of problems.\n\nTable 3 shows the literature excerpts with which respondents identified the most along with reasons. A student wrote, “Three men in a boat, I suppose. Firstly it was an assignment for our group and secondly, when I was younger and just being exposed to medical difficulties, I used to have lots of thoughts about my condition and made certain interpretations which weren’t true”. Among the books and excerpts with which participants identified the least was ‘Window on to Annapurna’ and ‘Three men in a boat’ (12 respondents each). The reasons mentioned for the first book were difficulty in identifying with the faith healing practices described, difficulty in understanding the excerpts and the respondents not having personal experience of life in less developed areas. For ‘Three men in a boat’ reasons mentioned were the excerpt was difficult to understand and the situation described was not believable. Among the suggestions to make the activity of interpreting literature excerpts more useful were using more literature in Nepali language and written by Nepali authors [14 respondents (20.6%)], students reading the entire book before the session [6 respondents (8.8%)], use of more literature excerpts [5 respondents (7.3%)], and more feedback to be provided by the facilitators and more time for the activity [4 respondents each (5.9%)]. It was also suggested by a respondent that the same literature excerpt could be provided to all the groups.\n\nThe median score of respondents with regard to enjoyment of the literature excerpts used and perceived effectiveness of the excerpts used were 4 (maximum score 5). Table 4 shows the median scores according to demographic characteristics of respondents. There were no significant differences in scores according to respondents’ characteristics. Respondents were asked whether they experienced any difficulty in putting the excerpts in the Nepalese context; 40 (58.8%) stated they faced no difficulties while 21 (30.9%) stated they had difficulties. Among the strategies used to overcome the difficulties were group work and consultation, using their imagination and putting themselves in the scenario, and taking help from the facilitators. Fifty-five respondents felt excerpts from works by Nepali authors and in the Nepali language should also be used. A student wrote, “Yes, it would be a great idea. The modules will be even more interesting and we can relate well. But excerpts from other nations should also be included. This will provide an overall vision on situations”. They felt excerpts related to medicine and excerpts from rural areas should be used. The excerpts can be obtained from different sources, and a few respondents suggested specific books that can be used while some felt students can themselves supply the excerpts. A large majority [62 respondents (91.2%)] felt literature excerpts should be continued to be used in future modules.\n\nTable 5 shows the suggested advantages of literature in medical education. A student stated, “Medical Studies are often facts, facts and only facts. We lack situations where we can acquire knowledge critically. Thus this can help improve critical thinking skills”. Respondents were asked whether they felt the use of English language excerpts in the module was appropriate. Fifty-one respondents (75%) felt they were appropriate. Among the reasons mentioned were English is the international language, the students were from English medium schools and were comfortable with English, the excerpts dealt with Nepal and English is the language of instruction. Eleven (16.2%) felt the excerpts were inappropriate. Cited reasons were they do not describe the Nepalese situation, Nepalese excerpts are available and English is not a commonly used language in Nepal. Forty-one respondents (60.3%) were easily able to understand the language used in the excerpts while 25 (36.8%) were not able to do so. Difficult words, tough language and low level of mastery of the English language were cited as reasons for difficulties.\n\nThe facilitators provided a brief introduction about the book and the author before distributing the excerpts. Fifty-eight respondents (85.3%) found this useful. The introduction often helped to put the excerpt in context, students knew more about the author and they could read the book if they were interested. Suggestions to further improve the use of literature excerpts were also obtained. Common among these were to also use Nepali language excerpts (16 respondents), use more excerpts (8), use excerpts with simpler language (6), use more excerpts from the medical field (5), provide photocopies of the excerpts before hand to the students (4), and provide more time for the activity (4 respondents).\n\n\nDiscussion\n\nParticipant feedback about the use of literature excerpts during the module was on the whole positive. They felt the excerpts used were not too difficult to understand and either reflected or could be extrapolated to the Nepalese scenario. They also wanted more scenarios by Nepalese authors and in the Nepal language to be used. They were strongly in favor of continuing the use of literature excerpts during future MH modules.\n\nMost of the students are self-financing and only seven students were scholarship students. Scholarship students are selected on the basis of ranks obtained in an entrance examination conducted by the Ministry of Education, Government of Nepal. Compared to the self-financing students they are usually of lower socioeconomic background and from rural areas. These students have to serve for two years in rural areas after graduation and are becoming an important source of support to Nepal’s health system17. KISTMC provides 10% of total seats on full tuition fees scholarship. In Nepal like in other parts of South Asia there are two media of instruction in schools. In vernacular medium schools all subjects are taught in the local language and English is taught as a second or third language. In English medium schools all subjects are taught in English and the native language is taught only as a second or third language. Most English medium schools are in the private sector and charge high tuition fees. Due to English being regarded as the international language and the language of science and education there is a high demand for English language education in the country. Most of our students were from English medium schools and from an urban background. They would have faced fewer problems in understanding and interpreting the English language excerpts. Medical education is expensive with most schools charging around 40,000 US dollars as tuition fees for the course.\n\nThe excerpts as stated were by foreign authors but were either about Nepal or dealt with health related topics. Students felt that these introduced them to different problems they may face in their future practice and also provided ideas about how to deal with these issues. Images of disease and death are common in literature and can serve to introduce students and doctors to experiences they may not have had themselves18. Narrative methods are widely used in medicine with patients telling their stories to doctors, who tell these stories to other doctors during case presentations and the patient is regarded as a book needing interpretation19. Study of literature can improve performance in the narrative aspects of medicine. We had used brief one-page excerpts in the module due to time and other constraints. Also literature was used as one of the modalities along with case scenarios, role-plays and paintings.\n\nIn California in the US a brief literature-based course was shown to contribute to greater student empathy and appreciation of the role of humanities in medical education20. At the University of Oxford in the United Kingdom, a special study module using literature was shown to have increased insights gained into patients and their experience of illness and empathy. Certain clinically important skills were also increased21. There have been many reviews highlighting the importance of MH and literature and its perceived advantages in undergraduate medical education. A recent review however, concludes that there is a shortage of studies describing long-term impact of MH on the development of medical proficiency22. The authors conclude that this may pose a threat to the continued development of MH as administrators and curriculum planners may demand objective evidence of its effectiveness. In Nepal at present to the best of our knowledge we are the only medical school offering a MH module and the first batch of students are now in their fourth year of study. Studying effectiveness in terms of professional behaviors will be important for continued growth of the discipline.\n\nThe books and the excerpts used have been already described. The excerpts were in English and most of the students were comfortable with the English language due to reasons previously mentioned. Two of the authors of this article (PRS and RMP) had previously explored the issue of English as the language of MH teaching23. In South Asia due to a variety of reasons among them the British colonial influence, English is the language of higher education and the medium of instruction in medical schools. Though Nepal was not ruled by a colonial power, it was strongly influenced by India especially in the field of education. English as the language of excerpts has both advantages and disadvantages. In many developing countries there are a number of languages spoken and it may be difficult to find a language acceptable to everyone23. In KISTMC nearly all our students are from Nepal and it may be possible to use Nepali language excerpts. This was not possible at MCOMS, Pokhara where there were students of different nationalities4. However, English language excerpts are easier to find and use compared to native language ones. In our previous modules at MCOMS4 and the faculty module at KISTMC5 participants had difficulty with the level of English in certain excerpts. These complaints were fewer this time as we had taken care to select excerpts with simpler language.\n\nA few students had volunteered to provide Nepali language excerpts and we can consider this during future modules. The excerpts were short and certain participants had problems putting the excerpt in context. Opinion is divided among facilitators and others about whether to select the majority of excerpts from medicine and related areas. Some feel that this is important as students will be practicing doctors once they qualify while others feel medicine, illness and death are a part of life and cannot be divorced from the larger scenario. Providing photocopies of the excerpts one day before to all students can be considered.\n\nA recent article has stated that MH could be considered as representative of western culture24. The authors state that MH programs in Asia promoted a quasi-Western approach to medical humanities (in history, philosophy, literature and art) and the diversity, sophistication and richness of different cultural traditions was uncomfortably marginalized. The authors further state that MH curricula concentrated on two genres of work. These were works in literature, arts and music valued for their insight and beauty and ‘experiential’ works written by both doctors and patients. The issue of indigenous cultural traditions being marginalized requires attention. In South Asia English is increasingly dominant as the language of education and culture. Western cultural values are strongly promoted through the mass media and the internet. How to involve more indigenous cultural expressions in MH modules can be a major challenge.\n\nOur experiences with the use of literature excerpts would be of interest to medical educators planning to introduce MH modules in other developing nations. At present these modules are few in number but many medical schools are interested in introducing a certain amount of MH teaching in the curriculum. In many developing nations the medium of instruction is either English or other colonial languages. Also in most developing nations only students from a science background are eligible to study medicine. In South Asia twelve years of schooling with the subjects of Physics, Chemistry and Biology during the last two years is necessary. Medical students are less exposed to literature and the humanities and sometimes have a negative perception of these disciplines.\n\nThe strength of the study is the high response rate. Our study had limitations. Student perception regarding the use of literature excerpts was only studied using a questionnaire. Other methods were not used. The questions included were arrived at by consensus among the authors. The questionnaire was pretested for comprehension and understanding among five third year students. We however felt that a few respondents had difficulty in properly comprehending certain questions. There is a certain amount of repetition in the responses obtained.\n\n\nConclusions\n\nThe use of literature excerpts was appreciated by the students. Most felt English language excerpts were acceptable but felt Nepali language excerpts could also be considered. Students strongly felt that the use of literature excerpts should be continued in future MH modules. This use will provide us with more information regarding student understanding and acceptance of the same. The type of excerpts to be used and its length have to be decided based on consensus among facilitators. Our experiences would be of interest to educators planning to introduce MH modules in other developing nations.",
"appendix": "Author contributions\n\n\n\nPRS was involved in conceptualizing the study, collecting the data, analyzing the results, reviewing the literature and writing the manuscript. KKS was involved in conceptualizing the study, collecting the data, reviewing the literature and writing the manuscript. AD helped in conceptualizing the study, collecting the data and writing the manuscript. AS was involved in collecting the data, reviewing the literature and writing the manuscript. RMP helped in collecting the data, reviewing the literature and writing the manuscript. All authors have read and approved the final submitted 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 thank all students who participated in the study. They thank Ms. Shova for help with conducting the sessions and Ms. Renu and Ms. Pooja for their logistic support on the study. They thank the college management, especially the Principal and the Director Academics for their continued support to MH programs in the institution.\n\n\nReferences\n\nShankar PR, Piryani RM: Four years of medical humanities in Nepal: What worked and what did not. Literature, arts and medicine blog. NYU School of Medicine. September 2010 Posted on 12/09/10. Reference Source\n\nShankar PR, Piryani RM, Upadhyay-Dhungel K: Student feedback on the use of paintings in Sparshanam, the Medical Humanities module at KIST Medical College, Nepal. BMC Med Educ. 2011; 11: 9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShankar PR, Piryani RM, Thapa TP, et al.: Our Experiences With ‘Sparshanam’, A Medical Humanities Module For Medical Students At KIST Medical College, Nepal. J Clin Diagn Res 2010; 4(1): 2158–2162. Reference Source\n\nShankar PR: A voluntary Medical Humanities module at the Manipal College of Medical Sciences, Pokhara, Nepal. Fam Med. 2008; 40(7): 468–70. PubMed Abstract\n\nShankar PR, Piryani RM, Karki BMS, et al.: A medical humanities module for faculty members at KIST Medical College, Imadol, Lalitpur. J Clin Diagn Res 2011; 5(7): 1489–1492. Reference Source\n\nDittrich LR (ed.): Ten years of medicine and the arts. 2001: Association of American Medical Colleges. Reference Source\n\nHunter KM, Charon R, Coulehan JL: The study of literature in medical education. Acad Med. 1995; 70(9): 787–94. PubMed Abstract\n\nSirridge M, Welch K: The Program in Medical Humanities at the University of Missouri-Kansas City School of Medicine. Acad Med. 2003; 78(10): 973–6. PubMed Abstract\n\nShapiro J, Duke A, Boker J, et al.: Just a spoonful of humanities makes the medicine go down: introducing literature into a family medicine clerkship. Med Educ. 2005; 39(6): 605–12. PubMed Abstract | Publisher Full Text\n\ndel Pozo PR, Fins JJ: The globalization of education in medical ethics and humanities: evolving pedagogy at Weill Cornell Medical College in Qatar. Acad Med. 2005; 80(2): 135–40. PubMed Abstract\n\nCharon R, Banks JT, Connelly JE, et al.: Literature and medicine: contributions to clinical practice. Ann Intern Med. 1995; 122(8): 599–606. PubMed Abstract | Publisher Full Text\n\nPeter Matthiessen: The snow leopard. London: The Harvill Press, 1989. Reference Source\n\nJerome K Jerome: Three men in a boat. London: Penguin Popular Classics, 1994. Reference Source\n\nJoy Stephens: Window on to Annapurna. Delhi: Book Faith India, 1995. Reference Source\n\nBroughton Coburn: Aama in America. Knopf Doubleday Publishing Group, 1996. Reference Source\n\nAbraham Verghese: The tennis partner. London: Vintage, 1999. Reference Source\n\nShankar PR: Scholarship students in private medical schools an important source of support to Nepal’s health system. Australa Med J 2011; 4(6): 279–80. Reference Source\n\nBolton G: Literature and medicine. Lancet. 2001; 357(9266): 1441–2. PubMed Abstract | Publisher Full Text\n\nMcLellan MF, Jones AH: Why literature and medicine? Lancet. 1996; 348(9020): 109–11. PubMed Abstract | Publisher Full Text\n\nShapiro J, Morrison E, Boker J: Teaching empathy to first year medical students: evaluation of an elective literature and medicine course. Educ Health (Abingdon). 2004; 17(1): 73–84. PubMed Abstract\n\nLancaster T, Hart R, Gardner S: Literature and medicine: evaluating a special study module using the nominal group technique. Med Educ. 2002; 36(11): 1071–6. PubMed Abstract | Publisher Full Text\n\nOusager J, Johannessen H: Humanities in undergraduate medical education: a literature review. Acad Med. 2010; 85(6): 988–98. PubMed Abstract | Publisher Full Text\n\nShankar PR, Piryani RM: English as the language of Medical Humanities learning in Nepal: Our experiences. The literature, art and medicine blog. April 2009 Posted on 22/04/2009. Reference Source\n\nHooker C, Noonan E: Medical humanities as expressive of western culture. Med Humanit. 2011; 37(2): 79–84. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "366",
"date": "22 Nov 2012",
"name": "Carol Ann Courneya",
"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": "365",
"date": "17 Dec 2012",
"name": "Amol R. Dongre",
"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": "364",
"date": "17 Dec 2012",
"name": "Avinash Supe",
"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
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https://f1000research.com/articles/1-49
|
https://f1000research.com/articles/1-48/v1
|
15 Nov 12
|
{
"type": "Case Report",
"title": "Case Report: Pellucid-like keratoconus",
"authors": [
"Mazen M Sinjab",
"Lara N Youssef",
"Lara N Youssef"
],
"abstract": "Purpose: To study the tomographic features of pellucid-like keratoconus (PLK), and to report a new sign on the pachymetry map (PM) in pellucid marginal degeneration (PMD).Patients and methods: A retrospective descriptive case series was performed in Damascus University in 2011. Clinical and tomographic findings of 15 eyes (9 patients) that had the claw pattern of the anterior sagital map (ASM) were reviewed. Patients were distributed into two groups: (1) 4 eyes were considered PMD since they had inferior corneal thinning on both slitlamp biomicroscopy and PM; (2) 11 eyes were considered as PLK since they did not show inferior corneal thinning. Patients were studied using slitlamp biomicroscopy and Scheimpflug-based tomography (Pentacam HR). The ASM, anterior elevation map (AEM) and PM were analyzed and compared to study the “kissing birds” sign, the “bell” sign, and cone location.Results: Patients’ average age was 25.93±8.05 (16–44 years). In total, 60% of patients were male. In group 1, the AEM in the best fit sphere (BFS) mode revealed no kissing birds sign, and the cone was central in 1 eye (25%) and paracentral in 3 eyes (75%). PM showed the bell sign in 4 eyes (100%). In group 2, the AEM in the BFS mode revealed the kissing birds sign in 2 eyes (18.2%), and the cone was central in 1 eye (9.1%), paracentral in 8 eyes (72.7%) and peripheral in 2 eyes (18.2%). PM didn’t show the bell sign in any eye.Conclusion: The claw pattern on the ASM is not a hallmark of PMD; it can be seen in PLK. Cone location does not relate to diagnosis. The “bell” sign on the PM is a deferential diagnostic sign in PMD.",
"keywords": [
"Keratoconus",
"Pellucid Marginal Degeneration",
"Corneal Tomography",
"Curvature map",
"Elevation map",
"Thickness map",
"Bell sign",
"Crab claw pattern",
"Kissing birds sign"
],
"content": "Introduction\n\nPellucid marginal degeneration (PMD) is an idiopathic, progressive, non-inflammatory, ectatic corneal disorder characterized by a peripheral inferior band of corneal thinning in a crescent-shaped pattern1, although PMD cases with areas of superior thinning have been reported2.\n\nSimilarities between PMD and keratoconus (KC) have led some ophthalmologists to consider PMD to be a peripheral form of KC2,3. Distinguishing between the two entities is of potential clinical importance since they differ markedly in prognosis and management. The management of PMD is unique since PMD is a progressive disease despite the fact that it is encountered in the third to fifth decade of life. Accordingly, corneal cross linking should still be one of the treatment options. When intracorneal rings (ICRs) implantation is indicated in the management of PMD, caution should be paid to the location of the inferior segment, since it passes through the inferior thinned area. Hence the need to calculate the depth of implantation depending on the thinnest point on the resumed passage, rather than on the thickness of the site of incision, in order to avoid deep corneal penetration.\n\nIn PMD, corneal tomographic analysis reveals a flattening in the vertical meridian, inducing a significant against-the-rule (ATR) astigmatism and a significant steepening around the area of maximum thinning3. This corneal configuration corresponds to a tomographic map that shows the classical claw pattern (Figure 1).\n\nNotice the marked flattening of the cornea along the vertical meridian and the marked steepening of the inferior corneal periphery, which extends into the mid-peripheral inferior oblique corneal meridians associated with against-the-rule astigmatism.\n\nAlthough corneal tomography is an important tool for the diagnosis of this corneal pathology, it should not be used as the only diagnostic criterion because it has been shown that this pattern is not always associated with the diagnosis of PMD; it might be seen with some other corneal ectatic disorders4. Therefore, pachymetric and biomicroscopic findings must also be considered for a reliable diagnosis.\n\nIn our study, we are reporting a \"bell-shaped\" sign on the pachymetry map (PM) in PMD which corresponds to the inferior thinning of the cornea observed by the slitlamp biomicroscopy. We are also reporting cases of KC with claw pattern on the sagital map but with neither the bell sign on the PM nor the inferior thinning on slitlamp biomicroscopy, identifying these cases to be “pellucid-like keratoconus (PLK).”\n\n\nPatients and methods\n\nA retrospective descriptive case series was performed in Damascus University in 2011. Clinical and tomographic findings of 15 eyes (9 patients) were reviewed and qualitatively analyzed. Inclusion criteria consisted of having a classic crab claw pattern on the anterior sagital curvature map (see Figure 1) taken by Pentacam® HR corneal tomographer (OCULUS Optikgeräte GmbH, Germany).\n\nPatients were distributed into two groups based on clinical and tomographic findings. The diagnosis of PMD was based on both corneal tomography and clinical findings of peripheral corneal thinning in an arcuate or crescentic pattern on slitlamp biomicroscopy (Figure 2 and Figure 3). Therefore, group 1 was considered PMD; group 2 was given the name of “pellucid-like keratoconus (PLK)” since the inferior thinning was not observed in this group. The PMD group (group 1) consisted of 4 eyes that had the claw pattern on the anterior sagital map (ASM), and an inferior corneal thinning on slitlamp biomicroscopy. The PLK group (group 2) consisted of 11 eyes that had the claw pattern on the ASM, but without an inferior corneal thinning on slitlamp biomicroscopy.\n\nA: Bell sign on the thickness map, it is an indicator of inferior corneal thinning; compare values between the inferior part of this cornea with other peripheral parts. B: Scheimpflug image showing the thinning (white arrow).\n\nNotice the abrupt narrowing of the slit beam in the area of inferior thinning (white arrow).\n\nAnterior sagital curvature, anterior elevation, and pachymetry maps were qualitatively analyzed and compared between groups 1 and 2. The anterior elevation map was studied using the best fit sphere (BFS) float mode to localize the cone and to identify the kissing birds sign. The cone was considered central, paracentral or peripheral when the apex of the cone was within the central 3 mm zone, within 3–5 mm zone or out of the central 5 mm zone respectively (Figure 4).\n\nThe cone is central when its apex is within the 3mm central circle, paracentral when it is in between 3 and 5mm central circles, and peripheral when it is out of the 5mm central circle.\n\n\nResults\n\nPatients’ average age was 25.93±8.05 (16–44) years. A total of 60% of the patients were male. Average age of patients was 23±7.31 (16–33) years in group 1, and 27±8.03 (19–44) years in group 2.\n\nTable 1 summarizes the results in both groups regarding the crab claw pattern, kissing birds sign, cone location, bell sign and inferior thinning on slitlamp biomicroscopy. In group 1, the cone was paracentral or central in 75% or 25% of cases respectively. Claw pattern, bell sign and inferior thinning were found in 100% of cases. In group 2, the kissing bird sign was present in 18.2% of cases, the cone was paracentral, peripheral or central in 72.7%, 18.2% or 9.1% of cases respectively. Bell sign and inferior thinning were absent.\n\n\nCase report\n\nA 27–year-old woman presented to the outpatient department with the complaint of blurred vision in both eyes. The uncorrected distance visual acuity (UDVA) was 0.7 (decimal) in the right eye (OD) and 0.2 (decimal) in the left eye (OS). On examination, the manifest refraction was -1.75D Cyl @65° OD, and-6.00D Cyl @105° OS. Corrected distance visual acuity (CDVA) was 1.0 (decimal) OD, and 0.7 (decimal) OS. Slitlamp biomicroscopy of the right eye revealed a clear cornea and normal features. The left eye showed inferior peripheral band of thinning extending from the 4 o’clock position to the 8 o’clock position. The intraocular pressure was normal in both eyes. Fundus examination revealed normal features. There were neither systemic nor local associations.\n\nCorneal tomography (Figures 5a and b) revealed inferior peri-limbal steepening with the crab claw pattern on the anterior sagital curvature map in both eyes. The anterior elevation map with BFS float mode revealed paracentral cones without the kissing birds sign in both eyes, the PM showed that the bell sign was present in the left eye and absent in the right eye. Therefore, diagnosis of PLK OD and PMD OS was made.\n\n(A): The right eye is PLK; notice the absence of the bell sign. (B): The left eye is PMD; notice the presence of this sign.\n\n\nDiscussion\n\nIn addition to careful clinical examination, corneal tomography has been recognized as an important and sensitive tool in detecting and managing ectatic corneal disorders, such as PMD and KC5,6. According to some reports, the typical sagital curvature map of the condition shows marked flattening of the cornea along a vertical axis and a steepening of the inferior cornea peripheral to the site of the lesion3. On the other hand, Lee BW et al. reported cases of ectatic corneal disorders with the same pattern4. Therefore, careful studying of corneal tomography with the main three maps is mandatory for diagnosing PMD and differentiating it from other ectatic corneal disorders. The main three maps consist of the anterior sagital curvature map, anterior elevation map and PM.\n\nIn our study, the crab claw pattern on the ASM was the inclusion criteria. Thereafter, cases were divided according to the presence or absence of the inferior corneal thinning on slitlamp biomicroscopy. Group 1 was identified as PMD when the thinning was present, and group 2 was identified as PLK when this sign was absent. We studied the other main maps qualitatively in both groups.\n\nOn the anterior elevation map, the kissing birds sign can be seen, and theoretically it should be always seen in PMD since it is a peripheral ectatic corneal disorder. In our study, we found that the presence of this sign was related to cone location. When the cone was peripheral, this sign was present; when the cone was central or paracentral, this sign was absent This sign appeared when the BFS float mode was used, while it disappeared when switching to the best fit toric ellipsoid (BFTE) float mode, as shown in Figure 6. In group 1, the cone was central or paracentral in 100% of cases, and the kissing birds sign was absent in 100% of cases. In group 2, the cone was peripheral in 18.2% of cases, in which the kissing birds sign was present.\n\nWhen it exists, it appears on the BFS float mode (A), and disappears when switching to the BFTE mode (B).\n\nCone location can be identified on the elevation maps or on the tangential curvature map, but not on the sagital curvature map7. In our study, we found no correlation between cone location and the differentiation between the two groups; i.e. the cone could be central, paracentral or peripheral in both entities. Therefore, neither the kissing birds sign nor the peripheral cone was a hallmark of PMD. Figure 7 is a PMD case without the kissing birds sign.\n\nNotice the bell sign on the corneal thickness map.\n\nThe PM showed thinning in PMD corneas towards the inferior part of the cornea, which was consistent with what was observed by the slitlamp biomicroscopy; a peripheral band of thinning of the inferior cornea from the 4 o'clock position to the 8 o'clock position and the light slit became very narrow abruptly in the inferior part of the cornea, which was the hallmark of the disease. This thinning was characterized with a special sign on the PM that can be called \"bell\" sign as shown in Figure 2. This sign was present in 100% of cases in group 1 and was absent in 100% of cases in group 2. Therefore, the hallmark of PMD was the bell sign on the PM, and this sign was due to inferior corneal thinning encountered in PMD.\n\nOn the other hand, there was no correlation between cone location and the thinnest area of the ectatic cornea. In group 1, although the bell sign was present and was an indicator of inferior thinning, the cone was central or paracentral in 100% of cases. On the other hand, the bell sign was absent in 100% of cases in group 2 although there were 2 cases (18.2%) with peripheral inferior cones.\n\nIn regard to the location of the thinnest point of the cornea, theoretically, the thinnest point should be inferiorly displaced in both PMD and in KC, but the amount of displacement on the Y coordinate should be much larger in PMD, especially in advanced cases (Figure 8: white arrows). This is not always true. In very advanced PMD, the cornea is severely distorted, particularly in the inferior part of the cornea, and the tomographers may miss many data from this part, and extrapolate the area with dark dots indicating that an important part of the data was missing in this area, as shown in Figure 9.\n\nThe thinnest location is largely inferiorly displaced (white arrows).\n\nThe red arrows point to the thinnest location. The white arrows point to cone location. Black arrow points to a black dotted area suggesting that some data were missing in the inferior cornea due to severe corneal distortion.\n\nIt is well known in the literature that PMD usually starts later in life than KC: it presents in the third to fifth decade of life3,5. In our study, the mean age of presentation was the third decade in both PMD and PLK groups. There was one patient with bilateral PMD and his age was 16 years at presentation. In a series of 58 patients reported by Sridhar and Mahesh8, there was an 8–year-old patient with PMD. Young ages were also seen in group 2; the age of the patients ranged from 19 to 44 years. Thus, patients' age is not an important issue when differentiating PMD from KC.\n\nPMD and KC are bilateral ectatic corneal disorders, although they can be asymmetric in severity between both eyes8. Nevertheless, some studies reported the occurrence of PMD in one eye and KC in the fellow eye9. In our study, we reported a case with PMD in one eye and PLK in the fellow eye.\n\nRegarding the case report, taking into account the clinical features, high ATR astigmatism, typical corneal tomography features and the presence of the bell sign on PM supported by peripheral corneal thinning, a diagnosis of PMD was made in the left eye and PLK in the right eye.\n\nIn conclusion, we reported cases of KC mimicking the tomographical appearance of PMD, and we called that pellucid-like keratoconus (PLK). Moreover, identifying features of PMD on corneal tomography and slitlamp biomicroscopy is very important since there are some tomographical similarities between PMD and PLK, especially in early stages of the former which misguides doctors to misinterpret PLK as PMD. We also reported a sign on the PM in PMD, and we called it the “bell” sign.\n\nFinally, the similarity between the two entities, in addition to what we found in the reported patient who has both entities, may suggest that PLK could represent a closely related disorder to PMD, just as Forme Fruste KC is to KC.",
"appendix": "Author contributions\n\n\n\nMazen Sinjab contributed to the study concept and design, analysis and interpretation of data, critical revision of the manuscript, and provided statistical expertise and supervision. Lara Youssef contributed as a co-author, helping with data collection and drafting 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 article was presented at the 2011 ESCRS conference in Vienna.\n\n\nReferences\n\nRabinowitz YS: Keratoconus. Surv Ophthalmol. 1998; 42(4): 297–319. PubMed Abstract | Publisher Full Text\n\nSridhar MS, Mahesh S, Bansal AK, et al.: Superior pellucid marginal corneal degeneration. Eye (Lond). 2004; 18(4): 393–9. PubMed Abstract | Publisher Full Text\n\nMaguire LJ, Klyce SD, McDonald MB, et al.: Corneal topography of pellucid marginal degeneration. Ophthalmology. 1987; 94(5): 519–24. PubMed Abstract\n\nLee BW, Jurkunas UV, Harissi-Dagher M, et al.: Ectatic disorders associated with a claw-shaped pattern on corneal topography. Am J Ophthalmol. 2007; 144(1): 154–6. PubMed Abstract\n\nKarabatsas CH, Cook SD: Topographic analysis in pellucid marginal corneal degeneration and keratoglobus. Eye (Lond). 1996; 10(Pt 4): 451–455. PubMed Abstract | Publisher Full Text\n\nSridhar MS, Mahesh S, Bansal AK, et al.: Pellucid marginal corneal degeneration. Ophthalmology. 2004; 111(6): 1102–1107. PubMed Abstract | Publisher Full Text\n\nHolladay JT: Detecting Forme Fruste Keratoconus With the Pentacam. Supplement to Cataract & Refractive Surgery Today 2008: 11–12. Reference Source\n\nWagenhorst BB: Unilateral pellucid marginal degeneration in an elderly patient. Br J Ophthalmol. 1996; 80(10): 927–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKayazawa F, Nishimura K, Kodama Y, et al.: Keratoconus with pellucid marginal corneal degeneration. Arch Ophthalmol. 1984; 102(6): 895–6. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "377",
"date": "19 Nov 2012",
"name": "Virender Sangwan",
"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 done study with a very good documentation of the case.",
"responses": [
{
"c_id": "80",
"date": "19 Nov 2012",
"name": "Mazen Sinjab",
"role": "Author Response",
"response": "Thank you Virender for your endorsement."
}
]
},
{
"id": "378",
"date": "26 Nov 2012",
"name": "Michael W Belin",
"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 appear not to be aware of the current literature which clearly distinguishes the tomographic patterns of inferior keratoconus from Pellucid. The limitation of curvature analysis and the importance of the pachymetric map have been previously well documented. The authors reference list is outdated and lacks articles that clearly describe the tomographic findings of true Pellucid: Walker RN et al. (2008), Belin MW et al. (2011) and Belin MW et al. (2012).The anterior curvature patters described as “crab claw” or many other descriptors are non-specific and highlight the limitations of curvature when trying to imply shape. Adding additional descriptors (e.g. bell shape) is counterproductive.The authors use the term “tomographic” without making a distinction between anterior curvature (classic topography) and tomography which includes both anterior and posterior corneal elevation and corneal thickness maps.The pachymetric “bell sign” is also a poor descriptor and is seen in this example because of the scale chosen. If the authors had chosen an appropriate scale that covered the full range of thickness, they would see the more classic inferior band that has been previously described and published.The case report is fraught with mistakes. First the authors keep stating that the best-fit-sphere was used but the machine is set for a toric-ellipsoid. The pachymetric map has not reliable data inferior, which is why no thickness readings are produced. One cannot diagnose true PMD in this case.All in all, the paper is poorly researched and the maps show a lack of understanding of the displays.",
"responses": [
{
"c_id": "79",
"date": "12 Dec 2012",
"name": "Mazen Sinjab",
"role": "Author Response",
"response": "Dear Prof Belin, Thank you so much for your valuable comments.Before I make any changes in my article, I would like to ask some questions. I have read the article “Scheimpflug Photographic Diagnosis of PellucidMarginal Degeneration” by Walker RN et al. (2008), I will refer to it as article number 1; and the article “What’s in a name: Keratoconus, Pellucid Marginal Degeneration and Related Thinning Disorders” by Belin MW et al. (2011), I will refer to it as article number 2. Unfortunately I could not have the third article which is “Simplified Nomenclature for Describing Keratoconus” by Belin MW et al. (2012). I would be very thankful if you could provide me with it and it would be so kind of you.The questions are:1. In article 2: the author stated: “in our original review article PMD was described as a typically bilateral, clear, inferior, peripheral corneal thinning disorder, characterized by a narrow band of corneal thinning (often approaching 20% normal thickness) separated from the inferior limbus by a relatively uninvolved area 1 to 2 mm in width.” However, nothing was mentioned in this article and in article 1 about the new definition of PMD and whether it is bilateral or can be unilateral. So the question is: can PMD be unilateral?2. In article 2 the author stated: “Ertan and Bahadir in 2006 described the use of the femtosecond laser and intrastromal rings to correct PMD. While no corneal thickness maps were presented, the posterior elevation maps were typical of KCN and not classic PMD.” But may I ask: what is typical of KCN and not classic PMD on the posterior elevation map? The same question about what the author mentioned about Kubaloglu and associates.3. How do you define that the cone is central, paracentral or peripheral on the elevation map with the BFS mode? I mean when do say the the cone is central (within 3 mm, 4mm, 5mm….) since this was not mentioned in both articles?4.In article 2, the author recommended that “extrapolated data should not be displayed” and thus the 9mm scale should be used, therefore according to this recommendation, the peripheral part of the cornea will not be displayed or estimated and the pachymetric changes of PMD will not be evaluated?! At the same time, the authors use the extrapolated areas in many of their talks during conferences and articles!5. In article 1: how could the authors recognize the thinning in case of focal edema? And how could they depend on Placido or even Scheimpflug in this very distorted cornea? They also stated:” a bilateral superior flattening” in figure 4 but only OS was displayed; What about OD, is it possible to see the flattening on OD despite edema even if it was focal and inferior?6. In article 1, page 2: the author stated:” thinning within 2 mm of the limbus, and a more normal corneal thickness inferior to the band of thinning.” But all of that lie in the extrapolated area which should not be displayed as the author recommended in article 2!Sincerely Yours, Mazen"
},
{
"c_id": "109",
"date": "13 Dec 2012",
"name": "Michael Belin",
"role": "Reviewer Response",
"response": "Dr. Sinjab, I sent you the requested article via this publisher.In reference to the findings of KCN vs PMD on the posterior elevation, Inf KCN shows the classic positive islands of elevation when using the standard BFS (sphere). Elevation maps are not overly useful in true PMD because the cornea is so flat in the central 8.0 mm zone that the reference surface is so flat as to not accentuate the pathology. This is why the pach map is the most important (opened up to show the full cornea)As far as which is a central cone, vs paracentral, vs peripheral. I do not believe there is anything established but I use the central 3.0 for “central” and outside 6.0 for “inferior” but it is often just a visual description.In general we limit our display to 9.0 mm as for routine screening this is easier. That is not the case for peripheral disease. In cases of true PMD, you have to show the entire cornea. It is true that the more peripheral the data the more likely it will be extrapolated but if the Pentacam produced a thickness reading it is useful for visual diagnosis. If the number is produced, but extrapolated, it means it did not meet the strict machine criteria. If no number (thickness) is produced then the map needs to be repeated as there is no useful data.In article 1 the thinning was easily seen in the other eye as this was bilateral. The Scheimplfug images were still reliable in the eye with the hydrops except for the far periphery so the central and paracentral flattening could still be seen. The reason both eyes were not shown in all displays is a limit of the journal for case reports."
},
{
"c_id": "110",
"date": "13 Dec 2012",
"name": "Mazen Sinjab",
"role": "Author Response",
"response": "Thank you very much Prof. Belin, and many thank to this journal that gave me the opportunity to learn from you. You were so kind to send me the article.But may I ask you: could PMD be unilateral?Sincerely Yours, Mazen"
},
{
"c_id": "150",
"date": "14 Dec 2012",
"name": "Michael Belin",
"role": "Reviewer Response",
"response": "UNILATERAL – I don’t know. I would guess that if PMD (as is KCN) is ultimately a disorder of collagen (i.e. genetic / biochemical) that the collagen would be abnormal in both eyes though clinical presentations may be unilateral or highly asymmetic."
}
]
},
{
"id": "379",
"date": "27 Nov 2012",
"name": "Natalie Afshari",
"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 the case report introduce the idea of pellucid like keratoconus (KC). They report cases of KC with a claw pattern on the sagital map but with neither the bell sign on the Pellucid nor the inferior thinning on the slit-lamp biomicroscopy. This idea is new and therefore worthy of publication.",
"responses": [
{
"c_id": "78",
"date": "27 Nov 2012",
"name": "Mazen Sinjab",
"role": "Author Response",
"response": "Dear Professor Afshari,During my past 16 years of practice as a refractive surgeon, I have seen more than 3000 cases of keratoconus and ectatic corneal disorders at my center and in the refractive clinic in the Damascus University hospital, and many of those cases were referred to me for a second opinion. This number seems to be large, but the cause behind it was that in Syria such diseases are relatively common due to close intermarriage, environment and to the high prevalence of vernal keratoconjunctivitis; this registers an incidence higher than the international one which is 1:2000, to be about 1:400 in Syria, and in some well known regions in Syria one can say 1:100. PMD consists of about 20% of the seen cases, and it is seen not only in adults but also in young patients, which gives the disease a more aggressive nature. The same can be said about KC, I have seen many cases with ages younger than 10 years old (the youngest was 4 years old!). Due to such a high number of cases that I have seen, I could recognize cases that lie in between KC and PMD although both entities have their own tomographic features; and that was the idea behind my study."
}
]
}
] | 1
|
https://f1000research.com/articles/1-48
|
https://f1000research.com/articles/1-46/v1
|
09 Nov 12
|
{
"type": "Research Article",
"title": "Major approaches in early diagnostics of common variable immunodeficiency in adults in Moscow",
"authors": [
"Alexander V Karaulov",
"Irina V Sidorenko",
"Anna S Kapustina",
"Irina V Sidorenko",
"Anna S Kapustina"
],
"abstract": "Common variable immunodeficiency (CVID) is a primary immunological disease characterized predominantly by hypogammaglobulinemia. The main clinical manifestations are severe recurrent infections that often lead to structural damage of affected organs. The early start of adequate intravenous immunoglobulin therapy has significantly improved the prognosis of this serious disorder. Patients with CVID are also predisposed to autoimmune and lymphoproliferative complications. This article deals with the features of this primary immunodeficiency in adults. Clinical manifestations, immunological features and treatment concepts were gathered during 21 years of observation of such patients in Moscow. The authors suggest early predictive clinical signs of CVID in adults.",
"keywords": [
"primary immunodeficiency",
"common variable immunodeficiency",
"recurrent infections of respiratory tract",
"malabsorption syndrome",
"autoimmune hematological disorders",
"intravenous immunoglobulin."
],
"content": "Background\n\nCommon variable immunodeficiency is a primary immunodeficiency (PID) characterized by variable age of onset of symptoms, recurrent severe bacterial infections, increased incidence of autoimmune disorders and malignancy1–3. CVID is the most frequent symptomatic primary immunodeficiency in adults1. CVID is usually diagnosed in patients presenting with hypogammaglobulinemia and a clinical history of recurrent and severe infections, mostly affecting the respiratory tract1–3. To confirm a diagnosis of CVID, it is important to exclude other primary antibody deficiency syndromes and secondary causes of hypogammaglobulinemia1.\n\n\nMaterials and methods\n\nWe observed 57 patients with CVID in 1990–2011. Written informed consent for publication of clinical details was obtained from all patients or their next of kin.\n\nWe conducted a comprehensive examination, treatment and, quality-of-life study of 27 males and 30 females aged from 18 to 74 (mean: 39±1, 95 years). The medical history was collected from all patients (emphasis being on the manifestation of first symptoms, mainly affected organs and systems), as was family history, the dynamics of clinical symptoms and, the effectiveness of the therapy. All patients were physically examined, and blood and urine tests were also conducted. We also used X-rays and computed tomography of the chest and sinuses, esophagogastroscopy, colonoscopy (indication), ultrasonography of the abdomen, kidneys and, thymus, research conducted on electrocardiogram (ECG) and, respiratory function. Patients were examined by an ear, nose and throat (ENT) specialist, gastroenterologist, pulmonologist, hematologist and, a rheumatologist.\n\nImmunological testing included the study of the following parameters of the immune system: the total number of leukocytes and lymphocytes, the percentage and absolute number of T-lymphocytes (CD3+-cells) and subpopulations of T-lymphocytes (CD4+-cells, CD8+-cells), natural killer (NK)-cells (CD16+-cells) and, B-lymphocytes (CD19+-cells) in peripheral blood. Levels of immunoglobulin classes A, M, G and, E in serum were determined by nephelometry. Immunophenotyping of peripheral blood lymphocytes was measured by cytometry using monoclonal antibodies. Autoantibodies were measured by indirect immunofluorescence. For detection of red-cell antibodies we used Coombs' reaction. The phagocytic link of immunity was investigated by determining the absolute and percentage content of neutrophils and monocytes, the absorptive activity of leukocytes, phagocyte chemiluminescence and bactericidal activity of leukocytes.\n\nAssessment of treatment efficiency was carried out by analyzing the dynamics of clinical and immunological parameters, measuring the duration of remission of the underlying disease and, studying the patient's of quality of life. Monitoring of serum immunoglobulin G (IgG) was carried out before the infusion of intravenous immunoglobulin (IVIG) to evaluate the achievement of adequate levels of IgG (not less than 5.0 g/l).\n\nAnalysis of the incidence of infectious complications and receiving antibiotics was carried out using the questionnaire \"Total well-being: the emergence of infectious complications\" filled prior to the IVIG infusion and, on the 2nd and 10th day after the transfusion medicine.\n\nAssessment of the safety of IVIG was performed on the basis of data on tolerability, and analysis of the patient’s vital signs and markers of renal function. Before each infusion of IVIG, as well as on the 2nd and 10th day after the transfusion the patients completed a questionnaire \"Total well-being: the emergence of reactions, possibly related to the introduction of IVIG\" pointing out any reactions that had arisen since the last replacement therapy.\n\nIn the case of a new drug being prescribed to the patient, the tolerability of IVIG was assessed during the first infusion by monitoring blood pressure, heart rate, body temperature, and studying the markers of renal function. Biochemical blood tests were conducted with a focus on the concentration of urea, electrolytes and creatinine before the first infusion, and on the second and fifth days after infusion.\n\nTo study patient quality of life, we used the Russian version of the general health questionnaire MOS SF-36 (36-item Measures of Sickness Short-Form Health Survey). The results were displayed on a scale from 0 to 100 points. A high rate showed a good quality of life of the respondent, a low showed a bad quality of life.\n\nOn the basis of this study, we worked out early predictive clinical signs of CVID for primary care.\n\n\nResults\n\nIn the observed group, duration of illness ranged from 3 to 62 years (mean 23.6±1,18 years). The first symptoms appeared in 28 patients (49.1%) before 15 years and in 29 patients (50.9%) over 15 years. The disease started in 10 patients in the first decade of life, in 25 patients in the second decade of life, in 13 patients in the third decade of life, in 4 patients at 30 to 40 years, and in 5 patients over 40 years.\n\nFamily history was burdened in 5 patients (8.8%): early death of children (with probable immunodeficiency) from infectious complications took place in 2 families; the brother (with unspecified immunodeficiency) of one patient died of toxoplasmosis; the son of one patient and the mother and son of the other patient have primary immunodeficiency – selective IgA-deficiency.\n\nThe main clinical manifestations were bacterial infections of the respiratory system; this occurred in 53 patients (93%). CVID began with recurrent purulent sinusitis in 57.9% of cases, ear infections in 26.3%, bronchitis in 54.4% and, pneumonia in 56.1%. In 4 patients (7%), the first clinical manifestation of CVID was damage to the gastrointestinal tract (enterocolitis and malabsorption syndrome). In 6 patients (10.5%), disease also began with autoimmune cytopenias: hemolytic anemia in 2, neutropenia in 1 and, thrombocytopenia in 3 patients (Table 1).\n\nBefore the diagnosis of PID and the use of replacement IVIG therapy, foci of chronic infection of the respiratory system formed in 54 patients (94.7%): chronic bronchitis 94.7%, chronic sinusitis 75.4% and, chronic otitis media 42.1%. Recurrent diarrhea and weight loss were observed in 47.4% patients. Septicemia, meningitis and, peritonitis were found in 15.8% cases. Severe clinical presentations required re-hospitalization including in intensive care units. Because of late assignment of IVIG therapy, chronic infection foci of the respiratory system formed in 94.7% of patients: bronchiectasis – 36.8%, local fibrosis – 50.9% (lobectomy was performed in 2 patients), chronic sinusitis – 75.4% (Table 2). Despite severe manifestations, a delay in diagnosis of CVID and consultation by immunologist was from 2 to 45 years (mean: 14.5 years).\n\nIn 24.6% of cases, autoimmune diseases were observed, including hemolytic anemia, thrombocytopenia, neutropenia (also in the manifestation of the disease), and nephritis, hepatitis, and scleroderma. A total of 47.4% of patients had recurrent herpes infection and, 47.4% of cases had evidence of non-malignant lymphoproliferation: an increase of peripheral lymph nodes, intestinal lymphoid hyperplasia and, splenomegaly, which occured separately or combined. In total, 15.8% of patients had malignancies: cancer of the stomach, colon, breast, ovarian, T-cell lymphoma, chronic lymphocytic leukemia and, multiple myeloma (Table 2). 40.4% of patients had manifestations of arthritis involving the knee, ankle, elbow and, wrist joints.\n\nThe initial investigation of immune status revealed reduction in the total IgG, IgA and, IgM levels in 89.5% of patients. In 2 cases, normal values of IgA were detected; in 4 cases normal values of IgM were shown. In all patients, IgG levels were below 4 g/l and in 36.8% only trace concentrations were identified (Table 3).\n\nThe content of B-lymphocytes (CD19 +) was within normal limits in 41 patients (71.9%), increased up to 21.1–30.9% (0.58–0.83×109/L) in 6 patients (10.5%) and, reduced to 1.9–4.9% (0.01–0.1×109/L) in 10 patients (7 women and 3 men). In 29 patients (50.9%) CD4+- cells were reduced to 16.4–24.7% (0.14–0.3×109/L). A total of 22 patients (38.6%) had elevated levels of CD8+-cells to 37.2–65.0% (0.77–1.82×109/L). 56.1% of cases (32 patients) showed a reduction of NK-cells to 1.8–4.4% (0.01–0.09×109/L).\n\nAll patients received combined therapy with mandatory regular IVIG infusion4,5. At the beginning of treatment, the patients received IVIG preparations at a dose of 0.3 g/kg body weight weekly for 1.5–2 months. After reaching the required level of IgG (5.0 g/l), patients received regular IVIG replacement therapy at a dose of 0.4–0.5 g/kg6,7. The introduction of a drug was conducted once every 4 weeks (an average of 1 per 30.5±1.5 days) intravenously.\n\nIVIG dose adjustment was carried out, taking into account the response to therapy, monitoring of foci of chronic infection, the presence of autoimmune disease and, malabsorption syndrome, and was conducted under the mandatory supervision of IgG levels in the serum, which was 5.0–8.0 g/l (mean 6.8±0.29 g/l) in all cases except for 4 patients, who did not comply with IVIG regimen, where it was 3.5–4.3 g/l.\n\nSide effects of IVIG therapy occurred in 16 (41%) patients. In total, 4 (10%) patients had systemic reactions within 20 minutes after the start of IVIG – lowering of blood pressure to 60/30 mm-Hg, bronchospasm and, vomiting. In 12 (31%) patients, after transfusion body temperature was recorded to rise up to 37.3–37.9°C. Additionally, chills, headache, dizziness and general weakness were observed. These symptoms characterize a reaction to the introduction of a large dose of protein, which is not an indication to stop administering the drug. Since IVIG is vital, individual selection of drugs was conducted, if necessary, patients received premedication with corticosteroids, antihistamines or inhibitors of prostaglandins, which were pre-introduced (20–30 minutes prior to transfusion of IVIG) to avoid possible adverse reactions.\n\nReplacement therapy in most cases is insufficient to control the foci of chronic infection, so in parallel with the introduction of IVIG, 40 (82%) of patients received broad-spectrum antibiotics. Due to this treatment the frequency of exacerbations of chronic foci of infection was significantly reduced. Four patients received acyclovir permanently because of the continuous recurrence of herpes infection. Immunosuppressive therapy was conducted in 7 patients with autoimmune thrombocytopenia and anemia. Hematopoietic growth factors were used in 5 patients with neutropenia, and 2 patients received ongoing treatment with growth factors. Due to the complex treatment, exacerbation of chronic bronchitis, sinusitis, otitis media and, herpes occured less often and less severely. The symptoms of malabsorption and reactive arthritis regressed, autoimmune cytopenia remission was achieved in all patients.\n\n\nConclusions\n\nDuring the first 5 years of observation in our clinic, in spite of regular IVIG therapy, 21% patients died due to late diagnosis of CVID, with very severe clinical presentations and complications. Therefore, we worked out early predictive clinical signs of CVID in adults on the basis of the recommendations of European Society for Immunodeficiencies (ESID):\n\n1) ≥ 4 bacterial respiratory tract infections (otitis, sinusitis, bronchitis, pneumonia) during 1 year;\n\n2) ≥ 2 radiologically confirmed pneumonias during 3 years;\n\n3) Recurrent or chronic bacterial respiratory tract infections combined with recurrent diarrhea;\n\n4) Bacterial infections of respiratory tract and/or recurrent diarrhea combined with severe generalized infections (septicemia, meningitis, osteomyelitis);\n\n5) Any of the above criteria combined with autoimmune diseases, especially autoimmune hemocytopenias (hemolytic anemia, thrombocytopenia, neutropenia).\n\n6) Chronic or recurrent bacterial infections of respiratory tract and/or malabsorption syndrome combined with malignancies, with bacterial infections preceding malignancies.\n\nThus, increasing awareness of PID among physicians is needed to reduce the number of undetected cases and to decrease the diagnostic delay. The developed approach and education of GPs has improved the diagnostics of predominantly antibody deficiencies in adults in Moscow.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from all patients or their next of kin.",
"appendix": "Author contributions\n\n\n\nAlexander Karaulov conceived the initial concept. Anna Kapustina drafted the manuscript. All authors were involved in data collection, data analysis and 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\nReferences\n\nBergbreiter A, Salzer U: Common variable immunodeficiency: a multifaceted and puzzling disorder. Expert Rev Clin Immunol. 2009; 5(2): 167–180. PubMed Abstract | Publisher Full Text\n\nCunningham-Rundles C, Bodian C: Common variable immunodeficiency: clinical and immunological features of 248 patients. Clin Immunol. 1999; 92(1): 34–48. PubMed Abstract | Publisher Full Text\n\nGeha RS, Notarangelo LD, Casanova JL, et al.: Primary immunodeficiency diseases: an update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee. J Allergy Clin Immunol. 2007; 120(4): 776–794. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDetková D, Español T: An update on treatment strategies for common variable immunodeficiency. Expert Rev Clin Immunol. 2009; 5(4): 381–390. PubMed Abstract | Publisher Full Text\n\nSewell WA, Buckland M, Jolles SR: Therapeutic strategies in common variable immunodeficiency. Drugs. 2003; 63(13): 1359–1371. PubMed Abstract | Publisher Full Text\n\nOksenhendler E, Gérard L, Fieschi C, et al.: Infections in 252 patients with common variable immunodeficiency. Clin Infect Dis. 2008; 46(10): 1547–1554. PubMed Abstract | Publisher Full Text\n\nQuinti I, Soresina A, Spadaro G, et al.: Long-term follow-up and outcome of a large cohort of patients with common variable immunodeficiency. J Clin Immunol. 2007; 27(3): 308–316. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "373",
"date": "09 Nov 2012",
"name": "Cem Akin",
"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": "374",
"date": "16 Nov 2012",
"name": "Anete Grumach",
"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 Russian experience with common variable immunodeficiency (CVID) patients. Although it is a descriptive report, there is restricted data published about Russian groups presenting primary immunodeficiency (PID) and this is a report from a State University. It helps provide an indication and raises awareness for the physicians with regards to PID diagnosis considering that 51 CVID cases were collected in 21 years of observation. No molecular studies were performed.",
"responses": []
},
{
"id": "375",
"date": "21 Nov 2012",
"name": "Francisco A Bonilla",
"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 only identified 57 patients with common variable immunodeficiency (CVID) over 21 years. This must represent only a fraction of the total number for such a large metropolitan area over such a long period of time. I suspect that many patients have gone undiagnosed and this leads me to question the general applicability of some of the authors’ findings.Furthermore, the authors do not include their case definition of CVID. There are many immunodeficiencies associated with hypogammaglobulinemia that would not be classified as CVID. The authors state that they studied quality of life in these patients, but they do not report any of that data here. The humoral and cellular immunologic data should be presented in a table with mean, standard deviation, median and range together with the authors’ normal ranges. The authors speak very generally about a variety of therapeutic measures applied in addition to IgG but doesn’t provide any quantitative (or often even qualitative) report of the response (or not) to these therapies. The mortality in these patents seems high. How does it compare with published assessment of mortality in CVID? Finally, the authors do not provide any evidence to support their conclusion that “The developed approach and education of GPs has improved the diagnostics of predominantly antibody deficiencies in adults in Moscow”.",
"responses": [
{
"c_id": "77",
"date": "05 Dec 2012",
"name": "Alexander Karaulov",
"role": "Author Response",
"response": "The article deals with data on the analysis of characteristics of adult patients with CVID in Moscow. The article reflects the work done during 20 years to improve the diagnosis of CVID and introduction of IVIG in Moscow.The diagnosis of CVID was verified on the basis of the typical clinical presentations and immunological deviations. CVID was diagnosed according to the criteria of this form of PID developed by ESID and PAGID. Other causes of hypogammaglobulinemia were excluded in these patients. For example, genetic typing was conducted to exclude X-linked agammaglobulinemia.The article focuses on the clinical manifestations of CVID, their characteristics, variety, the defeat of many organs and systems. The purpose of this scientific article is to draw attention of GPs to the problem of CVID. Due to awareness of the early clinical signs of CVID, GPs send patients to an immunologist. Thereby, a delay in diagnosis of CVID decreases. During past 10 years a delay in diagnosis of CVID in Moscow decreased from 14.5 to 6.4 years; due to early start of IVIG therapy quality of life has increased significantly – no operations on organs of respiratory tract in recent years.At the beginning of the formation of Moscow registry of adult patients with CVID there were only 4 alive patients over 18 years. Over the years of observation a number of adult patients has increased by more than 10 times. This is due to improvement of diagnosis of CVID and increase GPs, knowledge.More detailed characteristics of immunologic abnormalities in patients with CVID, the assessment of quality of life, more detailed description of the treatment strategy and the effectiveness of drugs will be presented in the following publications."
}
]
}
] | 1
|
https://f1000research.com/articles/1-46
|
https://f1000research.com/articles/1-47/v1
|
09 Nov 12
|
{
"type": "Research Article",
"title": "ALS-linked SOD1 in glial cells enhances ß-N-Methylamino L-Alanine (BMAA)-induced toxicity in Drosophila",
"authors": [
"Rafique Islam",
"Emily L Kumimoto",
"Hong Bao",
"Bing Zhang",
"Emily L Kumimoto",
"Hong Bao"
],
"abstract": "Environmental factors have been implicated in the etiology of a number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the role of environmental agents in ALS remains poorly understood. To this end, we used transgenic fruit flies (Drosophila melanogaster) to explore the interaction between mutant superoxide dismutase 1 (SOD1) and chemicals such as ß-N-methylamino L-alanine (BMAA), the herbicide agent paraquat, and superoxide species. We expressed ALS-linked human SOD1 (hSOD1A4V, and hSOD1G85R), hSOD1wt as well as the Drosophila native SOD1 (dSOD1) in motoneurons (MNs) or in glial cells alone and simultaneously in both types of cells. We then examined the effect of BMAA (3 mM), paraquat (20 mM), and hydrogen peroxide (H2O2, 1%) on the lifespan of SOD1-expressing flies. Our data show that glial expression of mutant and wild type hSOD1s reduces the ability of flies to climb. Further, we show that while all three chemicals significantly shorten the lifespan of flies, mutant SOD1 does not have a significant additional effect on the lifespan of flies fed on paraquat, but further shortens the lifespan of flies fed on H2O2. Finally, we show that BMAA shows a dramatic cell-type specific effect with mutant SOD1. Flies with expression of mutant hSOD1 in MNs survived longer on BMAA compared to control flies. In contrast, BMAA significantly shortened the lifespan of flies expressing mutant hSOD1 in glia. Consistent with a neuronal protection role, flies expressing these mutant hSOD1s in both MNs and glia also lived longer. Hence, our studies reveal a synergistic effect of mutant SOD1 with H2O2 and novel roles for mutant hSOD1s in neurons to reduce BMAA toxicity and in glia to enhance the toxicity of BMAA in flies.",
"keywords": [
"Autosomal-dominant mutations in the Cu/Zn-superoxide dismutase 1 (sod1) gene cause ~20% familial amyotrophic lateral sclerosis (fALS)1. To date",
"more than 140 mutations linked to fALS have been identified in sod1. Some of these mutants are enzymatically functional2",
"consistent with the idea that mutant sod1 results in a toxic gain-of-function rather than the loss of SOD1 activity3. This is further supported by studies of a mouse model where deletion of sod1 does not cause ALS-like motoneuron disease4",
"5."
],
"content": "Introduction\n\nAutosomal-dominant mutations in the Cu/Zn-superoxide dismutase 1 (sod1) gene cause ~20% familial amyotrophic lateral sclerosis (fALS)1. To date, more than 140 mutations linked to fALS have been identified in sod1. Some of these mutants are enzymatically functional2, consistent with the idea that mutant sod1 results in a toxic gain-of-function rather than the loss of SOD1 activity3. This is further supported by studies of a mouse model where deletion of sod1 does not cause ALS-like motoneuron disease4,5.\n\nBesides motoneurons, glial cells have also been known to play a role in SOD1-linked ALS. Using chimeric animals, Clement and colleagues showed that mutant SOD1 expressed in motoneurons survived significantly longer and delayed degeneration if the neighboring cells did not express mutant SOD16. Other investigators demonstrated that wild type MN neighbored with microglia and macrophages expressing mutant SOD1 underwent degeneration and contained ubiquitin-positive inclusions7,8. Yamanaka and colleagues showed that expression of wild type SOD1 outside of MN delayed the onset of the disease and extended the lifespan of transgenic mice up to 50%9. These experiments support the notion that fALS is a non-cell autonomous disease process where glial cells play a critical role in the progression of ALS.\n\nSeveral hypotheses have been proposed to account for non-cell autonomous mechanisms of ALS. Loss of the excitatory amino acid glutamate transporter (EAAT2) in astrocytes may cause glutamate-dependent excitotoxicity promoting disease progression10. Higher inflammatory response from microglia mediated by mutant-expressing astrocytes11 or inability to regulate glutamate receptor subunit 2 expression in motoneurons by mutant astrocytes12 have also been suggested as mechanisms for ALS development. Finally, release of unknown toxins by astrocytes as evidenced by rapid death of embryonic stem cell-derived motoneurons when co-cultured with mutant expressing glial cells also has been suggested13,14. These studies point to the importance of bilateral interactions between astrocytes and motor neurons in ALS development and progression.\n\nIn addition to genetic inheritance of fALS, environmental agents have also been identified as causes for ALS. β-N-methylamino-L-alanine (BMAA) is a non-specific amino acid that was found in the post-mortem brain samples of the people of Guam who developed amyotrophic lateral sclerosis/Parkinson-dementia complex (ALS/PDC) during the 1940s15,16. BMAA is generated by cyanobacteria and can be found in many aquatic and/or terrestrial ecosystems and is believed to accumulate in living organisms by a process called biomagnification17. Ross and colleagues reported behavioral changes in mice in response to intracerebroventricularly administered BMAA; these mice were hyper-excitable followed by whole body shaking/wobbling18. Intracranially injected BMAA caused injuries of hippocampal neurons in mice19. Using a cell culture system derived from spinal cord tissues of 13-day old mouse embryos, Rao and colleagues suggested that BMAA was selectively toxic to motor neurons via the AMP/Kainate receptor20. Lobner and colleagues have also proposed that BMAA is an agonist for NMDA and mGluR5 receptors because BMAA induces oxidative stress in cultured mouse cortical neurons21.\n\nIncreasing oxidative stress and reactive oxygen species (ROS) either by the herbicide paraquat or by excessive levels of hydrogen peroxide may also enhance neurodegeneration22–24. Paraquat has been implicated in Parkinson’s disease22,25,26 and in ALS. In a case-control study of ALS in northern Italy, Bonvicini and colleagues found that compared to age- and sex-matched controls, more ALS patients had experienced occupational pesticide exposure in excess of 6 months27. There was an increased risk of ALS among employees of the Dow Chemical Company who were exposed to the herbicide 2,4-dichlorophenoxyacetic acid versus other Dow employees28. Paraquat showed a decrease in ubiquitously expressed survival motor neuron 1 (SMN1) in the human cell line NSC34, implicating oxidative stress in the mechanism of ALS underlying SMN1 deficiency in familial ALS and potentially sporadic form of the disease29. More recently, TAR DNA-binding protein 43 (TDP-43) has been implicated in the etiology of sporadic ALS30. Using human neuron culture, Ayala et al. showed that H2O2 (10 µM) caused accumulation of TDP-43 in the cytosolic fractions, a condition presumably linked to pathology of ALS31.\n\nBMAA, paraquat, and hydrogen peroxide are also known to have debilitating effects on motor activity and longevity in fruit flies25,32,33. Pesticides were shown to cause complete or partial chromosome loss in repair-defective female Drosophila34. The effect of paraquat on longevity is best studied in fly models of Parkinson's disease35. Flies mutant for DJ-1 showed striking sensitivity to agents such as paraquat and rotenone36. Islam and colleagues have shown a significant loss of dopaminergic neurons when these flies were treated with either 500 µM rotenone or 10 mM paraquat37. Flies mutant for the glial lazarillo, which codes for the homologue of human Apolipoprotein D, also showed significantly reduced lifespan under paraquat treatment38. BMAA was found to be a glutamate agonist, reduced lifespan, impaired motor activity, and caused memory deficits in fruit flies39. Mekdara and colleagues recently also have reported a BMAA-induced decline in locomotion in a dose-dependent manner in Drosophila40. More recently, fruit flies fed with BMAA were shown to cause prolonged open state of the NMDA receptor channel41, suggesting a conserved molecular pathway for BMAA toxicity from mammals to flies.\n\nHow BMAA, paraquat, and H2O2 interact with mutant SOD1, however, remains unclear. In this report, we aim to understand the roles of mutant SOD1 expressed in different cell types in fly longevity when these flies are exposed to BMAA, paraquat, and hydrogen peroxide. We overexpressed mutant SOD1 in a cell-type specific fashion in flies and then treated them with 3 mM BMAA (in 3% sucrose), 20 mM paraquat (in fly food), or 1% hydrogen peroxide (in 3% sucrose). All three chemicals shortened the lifespan of the flies. However, flies with MN expression of mutant SOD1 lived longer on BMAA-containing food compared to control flies. Interestingly, flies with glial expression of mutant SOD1 died sooner on BMAA. Different from BMAA, H2O2 reduced lifespan of flies further in flies with mutant SOD1 expressed in any of the three cell types compared to controls. Unlike BMAA and H2O2, paraquat reduced longevity to a similar extent in both control flies and flies expressing mutant or wildtype SOD1. Hence, our results reveal a novel role for glial mutant SOD1 in mediating BMAA toxicity and a surprising role for motor neuronal mutant SOD1 in resisting BMAA toxicity. Furthermore, mutant SOD1 enhances H2O2 toxicity in shortening fly lifespan.\n\n\nMaterials and methods\n\nAll experimental flies were reared on cornmeal agar medium at constant room temperature (22°C) under a ~12 h/12 h light/dark cycle. Male flies were used in all experiments. F1 flies from Gal4 drivers>Canton S (CS) and Gal4 drivers>UAS-dsod1 crosses were used as control strain in all studies. Each food vial housed 10 flies.\n\nThe human sod1 transgenic lines (hsod1WT, hsod1A4V, and hsod1G85R), the wild type Drosophila SOD1 (dsod1), and motor neuron (D42) and glial (M1B) Gal4 drivers were described previously42. For hsod1G85R, four independent insertions were recombined to bring its expression level closer to that of hsod1wt, and hsod1A4V. The GAL4-UAS expression system was used to express sod1 transgenes in particular cell types. For motoneuron-specific expression, the D42-Gal4 driver line was used43,44. To express sod1 in glial cells alone M1B-Gal4 driver was used42. To express sod1 in both motoneurons and glial cells, both D42 and M1B Gal4 drivers were used.\n\nL-BMAA (B107) was purchased from Sigma (St. Louis, MO, USA) and dissolved in water. The BMAA solution was supplemented with 3% sucrose before treatment. Two pieces of 3M filter paper were placed in a clear plastic vial (2.5 × 9.5 cm2) and soaked with droplets of the BMAA/sucrose solution. The flies were transferred to new vials with newly soaked filter paper every 24 hours. Care was taken to avoid excessive accumulation of solutions to prevent the drowning of flies. The control flies were treated with 1, 3, 5 and 10 mM BMAA. However, for all the experiments of the transgenic flies 3 mM BMAA was used. Five and 35 day-old flies with undamaged wings were selected for the BMAA treatment. Flies under treatment and their control groups were monitored for survival every 24 hours. A fly was considered dead when it did not show any body movement after a few gentle tapings at the vial. When necessary, a light microscope was used to determine any movements of the limbs after a few gentle taps to confirm a fly's death. The numbers of dead flies were recorded for each day. Changes in longevity were computed by comparing with D42>UAS-dsod1 and D42>+ lines.\n\nMale sod1 transgenic and wild type flies aged 1–3 days were fed with standard cornmeal agar food containing 20 mM paraquat. The number of dead flies was recorded every 24 hours and the live flies were transferred to corresponding new vials with food and paraquat. The flies were observed until all flies were dead in experimental groups.\n\n30% hydrogen peroxide (EMD Chemicals, HX0635, Darmstadt, Germany) was diluted to 1% with a 3% sucrose solution. Approximately 100 male flies in groups of ten were aged to 5 days on standard cornmeal agar. They were then starved for six hours in clean plastic vials containing two pieces of filter paper soaked with 300µl of water. After 6 hours, starved flies were transferred to fresh vials containing filter paper soaked with either 300µl of the 1% H2O2/sucrose solution or 300µl of 3% sucrose solution as a control. Every 24 hours, the flies were transferred to fresh vials with newly soaked paper and monitored for survival. Flies were maintained at 22°C under a 12h/12h light/dark cycle for the duration of the experiment.\n\nWe modified the techniques of measuring motor activity from a previously published method45. Briefly, 59 day-old 10 male flies without any visible physical defect were selected and transferred to a vial with fresh food and harvested overnight (n=50 per genotype). These flies were not rendered to BMAA treatment. Next day, in a quiet area of the lab with normal light level, 10 flies were transferred to a clean and dry 100 ml glass cylinder. The flies were gently tapped down to the bottom of the cylinder and monitored for their climbing behavior in a 20-second period. These flies were assigned into three groups. Group 1: flies able to climb past 20 ml (35 mm); group 2: flies still climbing or roaming around in the bin in between 5 ml and 20 ml (7.5 mm -35 mm); group 3: flies that could not climb past 5 ml (7.5 mm).\n\nAll data were obtained from at least 3 experiments. All p values were based on 2-tailed t-tests or One-way ANOVA tests in the Prizm software and differences were considered significant if p<0.05. Error bars represent standard errors of the mean (SEM).\n\n\nResults\n\nClimbing is an innate negative gravitaxic behavior of Drosophila. Climbing activities have been used to measure the motor functions of fruit flies45. Transgenic mice overexpressing SOD1 showed late-onset progressive motor defects46. In flies, expression of mutant SOD1 reduced climbing capability42. Here, we tested the effect of mutant SOD1 overexpression in MN, glia, or in both on fly climbing and noticed a remarkable locomotive slowdown as the flies approached around 60 days. In a modified climbing assay, we assigned all our experimental flies a bin in a 100 ml cylinder depending on their location during a 20 second climbing trip. The fastest climber reached the top bin (above 35 mm), the sluggish group reached the middle bin (above 7.5 mm but below 35 mm), and the slowest group remained at the bottom bin (below 7.5 mm). We found that about 80% of the D42>UAS-sod1 flies reached the top bin (Figure 1A). On the contrary, less than 20% of the M1B>UAS-sod1 flies reached the top bin (Figure 1B). Overall, there is a significant shift of fly distributions to the bottom and middle portions of the graduated cylinder in M1B>UAS-sod1 flies. Interestingly, however, about 80% of the D42+M1B>UAS-sod1 flies also reached the top bin (Figure 1C). These results indicate significantly differential effects of SOD1 proteins in motor neurons and glial cells; mutant SOD1 in glia has a more profound negative effect on climbing in aging flies.\n\nHealthy looking 60 day-old male flies were selected for this assay. Ten flies were placed in a 100 ml glass cylinder. Following a brief and gentle tapping, the distribution of flies was then counted in 3 bins at different heights (above or at 35 mm height, between 7.5 mm and 35 mm, and 7.5 mm or lower). Climbing pattern of flies expressing SOD1 in motoneurons (A), glial cells (B), and co-expressing SOD1 in both motoneurons and glial cells (C) are shown. Flies expressing human SOD1 proteins in glial cells show a significant shift of distribution to the bottom of the cylinder compared to M1B>UAS-dsod1 (the control fly) and in comparison with their expression in motoneurons (A) or dual expression in MNs + glia (C). These observations suggest a significant impairment of climbing activities at 60 days in flies expressing human SOD1s in glia. The statistical significances were calculated using Prizm software, Two-way ANOVA and * p<0.01 and ** p<0.03 (n=50 for each genotype).\n\n\n\nSeveral reports have shown neurotoxic effects of BMAA in animal models15,18. More recently, Zhou and colleagues studied the dietary effects of BMAA in Drosophila32 and found BMAA as the most toxic among a few other excitatory amino acids to 1–3 day-old flies. BMAA shortened lifespan and resulted in neurological deficiencies. To examine the age-dependent effects of BMAA on longevity of flies, we fed 5 day-old wild type (Canton S) flies with 1, 3, 5 and 10 mM BMAA (Figure 2) and noticed a dosage-dependent decline in survival in both age groups. We opted to use 3 mM concentration in all subsequent experiments because of its intermediate effects on longevity.\n\nFive day-old male flies were reared in 1, 3, 5, and 10 mM BMAA diluted in 3% sucrose for up to 30 days (10 flies per vial). The 50% survival time for 1, 3, 5, and 10 mM BMAA was 19, 14, 10, and 4 days, respectively. The sham (0 mM BMAA) treated flies did not show any toxicity during the observation period. The statistics was performed using One-way ANOVA analysis of variance in Prizm software, p<0.01 (n=50 for 1, 3, and 5 mM BMAA groups; n=20 for the 0 mM BMAA group).\n\n\n\nTo determine a cellular target of SOD1 and its interaction with BMAA, H2O2, and paraquat we used the GAL4-UAS system to express SOD1 proteins in specific neuronal or glial cell types. We used D42-Gal443,44, M1B-Gal4 and a recombined D42+M1B-Gal4 drivers to express wild type human sod1 (hsod1wt) and two fALS-linked mutants of hsod1 (hsod1A4V, hsod1G85R) in motoneurons (D42>UAS-hsod1), glial cells (M1B>UAS-hsod1), and in both motoneurons and glial cells (D42+M1B>UAS-hsod1), respectively. The Drosophila native sod1 (driver>UAS-dsod1) and wild type (driver>CS) flies were also crossed to each driver and included as controls in all experiments. The male F1 flies were collected on day 1 after they emerged from the pupal case and 10 flies were housed per vial. These flies were fed with 3 mM BMAA in 3% sucrose solution at the age of 5 and 35 days for up to 30 days. The flies under treatment were monitored every 24 hours and the numbers of dead flies were recorded. The surviving flies were transferred to new food vials after every count. Our results show that the lifespan for 50% population alive (L50) for D42>UAS-hsod1A4V, D42>UAS-hsod1G85R, D42>UAS-hsod1wt flies was increased 33%, 41%, and 66%, respectively, when compared with the D42>UAS-dsod1 flies (Figure 3 A–C, and Table 1). Comparison of the same transgenic flies with the D42>CS flies treated with 3 mM BMAA also produced an increase in survival of the all D42>UAS-hsod1 flies (Figure 3).\n\nShown are survival rates of 5 day-old male sod1 transgenic flies under 3 mM BMAA treatment (10 flies per vial). Panels A, B, and C represent the survival rate of flies expressing mutant human A4V, G85R, wild type SOD1 proteins and control flies (D42>CS and D42>UAS-dsod1) in motoneurons using the D42-Gal4 driver. Flies expressing the human SOD1s (both the hSOD1WT, and mutants, hSOD1A4V and hSOD1G85R) survived longer compared to the control flies. At the 50% survival rate, the longevity is increased by 33%, 41% and 66% for A4V, G85R, wt SOD1, respectively, when compared to D42>dsod1 and D42>CS flies. Differences were considered statistically significant if p<0.05 and were calculated by Paired t-test method by using Prizm software. n=30 for D42>CS and D42>A4V, respectively; n=50 for the rest of genotypes.\n\n\n\nA minus (‘-’) indicates a decrease in survival rate.\n\nOn the contrary, the M1B>UAS-hsod1 flies were more sensitive to BMAA insult. The L50 for these flies was reduced by 19%, 30%, and 30% for M1B>UAS-hsod1A4V, M1B>UAS-hsod1G85R, M1B>UAS-hsod1wt, respectively compared to M1B>UAS-dsod1 flies (Figure 4 A, B, C and Table 1). However, to our surprise, the D42+M1B>UAS-hsod1 flies were found to be also capable of resisting BMAA toxicity in contrast to M1B>UAS-hsod1 flies. The resistance in this group was not as strong as it was in the D42>UAS-hsod1 group. The results show that the L50 for the D42+M1B>UAS-hsod1 flies was increased to 0%, 17%, and 22% for hSOD1A4V, hSOD1G85R, hSOD1wt, respectively in comparison with D42+M1B>UAS-dsod1 flies (Figure 5 A, B, C and Table 1). The analysis of the results from the 35 day-old flies showed that the extension or reduction in survival rates was consistent genotype-wide and in agreement with those found in 5 days old flies (Table 1 and Figure S1–Figure S3).\n\nShown are survival rates of 5 day-old male sod1 transgenic flies under 3 mM BMAA treatment (10 flies per vial). Panels A, B, and C represent flies expressing mutant human A4V, G85R, wild type SOD1 proteins, respectively, in comparison with control flies (M1B>CS and M1B>UAS-dsod1) in glia using the M1B-Gal4 driver. At the 50% survival rate, the longevity for A4V, G85R, and hSOD1 is reduced by 19%, 19%, 28%, respectively, compared to D42>UAS-dsod1 and D42>CS. Differences were considered statistically significant if p<0.05 and were calculated by a paired t-test method using Prizm software. n=30 for M1B>A4V and M1B>dsod1, respectively; n=40 for M1B>G85R; n=50 for M1B>CS.\n\n\n\nShown are survival rates of 5 day-old male sod1 transgenic flies (10 flies per vial). Panels A, B, and C represent flies expressing mutant human A4V, G85R, and wild type SOD1 proteins and control flies (D42+M1B>CS and D42+M1B>UAS-dsod1) in motoneurons and glia using D42-Gal4, M1B-Gal4, respectively. At the 50% survival rate, the longevity for A4V, G85R, and hSOD1 is increased by 0%, 17%, and 22% for A4V, G85R, and hSOD1 flies, respectively, compared to D42>UAS-dsod1 and D42>CS. Differences were considered statistically significant if p<0.05 and were calculated by a paired t-test method using Prizm software (n=50 for each genotype).\n\n\n\nShown are survival rates of 35 day-old male sod1 transgenic flies under 3 mM BMAA treatment (10 flies per vial). Panels A, B, and C represent flies expressing mutant human A4V, G85R, and wild type SOD1 proteins, respectively. Like 5 day-old time points (Figure 3 A–C), the SOD1 flies survived longer compared to the controls (CS and dSOD1). The 50% survival rate is increased by 10, 30 and 30% for A4V, G85R, wt SOD1, respectively, when compared to dSOD1 flies. Differences were considered statistically significant if p<0.01 and were calculated by a paired t-Test method by using Prizm software (n=30 for each genotype).\n\n\n\nShown are survival rates of 35 day-old male sod1 transgenic flies under 3 mM BMAA treatment (10 flies per vial). Panels A, B, and C represent flies expressing mutant human A4V, G85R, wild type SOD1 proteins. The 50% survival rate of A4V, G85R, and hSOD1 when compared with dSOD1 is decreased by 31, 38, and 38%, respectively. Differences were considered statistically significant if p<0.02 and were calculated by a paired t-test method by using Prizm software (n=30 for each genotype).\n\n\n\nShown are survival rates of 35 day-old male sod1 transgenic flies under 3 mM BMAA treatment (10 flies per vial). Panels A, B and C represent flies expressing mutant human A4V, G85R, wild type SOD1 proteins. The 50% survival rate of A4V, G85R, and hSOD1 when compared with dSOD1 is increased by 8, 30, and 15%, respectively. Differences were considered statistically significant if p<0.01 and were calculated by a paired t-test method using Prizm software (n=30 for each genotype).\n\n\n\nOur study feeding flies with H2O2 (1% in 3% sucrose) confirms previous published results on longevity33. In control flies (D42>+ or D42>UAS-dsod1), most flies survived the first day on H2O2, but gradually died within the next three-six days. We then tested whether mutant SOD1 expressed in MN or glia has any additional effects together with H2O2 on fly longevity. Our results show that all 5 day old flies began to die faster starting on the third day of treatment except D42>UAS-dsod1, and continued dying rapidly on day 4. On day 5, almost all flies, including D42>UAS-dsod1, were dead despite some resistance of dSOD1 on day 4 (Figure 6A). When mutant SOD1 was expressed in glia alone, we observed a similar survival pattern as of D42>UAS-dsod1 flies (Figure 6B). In flies with dual expression of mutant SOD1 in both MNs and glia, the subtle resistance by the dSOD1 flies observed in D42 and M1B flies diminished and they were dead sooner than other flies. However, on day 5 all flies were dead (Figure 6C). These results show that while human mutant SOD1 renders some resistance when expressed in either motor neurons alone or along with glia, these proteins adversely affect flies' survival when expressed in glial cells alone. Interestingly, though such resistance is very short in effect, Drosophila native SOD1 resists H2O2 toxicity in D42 and M1B flies, but not in D42+M1B flies. We also noted that our other control flies (Gal4 drivers>CS) were mostly similar to flies expressing mutant hSOD1. This makes it more difficult to interpret the data. The genetic argument is that dSOD1 would be a better control. Thus, we opted to consider the effects of mutant hSOD1s in comparison with dSOD1.\n\nFive day-old male flies expressing SOD1 proteins in motor neurons (A), glial cells (B) and in motoneurons together with glial cells (C) were treated with 1% H2O2diluted in 3% sucrose. The results indicate an enhancing effect of H2O2 on mutant SOD1 toxiciy in MNs or glia when compared to driver>UAS-dsod1, but not to driver>CS control. Statistical analysis was performed using a two-tailed ANOVA method in Prizm software and was considered significant if p<0.05 (n=80–96 for each genotype).\n\n\n\nParaquat is also known to shorten the lifespan of flies25,33. In a human cell line, NSC3, paraquat decreases expression of ubiquitously expressed Survival motor neuron resulting in increased oxidation29. In our laboratory, we reproduced the shortening of lifespan in wild type flies under treatment with parquat. Interestingly, however, in contrast to BMAA, we observed no significant effects of paraquat (20 mM paraquat contained in fly food) on the lifespan of the SOD1-expressing flies compared to the wild type control groups (Figure S4). The L50 was 11–12 days for D42>UAS-sod1, M1B>UAS-sod1, respectively, and L50 was 4–6 days for D42+M1B>UAS-sod1 in the paraquat experiment.\n\nFive day-old male flies expressing SOD1 proteins in motor neurons (A), glial cells (B) and in motoneurons together with glial cells (C) were treated with 20 mM paraquat in fly food (10 flies per vial). The results do not indicate any significant effect of paraquat in SOD1 flies of all genotypes. Total flies uses for D42Gal4>CS, 183; D42Gal4>UAS-dsod1, 194; D42Gal4>UAS-hSOD1A4V, 138; D42Gal4>UAS-hSOD1G85R, 206; D42Gal4>UAS-hSOD1, 231. M1BGal4>CS, 148; M1BGal4>UAS-dsod1, 171; M1BGal4>UAS-hSOD1A4V, 164; M1BGal4>UAS-hSOD1G85R, 144; M1BGal4>UAS-hSOD1, 200. D42+M1BGal4>CS, 148; D42+M1BGal4>UAS-dsod1, 156; D42+M1BGal4>UAS-hSOD1A4V, 177; D42+M1BGal4>UAS-hSOD1G85R, 286; D42+M1BGal4>UAS-hSOD1, 213.\n\n\n\n\nDiscussion\n\nIn this report we investigated the effects of BMAA, hydrogen peroxide, and paraquat on the lifespan of fruit flies and their interactions with mutant hSOD1 in search for a cell type-specific target for SOD1. As expected, all these chemicals significantly shortened the lifespan of wild-type and transgenic flies. However, significant reductions in lifespan of transgenic flies are cell type specific only for BMAA. In our studies, we added paraquat and hydrogen peroxide into fly food and in sucrose, respectively. The lifespan was significantly longer for flies fed with paraquat in the fly food than those fed with paraquat in sucrose33. This suggests that nutrients in the fly food are beneficial to flies in their resistance to paraquat toxicity, a finding consistent with a recent report33. Our results show that paraquat did not show any dramatic synergistic effects with mutant hSOD1 whether the mutant protein is expressed in MNs, glia, or in both. Further, expression of an enzymatically inactive form of mutant SOD1 (G85R)2 did not enhance the toxic effect of paraquat. Finally, expression of the Drosophila wild-type SOD1 (dSOD1) in MNs, glia, or in both cells does not afford any resistance to paraquat. These results suggest that SOD1 may not be the primary enzyme in flies to protect tissues from attacks by radical oxygen (-O-) produced by paraquat. It is possible that the systemic production of radical oxygen (-O-) by paraquat outweighs the dismutase activity of the SOD1 proteins expressed in MN and glial cells. Interestingly, dSOD1 briefly extends the lifespan of D42 and M1B but not of D42+M1B flies fed on H2O2. We do not know why there is a small difference between paraquat and H2O2 but speculate that the level of radical oxygen species may be significantly lower in H2O2 or that H2O2 causes lethality through mechanisms different from those by paraquat.\n\nAnother important finding of this study is the first demonstration of unique interactions between mutant hSOD1 and the environmental factor BMAA in a cell type-specific manner. In motoneurons mutant SOD1 appears to have protective roles in prolonging the lifespan of BMAA-treated flies whereas the same mutant SOD1 enhances the sensitivity to BMAA when expressed in glia. Notably, the protective effect on longevity is also observed for the enzymatically inactive G85R. These results suggest that the neuroprotective role of SOD1 against BMAA is unrelated to the dismutase activity of SOD1. Further, G85R expressed in glia does not further enhance the toxic effect of BMAA, arguing against the idea that BMAA shortens the fly lifespan via an oxidative stress mechanism.\n\nHow mutant SOD1 offers neuroprotection in motoneurons but enhances BMAA toxicity in glia remains unclear. It has been proposed that BMAA exerts toxic effects in murine cortical cell lines through activation of NMDA and mGluR5 receptors21. Bruijn and colleagues observed inclusions in astrocytes in a G85R transgenic mouse model, implicating astrocytes as primary targets for mutant-SOD1 mediated damage46. Trotti and colleagues suggest that the failure of astrocyte-mediated glutamate transport may be linked to neurodegeneration47. In an embryonic stem cell-based system motoneurons carrying either the non-pathological human sod1 transgene or the ALS-linked sod1 mutant G93A allele showed neurodegenerative properties when co-cultured with G93A glial cells14. Our data are consistent with the notion that mutant hSOD1 plays a major non-cell autonomous role in glia. We favor a ‘glutamate excitotoxicity’ hypothesis in which mutant SOD1 impairs glial function thereby potentiating the effect of BMAA on neurons.\n\nIn conclusion, our results provide a new understanding of SOD1 target cell type. While most research showing glial involvement in ALS was done in mouse model9,11,48, we are the first to show such involvement in a Drosophila model. The interactions of SOD1 and BMAA and H2O2 in our model represent a non-cell autonomous type of effect on the motor activity and overall survival of the transgenic flies.",
"appendix": "Author contributions\n\n\n\nRI, ELM, HB and BZ conceived and designed the experiments; RI, ELM, and HB conducted the experiments and analyzed the data. RI and BZ wrote the manuscript, and ELM and HB provided feedback on the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by NIH/NIEHS R21 grant (7R21ESO14441 to BZ) and by a Health research grant from the Oklahoma Center for the Advancement of Science & Technology (OCAST), (HR09-172S to BZ).\n\n\nAcknowledgements\n\nWe thank Dr. Nancy Bonini for the initial collaboration on establishing the fly model of SOD1-related ALS and her sharing of the transgenic lines.\n\n\nReferences\n\nRosen DR, Siddique T, Patterson D, et al.: Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993; 362(6415): 59–62. PubMed Abstract | Publisher Full Text\n\nBorchelt DR, Lee MK, Slunt HS, et al.: Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity. Proc Natl Acad Sci U S A. 1994; 91(17): 8292–8296. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCleveland DW, Rothstein JD: From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS. Nat Rev Neurosci. 2001; 2(11): 806–819. PubMed Abstract | Publisher Full Text\n\nBruijn LI, Miller TM, Cleveland DW, et al.: Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu Rev Neurosci. 2004; 27: 723–749. PubMed Abstract | Publisher Full Text\n\nBruijn LI, Houseweart MK, Kato S, et al.: Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science. 1998; 281(5384): 1851–1854. PubMed Abstract | Publisher Full Text\n\nClement AM, Nguyen MD, Roberts EA, et al.: Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice. Science. 2003; 302(5642): 113–117. PubMed Abstract | Publisher Full Text\n\nBoillee S, Vande VC, Cleveland DW: ALS: a disease of motor neurons and their nonneuronal neighbors. Neuron. 2006; 52(1): 39–59. PubMed Abstract | Publisher Full Text\n\nBoillee S, Yamanaka K, Lobsiger CS, et al.: Onset and progression in inherited ALS determined by motor neurons and microglia. Science. 2006; 312(5778): 1389–1392. PubMed Abstract | Publisher Full Text\n\nYamanaka K, Boillee S, Roberts EA, et al.: Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice. Proc Natl Acad Sci U S A. 2008; 105(21): 7594–7599. PubMed Abstract | Publisher Full Text | Free 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): 1604–1609. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYamanaka K, Chun SJ, Boillee S, et al.: Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nat Neurosci. 2008; 11(3): 251–253. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Damme P, Bogaert E, Dewil M, et al.: Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity. Proc Natl Acad Sci U S A. 2007; 104(37): 14825–14830. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNagai M, Re DB, Nagata T, et al.: Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nat Neurosci. 2007; 10(5): 615–622. PubMed Abstract | Publisher Full Text\n\nDi Giorgio FP, Carrasco MA, Siao MC, et al.: Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neurosci. 2007; 10(5): 608–614. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpencer PS, Nunn PB, Hugon J, et al.: Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science. 1987; 237(4814): 517–522. PubMed Abstract | Publisher Full Text\n\nCox PA, Sacks OW: Cycad neurotoxins, consumption of flying foxes, and ALS-PDC disease in Guam. Neurology. 2002; 58(6): 956–959. PubMed Abstract\n\nCox PA, Banack SA, Murch SJ, et al.: Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc Natl Acad Sci U S A. 2003; 100(23): 13380–13383. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoss SM, Spencer PS: Specific antagonism of behavioral action of \"uncommon\" amino acids linked to motor-system diseases. Synapse. 1987; 1(3): 248–253. PubMed Abstract | Publisher Full Text\n\nBuenz EJ, Howe CL: Beta-methylamino-alanine (BMAA) injures hippocampal neurons in vivo. Neurotoxicology. 2007; 28(3): 702–704. PubMed Abstract | Publisher Full Text\n\nRao SD, Banack SA, Cox PA, et al.: BMAA selectively injures motor neurons via AMPA/kainate receptor activation. Exp Neurol. 2006; 201(1): 244–252. PubMed Abstract | Publisher Full Text\n\nLobner D, Piana PM, Salous AK, et al.: Beta-N-methylamino-L-alanine enhances neurotoxicity through multiple mechanisms. Neurobiol Dis. 2007; 25(2): 360–366. PubMed Abstract | Publisher Full Text\n\nMcCormack AL, Thiruchelvam M, Manning-Bog AB, et al.: Environmental risk factors and Parkinson’s disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat. Neurobiol Dis. 2002; 10(2): 119–127. PubMed Abstract | Publisher Full Text\n\nBerry C, La Vecchia C, Nicotera P, et al.: Paraquat and Parkinson’s disease. Cell Death Differ. 2010; 17(7): 1115–1125. PubMed Abstract | Publisher Full Text\n\nKlein JA, Ackerman SL: Oxidative stress, cell cycle, and neurodegeneration. J Clin Invest. 2003; 111(6): 785–793. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJackson-Lewis V, Blesa J, Przedborski S: Animal models of Parkinson’s disease. Parkinsonism Relat Disord. 2012; 18(Suppl 1): S183–185. PubMed Abstract | Publisher Full Text\n\nLangston JW, Ballard P, Tetrud JW, et al.: Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science. 1983; 219(4587): 979–980. PubMed Abstract | Publisher Full Text\n\nBonvicini F, Marcello N, Mandrioli J, et al.: Exposure to pesticides and risk of amyotrophic lateral sclerosis: a population-based case-control study. Ann Ist Super Sanita. 2010; 46(3): 284–287. PubMed Abstract | Publisher Full Text\n\nBurns CJ, Beard KK, Cartmill JB: Mortality in chemical workers potentially exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) 1945–94: an update. Occup Environ Med. 2001; 58(1): 24–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTurner BJ, Parkinson NJ, Davies KE, et al.: Survival motor neuron deficiency enhances progression in an amyotrophic lateral sclerosis mouse model. Neurobiol Dis. 2009; 34(3): 511–517. PubMed Abstract | Publisher Full Text\n\nNeumann M, Sampathu DM, Kwong LK, et al.: Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science. 2006; 314(5796): 130–133. PubMed Abstract | Publisher Full Text\n\nAyala V, Granado-Serrano AB, Cacabelos D, et al.: Cell stress induces TDP-43 pathological changes associated with ERK1/2 dysfunction: implications in ALS. Acta Neuropathol. 2011; 122(3): 259–270. PubMed Abstract | Publisher Full Text\n\nZhou X, Escala W, Papapetropoulos S, et al.: BMAA neurotoxicity in Drosophila. Amyotroph Lateral Scler. 2009; 10(Suppl 2): 61–66. PubMed Abstract | Publisher Full Text\n\nPeng C, Zuo Y, Kwan KM, et al.: Blueberry extract prolongs lifespan of Drosophila melanogaster. Exp Gerontol. 2012; 47(2): 170–178. PubMed Abstract | Publisher Full Text\n\nWoodruff RC, Phillips JP, Irwin D: Pesticide-induced complete and partial chromosome loss in screens with repair-defective females of Drosophila melanogaster. Environ Mutagen. 1983; 5(6): 835–846. PubMed Abstract | Publisher Full Text\n\nCanet-Aviles RM, Wilson MA, Miller DW, et al.: The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc Natl Acad Sci U S A. 2004; 101(24): 9103–9108. PubMed Abstract | Publisher Full Text\n\nMeulener M, Whitworth AJ, Armstrong-Gold CE, et al.: Drosophila DJ-1 mutants are selectively sensitive to environmental toxins associated with Parkinson’s disease. Curr Biol. 2005; 15(17): 1572–1577. PubMed Abstract | Publisher Full Text\n\nIslam R, Yang L, Sah M, et al.: A neuroprotective role of the human uncoupling protein 2 (hUCP2) in a Drosophila Parkinson’s disease model. Neurobiol Dis. 2012; 46(1): 137–146. PubMed Abstract | Publisher Full Text\n\nSanchez D, Lopez-Arias B, Torroja L, et al.: Loss of glial lazarillo, a homolog of apolipoprotein D, reduces lifespan and stress resistance in Drosophila. Curr Biol. 2006; 16(7): 680–686. PubMed Abstract | Publisher Full Text\n\nZhou X, Escala W, Papapetropoulos S, et al.: beta-N-Methylamino-L-alanine Induces Neurological Deficits and Shortened Life Span in Drosophila. Toxins (Basel). 2010; 2(11): 2663–2679. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTom Mekdara N, Goto JJ, Choudhury S, et al.: A Novel Lenticular Arena to Quantify Locomotor Competence in Walking Fruit Flies. J Exp Zool A Ecol Genet Physiol. 2012; 317(6): 382–94. PubMed Abstract | Publisher Full Text\n\nGoto JJ, Koenig JH, Ikeda K: The physiological effect of ingested beta-N-methylamino-L-alanine on a glutamatergic synapse in an in vivo preparation. Comp Biochem Physiol C Toxicol Pharmacol. 2012; 156(3–4): 171–7. PubMed Abstract | Publisher Full Text\n\nWatson MR, Lagow RD, Xu K, et al.: A drosophila model for amyotrophic lateral sclerosis reveals motor neuron damage by human SOD1. J Biol Chem. 2008; 283(36): 24972–24981. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPhillips JP, Tainer JA, Getzoff ED, et al.: Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium. Proc Natl Acad Sci U S A. 1995; 92(19): 8574–8578. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYeh E, Gustafson K, Boulianne GL: Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila. Proc Natl Acad Sci U S A. 1995; 92(15): 7036–7040. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartinez VG, Javadi CS, Ngo E, et al.: Age-related changes in climbing behavior and neural circuit physiology in Drosophila. Dev Neurobiol. 2007; 67(6): 778–791. PubMed Abstract | Publisher Full Text\n\nBruijn LI, Becher MW, Lee MK, et al.: ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron. 1997; 18(2): 327–338. PubMed Abstract | Publisher Full Text\n\nTrotti D, Danbolt NC, Volterra A: Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol Sci. 1998; 19(8): 328–334. PubMed Abstract | Publisher Full Text\n\nIlieva H, Polymenidou M, Cleveland DW: Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J Cell Biol. 2009; 187(6): 761–772. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "453",
"date": "15 Nov 2012",
"name": "Joy Goto",
"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": "454",
"date": "26 Nov 2012",
"name": "Grace Zhai",
"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. One thing to note is that the three panels (A, B, and C) in Figures 3, 4, 5, S1, S2, and S3 should be combined, as it is redundant to show the same data three times in these figures.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-47
|
https://f1000research.com/articles/1-45/v1
|
08 Nov 12
|
{
"type": "Research Article",
"title": "Correlating data from different sensors to increase the positive predictive value of alarms: an empiric assessment",
"authors": [
"Yuval Bitan",
"Michael F O’Connor",
"Michael F O’Connor"
],
"abstract": "Objectives: Alarm fatigue from high false alarm rate is a well described phenomenon in the intensive care unit (ICU). Progress to further reduce false alarms must employ a new strategy. Highly sensitive alarms invariably have a very high false alarm rate. Clinically useful alarms have a high Positive-Predictive Value. Our goal is to demonstrate one approach to suppressing false alarms using an algorithm that correlates information across sensors and replicates the ways that human evaluators discriminate artifact from real signal.Methods: After obtaining IRB approval and waiver of informed consent, a set of definitions, (hypovolemia, left ventricular shock, tamponade, hemodynamically significant ventricular tachycardia, and hemodynamically significant supraventricular tachycardia), were installed in the monitors in a 10 bed cardiothoracic ICU and evaluated over an 85 day study period. The logic of the algorithms was intended to replicate the logic of practitioners, and correlated information across sensors in a way similar to that used by practitioners. The performance of the alarms was evaluated via a daily interview with the ICU attending and review of the tracings recorded over the previous 24 hours in the monitor. True alarms and false alarms were identified by an expert clinician, and the performance of the algorithms evaluated using the standard definitions of sensitivity, specificity, positive predictive value, and negative predictive value.Results: Between 1 and 221 instances of defined events occurred over the duration of the study, and the positive predictive value of the definitions varied between 4.1% and 84%.Conclusions: Correlation of information across alarms can suppress artifact, increase the positive predictive value of alarms, and can employ more sophisticated definitions of alarm events than present single-sensor based systems.",
"keywords": [
"Historically",
"desire for high performance and concern over legal liability has motivated the design of alarm systems in clinical medicine that are highly sensitive",
"but which also have a very high false positive rate1. False positive alarms have multiple causes",
"including ‘low threshold’ settings",
"motion interference",
"and false signals generated from a variety of clinical activities. Paradoxically",
"the high rate of false positive (80–99%) alarms trains practitioners to ignore alarms2",
"3. Alarm fatigue is a phenomenon where practitioners come to ignore alarms3. In many ICUs",
"the audible signals from the alarms built into their bedside monitors are disabled or silenced. This strategy has reduced the noise pollution associated with these systems without obviously decreasing their performance."
],
"content": "Introduction\n\nHistorically, desire for high performance and concern over legal liability has motivated the design of alarm systems in clinical medicine that are highly sensitive, but which also have a very high false positive rate1. False positive alarms have multiple causes, including ‘low threshold’ settings, motion interference, and false signals generated from a variety of clinical activities. Paradoxically, the high rate of false positive (80–99%) alarms trains practitioners to ignore alarms2,3. Alarm fatigue is a phenomenon where practitioners come to ignore alarms3. In many ICUs, the audible signals from the alarms built into their bedside monitors are disabled or silenced. This strategy has reduced the noise pollution associated with these systems without obviously decreasing their performance.\n\nPrevious literature4 points towards the need to reduce the total number of alarms that occur in working environments such as the ICU. One strategy to increase the clinical utility of such alarms is to specify alarm definitions that are less sensitive, but have a high positive predictive value (PPV). Based on Signal Detection Theory5 strategies to accomplish this could include higher thresholds for alarm conditions, and advanced alarms that might be less likely to be triggered by either artifact or clinical activity. Higher thresholds would alarm less often, but would also alert caregivers later in the course of a patient’s decompensation. Importantly, setting the threshold for an alarm at a higher value may not substantially change the rate of false alarms from artifacts. Alarms with a higher positive predictive value would be triggered less often, and would be much more likely to summon bedside caregivers to respond appropriately. The greatest risk from this strategy is that an alarm might not sound when a life threatening condition is present.\n\nAnother strategy to reduce the rate of false alarms is to increase the sophistication of the alarm software6, in effect, making the monitor analyze data across sensors to verify the alarm condition. For example, when a patient moves, she can disturb her EKG electrodes and produce an EKG signal that appears to be ventricular fibrillation. In this instance, the EKG alarms 'V fib'! Frequently, however, other sensors are generating information that could be used to suppress that false alarm.\n\nThe correlation of information across sensors may be especially effective in reducing artifact related false alarms. For example, either an arterial line or a pulse oximeter might detect a pulse in the above patient, which is impossible in the setting of V fib. By comparing information across sensors, smarter monitors might decrease the rate of false alarms and facilitate the early detection of other clinical problems. Similarly, a patient who is tachycardic should have a high heart rate on their EKG, pulse-oximeter, and arterial line (if one is present). Simply correlating information from these different sensors is likely to decrease the rate of false alarms without reducing sensitivity to a clinically important degree. The presence of alarms triggered by a single sensor is an artifact of device history, not deliberate design. Advanced software could be programmed to replicate the logic that caregivers utilize to discriminate real conditions from artifact.\n\nAnother strategy to increase response to alarms is to assess parameters that are clinically important in the context of the abnormal parameter. For example, tachycardia associated with a precipitous decline in blood pressure is almost always clinically more significant than tachycardia associated with no change or an increase in blood pressure. Advanced alarms which alert bedside caregivers to important patterns of change (clinical correlations) are far more likely to generate the desired clinical response than monitors that continually alarm for situations that represent little or no danger. Such alarms would have a high PPV, lower rate of false alarm, and are likely to elicit more purposeful responses from caregivers.\n\nIn this study, we utilized Philip’s Event Monitoring software to define alarm conditions that correlated information across sensors, and which were prospectively intended to have a high positive predictive value. The software being studied in this trial is intended to serve both of these purposes, and the data collected during this trial will inform its refinement.\n\nThe Clinical Study of the Event Surveillance Software/Event Alarming usability and functionality is a feedback collection and comparative multi-center study of the recently released Philips' D. O. software for Intellivue Monitors (MP70/90). The software was designed to detect scenarios that are either harmful or might predict a critical situation for the ICU patient.\n\n\nMethods\n\nCardiac surgery patients in a 10 bed Intensive Care Unit were eligible for Intellivue monitor data capture for the purpose of determining the incidence of true positive events as compared with false positive events. IRB approval was obtained and waiver of consent was granted. Event Surveillance software was installed into every monitor in the ICU, and operational in parallel with the institutional default alarms settings. Five clinically important alarm scenarios (‘smart alarms’) were programmed into the bedside monitors using the Event Surveillance software (Table 1).\n\nNotes on names in Table 1\n\n1. SVT + BP – Supraventricular Tachycardia and Blood Pressure – This is intended to indicate high heart rate with low blood pressure, as frequently occurs in patients with Atrial fibrillation and a rapid ventricular rate. Tachycardia associated with hypertension, as commonly occurs with light sedation, would not trigger this alarm.\n\n2. VTACH + BP – This is intended to indicate ventricular tachycardia with low blood pressure. This definition would be much less likely to be triggered by motion artifact than the EKG alarm is.\n\n3. LV SHOCK – This is intended to detect Left ventricular failure (cardiogenic shock).\n\n4. TPX & TPND – This is intended to detect either tamponade or tension pneumothorax.\n\n5. HYPOVL – This is intended to indicate low blood pressure from hypovolemia.\n\nThe first two (SVT+BP and Vtach+BP) definitions required the presence of an arterial line and EKG. The third and fourth (LV shock and tamponade) required a pulmonary artery catheter and an arterial line. Hypovolemia required the presence of a CVP monitor, and could be triggered by a blood pressure from either the arterial line or a non-invasive blood pressure cuff. If the requisite sensors were not present in a patient, then events and definitions related to that event were not analyzed for the purposes of this study. For example, if atrial fibrillation happened in a patient without an arterial line, it was ignored for the purposes of this study.\n\nWhen any alarm (factory installed or event surveillance software) is triggered, a log of monitor data from the event is stored in the central monitoring station. Every day, the log file of events from the previous 24 hours was reviewed with the ICU physician (attending or fellow), and all events were classified (Table 2).\n\n\nResults\n\nEvents were recorded for 85 days from Mid-May 2007 until Mid-November 2007 (Table 3). In total 564 patient days monitored were monitored.\n\nFor SVT + BP there were a total of 221 events over 35 patient days. There were 529 patient days where this event did not occur (i.e., no alarm and no false negative occurred).\n\nOut of the 221 events, 170 were True Positive events and 1 was a TP predict event (see Table 2 for abbreviations). 19 were FP Artifact and 22 were FP Insufficient Definition. Thus, out of a total of 221 alarms, 171 were true positive, for a PPV of 0.807.\n\nThe 171 TP events were concentrated on 10 patients (patient IDs: 31, 1, 22, 11, 10, 32, 19, 17, 8, 4). The 9 FN events happened to 7 patients. Ventricular Tachycardia with hypotension occurred only in one patient during the 564 recorded patient days, and there were no FP or FN events. Left Ventricular (LV) Shock occurred in 42 of the 564 patient days and among 6 patients in total. There were 8 FP Artifact events and only 1 FN with threat. Thus, the PPV here was 0.81. Tamponade had only one TP event, and 23 FP events (for 13 patient days), as well as 1 Non-threatening FN event in a total of 564 patient days.\n\nThe PPV was therefore 0.04. Hypovolemia had 8 TP events, as well as 21 FP events (for 10 patients) and 2 FN events. For Hypovolemia the PPV was 0.27.\n\n\nDiscussion\n\nNo alarm system in use or under development can perform perfectly. Hence, practitioners are compelled to trade-off among the kinds of failures that are acceptable to them. While there is ample literature that demonstrates that simple monitors generate vastly more false alarms than real alarms, the regulatory environment of most medical practice has generated regulations that require these alarms to be activated.\n\nIn the current study, the data we have collected thus far suggest that the SVT+BP trigger group is likely to be a useful alarm in clinical practice. The evidence is not quite as strong, but is encouraging for LV shock as well. The other events we were surveying for, tamponade, hypovolemic shock, and Vtach+BP were all sufficiently rare (by our definition) that we remain unable to evaluate the positive predictive performance of these trigger groups. While LV shock is commonplace in the ICU where this study was conducted, most patients were actively managed by their caregivers and rarely met the definition for LV shock we employed. Importantly, the absolute rate of false positive alarms for these groups was low (29%) compared to the approximately 80% rate reported in other studies2, consistent with our hypothesis that correlating information across sensors might decrease the rate of false positive alarms. Correlating information across sensors and simultaneously probing for important deflections from other sensors produced a dramatic improvement in alarm performance in this study.\n\nThe most important limitation to this approach is that event surveillance software utilizing multiple sensors requires that those sensors be present, operational, and free of artifact. There were multiple episodes of atrial fibrillation that occurred in patients who did not have an arterial line, and were hence not captured by event surveillance software, and not eligible for inclusion in this analysis. Dampening of the arterial waveform produced a situation in which the criterion for hypotension was satisfied in event surveillance software. This was principally a problem with the SVT+BP and hypovolemia definitions, but would confound any definition that relies upon accurate data from an arterial catheter. Another important failure came from artifact in the CVP. Failure to level can produce artifactually high or low values in the CVP. Infusions consistently produce artifactually elevated CVP measurements. These artifacts generated most of the false positives in the hypovolemia and tamponade definitions. The software used to conduct this study did not allow any parameter from a sensor to be used more than once in any definition, which precluded screening for these artifacts by excluding extreme values (e.g. CVP of 60mm Hg or -20 mmHg). The ability to examine a parameter more than once would have prevented many of the false positive activations of these definitions. The failure rate of definitions that require data from different sensors will be at least the sum of the artifact rate of those sensors. Logic that replicates how human operators process alarms can be employed using Event Surveillance software and similar software, and has the potential to significantly improve the performance of bedside monitors.\n\nThe event surveillance software employed in the present study could not access all of the information generated from all of the sensors in the monitor, which severely constrained the events that could be surveyed and the definitions that were generated. Successive generations of software, if they incorporate expanded ability to capture information, might be used to generate definitions that will be more useful than most of those used for the current study.\n\nThe most important limitation of the present study is that we were unable to deploy an independent observer in the ICU continuously, and thus had to depend upon bedside RNs and resident physicians to report episodes of the events we sought to capture. It is unlikely that we missed a large number of significant events, but precise estimation of the performance of these definitions would require this more reliable database. We hope that we will be able to obtain the resources to perform a successor study of this design at multiple sites. If all of the output from the clinical devices was recorded into a single massive database, that database could then be used to iteratively evaluate and refine different alarm definitions.\n\nEvent surveillance software utilizes the same audible and visible signals as the other alarms built into these monitors. Hence, study definitions with a very high true positive alarm rate were mixed in with the high rate of false alarms generated by the factory settings for each sensor. The number of false alarms from the individual sensors substantially outnumbers the alarms generated by event surveillance software. Until such time as different audible and visual alarms are utilized, it may be difficult or impossible to demonstrate an important difference in the response of bedside caregivers.\n\n\nConclusion\n\nCorrelation of information across sensors can be used to detect and suppress artifact in a manner similar to how human operators analyze data. Such simple algorithms can generate alarms with a much higher positive predictive value than the simple alarms associated with any of the individual sensors. Additionally, the ability to correlate information across sensors allows the monitor to process clinical information in a manner similar to human operators. The most important limitation to the correlation of information across sensors is that the failure rate becomes at least the sum of the artifact rate of the individual sensors. Nevertheless, these two approaches have the potential to significantly reduce false alarms, increase the positive predictive value of alarms, and make some progress reducing the ubiquitous problem of alarm fatigue in the ICU.",
"appendix": "Author contributions\n\n\n\nM. O’Connor and Y. Bitan conceived the study. M. O’Connor executed the study and gathered the data. Dr. Bitan analyzed the data and prepared the manuscript.\n\n\nCompeting interests\n\n\n\nBoth authors declare they have no competing interests.\n\n\nGrant information\n\n\n\n\nAcknowledgment\n\nThis work was performed at the Department of Anesthesia and Critical Care, The University of Chicago, Chicago, Illinois. The authors wish to thank Joachim Meyer for his insightful comments during the preparation of this paper. The authors would also like to thanks Berndt Duller for his help in analyzing the results of this study, and the technical support provided in installing the alarm definitions into the ICU monitors. The authors would also like to thank Leah Karl for her efforts on behalf of the study and Philips for supporting this study.\n\n\nReferences\n\nKerr JH, Hayes B: An \"alarming\" situation in the intensive therapy unit. Intensive Care Med. 1983; 9: 103–4. PubMed Abstract\n\nSchmid F, Goepfert MS, Kuhnt D, et al.: The Wolf is Crying in the Operating Room: Patient Monitor and Anesthesia Workstation Alarming Patterns During Cardiac Surgery. Anesth Analg. 2011; 112: 78–83. PubMed Abstract | Publisher Full Text\n\nLawless ST: Crying wolf: false alarms in a pediatric intensive care unit. Crit Care Med. 1994; 22: 981–5. PubMed Abstract\n\nBitan Y, Meyer J, Shinar D, et al.: Nurses’ reactions to alarms in the neonatal intensive care unit. Cogn Tech Work. 2004; 6: 239–46. Publisher Full Text\n\nGreen DM, Swets JA: Signal Detection Theory and Psychophysics. New York: Wiley, 1966. Reference Source\n\nTsien CL, Fackler JC: Poor prognosis for existing monitors in the intensive care unit. Crit Care Med. 1997; 25: 614–9. PubMed Abstract"
}
|
[
{
"id": "357",
"date": "15 Nov 2012",
"name": "Yan Xiao",
"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": "358",
"date": "19 Nov 2012",
"name": "Melanie Wright",
"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 scope and depth of the work is appropriate as something that would be presented as an abstract or pilot work, as the study is a collection of baseline data.There are no comparisons of other methods used to monitor patients, for example, did the authors turn off the single sensor alarms whilst performing this study? The authors also compare their presumed false alarm rates to rates presented in other studies, rather than actually capturing single sensor false alarm rates in this setting, and it is difficult to understand how one might place the use of the correlating data (for example SVT + BP to detect atrial fibrillation) within the context of other conditions that low BP and/or high HR/pulse might predict. How did they determine false negatives? Expert review of alarm logs does not instill me with confidence that they captured events that may have been missed. I think the limitations, appropriately described within the document, are great enough to question whether this research is yet at a level that is meaningful for a wide audience. However, the writing is good and the findings may be meaningful for others working in this developing area of research.",
"responses": []
},
{
"id": "360",
"date": "27 Nov 2012",
"name": "Gorazd Voga",
"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 ideology behind the research of this article is good and relevant. Despite the article having a few flaws, the work presented highlights an important topic that is worthy of further discussion.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-45
|
https://f1000research.com/articles/1-23/v1
|
05 Oct 12
|
{
"type": "Opinion Article",
"title": "Female circumcision: Limiting the harm",
"authors": [
"Mohamed Kandil"
],
"abstract": "Objective: To review the strength of evidence that links many health hazards to female genital cutting.Material and methods: Literature search in Medline/Pubmed and Google scholar.Results: Female genital cutting is still practiced secretly in both underdeveloped and developed countries due to prevailing strong traditional beliefs. There is insufficient evidence to support the claims that genital cutting is a harmful procedure if performed by experienced personnel in a suitable theatre with facilities for pain control and anesthesia. Cutting, however, is advised not to go beyond type I.Conclusion: Law makers around the globe are invited to review the legal situation in relation to female genital cutting. Proper counseling of parents about possible risks is a must in order to make informed decision about circumcising their daughters. The procedure should be offered to parents who insist on it; otherwise, they will do it illegally, exposing their daughters to possible complications.",
"keywords": [
"Female genital cutting/mutilation (FGC/M)",
"or circumcision as it was previously described1",
"is held responsible for a multitude of health risks. According to WHO",
"FGC/M is defined as “all procedures that involve partial or total removal of the external female genitalia",
"or other injury to the female genital organs for non-medical reasons”2."
],
"content": "Introduction\n\nFemale genital cutting/mutilation (FGC/M), or circumcision as it was previously described1, is held responsible for a multitude of health risks. According to WHO, FGC/M is defined as “all procedures that involve partial or total removal of the external female genitalia, or other injury to the female genital organs for non-medical reasons”2.\n\nThe legislations enacted in most countries to ban FGC had minimal effect on its prevalence3. In the most recent estimate carried out by the WHO in 2008, an average of between 100 and 140 million women have undergone FGC in the world and every year, 3 million female children are mutilated in Africa4.\n\n\nFemale genital cutting in medical literature\n\nI searched the English literature in Medline/Pubmed and Google Scholar for female genital cutting/mutilation and circumcision in the period from January 1980 until January 2012. The available studies showed that FGC may result in either physical and/or psychological injuries, immediate and/or late.\n\n\nAlleged health hazards\n\nThe three immediate complications are bleeding, pain and infection. They are not unique to FGC. They are liable to occur with any other type of female surgery, whether minor or major. Bleeding is liable to occur with the tiniest injury to the body, not only genitalia, and death may occur if not dealt with. Pain during genital cutting was attributed to non use of anesthesia or pain killers during the procedure5, something which is expected with any other similar situation. The procedure is illegal in most countries of the world and it is routinely performed at home using non-sterilized instruments. Infection is the normal sequel for any surgical interference performed in such an environment. We should ask ourselves what would be the percentages of these complications if FGC was performed in a well-equipped theatre by experienced personnel. They would probably not be different to any other surgical procedure.\n\nThe alleged late risks include a wide variety of complications. Scars and keloid formation may occur6. It is well known that the type of scar depends on the mode of healing, whether by primary or secondary intention. Healing with secondary intention and the formation of ugly scars occurs if the wound is left to heal on its own without repair. This pattern of healing is expected because the procedure is usually performed by the traditional illiterate birth attendant (IBA) at home. Epidermoid cysts may form probably due to cutting with non sharp instruments or imprecise cutting by the traditional IBA or un-experienced surgeon7. The occurrence of both complications can be minimized if the procedure is performed in a well-prepared theatre. Controversy exists as for sexual pleasure. Although many researchers reported that female genital mutilation interferes negatively with women’s sexual pleasure, others provided contradictory evidence and confirmed that women with types I and II cuttings were able to enjoy their sex lives8,9. Lightfoot-Klein10 conducted a study on infibulated females “type III cutting” in Sudan and, based on her findings, she stated that nearly 90% of all women said that they experienced orgasm or had experienced it at various periods in their marriage. Thabet et al. showed that women with type II cutting complain of defective sexuality compared to non circumcised women, while women with the more extensive type III cutting are not different to controls11. This is not logical. If FGC is responsible for defective sexuality, those with type III cutting should have the maximum suffering. The explanation for this contradiction is because sexual arousal is not only dependent upon clitoral stimulation. It involves the stimulation of nerve endings in and around the vagina, vulva, cervix, uterus and clitoris, with psychological response and mindset also playing a role12,13.\n\nThere are claims that women who have undergone genital cutting may have a feeling of inferiority14. This is apparent when these women immigrate to western societies which do not practice FGC. This psychological burden probably stems from the fact that their new societies consider FGC as abnormal contradicting the traditions and beliefs they have grown up with. There are other claims that infertility may also complicate FGC. Reasons are anatomic disfigurement due to excessive scarring after infibulation “type III” probably resulting from healing by secondary intention. Another cause is the associated infection; that might arise after FGC, to the internal genitalia causing inflammation and scarring and subsequent tubal block15. Infection again is due to the improper environment where the procedure was performed.\n\nThe WHO reported that obstetric complications are more likely to occur with genital cuttings and the risk increases with more advanced cutting16. This conclusion was based on a WHO collaborative prospective study which included 28,393 women attending for singleton delivery at 28 obstetric centers in Burkina Faso, Ghana, Kenya, Nigeria, Senegal, and Sudan. The WHO study and few others also showed that a higher percentage of cut women deliver by Cesarean section compared to uncut women due to an increased number of obstructed labors. There is a higher incidence of infant resuscitation, stillbirth, or neonatal death in mothers with FGC16–18. One of the major drawbacks of the WHO study is that the population studied is not representative for the whole population in the selected countries. In poor societies, only high-risk and complicated pregnancies are referred to hospitals. Such cases are more liable for adverse obstetric outcomes. This may have overestimated the rate of complications in women with FGC who attended hospitals to deliver. Claims for increased Cesarean deliveries in cut women were attributed to obstructed labor most likely due to excessive scarring at the pelvic outlet. The high Cesarean rate in this population cannot be attributed solely to obstruction due to excessive outlet scarring; obstructed labor may occur due to a variety of reasons. In fact, excessive scarring at the pelvic outlet is the easiest reason to deal with, using a generous episiotomy. The reason for increased stillbirth and/or neonatal death in mothers with FGC is probably related to the obstructed labor; whatever the reason is, it is not a direct complication of FGC.\n\n\nComments\n\nThe decline in FGC practice is not proportionate to the efforts exerted3. It is not easy to give up your traditions and cultural beliefs for what is considered, by many, to be an attempt to westernize societies in the third world. Many believe that national and international feminist organizations and child rights’ advocates have propagated misleading or unproven information through the media in order to force governments to prohibit the procedure. In fact, all the above-mentioned health hazards were concluded from studies that showed inconsistent findings. Some of them confirmed the hazards of FGC while others failed to prove them. It is the author’s view that none of these studies hold solid evidence to rely upon. These studies were either of retrospective design or studies depended on self-reported FGC and its health consequences. Such studies are imprecise and have low reliability19,20. Research including reported data about past experiences will always be threatened by the individual’s memory and the influence of exposure status on the recalling process21. The strongest evidence comes from randomized controlled trials followed by cohort studies. Data about health hazards linked to FGC were not derived from any of these studies. Such studies were never considered by the WHO or any other international health organization before the ban of FGM takes place in most countries. If a new test or a drug is to be prescribed for a patient, it should pass through a complicated series of tests and randomized comparisons before getting approved. The same occurs with any surgical procedure. No procedure can be considered superior to another or blamed for complications except after randomized controlled trials comparing the new to standard surgery. It therefore seems unrealistic to consider data about FGC not derived from randomized or cohort studies are true and conclusive.\n\n\nReligious and cultural views\n\nIn Islam and Judaism, male circumcision is a must while female is not. In Islam, if female circumcision is desired by parents, it should not go beyond type I FGC (Ia is removal of the prepuce and Ib is removal of the prepuce and clitoris) according to hadith “Sunna type of circumcision”. This type of female genital surgery is equated with male genital surgery22. In support of hadith, many studies showed that women with clitoridectomy “type I cutting” are less likely to develop gynecologic or obstetric complications compared to infibulated women “type III”6. Considering that the number of Moslems in the world ranks second, it seems logical to reconsider the legal attitude towards female circumcision and probably avoids the ban directed towards Sunna circumcision.\n\nThe ban against FGC seems to be gender based, especially because no similar act was taken against male circumcision. If male circumcision is considered safe by anti FGC groups, they should advise how to render FGC as safe as male circumcision instead of enforcing the ban against it.\n\nIt therefore seems that the prohibition of FGC for those who strongly believe in circumcision in the absence of solid scientific evidence does not respect their traditions and cultural beliefs. Women in societies which practice FGC and the practicing immigrant minorities living in the west consider that strength and identity partly come from the pain and difficulty which FGC causes, making them ‘strong’ and ‘desirable’ women23,24.\n\n\nFinal remarks\n\nTo conclude, law makers all around the globe are invited to review the legal situation of female circumcision. Parents, especially immigrants to the western world from the practicing societies, should be properly counselled for the possible complications, but should also be informed that these data were not derived from randomized controlled trials. Those who insist on circumcising their daughters should be allowed to do so, but advised not to exceed type I cutting; otherwise, they will go for it secretly and illegally by inexperienced personnel in a poorly hygienic environment with the possibility of complications.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nRahman A, Toubia N: Female Genital Mutilation: A practical Guide to worldwide Laws and Policies Worldwide. Published by Zed Books 2000. Reference Source\n\nWorld Health Organization. Fact sheet No 241, Geneva. February 2010. Last accessed 9 December 2011. Reference Source\n\nCottingham J, Kismodi E: Protecting girls and women from harmful practices affecting their health: Are we making progress? Int J Gynecol Obstet. 2009; 106(2): 128–31. PubMed Abstract | Publisher Full Text\n\nEliminating Female genital mutilation – An interagency statement (OHCHR, UNAIDS, UNDP, UNECA, UNESCO, UNFPA, UNHCR, UNICEF, UNIFEM, WHO), World Health Organization, 2008; pp.4, 22–28. Reference Source\n\nFemale genital mutilation. World Health Organization (2008). Last accessed date 27 November 2011. Reference Source\n\nKelly E, Hillard PJA: Female genital mutilation. Curr Opin Obstet Gynecol. 2005; 17(5): 490–4. Reference Source\n\nKroll GL, Miller L: Vulvar epithelial inclusion cyst as a late complication of childhood female traditional genital surgery. Am J Obstet Gynecol. 2000; 183(2): 509–10. PubMed Abstract | Publisher Full Text\n\nCatania L, Omar A, Vincenzo P, et al.: Pleasure and Orgasm in Women with Female Genital Mutilation/Cutting (FGM/C). J Sex Med. 2007; 4(6): 1666–78. PubMed Abstract | Publisher Full Text\n\nNussbaum MC: Judging Other Cultures: The Case of Genital Mutilation. Sex and Soc Justice. Oxford University Press 1999; pp.119–20. Reference Source\n\nLightfoot-Klein H: The Sexual Experience and Marital Adjustment of Genitally Circumcised and Infibulated Females in the Sudan. J Sex Res. 1989; 26(3): 375–392. Publisher Full Text\n\nThabet SM, Thabet AS: Defective sexuality and female circumcision: the cause and the possible management. J Obstet Gynaecol Res. 2003; 29(1): 12–9. PubMed Abstract | Publisher Full Text\n\nKomisaruk B, Carlos BF, Beverly W, et al.: The Science of Orgasm. JHU Press, 2006. For an interview with two of the researchers, see Exploring the Mind-Body Orgasm. Last accessed 26 Nov. 2011. Publisher Full Text\n\nMah K, Binik YM: Are orgasms in the mind of the body? Psychosocial versus physiological correlates of orgasmic pleasure and satisfaction. J Sex Marital Ther. 2005; 31(3): 187–200. PubMed Abstract | Publisher Full Text\n\nUtz-Billing I, Kentenich H: Female genital mutilation: an injury, physical and mental harm. J Psychosom Obstet Gynaecol. 2008; 29(4): 225–29. Last accessed: 9 December 2011. PubMed Abstract | Publisher Full Text\n\nAlmroth L, Elmusharaf S, El Hadi N, et al.: Primary infertility after genital mutilation in girlhood in Sudan: a case-control study. Lancet. 2005; 366(9483): 385–91. PubMed Abstract | Publisher Full Text\n\nWHO study group on female genital mutilation and obstetric outcome. Banks E, Meirik O, Farley T, et al.: Female genital mutilation and obstetric outcome: WHO collaborative prospective study in six African countries. Lancet. 2006; 367(9525): 1835–41. PubMed Abstract | Publisher Full Text\n\nLarsen U, Okonofua FE: Female circumcision and obstetric complications. Int J Gynecol Obstet. 2002; 77(3): 255–65. PubMed Abstract | Publisher Full Text\n\nHakim LY: Impact of female genital mutilation on maternal and neonatal outcomes during parturition. East Afr Med J. 2001; 78(5): 255–8. PubMed Abstract | Publisher Full Text\n\nGeneletti S, Richardson S, Best N: Adjusting for selection bias in retrospective, case-control studies. Biostatistics. 2009; 10(1): 17–31. PubMed Abstract | Publisher Full Text\n\nElmusharaf S, Elhadi N, Almroth L: Reliability of self reported form of female genital mutilation and WHO classification: cross sectional study. BMJ. 2006; 333(7559): 124–27. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHassan E: Recall Bias can be a Threat to Retrospective and Prospective Research Designs. Internet J Epidemiol. 2006; 2(3): 4. Publisher Full Text\n\nRichards D: Male Circumcision: Medical or Ritual? 3 JLM at 372; Parekh, op cit n 63, at 268; United Nations Centre for Human Rights, Harmful Traditional Practices Affecting the Health of Women and Children 1996; Fact Sheet No 23 (Geneva, 1995), p 8 (1996). Reference Source\n\nDirie MA, Lindmark G: The risk of medical complications after female circumcision. East Afr Med J. 1992; 69(9): 479–482. PubMed Abstract\n\nEl Saadawi N: The Hidden Face of Eve: Women in Arab World Women of Arab World, Translated and edited by Sherif Hetata. Published by Zed Books, London, 1980; pp.7–11. Reference Source"
}
|
[
{
"id": "312",
"date": "11 Oct 2012",
"name": "Ahmed Fetouh",
"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\nMy own personal stand is against female genital cutting except as a plastic surgery procedure for restricted indications.",
"responses": []
},
{
"id": "314",
"date": "16 Oct 2012",
"name": "Hisham Kandil",
"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 approve the validity of this opinion article with some minor remarks.I personally do not approve of female genital cutting as a general routine, however it is commonly practiced in rural areas of third world countries. Due to this, it is important to study how to best deal with the problem rather than totally deny it. The first degree procedure may be a first step towards avoiding further damage.I think that the final section ‘final remarks’ should be replaced with the title ‘conclusions’.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-23
|
https://f1000research.com/articles/1-44/v1
|
07 Nov 12
|
{
"type": "Research Article",
"title": "Respiratory events in patients undergoing laparoscopic gastric bypass surgery",
"authors": [
"Patrick Ziemann-Gimmel",
"Priscilla Hensel",
"Salam Abdo",
"John Koppman",
"Robert Marema",
"Priscilla Hensel",
"Salam Abdo",
"John Koppman",
"Robert Marema"
],
"abstract": "Background: The incidence of morbid obesity is increasing and has led to an increase in bariatric procedures and previous studies have shown that 71% of these patients suffer from obstructive sleep apnea (OSA). Patients with OSA have a higher rate of postoperative complications. We investigated if patients with OSA undergoing laparoscopic gastric bypass surgery have an increased risk of postoperative respiratory events. In this observational study we examined the data of 89 consecutive patients undergoing gastric bypass surgery.Methods: All patients scheduled for gastric bypass surgery between 7/28/2010 and 02/15/2011 were enrolled and managed according to our routine clinical protocol (48 with OSA / 41 without OSA (NOSA)). Depending on the patient’s preoperative compliance with CPAP therapy, they were further assigned into a compliant (OSAc) and noncompliant (OSAn) group. A respiratory event was defined as a deviation from the regular postoperative management.Results: Both OSA and NOSA groups were similar based on clinical characteristics and narcotic consumption. Fourteen patients (29.2%) suffered from a respiratory event in the OSA group and 8 patients (19.5%) in the NOSA group (p=0.29). Patients compliant with continuous positive airway pressure CPAP had a similar complication rate to patients without OSA (p=0.96). 53.8% of patients with OSA that were noncompliant with CPAP therapy (OSAn) had a respiratory event in the direct postoperative period. This is statistically significant in comparison to patients diagnosed with OSA that are compliant with CPAP (OSAc) (p=0.03)Conclusion: It may be beneficial to encourage OSA patients to use CPAP preoperatively to reduce postoperative respiratory events. Furthermore, adequately treated OSA may not be an independent risk factor for postoperative respiratory events.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: approved 1",
"approved with reservations 1",
"not approved 1]”."
],
"content": "Introduction\n\nThe incidence of morbid obesity is increasing and has led to an increase in bariatric procedures1. The incidence of obstructive sleep apnea (OSA) amongst patients undergoing bariatric surgery has been shown previously to be 71%2. The prevalence of moderate OSA (apnea hypopnea index – AHI≥15) in obese patients with a body mass index (BMI) of >40kg/m2 is 42–55% in men and 16–24% in women3. Patients with OSA have a higher rate of postoperative complications4,5.\n\nWe investigated if patients with OSA undergoing laparoscopic gastric bypass surgery have an increased risk of postoperative respiratory events and desaturations in the first postoperative night. In this observational study we examined the data of n=89 consecutive patients undergoing gastric bypass surgery.\n\n\nPatients and methods\n\nAfter Institutional Review Board (IRB) approval was obtained, data on all patients undergoing gastric bypass surgery in a community hospital was collected between 7/2010 and 2/2011. All patients schedule for gastric bypass surgery were enrolled and managed according to our routine clinical protocol. The need for consent was waived by our IRB due to the entirely observational nature of our study.\n\nThe clinical protocol includes a simplified Berlin questionnaire to detect sleep disordered breathing preoperatively. If patients were determined to be at risk for OSA they were evaluated by a pulmonologist to determine if a sleep study is indicated and continuous positive airway pressure CPAP therapy necessary.\n\nPatients with known OSA were encouraged to bring their CPAP equipment for perioperative use. Patients that used their CPAP device preoperatively were considered to be compliant (OSAc). Patients that were not using their CPAP device preoperatively were considered noncompliant (OSAn). All patients received oxygen via nasal cannula (2–4 l/min) and were monitored with continuous pulse oximetry and EKG after PACU discharge. Desaturations were defined as a pulse oximetry reading <90%. Time spent with an oxygen saturation below 90% during the monitoring period was defined as T90%. If patients showed frequent desaturations the oxygen flow was increased. If this was insufficient to resolve hypoxemia a mask or CPAP with oxygen was applied to increase the inspired fraction of oxygen and resolve possible obstruction.\n\nThe monitoring time started on the day of operation between 21:00–22:00 and ended between 07:00–07:30 the following morning. Pain was managed with intravenous hydromorphone PCA with standard settings (hydromorphone 0.2mg q 6min, 4h lockout 6mg, no basal rate). Hydromorphone consumption was recorded intraoperatively and during the monitoring period. Narcotics given after PACU discharge and before the monitoring time were not recorded.\n\nAll respiratory events were counted during the immediate postoperative period starting in PACU until the following morning. A respiratory event was defined as a deviation from the standard management protocol as described above. Application of an oral airway, oxygen mask or CPAP, prolonged postoperative ventilation and unplanned admission to the intensive care unit were considered respiratory events. CPAP is not routinely started in PACU in our institution. The administration of naloxone was considered a respiratory event as it is administered in patients that are considered to be hypoventilating. For all details please see Table 7.\n\nThe initial data was entered in an EXCEL spreadsheet and later transferred to a SAS data set for analysis. The categorical data was analyzed with the chi square test for independence. For small subsets for which there was insufficient data for the chi square test to be valid, data analysis was done with the two-tailed Fisher’s Exact Test.\n\n\nResults\n\n89 patients, 63 (70.8%) female and 26 (29.2%) male, underwent gastric bypass surgery. Patients were grouped according to the diagnosis OSA (OSA/n=48) and no diagnosis of OSA (NOSA/n=41). In the OSA group 29 (60.4%) patients were female and 19 (39.6%) male. In the NOSA group 34 (82.9%) patients were female and 7 (17.1%) male. The gender distribution is significantly different in both groups (p=0.02).\n\nThe BMI of patients in both groups was statistically not significantly different. Both groups had a similar number of smokers and patients on pain medication. In the OSA group significantly more patients were using antidepressants and anxiolytics. Patients who had OSA also had a significantly higher number of comorbidities (Table 1).\n\nSD – standard deviation.\n\nNS – non significant (p>0.05).\n\nBoth groups (OSA vs. NOSA) received similar amounts of midazolam (2.25mg vs. 2.20mg; p=0.802), fentanyl (198μg vs. 207μg; p=0.59) and hydromorphone (1.07mg vs. 0.85mg; p=0.344) intraoperatively (mean doses; p value). One patient in the OSA group and 2 patients in the NOSA group received ketamine intraoperatively. Two patients in the OSA group and one the NOSA group received morphine intraoperatively.\n\nPatients were monitored during the first postoperative night with continuous pulse oximetry and EKG. Data was collected at a central monitoring unit. The monitoring time did not differ significantly between groups (Table 2). Patients were monitored for an average of 9.67 hours (9.71 hours in the OSA group/9.62 hours in the NOSA group).\n\nSD – standard deviation.\n\nNS – non significant (p>0.05).\n\nData was unavailable for 5 patients: 3 patients in the OSA group and two patients in the NOSA group.\n\nThe hydromorphone consumption during the monitoring time did not differ significantly between the two groups (Table 3). Patients undergoing gastric bypass surgery required on average 2.13mg hydromorphone during the first night.\n\nSD – standard deviation.\n\nNS – non significant (p>0.05).\n\nData was unavailable for 6 patients: one patient in the OSA group and 5 patients in the NOSA group.\n\nTwo patients in the OSA group received extra analgesic medication. One received 30mg ketorolac intravenous and the other received his home dose of gabapentin (300mg) at night.\n\nThe total number of desaturations were compared in all groups. A desaturation was defined as oxygen saturation <90%. Patients noncompliant with CPAP showed significantly more desaturations than patients without OSA (NOSA). The other comparisons did not show statistical significance (see Table 4).\n\n* chi-square test.\n\nNS – non significant (p>0.05).\n\n# Fisher’s Exact test.\n\nPatients who presented with an oxygen desaturation below 90% during the monitoring time did not use more hydromorphone (2.2mg vs 2.0mg; p=0.66).\n\nT90% is defined as the time an oxygen saturation below 90% was measured during the monitoring time. The T90% was not significantly different in patients with OSA compared to patients that didn’t have a diagnosis of OSA. Patients that were compliant with using CPAP (OSAc) had a similar T90% compared to patients that didn’t have OSA (NOSA). Patients that were noncompliant with using CPAP (OSAn) showed a higher T90% than patients that were compliant with CPAP (OSAc) (p=0.19), but this finding was not statistically significant. This indicates that patients in all groups had similar T90%s (Table 5).\n\n* Significance between OSA and NOSA (Chi-square Analysis).\n\nSD – standard deviation.\n\nNS – non significant (p>0.05).\n\n$: significance between OSAc and OSAn (two-tailed Fisher’s Exact Test).\n\nData was unavailable for 3 patients: one patient in the OSA group and two patients in the NOSA group.\n\nUsing a chi square test, there was no statistically significant difference in the incidence of respiratory events in patients in the OSA group compared with in the NOSA group (p=0.29) (Table 6). Patients compliant with using CPAP (OSAc) had a similar incidence of respiratory events to patients without OSA (NOSA) (p=0.96). Patients noncompliant with using CPAP (OSAn) presented a statistically significant increased risk of suffering a respiratory event compared to patients compliant with CPAP (OSAc) (p=0.03).\n\n* Significance between OSA and NOSA (Chi-square Analysis).\n\nSD – standard deviation.\n\nNS – non significant (p>0.05).\n\n$: significance between OSAc and OSAn (two-tailed Fisher’s Exact Test).\n\nWe considered assisted or prolonged ventilation, administration of naloxone and unplanned admission to the SICU as serious events. There were 5 serious respiratory events in the OSA group and 2 in the NOSA group (patients 70, 107, 149, 69, 97 and 96, 129). For all details please see Table 7.\n\nOAW – oral airway, UAW – upper airway, PACU – post anesthesia care unit, SICU – surgical intensive care unit, c – compliant with CPAP, n – noncompliant with CPAP.\n\n\nDiscussion\n\nWe prospectively collected data on 89 consecutive patients undergoing gastric bypass surgery between 7/28/2010 and 2/15/2011. 48 patients were previously diagnosed with OSA. Sleep apnea in the surgical population is an independent risk factor for pulmonary complications6. This study was not controlled for BMI. Morbidly obese patients seem to have an increased risk of postoperative mortality7. It is unclear in the literature if bariatric patients with OSA have a higher incidence of postoperative complications5.\n\nHwang et al. found that sleep-disordered breathing (SDB) is associated with an increased risk of postoperative complications. The authors screened patients preoperatively. If patients showed clinical features suggestive of OSA they were selected for home nocturnal oximetry testing. They measured the number of episodes per hour of oxygen desaturation (oxygen desaturation index – ODI) of ≥ 4% (ODI4%) and the percentage of the study time spent with an oxygen saturation of <90% (T90%). A total of 172 patients aged 27 to 85 years were enrolled. They could show that an ODI4% ≥ 5 and a T90% were associated with an increased risk of postoperative complications5,8,9.\n\nThese findings are consistent with Liao et al., who reported in their retrospective study, that patients with OSA have an increased incidence of postoperative complications compared to matched controls9.\n\nIn contrast to the above findings there was no statistical difference in our study in respiratory events in the OSA compared to the NOSA group (p=0.29). In our study 29.2% (14/48) of patients with OSA had a respiratory event compared to 19.5% (8/41) in the group of patients without OSA (NOSA). Patients in both studies had a lower BMI than in our study and patients underwent a variety of different surgical procedures5,9. Interestingly the two patients with an ODI4% <5 in one study that suffered a complication were morbidly obese5. In the other study complications included mild and severe desaturations9. OSA is defined as a reduction or cessation of airflow with respiratory effort during sleep leading to oxygen desaturations10. It would be therefore anticipated to find a higher incidence of desaturations in the OSA group as compared to the control group. It is difficult to determine if mild desaturations with an oxygen saturation greater than 90% but less than 95% pose a significant immediate health risk and can therefore be counted as a complication, and it is unclear if the number of total respiratory “complications” in this study would have reached clinical significance9.\n\nJensen et al. reported that CPAP and bilevel positive airway pressure (BiPAP) use can be safely omitted after laparoscopic gastric bypass operation. The authors reported data on 1095 patients undergoing gastric bypass surgery. Out of 284 patients with a diagnosis of OSA, 144 used CPAP/BiPAP and 140 did not use CPAP/BiPAP. Patients received supplemental oxygen 2–4 l/min using a nasal cannula postoperatively. CPAP/BiPAP was not used after surgery and patients were instructed not to use it after discharge. The authors defined a respiratory complication as respiratory distress, pneumonia or reintubation within 30 days after gastric bypass operation. Data on overnight oxygen saturations were not published8. None of our patients required reintubation, developed pneumonia or respiratory distress postoperatively during the first 24h. CPAP treatment is primarily indicated to treat upper airway obstruction in patients with OSA during deep stages of sleep to prevent hypoxemia11. It can also be used to prevent reintubation in patients developing hypoxemia after surgery. In a randomized controlled trial the use of CPAP with oxygen to treat postoperative hypoxemia after abdominal surgery compared to oxygen alone decreased the need for reintubation and mechanical ventilation and appeared to be safe. As secondary endpoints, CPAP helped to prevent pneumonia, infection and sepsis but in this study, patients with sleep disorders or BMI>40 kg/m2 were excluded12.\n\nIn the literature only 25.8–50% of patients with OSA undergoing bariatric surgery are compliant with CPAP13. Patients with OSA who are noncompliant with CPAP seem to have a higher rate of complications compared to patients with OSA who are using CPAP9. OSA was shown to be a risk factor in adverse outcome in bariatric surgery14. In the present study 35 patients (72.9%) were using their CPAP and only 13 patients (27.1%) were noncompliant. 53% of patients with OSA that were noncompliant with CPAP therapy (OSAn) had a respiratory event in the direct postoperative period. This is statistically significant in comparison to patients diagnosed with OSA that are compliant with CPAP (OSAc) (p=0.03).\n\nIn the present study patients compliant with CPAP (OSAc) have a similar complication rate compared to patients that don’t have OSA (NOSA) (p=0.96). In the study by Liao et al. patients with OSA that were compliant with CPAP had a higher rate of complication than the control group. Patients in the OSA group had a higher BMI9 and this may have influenced the results. Morbidly obese patients are at greater risk of desaturations15. Obesity is an independent risk factor for sleep-disordered breathing (SDB) and the development of OSA16. In our study both groups had a similar BMI and were using similar amounts of opioids during the monitoring time. The mean BMI in two large multicenter studies was 46.9–47.0 kg/m214,17. The average BMI in our study was 47.1 kg/m2.\n\nOpioid consumption has a profound effect on SDB and affects sleep architecture.\n\nAnesthetic and analgesic agents used during the perioperative period can decrease pharyngeal tone, and depress ventilatory response to hypoxia and hypercapnia18. Midazolam and narcotic consumption during surgery and the monitoring period were similar in the OSA group and in the NOSA group. Narcotic consumption may lead to hypoventilation, hypercarbia and hypoxemia. By increasing the inspired concentration of oxygen hypercarbia induced hypoxemia can be prevented. All but one patient received oxygen via a nasal cannula with a flow to up to 4l/min. Incentive spirometry was also encouraged. 3 patients had to be treated with naloxone due to opioid induced hypoventilation (two patients in the OSA group and one in the NOSA group).\n\nIn a study by Ahmad et al., 40 patients underwent bariatric surgery. There was no difference in hypoxemic episodes in the group of patients diagnosed with OSA versus patients without OSA. 29 patients underwent gastric bypass surgery and 11 gastric banding. Surgical time for gastric bypass surgery compared to gastric band was longer (150–180min versus 116–125min), more invasive and more painful. Patients required higher doses of narcotic medication intra- and postoperatively (remifentanil 1060–1520μg vs 525–737μg; morphine 22–25.5mg vs 2–5.4mg). Patients undergoing gastric bypass surgery had a longer hospital stay than patients having gastric banding (60–73h versus 28–29h, respectively). The authors state in their discussion “the lack of a uniform surgical procedure could have affected the results”1. In other words the differences in surgical stress and narcotic consumption could have affected sleep architecture19. In our study, all patients underwent gastric bypass surgery by one of the two coauthors, (either J. Koppman or R. Marema).\n\nIn the study by Ahmad et al. oxygen saturation was measured for the first 24h following PACU discharge1. OSA is a sleep related disorder and may not influence daytime, awake oxygen saturation. Therefore differences that may be found at night may become insignificant when calculating long periods of wakefulness into a data set. This reduced the percent of time spent <90% saturation to 0.2–0.6%. The total monitoring time in our study was exclusively at night when one would expect the most desaturations. The patients in the present study showed the percentage time spent <90% saturation (T90%) of 6.05% in the OSA group and 5.14% in the NOSA group.\n\nAhmad et al. defined an ODI4% as a “hypoxemic” episode. The ODI showed a correlation to apnea-hypopnea-index (AHI) in the polysomnogram (PSG) which helped to determine the severity of OSA20. We defined clinically relevant hypoxemia as oxygen saturation <90%. The T90% was greater in patients who experienced complications compared to those without a complication (20.8% vs 9.9%). Patients in the ODI4% ≥5 group had significantly higher BMI, more comorbidities and underwent a variety of surgical procedures5. These factors may have influenced the results.\n\nThe small difference in desaturation (T90%) in the present study may be explained by the fact that we failed to identify patients with OSA in the NOSA group. Frey et al. found that the incidence of OSA is present in 71% of patients that have been evaluated for bariatric surgery2. In a multicenter study the incidence of OSA in patients undergoing gastric bypass surgery was 47%17. Application of CPAP or BiPAP was considered a respiratory event. 13 patients required CPAP/BiPAP postoperatively [7 (14.6%) patients in the OSA group and 6 (14.6%) patients in the NOSA group]. The STOP-BANG assessment has a high sensitivity (>90%) in detecting patients at risk for obstructive sleep apnea21. We did not screen the patients in this study with the STOP-BANG index. We collected only four of the 8 variables available (history of hypertension (HTN), BMI, gender and age). In the group of patients without OSA (NOSA, n=41) 27 (65.9%) patients had at least 3 positive answers. The number of patients with at least three positive answers would most likely be greater if we would have screened for all parameters of the STOP-BANG index. This may make it not very useful in identifying patients at risk for OSA in the bariatric population.\n\nAlso the application of oxygen may have prevented more desaturations in the first postoperative night. In the OSA group an average oxygen flow of 3.33l/min was administered. In the NOSA group an average oxygen flow of 2.75l/min was administered. We did not measure inspired oxygen concentration or oxygen flow provided overnight. Patients in one group could have been treated with higher oxygen concentration during the monitoring time influencing the results in this study.\n\nAlso the unequal gender distribution may have affected our results. In our study 70.8% of patients were female and 29.2% male. In the literature 77.5–83% of patients that undergo bariatric surgery are female1,2,14,17 whereas more male patients have OSA than female2 and we did indeed observe a higher percentage of male patients in the OSA group as compared to the NOSA (39.6% vs. 17.1%) group.\n\nIn the study by Ahmad et al. 74.2% in the OSA group were female and 25.8% male patients. In the NOSA group; all patients were female1.\n\n\nConclusion\n\nThe results in the present study suggest that morbidly obese patients with OSA have a similar rate of respiratory complications and desaturations compared to patients without OSA (NOSA) undergoing laparoscopic gastric bypass surgery. Patients with OSA that are noncompliant with CPAP (OSAn) have a statistically significant increase in respiratory complications compared to patients with OSA that are compliant in the use of CPAP (OSAc). The significant increase in desaturations and T90% in the OSAn group simply confirms the diagnosis of OSA. Our study suggest that it may be beneficial to educate and encourage patients with OSA in the use of CPAP to reduce postoperative respiratory events and that adequately treated OSA is not an independent risk factor for respiratory events22,23.",
"appendix": "Author contributions\n\n\n\nPatrick Ziemann-Gimmel helped design the study, conduct the study, enter and analyze the data and write the manuscript. Pricilla Hensel helped analyze the data and write the manuscript. Salam Abdo helped design the study, analyze the data and write the manuscript. John Koppman helped design and conduct the study, analyze the data and write the manuscript. Robert Marema helped design and conduct the study, analyze the data and write the manuscript. All authors approved the final manuscript.\n\n\nCompeting interests\n\n\n\nPatrick Ziemann-Gimmel received honoraria from Cadence and Baxter and is a shareholder in Cadence and Johnson & Johnson.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe results of this article were, in part, presented as a poster at the American Society of Anesthesiologists in Chicago 2011.\n\n\nReferences\n\nAhmad S, Nagle A, McCarthy RJ, et al.: Postoperative hypoxemia in morbidly obese patients with and without obstructive sleep apnea undergoing laparoscopic bariatric surgery. Anesth Analg. 2008; 107(1): 138–143. PubMed Abstract | Publisher Full Text\n\nFrey WC, Pilcher J: Obstructive sleep-related breathing disorders in patients evaluated for bariatric surgery. Obes Surg. 2003; 13(5): 676–683. PubMed Abstract | Publisher Full Text\n\nChung SA, Yuan H, Chung F: A systemic review of obstructive sleep apnea and its implications for anesthesiologists. Anesth Analg. 2008; 107(5): 1543–1563. PubMed Abstract | Publisher Full Text\n\nGupta RM, Parvizi J, Hanssen AD, et al.: Postoperative complications in patients with obstructive sleep apnea syndrome undergoing hip or knee replacement: a case-control study. Mayo Clin Proc. 2001; 76(9): 897–905. PubMed Abstract | Publisher Full Text\n\nHwang D, Shakir N, Limann B, et al.: Association of sleep-disordered breathing with postoperative complications. Chest. 2008; 133(5): 1128–1134. PubMed Abstract | Publisher Full Text\n\nMemtsoudis S, Liu SS, Ma Y, et al.: Perioperative pulmonary outcomes in patients with sleep apnea after noncardiac surgery. Anesth Analg. 2011; 112(1): 113–121. PubMed Abstract | Publisher Full Text\n\nNafiu OO, Kheterpal S, Moulding R, et al.: The association of body mass index to postoperative outcomes in elderly vascular surgery patients: a reverse J-curve phenomenon. Anesth Analg. 2011; 112(1): 23–29. PubMed Abstract | Publisher Full Text\n\nJensen C, Tejirian T, Lewis C, et al.: Postoperative CPAP and BiPAP use can be safely omitted after laparoscopic Roux-en-Y gastric bypass. Surg Obes Relat Dis. 2008; 4(4): 512–514. PubMed Abstract | Publisher Full Text\n\nLiao P, Yegneswaran B, Vairavanathan S, et al.: Postoperative complications in patients with obstructive sleep apnea: a retrospective matched cohort study. Can J Anaesth. 2009; 56(11): 819–828. PubMed Abstract | Publisher Full Text\n\nMeoli AL, Rosen CL, Kristo D, et al.: Upper airway management of the adult patient with obstructive sleep apnea in the perioperative period--avoiding complications. Sleep. 2003; 26(8): 1060–1065. PubMed Abstract\n\nHeinzer RC, Stanchina ML, Malhotra A, et al.: Lung volume and continuous positive airway pressure requirements in obstructive sleep apnea. Am J Respir Crit Care Med. 2005; 172(1): 114–117. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSquadrone V, Coha M, Cerutti E, et al.: Continuous positive airway pressure for treatment of postoperative hypoxemia: a randomized controlled trial. JAMA. 2005; 293(5): 589–595. PubMed Abstract | Publisher Full Text\n\nHuerta S, DeShields S, Shpiner R, et al.: Safety and efficacy of postoperative continuous positive airway pressure to prevent pulmonary complications after Roux-en-Y gastric bypass. J Gastrointest Surg. 2002; 6(3): 354–358. PubMed Abstract | Publisher Full Text\n\nFlum DR, Belle SH, King WC, et al.: Perioperative safety in the longitudinal assessment of bariatric surgery. N Engl J Med. 2009; 361(5): 445–454. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOverdyk FJ, Carter R, Maddox RR, et al.: Continuous oximetry/capnometry monitoring reveals frequent desaturation and bradypnea during patient-controlled analgesia. Anesth Analg. 2007; 105(2): 412–418. PubMed Abstract | Publisher Full Text\n\nStrobel RJ, Rosen RC: Obesity and weight loss in obstructive sleep apnea: a critical review. Sleep. 1996; 19(2): 104–115. PubMed Abstract\n\nBirkmeyer NJ, Dimick JB, Share D, et al.: Hospital complication rates with bariatric surgery in Michigan. JAMA. 2010; 304(4): 435–442. PubMed Abstract | Publisher Full Text\n\nGali B, Whalen FX, Schroeder DR, et al.: Identification of patients at risk for postoperative respiratory complications using a preoperative obstructive sleep apnea screening tool and postanesthesia care assessment. Anesthesiology. 2009; 110(4): 869–877. PubMed Abstract | Publisher Full Text\n\nKaw R, Michota F, Jaffer A, et al.: Unrecognized sleep apnea in the surgical patient: implications for the perioperative setting. Chest. 2006; 129(1): 198–205. PubMed Abstract | Publisher Full Text\n\nMagalang UJ, Dmochowski J, Veeramachaneni S, et al.: Prediction of the apnea-hypopnea index from overnight pulse oximetry. Chest. 2003; 124(5): 1694–1701. PubMed Abstract | Publisher Full Text\n\nChung F, Yegneswaran B, Liao P, et al.: STOP questionnaire: a tool to screen patients for obstructive sleep apnea. Anesthesiology. 2008; 108(5): 812–821. PubMed Abstract | Publisher Full Text\n\nWeingarten TN, Flores AS, McKenzie JA, et al.: Obstructive sleep apnoea and perioperative complications in bariatric patients. Br J Anaesth. 2011; 106(1): 131–139. PubMed Abstract | Publisher Full Text\n\nZiemann-Gimmel P: Obstructive sleep apnoea and perioperative complications in bariatric patients. Br J Anaesth. 2011; 107(2): 273. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "368",
"date": "11 Nov 2012",
"name": "Diederik Nieuwenhuijs",
"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": "369",
"date": "12 Nov 2012",
"name": "Sairam Parthasarathy",
"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 study with important findings regarding the increased risk for respiratory events in obstructive sleep apnea (OSA) patients who were not adherent to continuous positive airway pressure (CPAP) therapy.There are, however, limitations to this study in that the decision-makers were not blinded to the purpose of the study and the definition of “respiratory events” was all encompassing and included initiation of nasal CPAP for oxygen desaturations that failed to respond to O2 alone. Conceivably, due to the nature of the OSA and its treatment, it may have been prudent to have included only the following; application of an oral airway, prolonged postoperative ventilation, unplanned admission to the intensive care unit, post-operative pneumonia, and respiratory failure/re-intubation. Furthermore, Cox-proportional hazards analysis with time-to-event and adjustment for covariates could have increased the power and have adjusted for known confounders (such as anxiolytics) in this observational study.Lastly, a caveat for the finding that there was no difference in respiratory events between OSA and non-OSA group should be that there may not have been sufficient power to detect any differences. A power analysis for such comparison if performed prior to study initiation could have been helpful.",
"responses": [
{
"c_id": "76",
"date": "13 Nov 2012",
"name": "Patrick Zeimann-Gimmel",
"role": "Author Response",
"response": "Thank you Dr. Parthasarathy for your comments and criticism. The idea for this study was to determine if a OSA increases the postoperative risk for a respiratory event. In the current literature there is either a too broad definition of “complication” e.g. only pneumonia or re-intubation or patients were undergoing a variety of procedures with different narcotic requirements and different degrees of inflammation. We used a homogenous group of patients (gastric bypass) and decided to use a clinical approach. A deviation from the routine clinical protocol implies that health care providers spend more time treating patients who for example desaturate despite having an oxygen mask and have to then “fit” a CPAP in the middle of the night. Secondly, as you pointed out correctly, severe adverse outcomes such as pneumonia could have been included. Typically those develop later in the clinical course unless there would be direct evidence of aspiration in the perioperative period. Our goal was to collect data in the immediate postoperative “first night” where we believe sleep and its related breathing disorders are affected the most due to the degree of inflammation and higher narcotic requirements, anticipating the greatest difference in both groups. It is still a matter of discussion if there is a “REM rebound” on the third postoperative day leading to an increase in the apnea–hypopnea index. I completely agree that blinding increases the validity of results but this would be a difficult task in an observational study. Also a prior power analysis is clearly helpful to determine group size and to increase the validity of results. Unfortunately the data available is unclear in what the anticipated relative risk difference/reduction is in patients with OSA or without OSA. I also agree that a bigger sample size would have helped to validate the results but this in turn poses an ethical problem. The PI observes more respiratory events in patients non-compliant with CPAP. I saw it as my responsibility to improve patient care as soon as I saw a difference and we changed our clinical approach accordingly. This is the drawback of an observational study that protocols may change during the observational period. Also, only a few weeks after concluding the initial data acquisition we decided to change our postoperative analgesic treatment to a multimodal approach reducing significantly the narcotics. This would therefore have made a comparison difficult. Again thank you very much Dr. Parthasarathy for your comments."
}
]
},
{
"id": "371",
"date": "15 Nov 2012",
"name": "Rob Basner",
"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\nAlthough the subject matter is of great interest, there are many scientific concerns regarding the methodology, which greatly limit any clinical or scientific relevant conclusions to be made. Among these concerns is that the three groups are unbalanced regarding co-morbidities, and the documentation of type of anesthesia other than sedation and narcotic medications, and indeed specification of the type of surgery each patient underwent is lacking. For example, did any patients receive general anesthesia? Was there pre-operative intubation?In addition, there is also no analysis offered to determine power in order to assess significant differences among groups and similarly, no simultaneous comparison (e.g., ANOVA) among the 3 groups of interest; OSA, OSA (compliant and non compliant). Patient selection for CPAP was apparently not standardized, nor was the use of PAP post-operatively standardized or protocolized. It is also not clear from the study description how much O2 supplementation was provided and under what conditions (e.g., awake, sleep) was it allowed prior to considering a “deviation from the standard protocol”. Overall, there is a lack of standardization of the approach immediately, preoperatively, perioperatively, and post operatively emerging from the study description. Furthermore, there is no clear physiologic or clinical rationale regarding the use of CPAP pre-surgery being protective in this setting, a crucial consideration and an important conclusion one might have made given the gist of these results, particularly since the “no OSA” group likely contained OSA patients. “Compliance” itself was not documented either subjectively or objectively and many upper airway obstruction events were seen even in the “non OSA” group. Additionally, there was no apparent standardization of approach pre-surgically for the identification and treatment of OSA or for any other pulmonary co-morbidities.Therefore, there is no clear imperative here from which to draw conclusions regarding post operative complication risk, as the overall non-standardization of the methodology and the allowance of clinical bias and design unbalance compromises the scientific validity of such data. Thus, while the subject is of great interest and importance, adjudication regarding the importance of identification and pre-, peri-, and post- operative treatment of OSA in obese patients undergoing the particular bariatric procedure these patients underwent continues to warrant randomized clinical trials rather than retrospective data.",
"responses": [
{
"c_id": "75",
"date": "15 Nov 2012",
"name": "Patrick Zeimann-Gimmel",
"role": "Author Response",
"response": "I thank Dr. Basner for his comments and criticism.All patients underwent one single type of surgery: laparoscopic gastric bypass also known as Roux-en-Y. All patients undergo general anesthesia with endotracheal intubation. One of the reasons for general anesthesia with endotracheal tube is that the abdominal cavity is insufflated to a pressure of 15-20cm H2O with CO2. This creates a “cavity” so the surgeon has visibility and room to perform the procedure. So there is uniformity of surgical procedure and type of anesthesia.We agree completely with Dr. Basner, that a power analysis and possibly other statistical test could be applied to determine sample size or to improve statistical validity. In order to perform a valid power analysis one need to be able to estimate or “best guess” the difference that seems to be relevant. This becomes difficult when there is insufficient data available to guide this “best guess”.We agree that a standardized algorithmic approach to utilization of resources improves homogeneity of the results. Here a clinical approach was used: A patient becomes hypoxemic at any time during the hospital stay. Now interventions are initiated, as described in the methodology, in escalating order to resolve hypoxemia. We made the conscious decision to use a clinical approach to reflect clinical reality.Patients come treated and optimized by multiple different physicians with multiple different approaches to clinical problems eg. OSA and CPAP initiation. It is correct that potentially some patients were not diagnosed with OSA preoperatively and therefore were being “assigned” to the NOSA group. This could have influenced the results. Patients were screened of being at risk for OSA by a simplified Berlin questionnaire in the surgeon’s office. The awareness is very high that bariatric patients suffer from OSA and the threshold very low to refer a patient to a pulmonologist. The pulmonologist then determined if further testing eg. a polysomnogram is indicated. The surgery is delayed until the pulmonologist then optimized the patient’s medical condition and “clears” the patient for surgery.In science randomized clinical trials are base on and designed according to data gathered from studies of lower level of evidence eg. observational, retrospective or case-control studies. This study can serve as a starting point in the field of perioperative medicine taking care of a growing number of morbidly obese patients.There are certain ethical limitations that need to be considered, also, in the design of prospective randomized trials. Patients with a diagnosis of OSA should be treated accordingly even though there is limited evidence that preoperative CPAP therapy influences outcome.So how did we change our clinical practice? We continued with the screening process (high awareness/low threshold for referral) and if patients were newly diagnosed with OSA surgery was delayed so patients could get used to the CPAP therapy for 4-6 weeks. We also strongly emphasize to patients the importance of being compliant with CPAP preoperatively early on during the first info sessions."
}
]
}
] | 1
|
https://f1000research.com/articles/1-44
|
https://f1000research.com/articles/1-43/v1
|
06 Nov 12
|
{
"type": "Research Article",
"title": "Effect of obesity on ovarian reserve parameters in mid-reproductive age women",
"authors": [
"Hanan Altaee",
"Zaid Abdul Majeed Al-Madfai",
"Zainab Hassan Alkhafaji",
"Zaid Abdul Majeed Al-Madfai",
"Zainab Hassan Alkhafaji"
],
"abstract": "Background: The initiation and maintenance of reproductive functions are related to an optimal body weight in women. Body weight affects the ovarian reserve, which is basically an estimate of how many oocytes (eggs) are left in the ovaries.Objective: To study the relationship between obesity and serum and ultrasound markers of ovarian reserve in mid-reproductive age women (21–35 years old).Patients and methods: Twenty participants (“obese”) had a body mass index (BMI) of 30 to 35 kg/m2 and another 20 participants (“non-obese”) had a BMI 20–29 kg/m2. The obese women had a mean age of 27.9 years and the non-obese women had a mean age of 29.5 years. Blood samples were collected from all participants, anthropometric measurements were calculated, and transvaginal ultrasonography was performed to measure the antral follicle count (AFC) during the early follicular phase. The blood samples were assayed for antimüllerian hormone (AMH), follicle-stimulating hormone (FSH) and estradiol (E2).Results: There was no significant difference between the two groups regarding ovarian reserve markers and there is no significant correlation between these markers and BMI, except for serum E2 in the obese group.Conclusion: Obesity has no effect on the levels of serum FSH, AMH, or AFC indicating that obesity is unlikely to affect ovarian reserve in the mid-reproductive age group.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: approved 1",
"approved with reservations 1]”."
],
"content": "Introduction\n\nThe initiation and maintenance of reproductive functions are related to an optimal body weight in women. Underweight (BMI under 19 kg/m2), as well as overweight (BMI over 25 kg/m2) and obesity (BMI over 30 kg/m2) are associated with an increased risk of certain disorders1. In addition to conditions such as diabetes mellitus, hypertension, cardiovascular disease, pancreatitis, and musculoskeletal diseases, obese women are more likely to experience reproductive problems2,3, which include menstrual disorders, infertility, and maternal complications during pregnancy4,5. Overweight women, as distinct from obese women, are known to be at higher risk of menstrual dysfunction and anovulation. The mechanisms by which obesity causes or exacerbates subfertility are manifold, one suggested theory that Hyperandrogenemia results in granulosa cell apoptosis, while peripheral conversion of androgens to estrogen in adipose tissue inhibits gonadotrophin secretion6, or possibly due to altered secretion of pulsatile GnRH7. Obesity is also associated with polycystic ovary syndrome (PCOS), which is a heterogeneous condition characterized by oligo or anovulation, hyperandrogenism, menstrual irregularities and subfertility8,9. Overweight and obese subfertile women have a reduced probability of successful fertility treatment and their pregnancies are associated with more complications and higher costs10. Weight loss regularizes menstrual cycles and increases the chance of spontaneous ovulation and conception in anovulatory overweight and obese women. In women undergoing assisted reproductive technology, being obese or overweight has been associated with a need for higher doses of gonadotropins, increased cycle cancellation rates, and fewer oocytes retrieved than in women of normal weight11. Lower rates of embryo transfer, pregnancy, and live birth have also been reported in these women, as well as higher miscarriage rates12.\n\nThe term “ovarian reserve” refers to the quantity and quality of a woman’s current reservoir of oocytes, and is closely associated with reproductive potential. It is an indirect measure of a woman’s reproductive age13. Over the past two decades, a number of tests of ovarian reserve have been used to determine follicle number and quality and to predict the outcome of assisted reproduction procedures14. The woman’s age and assays of serum FSH in the early follicular phase were among the earliest and most useful parameters used for evaluation of ovarian reserve15,16. Several ultrasound parameters have been used for evaluation of ovarian reserve, including ovarian volume17,18 and the antral follicle count (AFC), with varying degrees of reliability19. Recently, serum antimüllerian hormone (AMH) levels have been introduced as a novel measure of ovarian reserve. AMH is a product of the granulosa cells in preantral and antral follicles20. Serum AMH levels decline with age and are correlated with the number of antral follicles and the ovarian response to hyperstimulation21.\n\nFew studies have evaluated the effect of obesity on ovarian reserve. The present study was conducted to examine the effect of obesity on ovarian reserve in women in the mid-reproductive age group. We assessed the effect of obesity on accepted markers of ovarian reserve, specifically levels of basal FSH, E2 and AMH, as well as the ultrasound marker of AFC.\n\n\nPatients and methods\n\nThis study was conducted in the fertility center of Al-Sader Medical City, in Al Najaf province/Iraq, from December 2010 to March 2011. All participating women gave written informed consent before beginning the study. We performed a cross-sectional comparative study of two age-matched groups of 20 participants (group A, obese women) had a BMI of 30–35 kg/m2, with mean age 27.9 years, and the other 20 participants (group B, non-obese women) BMI of 20–29 kg/m2 with mean age of 29.5 years, these serve as a control group. Blood samples were collected from all participants, and transvaginal ultrasonography was performed to measure the AFC during the early follicular phase. The blood samples were assayed for AMH, follicle-stimulating hormone (FSH) and estradiol (E2). Thyroid function test and serum testosterone as well as dehydroepiandrosterone serum levels were assessed as well. The women were seeking treatment for infertility because of tubal factor proved by hysterosalpingiography or laparoscopy.\n\nTo meet the inclusion criteria, women had to be in the mid-reproductive age (20–35 years) according to Stages of Reproductive Aging Workshop (STRAW)22, with an intact uterus and ovaries and to have a regular menstrual cycles for the previous three months, normal thyroid function and no evidence of hyperandrogenism by examination or hormonal assessment. Exclusion criteria were: current use of hormones or drugs that may affect ovarian function, smoking, pregnancy, lactation, previous ovarian surgery, clinical or ultrasound criteria suggesting polycystic ovarian syndrome or endometriosis, or any medical condition that might affect ovarian function. All participating women underwent a comprehensive history and thorough physical examination, calculation of BMI, assays of serum FSH, E2 and AMH, and had a transvaginal ultrasound examination for assessment of AFC. For calculation of BMI, height and weight were measured using the same scale for all participants. BMI was determined by the ratio of weight in kg divided by the height squared in metric units. Blood samples were withdrawn from the antecubital vein on cycle day 2, 3 of the menstrual cycle in all women. All samples were centrifuged at 2000 g for 15 minutes. Serum was separated and stored at -20°C until assayed. Measurement of serum FSH was performed using Mini VIDAS method (bioMérieux® France). Inter-assay Coefficient of Variance% (CV%) 4.7; Intra-assay CV% 5.9, lower limit of detection ≤ 0.1 mIU/ml within 95% probability. For E2 the kit we used (bioMérieux® France) with Mini VIDAS technique Inter-assay CV% 4.6; Intra-assay CV% 3.2. Lower limit of detection 9 pg/ml within 95% probability. Serum levels of AMH were determined by enzyme-linked immunosorbent assays (ELISA) using (Beckman coulter inc., USA) kit. The assays were done according to the manufacturer’s instructions. The detection limits of this assay were 0.08 ng/ml within 95% probability, and its intra-assay and inter-assay coefficients of variation were 5.6% and 4.5% respectively.\n\nTransvaginal ultrasound was performed during the early follicular phase (cycle day 2 or 3), by means of a transvaginal ultrasound scanner (Philips 11*E), with a 5 MHz probe. In each ovary, the total number of small follicles (2–8 mm) was counted. The total follicle count was the sum of the follicle counts in each ovary.\n\nStatistical analysis: descriptive statistics were expressed as mean and standard deviation. Student’s t-test was used to compare groups. Significant relationships between study parameters were evaluated by Pearson’s correlation coefficient. P-values < 0.05 were considered to be significant. Statistical analysis was performed using SPSS version 17.\n\n\nResults\n\nThe 20 women in group A (obese women) had a mean BMI of 32.45 kg/m2, with a range of 30 to 35 kg/m2, and the 20 non-obese women (group B) had a mean BMI of 24.9 kg/m2, with a range of 20 to 29 kg/m2. The mean age in the obese group was 27.9 years; with a range of 22 to 35 years. The mean age in the non-obese group was 29.5 years; with a range of 21 to 35 years. The mean BMI in the obese group (32.45 ± 1.57) was significantly higher than that of the non-obese group (24.9 ± 2.57) (P < 0.05). There was no significant difference between the two groups regarding age, serum levels of AMH or FSH, E2, or AFC. These data are shown in the (Table 1). There was no significant correlation between BMI and serum AMH, serum FSH and AFC in both groups; but significant positive correlation at P < 0.05 level was found between BMI and serum E2 in group A only, these results are shown in (Table 2).\n\nThe values are expressed as mean ± SD.\n\n* P less than 0.05.\n\n\nDiscussion\n\nObesity is an increasingly prevalent health hazard and causes many disorders of female reproduction23,24. In fact, overweight women have a higher incidence of menstrual dysfunction, anovulation, and infertility than other women of reproductive age25, even though altered pulsatile gonadotropin secretion is a well-defined mechanism in obese patients26.\n\nThis study was performed in obese and non-obese women20 with normal menstrual cycles who were referred to fertility center because of tubal factor infertility. Our aim was to examine the possible effects of body mass on some ovarian reserve markers, namely FSH, E2, AMH plasma levels and the number of ovarian follicles in the early follicular phase. The women included were normally ovulating obese and non-obese, with regular menstrual cycles and with neither clinical nor hormonal signs of hyperandrogenism in their mid-reproductive age.\n\nSeveral studies have suggested a negative effect of obesity on parameters of ovarian reserve. De Pergola and his coworkers suggested that overweight and obese fertile women, in comparison with women of normal weight, have lower serum levels of FSH, LH, inhibin B, and estradiol in the early follicular phase, with a possible direct inhibitory effect of body mass on gonadotropin and estradiol production, independent of age27. The difference between their study and our findings may be attributed to selection of BMI of the control group which was normal (BMI < 25 kg/m2), compared to our control group which included BMI > 25 kg/m2. Other investigators reported lower levels of AMH in obese women compared with normal weight women in the late reproductive age28. However, these studies documented that obesity had no effect on ovarian follicle count. They suggested that lower levels of AMH in obese late-reproductive age women result from physiologic processes other than decreased ovarian reserve27. Our results showed that there are no significant differences in serum levels of FSH, E2, AMH, and AFC between obese and non-obese women. There was no significant correlation between BMI and the serum or ultrasound markers of ovarian reserve. Accordingly, we are suggesting that obesity may have limited effect on ovarian reserve in mid-reproductive age women.\n\nThe fact that our results showed no effect of obesity on AMH levels, contrary to other studies, may be related to factors in our study population and limitations in other reports. Our group of obese women was limited to women with a BMI between 30 and 35 kg/m2. We did not include morbidly obese patients because we thought that this specific group of women may have a different endocrine profile that may not apply to women with lesser obesity.\n\nIn a study of women with polycystic ovary syndrome by Pigny and his team, they found that AMH levels were lower in obese than non-obese women, but the difference was not statistically significant29. Another study suggested no correlation between BMI and AMH in women with polycystic ovary syndrome and control subjects30. These data may support our findings. We did not find an effect of obesity on AFC, which has been suggested by others27,28. This supports our impression of a limited effect of obesity on ovarian reserve.\n\nZaidi and his colleagues showed that ovarian volume decreases with an increase in the BMI, indicating the possible decrease in fertility with an increase in a woman’s weight; their study group included normal weight and overweight, and includes a higher-age study group than ours31. This decrease may be due to age effect rather than BMI. They didn’t find any correlation between BMI and AFC, which is in consistent with our study results. In a study conducted in Tehran32 where 115 fertile women were included of a different age group (25–45-years old), they found that BMI had moderate positive correlation with FSH and a moderate negative correlation with estradiol and AFC, but after adjustment of age, BMI as an independent factor had no effect on ovarian reserve markers, a finding which supports our results.\n\nThe significant positive correlation of BMI with estrogen in obese women may be attributed to the contribution for estrogen from the conversion of androgens to estrogens by aromatase in adipose tissue, or may be due to subtle undetected lack of insulin that increases the blood cholesterol concentration. These effects are probably caused mainly by changes in the degree of activation of specific enzymes responsible for the metabolism of lipid substances including cholesterol, which is the precursor of estrogen33. The difference between our finding and that found by other researchers may be attributed to ethnic difference or life style factors34,35. The negative correlation between BMI and AMH have been confirmed by Pingy et al. but it doesn’t prove to be significant29.\n\n\nConclusion\n\nObesity doesn’t have an effect on the selected parameters of ovarian reserve among our cohort of mid-reproductive age women. However, this should be verified by larger studies with clear distinctions between normal, overweight, obese, and morbidly obese women, and between groups of different age groups.",
"appendix": "Author contributions\n\n\n\nHanan Altaee is the principal author who designed and implemented the study, and conducted the bulk of the research. Zeid Almadfa supervised the work and aided with the statistical analysis. Zainab Alkhafaji provided assistance with participant selection and conducted any vaginal ultrasound examinations.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgments\n\nWe would like to thank all the staff of fertility centre in Al-Sader teaching medical city/Al-najaf/Iraq, in particular, the hormonal analysis lab.\n\n\nReferences\n\nWorld Health Organization (WHO): Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health Organ Tech Rep Ser. 2000; 894: i–xii, 1–253. PubMed Abstract\n\nClark AM, Thornley B, Tomlinson L, et al.: Weight loss in obese infertile women results in improvement in reproductive outcome for all forms of infertility treatment. Hum Reprod. 1998; 13(6): 1502–1505. PubMed Abstract | Publisher Full Text\n\nHossain P, Kawaar B, Nahas EM, et al.: Obesity and diabetes in the developing world--a growing challenge. N Engl J Med. 2007; 356(3): 213–5. PubMed Abstract | Publisher Full Text\n\nOgden CL, Carroll MD, Curtin LR, et al.: Prevalence of overweight and obesity in the United States, 1999–2004. JAMA. 2006; 295(13): 1549–1555. PubMed Abstract | Publisher Full Text\n\nSneed ML, Uhler ML, Grotjan HE, et al.: Body mass index: impact on IVF success appears age-related. Hum Reprod. 2008; 23(8): 1835–1839. PubMed Abstract | Publisher Full Text\n\nMetwally M, Li TC, Ledger WL, et al.: The impact of obesity on female reproductive function. Obes Rev. 2007; 8(6): 515–23. PubMed Abstract | Publisher Full Text\n\nClark AM, Ledger W, Galletley C, et al.: Weight loss results in significant improvement in pregnancy and ovulation rates in ovulatory obese women. Hum Reprod. 1995; 10(10): 2705–12. PubMed Abstract\n\nPasquali R, Gambineir A: Polycystic ovarian syndrome: A multifaceted disease from adolescence to adult age. Ann N Y Acad Sci. 2006; 1092: 158–74. PubMed Abstract | Publisher Full Text\n\nDiamanti-Kandarakis E: Role of obesity and adiposity in polycystic ovarian syndrome. Int J Obest. 2007; 31(Suppl 2): S8–S13. PubMed Abstract | Publisher Full Text\n\nKoning AM, Kuchenbecker WK, Groen H, et al.: Economic consequences of overweight and obesity in infertility: a framework for evaluation the cost and outcomes of fertility care. Hum Reprod Update. 2010; 16(3): 246–54. PubMed Abstract | Publisher Full Text\n\nFedorcsak P, Dale PO, Storeng R, et al.: Impact of overweight and underweight on assisted reproduction treatment. Hum Reprod. 2004; 19(11): 2523–2528. PubMed Abstract | Publisher Full Text\n\nPandey S, Maheshwari A, Bhattacharya S, et al.: The impact of female obesity on the outcome of fertility treatment. J Hum Reprod Sci. 2010; 3(2): 62–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta S, Sharma D, Surti N, et al.: Ovarian reserve testing: systemic review of the literature. Arch Med Sci. 2009; 5(1A): S143–S150. Reference Source\n\nBroekmans FJ, Kwee J, Hendriks DJ, et al.: A systemic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update. 2006; 12(6): 685–718. PubMed Abstract | Publisher Full Text\n\nToner JP, Philput CB, Jones GS, et al.: Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril. 1991; 55(4): 784–91. PubMed Abstract | Publisher Full Text\n\nTan SL, Royston P, Campbell S, et al.: Cumulative conception and livebirth rates after in vitro fertilization. Lancet. 1992; 339(8806): 1390–4. PubMed Abstract | Publisher Full Text\n\nSyrop CH, Willhoite A, Van Voorhis BJ, et al.: Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril. 1995; 64(6): 1167–71. PubMed Abstract\n\nLass A, Skull J, McVeigh E, et al.: Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotropin for in vitro fertilization can predict poor response. Hum Reprod. 1997; 12(2): 294–7. PubMed Abstract | Publisher Full Text\n\nBancsi LF, Broekmans FJ, Ejkemans MJ, et al.: Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril. 2002; 77(2): 328–36. PubMed Abstract | Publisher Full Text\n\nDurlinger AL, Visser JA, Themmen AP, et al.: Regulation of ovarian function: The role of anti-Mullerian hormone. Reproduction. 2002; 124(5): 601–9. PubMed Abstract | Publisher Full Text\n\nde Vet A, Lanven JS, de Jong FH, et al.: Anti-mullerian hormone serum levels: a putative marker of ovarian aging. Fertil Steril. 2002; 77(2): 357–62. PubMed Abstract | Publisher Full Text\n\nSoules MR, Sherman S, Parrott E, et al.: Stages of Reproductive Aging Workshop (STRAW). J Womens Health Gend Based Med. 2001; 10(9): 843–848. PubMed Abstract | Publisher Full Text\n\nGrodstein F, Goldman MB, Cramer DW: Body mass index and ovulatory infertility. Epidemiology. 1994; 5(2): 247–50. PubMed Abstract | Publisher Full Text\n\nNorman RJ, Clark AM: Obesity and reproductive disorders: a review. Reprod Fertil Dev. 1998; 10(1): 55–63. PubMed Abstract | Publisher Full Text\n\nBolumar F, Olsen J, Rebagliato M, et al.: Body mass index and delayed conception: a European multicenter study on infertility and subfecundity. Am J Epidemiol. 2000; 151(11): 1072–9. PubMed Abstract\n\nGrenman S, Ronnemaa T, Irjala K, et al.: Sex steroid, gonadotropin, cortisol, and prolactin levels in healthy, massively obese women: correlation with abdominal fat cell size and effect of weight reduction. J Clin Endocrinol Metab. 1986; 63(6): 1257–61. PubMed Abstract | Publisher Full Text\n\nDe Pergola G, Maldera S, Tartagni M, et al.: Inhibitory effect of obesity on gonadotropin, estradiol, and inhibin B levels in fertile women. Obesity. 2006; 14(11): 1954–60. PubMed Abstract | Publisher Full Text\n\nSu HI, Sammel MD, Freeman EW, et al.: Body size affects measures of ovarian reserve in late reproductive age women. Menopause. 2008; 15(5): 857–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPigny P, Merlen E, Robert Y, et al.: Elevated serum level of anti-mullerian hormone in patients with polycystic ovary syndrome: relationship to the ovarian follicle excess and to the follicular arrest. J Clin Endocrinol Metab. 2003; 88(12): 5957–62. PubMed Abstract | Publisher Full Text\n\nPiltonen T, Morin-Papunen L, Koivunen R, et al.: Serum anti-Müllerian hormone levels remain high until late reproductive age and decrease during metformin therapy in women with polycystic ovary syndrome. Hum Reprod. 2005; 20(7): 1820–6. PubMed Abstract | Publisher Full Text\n\nZaidi S, Usmani A, Shokh IS, et al.: Ovarian reserve and BMI between fertile and subfertile women. J Coll Physicians Surg Pak. 2009; 19(1): 21–4. PubMed Abstract\n\nMoini A, Shafizadeh N, Vahid Dastjerdi M, et al.: The effect of Age on Ovarian Reserve Markers in Tehranian Women with Normal Fertility. Int J Endocrinol Metab. 2008; 6(2): 114–119. Reference Source\n\nGuyton AC, Hall JE: Metabolism and Temperature Regulation. In: Text Book of Medical Physiology. Guyton AC. (11th ed) WB. Saundras Company, Philadelphia, USA. 2006; unit XIII, 848. Reference Source\n\nManson JM, Sammel MD, Freeman EW, et al.: Racial differences in sex hormone levels in women approaching the transition to menopause. Fertil Steril. 2001; 75(2): 297–304. PubMed Abstract | Publisher Full Text\n\nRandolph JF Jr, Sowers M, Gold EB, et al.: Reproductive hormones in the early menopausal transition: relationship to ethnicity, body size, and menopausal status. J Clin Endocrinal Metab. 2003; 88(4): 1516–1522. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "355",
"date": "07 Nov 2012",
"name": "Richard A. 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",
"responses": []
},
{
"id": "741",
"date": "29 Jan 2013",
"name": "Angelique Goverde",
"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 cross-sectional comparative study of two age-matched groups, each of 20 participants, the first being obese (BMI 30-35 kg/m2), the second (control) group with BMI 20-29 kg/m2, in which the effect of obesity on markers of ovarian reserve in the early follicular phase was investigated. Cases and controls were attending the fertility clinic for tubal infertility and had regular menstrual cycles. Student’s t test was used to compare groups and Pearson’s correlation coefficient was used to evaluate relationships between study parameters.No differences between groups were found for AMH, FSH, E2 and antral follicle count (AFC); no correlation was found between BMI and AMH, FSH and AFC, but a significant positive correlation was found between BMI and E2.\n\nMy main concern is that the design and the study size of the work was insufficient to draw any conclusion whatsoever. No sample size calculations were reported, neither was type B error. Although BMI in the study cases was significantly higher than in control cases, the BMI range of the control group included overweight women as well, thus limiting the contrast between groups in this regard.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-43
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https://f1000research.com/articles/1-42/v1
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01 Nov 12
|
{
"type": "Case Report",
"title": "Case Report: Vocal cord collapse during phrenic nerve-paced respiration in congenital central hypoventilation syndrome",
"authors": [
"Mark C Domanski",
"Diego A Preciado",
"Diego A Preciado"
],
"abstract": "Objective: Phrenic nerve pacing can be used to treat congenital central hypoventilation syndrome (CCHS). We report how the lack of normal vocal cord tone during phrenic paced respiration can result in passive vocal cord collapse and produce obstructive symptoms.Methods: We describe a case of passive vocal cord collapse during phrenic nerve paced respiration in a patient with CCHS. As far as we know, this is the first report of this etiology of airway obstruction. The patient, a 7-year-old with CCHS and normal waking vocal cord movement, continued to require nightly continuous positive airway pressure (CPAP) despite successful utilization of phrenic nerve pacers. On direct laryngoscopy, the patient’s larynx was observed while the diaphragmatic pacers were sequentially engaged.Results: No abnormal vocal cord stimulation was witnessed during engaging of either phrenic nerve stimulator. However, the lack of normal inspiratory vocal cord abduction during phrenic nerve-paced respiration resulted in vocal cord collapse and partial obstruction due to passive adduction of the vocal cords through the Bernoulli effect. Bilateral phrenic nerve stimulation resulted in more vocal cord collapse than unilateral stimulation.Conclusions: The lack of vocal cord abduction on inspiration presents a limit to phrenic nerve pacers.",
"keywords": [
"Ondine’s curse",
"congenital central hypoventilation syndrome",
"phrenic nerve pacing",
"obstructive sleep apnea",
"vocal cord"
],
"content": "Introduction\n\nCongenital central hypoventilation syndrome (CCHS) is a rare disorder typified by the lack of ventilatory responsiveness to hypoxemia and hypercarbia. The patient with CCHS will typically have adequate conscious control of breathing when awake. However, when asleep, the anatomic nervous system fails to maintain respiration. Patients with CCHS may have other disorders of the autonomic nervous system such as Hirschsprung’s disease, lack of heart rate variability, poor temperature regulation, and diminished pupillary light response. Tumors of neural crest origin such as ganglioneuromas, neuroblastomas and ganglioneuroblastomas are also associated with CCHS1.\n\nCCHS is a rare clinical entity. CCHS was first described in 1970s by Mellins et al.2. As of 1999, it was estimated that there were only 200–300 patients with CCHS worldwide3.\n\nCCHS is caused by a heterogeneous mutation in PHOX2B. PHOX2B is highly conserved transcription factor found on chromosome 4p12. PHOX2B is expressed in both the central and peripheral autonomic nervous system during human development. A mouse model of CCHS showed that Phox2B -/- mice fail to develop the normal neuronal connections of several structures including the solitary tract4,5.\n\nChildren with CCHS typically present soon after birth with duskiness or cyanosis upon falling asleep. During sleep, falling oxyhemoglobin saturation and rising carbon dioxide levels fail to increase respiration or awaken the infant. The differential diagnosis includes discrete congenital myopathy, myasthenia gravis, altered airway anatomy, diaphragm dysfunction, congenital cardiac disease, a structural hindbrain or brainstem abnormality, Mobius syndrome, and inborn errors of metabolism1.\n\nEvaluation of suspected CCHS may include a muscle biopsy, chest x-ray, fluoroscopy of the diaphragm, bronchoscopy, electrocardiogram, Holter recording, echocardiogram, magnetic resonance imaging of the brain and brainstem, serum and urinary carnitine levels. Ophthalmological evaluation should assess for pupillary reactivity and optic disk anatomy. A rectal biopsy should be considered because of the association with Hirschsprung’s disease1.\n\nTreatment of the respiratory compromise in CCHS consists of a tracheostomy and adequate ventilatory support. Because of the persistent lack of response to hypoxemia and hypercarbia, frequent monitoring of pulse oximetry and end tidal CO2 may be prudent1. Once the child reaches appropriate age, insertion of diaphragmatic pacers may be considered6. Diaphragmatic pacers work by stimulating the phrenic nerve in the chest, resulting in diaphragm contraction and respiration. The energy for stimulation is provided by an external portable power source. Successful use of diaphragmatic stimulation may in some cases allow for tracheal decannulation1.\n\nNormal respiration is more than just appropriate diaphragmatic response to hypoxemia and hypercarbia. During inspiration, the vocal cords abduct. Classically, this \"V-shaped\" aperture is called the glottic \"chink\"7. If the vocal cords were not actively abducted during inspiration, they would be drawn together via the Bernoulli effect8.\n\n\nCase Description\n\nA seven year-old female presented with a history of CCHS and a current complaint of obstructive sleep apnea requiring continuous positive airway pressure (CPAP).\n\nIn the newborn period, the patient had been managed with a tracheostomy and traditional ventilatory support. An extensive workup failed to demonstrate any other major developmental abnormalities. In the preschool years, she received bilateral placement of intrathoracic diaphragmatic pacers that eventually allowed decannulation.\n\nDuring the day the patient ambulated with an external power source that was programmed to stimulate the pacing wires if she did not take any breaths in a predefined time period. This was in case the patient accidentally fell asleep. At night the device was set to provide her with continuous respirations. However, at night, the patient also required continuous positive airway pressure because of an obstructive component of her sleep apnea.\n\nFlexible nasal laryngoscopy showed no adenotonsillar hypertrophy. Pharyngeal and tongue anatomy was normal. Vocal cords movement was normal, including abduction during inspiration (Figure 1).\n\nThe patient was scheduled for evaluation in the operating room under general anesthesia. The patient received a preoperative dose of oral midazolam. In the operating room the phrenic pacers were turned off and general anesthesia was induced using inhalational agents. Muscle relaxants were not used. The patient was masked without difficulty. Direct laryngoscopy was performed without an endotracheal tube in place. No laryngeal structural pathology was found. Bronchoscopy using a rigid 4 mm Hopkins rod showed a well-healed tracheostomy site without any granulation or tracheomalacia (Figure 2).\n\nBecause of the patient’s diaphragmatic pacers, it was possible to observe how the patient \"normally\" breathed while asleep. While performing direct laryngoscopy, we proceeded to turn on the right and left phrenic nerve pacers individually and then together. Unilateral stimulation of the diaphragm resulted in respiration. No active stimulation such as myoclonus, abduction or abduction of the vocal cords was noted. However, during inspiration the true vocal cords were pulled medially. This was most evident at the start of inspiration. Expiration resulted in flutter of the vocal cords most prominent at the anterior commissure. Bilateral phrenic nerve stimulation produced greater respiratory efforts. This was accompanied by greater medial pull of the true vocal cords during inspiration along with audible stridor. Flutter of the vocal cords on expiration was greater as well (Figure 3).\n\nVocal cord collapse was greater during bilateral than unilateral diaphragmatic pacing.\n\nOnce the examination was complete, the patient was awoken in the operating room. The patient’s phrenic nerve pacers were turned on while positive pressure was provided via masking – allowing the child to breath while still under anesthesia. As the child awoke, she gradually took voluntary control of her respiration at which time the phrenic pacers automatically ceased.\n\n\nConclusion\n\nCCHS is a rare, multifaceted disorder involving severe central sleep apnea. Treatment is supportive, including aggressive respiratory support such a tracheostomy and sleep-time ventilatory support. Phrenic nerve pacing can obviate the need for external ventilator support, allowing the patient to be considered for decannulation. However, decannulation means that during sleep, air will pass by the vocal cords. As demonstrated in this case report, the vocal cords do not function normally in phrenic nerve-paced respiration. Because there is no central respiratory drive, there is no stimulation for the vocal cords to abduct on inspiration. Instead, during phrenic nerve pacer respiration, the vocal cords are drawn together via the Bernoulli effect and obstructive sleep apnea can result.\n\nAs many children with CCHS develop sequelae compatible of intermittent hypoxemia, passive True Vocal Chord (TVC) collapse is important to consider when evaluating their sleep apnea. The lack of normal vocal cord abduction on inspiration presents a limitation to diaphragmatic paced respiration. Our patient required CPAP while asleep for this reason.",
"appendix": "Author contributions\n\n\n\nMCD provided the description of the case and wrote up the manuscript. DP provided the editorial insight and the overall conclusion.\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\nAcknowledgment\n\nThe authors would like to thank Dr. Joseph Goodman for his assistance with this work.\n\n\nReferences\n\nIdiopathic congenital central hypoventilation syndrome: diagnosis and management. American Thoracic Society. Am J Respir Crit Care Med. 1999; 160(1): 368–73. PubMed Abstract | Publisher Full Text\n\nMellins RB, Balfour HH, Turino GM, et al.: Failure of automatic control of ventilation (Ondine's curse). Report of an infant born with this syndrome and review of the literature. Medicine (Baltimore). 1970; 49(6): 487–504. PubMed Abstract\n\nSilvestri JM, Chen ML, Weese-Mayer DE, et al.: Idiopathic congenital central hypoventilation syndrome: the next generation. Am J Med Genet. 2002; 112(1): 46–50. PubMed Abstract | Publisher Full Text\n\nAmiel J, Laudier B, Attie-Bitach T, et al.: Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nat Genet. 2003; 33(4): 459–461. PubMed Abstract | Publisher Full Text\n\nDubreuil V, Ramanantsoa N, Trochet D, et al.: A human mutation in Phox2b causes lack of CO2 chemosensitivity, fatal central apnea, and specific loss of parafacial neurons. Proc Natl Acad Sci U S A. 2008; 105(3): 1067–1072. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen ML, Tablizo MA, Kun S, et al.: Diaphragm pacers as a treatment for congenital central hypoventilation syndrome. Expert Rev Med Devices. 2005; 2(5): 577–585. PubMed Abstract | Publisher Full Text\n\nHicks M, Brugman SM, Katial R: Vocal cord dysfunction/paradoxical vocal fold motion. Prim Care. 2008; 35(1): 81–103, vii. PubMed Abstract | Publisher Full Text\n\nGates RK: The owner’s manual to the singing voice. Columbus, Ohio: The Ohio State University 2002. Reference Source"
}
|
[
{
"id": "362",
"date": "02 Nov 2012",
"name": "Nina Shapiro",
"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 review of a rare disorder, Congenital Central Hypoventilation Syndrome. The authors present a thorough presentation of the nature of this disorder, followed by an illustrative case of a patient who developed paradoxical vocal cord mobility during phrenic nerve stimulation.Utilization of phrenic nerve-paced respiration is carried out in effort to obviate the need for tracheotomy tube placement in these patients. However, the authors noted that this technique can lead to absence of vocal cord abduction on inspiration, which in turn leads to vocal fold closure and airway obstruction, secondary to the Bernouilli effect. Intraoperative photos are provided, as well as the outcome for this, and potentially future patients seen with this disorder.",
"responses": []
},
{
"id": "363",
"date": "11 Nov 2012",
"name": "Anastasios G Hantzakos",
"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\nInteresting paper! It would be further appreciated if the authors could add a section labeled “Discussion” where the details on the principles of PNP respiration and proposed solutions to prevent obstruction could be noted.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-42
|
https://f1000research.com/articles/1-41/v1
|
31 Oct 12
|
{
"type": "Case Report",
"title": "Case Report: Case report: a rare salivary gland tumor",
"authors": [
"Rateesh Sareen",
"Chandra L Pandey",
"Chandra L Pandey"
],
"abstract": "Salivary duct carcinoma is a distinctive primary neoplasm of the major salivary gland characterized by aggressive behavior with early metastasis, local recurrence and significant mortality. We report a 40 year old male with parotid swelling diagnosed as pleomorphic adenoma, who underwent parotidectomy with modified radical neck dissection and later, on routine histopathology, the swelling was reported as a salivary duct carcinoma, confirmed via immunohistochemistery. Given the relative low occurrence and known difficulty in making an accurate diagnosis using fine needle aspiration cytology, the possibility of salivary duct carcinoma in the appropriate clinical setting of elderly patients with parotid mass and facial palsy should be seriously considered.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: approved with reservations 2]”."
],
"content": "Introduction\n\nSalivary duct carcinoma is a distinctive primary neoplasm of the major salivary gland first described by Kleinsasser et al in 19681. The term was selected because of its resemblance to ductal carcinoma of the breast. It is characterized by aggressive behavior with early metastasis, local recurrence and significant mortality. Nearly 85% of cases occur in the parotid gland followed by submandibular gland2. The tumor has predilection for older men in the 6th to 7th decades of life3. A number of patients experience facial nerve palsy or paralysis and/or pain, and have cervical lymphadenopathy on presentation1. Familiarity with this entity is necessary to avoid false interpretation.\n\nA 40 year old Hindu male who had a 15 year history of smoking presented with a gradually increasing painless swelling on the left parotid region. On examination an 8 × 6 cm swelling was observed the A single Level II mobile lymph node of size < 1 cm was palpable. There was no facial palsy.\n\nAn ultrasonograph of the parotid region performed previously revealed a well defined hypoechoic mass (4.7 × 3.9 cm) with lobulation occupying the left temporomandibular joint with adjacent hypoechoic areas of varying sizes: 1.9 × 1.6 cm, 1.4 × 1.5 cm, and 1.1 × 1.2 cm. It was interpreted as a parotid mass. Fine needle aspiration cytology (FNAC) from the left parotid gland was done and reported as a pleomorphic adenoma. However, FNAC from the submandibular lymph node comprised of blood only.\n\nA repeat FNAC at our institute (Figure 1) showed moderately cellular aspirates with few clusters of normal salivary gland tissue along with epithelial cells showing overcrowding, with a mild-to-moderate pleomorphic population of medium sized cells with the vesicular nuclei having evenly distributed chromatin without conspicuous nucleoi. Cytoplasm was eosinophilic with ill defined borders. It was interpreted as an epithelial neoplasm.\n\nA CT scan of the face and neck showed that the left parotid gland had enlarged in size (9 × 6 × 4 cm), involving deep and superficial lobes, with replacement of normal glandular architecture by homogenous soft tissue. The adjacent musculo-fascial planes were preserved. Multiple enlarged discrete lymph nodes in the left parotid were noted. The left internal jugular vein was compressed and no intraluminal thrombosis was seen.\n\nIn order to reach a diagnosis, a frozen Level II lymph node was performed. (Figure 2) On frozen section, the lymph node architecture was not seen. Cells were singly scattered having scanty cytoplasm, enlarged hyperchromatic nuclei and condensed chromatin. It was not possible to identify the type of malignancy and was therefore reported as a high grade malignant neoplasm.\n\nOn routine histopathology, neck nodes were resected. Six out of seven lymph nodes showed a metastatic neoplasm comprising of sheets and lobules of pleomorphic cells with coarse clumped chromatin separated by fibrous septa. Mitotic activity increased, rosette formation was noted, and it was reported as a poorly differentiated carcinoma with basaloid phenotype. The presence of a high mitotic rate and of focal large, polypoid nuclei suggested an origin from the sebaceous gland.\n\nFinally, a total parotidectomy with modified neck dissection was performed. On gross examination the specimen comprised of:\n\n– Single gray soft tissue piece with skin tag (15 × 14 × 4 cm).\n\n– Skin (4.5 × 2 cm).\n\n– Salivary glands (4 × 2 × 2 cm) were grossly unremarkable.\n\n– Multiple lymph nodes at level II & III – (1.5 to 5.5 cm).\n\n– A deep lobe parotid gland.\n\n– Several gray soft tissue pieces (7 × 7 × 3 cm) with a level I lymph node.\n\n– A single gray soft tissue piece (4 × 4 × 1 cm) (Figure 3).\n\nMicroscopic analysis (Figure 4, Figure 5) showed that the tumor comprised of slightly pleomorhic ovoid cells with vesicular nuclei arranged in sheets and a trabecular pattern separated by fibrous septa. Mitotic activity was not increased. Focal area showed an acinar and comedo pattern. Perineural and lymphovascular invasion were seen. Infiltration into the salivary gland tissue was noted. Eight out of ten lymph nodes showed metastatic carcinoma. In view of metastasis to a lymph node, a diagnosis of high grade malignant epithelial neoplasm was suggested, which was later confirmed via immunohistochemistry as salivary duct carcinoma.\n\n\nDiscussion\n\nSalivary duct carcinoma (SDC) is an aggressive adenocarcinoma which resembles high-grade breast ductal carcinoma. It is also known as cribriform salivary carcinoma of excretory ducts, or high-grade salivary duct carcinoma. SDC represents 9% of salivary malignancies. The male: female ratio is at least 4:1 and most patients present after age 504. The parotid is most commonly involved, but submandibular, sublingual, minor salivary gland, maxillary and laryngeal tumours have been reported2. SDCs are usually firm, solid, tan, white or grey, with a cystic component. Infiltration of the adjacent parenchyma is usually obvious, but occasional tumours may appear to be circumscribed. SDC may also arise as the malignant component of a carcinoma ex-pleomorphic adenoma, so that the macroscopic features of pleomorphic adenoma may also be present. For SDC, perineural spread (60%) and intravascular tumour emboli (31%) are common4. SDC resembles intraductal and infiltrating mammary duct carcinoma, both architecturally and cytologically. The diagnostic “ductal lesion” comprises pleomorphic, epithelioid tumour cells with a cribriform growth pattern, “Roman bridge” formation, and intraductal comedonecrosis. Cytologically, these cells have abundant, pink cytoplasm and large pleomorphic nuclei with prominent nucleoli and coarse chromatin. The cytoplasm may also be densely eosinophilic, granular, or oncocytic. Mitotic figures are usually abundant. Goblet cells are not seen5,6.\n\nSDC is immunoreactive for low- and high-molecular-weight cytokeratin, and markers such as carcinoembryonic antigen (CEA), LeuM1, and epithelial membrane antigen (EMA)7. Strong nuclear reactivity for androgen receptors (AR) is reported in all SDC. As well as being positive for GCDFP-15, they are negative for S-100 protein, myoepithelial markers as well as estrogen and progesterone receptors8. The MIB1 proliferative index is high. Most SDCs show positive distinct membrane staining for HER-2/neu protein. Metastatic breast and squamous carcinomas, oncocytic carcinoma and mucoepidermoid carcinoma come in differential diagnosis because they also show similar immunohistochemistry profiles9–11.\n\nSDC is one of the most aggressive salivary malignancies. Sites for distant metastasis include lungs, bones, liver, brain and skin4. Sixty-five percent of patients die from the disease, usually within 4 years of diagnosis (ranging from 5 months to 10 years)11. The clinical course is characterized by early distant metastases. Tumour size, distant metastasis, and HER-2/neu overexpression are putative prognostic parameters for SDC, while expression of p53 protein, DNA aneuploidy, and proliferative activity do not correlate with outcome9,10. The clinical outcome for the mucin-rich variant of SDC is similar to that of conventional SDC12.\n\n\nConclusion\n\nGiven the known difficulty in making an accurate diagnosis of salivary duct carcinoma13, the identification of a tumor exhibiting variable nuclear grade with cribriform, papillary and comedo patterns in the appropriate clinical setting of elderly patients with parotid mass and facial palsy should suggest the diagnosis of this uncommon tumor after excluding a metastatic carcinoma.\n\n\nConsent\n\nWritten Informed consent for publication was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nRS and CLP contributed to the conception and design of the study. RS collected and analyzed the data and wrote up the manuscript. RS and CLP both approved 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\nMr Mudit Sharma Senior Technician, Department of Pathology and Bhagwan Mahaveer Cancer Hospital and Research Center, Jaipur, India for assisting in performing section cutting, staining and special stains.\n\n\nReferences\n\nLewis JE, McKinney BC, Weiland LH, et al.: Salivary Duct carcinoma. Clinicopathologic and Immunohistochemical Review of 26 Cases. Cancer. 1996; 77(2): 223–30. PubMed Abstract | Publisher Full Text\n\nEpivatianos A, Dimitrakopoulos J, Trigonidis G: Intraoral salivary duct carcinoma: a clinicopathological study of four cases and review of the literature. Ann Dent. 1995; 54(1–2): 36–40. PubMed Abstract\n\nBoson WL, Gomez RS, Araujo L, et al.: Odontogenic myxomas are not associated with activating mutations of the Gs alpha gene. Anticancer Res. 1998; 18(6A): 4415–4417. PubMed Abstract\n\nBarnes L, Rao U, Krause J, et al.: Salivary duct carcinoma. Part I. A clinicopathologic evaluation and DNA image analysis of 13 cases with review of the literature. Oral Surg Oral Med Oral Pathol. 1994; 78(1): 64–73. PubMed Abstract | Publisher Full Text\n\nHenley JD, Seo IS, Dayan D, et al.: Sarcomatoid salivary duct carcinoma of the parotid gland. Hum Pathol. 2000; 31(2): 208–213. PubMed Abstract | Publisher Full Text\n\nNagao T, Gaffey TA, Serizawa H, et al.: Sarcomatoid variant of salivary duct carcinoma: clinicopathologic and immunohistochemical study of eight cases with review of the literature. Am J Clin Pathol. 2004; 122(2): 222–231. PubMed Abstract | Publisher Full Text\n\nDelgado R, Vuitch F, Albores-Saavedra J: Salivary duct carcinoma. Cancer. 1993; 72(5): 1503–1512. PubMed Abstract | Publisher Full Text\n\nSkalova A, Starek I, Vanecek T, et al.: Expression of HER-2/neu gene and protein in salivary duct carcinomas of parotid gland as revealed by fluorescence in-situ hybridization and immunohistochemistry. Histopathology. 2003; 42(4): 348–356. PubMed Abstract | Publisher Full Text\n\nKapadia SB, Barnes L: Expression of androgen receptor, gross cystic disease fluid protein, and CD44 in salivary duct carcinoma. Mod Pathol. 1998; 11(11): 1033–1038. PubMed Abstract\n\nMartinez-Barba E, Cortes-Guardiola JA, Minguela-Puras A, et al.: Salivary duct carcinoma: clinicopathological and immunohistochemical studies. J Craniomaxillofac Surg. 1997; 25(6): 328–334. PubMed Abstract | Publisher Full Text\n\nBrandwein MS, Jagirdar J, Patil J, et al.: Salivary duct carcinoma (cribriform salivary carcinoma of excretory ducts). A clinicopathologic and immunohistochemical study of 12 cases. Cancer. 1990; 65(10): 2307–2314. PubMed Abstract | Publisher Full Text\n\nSimpsonn RHW, Prasad AR, Lewis JE, et al.: Mucin-rich variant of salivary duct carcinoma: a clinicopathologic and immunohistochemical study of four cases. Am J Surg Pathol. 2003; 27(8): 1070–1079. PubMed Abstract | Publisher Full Text\n\nKinnera V, Nandyala R, Yootla M, et al.: Salivary duct carcinoma of parotid gland. Internet J Pathol. 2009; 10(1). Reference Source"
}
|
[
{
"id": "353",
"date": "01 Nov 2012",
"name": "Pavel Dulguerov",
"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 general, This is a well written case report of salivary duct carcinoma.In this case, like most rare and poorly differentiated salivary neoplasms, the correct diagnosis was only possible on the final specimen despite FNA, frozen, and permanent section histopathology of the metastatic lymph nodes. While the immunohistochemistry is reviewed in the discussion, it is not specified how it was used in this particular case. This report will not alter the clinical management of this disease but it highlights the most salient points in the clinical diagnosis of salivary ductal carcinoma.",
"responses": []
},
{
"id": "354",
"date": "04 Nov 2012",
"name": "Giulio Cantu",
"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\nEvery report of rare cases must be appreciated, even if salivary duct carcinoma is not so rare (9% of salivary glands malignancies), and dozens of cases have been reported in the past.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-41
|
https://f1000research.com/articles/1-39/v1
|
30 Oct 12
|
{
"type": "Case Report",
"title": "Pleural effusion as the initial extramedullary manifestation of Acute Myeloid Leukemia",
"authors": [
"José Nieves-Nieves",
"Luis Hernandez-Vazquez",
"Dev Boodoosingh",
"Ricardo Fernández-Gonzalez",
"Rosángela Fernández-Medero",
"José Adorno-Fontánez",
"Edgardo Adorno-Fontánez",
"José Lozada-Costas",
"Luis Hernandez-Vazquez",
"Dev Boodoosingh",
"Ricardo Fernández-Gonzalez",
"Rosángela Fernández-Medero",
"José Adorno-Fontánez",
"Edgardo Adorno-Fontánez",
"José Lozada-Costas"
],
"abstract": "Leukemias rarely debut by pleural involvement as the first manifestation of the hematologic malignancy. This complication is most commonly seen in solid tumors such as carcinomas of the breast, lung, gastrointestinal tract and lymphomas. We present a case of a 66 year old male who presented with a pleural leukemic infiltration of his undiagnosed Acute Myeloid Leukemia that was not a complication of the disease extension, but the acute presentation of the illness. Progressive shortness of breath for two weeks, cough, clear sputum and weight loss were the initial complaints. Serum dyscrasia suggested a hematologic abnormality. A chest x-ray performed demonstrated a buildup of fluid with layering in the left pleural cavity. Diagnostic thoracentesis suggested an exudative etiology with cytology remarkable for 62% leukemic myeloblast. The diagnosis was confirmed by bone marrow biopsy with expression of the antigens CD 34+ and CD13+, with unfavorable cytogenetic prognosis and a trisomy 21 chromosomal defect. Chemotherapy was initiated, though no remission achieved with induction chemotherapy. Complications and disease progression precludes in the patient’s death. Although rare, due to the unusual presentation of the disease, this case clearly demonstrates the importance of biochemical analysis and cytopathology specimens obtained in pleural fluid.",
"keywords": [
"Acute Myelogenous Leukemia (AML) is a group of hematogenous neoplasms characterized by clonal proliferation of myeloid precursors with a reduced capacity to differentiate into more mature cellular elements1. As a result",
"there is an accumulation of leukemic blasts or immature forms in the bone marrow",
"peripheral blood",
"and occasionally in other tissues",
"with a variable reduction in the production of normal red blood cells",
"platelets",
"and mature granulocytes. The increased production of malignant cells",
"along with a reduction in these mature elements",
"result in a variety of systemic consequences including anemia",
"bleeding",
"and an increased risk of infection1. Less than 1 percent of patients present with prominent extramedullary disease2. These extramedullary manifestations can manifest simultaneously with",
"or precede",
"bone marrow involvement. Sites of isolated expression include bone",
"periosteum",
"soft tissues",
"and lymph nodes",
"and less commonly the orbit",
"intestine",
"mediastinum",
"epidural region",
"uterus",
"and ovary2. To our knowledge this is one of the few reported cases of pleural effusion as the initial manifestation of AML."
],
"content": "Introduction\n\nAcute Myelogenous Leukemia (AML) is a group of hematogenous neoplasms characterized by clonal proliferation of myeloid precursors with a reduced capacity to differentiate into more mature cellular elements1. As a result, there is an accumulation of leukemic blasts or immature forms in the bone marrow, peripheral blood, and occasionally in other tissues, with a variable reduction in the production of normal red blood cells, platelets, and mature granulocytes. The increased production of malignant cells, along with a reduction in these mature elements, result in a variety of systemic consequences including anemia, bleeding, and an increased risk of infection1. Less than 1 percent of patients present with prominent extramedullary disease2. These extramedullary manifestations can manifest simultaneously with, or precede, bone marrow involvement. Sites of isolated expression include bone, periosteum, soft tissues, and lymph nodes, and less commonly the orbit, intestine, mediastinum, epidural region, uterus, and ovary2. To our knowledge this is one of the few reported cases of pleural effusion as the initial manifestation of AML.\n\n\nCase report\n\nA 66 year old man with a long-standing history of mild to moderate asthma and arterial hypertension was evaluated for a worsening productive cough of clear sputum, dyspnea, wheezing, and unintentional weight loss of approximately thirty pounds. The patient denied fever, chills, hemoptysis, night sweats, chest pain, or exposure to sick contacts. His medications were frequent use of short acting β-agonist with minimal resolution of symptoms.\n\nOn physical examination, the patient was alert but in mild respiratory distress, afebrile without hemodynamic compromise. The cardiac examination was normal; pulmonary examination revealed diffusely decreased breathing sounds, inspiratory crackles, and dullness to percussion, decreased fremitus and egophony in up to two thirds of the left lung field. There was no use of accessory muscles and oxygen saturation was 90% with the patient breathing ambient air. Neither lymphadenopathy nor organomegaly was palpated. CBC was abnormal for hemoglobin 8.1 g/dL, platelet 60,000/µL, leukocyte count 87000/µL with 64% blast (Table 1). Arterial blood gases were pH 7.402, PCO2 38.3 mmHg, and PO2 67 mmHg; oxygen saturation was 89% without supplemental oxygen.\n\nA hematologic malignancy was suggestive due to the serum dyscrasia. Chest radiograph showed a large free flowing left pleural effusion (Figure 1). A diagnostic and therapeutic thoracentesis was performed with removal of approximately 1 liter of fluid. The symptoms resolved and biochemical analysis established an exudative etiology (Table 2). The cytopathology specimen obtained from the pleural fluid was positive for blast cells with 62% leukemic myeloblast. AML was confirmed by bone marrow biopsy with expression of the antigens CD 34+ and CD 13+ (Figure 2) with intermediate to unfavorable cytogenetic prognosis (Table 3).\n\nRatio of the pleural fluid lactate dehydrogenase and protein to serum lactate dehydrogenase and protein (Light’s criteria meeting exudative etiology).\n\nA karyotypic abnormality of Trisomy 21 was revealed through cytogenetic studies (Figure 3), which is the second most common chromosomal defect in AML. The patient was treated with Idarubicin combined with Cytarabine for the recently discovered AML and there was no re-accumulation of the pleural fluid. However, bone marrow aspiration was repeated to assess response to chemotherapy and he still presented with 62% of blasts cells. A new cycle of chemotherapy was started with Mitoxantrone, Etoposide and Cytarabine but only a partial response was obtained. Despite the therapeutic regimen, due to the severity of the disease and the poor cytogenetic prognosis, the patient’s condition deteriorated. In view of no significant response to therapy and dismal prognosis, supportive measures and palliative care was provided and eventually the patient died due to complications associated to AML.\n\n\nDiscussion\n\nPhysicians dealing with the diagnostic workup of pleural effusions rarely discover an underlying hematologic malignancy1. AML generally presents with symptoms related to complications of pancytopenia. Most patients have more subtle evidence of bone marrow involvement for weeks, or perhaps months, before the diagnosis can be made. Despite the pancytopenia, and/or coagulopathy, it is unusual for leukemias, either acute or chronic, to manifest with malignant pleural effusions as the initial presentation3–5. Usually this abnormal amount of fluid collection is a complication more commonly seen in solid tumors and lymphomas5.\n\nOur patient presented with AML and pulmonary involvement with signs and symptoms secondary to the pleural effusion itself rather than with classical appearance of the acute myeloid leukemia. An unusual case where neither the complications of the hematologic dyscrasia such as bleeding and recurrent infections, nor the physical findings of a swollen spleen, liver, or lymph nodes were the primary target organs that would lead to a presumptive diagnosis. This demonstrates the importance of the biochemical analysis and the cytopathology specimens obtained in pleural fluid since an early detection of any determined disease could guide effective therapy6,7. AML in this particular case, and prompt treatment could undoubtedly contribute in avoiding complications associated with the condition; an essential factor for improving quality of life. For this reason chest physicians should be aware of all possible pulmonary manifestations of hematologic malignancies.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the relative of the patient.",
"appendix": "Author contributions\n\n\n\nAuthors have contributed to the literature review, drafting of the manuscript, revisions of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests have been disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nJaffe ES, Harris NL, Stein H, et al.: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. World Health Organization Classification of Tumours, Volume 3. Lyon IARC Press 2001. Reference Source\n\nDores GM, Devesa SS, Curtis RE, et al.: Acute leukemia incidence and patient survival among children and adults in the United States, 2001–2007. Blood. 2012; 119(1): 34–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nByrd JC, Edenfield WJ, Shields DJ, et al.: Extramedullary myeloid cell tumors in acute nonlymphocytic leukemia: a clinical review. J Clin Oncol. 1995; 13(7): 1800–16. PubMed Abstract\n\nOhe K, Okamura T, Arima F, et al.: CD7 positive Acute Myelogenous Leukemia exhibiting pleural involvement as an initial manifestation. Rinsho Ketsueki. 1994; 35(6): 552–556 [Article in Japanese]. PubMed Abstract\n\nAlexandrakis MG, Passam FH, Kyriakou DS, et al.: Pleural Effusions in Hematologic Malignancies. Chest. 2004; 125(4): 1546–1555. PubMed Abstract | Publisher Full Text\n\nWan TS, Au WY, Chan JC, et al.: Trisomy 21 as the sole acquired karyotypic abnormality in acute myeloid leukemia and myelodysplastic syndrome. Leuk Res. 1999; 23(11): 1079–83. PubMed Abstract | Publisher Full Text\n\nCortes JE, Kantarjian H, O’Brien S, et al.: Clinical and prognostic significance of trisomy 21 in adult patients with Acute Myelogenous Leukemia and Myelodysplastic Syndromes. Leukemia. 1995; 9(1): 115–7. PubMed Abstract"
}
|
[
{
"id": "350",
"date": "21 Nov 2012",
"name": "Sharon Savage",
"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 case report describes a case of acute myeloid leukemia (AML) that presented with a large pleural effusion. This unusual presentation is important to recognize when evaluating patients with pleural effusions. This particular case report illustrates the importance of a cytopathology evaluation.The report itself would benefit from an updated reference on chromosomal abnormalities in AML, and it would also be helpful to know the doses of the chemotherapeutic agents and if the patient was treated on a clinical trial for newly diagnosed AML.",
"responses": []
},
{
"id": "351",
"date": "22 Nov 2012",
"name": "Stephen Nimer",
"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 points out an unusual clinical presentation of acute myeloid leukemia (AML) that illustrates the heterogeneity of presentations of extramedullary disease.While the diagnosis was clear at the onset, the patient had 64% circulating blasts and the pleural effusion was unusual and required evaluation. As monocyte subtypes of AML have a propensity for tissue invasion and the core binding factor (CBF) leukemia can be associated with granule cystic sarcomas, a bit more information on the immunophenotype of the AML would have been helpful in assessing this case. Also, given the many molecular markers that define the prognosis of AML patients, more details on any molecular studies that were done, or could have been done, would be helpful.",
"responses": []
},
{
"id": "352",
"date": "29 Nov 2012",
"name": "Adlette Inati",
"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 case of pleural effusion as an initial presentation of Acute Myeloid Leukemia (AML) in an adult patient with no physical exam findings suggestive of leukemia and or malignancy.The authors corroborated the exudative nature of this symptomatic s pleural effusion by flow cytometric analysis for CD34+ and CD13+ antigens and supported their diagnosis by bone marrow studies and cytogenetic analysis which showed a trisomy 21 pattern. While a chemotherapeutic regimen of Cytarabine and Idarubicine inhibited further progression and re accumulation of the pleural effusion, the patient’s condition deteriorated leading to his death. Given the dismal nature of the patient’s AML prognosis, hematologists need to be aware of this atypical and rare AML presentation. More importantly, pulmonary physicians should acquaint themselves with this clinical scenario because such patients present first to them and not to hematologists.Pleural effusion has been reported in association with solid tumors and sometimes as their presenting sign. A multivariate analysis by Faiz et al., Leukemia Lymphoma (2012) between 1997 and 2007 showed that 111 patients with acute leukemias had pleural effusions and the median overall survival in these patients was shorter than those with absence of pleural effusions. Other studies revealed rare occurrences of pleural effusion in AML patients.The authors need to highlight salient features of previously reported patients and compare them with their patient hoping to find predictive factors for development of such rare clinical scenarios and define better treatments.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-39
|
https://f1000research.com/articles/1-37/v1
|
26 Oct 12
|
{
"type": "Research Article",
"title": "Corticoid-associated complications in elderly",
"authors": [
"Besma Ben Ben Dhaou",
"Fatma Boussema",
"Zohra Aydi",
"Lilia Baili",
"Hédi Tira",
"Lilia Rokbani",
"Fatma Boussema",
"Zohra Aydi",
"Lilia Baili",
"Hédi Tira",
"Lilia Rokbani"
],
"abstract": "Background: Corticosteroids are widely prescribed products in the elderly particularly in systemic diseases and have been indispensable in controlling a variety of disease states. The various complications associated with this drug class warrant caution and monitoring with each formulation, especially with an older patient population.Aim: The aim of our study was to evaluate the frequency and type of side effects and complications of long-term corticosteroid therapy in the elderly.Methods: We conducted a retrospective study of 23 patients aged 65 and older hospitalized in the Internal Medicine Department of the Habib Thameur hospital from January 2000 to December 2004. Corticoid-related adverse effects were recorded throughout the follow-up period.Results: There were 20 women and 3 men aged 66 to 87 years with a mean age of 75.7 years. The diagnoses were 8 cases of temporal arteritis, 7 cases of rheumatoid arthritis, 3 cases of multiple myeloma, 2 scleroderma, 1 case of systemic lupus erythematosus, 1 case of retroperitoneal fibrosis and 1 case of psoriatic arthritis. We selected 66 complications. Infectious complications were found in 26 cases (39.3%), 11 cases (16.7%) of iatrogenic diabetes, arterial hypertension in 9 cases (13%), skeletal complications in two cases, psychiatric complications in two cases, ophthalmologic complications in one case.Conclusion: Despite lifestyle rules and adjunctive therapy, complications seem to be frequent. To minimize the disadvantages of prolonged corticosteroid treatment, regular monitoring and careful screening is imperative to detect and handle them in time.",
"keywords": [
"Corticosteroids have been in use for longer than 40 years. Over time",
"they have become indispensable in controlling a variety of disease states. Currently",
"glucocorticoids (GC) are available in numerous formulations: oral",
"topical",
"ophthalmic solutions and ointments",
"oral inhalers",
"nasal formulations",
"parenteral and rectal preparations. GC",
"especially when they are used throughout the course in geriatrics is not devoid of side effects and complications due",
"in part",
"to physiological changes of agin. The aim of our study was to evaluate the frequency and type of side effects and complications of long-term corticosteroid therapy in the elderly."
],
"content": "Introduction\n\nCorticosteroids have been in use for longer than 40 years. Over time, they have become indispensable in controlling a variety of disease states. Currently, glucocorticoids (GC) are available in numerous formulations: oral, topical, ophthalmic solutions and ointments, oral inhalers, nasal formulations, parenteral and rectal preparations. GC, especially when they are used throughout the course in geriatrics is not devoid of side effects and complications due, in part, to physiological changes of agin. The aim of our study was to evaluate the frequency and type of side effects and complications of long-term corticosteroid therapy in the elderly.\n\n\nMaterials and methods\n\nA retrospective study was performed in 23 patients aged 65 years older and collected in internal medicine department of the Habib Thameur hospital from January 2000 to December 2004. The adverse effects of corticosteroids were recorded throughout the monitoring period. We selected the following as inclusion criteria: an age greater than or equal to 65 years, at least one hospitalization during the included period, and the indication of a general glucocorticoid treatment on long-term excluding inhaled and topical corticosteroids. We noted the following for each patient: age, sex, medical history, reasons for hospitalization, clinical features, paraclinical explorations conducted, diagnosis retained. We systematically analyzed the prescription of treatment and the treatment protocol described by specifying: the type of drugs used, the type of use (oral or IV bolus), the period of the different phases (in particular the treatment time of attack), levels of degression and maintenance therapy, the dose used during each phase in mg/kg/day, the terms of degression, the occurrence of relapses and the evolutionary times and recurrence. We evaluated our patients, for side effects and complications that occurred during the evolution recalling the therapeutic adjuvant used. Data were entered using Excel software and analyzed using SPSS version 11.5.\n\n\nResults\n\nThe complete retrospective study of the 23 patients can be seen in Table 1.\n\nThere were 20 women and 3 men aged 66 to 87 years with a mean age of 75.7 years. The diagnoses were 8 cases of temporal arteritis, 7 cases of rheumatoid arthritis, 3 cases of multiple myeloma, 2 scleroderma, 1 case of systemic lupus erythematosus, 1 case of retroperitoneal fibrosis and 1 case of psoriatic arthritis. We selected 66 complications (Table 2). Infectious complications were found in 26 cases (39.3%): 2 viral infections, 7 fungal infections and 17 bacterial infections including 4 urinary infections, 3 bronchopulmonary infections, 3 skin infections, 2 Otorhinolaryngologic infections, 2 stomatological infections, 1 osteoarticular infection, 1 gastrointestinal infection and 1 case of reactivation of latent tuberculosis (Table 3).\n\nAmong the metabolic complications we identified 11 cases (16.7%) of iatrogenic diabetes that was aggravated by corticosteroids in 6 cases or discovered during treatment in 5 cases. High blood pressure (hypertension) aggravated or induced by corticosteroid treatment has been notified in 9 patients (13%). Osteoporosis is reported in 2 cases (3%), depression in 2 cases (3%), cataracts in 1 case.\n\n\nDiscussion\n\nAs in the literature, infectious complications were most common in our study followed by metabolic complications. Infectious complications were particularly found with systemic diseases such as vasculitis or giant cell arteritis, or connective tissue disorders such as rheumatoid arthritis or lupus erythematosus, infection being the leading cause of death in the latter disorder1. Thus, in a study on complications due to steroid therapy in 164 patients aged 75 and older and suffering from temporal arteritis, a total of 111 complications were reported, including infectious complications in 31 cases and 20 cases of pulmonary infection2. In contrast, a second study reviewing the complications of corticosteroid therapy in giant cell arteritis and polymyalgia rheumatica, including a total of 500 patients from 5 different studies, found a low number of infectious complications: in total, 9 patients including 7 cases of herpes zoster3. In rheumatoid arthritis, many infections are reported, even with steroids at a dose below 10 mg/day4. In the current literature, there are many observations describing severe infections that have been found (e.g. shingles, fungus ...)5. In a series5 about the adverse effects of corticosteroid therapy in rheumatoid arthritis, there were three more severe infections in the group treated with long-term corticosteroids compared with the group not receiving cortisone. In total in this series5, 8 patients with herpes zoster were identified, 5 of pneumonia, 4 of septic arthritis and 2 cases of urinary tract infections among a total of 22 severe infections. On the other hand, we must insist on the morbidity associated to long-term corticosteroids during systemic lupus erythematosus6,7. Although our sample is small, our results are comparable to those of Chevalet2, as we found that infections accounted for 39.3% of total complications of long-term corticosteroid treatment in our investigations.\n\nAmong the metabolic complications, we identified 11 cases (16.7%) of iatrogenic diabetes, and this was aggravated by corticosteroids in 6 cases or discovered during treatment in 5 cases. According to Agard8, diabetes affects 10% of patients with giant cell arteritis. Treatment based on a diabetic diet and insulin therapy should be preferred to oral antidiabetic drugs8. Corticosteroids, (even at low doses), can reveal diabetes, justifying minimum carbohydrate restriction, or can exacerbate pre-existing diabetes, temporarily insulin use9. High blood pressure (hypertension) aggravated or induced by corticosteroid treatment has been noted in 9 patients, at a rate of 13%. For 5 of the patients, hypertension was aggravated to a degree significant enough to warrant remission to previous antihypertensive treatment. For the other 4 patients, hypertension appeared, but was managed with the initiation of a low-sodium diet or mild antihypertensive therapy. Hypertension is more frequent during steroid treatment, but its relationship with dose and duration of treatment is unclear. Pre-existing hypertension appears to be a risk factor which is why thiazide diuretics are recommended in the first place10. Hypertension is the most important parameter to monitor. According to Agard8, it is often pre-existing in giant cell arteritis and is aggravated in 15 to 30% of cases which requires increasing the antihypertensive treatment. Among our 23 patients, we observed a case of femoral neck fracture in the 7th month of corticosteroid therapy in a woman of 72 years treated for temporal arteritis at a dose of 10 mg/day, and treated by nail plate with secondary loosening. This incident suggests an iatrogenic cause. A second patient had clinical features suggestive of vertebral fracture associated with radiographic images confirming the diagnosis. This complication is associated with taking long-term steroids, as in the patient's history; there was no case of previous vertebral collapse. According to some authors, the side-effects of corticosteroid therapy in giant cell arteritis are more common in patients aged 75 years or over receiving at least 40 mg/day prednisone treatment5. Furthermore, in a meta-analysis combining 11 studies collecting data from more than 1,000 patients, steroids was noted to cause side effects in 29% of patients, and cause a complication in 10% of rheumatology cases8. Glucocorticoid-induced osteoporosis is the most common complication of temporal arteritis for at least 10% of patients and 15% at 1 year of treatment of those aged over 75 years. Rheumatological complications related to corticosteroids are the most prevalent complication in those over 75 years. Within a group of 229 people treated with prolonged corticosteroid therapy, a prevalence of 46% of vertebral fractures was observed in the age group 70–79 years vs. 32% in that age group not using steroids, and respectively 60% versus 40% after 80 years. The subjects of 70–79 years have a vertebral slice fracture risk 5 times higher than those less than 60 years11. In two of our patients (8.6%), we observed the appearance of a depression with mood, character and behavior. According to some authors, psychiatric disorders do not appear to be increased with low doses of cortisone2. In a series involving subjects aged 75 years, psychiatric complications were reported in 13 cases including 7 cases of depressive disorders and, 6 cases of agitation with confusion or mania2. In 126 subjects with giant cell arteritis, 20 patients (16%) had psychiatric complications with corticosteroids. The onset of these disorders occur most often in the first month of treatment, taking on various forms such as: mood with irritability, sleep disorder, depression, manic states syndrome and anxiety disorders in vascular dementia12. The systematic implementation of a Mini Mental State or Geriatric Depression Scale in all patients receiving prolonged oral corticosteroids especially before the onset of rapid cognitive decline or psychiatric symptoms especially in patients treated for giant cell arteritis, should help target surveillance of subjects at risk12. We found one case of steroid-induced cataracts diagnosed in the 42nd month of corticosteroid therapy in a patient treated for rheumatoid arthritis. Eye problems, mainly represented by a posterior subcapsular cataract, and did not tend to regress to the withdrawal of corticosteroid therapy, even at low doses5. Cataract is deemed to be a complication of high-dose corticosteroids or related to the total dose and duration of treatment. This complication has also been reported with low dose corticosteroids, but it is uncommon. Its relative risk is 1.8 to 2.5 times higher with prolonged steroid treatment13. A mucocutaneous disorder was reported in one case, and these are linked to metabolic disturbances and obesity through fat overload or facial-nerve block. These treatment-related disorders are more difficult to prevent since they are often also linked to the condition being treated long-term14.\n\n\nConclusion\n\nGlucocorticoids are among the most commonly prescribed agents in clinical practice. Their varied physiological effects make them ideal agents for treating several disease states. Infectious and metabolic complications were the most common in our study. Physician education on risk factors might improve prescribing glucocorticoids in elderly patients. The knowledge of drug-use patterns is extremely important, particularly when treating a member of the aged-population who is a high-risk subject.",
"appendix": "Author Contributions\n\nAll authors participated in the completion of this work. They contributed in the practice study, interpretation of results and discussion.\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\nReferences\n\nAmoura Z, Amoura I, Bletry O, et al: Connectivites. In: Encyclopédie Médico-Chirurgicale: Thérapeutique Paris, Elsevier; 1994; 25-165-A-10: 1–12.\n\nChevalet P, Barrier JH, Glémarec J, et al: Horton's disease in elderly patients aged over 75: clinical course, complications of corticotherapy. Comparative study of 164 patients. Towards a reduced initial dose. Rev Med Interne 2001; 22 (7): 624–630 [Article in French].\n\nRecommendations for the prevention and treatment of glucocorticoid-induced osteoporosis. American College of rheumatology task force on osteoporosis guidelines. Arthritis Rheum 1996: 39 (11): 1791–1801.\n\nSoubrier M, Mathieu S, Payet S, et al: Elderly-onset rheumatoid arthritis. Revue Rhum 2010; 77 (4): 326–332.\n\nTreves R, Bertin P: Glucocorticoid therapy for rheumatoid arthritis. Ann Med Interne 2002; 153 (1): 53–60.\n\nCohen P, Guillevin L: Corticotherapy in systemic diseases. Ann Med Interne 1994; 145 Suppl 2: 23–28 [Article in French].\n\nGaujard S, Broussolle C, Cathebras P, et al: Systemic lupus erythematosus with disease onset after age 65. Rev Med Interne 2003; 24 (5): 288–294 [Article in French].\n\nAgard C, Barrier JH: Simple Horton's syndrome: modalities of treatment. Presse Med 2004; 33 (1): 41–50 [Article in French].\n\nDelauche-Cavallier MC: Polymyalgia rheumatica and giant cell arteritis. In: Encyclopédie Médico-Chirurgicale: Thérapeutique. Paris, Elsevier; 1987; 25171-A10: 1–6.\n\nHenzen C: Glucocorticoid treatment: risks and side effects. Form Med Suisse 2003; 19: 442–46.\n\nOrcel G: Glucocorticoid-induced osteoporosis: new approaches. Rhumatologie 1997; 66: 717–26.\n\nFauchais AL, Boivin H, Hachulla E, et al: Psychiatric complications of corticoid therapy in the elderly over 65 years of age treated for Horton disease. Rev Med Interne 2002; 23 (10): 828–833 [Article in French].\n\nRozenberg S: Corticosteroids and common lumbar spinal pathology. Rev Rhum 1998; 65: 719–26.\n\nLarbre JP, Lorca G: Corticosteroids: principles and rules of use. Rev Prat 1999; 49 (8): 893–898 [Article in French]."
}
|
[
{
"id": "348",
"date": "06 Nov 2012",
"name": "Boulos Haraoui",
"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 work carried out in this research article is in no way novel, and it provides very little in terms of being an important and relevant study.The study design uses a very small sample size (23 patients) and the inclusion criteria of the subjects is poor. Because of this weak study design, the work itself has little to no applicability.Other reasons why the work is of poor scientific value is that the work is;1. It is a retrospective analysis, therefore the collection of the data is not standardized.2. There are is a mix of different diseases (all treated with prednisone) which themselves carry their own cause of complications.3. The doses of the glucocorticoids were different in each case as was the length of the therapy.4. There was no control group for the study.",
"responses": []
},
{
"id": "349",
"date": "07 Nov 2012",
"name": "Yassine Amrani",
"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 significance of this retrospective study is pretty limited as the hypothesis (which is not novel) is applied to a target population that is highly heterogeneous in terms of the underlying conditions requiring the use of steroid therapy.The included patients suffer from auto-immune diseases (rheumatoid arthritis, SLE), cancers (multiple myeloma), temporal arteritis all of which could be affecting the risk of side-effects. In addition, treatment duration is also another factor susceptible of affecting the course of side effects that were seen. Some patients require lifelong therapy (10 mg/day) while others have been treated for only 18 months (and 4 weeks). Dosage and the type of corticosteroids (prednisone and dexamethasone) are not consistent among the different groups. To summarize these points, duration, dosage and the underlying causes should be carefully taken into account before setting up the inclusion/exclusion criteria since they all represent factors susceptible of greatly impacting on the patients’ response to therapy and susceptibility to side-effects. For example, patients with multiple myeloma are at high risk of developing bacterial infections irrespective of corticosteroid therapy. The other unknown parameter that has not been mentioned is the patients’ adherence to treatment. How was steroid adherence monitored, especially in patients with steroid-related neuropsychiatric adverse effects?Other concerns:The study question of the relative risk of adverse effects in elderly patients treated with long-term corticosteroids is interesting but not novel. Others concerns include the relatively small group size and the fact that there is no relative risk as a form of risk ratio (RR) and odd ratio (OR) attached to the study which, again would require the inclusion of a control group. Adding an equal number of hospitalized patients of the same age group but different diagnoses excluding the use steroid therapy would have been possibly a better control group.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-37
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https://f1000research.com/articles/1-35/v1
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22 Oct 12
|
{
"type": "Research Article",
"title": "Effect of storage levels of nitric oxide derivatives in blood components",
"authors": [
"Melissa A Qazi",
"Fabiola Rizzatti",
"Barbora Piknova",
"Nathawut Sibmooh",
"David F Stroncek",
"Alan N Schechter",
"Melissa A Qazi",
"Fabiola Rizzatti",
"Barbora Piknova",
"Nathawut Sibmooh",
"David F Stroncek"
],
"abstract": "Background: Potential deleterious effects of red blood cell (RBC) transfusions, especially from blood kept at length, have been ascribed to biochemical changes during storage, including those of nitric oxide (NO) metabolism.Study methods and design: In this study, NO metabolites, nitrite and nitrate, were quantified in RBCs and whole blood with time of storage. Whole blood (WB), leukoreduced (LR), and non-leukoreduced (NLR) components were obtained from healthy volunteer donors and stored in polyvinyl chloride bags for 42 days. Nitrite and nitrate were measured using reductive gas-phase chemiluminescence.Results: Nitrite concentrations initially decreased rapidly from about 150nmol/L, but stabilized at about 44nmol/L in room air for up to 42 days. Nitrate concentrations remained stable during storage at about 35µmol/L. Cells from bags maintained in an argon chamber showed decreased nitrite levels compared to those maintained in room air. Inhibition of enzymes implicated in the NO cycle did not alter nitrite levels.Conclusion: As erythrocytes may contribute to the control of blood flow and oxygen delivery through reduction of nitrite to NO under hypoxic conditions, the present findings provide insight into possible effects of blood transfusion. These measurements may explain some adverse effects of RBC transfusion and suggest ways of optimizing the preservation of stored blood.",
"keywords": [
"The field of transfusion medicine has experienced much controversy surrounding the safety and efficacy of current transfusion practices. Potentially deleterious effects that are seen with blood storage–including",
"but not limited to",
"declines in 2",
"3-DPG and ATP",
"as well as increases in potassium content and in free hemoglobin and iron (due to hemolysis of red cells)–have been termed the “storage lesion”1",
"2. Our limited understanding of the significance of this storage lesion or storage-induced physiological changes is at the root of a current debate about the efficacy of using long-stored blood. Reports of post-transfusion complications",
"such as multiple organ failure",
"sepsis",
"and even small general increases in morbidity and mortality",
"have fueled much concern1",
"2",
"but clinical investigation has yielded conflicting opinions about the actual impact of the age of blood on transfusion outcomes. Some studies suggest that transfusion with stored blood results in greater post-operative complications than transfusions with fresh blood because of the deleterious effects of storage3–5. However",
"several other studies have presented results that indicate no definitive difference between transfusion outcomes with fresh blood or aged blood6–8",
"or if there is an effect",
"it is probably small9",
"10. Infusing stored blood with augmented 2",
"3-DPG and ATP has resulted in improved transfusion outcomes11",
"and a similar approach is now being considered to target other potentially deleterious biochemical changes."
],
"content": "Introduction\n\nThe field of transfusion medicine has experienced much controversy surrounding the safety and efficacy of current transfusion practices. Potentially deleterious effects that are seen with blood storage–including, but not limited to, declines in 2,3-DPG and ATP, as well as increases in potassium content and in free hemoglobin and iron (due to hemolysis of red cells)–have been termed the “storage lesion”1,2. Our limited understanding of the significance of this storage lesion or storage-induced physiological changes is at the root of a current debate about the efficacy of using long-stored blood. Reports of post-transfusion complications, such as multiple organ failure, sepsis, and even small general increases in morbidity and mortality, have fueled much concern1,2, but clinical investigation has yielded conflicting opinions about the actual impact of the age of blood on transfusion outcomes. Some studies suggest that transfusion with stored blood results in greater post-operative complications than transfusions with fresh blood because of the deleterious effects of storage3–5. However, several other studies have presented results that indicate no definitive difference between transfusion outcomes with fresh blood or aged blood6–8, or if there is an effect, it is probably small9,10. Infusing stored blood with augmented 2,3-DPG and ATP has resulted in improved transfusion outcomes11, and a similar approach is now being considered to target other potentially deleterious biochemical changes.\n\nAmong the observed adverse effects of blood transfusions, reduced oxygen delivery and reduced vasodilatory capabilities of stored RBCs are considered especially critical factors1,2,12. It is now known that one of the primary vasodilators and regulators of blood flow is the endothelium-derived relaxing factor, nitric oxide (NO)13. Substantial production of NO occurs within tissues via several mechanisms. Initially, conversion of L-arginine to NO was thought to be primarily via endothelial nitric oxide synthase (eNOS)14–16 and to a lesser extent, via neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) enzymes17. It has recently been realized that, in addition to NOS synthesis, nitrite reduction to NO may be catalyzed by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin in blood and by other heme-proteins in various tissues18–23. Indeed, nitrite ions may be the major storage pool of NO bioactivity. On the other hand, erythrocytic hemoglobin is a major sink for the destruction of NO, and cell-free hemoglobin is an even more effective sink for NO24. Clearly the physiological and potentially pathological effects of red cell transfusions will be affected by any changes in these NO synthetic and destructive processes prior to, during, and immediately after red cell administration.\n\nCurrently, there is interest in the investigation of potential clinical consequences of changes in NO derivatives during storage, especially with respect to oxygen delivery and vasodilatory capabilities of transfused blood, as well as any association with transfusion-related complications. Two approaches to this have surfaced from our understanding of the metabolism of NO. In one, the nitrite/NO pathway is implicated-nitrite is a major storage pool of NO that can interconvert directly or indirectly with NO. In fact, studies on platelets have elucidated a functional role of nitrite as a modulator of platelet aggregation under hypoxic conditions25,26. In another approach, S-nitrosylated hemoglobin (SNOHb) is implicated-indeed, it was proposed that the amount of SNOHb is responsible for the quality of stored red cells, and that replenishing SNOHb would be therapeutically helpful, supposedly restoring the oxygen-transport and vasodilatory capabilities of RBCs. However, this theory has been questioned on several grounds20,27–29. Evaluation of NO availability with respect to hemoglobin-mediated reductive mechanisms thus appears warranted.\n\nIn the present study, we quantified the main nitric oxide metabolites–nitrite and nitrate–as a function of duration of blood storage. We investigated blood stored under standard blood bank protocols, as well as blood stored in an argon chamber, to prevent gas exchange with the surrounding air. We also addressed the role of enzyme inhibition in NO metabolite composition in stored blood. Our results demonstrated a stable nitrate concentration throughout storage but a gradual decline in nitrite concentration after the initial rapid destruction upon venisection. Stored blood consistently maintained low levels of nitrite until the end of storage. The potential implications of these results for blood transfusion therapies are discussed.\n\n\nMaterials and methods\n\nTen healthy volunteer donors enrolled in an Institutional Review Board approved protocol each provided one 450mL unit of blood for this study. Blood was drawn using the standard phlebotomy method and stored in polyvinyl chloride (PVC) bags for up to 42 days at 4°C, per standard blood bank protocol. An additional 20mL of whole blood was collected in 10.0mL Becton Dickinson (BD) Vacutainer®s for immediate processing. Each single unit of blood was split into units of WB, NLR, and LR blood components. Six units were used to measure changes in WB, LR, and NLR blood samples over 42 days. Three of these units were randomly designated for room air storage and the other three were for argon chamber storage. Supernatants from these six units were used to measure their respective nitrite and nitrate values when stored in either room air or the argon chamber, and small amounts of blood from each bag were used to measure the pO2 levels as well. The four remaining units were used for the enzyme inhibition studies, with 2 units for L-NAME and 2 units for acetazolamide. Each unit was split into LR and NLR control units and LR and NLR units for enzyme inhibitor administration.\n\nTo test immediate nitrite decay, a separate pool of donors was used.\n\nWhole blood collected using a citrate phosphate double dextrose anticoagulant (CP2D) PVC bag was split three ways–whole blood (WB), non-leukoreduced (NLR) blood component, and leukoreduced (LR) blood component (see Supplemental Figure 1 for an overview of the protocol). 100mL of WB was drawn into a new PVC bag. The remaining blood in the original CP2D unit was centrifuged and the plasma was removed; these RBCs were mixed with adenine-saline (AS-3) solution and comprised the NLR component. 150mL of this unit were filtered through an RCM1 Leukocyte Filter (Pall Corp., East Hills, NY) to obtain the LR component. All components were stored in identical AS-3 PVC bags. Blood separation occurred at 20 to 24°C and required approximately 1 hour.\n\nBlood components were either stored in room air for 42 days at 4°C - standard blood bank storage conditions, or in an air-tight chamber (Fisher Scientific, Vacuum Dessicator Cabinet, Suwanee, GA) for 42 days at 4°C in an argon atmosphere (Roberts Oxygen Co., 99% pure argon, Rockville, MD).\n\nSamples were collected for analysis of nitrite, nitrate, SNOHb, iron nitrosyl hemoglobin (HbNO), and methemoglobin (MetHb) in all components. Immediately upon venisection, baseline samples of heparinized WB and RBC were taken. Aliquots of blood were thereafter drawn from storage bags using a liquid transfer set (Charter Medical, Winston-Salem, NC) to maintain sterile conditions. Three replicates of each sample were collected in Eppendorf tubes everyday for the first seven days of storage, and every 2–3 days following that, until day 42. Upon collection, samples were placed on dry ice and maintained in a -80°C freezer until thawed on regular ice prior to chemiluminescent analysis. An Abbott i-STAT cartridge reader (Abbott Laboratories Inc., Portable Clinical Analyzer, East Windsor, NJ) was used to determine pH and pO2 levels (using CG8+ and G3+ cartridges) at the time of sample collection.\n\nA standard protocol was used to determine NO2- and NO3- levels in the three blood components30,31. “Stop solution” (K3Fe(CN)6, N-ethylmaleimide, water, NP40) was added to blood to maintain nitrite levels until sample analysis21. A 1:4 dilution of “stop solution” to blood was vortexed and placed on dry ice. At the time of sample analysis, a 1:1 dilution of 99.9% pure methanol and thawed sample was centrifuged for 2min at 13,000rpm; the supernatant was immediately injected into the chemiluminescent nitric oxide analyzer (NOA, Sievers, Model 280 NO analyzer, Boulder, CO) using helium as the carrier gas. The triiodide (I3-) ozone-based chemiluminescent assay was used to analyze nitrite levels. To analyze nitrate, deionized water (Millipore CQ-Gard, Bedford, MA) was added to blood to lyse cells. A 9:1 dilution of deionized water to blood was vortexed and placed on dry ice. At the time of sample analysis, a 3:1 dilution of pure HPLC grade ethanol and thawed sample was centrifuged, and the supernatant was immediately analyzed using the Vanadium(III)chloride chemiluminescent assay. The VCl3 reaction solution was maintained at 90°C with helium as the carrier gas. 1µM nitrite and nitrate solutions were used to generate standard curves for comparisons and adjustments of sample nitrite and nitrate concentrations.\n\nA standard protocol was used to determine SNOHb and HbNO levels in all components30,31. A thiol-stabilization solution (NEM-DPTA; K3Fe(CN)6, N-ethylmaleimide, Diethylenetriaminepenta acetic acid, NP40, water) was added to blood to maintain SNOHb and HbNO levels by inhibiting additional thiol reactions. A 4:1 dilution of NEM-DPTA to blood was vortexed and placed on dry ice. A 9:1 dilution of sample and 5% acid sulfanilamide (AS) was incubated for 5min; half was injected into the NOA (I3- assay) to give combined SNOHb and HbNO levels. The remaining sample was incubated with 50mM HgCl2, then incubated again with 5% AS, and injected into the NOA to give HbNO levels.\n\nBlood components were centrifuged at 13,000rpm for 5min and WB, NLR, and LR supernatants were collected. Aliquots of saline were collected directly from control PVC bags using a liquid transfer set. The aforementioned I3- and VCl3 assays were used for WB, NLR, and LR supernatants and saline samples. As samples were already separated from the blood pellet, treatment with methanol and ethanol was unnecessary.\n\nMethemoglobin (MetHb) analysis was performed at each sample collection. Pre-storage and storage values (up to 42 days) were evaluated using a CO-Oximeter (Radiometer America Inc., OSM3 Hemoxymeter, Cleveland, OH).\n\nFresh whole blood collected in heparinized Vacutainer®s was immediately sampled for pre-storage nitrite and nitrate levels using the aforementioned protocols. The first 5 hours of ex vivo NO2- decay was studied for WB and RBCs only. Aliquots were collected at time = 0, 5, 10, 15, 20, 30, 60, 120, 180, 240, and 300 (in minutes), where time points do not account for a 3–5 minute delay in receiving samples from the phlebotomist. 1mL of whole blood was centrifuged at 13,000rpm for 2min at each interval to obtain RBCs.\n\nBags of LR and NLR RBCs were stored for 7 days and infused once daily with one of three inhibitors: N-Nitro-L-arginine methyl ester hydrochloride (L-NAME, PBS; final concentration: 1mM, Sigma-Aldrich Co.)32 for NOS inhibition, acetazolamide (acetazolamide, DMSO; final concentration: 100µM, Sigma-Aldrich Co.)33 for carbonic anhydrase inhibition, and oxypurinol (oxypurinol, NaOH, Tris-Ringer buffer; final concentration: 0.1mM, Sigma-Aldrich Co.)34 for xanthine oxidase inhibition. All inhibitors were administered using a liquid transfer set. Controls were infused once daily with identical saline volume. Blood and supernatant samples were prepared 1 hour after infusion and tested per the aforementioned methods for nitrite analysis.\n\nData were recorded using the NOAnalysis 3.21 Liquid software. OriginLab 7 was used to analyze data and calculate the amount of NO detected, by evaluating the area under the peaks and comparing them to known standards. Nitrite and nitrate values were corrected by subtracting the amount of nitrite present in methanol and stop solution, and the amount of nitrate in ethanol and water, respectively. SNOHb was determined by subtracting HbNO levels from cumulative HbNO and SNOHb levels.\n\nGraphPad Prism 4 was used for statistical analysis and graphical representation of data (mean ± SEM). A one-way ANOVA test with the Bonferroni multiple comparison analysis was used to determine statistical significance. Results with a p-value of less than 0.05 were considered significant.\n\n\nResults\n\nNitrite levels showed the expected very rapid decay in both whole blood and red blood cells in the hours immediately following venisection (Figure 1). Blood was kept in air at room temperature for the duration of these measurements. At t = 0, the nitrite concentration in whole blood (Figure 1A) was about 150nM; by 60min, endogenous blood nitrite levels had decreased to about 85nM, and by 5 hours, nitrite levels had decreased to about 65nM. The values for the first 60 minutes are consistent with previously reported data21. Red blood cell preparations, in which the first measurements were delayed by the processing time (approximately 3–5 minutes after receipt from the phlebotomist), demonstrated comparable behavior (Figure 1B), but the higher initial values were lost during this time.\n\nBlood components were kept in room air at 24°C; number of donors, n=6 (A), n=4 (B). Time points above do not account for a 3–5 minute delay in receipt of blood from phlebotomist.\n\nNitrite underwent additional, but slower decay in all three product forms and their supernatants over 42 days of storage, but the nitrite concentration leveled off in room air samples at about 44nM (Figure 2A) in all three blood components. This gradual decrease in concentration and leveling was found in both air and argon chamber stored blood (Figure 2B). However, comparison of blood nitrite levels from air (Figure 2A) and the argon chamber (Figure 2B) reveals a noticeable depression in nitrite values in the chamber environment that is consistent throughout the storage period. RBCs stored in room air had nitrite concentrations of 42 ± 4nM on day 42; while the argon chamber samples decreased in concentration to 16 ± 3nM on day 42. WB stored in room air reached nitrite concentrations of 44 ± 8nM, while WB stored in the chamber reached nitrite concentrations of 26 ± 3nM by the end of the storage period (p>0.05). Under both storage conditions, nitrite levels were very similar (within the error of this assay) for the three types of cell preparations–whole blood, non-leukoreduced RBCs, and leukoreduced RBCs-for the duration of the experiment (Supplemental Figure 2). However, the higher values in room air as compared to chamber-stored samples suggest additional factors affecting production and/or destruction of nitrite ions. Supplemental Figure 2 presents curve-fitting of these data, displaying room air and chamber nitrite decay for the individual blood components. The same trends were seen in the nitrite concentrations of supernatants for each of the three blood components (Figures 3A and 3B). Nitrite concentration in supernatants was significantly lower than that in blood components, confirming nitrite localization in erythrocytes and the findings of previous studies21.\n\nBlood components stored in the three forms noted were kept for 42 days at 4°C in either room air (2A) or an argon chamber (2B), to emulate aerobic and hypoxic conditions, respectively; number of donors, n=3 (A), n=3 (B).\n\nFigure 3A shows the nitrite concentration in supernatants stored in room air, number of donors, n=3, while Figure 3B shows the same for supernatants stored in an argon chamber, number of donors, n=3. Nitrite concentrations in saline controls stored under both conditions are shown in Figure 3C, number of donors, n=6 (room air n=3, argon chamber n=3).\n\nIn saline stored in PVC bags, nitrite levels varied greatly based on storage conditions (Figure 3C). Saline stored in room air experienced a gradual increase in nitrite concentration from 62 ± 6nM on day 1 to 140 ± 7nM on day 42. Saline stored in the argon chamber remained relatively steady over the storage period, as expected, showing a slight increase in nitrite concentration from 36 ± 2nM on day 1 to 58 ± 5nM on day 42.\n\nIn contrast to the results with nitrite ions, nitrate levels in WB, in NLR and LR RBCs, and in supernatant samples remained steady for the duration of blood storage (Figure 4). Whole blood nitrate levels were slightly higher than either the non-leukoreduced or the leukoreduced RBC components stored either in air or in the argon chamber. In room air, WB nitrate concentration was about 47 ± 2µM, while NLR and LR nitrate concentrations were about 34 ± 2µM. For blood stored in the argon chamber, WB nitrate levels were lower than respective room air samples, with chamber nitrate remaining at about 37 ± 3µM. LR and NLR RBC nitrate levels in the chamber were unchanged when compared to LR and NLR RBCs stored in air, remaining at about 35 ± 2µM. Supernatant nitrate levels (measured after centrifugation of packed red cells or whole blood) followed the same trend as the blood samples, remaining steady for the duration of storage and exhibiting similar nitrate concentrations when stored in an argon chamber (data not shown).\n\nThe gas-phase chemiluminescence I3- assay was used to determine SNOHb and HbNO levels in blood components stored in both room air and chamber conditions. Figure 5 shows HbNO and SNOHb levels as detected by the NOA at two time points in the first hour following venisection. However, levels of HbNO and SNOHb were virtually undetectable 1 hour after blood collection and in stored blood thereafter.\n\nA CO-oximeter was used to measure MetHb levels in all three blood components stored in both room air and chamber conditions. Figure 6 shows the gradual increase in MetHb from nearly 0.5% to just above 1% during the storage period. MetHb concentration is expressed as percentage of total hemoglobin concentration.\n\nGas-phase chemiluminescence signals used to determine SNOHb concentration. The peaks from two samples in the first 20 minutes of storage are shown; SNOHb concentration is ascertained by subtracting the HbNO peak from the composite of SNOHb plus HbNO after treatment with HgCl2 and acid sulfanilamide. The values of SNOHb, near the sensitivity of the method, are less than 30nM, while HbNO is barely detectable. Neither peak was detected after 1hr of storage.\n\nRoom air at 4°C satisfied standard conditions for blood storage under current American Association of Blood Banks (AABB) Transfusion Medicine protocols; the argon chamber prevented gas exchange with ambient air and also mimicked blood storage under hypoxic conditions. Blood components stored in room air demonstrated a gradual rise in partial pressure of oxygen over the first three weeks and leveled off thereafter. pO2 of blood components stored in the argon chamber remained at the levels of venous blood initially drawn or even showed a small decrease during storage. The pH of the samples stored in air was measured over the 42 days and gradually decreased from about 7.4 to below 6.5 (Supplemental Figure 3). This is consistent with previous reports35. The time in which pH levels fell below 6.5 differed among the blood products, with NLR RBC pH falling after the first 13–16 days, LR RBC after 23–24 days, and WB after about 38 days of storage.\n\nNote: i-STAT apparatus does not detect pH levels below 6.5; values read as <6.5 were plotted at 6.5.\n\nIt has been suggested that xanthine oxidase may take on the role of the main enzyme responsible for converting nitrite to vasoactive NO in hypoxia36. In our study, however, several key NO-generating enzymes–NOS, carbonic anhydrase, and xanthine oxidase–were inhibited with L-NAME, acetazolamide, and oxypurinol, respectively. The amount of nitrite measured in the bags did not change as a result of NOS enzyme inhibition with L-NAME or carbonic anhydrase inhibition with acetazolamide (Supplemental Figure 4) over seven days of storage as compared to control LR or NLR red cells. The data for oxypurinol inhibition of xanthine oxidase were similar (data not shown).\n\nS4C and S4D show inhibition of carbonic anhydrase with acetazolamide in leukoreduced RBCs and in non-leukoreduced RBCs, respectively; number of donors, n=2. All bags were stored in room air at 4°C for 7 days.\n\n\nDiscussion\n\nThere is much concern, but also much controversy, about whether storage of red blood cells for transfusion decreases their therapeutic efficacy, or even leads to harm upon administration. The history of transfusion medicine has seen the development of technologies to allow increasingly extended storage of blood products, especially erythrocytes37; in the last century, such advances have revolutionized medical practice by allowing blood to become a readily available therapeutic agent. In recent years, these advances have been called into question by the perception of possible deleterious effects of long-stored blood as compared to relatively new units of blood. Such negative effects, which are said to be associated with a “storage lesion”, have been ascribed to some of the many biochemical changes that occur upon storage, including depletion of 2,3-DPG, ATP, and potassium ions, or even cellular changes, including increased rigidity or hemolysis, with resultant increases in free hemoglobin and iron in the transfusion recipients1,2,37. Although no significant hemolysis was seen in this study, a recent finding indicates that there may be some increase during the final week of storage38.\n\nClearly, many of these storage-related red cell changes, which could affect blood flow and oxygen delivery in the recipients, reverse rapidly upon infusion, and probably account for the fact that survival of 75% of transfused cells at 24 hours can be used as an achievable criterion for efficacy following storage39. (However, one might imagine that such in vivo repletion might not be rapid enough to avoid deleterious effects with very large volume transfusions). Indeed, the clinical evidence for blood age-associated transfusion complications is itself uncertain. It is clear that any such effects are relatively small compared to the efficacy of transfusions, which likely explains the difficulty of convincingly demonstrating them in small trials, where there are many confounding variables. However, in an era of therapy optimization, it is very important that we examine whether prolonged blood storage causes any changes, reversible or irreversible, that may interfere with the goals of transfusion medicine.\n\nThe importance of NO and the related oxides of nitrogen to determining blood flow and other biological phenomena has led to widespread focus on the role of NO in physiology and pathophysiology, and as of late, also during blood storage. It has long been recognized that both intraerythrocytic and free hemoglobin readily destroy NO. In a recently published reviews, a causal relationship between hemolysis-dependent changes in NO functionality and the storage lesion was presented, and the importance of contextual understanding of these changes through NO metabolites is addressed40,41. The recent recognition that red cells may transduce NO bioactivity by transporting NO in an endocrine-like fashion42 has focused interest on measuring parameters related to these processes in stored red cells, as they may balance the destructive mechanisms. Much recent work has focused on the ability of deoxyhemoglobin and other red cell-related proteins, xanthine oxidoreductase in particular43, to reduce red cell or plasma nitrite ions to NO. A recent study of NO-related metabolic changes during platelet storage also suggests the possibility of platelet consumption of nitrite26. An older hypothesis, in which NO forms S-nitrosohemoglobin (SNOHb) upon reaction with oxyhemoglobin but then dissociates upon deoxygenation, has some adherents, although recent experiments involving transgenic animal models seem to largely negate this theory44.\n\nNitrite in red cells and plasma may be considered as the primary storage form of NO because of its relative abundance in blood and increasing evidence of its physiological and pharmacological importance. Nitrite and nitrate ions are the major oxidation products of NO metabolism and are produced in the body by reaction with oxygen, hemoglobin, ceruloplasmin and possibly other molecules45. In addition, nitrite and nitrate are ingested with food; nitrite can also be produced from nitrate by salivary bacteria46. Measurement of nitrite levels in blood have been suggested as an index of NO bioavailability for epidemiological studies of cardiovascular disease severity47.\n\nOur approach to the question of NO bioavailability in stored red cells has been to measure nitrite and nitrate as a function of duration and conditions of storage as occurs in transfusion practice. Our major finding is that, although much red cell nitrite is rapidly lost during the first few hours of storage, nitrite levels continue to deplete, albeit at a much slower rate, to a clear plateau of about 44nM in the last two weeks of storage. This phenomenon is almost identical in red cells stored with or without leukodepletion and those stored as whole blood; this suggests that interactions with white blood cells and platelets do not significantly impact nitrite metabolic processes. However, while storage of blood in a closed chamber under argon did not change the progression of the nitrite depletion, this environment did lower overall nitrite values in a nearly uniform fashion, to a final concentration of about 20nM.\n\nRed cell nitrate levels remained virtually unchanged from day 1 to day 42, at around 35µM (approximately 1000X the final concentration of nitrite), and were identical for red cells stored as leukoreduced, non-leukoreduced, and whole blood. The storage conditions, whether room air or argon chamber, did not affect these measurements. As expected, metHb concentrations under all experimental conditions remained around 1%, as previously shown34. Levels of SNOHb during the first hour of analysis were barely detectable by gas-phase chemiluminescence after triiodide treatment, and were consequently unquantifiable.\n\nOur nitrite results, which showed that nitrite levels stabilized and remained stable at nonzero values, were surprising. From previous studies of the reaction of hemoglobin with nitrite in vitro, we would predict that nitrite would largely disappear over the course of storage. Although we cannot offer a complete explanation for the mechanism of nitrite retention in the red cell, our investigation of this effect has led to several conclusions. First, we addressed the potential role of several enzymes implicated in NO cycle–NOS, xanthine oxidase, and carbonic anhydrase. Nitrite levels did not change upon inhibition of these enzymes, indicating that they are not likely contributors to the maintenance of the low nitrite concentrations. Second, HbNO is also an unlikely source of nitrite, as it is nearly unquantifiable after twenty minutes, with a half-life of ~79 minutes23. Third, reduction of nitrate to nitrite also seems unlikely, since mammalian processes to account for such a reaction have not been described; however, the fact that nitrate remains steady does not necessarily exclude this process, as only a one-tenth of 1% change in nitrate could account for the maintenance of these nitrite levels. Finally, while bacteria are known to synthesize nitrite, bacterial contamination may be ruled out because standard blood storage protocol virtually eliminates the risk of such contamination. On the other hand, several explanations may account for at least part of this nitrite retention. Greater depletion of nitrite found in the argon chamber samples raises the possibility that nitrogen reactive species may enter the stored blood via diffusion from the atmosphere through the gas-permeable PVC bags, as confirmed by our saline solutions. Atmospheric nitrogen reactive species may therefore account for some of the nitrite that remains. Other potential mechanisms to explain nonzero values of nitrite at 42 days include the possibility of nitrite binding to RBC proteins (such as band 3 proteins or other members of the cytoskeletal protein scaffold via ionic interactions that could effectively shield nitrite from reacting with hemoglobin), hemoglobin–nitrite ionic interactions, and formation of nitrite-methemoglobin complexes.\n\nNO and its metabolic products are likely to play an important role in maintenance of banked blood, as effective agents for transfusion therapies. Nitrite seems to be a source of great NO bioactivity that remains present in the blood for the duration of storage. Nitrate is also present in relative abundance to nitrite throughout storage. The presence of these metabolites in red cells up to 42 days of storage provides the possibility that maintenance of NO/nitrite levels and NO bioactivity is likely to occur via the nitrate-nitrite-NO pathway.",
"appendix": "Author contributions\n\n\n\nMelissa A Qazi performed data collection, statistical analysis, and wrote the manuscript. Fabiola Rizzatti developed the project design, performed data collection and statistical analysis, and edited the manuscript. Nathawut Sibmooh and Barbora Piknova provided technical assistance with data collection and edited the manuscript. David Stroncek provided access to the NIH Blood Bank and Department of Transfusion Medicine facilities and assistance with DTM procedures. Alan N Schechter designed the project and edited the manuscript.\n\n\nCompeting interests\n\n\n\nAlan N Schechter has a patent on therapeutic use of nitrite salts. Melissa A Qazi, Fabiola Rizzatti, Barbora Piknova, Nathawut Sibmooh, and David Stroncek have no conflicts of interest to disclose.\n\n\nGrant information\n\nThe study was funded by and received research support from the Molecular Medicine Branch, NIDDK, NIH and the Department of Transfusion Medicine, Clinical Center, NIH.\n\n\nAcknowledgments\n\nWe would like to say a special thanks to Barbara Bryant for graciously offering her technical expertise and support during the completion of this work.\n\n\nReferences\n\nTinmouth A, Ferguson D, Yee IC, et al.: Clinical consequences of red cell storage in the critically ill. Transfusion. 2006; 46(11): 2014–2027. PubMed Abstract | Publisher Full Text\n\nKlein HG, Spahn DR, Carson JL: Red blood cell transfusion in clinical practice. Lancet. 2007; 370(9585): 415–426. PubMed Abstract | Publisher Full Text\n\nPurdy FR, Tweeddale MG, Merrick PM: Association of mortality with age of blood transfused in septic ICU patients. 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PubMed Abstract | Publisher Full Text\n\nYang BK, Vivas EX, Reiter CD, et al.: Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls, and nitrite in biological samples. Free Radic Res. 2003; 37(1): 1–10. PubMed Abstract | Publisher Full Text\n\nPelletier MM, Kleinbongard P, Ringwood L, et al.: The measurement of blood and plasma nitrite by chemiluminescence: pitfalls and solutions. Free Radic Biol Med. 2006; 41(4): 541–548. PubMed Abstract | Publisher Full Text\n\nYu LB, Dong XS, Sun WZ, et al.: Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T. World J Gastroenterol. 2005; 11(40): 6385–6388. PubMed Abstract\n\nMaren TH: Current status of membrane-bound carbonic anhydrase. Ann N Y Acad Sci. 1980; 341: 246–258. PubMed Abstract | Publisher Full Text\n\nBabbs CF, Salaris SC, Turek JJ: Cytochemical studies of hydrogen peroxide generation in postischemic hepatocytes. Am J Physiol. 1991; 260(1 Pt 2): H123–129. PubMed Abstract\n\nPicker SM, Radojska SM, Gathof BS: In vitro quality of red blood cells (RBCs) collected by multicomponent apheresis compared to manually collected RBCs during 49 days of storage. Transfusion. 2007; 47(4): 687–696. PubMed Abstract | Publisher Full Text\n\nCasey DB, Badejo AM Jr, Dhaliwal JS, et al.: Pulmonary vasodilator responses to sodium nitrite are mediated by an allopurinol-sensitive mechanism in the rat. Am J Physiol Heart Circ Physiol. 2009; 296(2): H524–533. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHess JR: An update on solutions for red cell storage. Vox Sang. 2006; 91(1): 13–19. PubMed Abstract | Publisher Full Text\n\nHess JR, Sparrow RL, van der Meer PF, et al.: Red blood cell hemolysis during blood bank storage: using national quality management data to answer basic scientific questions. Transfusion. 2009; 49(12): 2599–2603. PubMed Abstract | Publisher Full Text\n\nLockwood WB, Leonard J, Liles SL, et al.: Storage, monitoring, pretransfusion processing, and distribution of blood components. In: Roback JD, Combs MR, Grossman BJ, Hillyer CD, eds. Technical Manual, 16th edition. Bethesda, MD: AABB; 2008; 283–299. Reference Source\n\nGladwin MT, Kim-Shapiro DB: Storage lesion in banked blood due to hemolysis-dependent disruption of nitric oxide homeostasis. Curr Opin Hematol. 2009; 16(6): 515–523. PubMed Abstract | Publisher Full Text\n\nSchechter AN, Gladwin MT: Hemoglobin and the paracrine and endocrine functions of nitric oxide. N Engl J Med. 2003; 348(15): 1483–1485. PubMed Abstract | Publisher Full Text\n\nLi H, Samouilov A, Liu X, et al.: Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction.Evaluation of its role in nitric oxide generation in anoxic tissues. J Biol Chem. 2001; 276(27): 24482–24489. PubMed Abstract | Publisher Full Text\n\nIsbell TS, Sun CW, Wu LC, et al.: SNO-Hemoglobin is not essential for red blood cell-dependent hypoxic vasodilation. Nat Med. 2008; 14(7): 773–777. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLundberg JO, Weitzberg E, Gladwin MT: The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008; 7(2): 156–167. PubMed Abstract | Publisher Full Text\n\nKelm M: Nitric oxide metabolism and breakdown. Biochim Biophys Acta. 1999; 1411(2–3): 273–289. PubMed Abstract | Publisher Full Text\n\nKleinbongard P, Dejam A, Lauer T, et al.: Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans. Free Radic Biol Med. 2006; 40(2): 295–302. PubMed Abstract | Publisher Full Text\n\nKanias T, Wang L, Lippert A, et al.: Red blood cell endothelial nitric oxide synthase does not modulate red blood cell storage hemolysis. Transfusion. 2013; 53(5): 981–9. PubMed Abstract | Publisher Full Text"
}
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[
{
"id": "459",
"date": "23 Oct 2012",
"name": "Andre Dejam",
"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 great interest the article by Dr. Schechter’s group in DC on the effect of storage on levels of nitric oxide derivatives in blood components.I approve this article as it extends the current knowledge base in the field.",
"responses": []
},
{
"id": "460",
"date": "24 Oct 2012",
"name": "Jon O. Lundberg",
"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": "461",
"date": "25 Oct 2012",
"name": "Eddie Weitzberg",
"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": "462",
"date": "29 Oct 2012",
"name": "Malte Kelm",
"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 present study, Qazi et al. examined the effect of storage on levels of nitric oxide derivatives in blood components.They examined the biochemical changes in the nitric oxide metabolism in red blood cell transfusions and reported that nitrite concentrations initially decreased rapidly and stabilized afterwards for up to 42 days. Under hypoxic conditions in an argon chamber there are even less nitrite levels compared to room air.In my opinion the data provided by the authors is interesting as they show a long-term view on nitrite levels in red blood cell transfusions and provide valuable information about optimizing the preservation of stored blood. These findings definitely extend the current knowledge base in the field.",
"responses": []
},
{
"id": "463",
"date": "12 Nov 2012",
"name": "Jay Zweier",
"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 research article is well written and clear for the most part. I do however have 2 minor comments for the authors to think about:1. It would be interesting to include data on the time course and extent of hemolysis as this may contribute to toxicity and also the nitrite depletion.2. The presentation of Figure 5 is unclear. The authors should better describe and explain this figure. 2 sets of 2 peaks are seen at different times. Please explain the times shown for each peak. Please explain each peak and exactly what it is and how it was measured. It is noted that 2 samples are shown, which correspond to which peak? What is the difference between these 2 samples? How does this relate to the time shown? Were there quantitative standards that were run for HbNO and SNOHb? Could some portion of these small peaks arise from residual nitrite?",
"responses": [
{
"c_id": "106",
"date": "19 Nov 2012",
"name": "Barbara Piknova",
"role": "Author Response",
"response": "We thank Dr Zweier for his approval and comments.1. It is true that hemoglobin from RBCs lyzed during the storage over 42 day would significantly alter the amount of nitrite measured in preparation, as hemoglobin is a very potent nitrite oxidase. However, during our study we did not observe any significant hemolysis in our preparations (in fact, using spectrophotometry, Hb was below our detection limits in RBC supernatants) and data from other studies from NIH blood bank were also in favor of only minor hemolysis occurring during the careful handling of stored blood.2. Figure 5 is an example of our raw chemiluminescence data i.e. the output from the NOA analyzer. The principle and application of this method is described in details in Piknova B and Schechter AN (2011) Measurement of nitrite in blood samples using the ferricyanide-based hemoglobin oxidation assay. Methods Mol Biol. 704:39-56.In this method, reaction vessel is filled with nitrite/HbNO/SNOHb-reducing tri-iodide solution and connected to NOA analyzer. Carrier gas (He) passing through the reaction solution carries NO from reduced “NOx-compounds” into reaction chamber inside the NOA analyzer, where NO reacts with ozone (made by high-voltage generator from oxygen) and forms NO*. When excited NO* molecule returns to its main electron levels, a photon is emitted in red-light region and registered by photomultiplier. The reaction ratio is 1:1 in NO:NO* and also in NO*:photon, which allows to very precise quantification of NO and therefore, original nitrite/HbNO/SNOHb species present in sample. The specificity towards HbNO and SNOHb is achieved by different pre-treatments of sample and by subtraction of known components from composite peaks. In Figure 5 we tried to illustrate that peaks obtained for SNOHb+HbNO were already somehow on the detection limit of our method and that trying to quantify them would lead to significant errors. Therefore, we did not proceed to quantification of these compounds, as we know from our previous experiments that levels comparable with Figure 5 would be from ~20nM HbNO and SNOHb, which was really the low detection limit. Times shown on Figure 5 are for illustrative purposes only, subsequent injections of different samples into vessel are possible whenever the previous sample readings returns to the baseline."
}
]
}
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https://f1000research.com/articles/1-35
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https://f1000research.com/articles/1-33/v1
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19 Oct 12
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{
"type": "Research Article",
"title": "CA72-4 may contribute to real-time reconnaissance of gastric cancer",
"authors": [
"Jutta Keller",
"Ella Reiss-Sklan",
"Miri Refael",
"Viola Andresen",
"Yael Levy-Herman",
"Igor Ruvinsky",
"Jutta Keller",
"Ella Reiss-Sklan",
"Miri Refael",
"Viola Andresen",
"Yael Levy-Herman"
],
"abstract": "Objective: Data from prospective studies indicate a positive impact of gastric cancer screening programs on mortality associated with the disease. Unfortunately, endoscopic procedures, widely regarded as uncomfortable, face low patient compliance, thus underscoring the need for reliable biological markers capable of detection of tumor growth in bodily fluids. Furthermore, in light of the emerging patient-friendly, still devoid of histopathological capabilities, capsule endoscopy, gastric fluid may prove valuable for biomarker-assisted cancer diagnosis. We set out to determine whether CA72-4 measurement in gastric fluid may be of benefit for detection of gastric cancer.Design: Open prospective study.Setting: Sample collection was performed at a tertiary referral center for patients with gastroenterological diseases; immunological analysis was performed at the R&D facility of a commercial biotechnology company. Studies were part of an EU-FP6 project (NEMO).Patients: 176 patients referred for endoscopy due to gastrointestinal complaints.Interventions: Gastric juice was aspirated endoscopically according to standard operating procedures, volume and pH were measured immediately and samples stored at -80°C.Outcome measures: Concentration of CA72-4 tumor marker was evaluated by enzyme-linked immunosorbent assay (ELISA).Results: Median CA72-4 levels were about 4-fold higher in cancer patients compared with patients with normal gastric findings, gastric inflammation, intestinal metaplasia or other diseases (p=0.001). Multivariate linear regression analysis revealed that elevated CA72-4 was significantly predicted by gastric carcinoma adjusted for H. pylori status, age, smoking status, PPI dose, and pH of aspirate (R2=0.27, p<0.0001). In this model, diagnosis of gastric carcinoma had by far the greatest influence. At a cut-off level of 100 U/ml, CA72-4 had 75% sensitivity and 89% specificity for detection of gastric cancer.Conclusions: Based on our findings, CA72-4 level assessment in gastric fluid, featuring yet unmatched accuracy of malignant neoplasia detection may prove beneficial for gastric cancer screening.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: approved 1",
"approved with reservations 1]”."
],
"content": "Background and aims\n\nDespite continuous decline in incidence rates in both sexes, gastric cancer is still the fourth most common cancer worldwide, with 934,000 newly diagnosed cases per year1 and a vast annual death toll of more than 800,000, according to WHO 2004 statistics. With only little improvement over the past decades, the long-term survival from gastric cancer is poor, since patients are often diagnosed with advanced disease. In the USA, for example, five-year survival is 24%2. In a hope for an overall improvement in the forlorn statistics, worldwide efforts are invested to shorten the time-to-diagnosis. Stomach cancer screening has been first introduced in Japan in 1963, followed by The Republic of Korea in 1996, and has recently commenced in less developed countries, with Venezuela, Chile, and Costa Rica adapting the Japanese model to implement pilot screening routines. Notably, the fact that to date no randomized trial of stomach cancer screening has ever been conducted sets hurdles for reliable assessment of efficacy of such policy. Nevertheless, data from recent prospective studies shows reductions in mortality from gastric cancer among participants in screening programs in Japan and Costa Rica3–5. Importantly, in this regard, endoscopic procedures, although of undisputable great diagnostic value, face a lack of patient compliance, for being widely regarded as uncomfortable. Henceforth, to circumvent this obstacle, reliable biological markers, suitable for detection and monitoring of tumor growth in bodily fluids, have long been searched after, in both blood and gastric fluid. Understandably, being easily accessible, blood has long been the substance of choice for marker evaluation. However, as appears from the peer-reviewed sources, the vast majority of the proposed gastric cancer marker candidates share the lack of diagnostic potential. This flaw, expressed in low sensitivity and specificity parameters, obviously negates the idea of utilization of such markers in gastric cancer screening practice.\n\nAs new potential screening methodologies, such as capsule endoscopy6, arise, gastric fluid is becoming an attractive milieu for cancer diagnosis as it contains both secreted soluble and exfoliated cellular proteins from the entire gastric mucosa. Unfortunately, early reports on the diagnostic and prognostic utility of the preponderant indicators of neoplasia, such as carcinoembryonic antigen (CEA) and CA 19-9, yielded unsatisfactory results7–9, thus diverting researchers’ attention to other biomarkers, including CA72-4. Historically, following its identification [a sialosyl-2–6-α-N-acetylgalactosaminyl epitope of TAG-72 mucin;10–15], and characterization, the applicability of the marker to cancer detection was examined in several neoplastic conditions. As second generation high-affinity monoclonal antibody (recognizing a different epitope;16) has enabled establishment of a double-determinant immunoradiometric assay17,18 capable of detection of CA72-4 in bodily fluids of carcinoma patients, CA72-4 presence was assessed in the serum of patients diagnosed with gastrointestinal malignancies19–21. These studies disclosed a complementarity between CA72-4 and CEA, as the former was often elevated in samples from cancer patients in which the levels of the latter remained low. Moreover, serum CA72-4 in patients during post surgical follow-up was predictive of recurrent disease20. Several recently demonstrated lines of evidence also described CA72-4 as a potential serum and peritoneal wash-fluid marker of gastric malignant neoplasia, supreme to CEA (22; and references therein). In the present report we show that CA72-4, whose levels had to date been mainly assessed in serum samples23–26, has the potential to become a major biomarker in the gastric fluid-based gastric cancer diagnosis.\n\n\nMethods\n\nCollection of gastric juice samples and clinical data was performed at Israelitic Hospital (IH) in Hamburg, Germany, an academic hospital and tertiary referral center for patients with gastroenterological diseases. Immunological analysis was performed at the R&D laboratory of Novamed ltd., an ISO 9001-compliant facility, in Jerusalem, Israel.\n\nAll patients older than 18 years of age undergoing an esophago-gastro-duodenoscopy (EGD) for clinical reasons were generally eligible for the study. Study participants gave written informed consent before any study related procedures were performed.\n\nIn all volunteers the EGD was performed as indicated clinically and according to routine procedures, including biopsies and interventional therapeutic measures, e.g. dilation therapy, if indicated. However, juice samples were only collected and analyzed if investigators could intubate the stomach and thoroughly aspirate gastric contents under visual control into separate vials before taking biopsies or performing other measures that might have altered gastric contents (e.g. rinsing of the mucosa with saline in order to improve visibility of potential mucosal alterations).\n\nVolume and pH of each gastric juice sample were measured and recorded immediately, (pH was reassessed prior to immunological analysis). Subsequently, the pseudonymized samples were stored at -20°C until the end of the day and then transferred to -80°C for further storage until evaluation of biomarker concentration.\n\nClinical data may be of pivotal importance for correct identification of biomarkers and was collected prospectively from all study participants. Data were pseudonymized and included the following information: results of endoscopic and histologic investigations, age, sex, height, weight, symptoms, time of last food and fluid intake, diet (e.g. vegetarian), concomitant medication (in particular intake of proton pump inhibitors (PPI)), alcohol consumption, smoking habits and relevant previous and concomitant diseases including abdominal surgery.\n\nPatient groups were defined according to the results of the endoscopic and histologic investigations:\n\nA) Normal stomach: Normal EGD and normal histology according to representative biopsies from the antrum and the corpus.\n\nB) Gastric inflammation: Endoscopic diagnosis of gastric ulcer(s), erosions or gastritis and/or histologic findings of more than mild gastritis (endoscopic diagnosis of gastritis based on reddening and swelling of the mucosa was only accepted if confirmed histologically).\n\nC) Intestinal metaplasia: Histologic evidence of intestinal metaplasia, irrespective of other endoscopic and/or histologic findings (except gastric cancer).\n\nD) Gastric cancer: Endoscopic and histologic evidence of gastric cancer, no previous therapy.\n\nE) Miscellaneous: Other diseases of the stomach diagnosed endoscopically and/or by histology, or diseases of the esophagus or duodenum.\n\nAssays were performed by a laboratory technician blinded to patient clinical data, including diagnosis. Samples were analyzed, immediately following thawing, with CA72-4 ELISA assay (DRG instruments GmbH, Marburg, Germany), according to the manufacturer’s protocol, using 10 µl of sample. At least duplicate absorbance readings were obtained for each sample at 450nm wavelength using FL600 microplate reader with KC4™ data analysis software, (Bio-Tek® Instruments Inc., Winooski, VT, USA). Samples demonstrating high level of CA72-4 on the initial reading were assessed, in duplicates, up to 5 times on different days using a different assay plate and reagent set, depending on sample availability. Data collection, processing, and initial statistical analysis were performed on-site by a senior scientist, also blinded to patient clinical data. Data was then transferred to the IH site for detailed statistical analysis.\n\nStatistical analyses including ANOVA, Wilcoxon or Kruskal-Wallis test and univariate and multivariate linear regression analysis were performed using JMP software (version 6.0.3; SAS Institute Inc., Cary, NC, USA). Data are expressed as mean±SD or median with interquartile ranges depending on whether data were normally distributed or not. Multivariate linear regression analyses were used to investigate the influence of patient grouping and clinical parameters on CA72-4 concentrations in gastric juice. For the multivariate analyses, manual stepwise model building was performed and the following parameters were tested as predictors: age, gender, BMI, Helicobacter pylori (H. pylori) status (according to histology), smoking habits (never vs. active or ex-smoker), alcohol intake (never vs. current or ex-alcohol intake), PPI dose (in multiples of standard dose), endoscopic evidence of gastric bleeding and histologic diagnosis of gastric carcinoma, intestinal metaplasia, gastric inflammation and nonmalignant histologic alterations of the gastric mucosa.\n\n\nResults\n\nOverall 380 patients consented to participate in the study. Collection of gastric juice and clinical data was performed in 262 patients, in most of the others no juice samples could be obtained because the stomach contained insufficient amounts of fluid to be aspirated endoscopically or solids that could not be aspirated. In a minority of patients clinical data were insufficient for study purposes.\n\nCA72-4 concentrations were measured in 176 gastric juice samples, based on sample volume sufficiency, from subjects with normal stomach (N=28), gastric inflammation (N=58), intestinal metaplasia (N=26), gastric carcinoma and no previous therapy (N=8) and patients with miscellaneous diseases (N=56). 108 out of 176 patients were female. Mean age of the patients was 60.8±16.8 years, mean BMI was 25.1±4.9 kg/m2.\n\nCA72-4 concentrations in gastric juice ranged from 0.3 to 287 U/ml (Figure 1). Median CA72-4 concentration was 35.5 [19–68.5] U/ml. There was a marked and highly significant difference between the CA72-4 concentrations observed for the various diagnostic groups (p=0.0005). While CA72-4 concentrations in gastric juice were similar in patients with an endoscopically and histologically normal stomach, gastric inflammation, intestinal metaplasia of the gastric mucosa or miscellaneous diseases, patients with gastric cancer had markedly elevated CA72-4 levels (Figure 2). Compared with all other participants, median CA72-4 concentrations in gastric juice of cancer patients were increased about fourfold (144.5 [54.0–220.3] U/ml vs. 34.5 [18.1–64.0] U/ml, p=0.001) (Figure 2). Six out of eight patients with gastric cancer and 18 out of 168 patients with other diagnoses had CA72-4 concentrations above 100 U/ml. Thus, at this cut-off level elevated CA72-4 concentrations had 75% sensitivity and 89% specificity for detection of gastric cancer.\n\nIndividual values ranged from 0.3 to 278 U/ml. The box plot shows median CA72-4 concentration (35.5 U/ml) and interquartile ranges (19–68.5 U/ml).\n\nPatients with an endoscopically and histologically normal stomach, gastric inflammation, intestinal metaplasia of the gastric mucosa or miscellaneous diseases had similar CA72-4 concentrations. By contrast, in patients with gastric cancer CA72-4 concentrations were significantly elevated compared with all other groups (**p<0.001). The figure shows individual values and box-plot diagrams for the various groups. At a cut-off level of 100 U/ml (dotted line), elevated CA72-4 concentrations had 75% sensitivity and 89% specificity for detection of gastric cancer.\n\nIn the univariate linear regression analysis, the diagnosis of gastric cancer significantly predicted increased CA72-4 concentrations in gastric juice (p<0.0001). Age or smoking habits were predictors of borderline significance for increased CA72-4 levels in older subjects and smokers (p=0.051 and p=0.090, respectively).\n\nCA-72-4 levels were not significantly predicted by diagnosis of intestinal metaplasia (p=0.964), gastric inflammation (p=0.656), H. pylori status (p=0.874), gastric bleeding (p=0.491), sex (p=0.206), BMI (p=0.218), alcohol intake (p=0.857), PPI dose (p=0.252) or pH of the aspirate (p=0.426). If patients with gastric inflammation, intestinal metaplasia and miscellaneous diseases of the gastrointestinal tract were combined to one group of patients with pathologies other than gastric carcinoma, prevalence of this diagnosis did also not predict CA-72-4 concentrations in gastric fluid in the univariate analysis (p=0.226).\n\nMultivariate linear regression analysis confirmed that CA72-4 concentration in gastric juice was significantly predicted by diagnosis of gastric carcinoma adjusted for age, smoking habits, H. pylori status, PPI dose, and pH of the aspirate (R2=0.27, p<0.0001). In this model, diagnosis of gastric carcinoma had by far the greatest influence (p<0.0001). Age (p=0.033) and smoking status (p=0.002) were additional independent significant predictors of CA72-4 concentrations. The other parameters achieved only borderline significance (p=0.079 to p=0.138), but the overall significance of the model was reduced when these parameters were not taken into account.\n\nMultivariate linear regression analysis further revealed that CA72-4 concentrations were significantly predicted by pathologies other than gastric carcinoma adjusted for H. pylori and smoking status, age, PPI dose, and pH of the aspirate. However, gastrointestinal diseases other than gastric carcinoma had opposite (decreased levels in patients with such diseases) and much weaker effects (R2=0.07, p=0.028) on CA72-4 concentrations compared with diagnosis of gastric cancer. In this model, diagnosis of H. pylori and smoking status were independent significant predictors of CA72-4 concentrations (p=0.0384 and p=0.009, respectively), while the other parameters achieved borderline significance (p≥0.073).\n\n\nDiscussion\n\nThe combination of a relatively high incidence, especially in northeast Asia, and a frequently late stage diagnosis of gastric cancer, due to the patient- or physician-side misinterpretation of symptoms, has proven deadly over the years, making the disease the second most frequent cause of cancer death worldwide27,28. Generally, the cancer progression is aggressive upon late stage diagnosis with 5-year survival rates usually less than 30%. The existing means of screening are very accurate in providing a diagnosis, but face low patient-side compliance due to known discomfort associated with endoscopic procedures, thus imposing indirect constraints on the diagnostic value of fibreoptic endoscopy. The emerging capsule endoscopy, although holding a promise to circumvent the issue of compliance, still lacks diagnostic capabilities beyond visual identification of lesions. Thus, it will inevitably, require a reliable substitute for histological analysis, readily available through biopsy collection using a “conventional” endoscope, to outcompete the latter in screening efficiency, simplicity, and time-to-diagnosis. One such alternative is “lab-on-capsule” cancer marker-based molecular recognition of gastric malignancies. This option can be made possible through utilization of one or more marker(s) featuring high sensitivity and specificity in detection of gastric malignant tumors. In this work, we embarked upon identification of such a marker through assessment of the above mentioned parameters for a selected group of cancer markers, levels of which were measured in the gastric juice collected from patients with various disorders of the upper GIT, as well as in the gastric juice of patients in whom no disease was detected by either or both EGD and histopathology. During this study we have established several ELISA assays for markers previously implicated in relation with gastric cancer, such as: CEA7–9,23, pepsinogen II (PGII;29–33), regenerating islet-derived family, member 4 (RegIV;34,35), and cytokeratin 8 (CK8;36–38), and have also made use of commercially available assays for CA 19-97–9,23, Gastrin1730, and pepsinogen I (PGI;31–33). However, in our preliminary large-scale sampling analysis we failed to find any difference between the levels of these markers (including PGI/PGII ratio) in the gastric juice of cancer and non-cancer patients (data not shown). Quite the reverse, data generated in the course of this study points to a great diagnostic potential of CA72-4 direct measurements in gastric fluid. Notably in this regard, the CA72-4 concentrations in the gastric fluid of the majority of cancer patients were prominently elevated, compared to those measured in the gastric fluid obtained from patients with a completely normal stomach, patients with gastric inflammation, intestinal metaplasia or miscellaneous diseases of the upper gastrointestinal tract, including other malignant tumors.\n\nOne of the most obdurate hurdles to detection of cancer markers in gastric juice is the hostility of the gastric milieu imposed, among other factors, by high proteolytic activity and high acidity. Univariate linear regression analysis did not reveal a significant association between pH of gastric juice and CA72-4 concentrations. In multivariate linear regression analysis the “protective” effect of higher pH on CA72-4 concentrations was small, and failed to show statistical significance. In this regard, the apparent superiority of CA72-4 over other potential biomarkers in gastric juice may be speculated to stem from its relative stability in a wide range of pH, possibly due to the nature of the detected epitopes.\n\nAt a cut-off level of 100 U/ml CA72-4 had a sensitivity of 75% and a specificity of 89% for detection of gastric cancer. These data are also encouraging, although, with respect to the sensitivity parameter, the rather low number of cancer patients included in the study limits reliability of these findings and, generally, represents the most important drawback of our study. Gastric cancer was newly diagnosed in 8 out of 176 patients (5%), a proportion somewhat higher than that of cancer diagnoses expected in unselected patients undergoing EGD because of dyspeptic symptoms [1–2%;39). Notably, the IH is a tertiary referral center for patients with gastrointestinal diseases where gastric cancer patients are observed much more frequently. However, in most patients satisfactory endoscopic and histologic investigations have been performed prior to referral to IH. The experimental design did not allow performance of an EGD for reasons other than clinical, hence, certain patients could not be included in the study. Moreover, for final analysis of data only patients with newly diagnosed disease were selected in order to limit potential confounders. Interestingly, an ostensible decrease in CA72-4, measured in a single patient concomitantly with cancer detection (150 U/ml), was observed following chemotherapy (87 U/ml). Thus, in the future, effort should be invested in further exploration of this phenomenon and the possibility that CA72-4 may also be used to monitor treatment efficiency.\n\nContrary to sensitivity, the specificity data are based on analysis of a large number of gastric juice samples from patients with completely normal gastric findings or various gastric or other diseases, advocating for data reliability. We observed a trend towards lower intragastric CA72-4 concentrations in patients with pathologies other than gastric cancer and an accordingly high specificity of nearly 90% for CA72-4-based detection of gastric cancer at the cut-off level chosen. This is particularly encouraging for a putative screening marker, as low specificity would be associated with a large number of futile invasive diagnostic tests in patients that are, in fact, free from gastric cancer.\n\nIn conclusion, work will be needed to accurately establish the precise sensitivity and the cut-off level for cancer detection. However, more than a fourfold, and thus a highly biologically significant, increase in the mean value of CA72-4 in the cancer group patients in this study, compared to all other participants, along with the apparent high specificity of the marker-based cancer detection, underscore the validity of our findings, and suggest that CA72-4 may contribute to real-time detection of gastric cancer in the future.\n\n\nConsent\n\nStudy participants gave written informed consent before any study related procedures were performed.",
"appendix": "Author contributions\n\n\n\nJutta Keller – acquisition of data, study concept and design, analysis and interpretation of data, drafting of the manuscript, statistical analysis.\n\nElla Reiss-Sklan – acquisition of data, analysis and interpretation of data, statistical analysis.\n\nMiri Refael – acquisition of data.\n\nViola Andresen – statistical analysis.\n\nYael Herman-Levy – acquisition of data.\n\nIgor Ruvinsky – acquisition of data, study concept and design, analysis and interpretation of data, drafting of the manuscript, statistical analysis, study supervision.\n\nJutta Keller and Ella Sklan have contributed equally to this work.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported in part by CORDIS FP6 grant (#37362) for project LSHB-CT-2006-037362.\n\n\nReferences\n\nParkin DM, Bray F, Ferlay J, et al.: Global cancer statistics, 2002. CA Cancer J Clin. 2005; 55(2): 74–108. 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}
|
[
{
"id": "346",
"date": "26 Oct 2012",
"name": "Alex Boussioutas",
"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 have read this article with interest and I think it is a good paper, however I do have some reservations.The main one being there is no independent validation cohort and this is based on n=8 gastric cancers. I think if the authors tested their method on an independent cohort of samples (blinded) it would provide a very good validation.The other point is more pragmatic; do the authors still require an endoscopy to collect the gastric juice? If they will do this by less invasive means (e.g. nasogastric tube suction) they should evaluate the technique using that method. Otherwise if a patient is having an endoscopy you will be able to visualize a gastric cancer.",
"responses": [
{
"c_id": "70",
"date": "30 Oct 2012",
"name": "Igor Ruvinsky",
"role": "Author Response",
"response": "Dear Dr. Boussioutas,I absolutely agree with you in regard with the number of gastric cancer samples assessed in this study, and we expressed our reservations, accordingly, in the article.However, in our defense, I should stress the fact that this study was conducted as a part of a greater, FP6-sponsored, project (Nano based capsule-Endoscopy with molecular imaging and optical biopsy – NEMO) and, consequently, influenced by the time and budget constraints of the latter.Procedure-wise: one of the aims of the NEMO project was to to combine capsule endoscopy with nano-based molecular recognition of gastric malignancies (as well as with several other diagnostic tools and abilities), which required identification of high-value biomarker(s) alongside establishing a suitable detection platform. Ultimately, sampling and sample analysis would and, hopefully, will be performed on board of the capsule (a job for NEMO 2.0), with performance parameters of the detection platform attuned separately to each biomarker selected."
}
]
},
{
"id": "347",
"date": "26 Oct 2012",
"name": "Janusz Jankowski",
"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 essentially a good paper, however there are three areas where it needs to be improved.1. There needs to be a succinct but informative overview of the marker. In addition, could the authors present a Kaplan-Meier curve of the marker in pathological samples?2. The sensitivity and specificity ratios aren’t good enough. Despite the authors mentioning this in the discussion, both ratios ought to be well above 90% perhaps even 95% as both won’t be good enough for a screening test. The risk of missing a cancer is so huge otherwise.3. The authors need to follow the NIH sequence of validation of biomarkers and explain how they plan to proceed with further validation.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-33
|
https://f1000research.com/articles/1-34/v1
|
19 Oct 12
|
{
"type": "Case Report",
"title": "Curative treatment in a patient with gastric cancer stage IV: a case report",
"authors": [
"Andreas Diel",
"Ernst Rodermann",
"Hans-Friedrich Kienzle",
"Denis Meuthen",
"Ingo Meuthen",
"Andreas Diel",
"Ernst Rodermann",
"Hans-Friedrich Kienzle",
"Denis Meuthen"
],
"abstract": "A 39-year old patient with gastric adenocarcinoma stage IV failed to respond to preoperative chemotherapies containing 5-FU and cisplatin as well as 5-FU and irinotecan. After third-line chemotherapy with two cycles of docetaxel and cisplatin we confirmed a clinical partial response. A complete histologically confirmed remission was detected after complete resection of the tumor. Following two postoperative cycles of docetaxel and cisplatin, the tumor is still in complete remission after more than eight years.",
"keywords": [
"In November 2003",
"a 39 year old male patient was diagnosed with a locally advanced gastric adenocarcinoma. The diagnosis and tumor stage were confirmed by gastrocopy",
"endoscopic ultrasound (EUS = u-staging)",
"computer tomography (CT) scans of the thorax/abdomen and a bone scintigraphy. Gastroscopic biopsies revealed a Helicobacter pylori-negative gastric adenocarcinoma",
"diffuse type G3. According to the TNM/UICC staging system the carcinoma stage was cT4N3M0 or stage IV",
"respectively1. We found an invasion of the colon transversum and more than sixteen significantly enlarged lymph nodes (Figure 1a). We did not test the Her-2-neu status in 2003. Multdisciplinary treatment planning included preoperative chemotherapy followed by operation and postoperative chemotherapy in curative intention. The patient suffered from a severe hypertensive cardiomyopathy",
"thus he was not eligible for an anthracycline containing chemotherapy."
],
"content": "Case report\n\nIn November 2003, a 39 year old male patient was diagnosed with a locally advanced gastric adenocarcinoma. The diagnosis and tumor stage were confirmed by gastrocopy, endoscopic ultrasound (EUS = u-staging), computer tomography (CT) scans of the thorax/abdomen and a bone scintigraphy. Gastroscopic biopsies revealed a Helicobacter pylori-negative gastric adenocarcinoma, diffuse type G3. According to the TNM/UICC staging system the carcinoma stage was cT4N3M0 or stage IV, respectively1. We found an invasion of the colon transversum and more than sixteen significantly enlarged lymph nodes (Figure 1a). We did not test the Her-2-neu status in 2003. Multdisciplinary treatment planning included preoperative chemotherapy followed by operation and postoperative chemotherapy in curative intention. The patient suffered from a severe hypertensive cardiomyopathy, thus he was not eligible for an anthracycline containing chemotherapy.\n\na) before treatment December 2003 b) after partial remission May 2004 c) after complete remission November 2005.\n\nAfter six weeks on PLF (cisplatin 50 mg/m2 every two weeks, weekly folinic acid 500 mg/m2 and 5-FU 2000 mg/m2/24h), we found no response. We changed to the FLI-regiment (weekly folinic acid 500 mg/m2, 5-FU 2000 mg/m2/24h, irinotecan 80 mg/m2), there was no response after six weeks as well. After applying two cycles of DC (docetaxel 50 mg/m2 d 1 + 15, cisplatin 50 mg/m2 d 1 + 15, every four weeks), we detected a partial remission on endoscopy and abdominal CT (Figure 1b). In June 2004, a gastrectomy with a D2 lymph node dissection was performed resulting in a R0 status and a pathologically confirmed complete remission (CR). The patient was in good postoperative condition, he tolerated two further postoperative cycles of DC well without more than grade-2-toxicity. In September 2004, the patient underwent a laparotomy due to an acute bowel obstruction caused by adhesive bands after previous abdominal surgery. No carcinoma was detected macroscopically or histologically (Figure 1c). Up to this day, more than thirteen years after the diagnosis, the tumor is still in CR. The patient’s quality of life is not compromised in any way (ECOG performance status 0).\n\nUsing the TNM/UICC-classification of gastric cancer 2010, our patient would be classified as stage cT4bN3bM0, stage IIIc2.\n\n\nDiscussion\n\nThe 5-year median overall survival rate for stage III and IV patients receiving perioperative chemotherapy is 39.1% and 5.2% after surgical resection alone3–5. Since 2010, the standard of care in locally advanced gastric cancer (uT3-T4) is three praeoperative cycles of an anthracycin containing polychemotherapy like epirubicine, cisplatin and 5-FU (ECF) followed by three postoperative cycles. This kind of treatment improved 5-year survival from 23% with surgery alone to 36.3%6,7. There are no sufficient data from randomised phase III studies to strongly recommend perioperative chemotherapy for patients with uT2 carcinomas8.\n\nTwo preoperative cycles of PLF seem to be as effective as preoperative ECF9,10. In advanced gastric cancer, polychemotherapy offers a survival advantage over single-agent therapy. Comparing 5-FU/cisplatin-containing regiments with versus without anthracyclins and 5-FU/anthracyclin-containing combinations with or without cisplatin there is a significant survival benefit for 5-FU + anthracyclins + cisplatin. In this analysis, secondary resectability in locally advanced disease was not reported11.\n\nIn metastatic gastric adenocarcimomas and adenocarcinomas of the gastrooesophaeal junction 5-FU can be substituted by capecitabine without compromising the results, so capecitabine may be used instead of 5-FU in the perioperative setting in combination with cisplatin (XP) or with epirubicine and cisplatin (ECX)12–14. In metastatic disease oxaliplatin has been tested as a substitute for cisplatin in combination chemotherapy with similar results13,15. The use of oxaliplatin can be recommended for patients suffering from renal insufficiency or patients with severe adverse events after cisplatin treatment8. Irinotecan or Docetaxel combinations have not been established for the perioperative treatment of gastric cancer in 2004. Both drugs were used in combinations for palliative therapy only16,17.\n\nAt the ASCO meeting in 2011 the FLOT3 trial15 has been presented:\n\nPatients with untreated operable gastric adenocarcinoma received four preoperative cycles of FLOT (5-FU, leucovorin, oxaliplatin, docetaxel) and underwent surgical resection (D2 resection) followed by four postoperative cycles. Patients with limited metastatic disease (distant intra-abdominal lymph nodes and/or a maximum of one organ involved in metastatic disease, ECOG-PS < 1) received eight cycles and underwent surgical resection when a complete macroscopic resection seemed possible. Surgical treatment was conducted in 95.7% and 42.64% of the patients, respectively. For operable patients the median overall survival was not reached, for patients with limited disease the median overall survival was 18.6 months; there were also significant differences in progression-free survival (p < 0.001). Grade 3–4 toxicities were similar among the groups and occurred in 70.6% vs. 72.1% of the patients, respectively15.\n\nIn gastric adenocarcinoma or adenocarcinoma of the gastrooesophageal junction stage uT2 a perioperative chemotherapy can be considered (Level of evidence 1b), in stage uT3 and in resectable uT4a adenocarcinomas patients should receive a pre- and postoperative polychemotherapy (Level of evidence 1b)8.\n\nOur patient may be one of the rare cases with advanced gastric cancer cured by perioperative third line chemotherapy and surgery.\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\nIM found the case in his clinic. AD, ER, IM and HFK collected data. IM drafted the manuscript. DM rewiewed the literature and revised the manuscript critically. 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\nSobin LH, Wittekind C: TNM-classification of malignant tumors. 6th ed. New York: Springer2002. Reference Source\n\nSobin LH, Gospodarowicz MK, Wittekind C, et al.: TNM-classification of malignant tumors. 7th ed. Oxford: Wiley-Blackwell2010. Reference Source\n\nWang W, Li YF, Sun XW, et al.: Prognosis of 980 patients with gastric cancer after surgical resection. Chin J Cancer. 2010; 29(11): 923–930. PubMed Abstract | Publisher Full Text\n\nBoige V, Pignon JP, Saint-Aubert JB, et al.: Final results of a randomized trial comparing preoperative 5-fluorouracil (F)/cisplatin (P) to surgery alone in adenocarcinoma of stomach and lower esophagus. J Clin Oncol. 2007; 25(Suppl 18S): abstr 4510. Reference Source\n\nKelsen DP, Ginsberg R, Pajak TF, et al.: Chemotherapy followed by surgery compared with surgery alone for localized esophaegal cancer. N Engl J Med. 1998; 339(27): 1979–1984. PubMed Abstract | Publisher Full Text\n\nOkines A, Verheij M, Allum W, et al.: Gastric cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow up. Ann Oncol. 2010; 21(Suppl 5): v50–54. PubMed Abstract | Publisher Full Text\n\nCunningham D, Allum WH, Stenning SP, et al.: Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer. N Engl J Med. 2006; 355(1): 11–20. PubMed Abstract | Publisher Full Text\n\nMoehler M, Al-Batran SE, Andus T, et al.: German S3-guideline \"Diagnosis and treatment of esophagogastric cancer\". Z Gastroenterol. 2011; 49(4): 461–531. PubMed Abstract | Publisher Full Text\n\nSchuhmacher C, Schlag P, Lordick F, et al.: Neoadjuvant chemotherapy versus surgery alone for locally advanced adenocarcinoma of the stomach and cardia: Randomized EORTC phase III trial 40954. J Clin Oncol. 2009; 27(Suppl 15s): abstr 4510. Reference Source\n\nSchuhmacher C, Gretschel S, Lordick F, et al.: Neoadjuvant chemotheraphy compared with surgery alone for locally advanced cancer of the stomach and cardia: European organisation for research and treatment of cancer randomized trial 40954. J Clin Oncol. 2010; 28(35): 5210–5218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWagner AD, Unverzagt S, Grothe W, et al.: Chemotherapy for advanced gastric cancer. Cochrane Database Syst Rev. 2010; (3): CD004064. PubMed Abstract | Publisher Full Text\n\nKang YK, Kang WK, Shin DB, et al.: Capecitabine/cisplatin versus 5-fluorouracil/cisplatin as first-line therapy in patients with advanced gastric cancer: a randomised phase III noninferiority trial. Ann Oncol. 2009; 20(4): 666–673. PubMed Abstract | Publisher Full Text\n\nCunningham D, Starling N, Rao S, et al.: Capecitabine and oxaliplatin for advanced esophagogastric cancer. N Engl J Med. 2008; 358(1): 36–46. PubMed Abstract | Publisher Full Text\n\nOkines AF, Norman AR, McCloud P, et al.: Meta-analysis of the REAL-2 and ML17032 trials: evaluating capecitabine-based combination chemotherapy and infused 5-fluorouracil-based combination chemotherapy for the treatment of advanced oesophago-gastric cancer. Ann Oncol. 2009; 20(9): 1529–1534. PubMed Abstract | Publisher Full Text\n\nAl-Batran SE, Hartmann JT, Probst S, et al.: Phase III trial in metastatic gastroesophageal adenocarcinoma with fluorouracil, leucovorin plus either oxaliplatin or cisplatin: a study of the Arbeitsgemeinschaft Internistische Onkologie. J Clin Oncol. 2008; 26(9): 1435–1442. PubMed Abstract | Publisher Full Text\n\nPozzo C, Bugat R, Peschel C, et al.: Irinotecan in combination with CDDP or 5-FU and folinic acid is active in patients with advanced gastric or gastro-oesophageal junction adenocarcinoma: Final results of a randomised phase II study. Proc Am Soc Clin Oncol. 2001; 20: abstr 531.\n\nRoth AD, Maibach R, Falk S, et al.: Docetaxel-cisplatin-5FU (TCF) versus docetaxel-cisplatin (TC) versus epirubicin-cisplatin-5FU (ECF) as systemic treatment for advanced gastric carcinoma (AGC): A randomized phase II trial of the Swiss Group for Clinical Cancer Research (SAKK). J Clin Oncol. 2004; 22(Suppl (14s) (July 15 Supplement)): abstr 4020. Reference Source"
}
|
[
{
"id": "326",
"date": "08 Nov 2012",
"name": "Maria Alsina Maqueda",
"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 case report is interesting and worthy of publication. There are however a few revisions that should be made:1. In the Introduction section (2nd paragraph, 1st sentence), the chemotherapy scheme used in the patient is not the same as the one used by the study referenced; the scheme should be justified.2. In the Introduction section (3rd paragraph, last sentence) there are more recent references to justify the classification statement (Thuss-Patiente Eur J Cancer 2011, Kang Clin Oncol 2012 and Van Cutsem ASCO GI 2012).3. In the Discussion section (4th & 5th paragraphs) the authors refer to a study published in ASCO/JCO 2008, not 2011 which they have stated.4.In the Discussion section (5th paragraph) the authors describe a study extensively with compared with the rest of the article, maybe authors should justify why (the use of docetaxel?).5. The authors should discuss more extensively the use of the three lines with different efficacies in the neoadjuvant setting, and they should also address the issue of long gastric patient survivors.6. In the Reference section, references 9 and 10 references refer to the same study; it is the newest one should appear, and the older one can be removed.",
"responses": []
},
{
"id": "327",
"date": "09 Nov 2012",
"name": "Ian Beales",
"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 do discuss the FLOT3 trial at disproportionate detail compared to the rest of the discussion and probably a bit too excessively in response to the case reported. Whilst these data are interesting, it would be helpful to place these in a bit more context in relation to the case reported.There are some typographical and grammatical errors in the Discussion section (pre-operative and insufficient data in the first paragraph).The discussion about irinotecan and docetaxel combinations in the Discussion section: the authors could clarify whether they are primarily discussing the situation of current knowledge or as it stood in 2004. It would be most appropriate to discuss the current state of this combination in the article.",
"responses": []
},
{
"id": "328",
"date": "14 Nov 2012",
"name": "S Yousuf Zafar",
"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": "331",
"date": "19 Nov 2012",
"name": "Peter M Schlag",
"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 is a single case report without any new information and is without any scientific background.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-34
|
https://f1000research.com/articles/1-32/v1
|
19 Oct 12
|
{
"type": "Case Report",
"title": "Ankylosis of the hips and knees due to sickle cell disease",
"authors": [
"Saad Saleh Abdullah Al Elayan",
"Abdullah Al Hamdan",
"Abdullah Al Hamdan"
],
"abstract": "This is a case report of a 29-year-old Saudi male with sickle cell disease (SCD) with severe stiffness of his joints, mainly both knees and hips, secondary to complications of SCD. He was severely crippled: unable to sit, stand or walk, and was bedridden for 8 years when he was presented to us. Radiographs showed fusion of both knees and hips. There was no evidence of active osteomyelitis by Gallium scan. The patient’s hemoglobin S decreased to levels below 30% by exchange transfusion. Bilateral total hip replacement, as well as unilateral total knee replacement, was carried out to improve his level of function. There is only one reported case of such severe and multiple joint complications in a single patient suffering from SCD.The increased life expectancy that medical advances have offered to the sickle-cell patients has led to the appearance of sickle-cell-related complications, which were previously only seen rarely. These complications were successfully managed and the patient was able to move and transfer using a wheel chair.",
"keywords": [
"Sickle cell disease (SCD) is an autosomal recessive genetic disorder characterized primarily by chronic anemia and periodic episodes of pain",
"affecting millions throughout the world1. SCD patients are at increased risk of bony complications of the disease",
"such as osteomyelitis",
"osteonecrosis",
"osteopenia and ankylosis2. SCD is an important public health concern in the Kingdom of Saudi Arabia (KSA)",
"particularly in the Eastern and South West provinces3. Painful crises and avascular necrosis of the femoral head are common complications observed in these regions3. This case presentation is that of a patient with severe bony complications resulting from his SCD. Total bilateral hip and unilateral knee arthroplasty were performed to correct hip and knee ankylosis secondary to SCD. To our knowledge",
"total bilateral hip and unilateral knee arthroplasty has not been described previously in the literature except in one case of an African male with a similar presentation4."
],
"content": "Introduction\n\nSickle cell disease (SCD) is an autosomal recessive genetic disorder characterized primarily by chronic anemia and periodic episodes of pain, affecting millions throughout the world1. SCD patients are at increased risk of bony complications of the disease, such as osteomyelitis, osteonecrosis, osteopenia and ankylosis2. SCD is an important public health concern in the Kingdom of Saudi Arabia (KSA), particularly in the Eastern and South West provinces3. Painful crises and avascular necrosis of the femoral head are common complications observed in these regions3. This case presentation is that of a patient with severe bony complications resulting from his SCD. Total bilateral hip and unilateral knee arthroplasty were performed to correct hip and knee ankylosis secondary to SCD. To our knowledge, total bilateral hip and unilateral knee arthroplasty has not been described previously in the literature except in one case of an African male with a similar presentation4.\n\n\nCase report\n\nA 29-year-old Saudi male with SCD, was referred from the general surgery service to improve his poor physical condition. He was severely crippled and bed bound for 8 years with severe bilateral knee and hip dysfunction secondary to complications of SCD. Past history included an admission to hospital 8 years before for fever, swelling and severe pain of the right knee. Needle aspiration, as well as irrigation and debridement, was performed and the patient was diagnosed with septic arthritis. Since then he had developed progressive joint stiffness involving the dorsal and lumbar spines as well as the lower extremity.\n\nOn physical examination his knee range of motion (ROM) bilaterally was almost nonexistent with the knees held in full extension. His hips ROM was also severely limited, with no ROM. Loss of hip flexion was noted to result in the most severe functional loss with the hips fixed in full extension, adduction of 15° and 50° of external rotation. He required a 2-person assist as well as a walker to weight bear however he was unable to mobilize.\n\nSerial radiological studies were done. Radiological investigations demonstrated severe avascular necrosis and ankylosis of the hip (Figure 1) and severe erosion of the articular surfaces of the knees as well as ankylosis (Figure 2). Computed tomography (CT) scan of the hips and knees showed similar findings as that of the X-rays. Shoulders and spine X-ray were done for further assessment. Gallium bone scan demonstrated no evidence of active osteomyelitis. Our patient had a proper preoperative evaluation and blood transfusion to prevent adverse outcomes and sickle cell complications postoperatively5. We took him to surgery for bilateral cementless total hip replacement (THR) in one session.\n\nThe patient was operated on with an aim to provide movement at the hips and knees and to recover his ability to sit, stand, transfer, balance and improves personal hygiene. Bilateral THR was done successfully (Figure 3). The procedures were performed utilizing the Harding lateral approach. Particular attention was paid to padding bony prominences and skin care in view of his poor skin condition. Intraoperatively, the bone was noted to be very fragile. The patient was kept in the intensive care unit for two days post operatively for observation and pain control. Post bilateral THR, the patient had marked improvement of hip flexion bilaterally permitting easy functional sitting. Post surgery physiotherapy resulted in great improvement in his general physical activity.\n\nLeft total knee replacement (TKR) was performed one month after the THR (Figure 4). Intraoperatively, the bone quality was noted to be severely deficient. The quadriceps muscle was adherent to the femur and atrophied; therefore, V-Y quadricepsplasty was done. Soft tissue was mobilized carefully around the joint prior to component insertion. Intraoperatively, knee flexion approached 50°. Post-left-TKR rehabilitation was severely restricted secondary to poor bone quality. This prevented adequate ROM exercises and function, and ROM gains at the knee were minimal. Poor bone quality also limited the ability for the patient to progress to functional lower extremity weight-bearing activities. Postoperatively, rehabilitation, patient education, transfer training and functional rehabilitation were carried out. This allowed the patient to transfer safely and independently from bed to wheel chair without much pain, and improved the quality of life by changing the patient’s functional ability and allowing the freedom to mobilize independently with a wheelchair.\n\n\nDiscussion\n\nUnlike normal red blood cells (RBC), sickled cells, due to abnormal morphology, are unable to negotiate small blood vessels resulting in arterial occlusion and subsequent ischemia. This process can ultimately damage tissues and vital organs. The prevalence of avascular necrosis (AVN) in sickle-cell-disease patients is increasing, especially with the increased life expectancy in these patients. It is believed that the AVN results from repeated episodes of localized areas of epiphyseal, metaphyseal and diaphyseal bone marrow infarctions, resulting in ingrowths of new bone as well as diffuse sclerosis resulting from vascular occlusion by sludging of sickle cells in the sinusoids. In general, the bilateral simultaneous THR has demonstrated better functional outcome than the staged procedure in patients with the same condition, with no significant increase in the rate of dislocation or thromboembolic events6. A similar case was published in 2000: a Congolese sickle cell patient was referred to Belgium for management of severe stiffness of all his major joints. That patient was managed by bilateral staged girdle stone procedure initially, with a subsequent surgical site infection of the left hip. The infection was managed with appropriate antibiotics and debridement. Once the infection was treated, the patient had a staged bilateral THR procedure. The patient regained his ability to walk with crutches after the surgeries4. Another case was reported in 2008: a 25-year-old female with severe ankylosis of her hips and knees secondary to rheumatoid arthritis, and her function was severely limited. She underwent staged bilateral THR, followed by a staged bilateral TKR, the functional outcome was excellent in that case as well7.\n\nThe increased life expectancy that medical advances have offered to the sickle-cell patients has led to the appearance of sickle-cell-related complications, which were previously only seen rarely. Orthopedic surgeons should be aware of the optimal management options as well as possible operative and postoperative complications of patients with this disease.\n\n\nConsent\n\nWritten consent was obtained from the patient and the next of kin for the publication of the clinical details and clinical images related to the case report.",
"appendix": "Author contributions\n\n\n\nBoth the authors have contributed equally for the study, the corresponding author wrote 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 are thankful to the orthopedic team at the Prince Sultan Medical Military City, and especially thank Dr. Abdularahman Al Asmari and Dr. Mohammed Arshaduddin for their help.\n\n\nReferences\n\nModell B, Darlison M: Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ. 2008; 86(6): 480–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHammer M, Geuer KA, Aksoy S, et al.: Perioperative care for patients with sickle cell who are undergoing total hip replacement as treatment for osteonecrosis. Orthop Nurs. 2003; 22(6): 384–97. PubMed Abstract\n\nSadat-Ali M, Geeranvar SS, As-Suhaimi S, et al.: Orthopaedic complications in sickle cell disease. A comparative study from two regions in Saudi Arabia. Int Orthop. 1992; 16(3): 307–10. PubMed Abstract\n\nDumarey N, Martin P, Jayankura M, et al.: A ‘made in one piece’ skeleton in a 22-years-old man suffering from sickle cell anaemia. Clin Rheumatol. 2000; 19(4): 287–90. PubMed Abstract | Publisher Full Text\n\nFirth PG, Head CA: Sickle cell disease and anesthesia. Anesthesiology. 2004; 101(3): 766–85. PubMed Abstract\n\nTsiridis E, Pavlou G, Charity J, et al.: The safety and efficacy of bilateral simultaneous total hip replacement. An analysis of 2063 cases. J Bone Joint Surg Br. 2008; 90(8): 1005–12. PubMed Abstract | Publisher Full Text\n\nKarva AR, Board TN, Porter ML, et al.: Conversion of bilateral hip and knee ankylosis to total joint replacements. J Bone Joint Surg Br. 2008; 90(5): 668–73. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "320",
"date": "25 Oct 2012",
"name": "Maria Luisa Brandi",
"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 case report represents some very nice work.",
"responses": []
},
{
"id": "322",
"date": "29 Nov 2012",
"name": "Adlette Inati",
"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\nAvascular bone necrosis is a debilitating complication of sickle cell disease (SCD). Due to the prolonged life expectancy of SCD patients, the incidence of this complication is surging as it could be a manifestation of the disease chronicity.In this case report, The authors present a severe case of ankylosis of hips and knees secondary to SCD. Indeed, the severity of this presentation is unusual and may be partially due to lack of prevention and optimal early management. Although the clinical management of the patient was not discussed, it would be interesting to know if this patient was on hydroxyurea or on antithrombotic agents and if he was enrolled in a comprehensive SCD care facility. Moreover, since the patient has been bedridden for eight years, it is possible that thrombophilic factors may have further exacerbated the condition and its progression to multiple joints.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-32
|
https://f1000research.com/articles/1-31/v1
|
18 Oct 12
|
{
"type": "Case Report",
"title": "Systemic lupus erythematosus and Hodgkin disease",
"authors": [
"Besma Ben Dhaou",
"Fatma Boussema",
"Zohra Aydi",
"Lilia Baili",
"Lilia Rokbani",
"Fatma Boussema",
"Zohra Aydi",
"Lilia Baili",
"Lilia Rokbani"
],
"abstract": "We report on the rare association of Hodgkin’s disease with systemic lupus erythematosus. Four years after the diagnosis of systemic lupus erythematosus, the patient developed cervical mass and weight loss. Histological and subsequent clonality studies confirmed classical Hodgkin’s lymphoma. The awareness of the association of Hodgkin’s disease with systemic lupus erythematosus and its modes of presentation will help in the early diagnosis and management of such patients.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: approved 1",
"approved with reservations 1]”."
],
"content": "Introduction\n\nSystemic lupus erythematosus (SLE) is associated with lymphoproliferative diseases such as Hodgkin’s lymphoma (HL)1. Since there is considerable overlap between the features of SLE and HL there can be a great difficulty in diagnosing HL in the presence of SLE1.\n\n\nCase report\n\nA 35-year-old woman was followed from 2002 for SLE with neuropsychiatric, renal and hematologic involvements. She was treated with only glucocorticoids with favourable outcomes. In April 2006, when she was under steroid treatment of 10 mg/day, she was admitted for cervical mass and weight loss. Physical examination showed a left indolent, fixed and elastic cervical adenopathy. The biological assessment was normal. Computerized tomography of the chest and abdomen showed a left basicervical mass expanded to the anterior and superior mediastinum. Magnetic resonance imaging was suggestive of a thymoma (Figure 1, Figure 2). The cervicotomy showed a supraclavicular mass. Histological and subsequent clonality studies confirmed classical Hodgkin’s lymphoma (HL) of the nodular sclerosing type. Viral serology (for Epstein-Barr, herpes simplex, and herpes zoster) was negative. The diagnosis of Hodgkin’s disease stage Ia was retained and the patient was transferred to hematological department where she was treated by chemotherapy (adriamycin, bleomycin, vinblastine and dacarbazine) with favourable outcomes. She is being currently followed in our department and is in remission of her lupus and Hodgkin's disease.\n\n\nDiscussion\n\nThe relative risk of hematologic malignancy is estimated to be 60% higher in patients with SLE than in the general population, the reason being unknown1. Of all hematologic cases reported in patients with SLE, the most common is non-Hodgkin’s lymphoma followed by Hodgkin’s disease, leukemia, and then multiple myeloma1.\n\nThe initial presenting features of SLE and Hodgkin’s disease are similar, with fever, weight loss, and peripheral lymphadenopathy seen in most cases1. Our patient presented with weight loss and cervical adenopathy.\n\nPersistent large lymph nodes not responding to conventional therapy in SLE should be biopsied for alternative diagnosis (i.e., lymphoma)1.\n\nSeveral conditions and links have been identified that could potentially predispose patients with SLE to cancer (Table 1)2,3.\n\n* Growth and hormonal factors may play a role in autoimmunity as well as in malignancy3.\n\nA side-effect of immunosuppression is a possibility, as is intercurrent viral infection due to, for example, Epstein-Barr, herpes simplex, herpes zoster and polyoma viruses, which are potentially oncogenic. In our case, viral serology was negative.\n\nPatients who have had a renal transplant are known to have an increased risk of cancer4. They are, however, treated with much higher doses of immunosuppressive agents than patients with lupus. In the studies of Petterson et al.5, Abu-Shakra6 and Sultan et al.7, the use of cytotoxic agents was not related to the occurrence of malignancy. Our patient was treated only with corticosteroids. It may be that the disease itself confers an increased risk. Patients with SLE have defects in both their cellular and humoral immune systems.\n\nThe mechanisms of hematologic malignancies8 are thought to be related to the following:\n\nFailure or dysregulation of apoptosis as a result of mutated genes in SLE (Fas ligand).\n\nAccumulation of and mutations in B and T lymphocytes in the lymph nodes.\n\nT-cell immunodeficiency, allowing Epstein-Barr virus (EBV)-infected B-cell proliferation.\n\nExposure to immunosuppressive medications (possible increased risk of EBV infection in patients with SLE)9.\n\nLarge multicenter studies are required to adequately address the risk of developing malignancies in large cohorts of patients with SLE and to address issues such as associated risk factors and additional confounding factors, such as deprivation and exposure to therapy. The chance of detection of malignancy may vary due to factors such as access to health services, which vary widely and are not uniformly available, and may therefore underestimate the risk of malignancy.\n\n\nConclusion\n\nSLE has been associated with increased frequency of neoplasia, lymphoma, leukaemia and epithelial tumours. Hodgkin’s disease has been occasionally associated with SLE in adults. An awareness of the association of Hodgkin’s disease with SLE and the modes of presentation will help in the early diagnosis and clinical management of such patients.",
"appendix": "Author contributions\n\n\n\nAll authors participated in the completion of this work. They participated in the implementation of the clinical case and the proceeding discussion.\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\nBhalla R, Ajmani HS, Kim WW, et al.: Systemic lupus erythematosus and hodgkin’s lymphoma. J Rheumatol. 1993; 20(8): 1316–1320. PubMed Abstract\n\nBernatsky S, Clarke A, Ramsey-Goldman R: Malignancy and systemic lupus erythematosus. Curr Rheumatol Rep. 2002; 4(4): 351–358. PubMed Abstract | Publisher Full Text\n\nSliesoraitis S, Khan R, Rothman J: Methotrexate-induced Hodgkin disease in a patient with systemic lupus erythematosus. J Am Osteopath Assoc. 2009; 109(6): 325–328. PubMed Abstract\n\nLondon NJ, Farmery SM, Will EJ, et al.: Risk of neoplasia in renal transplant patients. Lancet. 1995; 346(8972): 403–6. PubMed Abstract\n\nPetterson T, Pukkala I, Teppo L, et al.: Increased risk of cancer in patients with systemic lupus erythematosus. Ann Rheum Dis. 1992; 51(4): 437–439. PubMed Abstract | Publisher Full Text\n\nAbu-Shakra M, Gladman DD, Urowitz MB, et al.: Malignancy in systemic lupus erythemmatosus. Arthritis Rheum. 1996; 39(6): 1050–4. PubMed Abstract | Publisher Full Text\n\nSultan SM, Ioannou Y, Isenberg DA: Is there an association of malignancy with systemic lupus erythematosus? An analysis of 276 patients under long-term review. Rheumatology (Oxford). 2000; 39(10): 1147–1152. PubMed Abstract | Publisher Full Text\n\nXu Y, Wiernik PH: Systemic lupus erythematosus and B-cell hematologic neoplasm. Lupus. 2001; 10(12): 841–850. PubMed Abstract | Publisher Full Text\n\nBernatsky S, Ramsey-Goldman R, Clarke A: Exploring the links between systemic lupus erythematosus and cancer. Rheum Dis Clin North Am. 2005; 31(2): 387–402 viii-ix. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "341",
"date": "30 Oct 2012",
"name": "Eoin McKinney",
"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": "342",
"date": "12 Nov 2012",
"name": "Frederic Geissmann",
"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 described a case of association of Hodgkin’s disease with systemic lupus erythematosus. This clinical report is interesting, if not novel, and reminds the clinician that persistent lymphadenopathy in SLE patients, can be due in some case to a curable malignancy.In my opinion, the report is too preliminary to allow the reader to conclude independently on the validity of the author’s findings. A revised version of this report should include the following points;1. To support their diagnosis and their conclusion, the authors should describe and document the clonality studies performed.2. A selection of histological/immunohistochemical stainings on which the diagnosis was based would also be useful.3. One proposed mechanism for the association of of Hodgkin’s disease with systemic lupus erythematosus is immunosupression due to treatment of SLE. The authors mentioned and discussed that viral serology for Epstein-Barr virus was negative, but did they investigate whether the EBV genone could be detected in pathological samples or the blood of the patients?4. Table 1 should be referenced and completed, so that reader can appreciate a) whether the ‘Potential links between systemic lupus erythematosus and malignancy’ rely on documented evidence, b) the level of that evidence, and c) the nature of the malignancies associated with SLE (i.e. Hodgkin’s disease only, or other cancers).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-31
|
https://f1000research.com/articles/1-30/v1
|
16 Oct 12
|
{
"type": "Commentary",
"title": "Common ground for biodiversity and ecosystem services: the “partial protection” challenge",
"authors": [
"Daniel P Faith"
],
"abstract": "New global initiatives require clarity about similarities and differences between biodiversity and ecosystem services. One argument is that ecosystem services capture utilitarian values, while biodiversity captures intrinsic values. However, the concept of biodiversity equally emerges from anthropogenic use values. Measures of biodiversity indicate broad option values, and so provide different information about future uses and benefits. Such differences nevertheless can be the basis for “common ground” for biodiversity and ecosystem services. Systematic conservation planning and related frameworks acknowledge such differences through effective trade-offs and synergies among different values of society. The early work on regional biodiversity trade-offs includes a little-explored aspect that could enhance this common ground. Regional planning here takes into account the “partial protection” of biodiversity provided by some land uses. Common-ground will be promoted by better integrating the ecosystem services and biodiversity conservation offered by ecosystems at the “natural end of the spectrum” with the partial protection and other benefits/services provided by more intensively-transformed places.",
"keywords": [
"\"Biodiversity\" and \"ecosystem services\" increasingly travel together as companion terms. Examples include the new \"Intergovernmental science-policy platform on biodiversity and ecosystem services\"",
"(IPBES)",
"the new Strategic Plan of the Convention on Biological Diversity (CBD)",
"and the emerging Global Biodiversity Observation Network (GEO BON). These new initiatives require clarity about the similarities and differences between biodiversity and ecosystem services. Some distinctions naturally emerge from our basic definitions – \"biodiversity\" refers to living variation",
"and \"ecosystem services\" refers to benefits to humans from natural ecosystems. However",
"biodiversity also has traditional links to benefits/values",
"and here comparisons with ecosystem services continue to raise important issues."
],
"content": "Introduction\n\n\"Biodiversity\" and \"ecosystem services\" increasingly travel together as companion terms. Examples include the new \"Intergovernmental science-policy platform on biodiversity and ecosystem services\", (IPBES), the new Strategic Plan of the Convention on Biological Diversity (CBD), and the emerging Global Biodiversity Observation Network (GEO BON). These new initiatives require clarity about the similarities and differences between biodiversity and ecosystem services. Some distinctions naturally emerge from our basic definitions – \"biodiversity\" refers to living variation, and \"ecosystem services\" refers to benefits to humans from natural ecosystems. However, biodiversity also has traditional links to benefits/values, and here comparisons with ecosystem services continue to raise important issues.\n\nBiodiversity sometimes is characterised as all about intrinsic, non-anthropogenic values, with ecosystem services then providing the links to human well-being. For example, Haines-Young and Potschin1 argue: \"Biodiversity has intrinsic value and should be conserved in its own right. However, the utilitarian arguments which can be made around the concept of ecosystem services and human well-being are likely to become an increasingly central focus of future debates about the need to preserve ‘natural capital’\". Similarly, Hardy2 argues: \"The idea of ecosystem services allows for acknowledging more than the \"intrinsic\" value of biodiversity by expanding the breadth of the conservation argument to include the \"utilitarian\" values of nature\". Thus, an argument is that only through ecosystem services do we move beyond biodiversity’s intrinsic values to also consider utilitarian values.\n\n\nCommon ground\n\nA recent statement by Reyers et al.3 that \"the concept of biodiversity emerges from an intrinsic context\" echoes earlier studies, including the previous assertion by Reyers and colleagues4 that \"biodiversity and ecosystem services are associated with different values (intrinsic vs. utilitarian)\" (see also5). However, Reyers et al.3 do suggest \"common ground\" based on biodiversity’s additional links to ethical, spiritual, and religious values. They argue that, because these are ecosystem services, conservation of ecosystems services sometimes captures biodiversity and its values (see also6,7).\n\nIn a response to Reyers et al., Faith8 points out that the concept of biodiversity equally emerges from anthropogenic use values, citing the early calls for conservation of diversity to ensure benefits \"for present and future use\"9, and the early references10 to \"option values\" (the value of biodiversity in providing uses, often unanticipated, for future generations; see also11,12). Thus, in contrast to recent perspectives, there is no requirement to add-in ecosystem services considerations in order to build a case for biodiversity conservation based on human-use values.\n\nReyers et al.13 agree that the concept of biodiversity emerges from anthropogenic values. However, they object to Faith’s observation8 that biodiversity and ecosystem services \"may differ in how well they capture current and future uses\". Reyers et al. correctly argue that ecosystem services include future uses. However, Faith argues that option values of biodiversity are broad in reflecting unknown benefits, including those from unknown elements or services14. In contrast, ecosystem services typically focus on option values related to possible future use of known services (e.g. future timber from a forest area). For example, DIVERSITAS links option value to the \"availability of a particular service for use in the future\". Broader option values are measured by estimating biodiversity (for discussion see14,15). Thus, biodiversity by its nature arguably contributes something additional, something different, concerning potential future uses.\n\nReyers et al.'s13 conclusion that \"some scientists focus on differences while others focus on similarity and common ground\" therefore is a concern. It implies that proposing differences is counter-productive to finding \"common ground\". However, I think any truly useful \"common ground\" for biodiversity and ecosystem services will build on differences. This is apparent in decision-support frameworks related to systematic conservation planning16 and \"regional sustainability analysis\"17 that seek trade-offs and synergies among the different values associated with biodiversity, ecosystem services, and other needs of society. Part of that common ground framework is now well-established. Measures of regional biodiversity are used to identify places with high versus low biodiversity marginal gains (\"complementarity\" values16 which vary depending on other allocations in the region). For a given locality, high complementarity, combined with high co-benefits (or \"negative costs\"18,19) and low opportunity costs of conservation, implies priority for conservation over alternative land uses having higher costs and smaller co-benefits (for related work, see20–27 and Insights from an Australian planning framework for biodiversity and ecosystem services.\n\n\nPartial protection\n\nThe early foundations of that regional biodiversity-plus-costs framework17,28–30 include some little-explored aspects that could enhance the common ground of biodiversity and ecosystem services: here, planning includes land/water uses offering ecosystem services or other benefits, combined with only \"partial protection\" of biodiversity (implying some lower complementarity value)17. Early examples17,19,28 illustrate cases where a partial protection option is allocated, and other cases where the non-conservation land use in a given place is preferred over the partial protection option because it maximises regional net benefits (see Partial degrees of protection and regional sustainability analysis).\n\nThe Millennium Ecosystem Assessment31 (MA) highlights this approach in the context of biodiversity policy options:\n\n\"...an integrated biodiversity trade-offs framework (Faith et al. 2001a, 2001b)32,33 suggests how such partial protection (for example, from private land) can contribute to the region’s trade-offs and net benefits\". However, the MA also observes that \"The great uncertainty is about what components of biodiversity persist under different management regimes, limiting the current effectiveness of this approach\"31.\n\nAs more information of this kind becomes available, case studies should explore applications, and evaluate interesting variants of the partial protection framework. Variants now include extensions to the original DIVERSITY-ED17,28–30, and TARGET (e.g.,19) partial protection approaches, to better accommodate multiple options for areas, and the related \"partial protection\" method in Marxan34.\n\nBecause partial protection accommodates otherwise-competing values, it helps establish an inclusive, \"common ground\", framework that acknowledges differences. Biodiversity measures can complement ecosystem services in indicating broader option (and other) values. Further, \"ecosystem services\", which conventionally refer to ecosystems at the \"natural end of the spectrum\"35, are complemented by more intensively-transformed places which sometimes provide partial protection along with other benefits/services.\n\nOf course, one could define \"ecosystem services\" to capture all these aspects, but making clear distinctions helps to avoid possible conceptual confusions arising when everything is forced under the ecosystem services umbrella (where any human benefit from any place becomes an \"ecosystem service\"; for related discussion, see4,7,14,36). Ecosystem services can point to co-benefits specifically from retained natural ecosystems (providing essentially \"full protection\" of the elements of biodiversity in that place), and be integrated into a broader decision-support framework that also considers the partial protection (or no-protection) options in a region.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nHaines-Young R, Potschin M: The links between biodiversity, ecosystem services and human well-being. Chapter six in Ecosystem Ecology: A New Synthesis, eds. DG Raffaelli and CLJ Frid. Cambridge University Press 2010. Publisher Full Text\n\nHardy D: Motivating Private Landowner Conservation to Maximize Ecosystem Services. River Basin Center, Odum School of Ecology, University of Georgia 2008. Reference Source\n\nReyers B, Polasky S, Tallis H, et al.: Finding a common ground for biodiversity and ecosystem services. Bioscience. 2012; 62(5): 503–507. Publisher Full Text\n\nEgoh B, Rouget M, Reyers B, et al.: Integrating ecosystem services into conservation assessments: a review. Ecol Econ. 2007; 63(4): 714–721. Publisher Full Text\n\nEgoh BN, Reyers B, Carwardine J, et al.: Safeguarding biodiversity and ecosystem services in the Little Karoo, South Africa. Conserv Biol. 2010; 24(4): 1021–1030. PubMed Abstract | Publisher Full Text\n\nMaguire LA, Justus J: Why Intrinsic Value Is a Poor Basis for Conservation Decisions. Bioscience. 2008; 58(10): 910–911. Publisher Full Text\n\nMace GM, Norris K, Fitter AH: Biodiversity and ecosystem services: a multilayered relationship. Trends Ecol Evol. 2012; 27(1): 19–26. PubMed Abstract | Publisher Full Text\n\nFaith DP: Biodiversity and ecosystem services: similar but different. Bioscience. 2012; 62(9): 785. Publisher Full Text\n\nIUCNWorld Conservation Strategy: living resource conservation for sustainable development. International Union for Conservation of Nature and Natural Resources (IUCN) 1980. Publisher Full Text\n\nMcNeely JA: Economics and biological diversity: developing and using economic incentives to conserve biological resources. IUCN, Gland, Switzerland. 1988; 232pp. Reference Source\n\nWilson EO: The Diversity of Life. Norton, New York 1992. Reference Source\n\nLarsen FW, Turner WR, Brooks TM: Conserving Critical Sites for Biodiversity Provides Disproportionate Benefits to People. PLoS One. 2012; 7(5): e36971. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReyers B, Polasky S, Tallis H, et al.: The Common ground of biodiversity and ecosystem services demonstrated: a response to Faith. Bioscience. 2012; 62(9): 785–786. Publisher Full Text\n\nFaith DP: Biodiversity. The Stanford Encyclopedia of Philosophy (Fall 2008 Edition), Edward N. Zalta (ed.) 2008. Reference Source\n\nHumphries C, Williams PH, Vane-Wright RI: Measuring biodiversity value for conservation. Annu Rev Ecol Syst. 1995; 26: 93–111. Publisher Full Text\n\nMargules CR, Pressey RL: Systematic conservation planning. Nature. 2000; 405(6783): 243–253. PubMed Abstract | Publisher Full Text\n\nFaith DP: Biodiversity and regional sustainability analysis. CSIRO, Canberra. 1995. Reference Source\n\nFaith DP, Walker PA: Integrating conservation and development: effective trade-offs between biodiversity and cost in the selection of protected areas. Biodivers Conserv. 1996; 5(4): 431–446. Publisher Full Text\n\nFaith DP, Walker PA: The role of trade-offs in biodiversity conservation planning: linking local management, regional planning and global conservation efforts. J Biosci. 2002; 27(4 Suppl 2): 393–407. PubMed Abstract | Publisher Full Text\n\nFaith DP, Carter G, Cassis G, et al.: Complementarity, biodiversity viability analysis, and policy-based algorithms for conservation. Environ Sci Policy. 2003; 6(3): 311–328. Publisher Full Text\n\nBoyland M, Nelson J, Bunnell FL, et al.: Creating land allocation zones for forest management: a simulated annealing approach. Can J For Res. 2004; 34(8): 1669–1682. Reference Source\n\nDavis FW, Costello C, Stoms D: Efficient conservation in a utility-maximization framework. Ecol Soc. 2006; 11(1): 33. Reference Source\n\nNaidoo R, Balmford A, Ferraro PJ, et al.: Integrating economic costs into conservation planning. Trends Ecol Evol. 2006; 21(12): 681–687. PubMed Abstract | Publisher Full Text\n\nChan KM, Shaw MR, Cameron DR, et al.: Conservation Planning for Ecosystem Services. PLoS Biol. 2006; 4(11): e379. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCameron SE, Williams KJ, Mitchell DK: Efficiency and concordance of alternative methods for minimizing opportunity costs in conservation planning. Conserv Biol. 2008; 22(4): 886–896. PubMed Abstract | Publisher Full Text\n\nChan KM, Hoshizaki L, Klinkenberg B: Ecosystem Services in Conservation Planning: Targeted Benefits vs. Co-Benefits or Costs? PLoS One. 2011; 6(9): e24378. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuck GW, Chan KMA, Klein CJ, et al.: Identifying spatial priorities for protecting ecosystem services [v1; ref status: indexed, http://f1000r.es/T0yHOY]. F1000 Research. 2012; 1: 17. Publisher Full Text\n\nFaith DP, Walker PA: Integrating conservation and development: incorporating vulnerability into biodiversity-assessment of areas. Biodivers Conserv. 1996; 5: 417–429. Publisher Full Text\n\nFaith DP, Walker PA, Ive J, et al.: Integrating conservation and forestry production exploring trade-offs between biodiversity and production in regional land-use assessment. For Ecol Manage. 1996; 85: 251–260. Publisher Full Text\n\nFaith DP, Walker PA: Regional sustainability and protected areas–biodiversity protection as part of regional integration of conservation and production. In: Pigram, J.J., Sundell, R.C. (Eds.), National Parks and Protected Areas: Selection, Delimitation and Management. Centre for Water Policy Research, University of New England, Armidale, 1997; pp. 271–296.\n\nMcNeely JA, Mooney HA, Oteng-Yeboah AA, et al.: Biodiversity. In: Ecosystems and Human Well-Being, Policy Responses. K. Chopra, et al., Eds. (Millennium Ecosystem Assessment, Island Press, Washington, DC) 2005; 3. : pp. 119–172. Reference Source\n\nFaith DP, Walker PA, Margules CR, et al.: Some future prospects for systematic biodiversity planning in Papua New Guinea – and for biodiversity planning in general. Pacific Conserv Biol. 2001; 6: 325–343. Reference Source\n\nFaith DP, Margules CR, Walker PA, et al.: Practical application of biodiversity surrogates and percentage targets for conservation in Papua New Guinea. Pacific Conserv Biol. 2001; 6: 289–303. Reference Source\n\nWatts ME, Ball IR, Stewart RR, et al.: Marxan with Zones: software for optimal conservation based land- and sea-use zoning. Envir Model Softw. 2009; 24(12): 1513–1521. Publisher Full Text\n\nDaily GC editor: Nature’s Services: Societal Dependence on Natural Ecosystems. Washington, DC: Island Press. 1997; 392 p. Reference Source\n\nBoyd J, Banzhaf HS: What Are Ecosystem Services? The Need for Standardized Environmental Accounting Units. RFF DP 06-02, Discussion paper. Resources for the Future 2006. Publisher Full Text"
}
|
[
{
"id": "318",
"date": "22 Oct 2012",
"name": "Gary Luck",
"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 brief but interesting opinion piece argues that biodiversity has both intrinsic value and human-use (utilitarian, instrumental) value.Previous studies have focused primarily on the intrinsic value of biodiversity and assigned utilitarian values to the ecosystem services generated by biodiversity. Yet, Faith argues that biodiversity has utilitarian value beyond those of ecosystem services because protecting biodiversity yields option values for humans from unknown future benefits. That is, protecting biodiversity maximises future options for benefits that may be used in the future, but which we are unaware of now. Current benefits are captured in the ecosystem-services concept, and option value for ecosystem services focuses on future use of current known services. Hence, both biodiversity and ecosystem services provide utilitarian values, but in different ways.In essence, Faith argues for the need to recognise the differences between biodiversity and ecosystem services, and he promotes the use of the ‘partial protection’ approach within a systematic planning framework. The partial protection approach applied within a given region would consider the various trade-offs from protecting biodiversity and protecting land or systems generating services to maximise regional net benefits.I agree with the need to recognise the different values and benefits of biodiversity and ecosystem services, and do not support subsuming biodiversity entirely within the ecosystem-services framework. The partial protection approach appears to offer promise in this area. This opinion piece is a reply to a reply and readers interested in following this argument should see citations 8 and 13 (in article reference list) and the original paper (Reyers, B et al. (2012) Finding common ground for biodiversity and ecosystem services).",
"responses": []
},
{
"id": "319",
"date": "22 Oct 2012",
"name": "Chris Margules",
"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 am pleased to approve this article for publication. I have the following three brief comments:1. It is important to acknowledge that partial protection, or protection of some components of biodiversity, is possible in places used predominantly for purposes other than biodiversity protection. The qualification of the MEA, noted by Faith, that it is difficult to measure such a contribution must also be acknowledged.2. More intensively transformed places may also contribute ecosystem services even though they protect little or almost no biodiversity. For example, the sediment free water that sugar cane plantations can deliver (although that water may contain dissolved nutrients).3. As a comment - an aside really - I wonder how anything can have non-anthropogenic values when those values are being assigned by people.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-30
|
https://f1000research.com/articles/1-29/v1
|
16 Oct 12
|
{
"type": "Research Article",
"title": "Donor awareness: key to successful voluntary blood donation",
"authors": [
"Rateesh Sareen",
"Gajendra N Gupta",
"Akanksha Dutt",
"Gajendra N Gupta",
"Akanksha Dutt"
],
"abstract": "Context: The current regulatory requirements for donor eligibility pose a challenge to blood centers in recruitment of voluntary blood donors, particularly in a developing country like India where awareness of the general population is low and myths about blood donation are prevalent. This study evaluates the reasons and rates of donor deferral in a tertiary hospital-based blood bank in western India.Aim: To find rates and reasons for deferral of voluntary blood donors in a city in western India.Settings and design: A retrospective study was done on blood donors during a 3-month period. Data collection was done by electronic records of blood donors.Materials and Methods: The study was conducted retrospectively at a tertiary care hospital in western India. All those who donated whole blood between 1st January 2011 and 31st March 2011 were included in the study. Data was collected using local blood bank software.Statistical analysis used: No statistical technique used as it is a data article.Results: 60.5% of donors were young, below 30 years of age. Donors were predominantly male (91.6%). Voluntary donors comprised 88% of the donors. Total deferral rate was 22.36%, with 17.29% permanent deferrals and 82.71% temporary deferrals. Main reasons for deferral were anemia 39.42%, low body weight 14.29%, hypertension 10.73%, age below 18 years 10.73% and history of medication 6.09%. The common causes of deferral between our study and other similar studies are the same.Conclusion: We concluded that majority of the donor population belongs to 18–30-year-old age group. This is encouraging with a voluntary blood donation initiative. Donor self exclusion and strict donor selection criteria application should be addressed by more proactive measures to make blood donation a safe and pleasurable experience.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: approved with reservations 2]”."
],
"content": "Introduction\n\nBlood donor selection criteria according to the guidelines of the National AIDS Control Organization are based on science, informed medical opinion and regulatory rules, its statistics show that annual rate of blood donation in India is about 7.4 million units, against requirement of 10 million units1. According to the Drugs and Cosmetics Act, not every person who walks into a camp/blood bank for blood donation is a donor. Donor by definition is a person who, after complete medical examination by the doctor, is declared fit for donation of blood. To make blood donation safe and increase the confidence of the masses in voluntary blood donation, many safety measures are implemented by the blood transfusion community. The most important of all safety measures is donor selection. Stringent, meticulous and serious donor screening is necessary to afford protection to blood donors and recipients2. The rates and reasons for donor deferral vary from region to region and from one center to another3.\n\n\nSubjects and methods\n\nWe obtained full ethics approval for this study from the Ethics Committee of the Santokba Durlabji Memorial Hospital and Research Center, and the study was conducted in our blood bank from 1st January to 31st March 2011. We included all individuals coming for whole blood donation in blood bank and camps. There were a total of 8700 individuals during the three-month study period, comprising 7970 (91.6%) males and 730 (8.4%) females. (Table 1) 90% of the donors was voluntary. Most of the donors were residents of our city or within 100 km radius. The volume of whole blood collected was according to donor weight: 50–60 kg – 350 ml and > 60 kg – 450 ml.\n\nThe table indicates the total of 8700 individuals for blood donation. It represents the percentage of male and female donor’s selection, and indicates which percentage were deferred donors.\n\nEach donor was selected by the medical officer after taking detailed medical history and general physical examination, which included body weight, temperature, pulse rate and regularity and blood pressure. Deferred donors were classified as temporary deferral or permanent deferral. We followed deferral criteria laid down in the Drugs and Cosmetics Act 1940 (the rules there under) and Technical manual - Director General Health Services and Drug Controller of India. Standard operating procedures based on national guidelines were used for donor selection and deferral. The cut off for hemoglobin was 12.5 gm/dl by fingerpick method. All donors were screened for Hemoglobin values using CuSO4 specific gravity method. Doubtful Hemoglobin values were confirmed by Hemocue 201+. In case of indoor donors, i.e. in blood bank, hemoglobin was estimated by Sysmex Kx21, fully automated complete blood count (CBC) counter.\n\n\nResults\n\nThe majority of blood donors were voluntary donors (Table 2). A total of 60.5% of donors were young between 18–30 years (Table 3). Out of 8700 volunteers, 7970 (91.6%) were male. Among the 730 female donors only 112 (15.34%) donors were selected whereas among male donors 6650 (83.44%) were selected. As for male donors, deferral rate was 19.85%. Overall deferral rate was 22.36%. The most common cause of deferral was anemia 764 (39.42%) both in males and females. The other causes in decreasing order of frequency were low body weight 277 (14.29%), under age 151 (7.79%), history of drugs/medications 118 (6.01%), recent blood donation 75 (3.87%), icterus 49 (2.53%) and menstrual bleeding 45 (2.32%) (Table 4). Among the causes of permanent deferral, cardiac problems along with hypertension were the most common, accounting for 208 (10.73%) of all causes. Uncommon causes include asthma 27 (1.39%), diabetes mellitus on insulin therapy 16 (0.83%), epilepsy 11 (0.57%), hepatitis 6 (0.31%), infection 40 (2.06%), malaria 31 (1.60%), tetanus or rabies vaccine 30 (1.55%) and other causes 37 (1.9%) including recent surgery, recent tattooing, dental procedure, fever, hypotension, low platelet count and renal disorder. There were 2 unsuccessful phlebotomies 0.001%. Farrales4 reported a higher rate of failed phlebotomies (0.5%) in their study and Custer et al.5 reported mis-collection (3.8%) in their study.\n\nThe table indicates the percentage of both male and female replacement and voluntary donors.\n\nThe table indicates the age distribution of the donors, and highlights that the majority of them are young.\n\nThe table highlights the permanent and the temporary deferral explanations amongst the donors.\n\n* Include Influence of alcohol, recent surgery, recent ear piercing, fever, recent tattooing, dental extraction, breast feeding and history of transfusion of blood/blood components.\n\n\nDiscussion\n\nThis study attempts to analyze the pattern of blood donation in a tertiary care hospital between Jan 1st 2011 to 31st March, 2011, in an emerging metropolitan city of western India.\n\nDonor rejection or deferral leaves a person with negative feeling about themselves as well as the blood banking system. However, there are definite advantages of elimination of donors in order to ensure the safety of blood donors as well as recipients of blood/blood products. Deferring donors also protects donors from possible adverse reactions and avoids consequent negative impact on donor motivation.\n\nIn our study the deferral rate was found to be 22.22% (1938). The lowest rejection rate was reported by Talonic6 (4%) in Papua New Guinea while Chaudhry7, Lim2, and Ranveet8, Unikrishnan9, Sunder10 reported 8–15% deferral rates in their studies. The comparatively higher deferral rate in our study was due to the stringent donor selection criteria, strict adherence to guidelines of the National Aids Control Organization and National Accreditation Board for Hospitals & Healthcare Providers. Women had a very high deferral rate in comparison to men reflecting the poor nutritional status of female population in our country. Similar observations were reported in studies in Manipal9, Delhi11 and Banglaru10. As reiterated in the national health policy of achieving 100% voluntary donation, our blood bank received 88% of its donors as voluntary donors in comparison to 12% replacement donors, which is way above the national average of 39.3%11. Voluntary donation adds to the quality and safety to blood donors and as there is no peer pressure on either the donor or the medical officer; evaluation of donors is purely on the basis of donor selection criteria. There were 335 (17.29%) permanent deferrals as against 1603 (82.71%) temporary deferrals. In a similar study, Custer et al.5 reported 68.5% temporary and 31.5% permanent deferrals. The lower numbers of permanent deferrals in our study are due to the majority of young donors. A total of 5263 (60.5%) donors were below 30 years of age (Table 2). It is due to active blood donation motivational activities and programs carried out by blood banks in educational institutes through lectures, presentations, posters and pamphlets that large number of young individuals are recruited, thereby strengthening long-term voluntary blood donation belts. In Shaz's study12, donors aged more than 60 years were allowed to donate, but in India, individuals above 60 years of age are not permitted to donate blood, but we received 913 (10.5%) donors who were between 50–60 years of age.\n\nThe minimum hemoglobin for blood donation is 12.5 mg/dl. Despite all efforts by the government towards reducing nutritional anemia, it is still very common in our country. This is evident from the finding that the most common cause of deferral in our study was low hemoglobin in 764 (39.42%) donors. Low hemoglobin was the commonest cause of deferral in most of studies by Sunder et al.11, Charles et al.13 and Agnihotri N14. The minimum cut-off hemoglobin level for blood donation is >12.5 gm% irrespective of sex. The second most common cause for deferral was body weight below 50 Kilograms 277 (15.48%), followed by under age 151 (7.79%). Body weight is related to poor health status of the general population and poor nutrition being common in low socio economic groups. Under-aged potential donors were unaware of basic requirements for blood donation, i.e. age, weight and hemoglobin percentage, indicating the importance of public awareness and education for successful voluntary blood donation. We mostly received voluntary donors, the majority of them in camps. Perhaps the organizations involved in recruiting donors are more enthusiastic in gathering people and pay less emphasis on the eligibility criteria, which adds to the large number of under-aged (151 [7.79%]) and underweight (277 [15.48%]) donors, thus resulting in deferrals. It is of utmost importance for organizations hosting blood donation camps to understand that blood donation is a science and meticulous donor screening is essential. The eligibility criteria for blood donation should be followed stringently; unnecessary gathering of people causes wastage of resources. Self exclusion by donors themselves is the answer to these problems. Self deferral by donors is only possible when our population is educated about selection criteria for blood donation.\n\nHypertension was the most common cause for permanent deferral 208 (10.73%). Hypertension was noted as common cause of deferral in a similar study by Bahadur et al.11–13. It was surprising that hypertension, along with cardiac problems, was roughly equally distributed in various age groups. In younger age groups, this might be due to apprehension and anxiety for donation, induced by fear of phlebotomy or fear of the sight of blood. Hypertension is a growing, undiagnosed, epidemic in our country, where people seek medical advice on appearance of signs and symptoms, and seldom go for annual checkups. Additionally, use of electronic blood pressure equipment, with more objective readings could have picked up more hypertensive donors. The use of drugs, particularly antibiotics, antihypertensive and analgesics, was a significant reason accounting for 118 (6.81%) of deferrals (Table 3).\n\nIndian studies from Chandigarh8 and Lucknow7 report jaundice as the most common cause of deferral. In a study by Halperin et al.15 the three most common causes of temporary deferral were low hemoglobin, cold/sore throat and fever, whereas in a study by Ranveet et al.8, underweight, underage and low hemoglobin level were the most common causes. Hence, donor deferral studies indicate that in each region there are unique sets of reasons for deferral.\n\nThe objective during donor selection should be blood collection as well as donor safety. Safety of donors is important as it helps in gaining confidence and winning the trust of future donors as well.\n\nDonor selection should be done with care, caution, sincerity and ethically, failing which we would be compromising donor safety and defeating the ultimate goal of 100% voluntary blood donation. In our country, where there are myths and social stigma attached with blood donation, we need to be very cautious in donor selection, as any harm to a donor would send the wrong message to the masses. Dorothy et al.16 supported the view that medical examination may actually serve as an incentive for future repeated donations.\n\nAnemia is the major cause of deferral. Referring such cases to physician for evaluation and treatment of anemia and asking them to donate at a later date is pivotal in ensuring donor recruitment and retention.\n\n\nConclusion\n\nThe study showed that most of the donors were between 18- to 30-years old. This is encouraging, as they could be motivated to become regular voluntary donors. Temporary deferral has a negative impact on blood donor return rate and subsequent donations7. It is necessary to follow strict donor selection criteria to make blood donation safe and win the trust of future donors. To strike a balance between donor selection and deferral, self exclusion by the donor is the key. This can be achieved by advertising campaigns, brochures, lectures, presentations, donor awareness programs and interaction with donors. The entire blood bank staff, especially medical officers, should share the responsibility of winning the confidence of donors and making blood donation a safe and pleasurable experience which will eventually increase voluntary blood donation, giving a permanent remedy to the shortage of blood in the country.",
"appendix": "Author contributions\n\n\n\nRS wrote the article and contributed to the conception and design, collection of data and data analysis. GNG aided in the design and final approval of the manuscript. AD contributed to the data analysis and interpretation, conception of the article and manuscript writing.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nWe would like to acknowledge Mr Pankaj Aggarwal, Mr Vinod Sharma, Ms Shilja Ahuja, who are the technical managers at the Blood Bank at the SDM hospital. We would also like to thank Mr Dinesh Chaturvedi for his help computer programming.\n\n\nReferences\n\nBlood safety and availability. World Health Organization. Fact sheet No 279, Geneva. June 2012. Reference Source\n\nLim JC, Tien SL, Ong YW: Main causes of pre-donation deferral of prospective blood donors in the Singapore Blood Transfusion Service. Ann Acad Med Singapore. 1993; 22(3): 326–31. PubMed Abstract\n\nGalea G, Gillon J, Urbaniak SJ, et al.: Study on medical donor deferrals at sessions. Transfus Med. 1996; 6(1): 37–43. PubMed Abstract | Publisher Full Text\n\nFarrales FB, Stevenson AR, Bayer WL: Causes of disqualification in a volunteer blood donor population. Transfusion. 1977; 17(6): 598–601. PubMed Abstract | Publisher Full Text\n\nCuster B, Johnson ES, Sullivan SD, et al.: Quantifying losses to the donated blood supply due to donor deferral and miscollection. Transfusion. 2004; 44(10): 1417–26. PubMed Abstract | Publisher Full Text\n\nTalonu T: Causes of volunteer blood donor rejection in Papua New Guinea. P N G Med J. 1983; 26(3–4): 195–7. PubMed Abstract\n\nChaudhary RK, Gupta D, Gupta RK: Analysis of donor-deferral pattern in a voluntary blood donor population. Transfus Med. 1995; 5(3): 209–12. PubMed Abstract | Publisher Full Text\n\nRanveet Kaur, Sabita Basu, Neelam Marwaha, et al.: A Reappraisal of underlying causes in donor deferral. Ann Natl Acad Med Sci. 2002; 38: 93–9.\n\nUnnikrishnan B, Rao P, Kumar N, et al.: Profile of blood donors and reasons for deferral in coastal South India. Australas Med J. 2011; 4(7): 379–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSundar P, Sangeetha SK, Seema DM, et al.: Pre-donation deferral of blood donors in South Indian set-up: An analysis. Asian J Transfus Sci. 2010; 4(2): 112–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBahadur S, Jain S, Goel RK, et al.: Analysis of blood donor deferral characteristics in Delhi, India. Southeast Asian J Trop Med Public Health. 2009; 40(5): 1087–91. PubMed Abstract\n\nShaz BH, James AB, Hillyer KL, et al.: Demographic variations in blood donor deferrals in a major metropolitan area. Transfusion. 2010; 50(4): 881–7. PubMed Abstract | Publisher Full Text\n\nCharles KS, Hughes P, Gadd R, et al.: Evaluation of blood donor deferral causes in the Trinidad and Tobago National Blood Transfusion Service. Transfus Med. 2010; 20(1): 11–4. PubMed Abstract | Publisher Full Text\n\nAgnihotri N: Whole blood donor deferral analysis at a centre in Western India. Asian J Transfus Sci. 2010; 4(2): 116–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHalperin D, Baetens J, Newman B, et al.: The effect of short-term, temporary deferral on future blood donation. Transfusion. 1998; 38(2): 181–3. PubMed Abstract | Publisher Full Text\n\nNguyen DD, Devita DA, Hirschler NV, et al.: Blood donor satisfaction and intention of future donation. Transfusion. 2008; 48(4): 742–8. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "405",
"date": "17 Oct 2012",
"name": "Cees Smit Sibinga",
"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\nUpon review, the title of the article and the actual research in the paper show a considerable discrepancy. The study itself seems to focus more on the reasons for deferral and not so much on awareness as stated in the title. Furthermore, there is no indication of the contents of an assumed awareness.Overall, the outcome provides some local demographic and some medical (deferral) information, which as such is nothing new or exciting. However, the awaking interest in a safe and sustainable blood supply in India deserves some peer appreciation.",
"responses": []
},
{
"id": "404",
"date": "24 Oct 2012",
"name": "Deborah Sesok-Pizzini",
"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 focuses on the challenges of blood donation in a tertiary care hospital in western India and presents a retrospective study of blood donor deferrals from a 3-month period from Jan 2011-March 2011.The study concluded that the deferral rate was 22.36% with a 17.29% permanent deferral rate for donors. Hypertension was the most common cause for a permanent deferral, and anemia was the most common cause for temporary deferral. Of interest, the paper concluded that the majority of donors were between 18-30 years of age.The limitations of this study are that it does not address issues of donor awareness and details about the recruitment process for these donors. Information is unavailable if these donors would in fact be return donors. Additional data, such as a survey given to donors, would be needed to understand the motivation of a regular voluntary donor and the impact of a temporary deferral. While this paper does give interesting information about deferral statistics, and meets the stated aim of the study, the results and conclusions do not really address donor awareness, and therefore conclusions are limited to assess the real impact on donor return rates in a voluntary donor program in India.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-29
|
https://f1000research.com/articles/1-28/v1
|
12 Oct 12
|
{
"type": "Case Report",
"title": "A hematologic condition expressed as a lung disease",
"authors": [
"Mónica Egozcue-Dionisi",
"José Nieves-Nieves",
"Ricardo Fernández-Gonzalez",
"Rosángela Fernández-Medero",
"Raúl Reyes-Sosa",
"José Lozada-Costas",
"Ramiro Pérez-Duardo",
"Román Vélez-Rosario",
"José Nieves-Nieves",
"Ricardo Fernández-Gonzalez",
"Rosángela Fernández-Medero",
"Raúl Reyes-Sosa",
"José Lozada-Costas",
"Ramiro Pérez-Duardo",
"Román Vélez-Rosario"
],
"abstract": "Pleural involvement secondary to Multiple Myeloma is considered a very rare complication. According to the literature only 1% of these patients develop a myelomatous pleural effusion. We present a case of a 39 year old man with multiple myeloma diagnosed six years prior to our evaluation, which developed progressive dyspnea, dry cough and right pleuritic chest pain two weeks prior to admission. On physical examination the patient had decreased breath sounds over the right posterior hemithorax accompanied by dullness to percussion. The chest radiogram was consistent with a right sided pleural effusion. Pleural fluid analysis revealed the presence of abundant abnormal plasma cells. The patient died four weeks after hospitalization. The presence of myelomatous pleural effusion is considered to be a poor prognostic finding, no matter at what disease stage it develops. So far no definite treatment has been shown to improve survival.",
"keywords": [
"Multiple Myeloma (MM) is a malignant neoplasm characterized by abnormal proliferation of plasma cells. The disease is typically manifested by anemia",
"pathologic fractures",
"hypercalcemia and renal failure. Pleural involvement in MM is very rare and seldom has been described in the literature. To our knowledge",
"approximately eighty cases have been mentioned in the largest case series reported. Pleural effusions can be either myelomatous or non-myelomatous",
"the former being the less common presentation. Most cases of myelomatous pleural effusions are due to IgA MM. We present a case of a patient with a pleural effusion secondary to IgG MM."
],
"content": "Introduction\n\nMultiple Myeloma (MM) is a malignant neoplasm characterized by abnormal proliferation of plasma cells. The disease is typically manifested by anemia, pathologic fractures, hypercalcemia and renal failure. Pleural involvement in MM is very rare and seldom has been described in the literature. To our knowledge, approximately eighty cases have been mentioned in the largest case series reported. Pleural effusions can be either myelomatous or non-myelomatous, the former being the less common presentation. Most cases of myelomatous pleural effusions are due to IgA MM. We present a case of a patient with a pleural effusion secondary to IgG MM.\n\n\nCase report\n\nA 39 year old man with hypertension, end-stage renal disease and chronic smoker, diagnosed with MM six years prior to our evaluation, came to our institution complaining of progressive dyspnea, fever, and dry cough of two weeks of evolution. He was treated with a course of oral antibiotics for five days with minor symptom improvement. On admission the patient was found with a temperature of 37.8°C, heart rate was 118/min., respiratory rate was 24 breaths/min., blood pressure was 120/74 mmHg, and oxygen saturation by pulse oximetry was 100% with a venturi-mask at 50% FIO2. Chest examination revealed multiple bilateral palpable plasmacytomas along the anterior and posterior hemithorax with decreased breath sounds below the right scapular area, and percussion dullness was heard on the right side. Antero-posterior chest radiogram showed a large right side pleural effusion with contralateral shifting of the mediastinal structures and patchy airspace opacities throughout the left lung (Figure 1). Complete blood count revealed pancytopenia with a WBC of 2.8 × 109/l, Hb of 8.4 g/dl and platelet count of 19 × 109. Blood chemistry showed a protein of 5.6 g/dl and lactate dehydrogenase of 721 IU/l. Calcium levels were within normal limits. Diagnostic and therapeutic thoracentesis was performed after platelet transfusion and a total of 900 ml of turbid, sero-sanguinous fluid was removed. Pleural fluid analysis was consistent with an exudate and the fluid cytology revealed the presence of abundant atypical plasma cells (Figure 2). Bacteria, fungi and acid fast smear and cultures of the pleural fluid were reported negative. The patient’s clinical condition was aggravated by bacteremia, septic shock and respiratory failure requiring mechanical ventilation. The patient was complicated by sepsis and died four weeks after hospitalization.\n\n\nDiscussion\n\nExtra-medullary involvement in MM is considered to be a rare complication of the disease1. Commonly involved sites are the nasal cavity, lung, pleura, thoracic wall, central nervous system, lymph nodes, spleen, skin and eyes. Involvement of serous cavities such as the pleural cavity, peritoneal cavity, cerebral-spinal space and pericardium is unusual, the pleural cavity being the most common site2. Pleural effusions occur in 6% of the patients with multiple myeloma and can be myelomatous or non-myelomatous3–5. Non-myelomatous pleural effusions can occur secondary to sepsis, pulmonary embolism, chronic renal failure and secondary neoplasm6. On the other hand, myelomatous pleural effusions have been described in only in 1% of the patients with MM and the diagnosis is based on the demonstration of monoclonal proteins in the pleural fluid by protein electrophoresis, finding monoclonal plasma cells in the fluid and/or histological examination of the pleura through biopsy7,8. Literature reveals that almost 40% of the cases of myelomatous pleural effusions are due to IgG type6.\n\nMultiple treatment regimens have been used including VAD regimen (vincristine, doxorubicin and dexamethasone), prednisolone, melphalan, etoposide, stem cell rescue and pleurodesis without a significant effect on mortality6,9. The use of bortezomib, a protease inhibitor, in refractory multiple myeloma has shown promising results. There is a single case of refractory MM and myelomatous pleural effusion treated successfully with intravenous and intrapleural bortezomib2,10.\n\n\nConclusion\n\nThere has been limited information in the literature regarding pulmonary manifestations of this hematologic malignancy. Pleural effusions can be present as an initial manifestation of the disease or as the disease progresses. As in our case, pleural involvement is associated with poor prognosis and high mortality rate no matter at what disease stage it appears. So far, there are no proven treatment regimens that can halt disease progression. Physicians should be aware of such a fatal complication as it predicts a very poor outcome. For this reason, additional studies towards the development of new treatment strategies should be considered.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the relative of the patient.",
"appendix": "Author contributions\n\n\n\nAuthors have contributed to the literature review, drafting of the manuscript, revisions of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGhosh S, Gopal R, Advani SH: Myelomatous pleural effusion. J Assoc Physicians India. 2006; 54: 738–9. PubMed Abstract\n\nKim YJ, Kim SJ, Min K, et al.: Multiple myeloma with myelomatous pleural effusion: A case report and a review of the literature. Acta Haematol. 2008; 120(2): 108–111. PubMed Abstract | Publisher Full Text\n\nRodriguez JN, Pereira A, Martínez JC, et al.: Pleural effusion in multiple myeloma. Chest. 1994; 105(2): 622–4. PubMed Abstract | Publisher Full Text\n\nSasser RL, Yam LT, Li CY: Myeloma with involvement of the serous cavities. Cytologic and immunochemical diagnosis and literature review. Acta Cytol. 1990; 34(4): 479–485. PubMed Abstract\n\nKintzer JS Jr, Rosenow EC 3rd, Kyle RA: Thoracic and pulmonary abnormalities in multiple myeloma. Arch Intern Med. 1978; 138(5): 727–730. PubMed Abstract | Publisher Full Text\n\nMalhotra KP, Agrawal V, Prasad N: Myelomatous pleural effusion: A diagnostic challenge. Indian J Cancer. 2010; 47(3): 351–352. PubMed Abstract | Publisher Full Text\n\nPalmer HE, Wilson CS, Bardales RH: Cytology and flow cytometry of malignant effusions of multiple myeloma. Diagn Cytopathol. 2000; 22(3): 147–151. PubMed Abstract | Publisher Full Text\n\nShirai T, Hashizume I, Kasamatsu N, et al.: A case of Bence-Jones protein-lambda positive multiple myeloma complicated by abnormal plasma cells in pleural effusion. Nihon Kokyuki Gakkai Zasshi. 1998; 36(2): 176–81. PubMed Abstract\n\nKamble R, Wilson CS, Fassas A, et al.: Malignant pleural effusion of multiple myeloma: Prognostic factors and outcome. Leuk Lymphoma. 2005; 46(8): 1137–42. PubMed Abstract | Publisher Full Text\n\nIannitto E, Minardi V, Tripodo C: Use of intrapleural bortezomib in myelomatous pleural effusion. Br J Haematol. 2007; 139(4): 621–622. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "313",
"date": "30 Oct 2012",
"name": "Artur Jurczyszyn",
"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": "315",
"date": "07 Nov 2012",
"name": "Sarah Waheed",
"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 failed to mention if this patient had ever received any chemotherapy previously and if they did what agents were used. Also what was the initial presentation of myeloma in this patient?The only imaging modality used was a Chest X-ray, it would have been helpful to know if a CT scan was done to evaluate if any pleural based mass was present along with the pleural effusion.",
"responses": []
},
{
"id": "316",
"date": "12 Nov 2012",
"name": "Hermann Einsele",
"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 response quality following high‐dose chemotherapy and autologous stem cell transplantation for multiple myeloma (MM) is clearly correlated with progression‐free and probably also overall survival. Thus a major goal of all protocols for the treatment of younger patients with MM is to achieve a chest radiogram (CR). Recent data of the Spanish myeloma study group reveal long‐term disease free survival in a subgroup of patients with CR after autologous stem cell transplant (SCT).Inclusion of novel agents (thalidomide, portezomib or lenalidomide) in the induction regimens prior to stem cell harvest and transplantation has allowed to achieve better remission quality prior to and after autologous SCT and to reduce treatment‐related mortality. Recently also protocols containing 2 novel agents have been used to improve induction regimens for patients with multiple myeloma.In this study the PETHEMA/GEM group has studied and compared 3 induction regiments VTD (2 novel agents), TD and VBMCP/VBAD (an intensive regimen with bortezomib). VTD showed the best equality of remission (CR rate 35% vs 14% with TD and 21% with VBMCP/VBAD/B. Also the PFS after VTD plus autologous SCT was significantly longer when compared two both other induction protocols. Unfortunately 14% of the patients in the VTD arm developed grade 3 and 4 PNP. Thus, future studies should use either bortezomib or proteosome inhibitors with a lower neurotoxicity.In addition, the VTD regimen in contrast to the GIMEMA study was not able to overcome the poor prognostic impact of high risk cytogenetics.Thus other treatment strategies e.g. allogeneic SCT, novel IMiDs or PI should be evaluated in these high risk MM patients (17p del, 7(4;14)).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-28
|
https://f1000research.com/articles/1-26/v1
|
11 Oct 12
|
{
"type": "Research Article",
"title": "Expression of erythropoietin in Indian tetraploid potato variety",
"authors": [
"Priti N Desai",
"Harish Padh",
"Priti N Desai"
],
"abstract": "With the advent of protein-based biotech drugs in the market, the quest for the “perfect” protein expression system, which is both economical and effective, has come into focus. Currently bacteria, yeast, insect cells, mammalian cells, transgenic animal and transgenic plants are widely used for the expression of therapeutic proteins. Among these, transgenic plants provide advantages in terms of low production cost, lower capital investment in infrastructure, and suitable post-translational modifications. The major limitation of plants as an expression host is the low level of transgene expression. To increase the expression of heterologous proteins in plants, a number of approaches have been used. One of the approaches is to increase the transgene expression by using tissue-specific promoter(s) which can concentrate the protein of interest in targeted tissues and, thus, prove advantageous in downstream purification. In the present report, a protocol for expression of heterologous protein erythropoietin in potato tuber using patatin, the tuber-tissue-specific promoter, was standardized. Expression vectors for production of the erythropoietin gene under tissue-specific promoter were successfully constructed. For production of a transgenic plant, tissue culture techniques for regeneration of the whole plant from single explants were standardized. Polymerase chain reaction (PCR) analysis was performed to confirm the stable integration of the erythropoietin gene in the potato plant by using sequence-specific primers.",
"keywords": [
"In the past decade",
"transgenic plant expression systems have emerged as a serious competitive force in the large-scale production of recombinant proteins. The first plant-derived heterologous proteins have already reached the market1",
"2 and detailed economic evaluations have demonstrated their competitiveness against established market sectors3",
"4. Several plant-derived recombinant therapeutic proteins which are in the final stage of clinical trials include human blood products",
"vaccine",
"antibody and growth hormones5. As with a number of products coming to the market",
"molecular farming in plants is finally coming of age. There have been technological developments on many levels",
"including transformation methods",
"control of gene expression",
"protein targeting and accumulation",
"the use of different crops as production platforms6 and modifications to alter the structural and functional properties of the product. One of the most driving factors has been yield improvements",
"as product yield has a significant impact on economic feasibility. To increase the final yield",
"a number of approaches can be used. One of them is to use a tissue-specific promoter",
"which can concentrate the protein of interest into targeted tissue and prove advantageous during downstream purification7."
],
"content": "Introduction\n\nIn the past decade, transgenic plant expression systems have emerged as a serious competitive force in the large-scale production of recombinant proteins. The first plant-derived heterologous proteins have already reached the market1,2 and detailed economic evaluations have demonstrated their competitiveness against established market sectors3,4. Several plant-derived recombinant therapeutic proteins which are in the final stage of clinical trials include human blood products, vaccine, antibody and growth hormones5. As with a number of products coming to the market, molecular farming in plants is finally coming of age. There have been technological developments on many levels, including transformation methods, control of gene expression, protein targeting and accumulation, the use of different crops as production platforms6 and modifications to alter the structural and functional properties of the product. One of the most driving factors has been yield improvements, as product yield has a significant impact on economic feasibility. To increase the final yield, a number of approaches can be used. One of them is to use a tissue-specific promoter, which can concentrate the protein of interest into targeted tissue and prove advantageous during downstream purification7.\n\nIn the present report, a protocol for expression of therapeutic proteins in potato tuber by using patatin, a tuber-specific promoter, was made. Indian tetraploid potato variety, Kufri Bahar was used for the expression of the therapeutic protein, as it is widely grown in Gujarat and other region of India. Potato is a vegetative propagated plant, which minimizes the spread of transgene contamination through pollen, and high tuber biomass makes it suitable for bulk production8.\n\nErythropoietin (EPO) was chosen as a protein of interest for tuber-specific expression, and to evaluate the capability of performing complex glycosylation in the potato plant. Erythropoietin is a highly glycosylated protein and glycosylation is necessary for its in vivo activity9. Presently, recombinant erythropoietin available on the market is produced from mammalian cells. Mammalian systems have disadvantages in term of cost, scalability and safety10,11. Recombinant human erythropoietin is widely used to treat anemia associated with chronic renal failure, rheumatoid arthritis, acquired immune deficiency syndrome (AIDS), and malignancies, as well as other types of anemia12.\n\nRecombinant EPO was expressed in tobacco BY2 cells. However, the expression level was very low (0.0026%) and expressed protein remained active only in vitro13. Thus, to increase the expression, researchers tried to express this protein in a whole plant using constitutive promoter (tobacco and Arabidopsis) where the side effect of over expression was observed14. Overexpression of EPO protein resulted in stunted vegetative growth, late flowering and male-sterility15.\n\nWhile in the present project we had chosen to express EPO protein in a tissue-specific manner, by using a tissue-specific approach, we aimed to decrease the metabolic burden to the whole plant and concentrate the protein in targeted tissue, where it can be easily purified. In the present study, potato tubers were selected as an organ of choice for the production of EPO since the same standard processes used in the starch industry may be adapted with little modifications to separate proteins. Also, the tubers, as most of the storage organs, offer a low hydrolytic profile, which facilitate protein stability.\n\nSeveral genes have been expressed in potato tubers using different techniques with varying degrees of success15–17. Considerable success has been achieved with the patatin promoter, which confers tuber specificity16,18. Most of the reports which use potato tuber as an expresser of therapeutic proteins had generally used the diploid potato variety19–23. However, we aim to use the Indian tetraploid potato variety Kufri Bahar, which is widely grown in Gujarat and other parts of India24.\n\n\nMaterials and Methods\n\nIn the present report, plasmids, bacterial strains and plants were used, as described in Table 1.\n\nEscherichia coli DH5α and Agrobacterium LBA4404 cultures were grown in luria broth (LB) medium. E. coli cultures were grown at 37°C while Agrobacterium cultures were grown at 28°C at 175 rpm. When necessary, antibiotics were added for E. coli (chloramphenicol [Sigma] 35mg/ml) and for Agrobacterium (chloramphenicol 35 mg/ml and rifampicin [Hi-media] 50mg/ml).\n\nAll enzymes which were used for cloning where purchased from MBI fermentas. Tuber-specific promoter patatin (B33) was excised from the vector pBinB33 using Eco RI and Bam HI Restriction Endonuclease (RE) sites and cloned into binary vector pCAMBIA1281Z using the same RE sites. pCAMBIA1281Z contained GUS as a reporter protein. The patatin gene was cloned upstream to the GUS gene at Eco RI and Bam HI RE sites, resulting in a plasmid named pPERDB33 (Figure 1a). For the expression of the EPO gene, the cDNA sequence of EPO was excised from the pIRsepo and cloned in to pPERDB33 in place of the GUS gene by using Nco I and Bst EII RE sites (Figure 1b). The resulting plasmid was named pPERDB33cEPO, which has the EPO gene downstream to the patatin promoter (Figure 1b). pPERDB33cEPO was then introduced in to E. coli DH5α for maintenance and further experimentation. pPERDB33cEPO was also introduced into Agrobacterium tumefaciens strain LBA 4404 by using triparental mating25.\n\na) Construction of vector containing patatin, pPERDB33 and, b) construction of vector containing EPO gene under patatin promoter.\n\nShoot cultures of the variety Kufri Bahar were established from sprouted buds excised from the tubers. The buds were surface sterilized with 0.1% mercuric chloride for 3 min followed by a rinse with 70% ethanol. After repeated washing with sterile distilled water, the buds were cultured on Murashige and Skoog (MS) medium supplemented with 0.1 µM GA3. The pH of the medium was adjusted to 5.7 and all the cultures were solidified with 0.8% agarose. The cultures were exposed to 16 hours light, which provided 1600 lux intensity of light, and maintained at 24 ± 2 °C temperature with 50–60% relative humidity. The buds developed into plantlets in 4 weeks. Nodes from these plantlets were cultured on MS medium containing 3% sucrose for raising the fresh cultures. Shoots from the fresh cultures were cultured into the MS medium supplemented with 8% sucrose and 6 µM BA to produce the microtuber. Inter-nodal segments, leaf discs and microtuber discs were routinely used for Agrobacterium-mediated transformation. To raise the shoots from inter-nodal segments, leaf discs and microtuber discs were used. Direct shoot regeneration was observed in all three explants in the two stage medium. In stage 1, all the explants were inoculated in PSRI 1 medium (MS medium + 1mg/l thiamine + 2% sucrose + 11µM zeatin + 1 µM NAA + 0.05 µM GA3 + 0.8% agar) for 20–30 days and then shifted to the PSRI 2 medium (MS medium + 1 mg/l thiamine + 2% sucrose + 9 μM zeatin + 0.1 μM NAA + 0.05 μM GA3). This medium was standardized for direct shoot regeneration from internodes by Millam (2006)26.\n\nInter-nodal segments, leaf discs and microtuber discs from in vitro grown plants were used for transformation and regeneration, essentially as described by Millam (2006)26 with some modifications. The procedure described by Millam (2006)26 for internodes was also used for leaf discs as well as microtuber discs. All three explants were incubated in liquid shoot regeneration medium (MS medium containing 1 mg/l thiamine + 2% sucrose + 11 µM zeatin +1 µM NAA + 0.05 µM GA3) for 2–3 hours in presence of 20 µM acetosyringone. After preconditioning, the explants were infected with Agrobacterium tumefaciens LBA4404 harboring the pPERDB33cEPO for 15 minutes. The explants were then blotted dry on sterile whatman No. 1 filter paper and were co-cultured on shoot regeneration medium containing 0.8% agar for 2 days. Following co-cultivation, the explants were transferred to shoot regeneration medium containing 0.8% agar and 500 mg/ml cefotaxime for 7 days for inhibition of Agrobacterium growth. After 7 days, explants were transferred to shoot regeneration medium containing 0.8% agar, 500 mg/ml cefotaxime and 7.5 mg/ml hygromycine for selection of transgenic shoots.\n\nAfter selection of transformed plant in antibiotic selection medium, genomic DNA was isolated by using fresh leaf from the regenerated shoots27. Genomic DNA was used as a template DNA for the amplification of the cEPO gene. After transformation, T-DNA was integrated into the plant nuclear DNA. In order to confirm the presence of cDNA sequence of EPO in plant nuclear DNA, gene specific PCR was performed by using EPO cDNA-specific primer, forward primer: 5’ CCACCACGCCTCATCTGTGAC 3’ and reverse primer 5’ TCTGTCCCCTGTCCTGCAGGC 3’. PCR amplification was performed in 50 µl reaction containing primers (50 ng each), Taq DNA polymerase (1 unit), 200 µM dNTP, 1 × PCR buffer and 50 ng of genomic DNA as template amplifying a 408 bp fragment of the EPO gene. The PCR cycling conditions were set as initial melting at 95°C for 5 minutes, followed by 30 cycles of amplifications with each cycle consisting of the following steps: 95°C for 15 seconds, 68°C for 20 seconds and 72°C for 1 minute.\n\n\nResults\n\nTo produce the EPO protein in a tuber-specific manner, patatin promoter was first excised and ligated into the backbone vector pCAMBIA1281Z to produce pPERDB33. The confirmation of pPERDB33 was done by RE digestion and agarose gel electrophoresis (figure 2).\n\nLane 1 contains Marker (lamda DNA digested with Hind III), lane 2 contains Eco RI and Bam HI digested plasmid from clone 23, lane 3 contains Eco RI digested plasmid from clone 23, lane 4 contains undigested plasmid from clone 23, lane 5 contains Eco RI and Bam HI digested plasmid from clone 26, lane 6 contains Eco RI digested plasmid from clone 26, lane 7 contains undigested plasmid from clone 26, Lane 8 contains undigested pCAMBIA 1281Z, lane 9 contains Eco RI digested pCAMBIA 1281Z, lane 10 contains Eco RI and Bam HI digested pCAMBIA 1281Z, lane 11 contains undigested pBinB33, lane 12 contains Eco RI digested pBinB33, lane 13 contains Eco RI and Bam HI digested pBinB33 and lane14 contains Marker (lamda DNA digested with Bst EII).\n\nAfter confirmation of the right clone having patatin prompter upstream to the GUS gene, the clone was maintained in E. coli. The Agrobacterium-mediated method was used for expression of the EPO gene in the potato plant. Plasmid pPERDB33, which contains the GUS gene downstream from the patatin promoter, was mobilized into Agrobacterium LBA4404 using tri-parental mating and confirmation of right clone, which was selected on the Luria agar plate containing rifampicin and chloramphenicol, and was done by the same method used for selection of the right clone in E. coli.\n\nTo clone the EPO protein into the vector pPERDB33 downstream to the patatin promoter the cDNA sequence of the human EPO gene was excised from plasmid pRISepo and ligated to the plasmid pPERDB33 in place of GUS. Confirmation of the right clone was done by RE digestion and agarose gel electrophoresis (Figure 3). The resultant plasmid which has the EPO gene downstream to the patatin promoter was named pPERDB33cEPO. pPERDB33cEPO was subsequently mobilized to the Agrobacterium LBA4404 using tri-parental mating and confirmed by RE digestion and agarose gel electrophoresis.\n\nLane 1 contains marker (lamda DNA digested with Hind III marker of MBI Fermentas), lane 2 contains pRIsepo digested with Nco I and Bst EII, lane 3 contains pPERDB33 digested with Nco I and Bst EII and lanes 4 to 8 contain PCR positive clones.\n\nThe resultant plasmid pPERDB33cEPO was then introduced into potato plants by using Agrobacterium-mediated transformation. Transformed plants were selected on the PSRI medium containing 7.5 mg/L hygromycin and 500 mg/L cefotaxime. After 1 month, transgenic shoot formation was observed from the microtuber discs in the antibiotic selection medium (Figure 4). However, shoot formation was also observed from inter-nodal explants, but they did not survive for a longer time in the antibiotic stress medium.\n\nGenomic DNA was isolated from the leaf of transformed plant. The relative quantity and quality of genomic DNA was analyzed using agarose gel electrophoresis. After transformation of the plant by the Agrobacterium method, the T-DNA segment containing the EPO gene was integrated into the plant nuclear genome. To investigate the stable integration of gene of interest in to plant nuclear genome, gene-specific PCR was done. Isolated genomic DNA from the transformed plant was used as template DNA. For gene-specific PCR analysis, EPO specifically primed along with the plasmid DNA containing the EPO sequence (pPERDB33cEPO) as a positive control was used.\n\nAfter PCR amplification, the PCR product was analyzed using 1.5% agarose gel (Figure 5). A 100 bp DNA ladder was used as marker. A 408 bp band corresponding to EPO amplicon was observed in the gel (Figure 5, lane 4), which was similar to the amplicon obtained from pPERDB33cEPO used as a positive control (Figure 5, lane 5). From this result it was confirmed that the EPO sequence was successfully integrated into the plant nuclear genome.\n\nLane 1 contains Genomic DNA isolated from transformed plant 1, lane 2 contains DNA ladder as a marker (100 bp MBI fermentas), lane 3 and lane 4 contain PCR product of transformed plants, lane 5 contains positive control (PCR product of pPERDB33cEPO), lane 6 contains negative control (PCR product of pBSSK which does not contain EPO gene), lane 7 contains negative control of PCR reaction (master mix) and lane 8 contains genomic DNA isolate from transform plants.\n\n\nDiscussion\n\nThe aim of the present study was to develop a protocol for production of heterologous proteins in potato tuber. Vectors for the production of the erythropoietin gene under tissue-specific promoter and tissue culture techniques for regeneration of a whole transgenic potato plant from single explants were standardized successfully. Direct shoot regeneration from a number of explants like leaf discs, in vivo tuber discs, in vitro tuber discs and internodes were successfully achieved. A very low plant transformation efficiency and a high percentage of necrosis were observed using Agrobacterium-mediated plant transformation. To reduce the necrosis of explants and increase the transformation efficiency time of co-cultivation, inoculum of the bacteria as well as the time for pre-selection were optimized. Hygromycin which was used in selection media caused necrosis of explants even at lower concentrations. Transgenic potato plants were confirmed with the gene-specific PCR, which proved that our gene of interest successfully integrated into the desired plant nuclear genome. However the PCR-positive plant was not able to form rooting when transformed to the rooting medium containing hygromycin. This may be because the hygromycin concentration was lethal when it directly came into contact with the shoot.\n\nOverall, the study demonstrates the feasibility of designing vectors to be used in creating transgenic potato plants for tissue-specific heterologous protein production. However, a lot remains to be done in optimizing the transformation and selection processes before the process can be used for large-scale production of heterologous proteins using tissue-specific expression in tuber of Solanum tuberosum.",
"appendix": "Authors’ contributions\n\nPND and HP reviewed the manuscript. PND was involved in drafting the manuscript and reviewing the literature. HP was responsible for supervising the production of the manuscript.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe present research work is funded by B.V. Patel PERD Centre, Ahmedabad, and Cadila Pharmaceuticals Ltd. Ahmedabad.\n\n\nAcknowledgements\n\nOne of the authors (Desai PN) acknowledges with thanks financial assistance from Cadila Pharmaceuticals Ltd., Ahmedabad and B.V. Patel PERD centre for providing the financial support. Authors also gratefully acknowledge Dr. Neeta Shrivastava for helping and providing access to the plant tissue culture facility.\n\n\nReferences\n\nHood EE, Witcher DR, Maddock S, et al:Commercial production of avidin from transgenic maize: characterization of transformant, prodcution, processing, extraction and purification. Mol Breed. (1997)3: 291–306.\n\nWitcher DR, Hood EE, Peterson D, et al:Commercial production of beta glucuronidase (GUS): a model system for the production of proteins in plants. Mol Breed. (1998)4: 301–312.\n\nKusnadi AR, Evangelista RL, Hood EE, et al:Processing of transgenic corn seed and its effect on the recovery of recombinant betaglucuronidase. Biotechnol Bioeng. (1998)60: 44–52.\n\nEvangelista RL, Kusnandi AR, Howard JA, et al:Process and economic evalution of the extraction and purification of recombinant beta-glucuronidase from transgenic corn. Biotechnol Prog. (1998)14: 607–614.\n\nMa JKC, Barros E, Bock R, et al:Molecular pharming for new drugs and vaccines. EMBO reporters. (2005)6: 593–599.\n\nTwyman RM, Stoger E, Schillberg S, et al:Molecular farming in plants: host systems and expression technology. Trends Biotechnol. (2003)21: 570–578.\n\nDesai PN, Shrivastava N, Padh H, et al:Production of heterologous proteins in plants: strategies for optimal expression. Biotechnol Adv, (2010)28(4): 427–435.\n\nMa JK, Drake PM, Christou P, et al:The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet, (2003)4(10): 794–805.\n\nTakeuchi M, Kobata A: Structures and functional roles of the sugar chains of human erythropoietins. Glycobiology. (1992)1: 337–46.\n\nDing XQ, Rao RV, Kuntz SM, et al:Impaired resensitization and recycling of the cholecystokinin receptor by co-expression of its second intracellular loop. Mol Pharmacol. (2000)58: 1424–1433.\n\nKawar ZS, Haslam SM, Morris HR, et al:Novel poly-Gal NAcbeta1-4GlcNAc(LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing beta1,4-N-acetyl galactosaminyltransferase and alpha1,3-fucosyltransferase. J Biol Chem. (2005)280: 12810–12819.\n\nLin K, Suggs S, Lin CH, et al:Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci USA. (1985)82: 7580–7584.\n\nMatsumoto S, Ikura K, Ueda M, et al:Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol Biol. (1995)27: 1163–1172.\n\nCheon BY, Kim HJ, Oh KH, et al:Overexpression of human erythropoietn (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis. Trans Res. (2004)13: 541–549.\n\nWarzecha H, Mason HS, Lane C, et al:Oral immunogenicity of human papillomavirus-like particles expressed in potato. J Virol. (2003)77(16): 8702–8711.\n\nArtsaenko O, Ketting B, Fiedler U, et al:Potato tubers as a biofactory for recombinant antibodies. Mol Breeding. (1998)4: 313–319.\n\nMason HS, Warzecha H, Mor T, et al:Edible Plant Vaccines: applications for prophylactic and therapeutic molecular medicine. Trends Mole Medi, (2002)8(7): 324–329.\n\nMason HS, Lam DM, Arntzen CJ, et al:Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci USA, (1992)89: 11745–11749.\n\nTacket CO, Mason HS, Losonsky G, et al:Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nature Med. (1998)4: 607–609.\n\nTacket CO, Mason HS, Losonsky G, et al:Human immune responses to a Novel Norwalk Virus Vaccine delivered in transgenic Potatoes. J Infect Dis. (2000)182: 302–305.\n\nRichter LJ, Thanavala Y, Arntzen CJ, et al:Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotechnol. 200018: 1167–1171.\n\nZhang B, Yang YH, Linn YM, et al:Expression and production of bioactive human interleukin-18 in transgenic tobacco plants. Biotechnol Lett. (2003)25: 1629–1635.\n\nPark Y, Cheong H: Expression and Production of Recombinant Human Interleukin-2 in Potato Plants. Protein Express Purif. (2002)25: 160–165.\n\nGopal J, Minocha JL, Sidhu JS, et al:Comparitive performance of potato crops raised from microtubers induced in the dark versus microtubers induced in light. Potato Res. (1997)40: 407–412.\n\nDraper J, Scott R, Armitage P, et al:Plant Genetic Transformation and Gene Expression. Blackwell Scientific Publications, Oxford. (1988), Pp3–68.\n\nMillam S: Potato, in Methods in molecular Biology: Kan Wang (344) Agrobacterium protocols, 2/e, 2. Humana Press Inc., Totowa, NJ (2006), pp 25–35.\n\nSharma AD, Gill PK, Singh P, et al:DNA Isolation from dry and fresh samples of polysaccharide-rich plants. Plant Mol Biol Repo. 200220: 415."
}
|
[
{
"id": "310",
"date": "17 Oct 2012",
"name": "Argelia Lorence",
"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\nAs expressed by the authors, the aim of this study was to develop a protocol for the production of erythropoietin (EPO), a pleiotropic cytokine with remarkable tissue-protective activities in addition to its well establish role in red blood cell production, in the Indian tetraploid potato variety Kufri Bahar.The recent FDA approval of Elelyso™, the first plant-made pharmaceutical for treatment of Type I Gaucher disease in humans, has renewed worldwide interest of using plants as viable production platforms for human and veterinary therapeutics.Although improving the production of EPO in plants is a topic of relevance, unfortunately the data described in this work is too preliminary and falls short from accomplishing the goal set by the authors. The key information lacking here is the level of expression of EPO achieved in this system, the demonstration that the expression level is stable over generations of these transgenics, and more importantly the proof that the expressed EPO is functional. The authors also failed to include in the introduction and to compare their strategy with the one of other groups who have successfully produced EPO in other plant expression systems Conley et al (2009)1 2 Conley et al (2010)3, Sperb et al. (2011)4, Conley et al (2011)5, Kitter et al. (2012)6; Nagels et al. (2012)7and Castilho et al. (2012)8.",
"responses": []
},
{
"id": "311",
"date": "24 Oct 2012",
"name": "Laura Privalle",
"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 idea presented by the authors is a good one and I was very interested in reading more about their success or their outcome. Unfortunately, I was quite disappointed as all that was presented in the paper was about a third of the story.They demonstrated that they could transform potato; however potato transformation is not new and of itself is an insufficient single result not deserving of publication. They did not demonstrate expression of erythropoetin or that using the patatin promoter would give sufficient expression to be of interest. Patatin is a major protein in potato but protein levels in potato are still quite low (~2%). The idea of using the starch extraction method to enrich the protein fraction is a good idea but again they did not present any follow-up that showed it would work for this particular protein of interest. When additional information is available it should be considered for publication but not before. In addition, I would recommend the authors seek a person more conversant in English to assist them in writing the next version.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-26
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https://f1000research.com/articles/1-10/v1
|
10 Aug 12
|
{
"type": "Research Article",
"title": "Murine Tim-1 is excluded from the immunological synapse",
"authors": [
"Jean Lin",
"Leo Chen",
"Lawrence P Kane",
"Jean Lin",
"Leo Chen"
],
"abstract": "The interaction between T cells and APCs bearing cognate antigen results in the formation of an immunological synapse (IS). During this process, many receptors and signaling proteins segregate to regions proximal to the synapse. This protein movement is thought to influence T cell function. However, some proteins are transported away from the IS, which is controlled in part by ERM family proteins. Tim-1 is a transmembrane protein with co-stimulatory functions that is found on many immune cells, including T cells. However, the expression pattern of Tim-1 on T cells upon activation by APCs has not been explored. Interestingly, in this study we demonstrate that the majority of Tim-1 on activated T cells is excluded from the IS. Tim-1 predominantly resides outside of the IS, and structure/function studies indicate that the cytoplasmic tail influences Tim-1 polarization. Specifically, a putative ERM binding motif (KRK 244-246) in the Tim-1 cytoplasmic tail appears necessary for proper Tim-1 localization. Furthermore, mutation of the KRK motif results in enhanced Tim1-mediated early tyrosine phosphorylation downstream of TCR/CD28 stimulation. Paradoxically however, the KRK motif is necessary for Tim-1 induced NFAT/AP-1 activation and co-stimulation of cytokine production. This work reveals unexpected complexity underlying Tim-1 localization and suggests potentially novel mechanisms by which Tim-1 modulates T cell activity.",
"keywords": [
"Antigen receptor",
"co-stimulatory",
"and signaling proteins adopt distinct patterns of localization and segregation upon T cell stimulation by peptide antigen presented by antigen-presenting cells (APC). Current models suggest that these patterns are critical for proper regulation of T cell activation. T cell recognition of an APC bearing cognate peptide drives the formation of a structure termed the immunological synapse (IS) or supramolecular activation cluster (SMAC)1. In a \"mature\" synapse",
"many proteins important for transduction of TCR signaling concentrate at the center of the contact site",
"the central supramolecular activation cluster (cSMAC)",
"between the T cell and APC. These proteins include CD3",
"CD28",
"ZAP-70 and PKC-θ1–3. At the IS",
"this concentration of signaling proteins may enhance signaling before engaged TCR’s are internalized",
"possibly to terminate signaling4."
],
"content": "Introduction\n\nAntigen receptor, co-stimulatory, and signaling proteins adopt distinct patterns of localization and segregation upon T cell stimulation by peptide antigen presented by antigen-presenting cells (APC). Current models suggest that these patterns are critical for proper regulation of T cell activation. T cell recognition of an APC bearing cognate peptide drives the formation of a structure termed the immunological synapse (IS) or supramolecular activation cluster (SMAC)1. In a \"mature\" synapse, many proteins important for transduction of TCR signaling concentrate at the center of the contact site, the central supramolecular activation cluster (cSMAC), between the T cell and APC. These proteins include CD3, CD28, ZAP-70 and PKC-θ1–3. At the IS, this concentration of signaling proteins may enhance signaling before engaged TCR’s are internalized, possibly to terminate signaling4.\n\nOpposite the immunological synapse is a region known as the distal pole complex (DPC). Many large adhesion and glycosylated molecules, such as CD43, are transported to this region4,5. Formation of the DPC is thought to be driven by ERM (ezrin, radixin, and moesin) family proteins6,7. The prevailing hypothesis is that the DPC serves as a reservoir for sequestering negative signaling molecules, such as CD43, away from the IS to allow for greater T cell activation8. However, the presence of a pool of active signaling molecules, including ZAP-70, PIP3, and STIM-1 and Orai1 suggests an additional positive role for the DPC9–11. While the precise function of the DPC is disputed, formation of the DPC does appear to impact T cell activation. For example, disrupting localization of proteins to the DPC with an ERM dominant negative construct can disrupt specific functions, including transcriptional activation and cytokine production6,12.\n\nTransmembrane immunoglobulin and mucin 1 (Tim-1) is a co-stimulatory molecule found on the surface of many immune cells. It was first identified in primates as a Hepatitis A virus cellular receptor (HAVCR1), although the mouse homolog does not bind HAV13. Variants in murine (and human) Tim-1 were later associated with asthma susceptibility14–16. Early work on the immune function of Tim-1 also revealed a role for Tim-1 as a co-stimulatory molecule on CD4+ T helper cells by enhancing inducible transcription, cytokine production, and proliferation17,18. Tim-1 has also been implicated in the regulation of B cells, CD8+ T cells, dendritic cells, NKT cells, and mast cells19–26. Tim-1 antibodies have demonstrated efficacy in the modulation of immune function in different models of disease, including asthma and organ transplantation17,19,22,27–30.\n\nAlthough much is known about the effects of Tim-1 on transcription factor induction and cytokine production, less is known about the sub-cellular localization of Tim-1, especially in T cells. The function of many molecules has not been fully appreciated until their localization was understood. For example, the role of PKC-θ in T cells was greatly enhanced by the discovery that it localizes at the SMAC in effector T cells and away from the IS in regulatory T cells31,32. Understanding Tim-1 localization in T cells may provide similar insights into its function, particularly since some controversy still exists about the role of Tim-1 in T cell signaling. While previous studies have implicated Tim-1 in enhancing T cell activation17,18,33,34, one report has suggested that Tim-1 might function in either a positive or negative fashion, depending on the strength of antibody ligation35. Another recent study demonstrated increased cytokine production by Tim-1 deficient T cells, suggesting that Tim-1 may also act as a negative regulator of T cell function, at least under some circumstances36. Thus, defining Tim-1 localization on T cells under different conditions may yield novel insights that help to resolve these apparently disparate findings.\n\nThe localization of Tim-1 has not been extensively explored. A previous report suggested that Tim-1 exists in vesicles in the cytoplasm of human embryonic kidney cells (293) and 300.19 pre-B cells37. Another study demonstrated that Tim-1 expressed on DO11.10 TCR transgenic T cells localized towards apoptotic thymocytes with exposed phosphatidylserine (PS), a Tim-1 ligand24. Other studies have suggested that human TIM-1 interacts with ZAP-70 and PI3K33 and may co-cap with CD335.\n\nAt this point, relatively little is known about the sub-cellular localization of Tim-1, especially in T cells. In particular, where Tim-1 distributes (or re-distributes) upon T cell activation is poorly characterized. In this study, we define the patterns of Tim-1 localization before and after T cell recognition of antigen/MHC, as well as the functional consequences of altering Tim-1 localization. Our studies have revealed unexpected complexity in the regulation of Tim-1 localization and its function in T cell activation. These findings may have implications for understanding the function of Tim-1 in regulating immune responses.\n\n\nMaterials and methods\n\nJurkat, D10, Raji, and CH27 cell lines were used and cultured as previously described38. The following antibodies were used: pSrc Y416 and pZAP-70 Y319 (Cell Signaling), PKC-θ (C-18, Santa Cruz), CD43 (clone S7, BD Pharmingen), M2-Cy3 (Sigma Aldrich), EEA1 (BD Transduction), M2 anti-flag (Sigma Aldrich), anti-human CD3 (Becton Dickinson), mouse CD3 and CD28 (BD Pharmingen), human CD28 (Life Technologies), Tim-1 Fc (eBiosciences), anti-TCR antibody C305 (Harlan), anti-Tim-1 antibodies (3B3 and 5F12), anti-Tim-4 antibodies (3A1, 3H11 and 5G3). Alexa fluor-conjugated secondary antibodies were from Life Technologies. Conalbumin was from Sigma Aldrich, and SEE from Toxin Technology.\n\nD10 cells were transiently transfected with up to 20 µg total of plasmid DNA by electroporation at 250V/950 µF and rested for 16 hours. Live cells were recovered the next day by spinning over a cushion of Lympholyte-M. D10 T cells (0.3×106) were combined with an equal number of conalbumin-loaded CH27 cells by centrifugation at 3000 rpm for 3 minutes, followed by incubation at 37 degrees for 5–40 minutes. The pellet was gently resuspended by pipetting 3 times with a large-bore 1 ml pipet tip. Cells were allowed to settle on a poly-l-lysine coated coverslip for 20 min before being fixed at a final concentration of 2% PFA. Cells were permeablized with 0.1% TX-100 and were blocked for 30 min in 10% anti-goat or anti-donkey serum. The following were used: M2-Cy3 (2 µg/mL), pSyk/ZAP-70 (1:100), PKC-θ (1:100), and EEA1 (1:100). Secondary antibodies were used at the following concentrations: anti-rabbit Alexa-647 – 1:1000, anti-mouse Alexa-488 – 1:2000, anti-mouse Alexa-555 – 1:2000, anti-rat-Cy3 – 1:1000. Mid-plane images were captured on an Olympus FluoView 1000. Images were exported as bit TIFFs and analyzed with Image J. Figures were assembled in Canvas 8. For live cell imaging, Jurkat T cells were co-transfected with Tim-1 GFP and ZAP-70 Tag RFP. Equal numbers of Jurkat and SEE loaded Raji cells (0.075×106 cells) were maintained at 37°C in Matek dishes and imaged on an Olympus FluoView 1000 or a Nikon A1.\n\nMature conjugates were identified by morphology and localization of ZAP-70 or PKC-θ at the interface between the B and T cell. Images were analyzed in Metamorph or Image J. When Tim-1 was localized opposite the IS, the cells were termed \"anti-synapse\". Tim-1 in conjugates that appeared close to the IS were termed \"front half\" of the cell. Tim-1 that appeared to be both opposite and near to the IS were termed \"unpolarized\". Finally, Tim-1 that had a predominantly intracellular and vesicular appearance was noted as \"punctate\".\n\nFor a select number of imaged conjugates that expressed Flag-Tim-1, two parameters were measured using Image J. First, the distance of Tim-1 from the synapse was determined as the angle between the center of the IS to the center of the Tim-1 region with the vertex of the angle set at the center of the cell. The extent of spread of Tim-1 was measured as the angle between the two edges of the Tim-1 region.\n\n8.7 micron aldehyde/sulfate latex beads (Life Technologies) were prepared according to manufacturer instructions. 80×106/mL beads were coated with 50 µg/mL anti-CD3 and 50 µg/mL anti-CD28.\n\nTim-1, Tim-1 Y276F, and Tim-1 cytoplasmic tail truncation were generated as described previously18. Tim1-GFP was generated by inserting the C57Bl/6 Tim-1 into pEGFP-N1. Site-directed mutagenesis was utilized to mutate a Flag-Tim-1 construct, using the QuikChange system (Stratagene). The KRK at position 244–246 of the C57BL/6 allele of Tim-1 was mutated to QGQ using the following primers: Forward: cc aggta catac ttatg caagg gcagt cagca tctct aagcg; Reverse: cgctt agaga tgctg actgc ccttg cataa gtatg tacct gg. The sequence was verified by automated sequencing. The ERM DN-GFP construct was a gift from Dr. Janis Burkhardt. ZAP-70 cDNA and vector containing Tag RFP were gifts from Dr. Steven Bunnell.\n\n0.5×106 Jurkat and D10 T cells were stained with 1 µg of M2 (anti-Flag) on ice and then stained with 1:200 Alexa-647 for surface staining. For intracellular staining cells were fixed in 1.5% paraformaldehyde at room temperature for 10 minutes. Cells were then permeablized on ice with ice cold methanol for 15 minutes before being washed and stained for M2 (anti-Flag). 0.5×106 CH27 or Raji cells were stained with 1–4ug of anti-Tim-1 or anti-Tim-4 antibodies on ice and then secondarily stained with 1:200 Alexa-647 on ice. Samples were read on a BD LSR II; FlowJo software was used to analyze data.\n\n20×106 Jurkat T cells were transfected with empty vector, Tim-1, or Tim-1QGQ. 1.5×106 cells were lysed using 1% NP-40 lysis buffer in addition with beta-glycerophosphate, sodium fluoride, sodium orthovanadate, AEBSF, aprotinin, leupeptin, pepstatin (Calbiochem/EMD Biochemicals). Lysates were run on a 10% SDS-PAGE gel before being transferred to PVDF membrane and blotted with anti-pY (4G10). Blots were developed with Super-Signal Pico ECL (Pierce) and imaged on a Kodak Image Station 4000MM.\n\nJurkat cells were transfected as described above with pCDEF3 (empty vector), WT Tim-1, Tim-1QGQ, or Tim-1 lacking the cytoplasmic tail truncation (Tim-1∆Cyto). Cells were re-suspended at 0.5×106 in PBS and placed on ice in the presence of anti-TCR (C305) at a dilution of 1:250 for 30 min. Cells were treated with 80 µM Dynasore for 20 minutes on ice to prevent clathrin-mediated TCR internalization. Cells were then incubated at 37°C for 0, 5, 10, 20, 30, 60, and 120 minutes. Immediately after the time points, cells were washed with ice cold PBS before staining with anti-human CD3 and M2 (to detect Flag Tim-1). Samples were read on a BD LSR II; data were analyzed using FlowJo software.\n\nJurkat T cells were co-transfected with empty vector, WT Tim-1, or Tim-1 QGQ, along with an NFAT/AP-1 luciferase reporter construct. Cells were allowed to recover for 16 hours before stimulating with the anti-TCR antibody C305 (1:1000) in the presence or absence of anti-CD28 (1:100) for 6 hours at 37°. D10 T cells were co-transfected with empty vector, WT Tim-1, or Tim-1 QGQ, along with an NFAT/AP-1 luciferase reporter construct. Cells were allowed to recover for 16–18 hours before stimulating with 1 µg/mL biotinylated anti-CD3, -CD4 and -CD28, plus streptavidin for six hours at 37°. Luciferase activity was determined as described previously39.\n\n0.5×106 Jurkat cells were stimulated with anti-TCR antibody C305 (1:1000), with or without CD28 (1:200) for 24 hours. Supernatants were taken and production of IL-2 was determined by ELISA (BD OptIA). D10 T cells (0.5×106) were stimulated with 1 µg/mL anti-CD3, -CD4 and -CD28 for 24 hours before supernatants were collected for measurement of IL-4 and TNF-α by ELISA. Comparisons were analyzed by paired Student’s t test, performed with Prism.\n\nThe chief limitation in these studies is the subjective nature of data collection and interpretation in the confocal imaging experiments. To determine true positive signal, cells that were stained with comparable amounts of secondary antibodies alone were compared to staining with primary and then secondary antibodies. In addition, the laser voltage was adjusted so that pixels were not saturated. Protein localization was observed in two different T cell lines, using both epitope- and GFP-tagged constructs. The core observation of WT mTim-1 exclusion from the immune synapse was observed over the course of dozens of experiments. Quantitation of WT and mutant Tim-1 localization was pooled from conjugates obtained in multiple separate experiments. Other experiments were performed at least three times, with statistical analysis being performed on replicates within a representative experiment.\n\n\nResults\n\nTo define patterns of Tim-1 localization on T cells, we transfected Tim-1 into the murine Th2 line D10, which does not expresses endogenous Tim-118. In contrast to studies that reported predominantly intracellular Tim-1 in non-T cells37, we found Tim-1 diffusely expressed on the surface of resting T cells (Figure 1A). However, when Tim-1 expressing T cells are activated by antigen loaded APCs; the pattern of Tim-1 localization is altered. Surprisingly, Tim-1 concentrates in a region opposite the immunological synapse, with the latter represented by PKC-θ or pZAP-70 (Y319). This localization is not epitope tag-dependent, since both C-terminus tagged Tim1-GFP and N-terminus tagged Flag-Tim1 localize opposite, or at least outside, the immunological synapse (Figure 1B). The majority of Tim-1 in T cell:APC conjugates (51.25% and 71% with Tim1-GFP and Flag-Tim1, respectively) appears in the \"back\" half of the cell, opposite, or at least away from, the immunological synapse (\"anti-synapse\"; Figure 1C–E). Tim-1 was present within the IS in only 1 conjugate (1.03% of the total). This appears to be a general phenomenon, as Tim-1 localization on Jurkat T cells interacting with APC’s is also found predominantly outside of the immunological synapse (57% of conjugates; Figure 1F).\n\n(A) Resting D10 T cells transiently co-transfected with Flag-Tim-1 and PKC-θ-GFP (green) were fixed, stained with anti-Flag-Cy3 (red), and visualized mid-plane by confocal microscopy. (B - upper panels) D10 T cells transiently transfected with Tim-1-GFP (green) were conjugated with conalbumin-loaded CH27 B cells. Endogenous PKC-θ was stained with PKC-θ (C-18) and Alexa-555-conjugated secondary antibody (red) as a marker of the IS/c-SMAC. (B - lower panels) D10 T cells transiently transfected with Flag-Tim1 (red) were conjugated with antigen loaded CH27 cells and stained with pZAP-70 and Alexa 488-conjugated secondary antibody (green) as a marker of the cSMAC. (C) Additional D10 T cell:APC conjugates showing the exclusion of Tim1-GFP from the IS. (D) Quantitation of the phenotype of Tim-1 GFP localization in D10:CH27 conjugates (n=66) from 15 experiments or (E) Flag-Tim-1 in D10:CH27 conjugates (n=31) from 6 experiments. (F) Quantitation of Tim-1 localization on Jurkat T cells making synapses with superantigen-loaded Raji B cells. (F, bottom) Schematic of system used to score conjugate phenotypes.\n\nTo further demonstrate that Tim-1 localizes predominantly away from the cSMAC, we performed live cell microscopy. Again, Tim-1 moved away from the nascent ZAP70-containing immunological synapse (Figure 2A and Movie 1).\n\n(A) Jurkat T cells transiently transfected with Tim-1-GFP (green) and ZAP70-Tag-RFP (red) were incubated with Raji cells pre-loaded with 1 µg/mL SEE and stained with Cell Tracker Blue (blue). Cells were incubated in a heated chamber for live cell imaging. (B) CH27 cells were stained with anti-Tim-1 (left) and anti-Tim-4 (right) antibodies and secondary antibody and analyzed by flow cytometry. (C) The presence of Tim-1 ligand(s) on CH27 (upper panels) and Raji (lower panels) B cells was revealed by staining with Tim1-Fc and secondary antibody, in the absence (left) or presence (right) of EDTA, followed by flow cytometry.\n\n\n\nWe also utilized a more reductionist system to examine the effect of anti-TCR and -CD28 on Tim-1 localization. Thus, Jurkat T cells expressing Tim1-GFP and ZAP70-TagRFP were mixed with latex beads coated with anti-CD3/CD28 antibodies. Here we observed that Tim-1 initially appears to concentrate near the bead along with ZAP-70. However, over time, most Tim-1 moves away from the beads (Movie 2). Overall, the pattern of Tim-1 localization is reminiscent of the distal pole complex8.\n\n\n\nSome proteins require the expression of their ligands on the APC in order to localize towards the IS. For instance, CD28 only localizes to the cSMAC in the presence of APCs expressing of one of its ligands – CD80 or CD8640. In agreement with the importance of ligands in receptor localization, it has been shown that Tim-1 faces apoptotic cells bearing one of its ligands, i.e. phosphatidylserine24. We tested whether the APCs used in our system contain a ligand for Tim-1. Variable results were obtained when probing for expression of known ligands for Tim-1, including Tim-1 itself and Tim-4, on the APCs that we employed, CH27 and Raji (Figure 2B and data not shown). While Tim-4 was consistently not detected on CH27 cells, Tim-1 staining was more variable. We did confirm that both types of APCs used in our studies express one or more surface ligands for Tim-1, as evidenced by binding of Tim1-Fc to the surface of the APCs (Figure 2C). Furthermore, Tim1-Fc binding to these cells was abolished in the presence of EDTA, demonstrating that Tim1-Fc binding to this/these still-undefined ligand(s), like the known Tim-1 ligands, requires divalent ions41,42. This finding is not entirely surprising since unidentified Tim-1 ligands have been suggested to exist in a previous study43. Thus, although known Tim-1 ligands (Tim-1/Tim-4) may or may not be expressed on the surface of the APCs used in our studies, one or more Tim-1 ligand(s) are present. Interestingly, this still does not result in Tim-1 localization towards the IS.\n\nNext, we determined the elements necessary for Tim-1 localization away from the IS. During conjugate formation, many proteins depend on motifs found in the cytoplasmic tail for proper localization. For instance, CD28 requires Y188 in its cytoplasmic tail for localization towards the IS44. Likewise, CD43, which moves opposite the immunological synapse and to the distal pole complex, requires its cytoplasmic tail for this localization6. Specifically, CD43 requires a membrane-proximal positively charged amino acid cluster (KRR) in its cytoplasmic tail for ERM binding and distal pole complex localization45. ERM family proteins are necessary for driving certain proteins, such as CD43 and Rho-GDI, towards the DPC6,46. Intriguingly, we noted a similar sequence in the juxtamembrane region of the Tim-1 cytoplasmic tail – a KRK motif at residues 244–246.\n\nTo determine the intrinsic requirements for Tim-1 exclusion from the IS, we examined the effect of three constructs on Tim-1 localization. Specifically, we tested the effect of Tim-1Y276F, a cytoplasmic tail truncation (Tim-1del.cyto), and Tim-1 244–246 KRK-QGQ (Tim-1QGQ) on Tim-1 localization (Figure 3A). As shown previously by our group, Y276 is critical for Tim-1 co-stimulatory function18. However, the Tim-1Y276F mutant construct still appears to localize opposite the immunological synapse (Figure 3B). To quantify the location and extent of spread of Tim-1 we examined two parameters. First, to determine the distance of Tim-1 in relation to the IS of T cell:APC conjugates, we measured the angle of Tim-1 from the center of the IS. Thus, if Tim-1 were concentrated directly opposite the synapse, Tim-1 would be 180° away from the IS. Second, we measured the extent of Tim-1 spreading on the cell surface. Wild type Tim-1 is predominantly found in the \"back\" half of the cell (>90 degrees away from the synapse with a median of 133.3°), opposite the immunological synapse, and is fairly tightly contained (spread of 20–180° with a median of 79.6°) (Figure 3B–C). Tim-1Y276F localization is similar to wild type Tim-1, in that in a majority of conjugates the protein is found more than 90° (median 136.6°) from the synapse and is spread over 20–120° with a median of 58.1° (Figure 3B–C). These findings suggest that the majority of Tim-1Y276F is concentrated opposite the synapse. Next, a Tim-1 cytoplasmic tail truncation was utilized. In contrast to WT or Y276F forms of the protein, Tim-1 with a cytoplasmic tail truncation is more likely to be present in the front half of the cell, closer to the IS with a median distance from the IS of 106.5° (Figure 3C). In about half of the conjugates analyzed, the Tim-1del.cyto construct was found in the front half of the cell (less than 90° from the IS), and in 28% of total conjugates Tim-1del.cyto even appears to cross into the IS (Figure 3C).\n\n(A) Murine Tim-1 cytoplasmic tail sequence. The vertical line indicates the location of the truncation in the delta-cyto construct. KRK is the putative ERM binding domain; Y276 is underlined. (B) D10 T cells transiently transfected with either Flag-Tim1Y276F (red) or Flag-Tim-1 cytoplasmic tail truncation (del.cyto; red) were mixed with conalbumin loaded CH27 cells (green). Cells were then stained with anti-Flag mAb directly conjugated to Cy3. (C) Quantitation of the angle from the IS to Tim-1 (top) and the extent of distribution of Tim-1 on the cell surface (bottom). (D - upper) Representative image of Tim-1QGQ-GFP and ZAP-70 RFP expressing D10 cells interacting with antigen-loaded CH27 B cells. (D - lower) Quantification of Tim-1QGQ localization in D10:CH27 and Jurkat:Raji conjugates from 12 and 13 experiments, respectively.\n\nThe greatest change in Tim-1 localization that we have observed thus far is seen when the positively charged, putative ERM-binding, motif in Tim-1 (244–246 KRK) is mutated. Rather than localizing diffusely on the surface of the T cells, Tim-1QGQ has a predominantly punctate (56.7% of D10 conjugates and 90% of Jurkat conjugates) appearance, consisting of mainly intracellular Tim-1, with some of this mutant even present in the IS (Figure 3 and Movie 3). Thus, the ability of Tim-1 to bind ERM proteins appears to be important for Tim-1 localization distal to the IS and within the DPC.\n\n\n\nGiven the dramatic effect on Tim-1 localization, we further characterized the Tim-1QGQ mutant. We observed that the Tim-1QGQ construct has lower surface expression than wild type Tim-1, even when higher concentrations of Tim-1QGQ plasmid are transfected. Although the total amount of plasmid transfected is the same (10 µg total), 10 µg of Tim-1QGQ plasmid yields less surface expression than 2.5 µg of WT Tim-1 plasmid (along with 7.5 µg empty vector). However, the total amount of Tim-1QGQ protein appears to be equivalent to WT when cells are permeablized (Figure 4A–B). This is consistent with our imaging, wherein Tim-1QGQ is not highly expressed on the cell surface but appears to distribute into intracellular pools within the cell (Figure 3D). Further, Tim-1QGQ does not co-localize with early endosomal antigen 1 (EEA1), suggesting that this pool of vesicular Tim-1 is not found in early endosomes (Figure 4C). The significant amount of intracellular Tim-1QGQ suggests either that Tim-1QGQ is rapidly recycled from the cell surface or that Tim-1QGQ is retained in a vesicular compartment within the cell.\n\nAnti-Flag staining of EV (empty vector), Flag-Tim-1, or Flag-Tim-1QGQ transfected D10 (A) or Jurkat T cells (B) as determined by flow cytometry. Surface staining of non-permeablized cells is on the left. Methanol permeabilization of T cells for intracellular Flag expression of EV (empty vector), Flag-Tim-1, or Flag-Tim-1QGQ transfected T cells, as determined by flow cytometry (right). (C) Representative image of Jurkat T cells transiently transfected with Tim-1QGQ-GFP (green) and co-stained for EEA1 and Alexa-555 (red) after conjugation to antigen loaded Raji cells from three experiments. (D) D10 T cells co-transfected with Flag-Tim-1 or Flag-Tim-1QGQ (red) and FERM-GFP (\"ERM-DN\") constructs and conjugated to antigen-loaded CH27 cells were stained with anti-Flag-Cy3 antibody and imaged by confocal microscopy. (E) To quantify the ERM DN and Tim-1 localization, a ten pixel line scan was drawn along the surface of the cell, with the intensity of staining represented as a percentage of the maximal pixel intensity. Top – WT Tim-1; Bottom – Tim-1QGQ.\n\nSince the KRK sequence in the Tim-1 cytoplasmic tail represents a putative ERM binding motif, we wanted to determine whether Tim-1 might interact with ERM proteins. Here, we used an dominant negative (DN) ERM construct, containing the N-terminal FERM domain (from ezrin) that binds proteins with ERM-binding motifs, along with a GFP moiety, but not the C-terminal actin-binding domain6. When cells are co-transfected with both Tim-1 and the ERM-DN, there is partial Tim-1 co-localization with the FERM-GFP (Figure 4D–E). This is consistent with a role for WT Tim-1 interacting with ERM proteins in the regulation of Tim-1 localization. However, mutation of the Tim-1 KRK motif diminishes the ability of the mutant to interact with the FERM-GFP construct, as compared to WT Tim-1 (Figure 4D–E), providing further validation of a possible interaction between Tim-1 and ERM family proteins.\n\nWe next determined whether Tim-1 localization affects Tim-1 co-stimulatory activity in conjunction with TCR and CD28. Interestingly, we were surprised to find that Tim-1QGQ promotes enhanced cellular tyrosine phosphorylation, as compared to wild type Tim-1 (Figure 5A). One of the tyrosine phosphorylated substrates induced in the Tim-1QGQ expressing cells is a band slightly above 50 kD. Since this would be consistent with Src family kinases (SFK), we were interested in determining whether this band was a phosphorylated SFK member. Using antibodies against the activating tyrosine (Y416 in Src), we were able to detect increased phosphorylation in Tim-1QGQ-expressing cells within minutes of TCR/CD28 stimulation (Figure 5A). Although not all tyrosine phosphorylation results in positive signaling, the increased phosphorylation at the activating tyrosine (Y416 in Src) in T cells expressing the Tim-1QGQ-expressing cells suggests that Tim-1QGQ may enhance early T cell signaling.\n\n(A) Jurkat T cells transfected with empty vector (EV), Flag-Tim-1, or Flag-Tim-1QGQ were stimulated with anti-TCR and anti-CD28 antibodies for the indicated times. Lysates were analyzed by SDS-PAGE and western blotting for pY (4G10), pSrc (Y416; analogous to Y394 in Lck), and β-actin. (B) Jurkat T cells were stimulated with anti-CD3 mAb for the indicated times. CD3 expression was measured with flow cytometry and mean fluorescence intensity (MFI) was determined in FloJo. Dynasore (DS, 80 µM) was used to prevent clathrin-mediated endocytosis after TCR/CD3 crosslinking.\n\nSince two previous reports demonstrated an association between Tim-1 and CD333,35, another possible explanation for the enhanced tyrosine phosphorylation in Tim-1QGQ expressing cells was that Tim-1QGQ might increase the levels of surface TCR/CD3 and/or slow the rate of TCR/CD3 internalization. To address this possibility, we stimulated Jurkat cells expressing WT or mutant Tim-1 with anti-CD3 antibody, and measured the levels of CD3 surface expression by flow cytometry. As expected, after antibody crosslinking, CD3 surface expression decreased over time (Figure 5B). T cells expressing WT Tim-1 or Tim-1QGQ displayed equivalent rates of TCR internalization, although starting levels of TCR/CD3 varied somewhat (Figure 5B). Thus, impairment of TCR/CD3 down-regulation does not appear to be the mechanism behind the increased tyrosine phosphorylation in T cells expressing Tim-1QGQ after CD3 crosslinking.\n\nNext, we examined the effects of altering Tim-1 localization on its ability to modulate inducible transcription and cytokine production. As we demonstrated previously, WT Tim-1 is able to co-stimulate the activity of an NFAT/AP-1 transcriptional reporter18,34. However, Tim-1QGQ was not able to enhance NFAT/AP-1 activation in D10 cells (Figure 6A). Furthermore, while WT Tim-1 can enhance cytokine production, Tim-1QGQ cannot (Figure 6B–D). Consistent with a role for ERM protein binding to the KRK motif in Tim-1, a dominant negative ERM construct also suppresses the ability of WT Tim-1 to enhance transcription or cytokine production (Figure 6). These findings suggest that Tim-1 interaction with ERM proteins, with proper subsequent localization of Tim-1, plays a role in Tim1-mediated transcriptional activity and cytokine production.\n\n(A) D10 cells were transfected with an NFAT/AP-1 reporter, along with empty vector, WT or mutant Flag-Tim-1 in the presence or absence of ERM-DN. The next day, cells were cultured for six hours in the presence or absence of CD3/CD4/CD28 stimulation before assaying for luciferase activity. (B) D10 T cells were transfected with empty vector, WT Tim-1, ERM-DN, or Tim-1QGQ. Cells were stimulated with anti-CD3 or anti-CD3/CD28 antibodies for 24 hours. Cell-free supernatants were collected and assayed for TNF-α production by ELISA. (C) D10 T cells were transfected with empty vector, Tim-1, ERMDN, or Tim-1QGQ. Cells were stimulated with anti-CD3 or anti-CD3/CD28 antibodies for 24 hours and IL-4 production was determined by ELISA. (D) Jurkat T cells were transfected with empty vector, Tim-1, ERMDN, or Tim-1QGQ. Cells were stimulated with α-TCR or α-TCR/CD28 for 24 hours before IL-2 production was determined by ELISA. Data are presented as average values, +/- standard deviation, of duplicate samples from an individual experiment.\n\n\nDiscussion\n\nHere, we have shown that in contrast to the majority of known co-stimulatory molecules and TCR associated signaling molecules, Tim-1 does not localize towards the immunological synapse. Rather, surprisingly, Tim-1 is excluded from the immunological synapse in an ERM-dependent manner. Our structure/function studies suggest that Tim-1 exclusion from the immunological synapse is an active process requiring more than one step. First, the Tim-1 cytoplasmic tail appears to be necessary for exclusion from the immunological synapse, since a cytoplasmic tail truncation results in greater amounts of Tim-1 in the SMAC. Second, specific residues in the cytoplasmic tail (i.e. KRK) are required for proper Tim-1 localization towards the distal pole complex. Furthermore, concentration of Tim-1 opposite the immunological synapse towards the anti-synapse, or distal pole complex, appears to influence both early signaling and Tim-1 induced enhancement of T cell function.\n\nWe find that Tim-1 is found mostly on the cell surface of T cells in the steady state. This is in contrast to previously published reports suggesting that Tim-1 is maintained in a mostly intracellular store and only becomes localized to the cell surface upon activation37. These discrepancies could be due to differences in cell type. Thus, the previously published report used HEK 293 cells and 300.19 pre-B cells. Also, since Tim-1 is a transmembrane protein, it is also possible that WT Tim-1 might reside in an intracellular compartment before being inducibly cycled to the surface, similar to CLTA-4. On T cells, Tim-1 localizes towards the interface with PS-expressing apoptotic thymocytes, a finding we were also able to confirm (data not shown)24. However, in our studies Tim-1 does not localize towards the interface with APCs bearing antigenic peptide and an unidentified Tim-1 ligand. This suggests that different Tim-1 ligands may have distinct effects on localization. Further examination of known Tim-1 ligands, such as Tim-4 and HAV, may help to clarify this issue. In addition, it will also be of interest to determine the identity of the as-yet-unknown ligand(s) expressed on the B cell lines that we have used as APC’s in our studies.\n\nRegarding the relationship of Tim-1 to TCR/CD3, there is some discrepancy between our findings and the recent literature. Thus, it has been suggested that hTIM-1 co-localizes with CD3 and ZAP-70 and that CD3 can be co-capped with mTim-133,35. These findings suggest that Tim-1 should be found at the IS with CD3 and ZAP-70. However, none of the previous studies investigated the kinetics of Tim-1 localization or the localization on T cells in conjugates with antigen-bearing APCs. We have shown that Tim-1 may at least partially co-localize with ZAP-70 in the presence of TCR/CD28 coated beads (and absence of any ligand for Tim-1). However, at later time points Tim-1 relocates away from the antibody coated beads. In addition, we have obtained preliminary data indicating that Tim-1 and ZAP-70 microclusters may co-localize (data not shown). This suggests that Tim-1 and ZAP-70 might interact at some early time point during T cell activation but that the interaction may not persist.\n\nThe question then arises of the functional importance of Tim-1 exclusion from the IS, possibly at the distal pole complex in T cells, and how it might relate to Tim-1 enhancement of NFAT/AP-1 activation and cytokine production. If Tim-1 is truly a co-stimulatory molecule, then why would it be excluded from the IS? While the predominant view in the field is that the region opposite the IS, or distal pole complex, serves as a reservoir for molecules that inhibit signaling, there is evidence that the DPC may also serve as an area for active signaling. Multiple reports in the literature have shown that certain active signaling molecules, including PIP3, ZAP-70, STIM1/Orai, and CD46, reside at least in part in the DPC9–11,47. Thus, Tim-1 may localize in the DPC in order to avoid being internalized and degraded at the immunological synapse. This may allow for extended time to interact with other signaling molecules, and in this way enhance signaling. Alternatively, Tim-1 may also enhance signaling by binding inhibitory molecules and moving them towards the DPC and away from the positively acting signaling molecules found at the immune synapse. However, it should be noted that one of the recent Tim-1 knockout studies suggested that Tim-1 deficient mice develop worse lung inflammation in a model of airway hyper-reactivity, although another knockout study did not demonstrate this36,48.\n\nAlso intriguing is the paradoxical difference between early signaling events in cells expressing WT Tim-1 or Tim-1QGQ. Surprisingly, Tim-1QGQ-expressing cells displayed enhanced tyrosine phosphorylation at early time points downstream of TCR and CD28 stimulation, compared with the effects of WT Tim-1. This may represent phosphorylation of inhibitory molecules and/or increased tyrosine phosphorylation of positive signaling molecules. Also, the punctate appearance of Tim-1QGQ could result from localization in endosomal compartments. Recent studies have highlighted the importance of endosomal vesicles carrying signaling molecules (e.g. LAT) into microclusters, in order to enhance the very earliest signaling events at the microclusters49. Thus, vesicular Tim-1QGQ localization could enhance early signaling before being rapidly transmitted to the SMAC for degradation. In this way, Tim-1QGQ may enhance very early signaling and be degraded before having an opportunity to enhance later events, such as cytokine production and transcriptional activity. Alternatively, the Tim-1QGQ mutant may be rapidly internalized, which would explain the reduced levels of surface expression. Proximal to the Tim-1 KRK motif is a YILM motif that is very similar to the CTLA-4 clathrin adaptor-binding motif (YVKM)50. It is therefore possible that the KRK-QGQ mutation (and subsequent reduced ERM protein binding) exposes this YILM motif and causes increased internalization. This would also be consistent with the fact that the Tim-1del.cyto construct, in which part of this motif is truncated (before the M), is not found in an intracellular, vesicular, compartment. WT Tim-1 may also briefly cycle through these endosomal compartments before being expressed more stably at the cell surface. Since a recent report has suggested that internalized/endosomal TCR can signal, it is possible that the increased early tyrosine phosphorylation in cells expressing Tim-1QGQ arises from this internal compartment51.\n\nRelevant for this discussion, recent reports have also implicated signaling from endosomes as contributing to lymphocyte activation49,51,52. Tim-1QGQ displays a predominantly punctate pattern, which is consistent with possible endosomal localization. Thus, another intriguing possibility is that during early signaling events, Tim-1QGQ in endosomes can enhance early signaling events downstream of the TCR.\n\nThe movement of proteins during T cell interaction with antigen presenting cells impacts T cell function. Here we have demonstrated that Tim-1 on T cells preferentially localizes opposite the immunological synapse when conjugated to antigen-bearing APCs. Our studies have begun to unravel the motifs and complexities involved with regulating Tim-1 localization. These findings may provide insight into the mechanism underlying the effects of Tim-1 on the immune response.",
"appendix": "Author contributions\n\n\n\nThe project was conceived by LPK and JL. Experiments were carried out by JL and LC. The manuscript was written by JL and LPK. Funding for the project was obtained by LPK.\n\n\nCompeting interests\n\n\n\nThe authors have no competing interests to disclose.\n\n\nGrant information\n\nThis work was supported by grants AI067544 and AI073748 from the NIH (to LPK).\n\n\nAcknowledgements\n\nWe thank S. Bunnell for fluorescent protein constructs, V. Kuchroo for anti-Tim-1 and anti-Tim-4 antibodies, R. DeKruyff for anti-Tim-1 antibody 3B3, and J. Burkhardt for the ERM-DN construct and helpful discussions.\n\n\nReferences\n\nMonks CR, Freiberg BA, Kupfer H, et al.: Three-dimensional segregation of supramolecular activation clusters in T cells. Nature. 1998; 395(6697): 82–86. PubMed Abstract | Publisher Full Text\n\nBromley SK, Iaboni A, Davis SJ, et al.: The immunological synapse and CD28-CD80 interactions. Nat Immunol. 2001; 2(12): 1159–1166. PubMed Abstract | Publisher Full Text\n\nBlanchard N, Di Bartolo V, Hivroz C, et al.: In the immune synapse, ZAP-70 controls T cell polarization and recruitment of signaling proteins but not formation of the synaptic pattern. Immunity. 2002; 17(4): 389–399. PubMed Abstract | Publisher Full Text\n\nHuppa JB, Davis MM: T-cell-antigen recognition and the immunological synapse. Nat Rev Immunol. 2003; 3(12): 973–983. PubMed Abstract | Publisher Full Text\n\nSperling AI, Sedy JR, Manjunath N, et al.: TCR signaling induces selective exclusion of CD43 from the T cell-antigen-presenting cell contact site. J Immunol. 1998; 161(12): 6459–6462. PubMed Abstract\n\nAllenspach EJ, Cullinan P, Tong J, et al.: ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synapse. Immunity. 2001; 15(5): 739–750. PubMed Abstract | Publisher Full Text\n\nDelon J, Kaibuchi K, Germain RN, et al.: Exclusion of CD43 from the immunological synapse is mediated by phosphorylation-regulated relocation of the cytoskeletal adaptor moesin. Immunity. 2001; 15(5): 691–701. PubMed Abstract | Publisher Full Text\n\nCullinan P, Sperling AI, Burkhardt JK, et al.: The distal pole complex: a novel membrane domain distal to the immunological synapse. Immunol Rev. 2002; 189: 111–122. PubMed Abstract | Publisher Full Text\n\nBarr VA, Bernot KM, Shaffer MH, et al.: Formation of STIM and Orai complexes: puncta and distal caps. Immunol Rev. 2009; 231(1): 148–159. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRandriamampita C, Mouchacca P, Malissen B, et al.: A novel ZAP-70 dependent FRET based biosensor reveals kinase activity at both the immunological synapse and the antisynapse. PLoS One. 2008; 3(1): e1521. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCostello PS, Gallagher M, Cantrell DA, et al.: Sustained and dynamic inositol lipid metabolism inside and outside the immunological synapse. Nat Immunol. 2002; 3(11): 1082–1089. PubMed Abstract | Publisher Full Text\n\nRoumier A, Olivo-Marin JC, Arpin M, et al.: The membrane-microfilament linker ezrin is involved in the formation of the immunological synapse and in T cell activation. Immunity. 2001; 15(5): 715–728. PubMed Abstract | Publisher Full Text\n\nManangeeswaran M, Jacques J, Tami C, et al.: Binding of hepatitis a virus to its cellular receptor 1 inhibits T-regulatory cell functions in humans. Gastroenterology. 2012; 142(7): 1516–25. e3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeigelstock D, Thompson P, Mattoo P, et al.: The human homolog of HAVcr-1 codes for a hepatitis A virus cellular receptor. J Virol. 1998; 72(8): 6621–6628. PubMed Abstract | Free Full Text\n\nMcIntire JJ, Umetsu SE, Akbari O, et al.: Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat Immunol. 2001; 2(12): 1109–1116. PubMed Abstract | Publisher Full Text\n\nMcIntire JJ, Umetsu SE, Macaubas C, et al.: Immunology: Hepatitis A virus link to atopic disease. Nature. 2003; 425(6958): 576. PubMed Abstract | Publisher Full Text\n\nUmetsu SE, Lee WL, McIntire JJ, et al.: TIM-1 induces T cell activation and inhibits the development of peripheral tolerance. Nat Immunol. 2005; 6(5): 447–454. PubMed Abstract | Publisher Full Text\n\nde Souza AJ, Oriss TB, O’Malley KJ, et al.: T cell Ig and mucin 1 (TIM-1) is expressed on in vivo-activated T cells and provides a costimulatory signal for T cell activation. Proc Natl Acad Sci U S A. 2005; 102(47): 17113–17118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDing Q, Yeung M, Camirand G, et al.: Regulatory B cells are identified by expression of TIM-1 and can be induced through TIM-1 ligation to promote tolerance in mice. J Clin Invest. 2011; 121(9): 3645–3656. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMa J, Usui Y, Takeda K, et al.: TIM-1 signaling in B cells regulates antibody production. Biochem Biophys Res Commun. 2011; 406(2): 223–228. PubMed Abstract | Publisher Full Text\n\nWong SH, Barlow JL, Nabarro S, et al.: Tim-1 is induced on germinal centre B cells through B-cell receptor signalling but is not essential for the germinal centre response. Immunology. 2010; 131(1): 77–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYuan X, Ansari MJ, D’Addio F, et al.: Targeting Tim-1 to overcome resistance to transplantation tolerance mediated by CD8 T17 cells. Proc Natl Acad Sci U S A. 2009; 106(26): 10734–10739. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXiao S, Zhu B, Jin H, et al.: Tim-1 stimulation of dendritic cells regulates the balance between effector and regulatory T cells. Eur J Immunol. 2011; 41(6): 1539–1549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee HH, Meyer EH, Goya S, et al.: Apoptotic cells activate NKT cells through T cell Ig-like mucin-like-1 resulting in airway hyperreactivity. J Immunol. 2010; 185(9): 5225–5235. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim HS, Lee CW, Chung DH, et al.: T cell Ig domain and mucin domain 1 engagement on invariant NKT cells in the presence of TCR stimulation enhances IL-4 production but inhibits IFN-gamma production. J Immunol. 2010; 184(8): 4095–4106. PubMed Abstract | Publisher Full Text\n\nNakae S, Iikura M, Suto H, et al.: TIM-1 and TIM-3 enhancement of Th2 cytokine production by mast cells. Blood. 2007; 110(7): 2565–2568. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSizing ID, Bailly V, McCoon P, et al.: Epitope-dependent effect of anti-murine TIM-1 monoclonal antibodies on T cell activity and lung immune responses. J Immunol. 2007; 178(4): 2249–2261. PubMed Abstract\n\nSonar SS, Hsu YM, Conrad ML, et al.: Antagonism of TIM-1 blocks the development of disease in a humanized mouse model of allergic asthma. J Clin Invest. 2010; 120(8): 2767–2781. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDegauque N, Mariat C, Kenny J, et al.: Immunostimulatory Tim-1-specific antibody deprograms Tregs and prevents transplant tolerance in mice. J Clin Invest. 2008; 118(2): 735–741. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUeno T, Habicht A, Clarkson MR, et al.: The emerging role of T cell Ig mucin 1 in alloimmune responses in an experimental mouse transplant model. J Clin Invest. 2008; 118(2): 742–751. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMonks CR, Kupfer H, Tamir I, et al.: Selective modulation of protein kinase C-theta during T-cell activation. Nature. 1997; 385(6611): 83–86. PubMed Abstract | Publisher Full Text\n\nZanin-Zhorov A, Ding Y, Kumari S, et al.: Protein kinase C-theta mediates negative feedback on regulatory T cell function. Science. 2010; 328(5976): 372–376. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBinne LL, Scott ML, Rennert PD, et al.: Human TIM-1 Associates with the TCR Complex and Up-Regulates T Cell Activation Signals. J Immunol. 2007; 178(7): 4342–4350. PubMed Abstract\n\nde Souza AJ, Oak JS, Jordanhazy R, et al.: T cell Ig and mucin domain-1-mediated T cell activation requires recruitment and activation of phosphoinositide 3-kinase. J Immunol. 2008; 180(10): 6518–6526. PubMed Abstract | Free Full Text\n\nXiao S, Najafian N, Reddy J, et al.: Differential engagement of Tim-1 during activation can positively or negatively costimulate T cell expansion and effector function. J Exp Med. 2007; 204(7): 1691–1702. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCurtiss ML, Gorman JV, Businga TR, et al.: Tim-1 Regulates Th2 Responses in an Airway Hypersensitivity Model. Eur J Immunol. 2012; 42(3): 651–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSantiago C, Ballesteros A, Tami C, et al.: Structures of T Cell Immunoglobulin Mucin Receptors 1 and 2 Reveal Mechanisms for Regulation of Immune Responses by the TIM Receptor Family. Immunity. 2007; 26(3): 299–310. PubMed Abstract | Publisher Full Text\n\nKane LP, Mollenauer MN, Weiss A, et al.: A proline-rich motif in the C terminus of Akt contributes to its localization in the immunological synapse. J Immunol. 2004; 172(9): 5441–5449. PubMed Abstract\n\nNarayan P, Holt B, Tosti R, et al.: CARMA1 is required for Akt-mediated NF-kappaB activation in T cells. Mol Cell Biol. 2006; 26(6): 2327–2336. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYokosuka T, Kobayashi W, Sakata-Sogawa K, et al.: Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation. Immunity. 2008; 29(4): 589–601. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSantiago C, Ballesteros A, Martinez-Munoz L, et al.: Structures of T Cell Immunoglobulin Mucin Protein 4 Show a Metal-Ion-Dependent Ligand Binding Site where Phosphatidylserine Binds. Immunity. 2007; 27(6): 941–951. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKobayashi N, Karisola P, Pena-Cruz V, et al.: TIM-1 and TIM-4 Glycoproteins Bind Phosphatidylserine and Mediate Uptake of Apoptotic Cells. Immunity. 2007; 27(6): 927–940. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXiao L, Fu ZR, Liu F, et al.: Suppression of allograft rejection by Tim-1-Fc through cross-linking with a novel Tim-1 binding partner on T cells. PLoS One. 2011; 6(7): e21697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSanchez-Lockhart M, Graf B, Miller J, et al.: Signals and sequences that control CD28 localization to the central region of the immunological synapse. J Immunol. 2008; 181(11): 7639–7648. PubMed Abstract\n\nYonemura S, Hirao M, Doi Y, et al.: Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol. 1998; 140(4): 885–895. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTakahashi K, Sasaki T, Mammoto A, et al.: Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein. J Biol Chem. 1997; 272(37): 23371–23375. PubMed Abstract | Publisher Full Text\n\nOliaro J, Pasam A, Waterhouse NJ, et al.: Ligation of the cell surface receptor, CD46, alters T cell polarity and response to antigen presentation. Proc Natl Acad Sci U S A. 2006; 103(49): 18685–18690. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarlow JL, Wong SH, Ballantyne SJ, et al.: Tim1 and Tim3 are not essential for experimental allergic asthma. Clin Exp Allergy. 2011; 41(7): 1012–1021. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPurbhoo MA, Liu H, Oddos S, et al.: Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse. Sci Signal. 2010; 3(121): ra36. PubMed Abstract | Publisher Full Text\n\nChuang E, Alegre ML, Duckett CS, et al.: Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression. J Immunol. 1997; 159(1): 144–151. PubMed Abstract\n\nYudushkin IA, Vale RD: Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3zeta in the endosomal compartment. Proc Natl Acad Sci U S A. 2010; 107(51): 22128–22133. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChaturvedi A, Martz R, Dorward D, et al.: Endocytosed BCRs sequentially regulate MAPK and Akt signaling pathways from intracellular compartments. Nat Immunol. 2012; 12(11): 1119–1126. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "203",
"date": "29 Aug 2012",
"name": "Wenxia Song",
"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 research described in this article is interesting, mostly well done, and worth publishing.My major concerns:1. The manuscript over interprets some of the data.2. The polarized distribution of Tim1 needs a confirmation using primary T cells.3. The effects of Tim1 mutations on the distribution of signaling molecules should be included to support tyrosine phosphorylation data.",
"responses": [
{
"c_id": "65",
"date": "30 Aug 2012",
"name": "Lawrence Kane",
"role": "Author Response",
"response": "Thanks for the feedback on our ms. Could you please clarify what you mean in point #1, regarding which data you think we should interpret more conservatively?"
},
{
"c_id": "66",
"date": "30 Aug 2012",
"name": "Wenxia Song",
"role": "Reader Comment",
"response": "That occurs at several places of the manuscript. A best example is the abstract where it was stated “Tim1-mediated early tyrosine phosphorylation” and “Tim-1 induced NFAT/AP-1 activation”. However, the data from your and other studies only suggest an involvement of Tim1 in the regulation of these activities, but not a direct mediation or induction role of Tim 1 in these activity. I suggest to tone those statements down."
}
]
},
{
"id": "204",
"date": "06 Sep 2012",
"name": "Terry Strom",
"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 findings concerning the role of TIM1 and domains thereof in the trafficking of T cells are original and important.The manuscript is clear and uses powerful tools. I do have minor reservations on the discussion concerning apparent differences between findings in this paper and those reported elsewhere. The authors use a simple reductionist tool kit. Normal T cells are not studied (for reasons that are well justified). Differences in the cell population used in this and the cited works may or may not prove important. Next is the choice reagents to conduct the analysis. In mouse systems there are anti Tim1 antibodies that stimulate and others that inhibit T cell activation. It would be fascinating to learn (not in this paper) whether this is due to effects on Tim1 localization in or opposed to the IS.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-10
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https://f1000research.com/articles/1-24/v1
|
09 Oct 12
|
{
"type": "Research Article",
"title": "Improving educational environment in medical colleges through transactional analysis practice of teachers",
"authors": [
"Marina Rajan",
"Thomas Chacko",
"Thomas Chacko"
],
"abstract": "Context: A FAIMER (Foundation for Advancement in International Medical Education and Research) fellow organized a comprehensive faculty development program to improve faculty awareness resulting in changed teaching practices and better teacher student relationships using Transactional Analysis (TA). Practicing TA tools help development of ‘awareness’ about intrapersonal and interpersonal processes.Objectives:To improve self-awareness among medical educators.To bring about self-directed change in practices among medical educators.To assess usefulness of TA tools for the same.Methods: An experienced trainer conducted a basic course (12 hours) in TA for faculty members. The PAC model of personality structure, functional fluency model of personal functioning, stroke theory on motivation, passivity and script theories of adult functional styles were taught experientially with examples from the Medical Education Scenario. Self-reported improvement in awareness and changes in practices were assessed immediately after, at three months, and one year after training.Findings: The mean improvement in self-'awareness' is 13.3% (95% C.I 9.3-17.2) among nineteen participants. This persists one year after training. Changes in practices within a year include, collecting feedback, new teaching styles and better relationship with students.Discussion and Conclusions: These findings demonstrate sustainable and measurable improvement in self-awareness by practice of TA tools. Improvement in self-'awareness' of faculty resulted in self-directed changes in teaching practices. Medical faculty has judged the TA tools effective for improving self-awareness leading to self-directed changes.",
"keywords": [
"There is an increasing awareness about the importance of educational environment in bringing about effective learning. \"The learning environment created in the training group is crucial in helping trainees take risks while feeling supported\"1. Student’s perception of learning",
"their teachers",
"the learning atmosphere",
"and their academic and social self-perceptions are major components of the DREEM (Dundee Ready Education Environment) questionnaire developed for studying the environment of medical education2. Many medical colleges in India are now taking initiatives to find out remedial measures to be taken to improve the learning environment3."
],
"content": "Context\n\nThere is an increasing awareness about the importance of educational environment in bringing about effective learning. \"The learning environment created in the training group is crucial in helping trainees take risks while feeling supported\"1. Student’s perception of learning, their teachers, the learning atmosphere, and their academic and social self-perceptions are major components of the DREEM (Dundee Ready Education Environment) questionnaire developed for studying the environment of medical education2. Many medical colleges in India are now taking initiatives to find out remedial measures to be taken to improve the learning environment3.\n\nDeveloping trusting relationships is one of the components of a model for self-directed learning1. The need for a comprehensive faculty development initiative in institutions is emphasized among the various strategies that are directed to improve teaching practices in medical schools. Faculty development projects are hence included for Curriculum Innovation Projects in FAIMER Fellowship programs. A \"Comprehensive faculty development, which is more important today than ever before, empowers faculty members to excel as educators and to create vibrant academic communities that value teaching and learning\"4.\n\nComprehensive faculty development could be done by using the Transactional Analysis (TA) theory of personality development (see What is TA). It can be the basis for bringing about faculty development by facilitating their understanding of their own teaching styles by practice of the TA tools. Transactional Analysis belongs to the humanistic school of psychology1. The International Transactional Analysis Association explains \"Educational transactional analysis is used by practitioners working in training centers, preschools, elementary and high schools, universities, and institutions that prepare teachers and trainers as well as in support of learners of all ages to thrive within their families, organizations, and communities.\" (see About TA Transactional Analysis), TA describes a theory of the structure of personality – Ego state (P-A-C-) model (see Key Ideas in TA) and how people communicate with each other based on this structure. Since education is an area of intensive interpersonal processes, practicing self-awareness using this model is known to help develop self-awareness (herein after referred to as ‘awareness’)5. The dynamics of how self-awareness leads to self-directed improvement in teaching is illustrated in figure 1.\n\nA survey conducted among students in our institution to find out their perceptions about the existing system of medical education and suggestions for improvement revealed many lacunae in the teacher-student relationships that form a barrier to learning and thus contribute to a poor learning environment (Marina Rajan Joseph, Student Perceptions about Learning Environment in a New Medical College in Kerala, Unpublished document). In this context, it was felt that an educational intervention study among the faculty to initiate interest among them about the need for an improvement in the learning environment within the institution is necessary. The author chose this as a Curriculum Innovation Project for her FAIMER fellowship.\n\n\nObjectives\n\n1. To increase personal ‘awareness’ about the interpersonal processes that takes place during teaching-learning among medical educators.\n\n2. To bring about a self-directed changes in the behavior of the teachers that makes them more student-centered and thereby contributing to a healthy educational environment.\n\n3. To assess the usefulness of the TA tools for the above purposes.\n\nIt was assumed that an improved self-awareness about the teaching learning processes and teacher student interactions among teachers would bring about a better understanding of lacunae in teacher student relationships and motivate teachers for self-directed corrective measures thereby resulting in an improved learning environment for the students.\n\n\nMethods\n\nThe project was presented to the institutional ethics committee and ratification obtained. Verbal informed consent was obtained from the participants.\n\nAn interventional study without a control group has been used. The author, a trained Transactional Analysis (Edu) trainer with five years of experience in training, conducted a course in TA with focus on intrapersonal and interpersonal processes, for medical faculty members in our college.\n\nSince it was an explorative study all faculty members who volunteered were included in the study.\n\nThe content of the program was designed based on the framework of 101 (Basic) courses in Transactional Analysis7. This included the Okay-Okay philosophy of TA, Structure of personality (the ego states or Parent – Adult – Child model), the Functional Fluency model, a new model in TA developed from the ego state model specifically for educators and trainers8, stroke theory on motivation- a theory which states that ‘Stroking’ or ‘stimulation’ is essential for health and that wherever positive stroking is lacking, there persons may seek negative strokes to affirm their existence-5, passivity theory which teaches about passive behavior which are said to be the barriers to effective problem solving and script theory which teaches about how early childhood decisions affect our style of function as adults.\n\nTwo colleagues trained in TA helped the author with the sessions, data collection and also provided structured and concurrent feedback to the trainer. Their feedbacks on the trainer and participants helped in maintaining objectivity during the training program. The program extended over 12 hours (2 days). The course was conducted ‘contractually’ (negotiating between the expectations of participants and what the trainer got to offer) and using ‘open honest transactions’ (objectively stating what is expected, and appropriate, descriptive rather than prescriptive feed backs) the two principles of TA practice.\n\nThe program was experiential with exercises and practice sessions for developing personal ‘awareness’ through practice of the Ego state tool9, stroking practice and role plays on types of interpersonal transactions. There were many one to one, small groups and large group discussions with opportunity for reflections. All theoretical aspects were illustrated using examples and case studies from everyday processes in Medical education. There participants introspected, shared insights and planned for future action based on insights developed.\n\nStudent’s perceptions about the present system of teaching and suggestions for improvement (students wanted teachers to be friendlier and teach more interactively and from practical experience) were also communicated to the teachers.\n\nSelf-assessment of the participant’s ‘awareness’ before training, immediately after, at three months and one year after the training program were collected using an adapted version of a retro pre-behavior assessment tool available free on the Business Balls website. This included awareness’ in eighteen different areas of interpersonal relations.\n\nThe improvement in awareness was assessed quantitatively using Epi Info. Mean improvement in awareness of the group at three different times after training and mean improvement in awareness about specific areas of behavior were calculated and compared. Program feedback and evaluation on the usefulness of the tool was also collected using a post test questionnaire. Consensus on the usefulness of the TA theory and its tools for improving self-awareness was calculated using the Consensus index10. Self-reported changes in practices have also been quantified.\n\nQualitative analyses of the group discussions and recommendations from action plans have also been included in the discussion.\n\n\nResults\n\nNineteen faculty members of different age and experience participated in the training (Table 1). After 3 months only 12 members and after 1 year only 11 members were available for follow up. Table 2 to Table 5 show the findings in mean improvement in awareness, areas in interpersonal processes where improvement has occurred, consensus on the usefulness of the TA tools and self-directed changes in practices resulting from awareness.\n\n* As can be seen from the 95% confidence limits of each value there is no statistically significant difference between the mean improvements recorded immediately after training, three months later or one year later.\n\n\nDiscussion\n\nThe mean improvement in self-awareness immediately after training persists at three months and one year after training (Table 2). It reflects the lasting quality of the awareness generated which is behavioral, a Kirkpatrick level three benefit (see Kirkpatrick Philosophy). Though other researchers5 have reported improvement in self-awareness after learning TA, there are not many reports on measuring and grading this awareness. In this study the greatest improvement in awareness was reported in three areas namely ‘being aware of my reaction to the behavior of others’, ‘being aware of what behavior modification I need to do’, and ‘knowing how to modify my behavior’ (Table 3). This is a very positive outcome of the training because in improving teacher student relationships, it is these three awareness competencies that help the teacher modulate behavior in the here and now of the teacher student interaction. It is also interesting that as time elapse after training, practice of awareness over a year has consistently improved the faculty’s self-perception about their ‘general interpersonal skills’. Research among student teachers has demonstrated that becoming aware of their own attitudes, beliefs and cognition helped them to achieve specific aims in professional practice5. It has been demonstrated among Russian university students that use of Transactional Analysis by them helped them to gain a high level of knowledge about effective relationships and ability to use this knowledge in their professional and personal lives11. The group’s consensus on usefulness of TA for medical teacher’s training has increased one year after training (Table 4). It probably reflects their personal experience of practicing the tools and perceived benefits thereof. The high degree of consensus among participants who are all doctors, on the usefulness of TA tools for Medical Teachers training, and on the usefulness of the PAC and ‘Functional Fluency’ models prompts future studies on these tools.\n\nDuring discussions the participants connected needs expressed by the students to the stroke theory on motivation. TA research work with College students has shown that greatest growth was reported in the area of stroking12. They identified lack of positive stroking in the medical education scenario as a probable reason for the lack of motivation they observed among many students, particularly considering the fact that in India students come to the medical college straight from schools. Positive stroking and Open Honest Transactions were identified by the participants as the most practical TA tools for improving teacher student relationships.\n\nEducation and psychology has a shared history and \"It has been the humanistic psychologists who have grappled mostly with the problems of learning\"1. She quotes Malcom Knowles who formulated the theory of adult learning and ‘andragogy’, as saying that \"Some of the most important contributions to learning theory have come from psychotherapy\". Carl Rogers famous for his innovative ‘Client-centered therapy’ applied the same to education and said that education is \"facilitation of learning\" and educator is \"facilitator of learning\" thus bringing in a ‘student centered’ approach to education.13. The transition from use of the term ‘Pedagogy’ to ‘Andragogy’ also reflects a transition from teacher-centered education to student-centered education14. The four desirable factors in teachers namely, ‘warmth, enthusiasm, use of discovery-learning methods, and high level of cognitive organization’15 are also qualities of educators who are student centered. Many faculty members in this study have adopted changes in similar practices within one year of attending the Transactional Analysis training (Table 5). It shows that comprehensive faculty development such as using the TA approach fosters development of many of the qualities of student centered among medical educators.\n\nThe participants developed an action plan at the end of the training. They requested more organized and formal training in pedagogical methods along with more inputs on interpersonal skills. They volunteered to organize an effectively functioning Medical Education unit that not only sharpens pedagogical skills but also leads to a better understanding of students, their learning processes and create a favorable educational environment in the institution.\n\n\nConclusion\n\nThese results suggest that practice of the ego state awareness using the PAC model helps to improve self-awareness and sustains it even up to a year after training. This awareness in turn is helping teachers to become aware of their own and student’s behavior in different situations and makes appropriate modifications. This in turn helps them to practice new teaching styles and improve teacher student relationships. Participants reported increasing confidence in the TA tools for medical teacher’s training. It suggests that continued and consistent inputs using the same may remarkably improve personal ‘awareness’ among teachers and thus the teacher-student relationship, leading to an improvement in the educational environment vital for promoting student learning.",
"appendix": "Author contributions\n\n\n\nMarina Rajan Joseph conceived this project, carried it out and wrote it up. Thomas V. Chacko MD refined and reviewed the article.\n\n\nCompeting interests\n\n\n\nThe author is a practicing and training Transactional Analyst and believes it is a useful technique for personal development. She has conducted the training. This may or may not have a bearing on the outcomes of the training and interpretations.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgement\n\nThe authors wish to acknowledge the support in design and conduct by the FAIMER community and financial support of the institutional administration for carrying out the project. We also acknowledge the physical and intellectual assistance rendered by TA practitioners Smt. Mary Koshy Computer Engineer and Smt Anjaly Vijayan Social work tutor in the conduct of the project, data collection and analysis. We are thankful to Drs. Amol Dongre and Ravi Shankar P for the constructive feedbacks on the writing.\n\n\nReferences\n\nGrant J: How the Philosophical Assumptions of Transactional Analysis Complement the theory of Adult Education. Transactional Analysis Journal. 2004; 272–276.\n\nWhittle S, Whelan B, Murdoch-Eaton DG: DREEM and beyond; studies of the educational environment as a means for its enhancement. Educ Health (Abingdon). 2007; 20 (1): 7. [Online] April 17, 2007. [Cited: August 30th 2010.]. PubMed Abstract\n\nReem A, Ramnarayanan K, Vinod P, et al.: Students' Perceptions of Learning Environment in Indian Medical School. BMC Med Educ. 2008; 8: 20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilkerson L, Irby DM: Strategies for improving teaching practices: a comprehensive approach to faculty development. Acad Med. 1998; 73 (4): 387–96. PubMed Abstract\n\nMarja KL, Susannah T: Student Teacher's Professional/Personal Through Academic study of Transactional Analysis. Transactional Analysis Journal. 2004; 253–271.\n\nInternational Transactional Analysis Association: T&CC Training and Examinations Handbook Section 4. The TA 101. Reference Source\n\nTemple S: Update on the Functional Fluency Model in Education. Transactional Analysis Journal. 2004; 34: 197–204. Reference Source\n\nSteiner C, Campos L, Pearl D, et al.: A Compilation of Core Concepts. Transactional Analysis Journal. 2003; 187. Reference Source\n\nDonald AL, Rick H, Martin KM, et al.: The Ego state Questionnaire – Revised. Transactional Analysis Journal. 2004; 90–95.\n\nWilliam JT, Jack R, Mark JW: A New Measure to Analyze Student Performance Using the Likert Scale. Information Systems Education Journal. 2008; 6(35): 3–9. Reference Source\n\nSmischenko O: The use of Transactional Analysis Theory in Teaching University Students the Psychology of Relationships. Transactional Analysis Journal. 2004; 249–252.\n\nSamuel G, Cynthia M, Brown EL: Transactional Analysis in the College Class Room. Transactional Analysis Journal. 2004; 243–248.\n\nRogers CR: Freedom to Learn. Columbus: OH Merrill, 1969; 104–105. Reference Source\n\nConner ML: Andragogy + Pedagogy. (Internet) 1997–2004. [Cited: September 15, 2010.] Reference Source\n\nGage NL: Teacher effectiveness and Teacher training: the search for a scientific basis. Palo Alto, CA: Pacific Books, 1972; 102. Reference Source"
}
|
[
{
"id": "308",
"date": "25 Oct 2012",
"name": "Judy McKimm",
"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 an interesting write up of a research project looking at how to introduce Transactional Analysis (TA) into faculty development activities to improve the learning environment.The methods are sound and relevant to the topic being researched and the findings consistent with other similar studies, giving evidence-based recommendations about using TA to improve practice.",
"responses": []
},
{
"id": "309",
"date": "26 Oct 2012",
"name": "Kirsty Forrest",
"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 describes a practical intervention for faculty development. Faculty development is currently the poor relation within medical education, and this study provides evidence of how to improve student learning by improving educator insight.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-24
|
https://f1000research.com/articles/1-20/v1
|
01 Oct 12
|
{
"type": "Research Article",
"title": "The efficacy of disinfectants on abattoirs’ Candida albicans isolates in Niger Delta region",
"authors": [
"Oluwayemisi A Olorode",
"G Chijioke Okpokwasli",
"G Chijioke Okpokwasli"
],
"abstract": "This study was conducted to evaluate the antimicrobial activities of common disinfectants- these are (parachlorometaxylenol) dettol, savlon purit and jik (sodium hypochlorite) on Candida albicans isolated from displaying and cutting tables in five different abattoirs in Port Harcourt (Niger Delta region); the abattoirs include Trans Amadi, Agip, Woji, Rumuokoro, and Rumuodara. This research was carried out between January 2005 and June 2006. Swab samples were collected from abattoirs cutting tables with sterile swab sticks and immediately transferred and cultured in the laboratory on a selective medium Sabouraud Dextrose Agar (SDA). The disinfectants’ concentrations were prepared at 10%, 20%, 40%, and 70%, in triplicates and the mean values calculated. 0.5 Mc Farland turbidity method of standardization and Agar diffusion method were used for disinfectants testing of the isolates. Statistical analysis of the data showed no significant difference in the effectiveness of these disinfectants at (p<0.05). In conclusion, this study has shown that savlon and dettol were the most potent antimicrobial agents at 10% concentration on Candida albicans isolates when compared with purit and jik in this study, hence they are good sanitizing agents to be applied on the abattoirs cutting tables, before meat products can be displayed for sale.",
"keywords": [
"Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in human1",
"2. The yeast Candida albicans is an opportunist fungal pathogen which can be present as a normal part of body’s microflora3. Candida albicans displays a variety of virulence factors which aid colonization and persistence in the body. One of the most important of these factors is the ability to adhere to host tissue using a variety of mechanisms. The importance of adherence may be illustrated by the ability of Candida albicans to adhere to various mucosal surfaces and to withstand forces that may lead to its removal from the body such as the bathing/washing action of body fluids. A hierarchy exists among Candida species indicating that the more common aetiological agents of candidiosis (C. albicans and C. tropicalis) are more adherent to host tissues in vitro than relatively non-pathogenic species such as C. krusei and C. guilliermondii4. Systemic fungal infections (fungemias) including those by Candida albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g. AIDS",
"cancer chemotherapy",
"organ or bone marrow transplantation). Candida albicans biofilms may form on the surface of implantable medical devices. In addition",
"hospital-acquired infections by Candida albicans have become a cause of major health concerns."
],
"content": "Introduction\n\nCandida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in human1,2. The yeast Candida albicans is an opportunist fungal pathogen which can be present as a normal part of body’s microflora3. Candida albicans displays a variety of virulence factors which aid colonization and persistence in the body. One of the most important of these factors is the ability to adhere to host tissue using a variety of mechanisms. The importance of adherence may be illustrated by the ability of Candida albicans to adhere to various mucosal surfaces and to withstand forces that may lead to its removal from the body such as the bathing/washing action of body fluids. A hierarchy exists among Candida species indicating that the more common aetiological agents of candidiosis (C. albicans and C. tropicalis) are more adherent to host tissues in vitro than relatively non-pathogenic species such as C. krusei and C. guilliermondii4. Systemic fungal infections (fungemias) including those by Candida albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g. AIDS, cancer chemotherapy, organ or bone marrow transplantation). Candida albicans biofilms may form on the surface of implantable medical devices. In addition, hospital-acquired infections by Candida albicans have become a cause of major health concerns.\n\nCalderone (2002) stated that Candida albicans is commensal and a constituent of the normal gut flora comprising microorganisms that live in the human mouth and gastrointestinal tract. C. albicans lives in 80% of the human population without causing harmful effects, although overgrowth of the fungus results in candidiasis (candidosis)5. Candidiasis is often observed in immunocompromised individuals such as HIV-infected patients. A common form of candidiasis restricted to the mucosal membranes in mouth or vagina is thrush, which is usually easily cured in people who are not immunocompromised. For example, higher prevalence of colonization of C. albicans was reported in young individuals with tongue piercing, in comparison to non-tongue-pierced matched individuals6. Long term untreated infection of Candidiasis in women can block the fallopian tubes hence resulting into infertility among couples. To infect host tissue, the unusual unicellular yeast-like form of Candida albicans reacts to environmental cues and switches into an invasive, multicellular filamentous form, a phenomenon called dimorphism1. Candida albicans produces a range of extracellular enzymes that facilitate adherence and/or tissues penetration. Phospholipase A, B, C and lysophospholipase may function to damage host cell membranes and facilitate invasion. C. albicans produces a range of acid proteinases which have been shown to aid adherence and invasion but which also play an important role in the degradation of the immunoglobulins IgG and IgA. The proteinases have a low pH optimum and this may assist the yeast colonization of the vagina, which is a low pH environment. Haemolysin production by C. albicans has also been documented and seems to be important in allowing the yeast access to iron released from ruptured red blood cells7. An important immune evasion tactic of this organism is the ability to bind to platelets via fibrinogen-binding ligands, which results in the fungal cell being surrounded by a cluster of platelets. Candida albicans is capable of giving rise to a variety of inter-convertible phenotypes which can be considered as providing an extra dimension to the existing virulence factors associated with this yeast. A number of switching systems have been identified and phenotypic switching may have evolved to compensate for the lack of variation achieved in other organisms that utilize sexual reproduction. Phenotypic switching allows the yeast to exploit micro-niches in the body and alters a variety of factors (example is antifungal drugs resistance, adherence, extracellular enzyme production), in addition to the actual phenotype and so may be considered as the ‘dominant’ or controlling virulence factor.\n\nAccording to Sheehan et al.8 investigation which stated that in terms of tissue colonization and invasion, adherence is the initial step in the process. Once the yeast has adhered, enzymes (phospholipase and proteinase) can facilitate adherence by damaging or degrading cell membranes and extracellular proteins. Hyphae may be produced and penetrate layers of cells using thigmotropism to find the line of least resistance. The passage through cells is undoubtedly aided by the production of extracellular enzymes9. Once endothelial cells are reached, enzymes may assist in the degradation of tissues and allow the yeast to enter the host’s bloodstream, where phenotypic switching or coating with platelets may be used to evade the immune system. While in the bloodstream the haemolysin may function to burst blood cells and release iron, which is essential for growth. Escape from the bloodstream involves adherence to the walls of capillaries and passage across the wall. This work is thus undertaken to evaluate the antimicrobial effectiveness of common disinfectants on Candida albicans isolated from five abattoirs displaying tables in Port Harcourt, Nigeria. This is to enable us determine the most potent of the test disinfectants to be used to sanitize these tables before any meat product is displayed for sale to avoid contamination of the meat products.\n\n\nMaterials and methods\n\nA selective medium Sabouroud Dextrose Agar (SDA) was used for the cultivation of Candida albicans. The following equipments were used-Microscope, Microscopy slides, cover slips, cotton wool, sterile swab sticks and blood serum, safranin, lugos iodine, gram differentials, crystal violet.\n\nFive different abattoirs (slaughter floor) in an open environment were sampled using sterile swab sticks, within Port Harcourt metropolis, Rivers State Nigeria. The abattoirs include Agip, Woji, Trans Amadi, Rumuokoro and Rumuodara. Samples were collected with sterile cotton swabs moistened with 1ml of 0.1% NaCl peptone solution, from a surface area of preferable 20cm, marked with sterile template and swabbed 10 times from top to bottom10. Then for wet areas dry sterile cotton swabs were used. The swabs were held in sterile forceps and the sampled surfaces were randomly collected every other week for a period of one and a half year starting from January 2005 to June 2006. The samples were taken to the laboratory immediately and cultured.\n\nGrowth media used for enumeration and characterization were selective according to the cultural requirements of fungi. Sarboroud Dextrose Agar (SDA) was used for Candida albicans cultivation and the same agar medium for stock culture. The medium was steam sterilised at 121°C for 15 minutes.\n\nThe spread plate method was used, serial dilutions of the swab sample was made by adding 1.0ml of phosphate buffer into the swab container and mixed thoroughly. From this, serial dilution was carried by adding 9ml of sterile distilled water to give 10-1, 10-2, 10-3, 10-4 and so on. 0.1ml was plated in triplicate onto sterile selective medium mentioned above. The sets of plates were then incubated at 37°C. Resulting pure colonies were transferred onto SDA agar for subsequent characterization and identification.\n\nPure cultures of fungi (yeast) Candida albicans isolated were characterized and identified on the basis of their cultural, morphological and biochemical properties and based on their macroscopic appearance on culture medium, and type of asexual spores produced and germ tube test, also identified by reference to Illustrated General of Imperfect Fungi11 and Fungi in Agricultural soil12. The test yeast used is Candida albicans.\n\nOrganisms used in this experiments were, Candida albicans. Candida albicans were grown in Sarboroud Broth overnight. Cultures were centrifuged at 512g (sigma model 3k-1) for 10mm and resulting cell pellets resuspended in 0.1% peptone.\n\nDisinfectants parachlorometaxylenol (PCMX) (dettol), savlon, purit and Sodium hypochlorite (jik) active were diluted in sterile distilled water prior to use to 10%, 20%, 40% and 70% concentrations these products were obtained from Reckit Benckiser Nigeria Limited Lagos, Nigeria, Johnson & Johnson, Lagos; CAP Plc (Chemical and Allied Products), Lagos respectively and used without further purification. The pH of the mixtures was controlled by the addition of HCL or NaOH as appropriate.\n\nApproximately 0.1ml of a yeast suspension approximately 1 × 109 bacteria (Cfu/ml) was added to the test disinfectants (10ml), mixed thoroughly and left at room temperature for a specified contact time starting from 0 min, 10 min, 20 min, 30 min, 40 min, 50 min and 60 min. This experiment occupied 21 test tubes each, for the experiment was carried out in triplicate. Following contact an aliquot (1ml) was transferred to universal quenching agent, UQA (9ml of a solution containing 1g peptone, 5g. Tween-80, 1g sodium thiosulphate and 0.7g lecithin 1-1 of deionised water pH7), for up to 60 min to inactivate the disinfectants. The quenched solution were serially diluted in sterile distilled water and survivors enumerated on SDA Agar (Oxoid) using 0.1ml spread plates. The colonies on the plates were counted after incubation at 37°C for 48hrs. Control test did not contain the disinfectants, but only the serially diluted suspension was plated and counted.\n\nDettol’s composition include Chlorxylnol B.P.C. 4.8% w/v, Oleum Pini Aromaticum 9.0% v/w Denatured spirits 11.3% w/v, sapo vegetails 5.8% w/v, Saccharum Ustumqs, aqua ad 100vol. Purit contains Chlorhexidine Gluconate B.P. 0.3% w/v and Centrimide B.P. 3.0% w/v. Savlon constitutes n-Propyl alcohol 2.84% m/v, preservative Chlorhexidine gluconate 0.3g and Centrimide 3.0g all in 100ml. Hypochloride contains sodium hypochlorite as an active ingredient.\n\nRideal-Walker Phenol co-efficient experimental test method for the evaluation of test disinfectants as jointly reported by S. Rideal and J.T.A. Walker in 190313 was the first real attempt to evaluate the effectiveness of disinfectants on a qualitative basis. The standard procedure for this test was published by the British Standard Institution.\n\nThe test organism was Salmonella typhi NCTC 786 obtainable from the National Collection of typed cultures in London. It was supplied in the freeze-dried form and reactivated by weekly sub-culture on a Rideal-Walker agar slope, incubated at 37°C for 24hrs and then stored at room temperature until the time for another sub-culture. Only cultures that were 22 to 26 hours old and only those from the 3rd to the 14th subculture were used for the test.\n\nThe standard phenol solution contained 1g of phenol in 95, 100, 105, 110 and 115ml of solutions made with sterile distilled water. For the test disinfectants dettol, purit, savlon and jik, four dilutions were made, varied in arithmetic series, changing by the unit of 50; these are 1:200, 1:250, 1:300 and 1:350.\n\nA volume of 5ml of each of the dilutions of test disinfectants and standard phenol was placed in separate sterile test tubes. A 24 hour broth culture of the test organism was also prepared. The dilutions and the culture were placed in a water bath maintained at 17–18°C. When the contents of the tubes and the culture had attained the operational temperature, 0.2ml of the culture was transferred to each of the dilution and shook gently to begin the action of the germicide on the cells. At 2½ minute interval following the incubation of the tubes, a standard loopful of the reaction mixture was transferred to 5ml of sterile nutrient broth (the recovery medium) in a tube. In this way, each reaction mixture was sub-cultured into four separate test tubes of the recovery medium at intervals of 2½, 5, 7½ and 10 minutes respectively. The tubes of recovery medium were incubated at 37°C for 48 hours after which the presence or absence of growth in each tube was recorded.\n\n\nResults\n\nFigure 1a shows the graph of disinfectants resistant Candida isolates at 10% concentration. From this graph it could be deduced that savlon was the most potent disinfectant to Candida species, followed by purit, then dettol, while Sodium hypochlorite was the least potent disinfectant to Candida isolates. From this result, we can conclude that savlon was the best disinfectant to be used to disinfect Candida contaminants on any inanimate objects.\n\nDisinfectant-resistant Candida isolates at 10% (a), 20% (b), 40% (c) and 70% (d) concentration.\n\nFigure 1b showed the graph of disinfectants resistant Candida isolates at 20% concentration. From this graph, it could be deduced that Candida species, were more sensitive to both savlon and purit than to dettol and jik (Sodium hypochlorite). Candida species were more sensitive to purit and savlon, less sensitive to dettol and least sensitive to Sodium hypochlorite.\n\n\nDiscussion and conclusion\n\nLaboratory testing of disinfectants was carried out by assessing the sensitivity of various Candida albicans strains to biocidal agents using microbial suspension test. Similar observations have been made on bacterial suspensions such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus faecalis, Proteus mirabilis and Escherichia coli on disinfectants14. Disinfectants used for the test are chlorine based and these included dettol, savlon, purit and jik containing sodium hypochlorite.\n\nFrom the results of this investigation, it was discovered that Candida albicans species were readily isolated from raw meat and from the equipments used in abattoirs. Similar observation has been made by Haniel (1989)15 from his investigation and he stated that undetected Salmonella species in instrument or food products such as raw meat could pose a health risk, because they are not heat treated and raw animal tissues may harbor many potentially pathogenic organisms, including bacteria and yeasts e.g. (Salmonella, Escherichia coli, Candida species) and parasites. For these reasons, the Food and Dietary Association, FDA has promulgated a regulation specifying that meat scraps or other similar animal products are adulterated when they are found upon examination to be contaminated with Candida and Salmonella microorganisms. According to the investigation of Kirk (2005)16 from Minnesota Department of Agriculture, title 21 code of Federal Regulations (CFR) Part 500.35 stated that not only are the animals consuming the product at risk of infection by organism contained in the raw tissues, but the people handling the product are also at risk. He then stated that adequate heat treatment is the most effective and efficient means of mitigating this risk. Because he reported some processes currently used, such as freezing or freeze drying, do not achieve the same degree of effectiveness as heat treatment. Candida species were readily isolated from the tables where meat carcasses are displayed. Similar observation was made by dEnfert and Hube (2007)2 where he stated that these fungi and bacteria are potentially fatal to human especially when ingested and when the immune system is compromised. Ingestion of these yeasts in meat products could be very dangerous or when it has a contact with urinogenital tracts, during defecating or urinating, from contaminated hands and fingers. These fungi cause vomiting and diarrhea, many ailments such as urinary tract infection, even the long term effects in the tracts could result to blockage of fallopian tubes in women and sterility in men. This could be severe in immunocompromised infected people, while healthy people can recover from these infections. Symptoms of candidiasis usually occur within two to ten days of ingesting the yeast (Cotter and Kavanagh, 2000).\n\nCandida albicans strains were isolated in number virtually from all the studied samples which are cutting tables. Similar observation was made by Gardner (1993)17, and dEnfert and Hube, (2007)2. The authors worked on the importance of Quality Assurance and Food Safety in Modern Food Production System, they stated that in countries that have implemented a consistent mandatory meat inspection, this harvest food safety procedure and the more and more stringent rules for post-harvest safety measures improving the hygiene standards during slaughter, meat process and distribution have led to a remarkable decline of meat related food-borne disease. And in respect to meat management, it is advised that any animal that arrives at the abattoir should be slaughtered immediately, but animals which have not been slaughtered within 12 hours of the arrival should be fed, and subsequently be given moderate amounts of food at appropriate intervals. Also suitable drinking water should be available to the animals on their arrival and at all times to animals in lairages unless they are to be slaughtered immediately. There has been criticism of the methods of preparation hutching, and killing within some slaughter houses and in particular of the speed with which the slaughter is sometimes conducted18–21 (also see PETA video taken inside AgriProcessors Inc. in Iowa in 2004. Warning: graphic images). There has also been criticism of the methods of transport of the animals, who are driven for hundreds of miles to slaughter houses in conditions that often result in crush injuries and coma en route, a good condition for microbial contamination and infection22,23. Vibrio cholera the causative agent of cholera, is sensitive to the low pH found in the human stomach, a high infectious dose of 108 bacteria is required for the onset of severe cholera; however the infectious dose can drop to 104 bacteria in individuals who produce less stomach acid including young children, the elderly and those who take antacids.\n\nFrom the overall report of this study, dettol at 10% is an appropriate disinfectant or sanitizer that can be used to sanitize the abattoirs cutting tables before any meat products are displayed for sale and should be rinsed off with sterile and clean water after 10 minutes of application. The need for good quality water becomes more and more pressing in the face of the increasing pollution of our water bodies by human activities such as those related to abattoirs services, agriculture and industries. Good water is essential for washing the slaughtered animals, displaying tables, equipments use such as knives, apron, slaughter floor, besides animals themselves need portable water for drinking before slaughter while kept in lairage (see: \"Molecule of the Month: Dettol\" and \"'Dettol Man’ cleans himself to death\"). Revankar and Lester (2007)24 stated that overuse of dettol could provide a selective environment in which existing resistant bacteria can grow with little competition. In Nigeria, there is a need for the receiving streams around these abattoirs to be treated because people living in these environments defecate into the water bodies, this act should be discouraged since these are flowing rivers and could serve as sources of drinking and cooking to neighboring villages nearby. This could be the cause of high Vibrio isolates in these abattoirs.\n\nThis law of meat inspection makes food safer and is the principle of food hygiene being the most important means of protecting consumer against food-borne health risks, in order to reduce food infection. Nevertheless, the receiving streams around these abattoirs should be treated because people living in abattoirs environment defecate inside these water bodies, this act should be discouraged since, they are flowing rivers and could serve as sources of drinking water to neighboring villages where their flow goes. This may be one of the reasons why greater numbers of Candida species were isolated from these study abattoirs.\n\nThe federal government and local government should be blamed for these vices, because the abattoirs’ chairman, the butchers, the sellers and the inhabitants of these areas have the propensity to live in such a stench environment due to lack of both intuitive and acquired knowledge in boosting the standard of hygiene in abattoirs. Therefore, the government should enforce a legislative law with respect to the standard of hygiene that should be practiced among the abattoirs users, and a circular should be passed round in such a way that everybody should be a participant and there should not be a room for exculpation by any member whatsoever. And anyone that violates this rule should be brought to face the music.\n\nIt is advised, due to the overall report of this study, Candida albicans is more sensitive to savlon than to other test disinfectants in Port Harcourt abattoirs, hence savlon should be recommended as a potent sanitizer on the abattoirs’ displaying tables and in Nigeria as a whole. Savlon was recommended for destroying Candida albicans contaminants within 10 to 20 minutes of exposure to this disinfectant. Phenol co-efficient experimental calculation showed that phenol was less potent than dettol having phenol co-efficient of 7, on Salmonella sp. 2 for Dettol on Pseudomonas sp. but having 1 on Pseudomonas sp. purit, and savlon were more potent than phenol having phenol co-efficient of 1.7, 2 on Salmonella sp. and Pseudomonas sp. respectively, but equal in phenol co-efficient with phenol on Staphylococcus sp. of 1. According to the investigation of De bruin (1976)25 who stated that phenol is generally protoplasmic poison and was the first antiseptic to be employed by Joseph Lister (1912–1927) so it does not make it more potent than the newly produced antiseptics or disinfectants due to the development science and technology. Though, Keswick et al. (1985)26 stated that chemical agents used as germicides destroy microbial pathogens using a variety of biochemical mechanisms, including cell membrane.\n\nThe results of this study are in agreement with that of Le Chevalier et al. (1998)27 which stated that pathogens resistance to chemical disinfectants is typically an inherent characteristic and not one that is acquired or initiated due to exposure to disinfectants. He also stated that mechanisms of resistance of microorganisms have evolved with specialized structures aiding in their survival. Certain Candida albicans strains produce a gelatinous material known as proteins to form a biofilm. Biofilm help organisms stick to environmental surfaces and physically protect them from exposure to harmful disinfectants or other detrimental environmental conditions28. This slimy substance can be 100 or more times the mass of the bacterial cells. While the bacteria that initiate biofilm are often not harmful, pathogenic bacteria may also stick to the biofilm and share the protective nature of the environment Hardie et al. (2009)29. Candida species was very sensitive to most of the tested disinfectants in this study. These sensitivity and resistant attributes exhibited by these microbial isolates may be due to some of the innate characteristics stated above. The high incidence of water and food borne sanitation diseases is one of the major problems confronting our country, Nigeria, due to unhygienic abattoir environments.\n\nIn conclusion, the result of this research has shown that for our meat products to be free from Candida contaminants, savlon is an effective sanitizer on abattoirs cutting tables and should be applied at 10% concentration for 10 minutes and then rinsed off with sterile water before meat products are displayed for sale. This automatically reduces the yeast load on these objects.\n\nThe following recommendations are made as follows:\n\nThe federal government should set up a group of Environmental Health workers which will comprise of Environmental Microbiologists, Veterinary Doctors, Medical Doctors and Medical Laboratory Scientists that would play a significant role in the promotion of the health of the people. Adequate sanitation and safe water are fundamental prerequisites for Nigeria abattoirs as a whole for better health, otherwise other programs may not succeed and there can be lasting improvement of public health. These should be vigilantly implemented by these personnel listed above including sanitary engineers and inspectors, and should be directly involved in this exercise.\n\nIt was discovered from this study that every abattoir has a receiving stream and this stream or river could serve as a source of drinking water to some rural dwellers around the vicinity, hence the butchers and other inhabitants in abattoirs defecate into this same river. The government should abrogate these vices by providing good toilet facilities for them, for water quality control, proper excreta disposal and Food sanitary inspection should be practiced (Food Law internet Project (FLIP) 2000. Food safety Administration. Authorized information provided to the WHO. University of Reading UK, Michigan State University, U.S.A)30.",
"appendix": "Author contributions\n\n\n\nOAO wrote the article. GCO was OAO’s PhD supervisor and assisted in the provision of some of the materials, such as glassware etc. which was used in the 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 thank Rotimi Gbolahan Olorode for his financial support and for providing a good atmosphere during the computerization period of the work. Appreciations go to the Managing Director and the staff of Medi Test Medical Laboratory, Port Harcourt Office, Rivers State, Nigeria. We also thank the chairman and members of the Association of Slaughterhouse operators (Port Harcourt branch) for the opportunity to collect the necessary samples used for the study.\n\n\nReferences\n\nRyan KJ, Ray CG (editor): Sherris Medical Microbiology (4th ed). McGraw Hill 2004. Reference Source\n\nd'Enfert C, Hube B (editor): Candida: Comparative and Functional Genomics. Caister Academic Press, 2007; 5. Reference Source\n\nCotter G, Kavanagh K: Adherence mechanisms of Candida albicans. Br J Biomed Sci. 2000; 57(3): 241–249. PubMed Abstract\n\nBennett JW: Mycotechnology: the role of fungi in biotechnology. J Biotechnol. 1998; 66(2–3): 101–107. PubMed Abstract | Publisher Full Text\n\nCalderone RA (editor): Candida and Candidiasis. ASM Press, USA pages 4 2002. Reference Source\n\nZadik Y, Burnstein S, Derazne E, et al.: Colonisation of Candida prevalence among tongue-pierced and non-pierced immunocompetent adults. Oral Dis. 2010; 16(2): 172–175. PubMed Abstract | Publisher Full Text\n\nRossoni EM, Gaylade CC: Comparison of sodium hypochlorite and paracetic acid as sanitizing agents for stainless steel food processing surfaces using epifluorescence microscopy. Int J Food Microbiol. 2000; 61(1): 81–85. PubMed Abstract | Publisher Full Text\n\nSheehan DJ, Hitchcock CA, Sibley CM, et al.: Current and emerging azole antifungal agents. Clin Microbiol Rev. 1999; 12(1): 40–79. PubMed Abstract | Free Full Text\n\nLewis RE, Klepser ME: The Changing face of nosocomial Candidemia: Epidemiology, resistance and drug therapy. Am J Health Syst Pharm. 1999; 56(6): 525–533. PubMed Abstract\n\nDownes FP, Ito K: Compendium of Methods for the Microbiology examination of foods. 4th Edition. America Publisher: Health Association, Washington 2001. Reference Source\n\nBarnett HL, Hunter BB: Illustrated General of imperfect Fungi, (3rd edn). Burgess Publishing Company Minneapolis page 331 1972. Reference Source\n\nDomsch KH, Gams W, Hudson PS: Fungi in Agricultural soils. Longman, London: page 219 1972. Reference Source\n\nGerald R: The testing of disinfectants. Int Biodeterior Biodegradation. 1998; 41: 269–272. Publisher Full Text\n\nLambert RJ, Johnson MD, Simons EA, et al.: Disinfectants Testing: Use of Bioscreen Microbiological Growth Analyser for Laboratory biocide screening. Lett Appl Microbiol. 1998; 26(4): 288–92. Unilever Research, Colworth Laboratory, Shambrook, Bedfordshire, UK. PubMed Abstract | Publisher Full Text\n\nHaniel EL: Liquid Chemical Germicides. University of Tennessee, Knoxville, TN 1989.\n\nKirk E, Smith DWM: Bacteria in Raw Meat, page 2 Good Medicine Publish, USA 2005.\n\nGardner JF: Antiseptics and Disinfectants. Applied Science Publishers Ltd, London pages 2–3 1993. Reference Source\n\nStevenson P: Animal welfare problems in UK slaughterhouses. Compassion in World Farming 2001. Reference Source\n\nTorres B: Making a Killing The Political Economy of Animal Rights pages 3–4, AK Press 2007. Reference Source\n\nEisnitz GA: Slaughterhouse. Prometheus Books 1997. Reference Source\n\nHershaft A: Slaughterhouse Review of Gail Eisnitz’s Slaughterhouse, pages 46–48, Prentice-Hall, Inc., USA 2008.\n\nVansickle J: Quality Assurance Program Launched. National Hog Farmer, February 15, 2002, which reports that each year 420,000 pigs are crippled and 170,000 killed during transport to slaughterhouses, cited in Williams, Erin E. and De Mello, Margo. Why Animals Matter, Prometheus Books, 2007, page 9 2002. Reference Source\n\nHelen E, Mary A: Microbial Resistance to Disinfectants. A.R. Kelly (ed). 1989; 44. : 4. Arizona University Press, Tucson.\n\nRevankar SG, Patterson JE, Sutton DA, et al.: Disseminated phacohyphomycosis: Review of an emerging mycosis. Clin Infect Dis. 2002; 34: 467–76. PubMed Abstract | Publisher Full Text\n\nDe bruin DB: Mechanism of toxicity for various compounds. J. Kenneth (ed.). A Bacterial Bioassay for Assessment of Waste Water Toxicity, 1976; 383–390.\n\nKeswick BH, Satterwhite TK, Johnson PC, et al.: Inactivation of Norwalk virus in drinking water by chlorine. Appl Environ Microbiol 1985; 50(2): 261–4. PubMed Abstract | Free Full Text\n\nLe Chevallier MW, Cawthon CD, Lee RG, et al.: Inactivation of biofilm bacteria. Appl Environ Microbiol. 1998; 54(10): 2492–9. PubMed Abstract | Free Full Text\n\nCornelis P: Pseudomonas: Genomics and Molecular Biology 1st ed. Caister pages 11–13, Academic Press; London 2008. Reference Source\n\nHardie H, Cornelis P: The Secreted Proteins of Pseudomonas aeruginosa: Their Export Machineries, and How They Contribute to Pathogenesis’. Caister Academic Press, 2009; 42–44, 978–979.\n\nFood Law Internet Project (FL.IP). Food safety Administration. Authorized information provided to the W.H.O. University of Reading UK, Michigan State University, U.S.A, 2000; pages 4–6. Reference Source"
}
|
[
{
"id": "301",
"date": "20 Nov 2012",
"name": "Ifeoma Enweani",
"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": "300",
"date": "08 May 2013",
"name": "Astrid Mayr",
"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 think this is an interesting manuscript which provides good information about basic hygienic issues, like disinfection and prevention of infection.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-20
|
https://f1000research.com/articles/1-17/v1
|
27 Sep 12
|
{
"type": "Research Article",
"title": "Identifying spatial priorities for protecting ecosystem services",
"authors": [
"Gary W Luck",
"Kai MA Chan",
"Carissa J Klien",
"Kai MA Chan",
"Carissa J Klien"
],
"abstract": "Priorities for protecting ecosystem services must be identified to ensure future human well-being. Approaches to broad-scale spatial prioritization of ecosystem services are becoming increasingly popular and are a vital precursor to identifying locations where further detailed analyses of the management of ecosystem services is required (e.g., examining trade-offs among management actions). Prioritization approaches often examine the spatial congruence between priorities for protecting ecosystem services and priorities for protecting biodiversity; therefore, the spatial prioritization method used is crucial because it will influence the alignment of service protection and conservation goals. While spatial prioritization of ecosystem services and prioritization for conservation share similarities, such as the need to document threats and costs, the former differs substantially from the latter owing to the requirement to measure the following components: supply of services; availability of human-derived alternatives to service provision; capacity to meet beneficiary demand; and site dependency in and scale of service delivery. We review studies that identify broad-scale spatial priorities for managing ecosystem services and demonstrate that researchers have used different approaches and included various measures for identifying priorities, and most studies do not consider all of the components listed above. We describe a conceptual framework for integrating each of these components into spatial prioritization of ecosystem services and illustrate our approach using a worked example for water provision. A fuller characterization of the biophysical and social context for ecosystem services that we call for should improve future prioritization and the identification of locations where ecosystem-service management is especially important or cost effective.",
"keywords": [
"conservation policy",
"ecosystem-service management",
"ecosystem-service policy",
"ecosystem-service prioritization",
"spatial prioritization"
],
"content": "Introduction\n\nEcosystem services (ES) are vital for human well-being1. Much attention has been devoted to mapping and quantifying ES to achieve the dual goals of protecting biodiversity and human well-being. A growing number of broad-scale mapping studies aim to identify priority regions for conducting more localised place-based management of ES [e.g.,2–5]. Place-based management requires intensive collection of detailed socio-economic and biophysical data, and close collaboration with stakeholders for effective decision making6,7. Given limited resources and information, and increasing threats to ecosystems, it is not possible to do these comprehensive analyses everywhere in a timely manner. We argue that there is currently an under-appreciated, but vital role for spatial prioritization of locations in which place-based management should occur so that attention is focussed on those locations where resource investment will yield the greatest return for human well-being. Indeed, data are deficient in most locations for informing comprehensive and accurate analyses of trade-offs in ES management, and spatial prioritization is a crucial precursor to attempting such trade-off analyses so that data mining efforts occur in the most critical locations. Moreover, prioritization is essential because much ES management is conducted by government or non-government organizations (NGOs) that could potentially operate in many places.\n\nGiven the important role that broad-scale prioritization can play in guiding decisions about where to conduct place-based ES management, a critical assessment of current prioritization approaches is warranted. Some schemes for identifying spatial priorities for managing ES are simple characterizations of biophysical processes and social demand, with little consideration of important information such as the availability of alternatives to ES for meeting human needs, threats to service provision, and the costs of management actions. Although fundamentally different to spatial prioritization for biodiversity conservation, spatial prioritization of ES may be guided by some of the key principles of the former. Spatial prioritization for conservation is well established and may be applied at coarse (e.g., biodiversity hotspots or priority ecoregions;8) or fine scales, identifying locations or actions in locations that are relatively more important for protecting biodiversity than other actions or other locations9. As with spatial prioritization of ES, spatial prioritization for conservation may help to identify locations where more detailed systematic conservation planning should be conducted, and is just one component of the planning process10,11.\n\nSpatial prioritization of ES differs from spatial prioritization for conservation because ES are valued primarily for their worth to humans, can be transferable across space (may not need to be protected at a specific location), are sometimes substitutable by human engineering, and service beneficiaries define the success of management actions. Yet, as with spatial prioritization for conservation, spatial prioritization of ES can guide decsions about local-scale planning and inform the allocation of resources from management agencies (e.g., World Wildlife Fund;12). Moreover, spatial prioritization for conservation is a useful starting framework for ES prioritization because the former is well entrenched in planning discourse13 and yields valuable lessons for ES management14.\n\nCurrent approaches to identifying spatial priorities for managing ES apply different prioritization methods (see Table 1), and developing more consistent and comprehensive methods is an important goal for future prioritization studies. We review past approaches to spatial prioritization of ES, identifying key aspects that should be considered in future analyses. At appropriate places we discuss the relevance of spatial prioritization for biodiversity conservation to spatial prioritization of ES because certain aspects, such as accounting for costs and threats, are common to both. We then demonstrate the importance of these aspects through a conceptual framework for prioritization that outlines an approach for managing the most vital ES for the least cost where they are most needed15. We illustrate the framework with a worked example using the ES of water provision. Egoh et al.14 reviewed the extent to which ES were included in conservation assessments (≈ identifying spatial priorities). Our work differs from Egoh et al. by assessing how ES priorities have been identified and how methods for prioritization should be improved. It also complements discussions of other aspects of ES management such as how to operationalize ES on the ground16, developing appropriate payments for services schemes (e.g.,17,18) or how to manage service provision at specific sites [e.g.,19,20].\n\nShown are the ecosystem services included in the study and how the authors expressed supply/benefits, demand, threats, costs or availability of alternatives to service provision. Blank cells represent a lack of information. A consistent typology for ecosystem services is not presented in the table because we have presented the ecosystem-service labels that were used in the original study.\n\nTable 1 : Notes\n\n1. Holland et al.48 used four indicators of river status – environmental quality index, taxon richness, habitat quality assessment and habitat modification index – to represent the capacity of river systems and catchments to provide freshwater ecosystem services. The authors argue that changes in the value of these indices reflect changes in the capacity of river systems to provide services such as maintaining water quality, controlling sedimentation and erosion, mitigating floods, cycling nutrients, and filtering pollutants.\n\n2. Carbon stored in soils and vegetation. The authors conducted analyses at different grain sizes (4 km2 and 100 km2) and different spatial extents (Britain/England and 100 × 100 km squares across Britain) and examined variation across regions within Britain.\n\n3. Annual income.\n\n4. The gross margin is the value of outputs minus variable costs and subsidy payments.\n\n5. Recreational use of the countryside.\n\n6. The number of day leisure visits as a measure of the recreational value of particular rural locations (this measure could be interpreted as the demand for recreational services).\n\n7. Amount of carbon sequestered each year.\n\n8. Nitrogen and phosphorus removed in particular landscapes.\n\n9. Capacity of land to retain sediment.\n\n10. Combining information on nest sites, floral resources and bee flight ranges to estimate pollinator abundance and likely visitation to agricultural areas.\n\n11. The authors set targets to address the issue of demand (e.g., capturing 50% of total carbon stored in an ecoregion).\n\n12. Costs are represented by the suitability of areas for conservation based on numerical values that reflect the degree of impediments to conservation success. For carbon storage it is a flat cost; the area of the planning unit.\n\n13. Averted risk of extreme floods.\n\n14. The fraction of total flood control value, as a function of the number of housing units in the floodplain.\n\n15. Production of forage for grazing rangeland stock.\n\n16. Dollar value of forage production.\n\n17. The target was 75% of forage production value.\n\n18. The sum of weighted values associated with developed land, agriculture, road density and length of human-induced patch edges.\n\n19. Provision of recreation opportunities.\n\n20. Quantity of suitable habitat in addition to accessibility issues and rights to access.\n\n21. A baseline target (assumed minimum requirement) of 12 days of outdoor recreation per person per year.\n\n22. Crop pollination by natural pollinators.\n\n23. The dollar value of agricultural crops benefitting from pollination.\n\n24. 75% of feature value across the ecoregion.\n\n25. The supply of fresh water.\n\n26. 40% of total freshwater use.\n\n27. The authors pursued two approaches, a target-based approach and incorporating ecosystem services as extra costs or benefits in the cost layer.\n\n28. This is a species-based approach so the priorities are based on species and their distribution across the landscape.\n\n29. For example, positive or negative economic value.\n\n30. The magnitude of threats affecting each species based on major land uses. The loss of a species is equivalent to the loss of the service(s) that species provides.\n\n31. Median annual simulated run-off.\n\n32. Groundwater contribution to surface run-off.\n\n33. Hotspots mapped as areas with severe erosion potential and vegetation and litter cover of at least 70% where maintaining the cover is essential to prevent erosion.\n\n34. Soil depth and leaf litter.\n\n35. The authors assessed various scenarios for capturing ecosystem services based on incidental protection through the conservation of biodiversity or the inclusion of spatially explicit data on service distribution using Marxan. In Egoh et al.40, the authors set different target thresholds for capturing certain percentages of service provision for surface water supply, water flow regulation, carbon storage, soil retention and soil accumulation.\n\n36. The authors estimated the amount of each ecosystem service provided by vegetation types under intact and degraded conditions. Measuring the difference between the two is indicative of the threat of degradation to service provision.\n\n37. The cost of conserving a planning unit was equivalent to the value of irrigated cropping or grazing. The opportunity costs of conservation were addressed in terms of lost production. The authors included spatial variability in costs because values are per planning unit. In Egoh et al.40, catchment area is used as a cost layer (larger areas = greater cost).\n\n38. By natural vegetation.\n\n39. The authors examined the relationship between fodder provision and stocking rates to determine the stocking rates that can be implemented without degrading the environment (i.e., sustainable stocking rates). Hence, over-stocking is considered implicitly as a threat to vegetation condition.\n\n40. Groundwater recharge.\n\n41. For example, for flood mitigation. The authors also examined opportunities for service enhancement.\n\n42. Incorporating the density of people who rely on the service (beneficiaries) as density per watershed, and the water–production efficiency as water supply divided by area of watershed.\n\n43. Water supply relative to demand adjusted for the need to redistribute supply within watersheds. Watersheds were supply does not (or only just) meets demand were prioritized.\n\n44. Amount of vegetation cover and rate of vegetation loss with mid-range values designated as priorities.\n\n45. A proxy was used representing resource and maintenance costs (e.g., land acquisition, infrastructure and labour) and considering watershed-level measures of income, population size and area.\n\n46. Financial capacity to pay for alternatives to service provision such as dams and filtration plants.\n\n47. Includes the trade-off between a high level of flood activity (number of floods, duration of floods and area affected) and a high level of impact on human populations (deaths and displacement, and human population density in watershed), and the costs of service protection.\n\n48. As a proportion of all land. The authors examine also the opportunities for service enhancement through landscape restoration.\n\n49. The authors used expert opinion to estimate possible land transformation within the next 5 years. This identified negative and positive changes to service provision.\n\n50. Based on stakeholder preference.\n\n51. The inclusion of stakeholders in the ranking process addresses to a degree the demand for services and/or the value of services to beneficiaries. This is an explicit incorporation of beneficiaries in the process.\n\n52. Based on land management and stakeholder perception.\n\n53. The authors compared the ecosystem-service values to the cost of conserving the natural habitat that underlies their provision. The opportunity cost was calculated as the expected agricultural value of each forested parcel of land.\n\n54. To estimate the economic value of bushmeat the authors used the local market price of a kilo of beef since domestic meat is a possible substitute for bushmeat. This approach implicitly recognises alternatives to service provision.\n\n55. Value for new pharmaceutical products.\n\n56. The authors assumed imminent deforestation outside of core protected areas.\n\n57. Net annual rate of atmospheric carbon added to existing biomass carbon pools (measured using a proxy).\n\n58. The authors’ maximized service provision for a given ecoregion area constraint using optimization methods. Incorporating the issue of area constraints addresses costs, and the maximization goal gets somewhat at demand.\n\n59. Annual production of livestock from grazing on unimproved natural pastures (expressed as tons of meat).\n\n60. Beneficiaries were at the point of production only (where economic benefits are realized). The authors identified production peaks of water provision and grassland production in densely populated biodiversity hotspots, indirectly addressing the issue of spatial variability in demand.\n\n61. Water availability and water use.\n\n62. Only the key points are captured here, see the publication for full details.\n\n63. Scenario analyses explore implications of possible future landscape changes.\n\n64. Estimated through soil loss. Regions with lower potential soil loss were a priority, which implicitly recognises the importance of threats.\n\n65. Water-supply function and flow regulation (mean annual catchment runoff and mean annual groundwater recharge).\n\n66. Identified beneficiaries in the biome through a literature review and expert consultation.\n\n67. Mean carrying capacity of the land incorporating climate, soil type and vegetation.\n\n68. Areas that tourists can see within a 10 km buffer surrounding the major tourist driving routes (see Reyers et al.55).\n\n69. Landslide, flood and drought prevention functions.\n\n70. Landslide prevention considered in terms of landslide hazard; the more hazardous an area the more important it is to keep forest in place (an alternative perception of ‘demand’). Drought and flood prevention reflects water retention capability of forest.\n\n71. Estimated using the proximity to settlements and roads (measures of access for deforestation), and distribution of the number of commercial species of trees (a measure of forest desirability for logging).\n\n72. Carrying capacities for domestic stock expressed as the number of hectares required per large stock unit (hectares values were determined for pristine examples of habitat types).\n\n73. The authors compared the potential delivery of ecosystem services from ‘pristine’ locations to that provided by degraded locations, estimating how landscape degradation may diminish the capacity of locations to provide a given service (an indirect assessment of threat).\n\n74. The authors mapped areas vulnerable to erosion and classified them as high, medium and low erosion hazard. Habitat types provide erosion control where there is a high threat of erosion owing to factors such as topography, rainfall and soil (indirectly addressing the issue of threat).\n\n75. Millions of cubic meters of groundwater recharge per 1-km2 grid cell.\n\n76. A related study by Wendland et al.18 included costs, threats and demand, but it is unclear if these are included in the measure of hydrological importance used in Rogers et al.31.\n\n77. Provision of drinking water to downstream users and irrigation for rice paddies.\n\n78. The authors examined the threats to the biological value of key biodiversity areas (KBAs) based on a ‘human pressure index’ calculated from measures of human population density, road density, fire prevalence and agricultural suitability. They did not directly examine threats to ecosystem-service provision, but did this indirectly by looking at threats to the protection of KBAs, which were ranked based on their hydrological service value.\n\n79. The carbon density of living biomass.\n\n80. The number (and type) of services is a little ambiguous; it appears to be between 9 and 13 depending on the analysis. The authors also conducted analyses at three different spatial scales.\n\n81. Ecosystem service values were expressed in dollar values of land units based on land cover and the services provided by particular land covers.\n\n82. The authors deal with threat(s) to service provision indirectly by modelling the change in ecosystem service value with two alternative development scenarios.\n\n83. The authors calculated the ecosystem-service values ($ value) for 17 different services and recognised variation in the spatial dependencies of services.\n\n84. The authors assessed the vulnerability of biodiversity (‘threat’) and then determined the ecosystem-service value captured in biodiversity templates where low vulnerability is a priority and high vulnerability is a priority.\n\n85. The authors calculated water availability (total and per person) and mapped supply and demand ratios.\n\n86. Water availability per person was referenced against an accepted minimum target (1000 m3) set by the United Nations (hence, this target represents ‘demand’). The authors also calculated the percentage of the population with access to improved water and improved sanitation, and under five mortality per 1000 births.\n\n87. The percentage contribution of carbohydrate and protein-supplying crops to total dietary intake.\n\n88. Service provision is compared to recommended minimum daily intake (2100 kcal per person) and minimum daily intake of protein.\n\n89. Lost agricultural production.\n\n90. Opportunity costs for agriculture and stock.\n\n91. The authors did not calculate water quantity, but used a proxy for the supply of sediment-free water based on population data, land cover and water flow direction.\n\n92. The authors measured downstream users through the downstream populations’ need for quality drinking water, downstream area of irrigated rice fields, and downstream area of mangroves.\n\n\nComponents of spatial prioritization\n\nThe following are key elements to any conservation prioritization problem: biodiversity features [assets] that need protection (e.g., species or habitats); processes that threaten these features (e.g., habitat loss); a set of actions that may be effective at abating the threats (e.g., manage invasive species); and financial information specifying the cost of implementing each action, and the available conservation budget11. ES prioritization shares these elements; that is, identifying ecosystem features that supply services, threats to service provision, potential actions to ensure future supply of services, and the costs of these actions. Yet, prioritization of services requires at least the following additional considerations: the availability of alternative means of providing benefits supplied by services; the capacity of an ES to meet human demands; and scale of, and site dependency in, the delivery of services.\n\nWhile each of these factors may contribute to the economic valuation of an ES (i.e., captured by a metric such as dollar value) such complete and site-specific economic values are rare. Studies that estimate the financial value of ES facilitate the appreciation of services in widely understood terms, but this approach has well recognised limitations including the fact that financial values under-represent benefits to the poor as they have less capacity to pay than rich people21–23. Therefore, it is important to explore alternative approaches to identifying spatial priorities for ES management that circumvent some of the limitations of using financial values.\n\nQuantifying the benefits of protecting the supply of ES is generally most appropriately assessed in terms of the difference between protecting supply and not protecting supply. The advantages of protecting ES supply may be represented as benefits expressed in dollar values or avoided ecosystem damage (e.g., prioritizing locations with high soil erosion potential, but where vegetation cover ensures soil retention;24), or through quantifying the supply of services, often in biophysical units. The latter is the most common approach in broad-scale prioritization studies (Table 1). Biophysical quantities can include, for example, the amount of carbon stored in particular ecosystem types, water availability or supply, or fodder production. However, it is crucial to address also the issue of the level of biophysical quantity demanded by service beneficiaries. We refer to the level of human need for a service as ‘demand’, but recognise that this level changes with context and differs from the economic perspective of demand as the amount of a good or service that can be purchased at a given price.\n\nSimply increasing the quantity of a given service may/may not be appropriate depending on human need. It could also divert funds from more necessary actions because if the quantities of certain ES are adequate and not under threat, investment in the protection of these services could be a lower priority compared to services currently unable to meet human needs (see ‘Target setting and the capacity to meet demand’). Luck et al.15 explicitly addressed this issue by prioritizing locations for managing ES based on the human need for the services of water provision and flood mitigation. This directly links the quantity of service provided with the needs of beneficiaries and better identifies where needs are not being met.\n\nThe benefits of managing for ES vary across space and time, reflecting, for example, variation in human need and the capacity to pay for human-derived alternatives. This spatio-temporal variation is decidedly complex, influenced by factors such as the type of service being considered, market fluctuations, and the changing needs of beneficiaries. This dynamism magnifies the complexity of ES prioritization beyond that of biodiversity prioritization. For example, Wilson et al.11 note that the benefit-protection function in conservation planning is asymptotic in that benefit accumulation is less and less with the protection of more land. While the same is true for some ES25, the shape of the curve will vary over time and space with beneficiary demand driven by, among other things, markets and changing needs. Moreover, owing to global markets, it can be extremely difficult to identify who benefits from a given service. It is less problematic to focus on the immediate beneficiaries of service provision (e.g., growers benefiting from crop pollination) rather than also considering those individuals that benefit from the products of services (e.g., consumers of crop commodities;26). In some cases, it may be sufficient to recognise simply that the benefits from the provision of a particular service are globally widespread and diffuse (e.g., carbon storage).\n\nConservation planners may quantify threatening processes that increase the risk of biodiversity loss27 and a similar focus on threats to ES provision is an appropriate way to incorporate threats into service prioritization. It is also important to recognise the fundamental difference between the vulnerability of an ES to threat(s), and the level of threat a particular service is under. Some services may be particularly vulnerable to threats (e.g., crop pollination reliant on a single pollinator species), but not currently threatened, whereas other services may be resilient to a range of threats, but at risk of decline owing to the magnitude of threat(s).\n\nDespite its importance, few ES prioritization schemes to date have explicitly incorporated threats (Table 1). Egoh et al.3 documented biophysical quantities of ES provided by intact and degraded vegetation, which implicitly addresses threat to service provision through landscape degradation. Others examined changes in quantities or dollar values of services through modelling alternative future land-use scenarios, recognising that some scenarios (e.g., extensive development) represent a greater threat to service provision than others28–30. A more explicit approach to incorporating threats is to document the likelihood of decline or loss of service-providing ecosystems through, for example, human development or habitat loss18,31.\n\nAddressing threats to ES is most important when service provision is not substitutable across space (i.e., site dependency is high because the service must be provided in a specific location; e.g., storm protection), there are no human-derived alternatives to service provision or these alternatives are expensive relative to the capacity of local communities to pay for the alternatives, or ecosystem changes are irreversible (e.g., species extinction).\n\nConservation planners list a variety of costs that should be considered when assessing options for protecting biodiversity32. These range from acquisition costs (e.g., purchasing land for conservation) and management costs (e.g., maintaining conservation areas), through to social costs (e.g., the number of people displaced from conservation areas;11,33). Costs will vary across space and must be linked to actions to improve planning relevance9. For example, if the action required is land acquisition then a relevant cost is land price; if the action is management of a conservation area then a relevant cost would be the salaries of conservation managers.\n\nThe management of ES attracts similar costs dependent on the type of action required to protect the service. Indeed, some ES prioritization schemes incorporate opportunity costs in a similar way to biodiversity prioritization, recognising that managing ecosystems for service provision can yield the same opportunity costs as protecting ecosystems for biodiversity (e.g., when an area cannot be used for production3,18,34; Table 1). Costs may also be incorporated through the use of proxies for resource and maintenance expenses (see ‘An example of spatial prioritization’).\n\nIt is important to identify the assignation of costs (who pays) and benefits in both biodiversity conservation and ES prioritization35. For example, designation of a conservation area yields benefits that are primarily public, notwithstanding, for example, income generated from nature tourism, but sometimes at a cost to private interests (e.g., opportunity cost of lost revenue from production). Managing an area for the delivery of ES can yield relatively greater private benefits, particularly for service beneficiaries, with costs borne by both public and other private interests. For example, a forest designated for timber harvest will yield financial benefits to logging companies at a cost to the public (e.g., through lost carbon storage) and other private interests (e.g., those interested in using the forest for ecotourism). Ensuring greater equity in the distribution of benefits and costs from services provided by public or private assets may be achieved through various mechanisms such as government regulation, self-regulation (enforced by societal norms), or market approaches like cap and trade or payments for ES36,37. Yet, the appropriateness of a particular mechanism depends on the characteristics of the service being targeted (e.g., who generates the service, management jurisdiction, and provider–beneficiary spatio-temporal dynamics; see Kinzig et al.37).\n\nThe availability of human-derived alternatives to the provision of ES is a vital consideration in service prioritization. These alternatives can include, for example, a water filtration plant to cover the filtration services of wetlands or pesticides to cover biological control. The availability of alternatives and the capacity of relevant human communities to pay for these alternatives can influence the treatment of other factors such as benefits, threats, actions and costs. For example, managing a particular service may be given lower priority if human-derived alternatives are readily available and affordable, although the associated costs of these alternatives must be considered also (e.g., the health costs of increasing pesticide use). Only a few studies that attempt ES prioritization address the issue of availability of alternatives (Table 1). As part of the prioritization process, the availability and cost of alternatives should be considered simultaneously with the list of potential actions for service protection or enhancing service provision.\n\nSetting targets is common in conservation planning and can be a requirement for assessing the capacity of selection procedures to meet conservation objectives38. In most cases, setting a target is equivalent to meeting a baseline threshold. Target setting in ES prioritization is rare and has, to the best of our knowledge, only occurred in four published studies3,4,39,40(Table 1). For example, Chan et al.39 set a baseline target (assumed minimum requirement) of 12 days of outdoor recreation per person per year and determined the space required to provide that level of service from data on park visitation. Chan et al.39 also stipulated that targets had to be met in different stratification zones within the study area, which accounted somewhat for the site dependency of service production and variability in the spatial distribution of beneficiary needs.\n\nWhile target setting is one approach to assessing the capacity of ecosystems to meet the demands of beneficiaries, provision–demand relations have been variously dealt with in the literature (Table 1). For example, some studies included data on water use when calculating water provision capacity [e.g.,15,26], while others measured downstream need for water of a given quality through the calculation of population densities and areas of irrigated rice and mangroves18. Van Jaarsveld et al.41 calculated water and food provision relative to accepted minimum standards for human consumption. The need and approach to calculating demand for service provision will vary depending on the service of interest. For example, it is generally considered unnecessary to calculate spatially explicit demand for carbon storage because this service benefits the global community and demand is not spatially variable.\n\nSite dependency in the provision of an ES reflects the level of need for a particular service to be provided in a particular location in order to deliver benefits to a given set of beneficiaries. This can be interpreted also in the context of the scale of service provision (e.g., local to global). For example, storm protection from mangroves has high site dependency in provision – mangrove forests must occur in locations where local communities are threatened by storm activity. This should not be confused with the substitutability of the service; that is, whether human-derived alternatives (e.g., sea walls) or other coastal vegetation types can provide a similar service. In contrast, global climate regulation through ecosystems storing carbon has lower site dependency in provision because it does not have to occur at a particular location (i.e., there are various options for managing ecosystems to store carbon). However, there is still some level of site preference because certain ecosystems (e.g., rainforests) store more carbon than others. Site dependency and scale varies also in the use of the service. For example, the beneficiaries of biological control in agro-ecosystems generally occur at the local to regional scale, if the emphasis is on growers, whereas the beneficiaries of climate regulation occur at the global scale.\n\nVariation in the site dependency and scale of the provision and use of ES has major implications for the valuation of services, which must consider spatially explicit and scale-dependent relationships in production–consumption flows42. Such relationships also have important implications for prioritization strategies. High site dependency could result in certain locations that generate that service being classified as irreplaceable. For example, Bohensky et al.43 identified irreplaceable land units for food and water provision to meet pre-determined targets of caloric intake for a given population. When services have lower levels of site dependency in production there is greater flexibility in site selection during the prioritization process (all else being equal).\n\n\nAn example of spatial prioritization\n\nThe relationships among the various components of our conceptual framework for spatial prioritization of ES are presented in Figure 1. We illustrate our approach in this section using a worked example based on data published in Luck et al.15 focussing, for the sake of simplicity, on a single ES: water provision.\n\nThe global analysis of Luck et al.15 identified watersheds that are a priority for protecting particular ES. The first step in the analysis was to quantify the benefits and supply of the service. The benefits of protecting the supply of potable water was measured through human population density in each watershed; that is, there were greater benefits to protecting supply in watersheds with higher population density compared to those with lower density. Water supply was measured using a global hydrological model, and ‘water-production efficiency’ was calculated for each watershed by dividing supply in each watershed with watershed area.\n\nThe costs of actions to manage water provision were represented using a proxy for resource (e.g., land acquisition and infrastructure) and maintenance (e.g., labour) costs. This proxy incorporated data on total income in the watershed (per capita gross national income), population size and watershed area. Resource costs were assumed to scale positively with per-capita wealth and population density (assuming that land and infrastructure prices are generally higher where population density is higher), while maintenance costs were assumed to also scale positively with per-capita wealth. Finally, the cost-effectiveness of protecting the service in each watershed was calculated by dividing human population density and water supply (benefits) by the cost.\n\nThe capacity to meet demand was measured using values for water supply and water withdrawals in each watershed. It also considered regional water deficits (withdrawals > supply) and the proportion of total supply that remained once demands were met, adjusting the watershed-level capacity measure downwards proportional to the need to move water to regions (within a watershed) where supply did not meet demand. It was assumed that managing the service of water provision was most important in watersheds where supply barely meets or is short of demand, and less important when supply greatly exceeded demand.\n\nTo estimate threat to water provision, expected vegetation cover in each watershed was used, recognising the link between vegetation and water provision, filtration and the maintenance of water quality (although this link is decidedly complex; see Luck et al.15 for details). Vegetation cover and type in a watershed may be indicative of the capacity of the watershed to provide potable water naturally, and change in vegetation cover can be considered a proxy for threat to water provision. To quantify this threat, the following data were used: the proportion of each watershed covered in tree, shrub and herbaceous vegetation; the annual rate of change in vegetation cover (over a proceeding 5-year period); the time span over which change in cover would be predicted (e.g., 20 years); and the proportion of the watershed that was protected (assuming vegetation in protected areas could not be cleared). Watersheds with mid-range values of vegetation cover, rates of vegetation loss and/or area protected were considered priorities for water provision management, because, for example, watersheds with low cover and high rates of loss would require large investments in ES management relative to return, whereas watersheds with high cover and low rates of loss are under less threat to the disruption of the service.\n\nThe final consideration in spatial prioritization is the availability of alternatives to the provision of the service via ecosystems. Improvements in the supply of potable water may be made through the construction of dams and building of filtration plants, for example, rather than ecosystem management. The availability of these alternatives is often a function of the capacity of local communities to pay for them, in addition to other constraints (e.g., topographic suitability for dam construction). Therefore, Luck et al.15 used the gross national income per capita of countries spanning each watershed as an indicator of the capacity of communities reliant on the watershed to pay for alternatives to natural water provision.\n\nThe above components were combined into a single index representing the relative importance of each watershed for protecting water supply. This example, and our prioritization framework generally, is appropriate when planning units are large and there are a variety of available options for managing services, and it is difficult to express the components of prioritization precisely. Our framework treats the supply of services, threats and costs in ES prioritization inclusive of beneficiary demand, capacity to meet demand, and availability of alternatives to ES provision.\n\n\nDiscussion\n\nOur review of current approaches to identifying spatial priorities for managing ES found that the important components of prioritization (benefits, costs, threats, availability of alternative, and capacity to meet demand) were treated in substantially different ways or sometimes omitted completely (although not all components are applicable in every context). Moreover, few studies explicitly addressed the issue of site dependency and scale in the provision of services and/or location of beneficiaries. Accordingly, there is substantial scope for improving ES analyses aimed at identifying spatial priorities for managing services.\n\nIf ES benefits were commodities in perfect markets, the price of such benefits would reflect all of the spatial prioritization components identified above. Yet, many ES benefits are not commodities, and for those that are, the associated markets are far from perfect, suffering from numerous market failures including monopsony (single buyers, as in reverse auctions), oligopoly (few sellers, as in many payments-for-ES schemes), externalities and information asymmetries. This means that market prices will not generally reflect all of the components of prioritization appropriately. Stated-preference non-market valuation approaches can be informative in certain settings where markets do not apply, but they are generally of limited utility reflecting the various dimensions of value/social priority44,45. Accordingly, even where economic valuation data are available, it will still be appropriate for ES prioritization exercises to separately integrate some of the components we identify.\n\nAlthough we have focussed on the mechanics of prioritization, the following issues must be addressed prior to such analyses: 1) identification of the ES to be included; 2) capacity to access spatially explicit data; and 3) data quality (Figure 1). Identifying important ES should occur through in-depth consultation among scientists, policy-makers, managers and stakeholders (especially service beneficiaries; see Fisher et al.46). For example, if prioritization was required across a particular country, federal management agencies and relevant stakeholders may engage in a process of identifying those services most important to the well-being of the country. ‘Importance’ may be a factor of the total financial value of a service (e.g., agricultural production) and/or the societal need for a service (e.g., provision of potable water) and assessed through appropriate valuation approaches. Hence, a priority list of which services to focus on is required prior to deciding on where to invest in ES management. The selection of different services will influence the approach to spatial prioritization because ES management will have different objectives (e.g., improving timber harvest or maintaining water supply). However, the major steps we outline here will be relevant in most cases.\n\nApplication of our approach requires spatially explicit data on where services are produced, benefits, costs and threats. Data on spatially explicit production is usually obtained through mapping the location of ecosystems that provide services (e.g., grasslands that support livestock). Mostly, this involves maps of vegetation types or water sources (Table 1). Benefits are represented spatially generally via the biophysical quantity of a given service produced by a given location (e.g., carbon stored) and/or its financial value. To represent costs spatially, researchers have used simply the area of the planning unit (e.g.,39, for some services) or current land values [e.g.,34]. Documenting costs is problematic because of spatio-temporal variation in financial values, which is possibly why some researchers have resorted to more simple rules-of-thumb (e.g., assuming that managing larger areas yields greater costs). Also problematic is spatially explicit measures of threat, which have been represented by, for example, maps of land-use or historical or potential land-cover change and how these relate, spatially, to the location of service provision [e.g.,30,47] (Table 1).\n\nFinally, the type of data used in prioritization will greatly affect outcomes. For example, Anderson et al.2 demonstrated that variation in the resolution (≈ grain size) and/or spatial extent of a prioritization analysis influenced the level of congruence between biodiversity and ES priorities. Moreover, data quality may be poor for certain services and certain components of prioritization. For example, crude proxies or indicators may be required for services for which it is difficult to obtain accurate, spatially explicit measures of supply and demand (e.g., flood mitigation; see Holland et al.48). Our framework, which promotes the use also of data on threats, costs, alternatives and site dependency, may help to alleviate this issue because prioritization could be based just on those components for which data quality is acceptable.\n\nThe most appropriate metric to represent the supply of the service will be context dependent, but the use of biophysical quantities will be suitable in most cases. For example, if the service is storm protection then a suitable metric may be the area of mangroves that needs to be maintained to deliver a given level of protection [e.g.,25]. ES supply should be assessed relative to the demand for the service, which can be measured using a target-based approach, through current or projected use of the service or its products, through demonstrated need for the service (e.g., historical impacts of storms) or through meeting an accepted minimum standard (e.g., acceptable losses due to storm damage). Quantifying demand for a service requires the implicit or, preferably, explicit identification of beneficiaries, which may be immediate beneficiaries (e.g., residents of coastal villages threatened by storms) and/or ‘non-immediate’ beneficiaries (e.g., consumers of goods produced by the villages).\n\nThe application of prioritization frameworks generally involves multiple services across many planning units and priorities for different services are not necessarily congruent2. This requires an analysis of trade-offs between services in managing land/sea-space for service provision5. Moilanen et al.49 addressed this issue using a multi-objective prioritization approach for biodiversity and ES based on the conservation planning software Zonation. The authors argued that regional variation in land-use priorities meant that spatial separation of land uses may reduce management conflicts. Moreover, areas that are a priority for multiple ES could be used in trade-offs assuming the areas were not critical priorities for every service.\n\nIn multi-objective prioritization frameworks that consider spatial separation of ES management or trade-offs in land-use priorities, it is vital to address site-dependency and scale of service provision and location of beneficiaries. As we argue above, there is little flexibility in managing for the provision of services that are delivered locally to in situ beneficiaries (e.g., flood mitigation). Avoiding inappropriate management decisions and trade-offs rests entirely on taking a comprehensive approach to identifying priorities. Here we describe the major factors that must be considered in ES prioritization and argue that addressing as many of these factors as possible will improve the outcomes of multi-objective prioritization frameworks that aim to promote human well-being through the protection of services.\n\nDeveloping comprehensive methods for identifying ES priorities is much more than just an academic exercise. Governments and NGOs across the world are increasingly including the protection of ES into their policy directives. For example, the governments of China, Costa Rica and Mexico pay landholders that engage in management that protects the supply of hydrological services50–52. Vital to this process is identifying locations that offer the greatest return on investment. This requires a systematic and thorough approach to identifying spatial priorities for protecting ES.",
"appendix": "Author contributions\n\n\n\nGL and KC conceived the study. GL summarised the information in Table 1 and prepared the first draft of the manuscript. All authors revised subsequent drafts of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nWe declare no competing interests.\n\n\nGrant information\n\nThe contribution of GL and CK was supported by the Australian Research Council’s Future Fellowship (project number FT0990436G) and Postdoctoral Fellowship (project number DP110102153) programs, respectively. The contribution of KC was supported by the Canada Research Chairs program and the Canadian Foundation for Innovation/British Columbia Knowledge Development Fund (Leaders Opportunity Fund).\n\n\nReferences\n\nMA (Millennium Ecosystem Assessment)Ecosystems and human well-being: synthesis. Island Press, Washington D.C. 2005. 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Publisher Full Text\n\nPhua MH, Minowa M: A GIS-based multi-criteria decision making approach to forest conservation planning at a landscape scale: a case study in the Kinabalu Area, Sabah, Malaysia. Landsc Urban Plan. 2005; 71(2–4): 207–222. Publisher Full Text\n\nStrassburg BBN, Kelly A, Balmford A, et al.: Global congruence of carbon storage and biodiversity in terrestrial ecosystems. Conservation Letters. 2010; 3(2): 98–105. Publisher Full Text\n\nTurner WR, Brandon K, Brooks TM, et al.: Global conservation of biodiversity and ecosystem services. BioScience. 2007; 57(10): 868–873. Publisher Full Text\n\nVenter O, Laurance WF, Iwamura WF, et al.: Harnessing carbon payments to protect biodiversity. Science. 2009; 326(5958): 1368. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "306",
"date": "29 Sep 2012",
"name": "Elena M. Bennett",
"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 presents an interesting conceptual framework that researchers as well as governments or other stakeholders could use to identify priority areas for protecting ecosystem services.The authors suggest the following key elements: (1) determine the services of interest, (2) quantify the demand for the service(s) and capacity of the ecosystem to meet demand through supply of the service (3) identify the threats and level of threat, and (4) estimate the cost of protecting the service as well as any alternative means of providing the service. The authors then review the literature in which priorities have been assessed and identify which steps have been taken and which not in each study or effort. I hope this paper will increase our attention to key aspects of ES assessment that are often ignored, especially the interaction among all of these key factors.",
"responses": []
},
{
"id": "307",
"date": "04 Oct 2012",
"name": "David Norton",
"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 provides a conceptual framework for prioritizing areas for protecting ecosystem services (ES) such as water provision.The article explores similarities and differences between ES prioritization and biodiversity prioritization, and highlights several key components that need to be considered in prioritizing areas for protecting ES. Two key components are the need to consider the costs of protecting areas and the importance of considering human-derived alternatives. The conceptual framework is then applied to a case study based on water provision.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-17
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https://f1000research.com/articles/1-18/v1
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27 Sep 12
|
{
"type": "Case Report",
"title": "Folie a deux: a case report",
"authors": [
"Sobia Haqqi",
"Nisreen Ali",
"Nisreen Ali"
],
"abstract": "Folie a deux, to date, remains a rare, yet a challenging psychiatric diagnosis. We discuss two cases that were identified in our out-patient clinics. One case was lost to follow up, while the other one showed improvement over time with appropriate management. Conclusion: As with any rare disorder, recognition and correct referral for rare diagnosis like folie a deux is of paramount importance.",
"keywords": [
"A fascinating",
"rare and poorly understood psychological disorder",
"folie a deux",
"was first described by Lasègue and Falret in 18771. It is characterized by the transference of delusions from an individual (the primary)",
"who already suffers from a psychotic disorder",
"to a person or persons (the secondary) who are in close association and relative social isolation with the primary2."
],
"content": "Introduction\n\nA fascinating, rare and poorly understood psychological disorder, folie a deux, was first described by Lasègue and Falret in 18771. It is characterized by the transference of delusions from an individual (the primary), who already suffers from a psychotic disorder, to a person or persons (the secondary) who are in close association and relative social isolation with the primary2.\n\nThe relationship shared between the primary and the secondary is that of domination and submission, inducer and recipient respectively.\n\nA review of literature showed that delusional disorders and schizophrenia were the most commonly diagnosed psychotic disorders in the primary. Delusions of persecution and grandiose accounted for most3.\n\nFamily history and genetic association are considered strong factors for the development of folie a deux3,4.\n\nLiterature surrounding this disorder mainly consists of a plethora of case reports. Subsequently, it is due to these reports that there seems to be a better understanding regarding this elusive disorder.\n\nWe came across two interesting cases. In the first case, two sisters were brought in by their extended family. According to their Uncle, they were living in a one-room house with their mother who had died recently from some underlying medical illness. These three had lived in isolation for almost their entire life, with no contact with the outside world except through a domestic help to run house hold errands. On initial presentation to the psychiatry out-patient clinic, the main concern seemed to be disheveled appearance and weak physical health. Only later on, after complete psychiatric interviewing paranoid delusions against their relatives were confirmed as being shared by both. Further probing revealed that the delusions were being shared with the deceased mother. Due to some tribal reasons, the sisters were forced to leave the city and were lost to follow up.\n\nThe second case which the principal author is currently following, involves a mother and a teenage son. The mother was referred to the psychiatry out-patient clinic from the medical out-patient clinic after complete medical evaluation regarding her presenting complaints of vague aches and pains.\n\nOn initial assessment, the lady seemed guarded with her history, made no eye contact and seemed not much interested in talking about her aches and pains. She presented, almost a week later, this time accompanied with her teenage son. Shared delusions of persecution towards the husband/father were revealed on psychiatric assessment of both mother and son. The mother seemed to be the dominant psychotic individual and the son seemed to be the passive recipient.\n\nThe mother was started on atypical anti-psychotic Risperidone. The son had already plans to pursue higher studies in another city to which he was encouraged to go.\n\n\nDiscussion\n\nThe primary source of knowledge regarding shared psychiatric disorders mainly consists of a plethora of case reports. This rare disorder is identified on the basis of certain established diagnostic criteria.\n\nThese include; delusion develops in an individual in the context of a close relationship with another person(s), who has an already established delusion (Criterion A), and delusions are similar in content (Criterion B), the disturbance is not accounted for by another psychotic disorder or physiological effects of a substance or any general medical condition (Criterion C)5.\n\nAfter a complete psychiatric anamnesis, clinical interview, psychological testing and Mental State Examination we diagnosed our patient as folie a deux.\n\nA patient of folie a deux can present with a complaint that may be completely unrelated to his/her psychiatric condition. As reported in another case as well6, our patient complained of vague aches and pains, and only after a thorough history and examination of mother and her son were we able to make a diagnosis.\n\nAccording to Lazarus certain conditions have to be present for the development of folie a deux, these include an intimate emotional association between the primary and secondary and a genetic predisposition to psychosis such as blood relations with the primary case7. The relationship of mother and child is usually long standing and resistant to change. This fosters an environment for folie a deux to develop as the 13 year old son; the secondary individual in this case, was predisposed through genetic and environmental factors for sharing his mother’s paranoid delusions.\n\nThe classification of shared delusional disorder by Gralnick suggests four types. Folie impose, folie simultanée, folie communiqué, folie induite8.\n\nOur case report is in concordance with the description of folie impose. Here the dominant individual, who usually suffers from a psychotic illness, imposes his/her delusion on the secondary more submissive individual and in this situation separation of the two is crucial for cure of the secondary8.\n\nAs is stated through literature, the most common psychiatric disturbance that the primary suffers from is schizophrenia and the most common delusions are of persecution3. In the case aforementioned the mother was diagnosed with schizophrenia, and paranoid delusions which were shared by the son.\n\nAlmost all cases in literature involve members of a nuclear family9. In this 70 percent are between mother and child, husband and wife or siblings10.\n\nIn these relationships the primary usually professes certain characteristics that place him/her in the position of dominance. As suggested by other case reports as well, the inducer is usually advanced age, superior intelligence with a forceful aggressive character while the induced is younger in age, paranoid, dependant and less intelligent than the primary11,12.\n\nThe relative isolation further augments the process of the transference of the delusions, and the course can usually be chronic, as there is lack of intervention and identification of the disease by other people around.\n\nThe secondary, provoked by his dependence and submissive passive personality accepts the delusions than risk losing the intimate relationship with the primary13, further resulting in seclusion and separation from reality.\n\nThis type of environment breeds a mistrustful and hostile relation with the rest of the world and this further leads to paranoia which can then cause delusions of persecution as in our case.\n\nPrognostic and therapeutic factors in folie a deux are complicated by the fact that many patients are lost to follow up. Many patients present individually which may further make identification and treatment difficult. Furthermore this relatively rare disorder has no systematic treatment regimen found to be effective. Inconsistencies in literature regarding treatment modalities exist between either one of two; Separation as the sole treatment for the secondary, or psychotherapy and medical intervention in conjunction with separation3, for cure of the secondary.\n\nMost authors concur to separation being crucial for the basis of any intervention in the management of this disorder14–16.\n\nIn our case the mother was put on atypical anti psychotics and the son was sent to live with his relatives in another city, with no pharmacological intervention. The son showed remarkable improvement with waning of the delusions and improvement in academics.\n\nOn subsequent follow up visits mother is compliant and showed no side effects of medication. Improvement was noted in her attitude towards self care with weakening of delusional ideology. However she has limited insight so far as to the nature of her psychiatric illness. Compliance and full remission is the target for now.\n\n\nConclusion\n\nAs with any rare disorder, recognition and correct referral is of paramount importance. With timely intervention and regular follow up, Folie a deux has good prognosis. It is thus essential for psychiatrists to understand the psychodynamics of the disease and plan for long term management.",
"appendix": "Author contributions\n\n\n\nSobia Haqqi: Found the cases in her clinics, collected data, write up. Nisreen: Write up under supervision from Sobia Haqqi.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nLasègue C, Falret J: La folie à deux. Ann Med Psychol. 1877; 18: 321–355. Reference Source\n\nAmerikan Psikiyatri Birliği. DSM-IV-TR Ruhsal Bozuklukların Tanısal ve Sayımsal El Kitabı. E Köroğlu (Çev. ed.), Ankara, Hekimler Yayın Birliği. 2007; s.470–473. Reference Source\n\nArnone D, Patel A, Tan GM: The nosological significance of Folie à Deux: a review of the literature. Ann Gen Psychiatry. 2006; 5: 11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSadock BJ, Sadock VA, Pedro R, et al.: Kaplan and Sadock’s: Synopsis of Psychiatry: Behavioral Sciences/Clinical Psychiatry. 9th ed. Philadelphia. Lippincott Williams and Wilkins, 2003; 517–518. Reference Source\n\nThe Diagnostic and Statistical manual of Mental Disorders. Fourth Edition. DSM IV. Reference Source\n\nJolfaei AG, Isfahani MN, Bidaki R: Folie à deux and delusional disorder by proxy in a family. J Res Med Sci. March 2011; 16(Suppl 1): S453–S455. PubMed Abstract | Free Full Text\n\nLazarus A: Folie a deux: psychosis by association or genetic determinism? Compr Psychiatry. 1985; 26(2): 129–35. PubMed Abstract | Publisher Full Text\n\nGralnick A: Folie à deux: the psychosis of association. Psychiatry Q. 1942; 16: 230–63. Publisher Full Text\n\nSharon I, Sharon R, Eliyahu Y, et al.: Shared delusional disorder. Accessed November 10, 2004.\n\nPublic Law Research Institute, Hastings College of Law at the University of California The temporary insanity defense in California. Accessed November 17, 2004.\n\nMentjox R, van Houten CA, Kooiman CG: Induced psychotic disorder: Clinical aspects, theoretical considerations, and some guidelines for treatment. Compr Psychiatry. 1993; 34(2): 120–126. PubMed Abstract | Publisher Full Text\n\nManschreck TC: Delusinal disorder and shared psychotic disorder. In: Sadock BJ, Sadock, editors. Comprehensive Textbook of Psychiatry. Seventh Edition. Philadelphia. Lippincott Williams and Wilkins, 2000; p. 1257. Publisher Full Text\n\nEnoch MD, Ball HN: In: Uncommon psychiatric syndromes. 4th ed. Enoch MD, Ball HN, editors. New Delhi: Arnold Viva; 2004; pp. 179–208.\n\nDodig-Curković K, Curković M, Degmecić D, et al.: Shared psychotic disorder (\"folie a deux\") between mother and 15 years old son. Coll Antropol. 2008; 32(4): 1255–8. PubMed Abstract\n\nPatel AS, Arnone D, Ryan W: Folie a deux in bipolar affective disorder: a case report. Bipolar Disord. 2004; 6(2): 162–5. PubMed Abstract | Publisher Full Text\n\nMouchet-Mages S, Gourevitch R, Lôo H: Folie à deux: update of an old concept regarding two cases. Encephale. 2008; 34(1): 31–7. [Article in French]. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "274",
"date": "01 Oct 2012",
"name": "Driss Moussaoui",
"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 this case report and found it interesting. This kind of psychiatric pathology is mostly found in countries with strong social bonds between the members of the community, including the family.I had a case of shared hallucinations between father and wife/daughters. When the father said “do you hear the noise?”, they said yes, and not only to please him. For women of this family, it was impossible to consider that the father may say wrong things.Therefore, I suggest to accept this report, with a slight look at the references #2 and #5 that should be revised and completed.",
"responses": []
},
{
"id": "275",
"date": "08 Oct 2012",
"name": "Alfredo Carlo Altamura",
"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/1-18
|
https://f1000research.com/articles/1-15/v1
|
21 Sep 12
|
{
"type": "Case Report",
"title": "Insulinoma presenting as refractory seizure disorder",
"authors": [
"Pamela Correia",
"Roopal Panchani",
"Rajeev Ranjan",
"Chandrashekhar Agrawal",
"Roopal Panchani",
"Rajeev Ranjan",
"Chandrashekhar Agrawal"
],
"abstract": "Hypoglycaemia can lead to acute disorders of cognition, consciousness, epilepsy, transient ischemia, psychosis and chronic disorders of dementia and neuropathy. Misdiagnosis and delay in treatment are common and prolonged hypoglycemia can lead to permanent neurological deficit or fatal coma. Hypoglycemia caused by an insulinoma is a readily treatable condition that should be considered in the differential diagnosis of intractable seizures. The following case report highlights the need for careful reassessment of all seizures that are atypical and refractory to medication.",
"keywords": [
"Hypoglycemia",
"seizures",
"insulinoma"
],
"content": "Case report\n\nA 29 year old man had a one year history of multiple seizure episodes, which were as frequent as 3–4 episodes in a week despite being on regular antiepileptic medication of 3 drugs including carbamazepine, sodium valproate and clobazam. All the episodes were uncannily similar in that they always occurred on awakening and in the early hours of morning, between 5.00 to 7.00 am. The attacks were episodic stereotyped confusional spells characterized by abnormal posturing, perioral and eyelid twitching and unresponsiveness. The attacks would usually last from a few minutes to about half an hour. Upon initial OPD visits, compliance was checked and metabolic causes were ruled out. MRI Brain was done which was normal. No interracial epileptic activity was observed during prolonged video EEG monitoring.\n\nFive months later, seizures started occurring at increased frequency. On presentation to our hospital he was brought early morning in an unconscious state and was found to have hypoglycemia (48 mg/dl) which recovered immediately after treatment. On further questioning, it was apparent that his previous seizures tended to occur in early morning or several hours after the meal and that the post seizure confusion state could be shortened if he ate something during the confusion period. The patient had gained 10 kg weight over the period of last 6 months. The subsequent endocrine evaluation suggested insulinoma. Contrast enhanced CT revealed a solitary insulinoma in the head of pancreas. (Figure 1 and Figure 2) The patient underwent a surgical removal of the insulinoma with a Whipple’s resection procedure. A benign insulinoma was confirmed on histopathological examination. Following the procedure he has been totally seizure free and asymptomatic for last 6 months.\n\n\nDiscussion\n\nInsulinomas are the commonest hormone-secreting tumor of the gastrointestinal tract; the incidence is 4 cases/million/year. Tumors may occur as a unifocal sporadic event or in 5–10% of patients with MEN-1. 10% are metastatic, and a further 10% are multiple but behave as benign tumors. Insulinomas are found throughout the pancreas and are small (usually 10–20 mm). The median interval from onset of symptoms to the diagnosis of insulinoma is 2 years with a wide range of one month to 30 years as reported by Service F. and collegues1. Presentation is usually insidious with neuroglycopenia and fasting hypoglycemia.\n\nDiagnosis relies on key neuroglycopenic and sympathetic symptoms together with biochemical confirmation by documenting blood glucose levels below 50 mg/dl during monitored symptomatic episodes (that improve with oral intake), elevated C-peptide levels (>200 pmol/L), increased serum insulin levels (>5 µmol/mL), and absence of plasma sulfonylurea.\n\nMany patients with an insulinoma do not report the adrenergic symptoms of hypoglycemia and present with neurological or psychiatric manifestations that often lead to misdiagnosis. The delay in diagnosis is due to several factors. Firstly, the symptoms of insulinoma lack specificity, including various seizure disorders, personality change, bizarre behavior, amnesia, convulsions, and incidentally dystonia and polyneuropathy; these symptoms are similar with many common neurological and psychiatric disorders. Secondly, fasting blood glucose level can be normal in some patients. Thirdly, hypoglycemia itself induces neuroglycopenic and autonomic unawareness.\n\nSeizure disorder has been described in few contemporary cases of persistent hypoglycemia later on diagnosed as insulinoma two of them reported by Akanji A. and colleagues in 1992 followed by Basil C. and Pack A. in 20012,3. Of late Wang S. and his colleagues described recurrent episodes of automatism, confusion and convulsion with electroencephalography (EEG) findings resembling the pattern in complex partial seizures in a case of insulinoma4. Jaladyan V. and Darbiyan V. described a case of a girl presenting with drug-refractory myoclonia and generalized tonic-clonic seizure (GTCS) initially misdiagnosed as having juvenile myoclonus epilepsy (JME) before insulinoma was detected5. O’sullivan S. and Redmond J. also presented a case of insulinoma misdiagnosed as late onset refractory epilepsy6.\n\nAs in our case, the interpretation of clinical symptoms and normal EEG with seizures refractory to treatment was indicative of any toxic or metabolic cause or poor compliance to treatment. The possibility of hypoglycemia was excluded by three normal blood glucose measurements. Furthermore hypoglycemia due to an insulinoma especially in a young age group comes little lower in the list of other possible causes. Insulinoma, responsible for profound hypoglycemia usually has its onset in middle or older age. Symptoms are usually prominent following prolonged fasting states, especially in the morning as in our patient, but also in late afternoon.\n\nInsulinoma can also mimic other neurological diagnoses apart from seizures. In Daggett and Nabarro's review of 252 reported cases the most common neurological symptoms were confusion, coma, and seizures7. Non-specific headache, generalized weakness, paraesthesia, and stroke-like paralysis were less frequent and dizziness and dysarthria were uncommon. Newman and Kinkel reported a diabetic woman who developed two episodes of limb choreoathetosis and opisthotonus associated with hypoglycemia8. Winer et al. described a woman with an insulinoma who, when recovering from hypoglycemic attacks developed abnormal posturing of her body9. Chronic neuropathic and dementing syndromes due to hypoglycemia have also been described by Danta G. and Snook J. and his colleagues10,11. In a prospective survey done by Harrington M., two of 25 patients referred to neurologists with \"funny turns\" were found to have an insulinoma12.\n\nThis case highlights the importance of considering hypoglycemia in atypical neurological or psychiatric episodes and emphasizes the role of inpatient evaluation of refractory epilepsy. Neuroglycopenia should be considered in all patients with seizures, and other neuropsychiatric symptoms, especially if they do not conform to diagnostic criteria or respond to standard treatment. Taking a full history (including relationship of attacks to food, non-stereotyped or atypical attacks, and poor response to treatment) and clinical suspicion are the key to making a diagnosis of insulinoma. Once suspected, confirming the diagnosis with a 72 h fast is relatively simple.\n\nBenign insulin secreting adenomas are a potentially curable cause of seizures but may be fatal if unrecognized. Our case reiterates the importance of evaluating the metabolic cause of refractory seizure disorders. Although sometimes insulinoma shares some common semiological and EEG features, the possibility of atypical causes like insulinoma associated seizures should be considered in patients with some clues to diagnosis like close relationship to food intake, history of weight gain atypical attacks, seizures refractory to treatment. Seizures that present with such a particular prototypical pattern should arouse a strong clinical suspicion. Hence, early diagnosis and treatment can free many a patient from burdensome multiple antiepileptic treatments.\n\n\nConsent\n\nWritten informed consent was obtained for publication of their clinical details and images was obtained from the patient.",
"appendix": "Competing interests\n\n\n\nNone of the authors have any competing interests or disclosures.\n\n\nAcknowledgements\n\nDept of Pathology and Radiology for their support in the diagnosis of the patient at Sir Ganga Ram Hospital, New Delhi.\n\n\nReferences\n\nService FJ, Dale AJ, Elveback LR, et al.: Insulinoma, clinical and diagnostic features of 60 consecutive cases. Mayo Clin Proc. 1976; 51(7): 417–429. PubMed Abstract\n\nAkanji AO, George AO, Olasode BJ, et al.: Insulinoma in pregnancy presenting as a seizure disorder: a case report. East Afr Med J. 1992; 69(2): 117–119. PubMed Abstract\n\nBazil CW, Pack A: Insulinoma presenting as seizure disorder. Neurology. 2001; 56(6): 817–8. PubMed Abstract | Publisher Full Text\n\nWang S, Hu HT, Wen SQ, et al.: An insulinoma with clinical and electroencephalographic features resembling complex partial seizures. J Zhejiang Univ Sci. 2008; 9(6): 496–499. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJaladyan V, Darbinyan V: Insulinoma misdiagnosed as juvenile myoclonic epilepsy. Eur J Pediatr. 2007; 166(5): 485–487. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Sullivan SS, Redmond J: Insulinoma presenting as refractory late-onset epilepsy. Epilepsia. 2005; 46(10): 1690–1691. PubMed Abstract | Publisher Full Text\n\nDaggett P, Nabarro J: Neurological aspects of insulinomas. Postgrad Med J. 1984; 60(707): 577–581. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNewman RP, Kinkel WR: Paroxysmal choreoathetosis due to hypoglycemia. Arch Neurol. 1984; 41(3): 341–342. PubMed Abstract | Publisher Full Text\n\nWiner JB, Fish DR, Sawyers D, et al.: A movement disorder as a presenting feature of recurrent hypoglycaemia. Mov Disord. 1990; 5(2): 176–177. PubMed Abstract | Publisher Full Text\n\nDanta G: Hypoglycemic peripheral neuropathy. Arch Neurol. 1969; 21(2): 121–132. PubMed Abstract | Publisher Full Text\n\nSnook JA, Vander Star R, Weller RO: Insulinoma producing progressive neurological deterioration over 30 years. Br Med J. 1986; 293(6541): 241–242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarrington MG, McGeorge AP, Balantyne JP, et al.: A prospective survey for insulinomas in a neurology department. Lancet. 1983; 1(8333): 1094–1095. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "455",
"date": "29 Sep 2012",
"name": "Francesco Minuto",
"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\nOn viewing this seems like properly done research. The work itself is not really uncommon, neither new but it is worth publishing.",
"responses": []
},
{
"id": "456",
"date": "02 Oct 2012",
"name": "Niki Karavitaki",
"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/1-15
|
https://f1000research.com/articles/1-16/v1
|
21 Sep 12
|
{
"type": "Opinion Article",
"title": "Is the pan-genome also a pan-selectome?",
"authors": [
"Francisco Rodriguez-Valera",
"David W Ussery",
"Francisco Rodriguez-Valera"
],
"abstract": "The comparative genomics of prokaryotes has shown the presence of conserved regions containing highly similar genes (the 'core genome') and other regions that vary in gene content (the ‘flexible’ regions). A significant part of the latter is involved in surface structures that are phage recognition targets. Another sizeable part provides for differences in niche exploitation. Metagenomic data indicates that natural populations of prokaryotes are composed of assemblages of clonal lineages or \"meta-clones\" that share a core of genes but contain a high diversity by varying the flexible component. This meta-clonal diversity is maintained by a collection of phages that equalize the populations by preventing any individual clonal lineage from hoarding common resources. Thus, this polyclonal assemblage and the phages preying upon them constitute natural selection units.",
"keywords": [
"Bacterial and archaeal genomes show a surprising diversity in gene content even in otherwise very similar strains1",
"2. Some parts of the genome are shared and keep a high sequence similarity. The 95% average nucleotide identity (ANI) appears as a kind of “magic number” that fits the definition of most classical species",
"replacing the pre-genomic 70% DNA-DNA hybridization “golden rule”3. This is the ‘core’ of prokaryotic genomes (surprisingly similar figures hold for Bacteria and Achaea in spite of their highly divergent molecular biology)4",
"5."
],
"content": "The pan-genomic world\n\nBacterial and archaeal genomes show a surprising diversity in gene content even in otherwise very similar strains1,2. Some parts of the genome are shared and keep a high sequence similarity. The 95% average nucleotide identity (ANI) appears as a kind of “magic number” that fits the definition of most classical species, replacing the pre-genomic 70% DNA-DNA hybridization “golden rule”3. This is the ‘core’ of prokaryotic genomes (surprisingly similar figures hold for Bacteria and Achaea in spite of their highly divergent molecular biology)4,5.\n\nHowever, the most remarkable finding of prokaryotic genomes is the presence of other genomic regions that are extremely variable and differ in gene content and synteny from one strain to another6. One consequence is that the diversity of genes found in a single prokaryotic species is stunning; for example in Escherichia coli with about 5000 genes per genome, it is estimated that there could be about 45,000 different gene families in its pan-genome7. The ‘open-ness’ of a bacterial pan-genome for a species ranges from roughly twice the size of an individual genome, to more than ten-fold6,8. Thus, the genetic diversity hidden in the prokaryotic domain is much higher than initially suspected. This raises several questions regarding the biology and evolution of prokaryotic cells. How is this enormous diversity generated and, even more importantly, maintained? How does it impact an organism’s survival strategies and adaptation potential? These questions are fundamental gaps in our knowledge of the largest and oldest group of organisms on the planet.\n\nThe availability of multiple genomes from the same bacterial species has advanced greatly our knowledge of prokaryotic pangenomes2,8. Furthermore, the availability of large metagenomic datasets permits the analysis of the presence or absence of parts of the genomes of microbes that are known to be abundant in a specific habitat9–12; this bypasses the limitations and biases of pure culture retrieval of strains. We can begin to see general trends now in the pools of genes in the core and ‘flexible’ components of prokaryotic pan-genomes.\n\n\nThe flexible pool and the cell surface\n\nOne major problem of the flexible pool is its remarkable diversity, which makes it hard to classify its genes. Being less widely distributed, they are more difficult to annotate and often appear as hypothetical proteins. However, as more genomes are sequenced, patterns start to be discernible. Particularly informative are the clusters of ‘flexible genes’ collected in genomic islands (GIs), which contain groups of contiguous genes, making functional inference much more reliable. Much of the flexible pool is collected in GIs of 10Kb or more. In this paper we will focus on some of these islands that are present in most (or all) strain genomes but containing different genes (i.e. they are in the same genomic context and code for the same type of function or structure but the genes are only distantly related, if at all). For the sake of clarity we will designate these genomic islands found in many strains but containing different genes ‘flexible Genomic Islands’ (fGIs).\n\nOne kind of fGI that appears to have universal distribution encodes for the synthesis of exposed structures of the prokaryotic cell. One of the most remarkable examples of this phenomenon is the gene cluster that codes for the synthesis of the O-chain of the Gram-negative lipopolysaccharide (LPS;13). Classically known as the ‘O-antigen’, the diversity of this exposed envelope in Salmonella or Escherichia has been known for many years14,15. The O-chain is a repeat-unit polysaccharide, the monomeric repeat has generally between two and six sugar residues. O-chains are extremely variable in the nature, order and linkage of the different sugars13,16. This complex polysaccharide is very important for the survival of the cell, providing the appropriate hydrophilic envelope to allow nutrient imports towards the cell17. After subculture in the laboratory many strains lose part of the polysaccharide, originating “rough” mutants, which probably have diluted the critical importance that it had for the cell’s lifestyle. However, the importance of the O-chain for antibiotic susceptibility is long known, illustrating how the permeability properties of the cell vary with small changes affecting this structure18. Further, the gene cluster for such an important cell component is extremely variable. This diversity has classically been explained by the advantage of the concomitant antigenic variation that could prevent the host from identifying, and eventually expelling, all the strains of one of these species. However, even accepting this simplistic inference from host-microbiome interaction, the variability found in free-living bacteria is comparable (if not higher) than those of specialized pathogens or symbionts17.\n\nAs an example, Figure 1 shows the fGIs detected in the genomes available of Candidatus Pelagibacter ubique, probably the most abundant pelagic marine microbe. Further, in addition to the O-chain gene cluster, all known exposed structural motives that can vary reflect similar genomic patterns of variation. For example, capsular or slime layer polysaccharides5,19, the teichoic acids of Gram-positives20, or the S-layer glycoproteins of archaea4,21 all seem to be located in fGIs. Other exposed structures that are also typical components of the flexible gene pool are flagella, pili and porins. An alternative way to view the diversity found in all these cell components is that they all are important phage recognition targets.\n\n\nPhages, phages everywhere\n\nViruses and their hosts are extremely entangled entities. In many environments there are on average about 10 phages for every one bacterium22, which means that bacteria are under constant attack. There are millions of viruses in every drop of ocean water; on average, it is estimated that about a mole of viruses (6x1023) attack bacteria every minute in the oceans5,6. Some estimates suggest that a quarter of newly photosynthesized carbon in marine environments travels through the ‘‘viral shunt’’, moving it directly to dissolved organic carbon before grazers or other consumers can access it6,7. The diversity of bacteriophages is quite large, and dynamic, changing with time in a given environment23. Presumably, this change in virus diversity reflects changes in bacterial diversity, since phages are obligate parasites, and many phages can only infect specific bacterial strains. It has been estimated that 60–70% of the bacterial genomes sequenced to date contain prophages8,9 (Rob Edwards, Personal communication). About two-thirds of all sequenced proteobacteria (no 'l') Gamma-proteobacterial and low GC Gram-positive bacteria harbor prophages10; thus the phages are also part of the pan-genome for many of these organisms.\n\nThe surface of a cell is what is presented to the world (both friend and foe alike); in environments where bacteria are under constant attack from viruses, the need to often change the shape and appearance of surface proteins is compelling. Thus a good evolutionary strategy would be to vary these proteins, both by changing their structures, but also by distributing them amongst other bacteria within the population, where possible.\n\nCan the need to diversify phage receptors explain the enormous diversity of the pan-genome? Certainly not, but it could be responsible for a large part. However, the other major component of the flexible pool might also be preserved via phage predation12.\n\n\nThe flexible pool and niche partitioning\n\nMany components of the flexible pool are involved in niche partitioning: 1) transport of substrates and the cognate metabolic pathways required for their assimilation by the cell, 2) regulation, such as two component systems involved in fine tuning the response to environmental stimuli 3) respiratory chains and or protective mechanisms involved in different oxygen or light relationships. This has been found to apply to both bacteria11,24–26,27 and archaea4. For heterotrophic osmotrophs the transporter baggage carried in the genome is determined largely by lifestyle and niche specialization. Accordingly the transporters found in different clonal lineages are extremely variable and are typical components of the flexible gene pool. In a remarkably parallel way, hli genes (coding for high-light inducible proteins) present in marine picocyanobacteria might influence the fine light qualities exploited by these widespread phototrophs and are also typical components of the flexible pool of these microbes28. A similar story is depicted by the tonB receptors involved in the transport of micronutrients29.\n\nIt is important to emphasize here that fGIs related to phage sensitivity such as the O-chain of the LPS and fGIs related to niche specialization are genetically linked in a single replicon so that negative selection by phage predation would compensate automatically positive selection by overly efficient exploitation of resources. For example, a sudden increase in the concentration of nutrients that are exploited by a clone that might lead to a major clonal expansion would be kept in check by the increase in the concentration of the linked phage receptors12. This mechanism of population control although negative for the short term prevalence of the clone might be good for its long term survival since it maintains the complexity of the community and its endurance.\n\n\nThe pan-selectome\n\nThe unit of selection has been a major conundrum in evolutionary biology30. Historically the proposals have been, according to times and fashions, going from the gene31 to the community32 or even the planet33. In 1997 Ernst Mayr defined the unit of selection as “a discrete entity and a cohesive whole, an individual or a social group, the survival and successful reproduction of which is favored by selection owing to its possession of certain properties”34.\n\nWe would like to advance here the idea that in nature, the selection unit in a prokaryotic community or assemblage is an ensemble of clonal lineages that share the same (or highly similar) core genome but differ in the flexible gene complements regions (i.e. the selection unit at the genomic level would be the pan-genome) Box 1. These meta-clonal populations are maintained and equalized by phage predation12 in an analogous way as the immune system in a mammal maintains in check tumors. Thus, phage populations should be considered as belonging to the same selection unit, not only because they are part of the pan-genome (which they often are) but because they are instrumental for its long term preservation (Figure 2). Furthermore, the different clonal lineages as retrieved by pure culture have little chance of succeeding in nature (or in complex biotechnological processes such as dairy or wine production or sewage treatment).\n\nThe genome is indicated as a solid blue circle (core) with two flexible genome islands fGIs (see text) in different colors to indicate different genes but coding for the same functions. The two fGIs indicated code for the O-chain of the lipopolysaccharide (in a Gram negative bacterium) and for a set of transporters. In reality there are many more fGIs (often four or five or more); further many differential transporters or other niche exploitation related features can be coded in small flexible islands or islets interspersed along the core, but they are all genetically linked to the O-chain and other surface related fGIs that are major recognition targets for phages. Three types of phages and receptors have been indicated, by different geometric forms. This set of cellular clones and phages is in equilibrium since the disproportionate increase of cells or phages is prevented by the density dependent kinetics of phage infection. For example if one clone increases over a certain threshold it will be over-preyed by its phages, until population returns to normal following a classical Lotka-Volterra predator/prey equilibrium. This is favorable for the meta-clonal population, since it keeps different lineages with complementary ecological skills acting in tune. This meta-clonal/viral population can be selected as an unit to exploit similar environments such as the water column in the ocean that is very similar worldwide.\n\nAlthough this idea invokes elements of group selection, a controversial theory of evolutionary biology35, it is being proposed to explain evolution of prokaryotic populations, about which very little evolutionary theory has been solidly established, particularly outside of the walls of the test tube.\n\n\nData supporting group selection in prokaryotes\n\nOne of us proposed a Constant-Diversity model based on the distribution of gene functions in the genomic islands that under-recruit in metagenomes, where the core genome recruits at high similarity12. Since then, several independent workers have found data supporting this notion.\n\nOne of the most convincing demonstrations that different fGIs in prokaryotes are largely involved in phage sensitivity is the work of Avrani et al.36, in which the resistance to phages in several isolates of Prochlorococcus could all be assigned to mutations taking place in Genomic Island 4 of this microbe identified previously9, as involved in the synthesis of the O-chain of the lipopolysaccharide. By measuring the frequencies of mutants in metagenomic datasets the authors conclude: “abundant Prochlorococcus populations belonging to a single ecotype with common physiological and ecological characteristics are actually an assortment of subpopulations with different susceptibilities to co-occurring phages” and “Thus, large numbers of taxonomically identical organisms, fulfilling the same ecological role, are probably maintained in the environment as a result of micro-diversity in phage susceptibility regions”36. A similar situation is found in Synechococcus where also resistant mutants were found to have altered genes in the O-chain region37.\n\nMany other recent developments support the coexistence of complex populations of phages and their hosts in natural communities23,38–41. There is also evidence that phages contribute to keep high levels of host diversity42 and that diversity promotes productivity43.\n\nRecently there have been other alternatives used to explain the diversity of pan-genomes by some type of kin selection, such as the so-called Black Queen hypothesis44 in which some genes present in certain lineages can supply the functions for other clonal lineages. Along the same lines, Teusink et al. describe a “game theory” explanation for the coexistence of proteolytic and non-proteolytic strains in dairy multi-starter cultures45. However, these models only make even more critical the role of phages to keep the proper ratios among the different cooperating lineages.\n\n\nConclusion\n\nWe are proposing here a way of thinking about prokaryotic communities in which cells with a core above 95% ANI, but with a wide diversity of flexible genome complement, and phages praying on them, form an evolutionary selection unit, the “prokaryotic selecton”. This has important repercussions for the evolutionary biology of prokaryotes.\n\nThe species as an evolutionary unit is at the centre of the modern Neo-Darwinian synthesis. Species are not just considered as a taxonomic level of classification but as a kind of biological entity or level of organization beyond the cell or the individual46. However, this “natural species concept” has been difficult to transfer to prokaryotes due to the lack of sexual reproduction and the unpredictable levels of recombination (particularly when including the illegitimate one) of prokaryotic genomes47. If the pan-selectomes described here are the units of selection, they might also fulfill the requirements to be considered natural evolutionary units. However, this requires a mechanism that provides the discontinuities in genetic diversity found in nature. Metagenomic data show that there are discontinuities located at ca 95% ANI, beyond which a gap indicates an empty space in the sequence diversity space48. Of course this only applies to the core genome of the meta-clones but still reflects a coherence that requires an evolutionary drive reminiscent of the breeding barriers found in animal species for example. How could the meta-clones explain such discontinuities? This critical question remains to be answered. However, we would like to advance one hypothesis that we call “the maverick hypothesis”. Let’s assume that a meta-clone of bacteria and phages is established somewhere, for example, exploiting the degradation of chitin, a common component in the water column of the ocean. A pan-genome evolves that allows for the efficient exploitation of the multiple resources associated to this polysaccharide (other accompanying organic molecules, phosphorus and nitrogen source etc.). The populations of this microbe have also a complement of phages to keep a well-equilibrated consortium. The physical proximity, near where the resources (such as zooplankton remains) are found facilitates genomic homogenization by homologous recombination. The pools of genes in the flexible genome can diverge enormously but the core will remain relatively coherent. The rise of a “maverick” that would try to form a monoclonal population diverging away from the homogenizing influence of the rest would be prevented by the excessive phage predation pressure coupled to less efficient exploitation of resources. This trend in the long run might be enough to provide the discontinuities required to form a natural species-like entity.",
"appendix": "Author contributions\n\n\n\nThe two authors contributed to this manuscript; both were involved in writing multiple drafts. Francisco Eduardo Rodriguez Valera wrote Box 1, and prepared Figure 2, and Dave Ussery made Figure 1.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nPart of this work was supported by the Center for Genomic Epidemiology (09-067103/DSF). Additional funding is from projects MAGYK (BIO2008-02444), MICROGEN (Programa CONSOLIDER-INGENIO 2010 CDS2009-00006), CGL2009-12651-C02-01 from the Spanish Ministerio de Ciencia e Innovación, DIMEGEN (PROMETEO/2010/089), ACOMP/2009/155 from the Generalitat Valenciana and MACUMBA from the European Commission.\n\n\nReferences\n\nPasserini D, Beltramo C, Coddeville M, et al.: Genes but not genomes reveal bacterial domestication of Lactococcus lactis. PLoS One. 2010; 5(12): e15306. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nDay MD, Beck D, Foster JA: Microbial Communities as Experimental Units. Bioscience. 2011; 61(5): 398–406. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSugimoto T: Darwinian evolution does not rule out the gaia hypothesis. J Theor Biol. 2002; 218(4): 447–455. PubMed Abstract | Publisher Full Text\n\nMayr E: The objects of selection. Proc Natl Acad Sci U S A. 1997; 94(6): 2091–2094. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorrello ME: The rise, fall and resurrection of group selection. Endeavour. 2005; 29(1): 43–47. PubMed Abstract | Publisher Full Text\n\nAvrani S, Wurtzel O, Sharon I, et al.: Genomic island variability facilitates Prochlorococcus-virus coexistence. Nature. 2011; 474(7353): 604–608. PubMed Abstract | Publisher Full Text\n\nMarston MF, Pierciey FJ Jr, Shepard A, et al.: Rapid diversification of coevolving marine Synechococcus and a virus. Proc Natl Acad Sci U S A. 2012; 109(12): 4544–4549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMenge DN, Weitz JS: Dangerous nutrients: evolution of phytoplankton resource uptake subject to virus attack. J Theor Biol. 2009; 257(1): 104–115. PubMed Abstract | Publisher Full Text\n\nKashiwagi A, Yomo T: Ongoing phenotypic and genomic changes in experimental coevolution of RNA bacteriophage Qbeta and Escherichia coli. PLoS Genet. 2011; 7(8): e1002188. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShapiro OH, Kushmaro A, Brenner A: Bacteriophage predation regulates microbial abundance and diversity in a full-scale bioreactor treating industrial wastewater. ISME J. 2010; 4(3): 327–336. PubMed Abstract | Publisher Full Text\n\nAlbertsen M, Hansen LB, Saunders AM, et al.: A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal. ISME J. 2012; 6(6): 1094–1106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaterson S, Vogwill T, Buckling A, et al.: Antagonistic coevolution accelerates molecular evolution. Nature. 2010; 464(7286): 275–278. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCardinale BJ: Biodiversity improves water quality through niche partitioning. Nature. 2011; 472(7341): 86–89. PubMed Abstract | Publisher Full Text\n\nMorris JJ, Lenski RE, Zinser ER: The Black Queen Hypothesis: evolution of dependencies through adaptive gene loss. MBio. 2012; 3(2): e00036–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTeusink B, Bachmann H, Molenaar D: Systems biology of lactic acid bacteria: a critical review. Microb Cell Fact. 2011; 10(Suppl 1): S11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Queiroz K: Ernst Mayr and the modern concept of species. Proc Natl Acad Sci U S A. 2005; 102(Suppl 1): 6600–6607. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDoolittle WF: Population genomics: how bacterial species form and why they don't exist. Curr Biol. 2012; 22(11): R451–453. PubMed Abstract | Publisher Full Text\n\nD Caro-Quintero A, Konstantinidis KT: Bacterial species may exist, metagenomics reveal. Environ Microbiol. 2012; 14(2): 347–355. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "270",
"date": "28 Sep 2012",
"name": "Ben Busby",
"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 clearly written and interesting opinion that we mostly agree with.Together with the recent studies of the CRISPR system and Patrick Forterre’s just-published provocative concept of the “virocell”, this paper reflects – and promotes – a growing appreciation of the importance of bacteriophages in shaping the bacterial (prokaryotic) world. While we believe that the relationships between bacteria and their phages are even more complex than discussed here (e.g. phage lysogeny likely plays a greater and more complex role) this manuscript is definitely a good step in the right direction.",
"responses": []
},
{
"id": "271",
"date": "05 Oct 2012",
"name": "Granger Sutton",
"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 work on pan-genome analysis tools and found the article well written and interesting.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-16
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https://f1000research.com/articles/1-2/v1
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16 Jul 12
|
{
"type": "Research Article",
"title": "Considerations for clinical read alignment and mutational profiling using next-generation sequencing",
"authors": [
"Gavin R Oliver"
],
"abstract": "Next-generation sequencing technologies are increasingly being applied in clinical settings, however the data are characterized by a range of platform-specific artifacts making downstream analysis problematic and error prone. One major application of NGS is in the profiling of clinically relevant mutations whereby sequences are aligned to a reference genome and potential mutations assessed and scored. Accurate sequence alignment is pivotal in reliable assessment of potential mutations however selection of appropriate alignment tools is a non-trivial task complicated by the availability of multiple solutions each with its own performance characteristics. Using BRCA1 as an example, we have simulated and mutated a test dataset based on Illumina sequencing technology. Our findings reveal key differences in the performances of a range of common commercial and open source tools and will be of importance to anyone using NGS to profile mutations in clinical or basic research.",
"keywords": [
"Since emergence in 2005",
"next-generation sequencing (NGS) technologies have proven prolific tools in the research setting",
"permeating a variety of scientific disciplines and demonstrating a range of applications that seems to be limited only by the imagination of the sequencing community. The technology continues to develop at a rapid pace with established instrument manufacturers regularly augmenting their product portfolios and an increasing number of start-up companies promising to disrupt the market. Beyond basic research applications",
"NGS technologies are now increasingly being applied in the clinical environment",
"driven partly by their rapid maturation and the arrival to market of smaller",
"cheaper sequencing platforms."
],
"content": "Introduction\n\nSince emergence in 2005, next-generation sequencing (NGS) technologies have proven prolific tools in the research setting, permeating a variety of scientific disciplines and demonstrating a range of applications that seems to be limited only by the imagination of the sequencing community. The technology continues to develop at a rapid pace with established instrument manufacturers regularly augmenting their product portfolios and an increasing number of start-up companies promising to disrupt the market. Beyond basic research applications, NGS technologies are now increasingly being applied in the clinical environment, driven partly by their rapid maturation and the arrival to market of smaller, cheaper sequencing platforms.\n\nThe potential clinical application of NGS has a broad scope ranging from full human genome profiling1 to investigation of the microbiome2 and includes applications such as biomarker discovery, patient diagnosis, prediction of drug response and patient stratification for clinical trials. Such applications often involve the targeted profiling of genes known to be of clinical relevance. These genes harbor diagnostically relevant variants including single nucleotide polymorphisms (SNPs), and small insertions and deletions (INDELs). Individual genes have previously been interrogated in clinical testing using traditional techniques such as Sanger sequencing however NGS technologies have already begun to supplant the previous tools of choice in these areas, offering increased speed and throughput with reduced running costs.\n\nDespite many successes and increasing uptake, the data generated by NGS analyzers is not perfect, with each platform yielding characteristic errors and biases. Furthermore, NGS technologies produce reads that are much shorter than those traditionally produced by Sanger sequencing methods and this can complicate matters further, especially in genomes containing a large proportion of repetitive elements3. The effect of these problems is most visible in large scale studies such as genome-wide sequencing where a recent study reported a 1 million variant platform-based discrepancy for a single genome4. This fact bestows responsibility on both algorithm and software developers and downstream users to develop deep understanding of the various data types and their idiosyncrasies and to apply this appreciation in their analysis and interpretation, in order to correct or compensate for potential errors. Despite forming an area of active research, data interpretation remains an issue and is no doubt a factor in feeding the inertia of many clinical facilities that are reluctant to adopt the new technologies5.\n\nTwo major computational steps in variant detection from NGS data are read alignment whereby the data are mapped to corresponding locations on a target genome, and mutation calling whereby nucleotides differing from the target genome are assessed and scored on their likelihood of representing a genuine mutation versus an error. While these two stages of analysis may be supplemented with various pre or post processing techniques they represent the most crucial steps and therefore the area of most active software and algorithm development.\n\nAligners of choice have begun to emerge6,7 however their strengths are often application specific and different tools are recommended depending on the sequencing platform and individual study goals. Aligners generally fall into the categories of being based on either hash tables or suffix trees8. Suffix tree-based aligners such as Heng Li’s BWA9 are characterized by their speed and memory-efficiency whilst generally achieving lower sensitivity than hash-based counterparts such as Stampy10. There is thus a necessary trade-off between speed and sensitivity at the read alignment stage with speed often being prioritized due to the volumes of data produced by NGS technologies and the corresponding time required for analysis. Aligners can be further classified as gapped or ungapped based on their ability to produce successful alignments in the presence of small INDELs. Aligners including BWA and Stampy have been shown to produce alignments with a fair degree of success for reads containing a range of INDEL sizes10 however such abilities will vary based on a range of complicating factors including size and location of the INDEL. As well as generating continuous controversy11, the BRCA1 gene presents a particularly interesting set of alignment challenges due to a disproportionately high concentration of INDELs greater than eight nucleotides in length. In fact, 3% of known deleterious mutations in the Breast Cancer Information Core (BIC) database12 fall into this category and represent a significant over-representation when compared to a healthy genome. Furthermore the BRCA1 gene contains multiple areas of high shared identity in the form of tandem repeats, posing another difficulty in achieving accurate read mapping. Challenges like this pose particular difficulties in the clinical setting where errors have the potential to translate to misdiagnosis or mistreatment, directly affecting and endangering the lives of patients. No gold-standard clinical alignment tools yet exist and numerous publicized examples of early translational work appear to base their choice of tool on user-friendliness or availability of a graphical-user-interface rather than assessments of performance. We have investigated the performance of a range of popular alignment tools and assessed their ability to accurately detect known mutations. Several of these tools are already being used as components of diagnostic workflows in the clinical setting. Here we present data generated using simulated reads derived from the human BRCA1 gene. Our findings demonstrate the widely varying abilities of common read alignment tools and their impact on downstream variant calling. Furthermore the results suggest a need for careful and thorough evaluation of all tools being used in a particular analysis pipeline through simulation and analysis of data of known constitution.\n\n\nMethods\n\nA range of open source and commercial alignment tools were selected for assessment based on their reported ability to facilitate detection of both SNPs and INDELs as well as frequency of citation in both scientific and commercial literature. The aligners included in the comparison were: BWA (0.5.9-r16), Bfast (0.7.0a)13, Smalt (0.5.8), Stampy (1.0.14), Mosaik (2.1.33), CLC Genomics Workbench’s (5.0.1) NGS and Beta aligner, Novocraft’s Novoalign (V2.07.18)14, Omixon’s Variant Toolkit (2.1.3), Bowtie 2 (2.0.0-beta5)15 and Softgenetics Nextgene (2.2) aligner.\n\nStampy was used to simulate sixty-seven groups of 200,000 90bp paired-end FASTQ Illumina reads from the human BRCA1 gene (hg19) with an appropriate error profile. Each sequence grouping was mutated in-silico with custom scripts used to introduce a combination of 20 SNPs and 13 INDELs. These were selected from a test set of 2211 (1299 unique) known BRCA1 variants containing 1340 SNPs, 320 insertions and 551 deletions.\n\nReads were aligned to hg19 chromosome 17 on a HP DL585 G6 server with 4 six-core AMD Opteron 2.8Ghz processors and 256GB of RAM. Multi-threading with the maximum number of threads supported by the aligner was utilized. Each aligner was run with parameters as close as possible to default. Each aligner was run in both single-end and paired-end alignment mode with half of the paired-end reads being used to simulate a single-end read dataset. Run-times were benchmarked based on the wall-clock time taken to produce a SAM format alignment for all 67 sets of FASTQ reads.\n\nEach SAM formatted file was converted to BAM format and processed to ensure downstream compatibility with GATK16 using a combination of tools from the Picard collection. (SamFormatConverter, AddOrReplaceReadGroups, SortSam and BuildBamIndex respectively). BAM files were then processed in a GATK-based pipeline. The pipeline consisted of local realignment around INDELs (RealignerTarget Creator and IndelRealigner), quality score recalibration (CountCovariates and TableRecalibrator) and finally variant calling (Unified-Genotyper). The wrapper scripts sam2bam.sh and gatk.sh are provided and can be used to recreate the processing steps from alignment files (SAM format) to variant call (VCF format).\n\n\nResults/discussion\n\nThe reads and mutation panel utilized here represent a challenging and multi-functional test set with widely varying INDEL sizes (Table 1) and an extensive range of SNPs providing a useful means of assessing the aligners’ single and paired-end modes. The reads were created to contain only known mutations from the human BRCA1 gene thus facilitating downstream assessment of mutation profiling accuracy whilst remaining comparable to real-world data. Reads were simulated to closely match the error profile of Illumina’s sequencing technology enabling a further level of realism to be captured in the simulated test-set. Only homozygous variants at relatively high levels of sequence coverage (70–140x) were included in the test set to ensure testing of the alignment tools’ abilities rather than the quality of the downstream variant calling methods.\n\nA representative range of SNPs and small INDELs corresponding to known mutations were distributed between 67 groups of simulated Illumina reads in order to test the read aligners’ abilities to accurately align mutation-containing reads.\n\nRun-times varied widely from seconds to hours and are shown in Table 2. Novocraft’s Novoalign software performed fastest and was closely followed by BWA and Bowtie 2 in both single and paired-end mode whilst Bfast’s paired-end mode represented the slowest run-time by almost half a day. Nextgene was excluded from this comparison due to the fact it is Windows-based software and it was not possible to assess run-times on the same hardware as for the other aligners. The criticality of program speed is largely dependent on end-application. For example, targeted sequencing experiments will generally involve much smaller data volumes than whole human genome sequencing. It should also be borne in mind that read generation by the sequencing machine itself is a rate limiting step and sequence generation and alignment steps can be run in parallel. With the exception of the paired-end run with Bfast, most alignment times recorded here might be considered manageable for most purposes.\n\nEach aligner was tested by aligning all 67 groups of simulated Illumina reads in single and paired-end mode. The maximum number of threads utilizable by each aligner were used in testing.\n\nThe greatest overall sensitivities of detection were achieved by Novoalign and Omixon’s Variant Toolkit in paired-end and single-end mode respectively (Figure 1, Table 3). Stampy also enabled highly sensitive mutation detection with Bfast performing least favorably. Sensitivities were also assessed based on the category of mutation (Table 4).\n\nGraphical representation of the total percentage of mutations detected across of 67 groups of simulated Illumina reads in single-end and paired-end mode.\n\nThe total percentage of mutations detected by each aligner across the 67 groups of simulated Illumina reads was recorded for single and paired-end run-modes. Each read group contained 13 INDELs of varying sizes as well as 20 SNPs.\n\nTotal percentage of SNPs and INDELs detected across the 67 groups of simulated Illumina reads by each aligner in single and paired-end mode.\n\nSome aligners such as Smalt, Bowtie 2 and CLC were clearly seen to perform more strongly on detection of SNPs than INDELs in general. Nextgene performed similarly for SNPs and deletions but had lower sensitivity of insertion detection. BWA showed obvious decreases in ability to detect INDELs when shifting from paired-end to single-end mode. In contrast, Novoalign, Omixon and Stampy performed well regardless of run-mode or mutation type. Overall Novoalign was the best performer in paired-end mode while Omixon achieved the highest sensitivities in single-end mode. In a clinical environment, sensitivity will likely represent the most important metric in evaluating alignment software, however other factors may also be of importance, depending on the test in question. Specificity values are not included here as they were non-discriminatory in this context due to low numbers of false positives relative to the high number of true negatives.\n\nControlling the rate of false positive results is clinically important in avoiding unnecessary treatment, expense and patient anxiety. The number of incorrectly identified mutations varied widely dependent on aligner and run-mode (Figure 2, Table 5). The highest rates were obtained using Bfast, followed by CLC Bio’s Beta aligner, Nextgene, Mosaik, Stampy, and Bowtie 2 while Novoalign and Smalt were the strongest performers followed closely by Omixon and CLC. Number of false positives alone is of limited utility in assessing aligner performance, however positive predictive value (PPV) provides a useful metric which combines counts of both true and false positives in a single value (Table 6). PPV was calculated based on the equation:\n\n\n\nEach read group contained 20 SNPs and 13 INDELs.\n\nEach read group contained 20 SNPs and 13 INDELs.\n\nIn this case PPV is the quotient of the total number of true positives divided by the sum of the total number of true positives and true negatives.\n\nNo inferences were made about prevalence in calculating the values. Novoalign had the highest PPV in both single and paired-end mode. Smalt, CLC and Omixon’s Variant Toolkit also performed strongly on this metric. Notably Stampy performed relatively poorly in contrast to its high sensitivity.\n\nNotably Bfast, Nextgene, Stampy, Mosaik and Bowtie 2 all showed obvious increases in the number of false positives detected in paired-end mode. Conversely, switching from paired-end to single-end reads had varying adverse effects on the sensitivities of all aligners except for Nextgene. The worst affected was BWA which retained all SNP calls but demonstrated a clear dependency on the information offered by paired-ends by losing 13% of INDEL calls. Notably Nextgene and Omixon’s Variant Toolkit were the only software to retain all INDEL calls when switching from paired to single-end mode. Omixon’s Variant toolkit was the most sensitive of all the aligners in single end mode with a 99.14% overall detection sensitivity.\n\nThis strong performance in single-end mode is relevant not only from a diagnostic standpoint, but also from a clinical cost-saving perspective as paired-end protocols ultimately incur extra costs per run vs single-end protocols. While paired-end reads generally represent a saving in terms of cost per megabase, they effectively double sequencing output and this may not be a cost-effective option depending on the logistics of the individual run. Furthermore, researchers who outsource sequencing will often see the available protocol for their relatively small sequencing project dictated by the larger projects they are multiplexed alongside.\n\nNot unexpectedly there appeared to be a general trend of INDEL size affecting most aligners ability to facilitate downstream calling (Figure 3). The size of the effect varied by aligner with BWA and Novoalign showing good detection rates for all but the largest deletions while others such as Bowtie 2 and the CLC aligners were not successful far beyond a 10bp INDEL size. Nextgene showed better sensitivity for insertions than deletions. Stampy and Omixon’s Variant Toolkit were the only two aligners to detect the largest deletions. This highlights a need for those involved with testing and analysis to develop an appreciation of the various mutations that might exist in their target genes and to select their analysis tools appropriately. INDELs in the range represented by the BRCA1 mutation panel have real-world relevance in genetic disorders and strong aligner performance on larger INDELs appears to be an exception rather than a rule.\n\n\nSummary and conclusions\n\nUsing a simulated, targeted sequencing scenario with Illumina read data, the work presented here highlights several important considerations regarding aligner choice in studies involving profiling of mutations. Furthermore the data presented goes some way to characterizing the performance of a comprehensive selection of commonly used aligners and should represent a useful resource for anyone focused on similar scientific studies.\n\nWhilst the dataset used in this study was engineered to include a challenging range of mutations and efforts were made to simulate the error profile of Illumina sequencing technology, it is nevertheless a simplified representation of real-world data. Experimental artifacts such as PCR stutter have the potential to present further challenges to alignment algorithms and there is no consideration of such issues here. Furthermore, the test dataset used in this study produced a uniform, high coverage of the target gene and only homozygous variants were simulated. Finally, the use of only a single variant-caller in the study means that some of the errors encountered may not be due to alignment issues. The aim is to follow up the current study by focusing on an expanded gene-set, alternative variant-callers, homo and heterozygous mutations and different sequence formats. Nonetheless, this focused study demonstrates the utility of simulated data in assessing program performance.\n\nWith the exception of Bfast, all aligners performed relatively well on the BRCA1 dataset. Clinical applications necessitate the use of the most highly accurate solutions, however. Only Novoalign, Omixon’s Variant Toolkit and Stampy achieved 99% or greater sensitivity in both paired-end and single-end mode. Omixon and Stampy were the only two aligners to detect the longest deletions in the dataset however Stampy’s performance was let down by a false positive rate which would be considered unacceptably high for most applications. While Novoalign did not detect the largest deletions, it was the fastest aligner and the most sensitive in paired-end mode. Nevertheless, assuming the longer run-times are not an issue, Omixon’s superior sensitivity in single-end read mode likely makes it the best option when paired-end protocols are not possible. It should also be mentioned that as a freely available, open-source tool, BWA’s paired-end performance is laudable and goes some way to justifying its widespread use and popularity.\n\nWhile the tests here produce some clear winners, they also serve to highlight that program performance can vary widely based on the fine-details of a particular run. Even the strongest overall performer can be found lacking in some respects and this means that researchers should be vigilant in their selection of tools for a particular application. In certain instances it may even be necessary to combine two or more approaches to ensure that all relevant aspects of a given dataset are sufficiently characterized and any approach will still require some level of visual inspection and quality control in a clinical setting. The data presented here should facilitate and expedite selection of the correct aligner for a particular task but they do not obviate the requirement for careful consideration nor further testing and analysis on the part of the end-user.",
"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\nLupski JR, Reid JG, Gonzaga-Jauregui C, et al.: Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy. N Engl J Med. 2010; 362(13): 1181–1191. PubMed Abstract | Publisher Full Text\n\nNIH HMP Working GroupPeterson J, Garges S, Giovanni M, et al.: The NIH Human Microbiome Project. Genome Res. 2009; 19(12): 2317–2323. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTreangen TJ, Salzberg SL: Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet. 2011; 13(1): 36–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoore B, Hu H, Singleton M, et al.: Global analysis of disease-related DNA sequence variation in 10 healthy individuals: implications for whole genome-based clinical diagnostics. Genet Med. 2011; 13(3): 210–217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDahl A, Mertes F, Timmermann B, et al.: The application of massively parallel sequencing technologies in diagnostics. F1000 Biol Rep. 2010; 2: 59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNielsen R, Paul JS, Albrechtsen A, et al.: Genotype and SNP calling from next-generation sequencing data. Nat Rev Genet. 2011; 12(6): 443–451. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPattnaik S, Vaidyanathan S, Pooja DG, et al.: Customisation of the Exome Data Analysis Pipeline Using a Combinatorial Approach. PLoS One. 2012; 7(1): e30080. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Homer N: A survey of sequence alignment algorithms for next-generation sequencing. Brief Bioinform. 2010; 11(5): 473–483. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010; 26(5): 589–595. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLunter G, Goodson M: Stampy: A statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 2011; 21(6): 936–939. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalzberg SL, Pertea M: Do-it-yourself genetic testing. Genome Biol. 2010; 11(10): 404. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzabo C, Masiello A, Ryan JF, et al.: The breast cancer information core: Database design, structure, and scope. Hum Mutat. 2000; 16(2): 123–131. PubMed Abstract | Publisher Full Text\n\nHomer N, Merriman B, Nelson SF: Bfast: an alignment tool for large scale genome resequencing. PLoS One. 2009; 4(11): e7767. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHercus C: Novoalign v2. 2011; 07.\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\nMcKenna A, Hanna M, Banks E, et al.: The genome analysis toolkit: a mapreduce framework for analyzing next-generation dna sequencing data. Genome Res. 2010; 20(9): 1297–1303. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "150",
"date": "25 Jul 2012",
"name": "Thomas 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",
"responses": []
},
{
"id": "151",
"date": "26 Jul 2012",
"name": "Vera Kalscheuer",
"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": "152",
"date": "27 Jul 2012",
"name": "Mihaela Pertea",
"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\nIn this paper the author sets out to investigate the performance of several alignment tools and to assess their ability to accurately detect known mutations when used in a variant calling pipeline. This is an important issue to address before designing a particular analysis pipeline for variant detection. However, this paper makes multiple very strong claims about the superiority of various alignment algorithms based on highly flawed computational experiments. Overall the results are at best misleading, and many of the conclusions are simply wrong.Our concerns are related to the following issues:1. Concerns about the experimental design:The experiment claims to measure the accuracy, and in particular the sensitivity and FDR rate, for many sequence aligners. Unfortunately, it simply doesn’t measure anything of the sort. Instead, it measures the sensitivity and FDR of the GATK SNP pipeline, a complex series of programs with many, many parameters, with different aligners fed into the very first step of GATK. GATK is exquisitely sensitive to these parameters; in our experience we can easily increase the number of SNPs by a factor of 5-fold simply by varying its parameters, REGARDLESS of the alignments provided at the front end. Unless the author optimizes GATK for each aligner – which he explicitly did not do – these results are simply invalid. Thus the whole experiment is deeply flawed.It is not sufficient, in a benchmarking test like this one, to use only default running parameters (as the author says he did), and to make no effort at careful evaluation of what would be the best parameters to use for each aligner in that specific experiment. If the author wishes to compare aligners as part of a complex pipeline (GATK), he needs to do much more work than the simple push button runs he did here.The whole point of simulated data ought to be that one can check each read and see if it was aligned to the correct place. This should be easy to do as all the reads are simulated and therefore their location is known a priori. If (and only if) the author compared the alignments to the true alignment, then he could report valid findings about the sensitivity at finding SNPs, indels, etc. He did not do this, which is somewhat astonishing. As it stands, the main results – including Tables 3 and 4 and Figure 1 – are simply wrong.Also, in order to make his results reproducible, the author should provide the alignment results for all programs, as well as the exact command lines used for each aligner. Just specifying that he ran the aligners with parameters “as close as possible” to defaults is not enough.2. Running time evaluations:Another major conclusion of the paper concerns run times, which the author reports in a separate section (3.2). An obvious flaw here is that running the aligners on such small datasets (each only 200,000 reads) cannot properly differentiate the relative running times of the different programs, especially the faster ones. Exome sequencing, a very common experiment today, generates roughly 100 million reads per experiment – 500 times larger than each sample data used here. Whole-genome data sets are much larger. To provide any realistic run time findings, the author needs to load at least an exome-sized data set and run it. He doesn’t need to use simulated reads – many exomes are publicly available. Since he is only measuring run time, he doesn’t need to worry about the sensitivity of these alignments, just speed.If the author wants to report findings about run-time, he needs to scrap this experiment and run a more realistic data set. If 100 million reads, not large by today’s standards, swamps the ability of any aligner to handle it, then he can report that.Other comments in the alignment section are not justified. For example, claiming that “most alignment times recorded here might be considered manageable for most purposes” seems to be little more than the author’s unsupported opinion, based on a relatively tiny number of reads.3. Other significant concerns:a). The author used the Stampy package to simulate the reads from the BRCA1 region. What was the reason that this particular read simulator was used, and not another one that is independent from all aligners involved? E.g., the Mason simulator is considered to be relatively realistic. The Stampy simulator might give an unfair advantage to the Stampy aligner.b). Why did the author align the simulated reads only to chromosome 17? If this is supposed to simulate a targeted sequencing experiment, why not just align to the BRCA1 region, which is far, far smaller than the entire chromosome? A much more realistic design would be to align to the whole human genome, which is normally done for real data where contamination from other parts of the genome is common. The author should also specify how he obtained the index required by the different aligners, and how long it took to create such an index (from the running times of the programs presented in the paper I assume this time was not included).c). The way the programs were run is completely unclear, since no command line options are provided. Besides a step required to create an index (see above), some of the aligners require two steps to be run (e.g. BWA requires both an ‘aln’ and a ‘samse/sampe’ commands to be run; Stampy can be run in a hybrid version with a BWA option first). Were both of these steps included in the running times presented? Most of these programs have many options that can increase their sensitivity at the cost (sometimes small, sometimes not) of increased run time.d). The author makes a technical error in classifying aligners into two categories, “based on either hash tables or suffix trees.” The Burrows-Wheeler Transform (the basis of Bowtie, BWA, and SOAP2) is simply not a suffix tree. Further, it is not only simplistic but incorrect to state that hash-based programs are generally more sensitive, while the ones based on suffix trees are faster. That is wrong in multiple ways; there are many examples of hash-based approaches that are fast but not sensitive, and suffix-tree approaches don’t have to be faster. These features (speed/sensitivity) depend much more on the numerous implementation details, of which the author appears to be unaware.e). The two wrapper scripts (sam2bam.sh and gatk.sh) that the author mentions that he made available do not seem to be present.f). Each of the 67 data sets presented in the paper include 20 SNPs and 13 indels. Why use 67 data sets? And why have exactly the same number of SNPs and indels in each one? What criteria were used to include these particular numbers of SNPs and indels? Since each data set is representative for only one variant of the BRCA1 gene, how likely it is that in real data these 20 SNPs and 13 indels will appear at the same time in the gene? This is an unrealistic data set that has a bizarrely skewed bias.g). The author states – when referring to Figure 3 – that the size of the indels influences their detection rates. He specifically says that the “size of the effect varied by aligner with BWA and Novoalign showing good detection rates for all but the largest deletions.” This statement is simply not correct: BWA cannot find large deletions (by design). Neither can Bowtie. However, GATK can find larger deletions in some cases, even if the input alignments don’t detect them. There are also entirely separate programs (e.g., Pindel) designed to find larger indels, and researchers looking for large indels know about these programs (and use them). This whole discussion again reflects the fundamental flaw in the experimental design: the author is measuring GATK’s performance, not the performance of the aligners. In addition, the author’s interpretation of Figure 3 seems biased, and is not supported by the data in the figure itself.4. Minor concerns:a). PPV is defined differently in the main body of the paper and in Table 6′s caption.b). The author needs to include citations or at least web addresses for all the aligners presented in the paper.c). I assume GLG in Table 5 is in fact CLC.d). Where did the author collect the known mutations for the BRCA1 gene from? He needs to provide citations.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-2
|
https://f1000research.com/articles/1-14/v1
|
07 Sep 12
|
{
"type": "Opinion Article",
"title": "Progress and challenges in the computational prediction of gene function using networks",
"authors": [
"Paul Pavlidis",
"Jesse Gillis"
],
"abstract": "In this opinion piece, we attempt to unify recent arguments we have made that serious confounds affect the use of network data to predict and characterize gene function. The development of computational approaches to determine gene function is a major strand of computational genomics research. However, progress beyond using BLAST to transfer annotations has been surprisingly slow. We have previously argued that a large part of the reported success in using \"guilt by association\" in network data is due to the tendency of methods to simply assign new functions to already well-annotated genes. While such predictions will tend to be correct, they are generic; it is true, but not very helpful, that a gene with many functions is more likely to have any function. We have also presented evidence that much of the remaining performance in cross-validation cannot be usefully generalized to new predictions, making progressive improvement in analysis difficult to engineer. Here we summarize our findings about how these problems will affect network analysis, discuss some ongoing responses within the field to these issues, and consolidate some recommendations and speculation, which we hope will modestly increase the reliability and specificity of gene function prediction.",
"keywords": [
"A central challenge in genomics is the determination of gene function. As data sets characterizing genes grow in size and complexity",
"it seems self-evident that computation can assist in inference as to gene function. However",
"despite extensive work over the past decade",
"computational determination of gene function has made only uncertain progress. With the important exception of the use of sequence similarity",
"it is still uncommon for experimental researchers to use computational gene function prediction methods as a starting point in a study. Instead",
"such methods seem more commonly used by computational methods developers",
"and by experimentalists who are seeking post-experiment interpretation of a result (with the attendant danger of confabulation). While there are exceptions",
"in the past few years we have been struck by the gap between the proliferation of function prediction methods and the rate of discovery of gene function",
"particularly for genes which are not already well characterized",
"despite the enormous increase in the amount of available data."
],
"content": "Background\n\nA central challenge in genomics is the determination of gene function. As data sets characterizing genes grow in size and complexity, it seems self-evident that computation can assist in inference as to gene function. However, despite extensive work over the past decade, computational determination of gene function has made only uncertain progress. With the important exception of the use of sequence similarity, it is still uncommon for experimental researchers to use computational gene function prediction methods as a starting point in a study. Instead, such methods seem more commonly used by computational methods developers, and by experimentalists who are seeking post-experiment interpretation of a result (with the attendant danger of confabulation). While there are exceptions, in the past few years we have been struck by the gap between the proliferation of function prediction methods and the rate of discovery of gene function, particularly for genes which are not already well characterized, despite the enormous increase in the amount of available data.\n\nIn a recent series of papers1–3, we lay out a case that much research for the analysis of gene function from network-like data (using Guilt By Association; GBA) is based on somewhat shaky premises. The guilt by association principle states that genes with similar functions will tend to be associated (or possess similar properties), allowing previously unknown functions of a gene to be statistically inferred given some prior knowledge about other genes, and association data. Our studies were specifically motivated by challenges we encountered in applying GBA to real-life gene function prediction problems. We uncovered a range of underlying biases that caused the results of GBA to be misleading, which turned out to be pervasive yet previously undocumented. We believe the biases we have described are part of the reason for the relatively limited success of computational GBA. In those papers we described control experiments and other considerations that, we hoped, would help the field move forward. At the same time, we recognized that the challenges were profound and could not identify a one-size-fits-all solution that avoids the biases yet still yields demonstrable and useful gene function prediction performance. In the months since our publications appeared, we have had the opportunity to discuss our findings with many colleagues, and realized that there was a need to summarize and unify the arguments we made. In this commentary we begin by briefly reviewing the key findings of our studies, expand on some points, and address some of the issues that have come up in the meantime. Our aim is to spark further discussion and we hope take advantage of this venue to provide updates and additions.\n\nIn discussing GBA, we are specifically referring to the use of the inference as a computational tool to predict gene function from large data sets. As a biologically-motivated principle used to infer gene function on a gene-by-gene basis, GBA is not controversial and long predates the advent of “gene network analysis” approaches. In addition to its potential use for predicting gene function, the GBA principle is also used as “independent” verification of experimentally derived target gene groups or data. In these cases, the ability of an algorithm to learn a group of genes using network data is taken to provide some measure of confidence that the gene set (or possibly network data) has some functional meaning. Indeed, this application is probably more common than the use of GBA for making “de novo” predictions.\n\nTo be more concrete about what we mean by GBA, we are concerned with a large class of computational approaches aimed at predicting gene function, which all take as input four things:\n\n1. A set of candidate genes, which may be all genes in the genome or a more focused set such as those in a candidate genetic locus. The latter case is often referred to as the “disease gene candidate prioritization” task4 but the distinction is not important for our discussion.\n\n2. One or more target gene groups of interest typically defined around a function, such as “synaptic plasticity”, “involved in breast cancer” or “required for the stress response”. We wish to use GBA to assign one or more of the candidate genes to the target gene group. From the point of view of the candidates, GBA assigns a novel target group membership (function) to a particular gene. Operationally, a target group is defined by the set of genes which are already known to have the given property such as membership in a Gene Ontology (GO) group. A target is used even in approaches lacking an explicit target group: we want to be able to act on what we learn, so it has to fit into some pre-existing scheme. Thus while a sequence similarity search does not require a target gene group, interpretation of the results uses such information.\n\n3. Data that contains information about associations or similarities among the target and candidate genes. These data are often represented or thought of as a network, but this is not necessarily explicit. Our studies relied largely on coexpression, protein interactions, and genetic interactions, but we also performed experiments with “networks” constructed from sequence patterns, phylogenetic profiles, and phenotype and disease association profiles. Though we use the term “network” it is important to distinguish between this type of abstract network used for inference, and gene networks that represent physiochemical interactions in the cell, though the line between these can be fuzzy.\n\n4. An algorithm for transferring functional labels from the target genes to the previously unlabeled candidate genes.\n\nIn this article, we use the term “GBA” to refer to the combination of these four factors, not to any one of them. The output of GBA, for a given target gene group (function) is usually a ranking of the candidate genes, where the genes ranked most highly are those which the algorithm predicts are most likely to belong to the target group, given the data. Many (perhaps most) studies undertake evaluations using the Gene Ontology5 as targets under a cross-validation scheme. Performance is typically evaluated using the area under ROC curves, or precision-recall curves. Some studies undertake experimental validation of a subset of “novel” predictions, often focusing on a particular phenotype or function of interest.\n\nIn a previous study2, we show that algorithms that use “label propagation” or related approaches can be replaced by methods that, given a sparse network, first propagate edges (so that indirect edges become lower-weight edges in the network), and use a “simple guilt by association” method (neighbor voting). For coexpression data, our data indicate that if possible the original kernel (coexpression matrix) should be used, rather than a sparsified representation. We also showed that coexpression data behaves much like protein interaction data when enough data is combined.\n\nIn another study1, we analyzed the effects of bias in the prevalence of genes across target gene sets. By prevalence we mean the number of target gene sets a gene belongs to; this can be thought of as a type of gene multifunctionality. We show that a list of genes ordered by prevalence in GO (and other schemes that might be viewed as alternatives such as KEGG) performs comparably or better to real machine learning algorithms over many prediction tasks, despite lacking any specificity to the particular learning task. In another paper we provide anecdotal evidence that such effects are likely to be at play in disease candidate gene prioritization6. Further, node degree in many networks is correlated with the number of GO terms a gene has, so that much of the performance as evaluated with ROC curves can be explained by algorithms simply assigning all functions to high-degree nodes. We showed that this problem is not readily fixed by various node-degree weighting schemes including filtering out high-node degree genes.\n\nIn our most recent work3, we assessed the use of GBA within network data and show that a large fraction of the apparent performance not explained by pure node degree effects are due to the impact of a very small number of edges in the networks, from which it is very difficult to glean generalizable performance. The exception were protein complexes, which display a clique-like structure in many networks, but again it is impossible to generalize from such patterns to make new (non-trivial) predictions.\n\nWe realize, and tried to document1–3 that many researchers in the field have at least a vague sense that something is wrong. In that sense, what we are saying is not news. Our contribution is that we have attempted to document precise explanations for problems which have gone unnoticed for a long time. In the following sections we first summarize the problems we see with GBA, go on to describe why the problems are difficult to fix; provide some suggestions for best practices; and finally close with some speculation.\n\n\nA few problems with GBA\n\nPrior knowledge about gene function is very biased toward well studied genes. One of our most important claims is that the Gene Ontology (or any of its relatives, which encompasses most such schemes) aligns to the data in ways that are not helpful2,3. It may be that our collective human-constructed “ontology” of gene function, as powerful as it has been at organizing information, just doesn’t have a sufficiently general relationship with biology as we measure it in the lab. This means that the tasks we are able to learn are so strongly biased by relatively uninteresting interactions between data and the target that more biologically specific signals are probably being missed. In other words, it may be a case where true-but-uninteresting prediction is all but impossible to avoid or improve upon. An interesting side effect of thinking that GO (et al.) is not the ideal target is that the “best GBA method” (by some currently unknown standard) might actually perform badly on GO. Evaluation of such a method (if it exists), other than by exhaustive experimental validation, is currently impossible. We also caution that some of our other points are confounded by our own reliance on existing schemes (including GO), so we leave the possibility open that there is a leap waiting to be made.\n\n“Good” predictions tend to be generic predictions. Our results imply that many predictions will be generic2 so the most likely candidate genes will tend to be a gene that has numerous other functions – whether this is already known or not. The gene that is predicted to be involved in muscle development might also be involved in twenty other processes. It is clearly of interest to make predictions that are functionally specific, or at least to know how specific they are. If one is to claim that a GBA approach is “good”, one of the criteria might be that the gene doesn’t have functions that weren’t predicted. Ideally in a validation, one should show that disruption of the gene does not affect other functions that were not predicted, thus providing some measure of specificity. We recognize that our suggestion of additional costly and difficult control experiments is unlikely to be popular. However, we fear that the current situation feeds a vicious cycle where GBA continues to seem to work (somewhat) in a way that is misleading to computational method developers and also driving even stronger biases in biological knowledge.\n\nHigh performance in cross-validation doesn’t help us find what “works”. In most studies, cross-validation is used to estimate which functions are learnable. The hope is that that high cross-validation performance for a particular function will generalize to novel predictions for that function, and therefore will hold up in experimental validation. However, our results indicate that while there are “good” pieces of information in network data, the presence of one such piece of information for a given function does not imply the presence of more to be discovered3. This graininess in network information (or lack of “systemic” functional encoding) may mean that a limited number of experimental validations do not validate a method as a general-purpose way to determine function. We see it as a substantial challenge to assess what works. For example, how do we define data that is “good” for GBA in a way that will generalize to novel predictions rather than simply tell us “what worked, worked” (maybe only that one time).\n\nUnbiased data is desirable but (probably) non-existent. One way to avoid some problems is to only use data from experiments which analyze all genes in the genome at once. This is not always possible, and even when it is, there are still often biases towards (for example) genes which are conserved, have readily cloneable nucleotide sequences, are expressed at readily detectable levels, have well-annotated gene structures, have immunogenic products (so they yield good antibodies), are suppressible by siRNA, are non-essential, and so on. We refer to these as biases because any correlation between the prevalence of genes in a dataset and their prevalence within GO (number of annotations) will generate apparently effective GBA in which a subset of genes or connection will dominate results with either little specificity or little generalizability. Such factors should be at least considered as confounds, and representation or prevalence discrepancies should ideally held as constant as possible when data sets are integrated. We feel the terminology “unbiased” should be avoided except in relative terms, or that at least it should be clearly stated which biases are being referred to.\n\nEven if GBA works, it may not work well enough for experimental follow-up. It is very unclear what degree of precision is obtained in “real-life” applications of GBA in which experimental validation is performed. Obviously it varies, but our impression is that it is much worse than what biologists would usually refer to as “statistically significant” – that is, 95% confidence in a single prediction. In general, significance from computational methods arises from aggregate performance of groups of genes, but this is less useful in providing (potentially costly) experimental targets. In a recent study, a false discovery rate of 87% was reported7. Another study reported a more impressive but still high false discovery rate of 44%8. Clearly such rates are sufficient to be of use (a bona fide new drug target is worth a lot of trouble), but in our experience they are also low enough to discourage many biologists from routine follow-up of computational predictions. We note that neither of the studies just mentioned were formal assessments in the sense that the benchmark was not held in escrow by a third party. In addition, they only validate predictions for a couple of functions, so it may be risky to generalize to other functions. Additional assessments that take a formal approach would help advance the field, but designing such challenges and evaluation is not trivial.\n\n\nWarning against easy-fixes\n\nIt is easy to hide the problems. Many of the problematic aspects of an analysis are most easily observed due to their side-effects. For example, one of our observations is that, according to many commonly used metrics, a sizeable fraction of performance can be explained by simply predicting high node degree genes often (because high node degree genes are often multifunctional). It is trivial to remove the appearance of this problem, particularly by alterations in the metric or network (simply add random connections to make node degrees equivalent). However, the underlying problem (overly generic results contribute to performance) could remain with the issue simply having been hidden from view. Similarly, some problems are made clearer when using ROC while others are revealed by using precision-recall (ROC being susceptible to overly generic predictions, while precision-recall is affected by non-generalizable one-off observations). Unfortunately, one possibility is that some algorithm’s bad behavior will simply become harder to observe in the face of better characterization of the problems.\n\nImproving GBA cannot be done by enforcing a better match between data and targets. It may be tempting to consider cross-validation performance as the most important metric of a method’s utility, but this is a very dangerous assumption (as is well-appreciated in the machine learning field). In one version of this point of view, poor cross-validation performance is viewed as meaning that the data are not good enough, and data should therefore by chosen based on what works best. Thus some researchers have proposed to restructure or re-weight data that better matches the gold standard (e.g. GO)9–11. A closely related tactic is using the Gene Ontology as a data source12. The potential for overfitting/overtraining and logical circularity encompassed by such approaches should be abundantly clear. In particular, selecting the best performing datasets may tend to increase the biases which do, indeed, yield high (but useless) performance. Similarly, one might consider changing the ontologies so that they better match guilt by association results. That is, instead of changing the data change the target groups (thus far we have only heard this raised as a possible strategy). We can only think of this as “moving the goal posts” and extremely risky if all parties are not aware of the effect this would have on predictions. We think the separation of GBA as a top-down principle (in algorithms) must be maintained from bottom-up observations (from which derive ontologies and data). Otherwise GBA’s potential for making new discoveries will be crippled.\n\n\nSuggestions\n\nWe shouldn’t confound discussions about performance with novel or non-standard metrics. As important as settling on a method is settling on an evaluation metric. Everybody’s approach works best by some metric. Introducing novel or ad hoc metrics to go with a novel prediction method simply muddies comparisons. In our experience, biologists are most interested in precision, less interested in recall, and generally not receiver operating characteristics (whether they know the jargon or not). Reports of performance should be corrected for multiple testing if multiple metrics are used.\n\nIgnoring “low quality” GO evidence codes is not as straightforward as it seems. It is common to exclude “IEA” (inferred by electronic annotation) GO annotations from computational analyses. The rationale usually given is that IEA annotations are unreliable (this is somewhat ironic since what GBA methods are trying to do is provide IEA annotations). In any case, hiding such annotations from training and test data can lead to the possibility that one can merely reconstruct them in a manner that, in effect, the GO consortium has already done (predominantly by leveraging sequence similarity). In addition, many high-quality annotations in GO started out as “IEA” and were specifically targeted for further manual curation. This throws into question the purity of “rollback” validations that consider only manual annotations from before a certain time limit during training, and test on more recent annotations. On the balance it is probably best to include IEA annotations throughout and that to be of real value, function predictions should go beyond what GO is already providing.\n\nFunction prediction should not only be information retrieval. We wish to make a distinction among different types of “automated annotation”. There are many pieces of functional data in the biomedical literature that have not been converted to (for example) GO annotations, so they are difficult to access. It is an interesting task to attempt to mine such information from the literature directly or indirectly. However, this is not prediction, but information retrieval. This basically ensures that in some sense the most confident predictions made will tend not to be novel, but capture information that is already known. We feel this task is different from cases where the function prediction is “de novo”, without using existing functional annotation. We realize there is no firm line between a supposed piece of information retrieval and a prediction, and it is an interesting question whether it is even possible to make de novo predictions (or even what that would mean in a pure sense), because information has the potential to leak between data types in insidious ways. For example, expression microarrays are biased against the representation of poorly-characterized genes.\n\nAssessments of performance should use well-chosen priors. A theme that has emerged is the urgency to more carefully assess null performance in the evaluation of gene function prediction. In particular, we should be determining the performance of algorithm learning from the data relative to the performance using well-chosen priors. This is rather obvious because methods can exploit the choice of prior, but less obvious because current methods of validation tend to miss this point. We should be thinking of ways to cheat at prediction (yielding apparently good but useless performance) and taking this as the baseline upon which we must improve. Usually if machine learning algorithms can cheat in this sense, they will (without any collusion on the part of their designers). Information leakage between training and test data, despite the best intentions, is very hard to avoid. We showed that current approaches inadvertently strongly exploit the choice of prior, and this makes prediction methods much less useful than is commonly claimed. This decrease in utility comes both from limitations in functional specificity (many predictions will be very general such as “growth”), prediction specificity (many predictions will just be wrong), and “rich get richer” effects (genes that have lots of functions will be proposed to have more). We urge investigators to include assessments of the impact of node degree and of critical edges on their predictions.\n\nData drives performance more than algorithms. We presented evidence that the variance in apparent prediction performance among approaches is largely explained by differences in the data, not the algorithm. Differences in algorithms play a relatively minor role. This is a banal observation in machine learning at large, but in gene function prediction we are only learning this lesson now. Besides the experiments presented in2, we point to the close performance of different methods in the Mousefunc assessment13. A recent evaluation of gene network inference methods (which largely rely on guilt by association), the DREAM5 challenge, further supports our claim. Simple “off-the-shelf” methods performed competitively with other more complex approaches, and the best methods were distinguished by which of the available data were used14. This is not to say that one can’t do badly at such an assessment, but that the upper bound is rapidly reached by a reasonably good algorithm. We feel it is unlikely that substantial progress will be made by working on developing new algorithms, and that the focus should be on the data. This is a sufficiently important issue that in our opinion, it would be interesting to consider basing critical assessments on a single method (or aggregate of methods), and letting data vary as desired.\n\nContext specificity of data can help. One of the factors that cause functional predictions to be generic is that they often use data that combined numerous experimental conditions; protein interaction networks constructed by aggregated data sources are chief among these. It would be helpful to be able to show that a function prediction is appropriately context-specific. For this reason, we predict that analyses that use context-specific data will increase. One example of data that is in principle context-specific is gene co-expression. Co-expression occupies an unusual place in high-throughput analyses. For researchers focused on particular biological problems, it is a relatively easy way of obtaining context specific data. In contrast, to researchers focused on computational methodologies it is often regarded as a noisy data source which poorly recapitulates known biological function as catalogued in GO. However, many of our results support the view that co-expression data is much more difficult for algorithms to “cheat” on, since it less specifically captures literature biases. Other forms of data specificity (e.g., different environments or phenotypes used for assaying genetic interactions) are also strongly desirable to determine the robustness of results. Of course, even with such data it remains important to perform controls for specificity (e.g., constructing control networks, checking control gene sets that matched with respect to properties that allow cheating).\n\n\nSpeculation\n\nBiases affect network analyses besides functional prediction. Because we suspect most variance in GBA performance is data driven, we believe the biases we have discussed in GBA are also partially data driven. That is, they are not just a feature of the GBA method, but of the data. An example of another type of analysis is predicting interactions themselves, as opposed to functions. We strongly doubt whether “association by guilt” is fundamentally different from guilt by association. For example, we would expect some predicted connections to be trivial (e.g., interacting promiscuous proteins) with protein complexes again being a special case (i.e., filling in a few missing connections from a fully connected sub-graph). We likewise suspect that our finding that many function prediction algorithms act as if they are reconstructing filtered values through indirect connectivity1 may apply to predicting connectivity itself; perhaps the best predicted connections are simply those that were “nearly known” to begin with. Another topic we have not considered extensively is unsupervised approaches, exemplified by the WGCNA coexpression graph clustering method15. These methods are typically combined with looking for clusters that are enriched for certain gene functions. We consider it likely that such approaches are subject to many of the same problems as the supervised methods, including biases toward highly-annotated and highly-connected genes.\n\nGO is here to stay. As outlined above, it is unlikely that we will have a true replacement for GO any time soon, in part because there is no clear idea of what it would be based upon. Currently available alternatives to GO (as a source of functional gene sets) such as KEGG mainly resemble it in terms of annotation biases. In the meantime, the appropriate use of GO is not at all settled (e.g.,16) and we expect that awareness of the limitations of GO as a target for function analysis will have an increasing impact.\n\n\nConclusions\n\nDespite all the problems and limitations we describe, we believe there are still good reasons to be optimistic about the future of GBA. While claims of being able to predict function globally should be treated with greater skepticism, focused analyses will likely continue to pay gradual dividends. Similarly, while validation of globally applicable methods may succeed in focused projects, the methods themselves are very unlikely to be universally successful and claims within the literature should be tempered by more detailed and explicit discussion of exact limitations.",
"appendix": "Author contributions\n\n\n\nPP and JG conceived and wrote the article. PP wrote the first draft.\n\n\nCompeting interests\n\n\n\nThe author declares no competing interests related to this article.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nWe are grateful to numerous colleagues who took the time to discuss our work with us, which has helped us see where we have left some issues unclear or worth elaborating on. We thank Pavlidis lab members for comments on the manuscript.\n\n\nReferences\n\nGillis J, Pavlidis P: The role of indirect connections in gene networks in predicting function. Bioinformatics. 2011; 27(13): 1860–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGillis J, Pavlidis P: The impact of multifunctional genes on \"guilt by association\" analysis. PLoS One. 2011; 6(2): e17258. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGillis J, Pavlidis P: \"Guilt by association\" is the exception rather than the rule in gene networks. PLoS Comput Biol. 2012; 8(3): e1002444. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoreau Y, Tranchevent LC: Computational tools for prioritizing candidate genes: boosting disease gene discovery. Nat Rev Genet. 2012; 13(8): 523–36. PubMed Abstract | Publisher Full Text\n\nAshburner M, Ball CA, Blake JA, et al.: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25(1): 25–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQiao Y, Harvard C, Tyson C, et al.: Outcome of array CGH analysis for 255 subjects with intellectual disability and search for candidate genes using bioinformatics. Hum Genet. 2010; 128(2): 179–94. PubMed Abstract | Publisher Full Text\n\nMcGary KL, Park TJ, Woods JO, et al.: Systematic discovery of nonobvious human disease models through orthologous phenotypes. Proc Natl Acad Sci U S A. 2010; 107(14): 6544–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHess DC, Myers CL, Huttenhower C, et al.: Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis. PLoS Genet. 2009; 5(3): e1000407. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee I, Li Z, Marcotte EM: An improved, bias-reduced probabilistic functional gene network of baker's yeast, Saccharomyces cerevisiae. PLoS One. 2007; 2(10): e988. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHibbs MA, Hess DC, Myers CL, et al.: Exploring the functional landscape of gene expression: directed search of large microarray compendia. Bioinformatics. 2007; 23(20): 2692–9. PubMed Abstract | Publisher Full Text\n\nMostafavi S, Ray D, Warde-Farley D, et al.: GeneMANIA: a real-time multiple association network integration algorithm for predicting gene function. Genome Biol. 2008; 9(Suppl 1): S4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTranchevent LC, Barriot R, Yu S, et al.: ENDEAVOUR update: a web resource for gene prioritization in multiple species. Nucleic Acids Res. 2008; 36(Web Server issue): W377–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPena-Castillo L, Tasan M, Myers CL, et al.: A critical assessment of Mus musculus gene function prediction using integrated genomic evidence. Genome Biol. 2008; 9(Suppl 1): S2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarbach D, Costello JC, Küffner R, et al.: Wisdom of crowds for robust gene network inference. Nat Methods. 2012; 9(8): 796–804. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhao W, Langfelder P, Fuller T, et al.: Weighted gene coexpression network analysis: state of the art. J Biopharm Stat. 2010; 20(2): 281–300. PubMed Abstract | Publisher Full Text\n\nThomas PD, Wood V, Mungall CJ, et al.: On the Use of Gene Ontology Annotations to Assess Functional Similarity among Orthologs and Paralogs: A Short Report. PLoS Comput Biol. 2012; 8(2): e1002386. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "265",
"date": "11 Sep 2012",
"name": "Yves Moreau",
"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 is an opinion piece based on previous peer-reviewed work of the authors. As such it appears of appropriate quality.",
"responses": []
},
{
"id": "266",
"date": "25 Sep 2012",
"name": "Jonathan D Wren",
"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 synthesize, summarize and clarify their prior work and observations on predicting gene function using linked (networked) data to infer properties of unannotated/uncharacterized nodes on the basis of their surrounding nodes.Their observations and cautionary notes are important for people attempting to infer function using this “guilt by association” (GBA) approach, or, as it might alternatively be called in machine learning, the multi-label classification problem.Some of the problems brought up, though, may not be readily surmountable, such as the bias in annotation whereby some genes are extremely well annotated and some categories/labels are far more frequent than others. Insofar as science proceeds by building upon prior observations, the use of existing gene knowledge to infer new knowledge will only be able to frame predicted gene function in terms of previously characterized functions. e.g., an uncharacterized gene predicted to affect DNA repair cannot have its precise role/purpose in DNA repair predicted, but rather can only be associated with previously characterized DNA repair phenotypes (e.g., non-homologous end joining, double-strand break repair, etc). Additionally, its not clear what the gap is between how frequent a phenotype (e.g., angiogenesis) is empirically and how frequent it is among predicted functions. This is an important limitation of the method, but insofar as existing data can be used to guide experimentation, it is still useful as long as one takes into account the authors’ caution that “good” algorithmic predictions tend to be generic.In this reviewer’s experience, and with his own particular approach to the problem, the use of GBA has led to successful characterizations of several genes (PMIDs: 22187488, 21868574, 19646878, and one recently accepted for publication in Neurosurgery). This does not invalidate the authors’ points, which are well taken by this reviewer, nor is it proof of efficacy. However, it is offered as non-systematic evidence that GBA has been useful/successful in guiding experimentation. Systematic approaches and appropriate negative controls are important, as the authors suggest, and need to be done. The field is relatively young, however, and I think the observations by Pavlidis and Gillis discussed here are important to help separate the promise and the peril of research in this area, as I believe the GBA approach (in principle, not necessarily in practice) holds the potential to aid experimental science. Their observations are important to consider in terms of interpreting prediction results and guiding future research in predicting gene function.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-14
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https://f1000research.com/articles/1-6/v1
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26 Jul 12
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{
"type": "Case Report",
"title": "Sirenomelia in a Cameroonian woman: a case report and review of the literature",
"authors": [
"Frederick LI Morfaw",
"Philip N Nana",
"Philip N Nana"
],
"abstract": "Sirenomelia is a rare congenital malformative disorder characterized by fusion of the lower limbs giving a characteristic mermaid-like appearance to the affected foetus. We report a case of sirenomelia occurring in a 19 year old Cameroonian woman following premature rupture of membranes and associated cord prolapse. This is the first documented case in this country. We highlight some of the cultural myths associated with this disorder and discuss our findings relative to the present literature and related controversies on its etiopathogenesis.",
"keywords": [
"Sirenomelia is an extremely rare congenital malformative disorder",
"which is often fatal. Its incidence is estimated at about 1 in 100 000 pregnancies1. The most prominent yet inconstant feature of this malformative disorder is the complete or partial fusion of the lower limbs. The resultant infant bears a resemblance to the mermaid of ancient Greek mythology2. Hence this disorder has equally been referred to as symmelia",
"sympodia monopodia",
"sympus",
"but most commonly as the ‘mermaid syndrome’ since the fusion of the lower limbs gives a characteristic mermaid-like appearance3. In the African context",
"such mermaid-like babies are referred to as ‘mammy-water babies’",
"and bear an evil connotation associated with witchcraft and sorcery. This anomaly predominantly affects males (sex ratio 2.7:1)4",
"and is frequent among one of two monozygotic twins. It is usually incompatible with life",
"yet there are a number of reported cases of surviving infants with this condition in the English literature5–9. Its ethiopathogenesis remains controversial."
],
"content": "Introduction\n\nSirenomelia is an extremely rare congenital malformative disorder, which is often fatal. Its incidence is estimated at about 1 in 100 000 pregnancies1. The most prominent yet inconstant feature of this malformative disorder is the complete or partial fusion of the lower limbs. The resultant infant bears a resemblance to the mermaid of ancient Greek mythology2. Hence this disorder has equally been referred to as symmelia, sympodia monopodia, sympus, but most commonly as the ‘mermaid syndrome’ since the fusion of the lower limbs gives a characteristic mermaid-like appearance3. In the African context, such mermaid-like babies are referred to as ‘mammy-water babies’, and bear an evil connotation associated with witchcraft and sorcery. This anomaly predominantly affects males (sex ratio 2.7:1)4, and is frequent among one of two monozygotic twins. It is usually incompatible with life, yet there are a number of reported cases of surviving infants with this condition in the English literature5–9. Its ethiopathogenesis remains controversial.\n\nWe report here the first documented case of sirenomelia in Cameroon, and discuss our findings in relation to the present literature and related controversies of its etiopathogenesis.\n\n\nCase report\n\nMiss N.N, 19 years old G2P0010, a single student at 38 weeks gestation according to her last menstrual period was referred to the Yaoundé Central Maternity for the management of cord prolapse at term. Her history revealed premature rupture of membranes 4 days prior to her consultation, with continuous per vaginal flow of clear liquor. She declares that foetal movements had been present prior to membrane rupture. She eventually developed uterine contractions 4 days later with an associated cord prolapse visible at the introitus. This pushed her to consult at a health centre from where she was referred to our hospital.\n\nIn her past medical history, she was not diabetic nor did she bear any known chronic pathologies. There was no family history of diabetes nor malformations. Her first pregnancy two years earlier ended up in a clandestine voluntary termination of pregnancy by endo-uterine aspiration in the first trimester without any recorded complications. Her present pregnancy resulted from a non-consanguineous union with a 23 year old student. This pregnancy was very poorly followed up in a local health centre with two antenatal consultations. The few tests that were carried out did not reveal any anomalies, but no ultrasound was done. The evolution of the pregnancy so far had been uneventful without any history of teratogenic drug intake or traditional concoctions. On admission the patient was hemodynamically stable, afebrile, with a symphysis-fundal height of 26 cm, absent foetal heart tones, a foetus in cephalic presentation, and a non-pulsating third degree cord prolapse. The working diagnosis of a prolonged rupture of membranes complicated by third degree cord prolapse and intrauterine foetal death was made. The patient was induced using oxytocin and was placed on prophylactic parenteral antibiotics. Labour evolved normally and within 5 hours she expelled a dead first degree macerated foetus with the following morphological abnormalities: (see Figure 1–Figure 6).\n\nDistended abdomen\n\numbilical cord with a single artery\n\nUndetermined sex (absent external genitalia with only a 2×3 cm tag marking the position of the genitalia\n\nImperforate anus\n\nabsence of a urinary meatus\n\nfused lower segment of the body below the pelvis into a single lower limb, with two feet fused posteriorly giving a single flipper-like foot with eleven toes spread out in a fan-like pattern (Mermaid-like or ‘mammy-water’). The foot was oriented anteriorly relative to the trunk, and external palpation gave the impression of probably two femurs and two tibias.\n\nThere was complete placental delivery and uterine revision was done removing membranous debris.\n\nThe birth weight of 2.3Kg at 38 weeks gestation reflected intrauterine growth restriction.\n\nTraditional and cultural beliefs precluded autopsy, and the corpse was handed over to the family for burial. The patient was maintained on parenteral antibiotics for 48 hours then oral relay was done on day 2 post-partum. She received adequate post-partum counselling and was discharged on that day.\n\n\nDiscussion\n\nSirenomelia or mermaid syndrome is an abnormal development of the caudal region of the body involving varying degrees of fusion of the lower limbs with or without bony defects. It is usually associated with other visceral defects such as hypoplastic lungs, cardiac agenesis, absent genitalia, digestive defects, absent kidney and bladder, vertebral and central nervous system defects10,11. The prognosis is usually poor with survival depending on the nature of the visceral anomalies. Death usually results from obstructive renal failure due to renal agenesis or dysgenesis, with survival depending on adequate kidney functioning and renal outflow12. In our patient however we could not ascertain the nature of the full foetal anomalies given that an autopsy was never performed. Moreover, it was a poorly followed up pregnancy without any ultrasonographic evaluation. Furthermore, the third degree cord prolapse was an obvious cause of fetal death. The history was suggestive of foetal movements in the few days prior to membrane rupture, thus this foetus could have been alive prior to membrane rupture.\n\nThe etiology of sirenomelia is unknown and no teratogens have been found in humans13. Certain risk factors however exist. The syndrome has been associated with maternal diabetes mellitus, which is considered to be the most important risk factor14. There is a 200–250 reported relative risk of occurrence in diabetes14. Davari et al. report that up to 22% of these foetuses would have diabetic mothers14. However, other authors refute this association, precising that only about 0.5–3.7% of sirenomelia cases occur in diabetic mothers15–17. Thus the association between maternal diabetes and sirenomelia has therefore been described as weak18. Our patient was not known to be diabetic.\n\nThe syndrome is also reported to be associated with twins. Reports indicate a 100–150 times higher incidence in monozygotic twins relative to dizygotic twins or singletons18. Moreover, about 20% of cases are derived from products of twin pregnancies15. In our patient, this was not a twin gestation, and there was no family history of twinning. A major concern in this syndrome is the possibility of genetic transmission. Although Lynch et al.19 recognised an autosomal form of caudal dysgenesis, no chromosomal abnormalities are found in sirenomelia and it does not recur in families10. This was a reassuring feature for our patient and should serve as a counseling feature for mothers bearing babies with this distressing anomaly. Yet genetic counseling should still be proposed given the reported risk of reoccurrence of 3–5%13.\n\nThe etiopathogenesis of this syndrome has been subject to a lot of debate over time. Numerous theories have been proposed to explain its origin.\n\nFrom an embryological point of view, the sequence of events leading to sirenomelia (sirenomelia sequence) results from an ‘embryological insult’ involving the caudal mesoderm occurring between days 28–32 of foetal life12. By this gestational age, the cloaca is already formed, the kidneys are found in the pelvis while the gonads are intra-abdominal13. Hence any developmental abnormalities of the caudal extremity would affect equally the kidneys, the bladder, the terminal bowels, the pelvic bones as well as the genitalia (excluding the gonads which are intra-abdominal)13. In this sequence, there is renal agenesis, absent genital organs, anal imperforation, absent rectum and dysgenesis/agenesis of the sacrum. There could be vertebral dysgenesis, lower limb atrophy and inconstant lower limb fusion13.\n\nStevenson et al.20 proposed the vascular steal theory to explain the development of abnormalities on the caudal extremity. This theory suggests that there is shunting of blood via an abnormal abdominal artery arising from high up in the aorta towards the placenta. Consequently there is hypoplasia of the vasculature distal to the artery leading to nutritional deficiency of the caudal half of the body21. Hence there may be complete/incomplete agenesis of the caudal structures described above, except the gonads which are intra-abdominal. The single umbilical artery in our patient favours this theory. However, Jaiyessimi et al.18 reported a case of sirenomelia without this vitelline artery steal, indicating that factors other than vitelline artery steal could be responsible for sirenomelia in humans.\n\nA third theory regards sirenomelia as part of the caudal regression syndrome (CRS). This is a rare congenital defect characterized by a broad spectrum of lumbosacral agenesis. This syndrome was described by Duhamel22 to include genitourinary and vertebral anomalies. Caudal regression syndrome is mainly characterized by sacrum dysgenesis, altered spinal cord, urinary incontinence of variable intensity and misplaced lower limbs. Renal dysgenesis and imperforate anus are an inconstant feature. Some authors consider sirenomelia to be the most extreme form of this relentless condition. It is however worth noting that authors such as Pinette et al.12 still describe as speculative the distinction between caudal regression syndrome and sirenomelia.\n\nA fourth theory syndrome described in the literature regards sirenomelia as part of the VACTERL syndrome. VACTERL syndrome involves vertebral, anal, cardiovascular, tracheal esophageal, renal and limb dysgenesis. There is a major overlap in the phenotypic manifestations of sirenomelia and VACTERL18. In most cases, the distinction between sirenomelia sequence and VACTERL lies within the severity of the component defects, and the single lower limb in sirenomelia can be regarded as an indicator of other severe malformations, especially in the gastrointestinal and genito-urinary systems18. In our patient the lack of an autopsy did not permit any assertions to be made as to the relationship with VACTERL.\n\nA fifth theory is the pressure theory which stipulates that external forces acting on the caudal extremity of the embryo causes its hypoplasia13. Attempts at supporting this theory were made by Gardner et al.23, who stipulated that excessive rotation of the neural tube at its caudal end provoked a lateral rotation of the mesoderm causing fusion of the lower limbs, and closure of the primitive bowel and urethra. This theory is however not widely accepted13.\n\nOther less pertinent theories also exist. Yet the overlap in these syndromes/theories waters the debate in the scientific world as to the uniqueness or diversities of these syndromes.\n\nStocker and Heifetz15 classified the sirenomelia sequence into 7 types as shown be in Table 1.\n\nWe did not have any radiographs and could therefore not classify our patient into any of these categories with certainty even though external palpation was in favour of a type I.\n\nThe past medical history of the patient could identify patients at risk. Antenatal diagnosis is possible on ultrasound and x-ray. Ultrasound is however the predominant diagnostic tool. Moreover, there is no open defect which could cause abnormal increases in alpha foeto-protein levels, and strange as it may be, chromosomes are usually normal14.\n\nThe use of ultrasound in diagnosis is however not without difficulty. However, in sirenomelic foetuses, bilateral renal agenesis causes severe oligohydramnios thus limiting ultrasound evaluation of the limbs in the second and third trimesters24,25. However, in earlier gestational ages, the amniotic fluid volume may be sufficient to detect abnormal lower limbs. In such cases, we may notice in addition to abnormal lower limbs, bilateral renal dysgenesis, absent bladder, undetermined external genitalia, anorectal agenesis and lumbosacral agenesis4. Other abnormalities may touch the cardiovascular system and abdominal walls. Ultrasound features permitting confirmation of the diagnosis include lack of tibia/fibula, a single femur, convergent femoral bones, bilateral renal agenesis, polycystic kidneys/renal agenesis, obstructive uropathy and intra-uterine growth retardation21. Antenatal confirmation of the diagnosis justifies a therapeutic termination of the pregnancy.\n\nAt delivery, clinical evaluation is usually sufficient to confirm the diagnosis. In our case, the diagnosis was obvious given the fused lower limbs, single umbilical artery, and imperforate anus. However radiographic images are important if we are to be able to classify the condition according to the Stocker and Heifer classification (Table 1)15. An autopsy permits determination of the extent of the associated anomalies, but is usually of limited use in the African context where cultural norms and beliefs largely precludes its practice.\n\nSirenomelia carries with it a very poor prognosis. Survival is largely dependent on the extent of visceral anomalies, especially obstructive renal failure due to renal agenesis/dysgenesis12. In the case of antenatal diagnosis, a voluntary termination of pregnancy is advisable in order to avoid the physical and psychological stress to parents and the family. This decision however depends on the gestational age of the pregnancy, the severity of the malformations and of course the desires of the parents13.\n\nRecent reports indicate that about 50% of these infants are born alive after 8–9 months gestation13. However most of them die within 5 days of life15. The management of sirenomelia is difficult and expensive, and the outcome is unpredictable13. The main therapeutic modalities are surgical and medical, aimed mainly at maintaining adequate renal function. Surgery to correct the anomaly and separate the fused limbs is usually not a priority as there is no guarantee of its success, and it carries with it an increased risk of compromising the life of an already delicate infant.\n\nThere have been reports of surviving sirenomelic foetuses5–9. Pertinent amongst these is the case of the surviving infant with sirenomelia associated with absent bladder reported by Stanton et al.9. This infant underwent 5 surgeries before the age of 4 years and continues to be bedridden and dependent. In the case described by Pinette et al.12, the infant received a renal transplant using a cadaveric donor kidney. By publication time, this infant was 5 years old with normal renal and cognitive development for her age. However, separation of the lower limbs was indefinitely delayed due to concerns regarding disruption of blood supply to abdominal organs and the transplanted kidney.\n\nThese reflect the constraints, both financial and physical to conservatively manage sirenomelia and reiterate the importance of antenatal diagnosis and voluntary termination of pregnancy especially in resource limited settings like ours.\n\n\nConclusion\n\nSirenomelia remains a rare but peculiar syndrome. Its antenatal diagnosis is possible albeit difficult by ultrasound. Controversies on its etiopathogenesis persist even though it is increasingly believed to be distinct from the caudal regression syndrome as hitherto thought. The associated visceral anomalies are usually incompatible with life. However surviving sirenomelic foetuses have been described with costly conservative management and mediocre results. In the African context, these mermaid-like foetuses are described as ‘mammy-water babies’. This carries with it the connotation of sorcery and witchcraft with which no family wishes to be identified. Therefore antenatal diagnosis and termination of pregnancy is advisable. Knowledge of this rare syndrome is important to dissipate cultural myths whenever it occurs, and free the family from stigmatization.\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\nF.M. received the patient and discussed the case history and management with P.N. F.M. wrote the first draft of the article. F.M. and P.N. read and revised several versions of the manuscript. Both authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nThe authors do not declare any competing interest.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMartinez-Frias ML, Garcia A, Bermejo E, et al.: Cyclopia and sirenomelia in a liveborn infant. J Med Genet. 1998; 35(3): 263–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMessineo A, Innocenti M, Gelli R, et al.: Multidisciplinary surgical approach to a surviving infant with sirenomelia. Pediatrics. 2006; 118(1): e220–e223. PubMed Abstract | Publisher Full Text\n\nKulkarni ML, Sureshkumar C, Sindhur PS, et al.: Sirenomelia with spina bifida. Indian Pediatr. 1994; 31(1): 51–5. PubMed Abstract\n\nValenzano M, Paoletti R, Rossi A, et al.: Sirenomelia. Pathological features, antenatal ultrasonographic clues, and a review of current embryogenic theories. Hum Reprod Update. 1999; 5(1): 82–6. PubMed Abstract | Publisher Full Text\n\nSavader SJ, Savader BL, Clark RA, et al.: Sirenomelia without Potter syndrome: MR characteristics. J Comput Assist Tomogr. 1989; 13(4): 689–91. PubMed Abstract | Publisher Full Text\n\nMurphy JJ, Fraser GC, Blair GK, et al.: Sirenomelia: case of the surviving mermaid. J Pediatr Surg. 1992; 27(10): 1265–8. PubMed Abstract | Publisher Full Text\n\nClarke LA, Stringer DA, Fraser GC, et al.: Long term survival of an infant with sirenomelia. Am J Med Genet. 1993; 45(3): 292–6. PubMed Abstract | Publisher Full Text\n\nMcCoy MC, Chescheir NC, Kuller JA, et al.: A fetus with sirenomelia, omphalocele, and meningomyelocele, but normal kidneys. Teratology. 1994; 50(2): 168–71. PubMed Abstract | Publisher Full Text\n\nStanton MP, Penington EC, Hutson JM, et al.: A surviving infant with sirenomelia (Mermaid syndrome) associated with absent bladder. J Pediatr Surg. 2003; 38(8): 1266–8. PubMed Abstract | Publisher Full Text\n\nTang TT, Oechler HW, Hinke DH, et al.: Limb body-wall complex in association with sirenomelia sequence. Am J Med Genet. 1991; 41(1): 21–5. PubMed Abstract | Publisher Full Text\n\nRodriguez JI, Palacios J, Razquin S, et al.: Sirenomelia and anencephaly. Am J Med Genet. 1991; 39(1): 25–7. PubMed Abstract | Publisher Full Text\n\nPinette MG, Hand M, Hunt RC, et al.: Surviving sirenomelia. J Ultrasound Med. 2005; 24(11): 1555–9. PubMed Abstract\n\nFadhlaoui A, Khrouf M, Gaigi S, et al.: The sirenomelia sequence: a case history. Clin Med Insights Case Rep. 2010; 3: 41–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavari F, Googol N, Kaveh M, et al.: Sirenomelia (mermaid syndrome) in an infant of a diabetic mother. Acta Medica Iranica. 2003; 41(1): 69–72. Reference Source\n\nStocker JT, Heifetz SA: Sirenomelia. A morphological study of 33 cases and review of the literature. Perspect Pediatr Pathol. 1987; 10: 7–50. PubMed Abstract\n\nDuncan PA, Shapiro LR, Klein RM, et al.: Sacrococcygeal dysgenesis association. Am J Med Genet. 1991; 41(2): 153–61. PubMed Abstract | Publisher Full Text\n\nLynch SA, Wright C: Sirenomelia, limb reduction defects, cardiovascular malformation, renal agenesis in an infant born to a diabetic mother. Clin Dysmorphol. 1997; 6(1): 75–80. PubMed Abstract | Publisher Full Text\n\nJaiyesimi F, Gomathinayagam T, Dixit A, et al.: Sirenomelia without vitelline artery steal. Ann Saudi Med. 1998; 18(6): 542–4. PubMed Abstract\n\nLynch SA, Bond PM, Copp AJ, et al.: A gene for autosomal dominant sacral agenesis maps to the holoprosencephaly region at 7q36. Nat Genet. 1995; 11(1): 93–5. PubMed Abstract | Publisher Full Text\n\nStevenson RE, Jones KL, Phelan MC, et al.: Vascular steal: the pathogenetic mechanism producing sirenomelia and associated defects of the viscera and soft tissues. Pediatrics. 1986; 78(3): 451–7. PubMed Abstract\n\nSirtori M, Ghidini A, Romero R, et al.: Prenatal diagnosis of sirenomelia. J Ultrasound Med. 1989; 8(2): 83–8. PubMed Abstract\n\nDuhamel B: From the Mermaid to Anal Imperforation: The Syndrome of Caudal Regression. Arch Dis Child. 1961; 36(186): 152–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGardner WJ, Breuer AC: Anomalies of heart, spleen, kidneys, gut and limbs may result from an overdistended neural tube: a hypothesis. Pediatrics. 1980; 65(3): 508–14. PubMed Abstract\n\nChenoweth CK, Kellogg SJ, Abu-Yousef MM, et al.: Antenatal sonographic diagnosis of sirenomelia. J Clin Ultrasound. 1991; 19(3): 167–71. PubMed Abstract | Publisher Full Text\n\nvan Zalen-Sprock MM, van Vugt JM, van der Harten JJ, et al.: Early second-trimester diagnosis of sirenomelia. Prenat Diagn. 1995; 15(2): 171–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "257",
"date": "26 Jul 2012",
"name": "Laxmi Baxi",
"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. However there are a few things that need addressing:1. There should be no judgmental statements.2. The author(s) should not confuse between gene and chromosomes.3. There should be more emphasis on scientific data and information.4. If the author(s) do need to address historical data, then it should be in full.It is too bad there isn’t any pre- or post-birth imaging, however the photographs presented are interesting. There also seems to be a repetition of some facts, which need to be curtailed.",
"responses": [
{
"c_id": "58",
"date": "12 Sep 2012",
"name": "Frederick Morfaw",
"role": "Author Response",
"response": "Thank you for your very pertinent comments. With these taken on board we have now reviewed the work and made changes accordingly in a new version of the article."
}
]
},
{
"id": "258",
"date": "27 Jul 2012",
"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\nThis is an interesting article with some quaint comments indicative of local community attitudes to bizarre abnormalities, which increase the appeal of the article.The author(s) should consider being more specific about the actual incidence of the abnormality itself and the actual incidence associated with twins and diabetes. The author(s) should also review the level of detail under the ‘Ethiopathogenesis’ and ‘Classification’ subheadings, as I believe that these sections should be shortened/abbreviated if it is to fulfill the criteria of a ‘Case Report’ presentation.",
"responses": [
{
"c_id": "100",
"date": "12 Sep 2012",
"name": "Frederick Morfaw",
"role": "Author Response",
"response": "Thank you for your very pertinent comments. With these taken on board we have now reviewed the work and made changes accordingly in a new version of the article."
}
]
}
] | 1
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https://f1000research.com/articles/1-6
|
https://f1000research.com/articles/1-13/v1
|
30 Aug 12
|
{
"type": "Case Report",
"title": "Case report: Intradiscal oxygen ozone therapy in uncontained lumbar disc herniation",
"authors": [
"Arockia Doss"
],
"abstract": "Percutaneous intradiscal oxygen ozone (O2O3) for contained lumbar disc herniation (LDH) is used as a minimally invasive alternative to surgery. This article reports excellent benefit in two patients with uncontained LDH following treatment with minimally invasive percutaneous intradiscal injection of O2O3. There is an urgent need for more research and awareness into this less expensive non-surgical out-patient treatment.",
"keywords": [
"Please note that the refereeing status of this article was changed from “indexed” to “[v1",
"ref status: approved 1",
"approved with reservations 1]”."
],
"content": "Introduction\n\nSymptomatic lumbar disc herniation (LDH) is common with a life-time incidence of 2% with most occurring at the L4/5 level in the 30 to 50 age group. LDH affects mobility, function, quality of life and costs highly to society1,2. Treatment options include simple analgesics, physical therapy and corticosteroid injections, as well as percutaneous and conventional discectomy. Long term outcomes, complications and occasionally suboptimal results of open disc surgery have lead to the development of percutaneous techniques3. Percutaneous minimally invasive treatments for LDH aim for chemical, mechanical or thermal methods to decompress the disc. These are based on a study by Hijika et al. (1975) that stated that, ‘reduction of intradiscal pressure reduced irritation of the nerve root and the pain receptors in the annulus and peridiscal area’3–8.\n\nPercutaneous techniques are typically reserved for contained small to medium sized LDH. This report describes the use of minimally invasive percutaneous intradiscal oxygen ozone (O2O3) gas in two patients with severe low back pain and sciatica due to compressive uncontained LDH.\n\n\nCase 1\n\nA 46 year old, non-smoker, male plumber and gas fitter with body mass index (BMI) of 27.3, presented in January 2011 with discogenic type low back pain, left lower limb sciatica to the calf, lateral foot, toes and hyperalgesia to the sole of the foot, which were managed with manual therapy and oral analgesics. In June 2011 symptoms returned after he had been playing with his son and was admitted to the emergency department due to severe low back spasm. An MRI showed a large uncontained L4/5 disc herniation compressing the spinal canal and the left L5 root compatible with his symptoms (Figure 1a,b). He refused surgery and was referred for percutaneous intradiscal O2O3 treatment in June 2011 with an Oswestry Disability Index (ODI) of 24% suggesting mild to moderate disability. He had been on oral Tramadol 200mg BD and Naproxyn 500mg PRN. There was weakness to resisted left big toe dorsiflexion due to L5 nerve compression. Following written informed consent explaining the risks and outcomes, under CT guidance using a sterile technique, and local anaesthetic in a prone position, 20cc medical grade Oxygen Ozone (27µg/ml) was injected into the nucleus pulposus of the disc through a 22g spinal needle (Figure 1c,d). The needle was withdrawn out of the disc and Celestone Chronodose (5.7mg) was injected into the anterior epidural space. Two months post intradiscal O2O3, ODI was 8% (77% reduction from pretreatment) and at seven months his pain had completely resolved. He returned to normal activities with cessation of all analgesics. Mild paraesthesia of the left L5 distribution persisted with resumption of usual work and activities at this time. MRI at seven months showed dramatic resolution of disc herniation and decompression of the spinal canal (Figure 1e,f).\n\nFigure 1. Case 1, 1a: Pretreatment sagittal T2 weighted MRI shows large left paracentral uncontained disc herniation at L4/5 compared with normal disc margin at the levels above and below, 1b: Pretreatment axial T2 weighted MRI with arrow pointing to posterior displacement and compression of thecal sac due to the disc herniation, 1c&d: CT guided intradiscal O2O3 injection in another patient illustrates needle placement in the nucleus pulposus of disc and distribution of O2O3 in the disc, epidural and paraspinal spaces, 1e&f: follow up MRI seven months after intradiscal O2O3 injection shows complete resolution of disc herniation, note the reduction in disc height and hydration in 1e compared to 1a and restoration of normal thecal sac margin highlighted by the arrow in 1f in comparison to 1b.\n\n\nCase 2\n\nA 30 year old mother with BMI of 30.1, lifelong non smoker, suffered debilitating low back pain for about 8 years. Intermittent exacerbation of pins and needles and right lower limb sciatica were treated with spinal corticosteroids, nonsteroidal antiinflammatories and opiates. Acute episodes needed admission to emergency department. Prior to referral for consideration of percutaneous intradiscal O2O3 treatment as a last option, she had been reviewed by spinal surgery and chronic pain management. She was on Tramadol 200mg, Panadeine Forte, Lyrica and OxyNorm. At presentation for percutaneous treatment, ODI was 66% (moderately severe disability), there were normal reflexes and power of lower limbs, dysaesthesia to right lateral foot and right paravertebral spasm. MRI showed a 12mm LDH with an extrusion compressing the right L5 root and L4/5 disc degeneration (see Figure 2). Following written informed consent explaining the risks and outcomes, under CT guidance, in a prone position using local anaesthetic and sterile technique, 10cc O2O3 (27µg/ml) gas was injected into the nucleus pulposus of the L4/5 disc through a 22G spinal needle. The needle was withdrawn and O2O3, followed by Triamnicalone (40mg), was injected around the right L5 nerve. At 4 weeks follow up there was dramatic improvement with no low back or radicular pain. At nine months telephone interview ODI was 4% (improved by 93% from pretreatment ODI), with complete cessation of all medications and the occasional niggling pain.\n\n\nDiscussion\n\nMinimally invasive percutaneous intradiscal injection of O2O3 has been widely used in Europe for LDH since 19969. It is proposed that O2O3 acts by ‘mummifying’ the disc reducing disc volume with an effect of a discectomy. The chemical properties of oxygen ozone, by way of reaction of the hydroxyl radical with carbohydrates and amino acids leading to the breakdown of nucleus pulposus with rapid disappearance of herniated disc material3, may explain this phenomenon of ‘chemical discectomy’. The MRI in Figure 1 shows reduction in disc height and marked resolution of the herniated disc material after O2O3 treatment. This supports the phenomenon of ozone induced chemical discectomy. O2O3 also down-regulates nerves and has an anti-inflammatory effect10.\n\nIn a randomized controlled trial, Gallucci et al. showed that intraforaminal and intradiscal injections of a steroid, anaesthetic and Oxygen Ozone are more effective at six months than injections of only a steroid and anaesthetic at the same sites11. Leonardi et al. showed in 600 patients with LDH and sciatica treated with intradiscal oxygen ozone at least 70% achieved excellent or good outcome. In addition, a combined intradiscal oxygen ozone and concomitant periganglionic cortisone and oxygen ozone injection achieved a cumulative effect that enhances overall outcome of treatment of pain caused by disc herniation with a satisfactory therapeutic outcome in 78% of patients at six months follow up in 300 patients10. Muto et al. showed that between 1996 and 2003 of 2200 patients with low back pain or sciatica due to LDH, CT guided intradiscal oxygen ozone injection achieved an 80% success in 1750 patients at 6 months follow up. Success rate dropped to 75% in 1400 patients followed up to 18 months. Reduction in size of herniated disc was seen in 63% of followed up patients. Failure was mostly due to calcific herniated disc, spinal canal stenosis, recurrent disc herniation and multilevel degenerative disease9. A meta-analysis of twelve studies showed that oxygen ozone treatment in herniated discs is an effective and extremely safe procedure, with impressive improvement in pain and function in view of the broad inclusion criteria of patients ranging from 13 to 94 years with all types of disc herniations. It also showed that pain and function outcome scores were similar for LDH treated with surgical discectomy but the complication rate is much lower (<0.1%)12. A randomized controlled trial is underway comparing microdiscectomy and percutaneous O2O3 intradiscal therapy13.\n\nLu et al. showed that percutaneous ozone injection is safe in large LDH in 58 patients with overall efficacy of 91.4% with excellent and good outcome in 63.8% and 27.6% respectively14. Wu et al. used a combination of O2O3 with collagenase injection and compared this with surgery for uncontained LDH and followed them up for 12 months. They showed non-statistically significant difference between the two groups at 3 and 12 months, though the surgical group had a statistically significant greater improvement in the first few weeks15.\n\nThe total health care cost for disorders of intervertebral discs and bones in the spinal column was estimated at US$ 25.8 billion16. Cost per procedure of intradiscal O2O3 is free from recurring operative instrumental consumable cost. Oxygen Ozone is procured from inexpensive medical grade oxygen that is processed through an oxygen generator, obtained as a one off capital non-recurring expenditure. Percutaneous intradiscal treatment is an outpatient procedure that attracts far less expense compared to procedures that require hospitalization. There are other cost savings for the individual, community and health systems due to reduced recuperation time.\n\nIn summary, Oxygen ozone is a minimally invasive, effective, low risk treatment for refractory LDH with potential to reduce health care costs and there is an urgent need for more research and awareness into this non surgical outpatient treatment.\n\n\nConsent\n\nConsent was obtained from both patients to publish this anonymized case report.",
"appendix": "Competing interests\n\n\n\nI Dr Doss have no competing interests in this paper to any agency or third party. I have not received any financial or non financial incentives in relation to this article.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI wish to thank the patients, their next of kin for consenting to undergo this procedure. I also thank Drs Chris Wallman, Richard Obadua and Norman Pinsky for referring these patients for this procedure.\n\n\nReferences\n\nKelekis AD, Somon T, Yilmaz H, et al.: Interventional spine procedures. Eur J Radiol. 2005; 55(3): 362–383. PubMed Abstract | Publisher Full Text\n\nShah RV, Everett CE, McKenzie-Brown AM, et al.: Discography as a diagnostic test for spinal pain: a systematic and narrative review. Pain Physician. 2005; 8(2): 187–209. PubMed Abstract\n\nKelekis AD, Filippiadis DK, Martin JB, et al.: Standards of practice: quality assurance guidelines for percutaneous treatments of intervertebral discs. Cardiovasc Intervent Radiol. 2010; 33(5): 909–913. PubMed Abstract | Publisher Full Text\n\nSingh V, Derby R: Percutaneous lumbar disc decompression. Pain Physician. 2006; 9(2): 139–146. PubMed Abstract\n\nChoy DS, Ascher PW, Saddekni S, et al.: Percutaneous laser disc decompressions. A new therapeutic modality. Spine (Phila Pa 1976). 1992; 17(8): 949–956. PubMed Abstract | Publisher Full Text\n\nShields CB: In defence of chemonucleolysis. Clin Neurosurg. 1986; 33: 397–405. PubMed Abstract\n\nEckel TS: Intradiscal electrothermal therapy. In: Handbook of diagnostic and therapeutic spine procedures. A.L. Williams and F.R. Murtagh, Editors. Louis St: CV Mosby, 2002; 229–244.\n\nGangi A, Dietemann JL, Ide C, et al.: Percutaneous laser disk decompression under CT and fluoroscopic guidance: indications, technique, and clinical experience. Radiographics. 1996; 16(1): 89–96. PubMed Abstract | Publisher Full Text\n\nMuto M, Andreula C, Leonardi M, et al.: Treatment of herniated lumbar disc by intradiscal and intraforaminal oxygen-ozone (O2-O3) injection. J Neuroradiol. 2004; 31(3): 183–9. PubMed Abstract | Publisher Full Text\n\nAndreula CF, Simonetti L, de Santis F, et al.: Minimally invasive oxygen-ozone therapy for lumbar disk herniation. AJNR Am J Neuroradiol. 2003; 24(5): 996–1000. PubMed Abstract\n\nGallucci M, Limbucci N, Zugaro L, et al.: Sciatica: treatment with intradiscal and intraforaminal injections of steroid and oxygen-ozone versus steroid only. Radiology. 2007; 242(3): 907–13. PubMed Abstract | Publisher Full Text\n\nSteppan J, Meaders T, Muto M, et al.: A meta analysis of the effectiveness and safety of ozone treatments for herniated lumbar discs. J Vasc Interv Radiol. 2010; 21(4): 534–548. PubMed Abstract | Publisher Full Text\n\nKovacs Foundation: The Effect of ozone therapy for Lumbar Herniated Disc. In: ClinicalTrials.gov [cited 2012 Aug 30]. Reference Source\n\nLu W, Li YH, He XF, et al.: Treatment of large lumbar disc herniation with percutaneous ozone injection via the posterior-lateral route and inner margin of the facet joint. World J Radiol. 2010; 2(3): 109–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu Z, Wei LX, Li J, et al.: Percutaneous treatment of non-contained lumbar disc herniation by injection of oxygen-ozone combined with collagenase. Eur J Radiol. 2009; 72(3): 499–504. PubMed Abstract | Publisher Full Text\n\nThe Cost Burden of Disease US and Michigan. Issue Brief January. 2010. Reference Source"
}
|
[
{
"id": "303",
"date": "18 Sep 2012",
"name": "Wally Kos",
"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 think the case report(s) are fine, the treatment methods complying with standard practice & reflecting sound & safe practice.Whilst not a scientific paper, it brings attention to a therapy which is poorly understood in the major part of the English speaking world. It could open up personal inquiry to the many papers published out of Europe & India.",
"responses": []
},
{
"id": "302",
"date": "18 Sep 2012",
"name": "Nikolai Bogduk",
"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\nCase reports should be used to publicise unusual or rare events that for epidemiological reasons cannot be reported in better, more informative formats such as case series and controlled trials.There is nothing new or unusual about ozone therapy for disc herniation. There is already an abundant literature, with many case series, that describes this intervention. A report of two cases does nothing to enhance this literature. The science of this intervention can be advanced only by a randomised controlled trial.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-13
|
https://f1000research.com/articles/1-11/v1
|
14 Aug 12
|
{
"type": "Data Article",
"title": "Freely available compound data sets and software tools for chemoinformatics and computational medicinal chemistry applications",
"authors": [
"Ye Hu",
"Jürgen Bajorath",
"Ye Hu"
],
"abstract": "We have generated a number of compound data sets and programs for different types of applications in pharmaceutical research. These data sets and programs were originally designed for our research projects and are made publicly available. Without consulting original literature sources, it is difficult to understand specific features of data sets and software tools, basic ideas underlying their design, and applicability domains. Currently, 30 different entries are available for download from our website. In this data article, we provide an overview of the data and tools we make available and designate the areas of research for which they should be useful. For selected data sets and methods/programs, detailed descriptions are given. This article should help interested readers to select data and tools for specific computational investigations.",
"keywords": [
"For compound data mining and the development and evaluation of chemoinformatics methods",
"public domain databases have become indispensable resources. Currently",
"major public repositories include PubChem1",
"BindingDB2",
"ChEMBL3",
"and ZINC4. While the former three databases contain compounds and bioactivity data",
"the latter collects commercially available compounds that are typically not annotated with activity information. Bioactivity data are usually obtained from original literature or patent sources. From these databases",
"screening data sets (PubChem) and compound activity classes (BindingDB",
"ChEMBL) can be extracted. Benchmarking of newly developed computational methods typically depends on the availability of such activity classes. Many compounds and measurements are now also shared between these databases. In addition",
"there are also a number of smaller public and commercial compound databases",
"which we do not consider here for the purpose of our discussion (with one exception",
"see below)."
],
"content": "Introduction\n\nFor compound data mining and the development and evaluation of chemoinformatics methods, public domain databases have become indispensable resources. Currently, major public repositories include PubChem1, BindingDB2, ChEMBL3, and ZINC4. While the former three databases contain compounds and bioactivity data, the latter collects commercially available compounds that are typically not annotated with activity information. Bioactivity data are usually obtained from original literature or patent sources. From these databases, screening data sets (PubChem) and compound activity classes (BindingDB, ChEMBL) can be extracted. Benchmarking of newly developed computational methods typically depends on the availability of such activity classes. Many compounds and measurements are now also shared between these databases. In addition, there are also a number of smaller public and commercial compound databases, which we do not consider here for the purpose of our discussion (with one exception; see below).\n\nImportantly, depending on the scientific questions under investigation, it is often required to design and assemble data sets with specific features. Such data sets, which are usually reported as a part of a publication describing a computational analysis, a new method, or a benchmark investigation, are only infrequently made available to the public. Herein, we describe data sets originating from our laboratory that can be freely obtained. In addition, we also provide information about software tools developed by us that are available via the same website.\n\n\nObjectives\n\nThe data sets and software tools reported herein have been generated for research activities that essentially fall into four different areas, as reported in Table 1. Area A comprises virtual screening and machine learning applications and is a core area of chemoinformatics. Areas B and C represent molecular selectivity analysis and visualization of structure-activity relationships (SARs), respectively. Furthermore, area D summarizes data mining activities with a focus on structure-activity or -selectivity relationships. Areas B-D are equally relevant for medicinal chemistry (and also chemoinformatics). In addition, especially area B is also relevant for chemical biology. By describing these tools in context, it is hoped that their accessibility to researchers in these areas might be further increased.\n\nA list of 30 entries providing data sets and/or methods/programs is shown. For each entry, research area indices are assigned as described in the text, i.e. area ‘A’ indicates virtual screening (similarity searching), fingerprint engineering and machine learning; area ‘B’ represents molecular selectivity analysis, area ‘C’ SAR visualization, and area ‘D’ structure-activity or -selectivity relationship-oriented data mining. In addition, publication information is given. For compound data sets, short descriptions are provided. Selected compound data sets are highlighted in red and discussed in the text.\n\n\nMaterials and methods\n\nData sets reported herein were mostly, but not exclusively, assembled from BindingDB and ChEMBL on the basis of defined selection criteria (as specified in the original publications). These sets contain compound structures, provided as SMILES5 strings or SD files6, and -whenever appropriate- associated bioactivity information. Some of the older activity classes that are still available from our website have originated from the license-restricted Molecular Drug Data Report (MDDR)7. Therefore, these data sets do not contain compound structures, but only compound identifier information (because a user must obtain a license to access the database). We do no longer license commercial or otherwise restricted databases and will remove corresponding entries from our download section in the near future (to ensure that all compounds are freely available). For the time being, all the information can be accessed. Scripts and programs available from our site generally represent a new computational method or analysis protocol and were implemented in-house in different scripting and programming languages, as specified in the respective entries. Source code is provided. All data sets and programs can be obtained via following URL: http://www.limes.uni-bonn.de/forschung/abteilungen/Bajorath/labwebsite/downloads. The download section is updated several times per year with materials reported in new publications.\n\n\nResults and discussion\n\nTable 1 lists all 30 currently available entries including data sets and/or methods/programs. In each case, research area indices are assigned and publication information is provided. Eleven entries are assigned to area A and one, nine, and five entries to area B, C, and D, respectively. In addition, three entries are assigned to areas A and B and one to B and D. Compound data sets originated from 22 different studies and methods from four. In addition, for four other investigations, both methods and data sets are provided.\n\nFor compound data sets, short descriptions are provided in Table 1. Furthermore, method/program descriptions are given in Table 2. In a number of instances, data entries contain sets of (filtered) compound activity classes (ACs) to ensure reproducibility of results reported in a specific publication. These sets were often directly taken from BindingDB, ChEMBL, the MDDR, or original literature sources and might not be of above-average interest. Nevertheless, for benchmarking of virtual screening methods, these sets are useful. However, other data sets have been especially designed for novel applications. In the following, selected data sets and methods are described in more detail that might be of particular interest for investigators in the designated (or other) research areas. The indices of these entries are highlighted in Table 1 and Table 2.\n\nEight entries with methods/programs are listed. For each entry, a brief description is provided. Selected entries are highlighted in red and discussed in the text.\n\nEntry 1: The ACs in this set are designed to have increasing intra-class structural diversity and hence represent test cases of increasing degrees of difficulty for the evaluation of ligand-based virtual screening (LBVS) methods.\n\nEntry 4: In 26 so-called selectivity sets, compounds are organized on the basis of differential potency against pairs of targets as a measure of selectivity. These sets were originally designed to evaluate an extension of standard similarity searching termed selectivity searching. The sets can be used as test cases for any methods that evaluate or predict molecular selectivity, similar to entries 5 and 6. As such, these data sets are relevant for computational chemical biology.\n\nEntry 5: Selectivity sets focusing on the biogenic amine G protein coupled receptor (GPCR) family.\n\nEntry 6: Eighteen sets with further refined selectivity criteria targeting four different protein families.\n\nEntry 7: Twenty-five different sets are provided that contain compounds with increasing topological complexity and molecular size. These compound sets were designed to evaluate molecular complexity effects in similarity searching. They can be utilized to examine the complexity and/or size dependence of a computational method.\n\nEntry 17: Sets of matched molecular pairs (MMPs) are given that were systematically extracted from BindingDB and ChEMBL. An MMP is defined as a pair of compounds that only differ by the exchange of a single fragment (substructure).\n\nEntry 24: On the basis of systematic similarity search profiling of ChEMBL, 50 ACs were selected. These sets represent meaningful test cases for benchmarking of LBVS methods. The ACs were assembled because they were neither too \"easy\" nor too \"difficult\" for standard similarity searching using different molecular fingerprints.\n\nEntry 25: This database contains a collection of known active reference compounds, newly identified actives (hits), and screening database information extracted from original literature sources reporting prospective LBVS applications. Only studies were considered that provided sufficiently detailed information to reproduce the search calculations. These studies were identified in a systematic survey of published LBVS applications. The database provides an alternative benchmark system for LBVS. For example, on the basis of these compound sets, it can be determined whether a new methodology is capable of reproducing the results of successful prospective virtual screens using other approaches (i.e., screens that have identified structurally novel and experimentally confirmed hits).\n\nEntry 30: Sets of activity cliffs are provided that belong to five newly introduced structural categories. These cliffs were systematically extracted from ChEMBL (latest release). An activity cliff is defined as a pair of structurally similar or analogous compounds with a large difference in potency. Accordingly, activity cliffs typically represent a rich source of SAR information.\n\nEntries 1, 4–7, 9, 11, and 12 (assembled until 2010) only contain MDDR compound identifiers, but no structures, due to license restrictions, as commented on above.\n\n\nSelected methods and programs\n\nEntry 10: A graphical data structure termed combinatorial analog graph (CAG) is introduced to systematically organize analog series on the basis of substitution patterns and identify subsets of analogs having high in SAR information content.\n\nEntry 14: A further extended and refined CAG implementation for the study of SARs across multiple targets.\n\nEntry 15: SARANEA (a semantic construct of SAR and \"Araneae\", i.e., the scientific order of spiders) is a collection of different tools for graphical and numerical SAR analysis. It contains the network-like similarity graph (NSG), an SAR network (reminiscent of \"spider webs\") in which compounds are nodes and edges structural similarity relationships. In addition, nodes are annotated with different levels of SAR information. Several NSG variants have been introduced for different aspects of SAR exploration. The SARANEA tool collection was designed for large-scale SAR data mining and analysis, comparison of global and local SAR features, and the study of structure-selectivity relationships.\n\nEntry 16: A program to calculate and display three-dimensional activity landscapes of compound data sets. An activity landscape is defined as any graphical representation that integrates molecular similarity and activity relationships. A 3D activity landscape can be conceptualized as a 2D projection of a chemical reference space (in which compound dissimilarity increases with inter-compound distance) with an interpolated potency surface added as the third dimension.\n\nEntry 18: The similarity-potency tree (SPT) is a graph representation that organizes compound neighborhoods in large data sets on the basis of structural nearest neighbor relationships and reveals chemically interpretable SAR information. This data structure can be understood as a compound-centric activity landscape view. A basic SPT implementation is also available as a part of SARANEA.\n\nEntry 27: \"Scaffold hopping\", i.e., the detection of active compounds having different structural frameworks (core structures), is the ultimate goal of LBVS and its primary measure of success. However, the evaluation of the scaffold hopping potential of different LBVS methods is complicated by the fact that scaffold hops can involve similar or different core structures, which is generally not taken into account in the statistical assessment of benchmark investigations. An algorithm is presented that calculates the structural distance between any two scaffolds, regardless of their chemical composition or size. Application of this method makes it possible to quantify the degree of difficulty involved in computational scaffold hopping exercises.\n\n\nConclusions\n\nHerein we have given an overview of specialized compound data sets and methods/programs that have originated from different research projects in our laboratory and that are made freely available to others with interests in chemoinformatics, computational medicinal chemistry, and chemical biology. These tools were presented and described in context. We hope that this report will further alert investigators in our and other scientific fields to available resources for specific computational applications and help to select data sets and tools that are relevant for given research topics. It is also hoped that the introduced methodological concepts will further evolve through wide use by others.",
"appendix": "Author contributions\n\n\n\nJB conceived the study, YH collected and organized the data and information, YH and JB wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests declared.\n\n\nGrant information\n\n\n\n\nAcknowledgments\n\nWe thank current and former members of our research group who have contributed to development of the data sets and methods/programs reported herein.\n\n\nReferences\n\nWang Y, Xiao J, Suzek TO, et al.: PubChem: a public information system for analyzing bioactivities of small molecules. Nucleic Acids Res. 2009; 37(Web Server issue): W623–W633. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu T, Lin Y, Wen X, et al.: BindingDB: A Web-accessible database of experimentally determined protein−ligand binding affinities. Nucleic Acids Res. 2007; 35(Database issue): D198–D201. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaulton A, Bellis LJ, Bento AP, et al.: ChEMBL: A large-scale bioactivity database for drug discovery. Nucleic Acids Res. 2012; 40(Database issue): D1100–D1107. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIrwin JJ, Sterling T, Mysinger MM, et al.: ZINC: A free tool to discover chemistry for biology. J Chem Inf Model. 2012; 52(7): 1757–1768. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeininger D: SMILES, a chemical language and information system. 1. Introduction to methodology and encoding rules. J Chem Inf Comput Sci. 1988; 28(1): 31–36. Publisher Full Text\n\nDalby A, Nourse JG, Hounshell WD, et al.: Description of several chemical structure file formats used by computer programs developed at Molecular Design Limited. J Chem Inf Comput Sci. 1992; 32(3): 244–255. Publisher Full Text\n\nMolecular Drug Data Report (MDDR), San Diego, CA, USA.\n\nTovar A, Eckert H, Bajorath J: Comparison of 2D fingerprint methods for multiple-template similarity searching on compound activity classes of increasing structural diversity. ChemMedChem. 2007; 2(2): 208–217. PubMed Abstract | Publisher Full Text\n\nWang Y, Godden JW, Bajorath J: A novel Descriptor histogram filtering method for database mining and the identification of active molecules. Lett Drug Design Discov. 2007; 4(4): 286–292. Publisher Full Text\n\nStumpfe D, Ahmed HE, Vogt I, et al.: Methods for computer-aided chemical biology, part 1: design of a benchmark system for the evaluation of compound selectivity. Chem Biol Drug Des. 2007; 70(3): 182–194. PubMed Abstract | Publisher Full Text\n\nVogt I, Ahmed HE, Auer J, et al.: Exploring structure-selectivity relationships of biogenic amine GPCR antagonists using similarity searching and dynamic compound mapping. Mol Divers. 2008; 12(1): 25–40. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Geppert H, Bajorath J: Methods for computer-aided chemical biology, part 3: analysis of structure-selectivity relationships through single- or dual-step selectivity searching and Bayesian classification. Chem Biol Drug Des. 2008; 71(6): 518–528. PubMed Abstract | Publisher Full Text\n\nWang Y, Geppert H, Bajorath J: Random reduction in fingerprint bit density improves compound recall in search calculations using complex reference molecules. Chem Biol Drug Des. 2008; 71(6): 511–517. PubMed Abstract | Publisher Full Text\n\nNisius B, Göller AH, Bajorath J: Combining cluster analysis, feature selection and multiple support vector machine models for the identification of human ether-a-go-go related gene channel blocking compounds. Chem Biol Drug Des. 2009; 73(1): 17–25. PubMed Abstract | Publisher Full Text\n\nAhmed HE, Geppert H, Stumpfe D, et al.: Methods for computer-aided chemical biology. Part 4: selectivity searching for ion channel ligands and mapping of molecular fragments as selectivity markers. Chem Biol Drug Des. 2009; 73(3): 273–282. PubMed Abstract | Publisher Full Text\n\nPeltason L, Weskamp N, Teckentrup A, et al.: Exploration of structure-activity relationship determinants in analogue series. J Med Chem. 2009; 52(10): 3212–3224. PubMed Abstract | Publisher Full Text\n\nNisius B, Bajorath J: Molecular fingerprint recombination: generating hybrid fingerprints for similarity searching from different fingerprint types. ChemMedChem. 2009; 4(11): 1859–1863. PubMed Abstract | Publisher Full Text\n\nBatista J, Tan L, Bajorath J: Atom-centered interacting fragments and similarity search applications. J Chem Inf Model. 2010; 50(1): 79–86. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Exploring target-selectivity patterns of molecular scaffolds. ACS Med Chem Lett. 2010; 1(2): 54–58. Publisher Full Text\n\nWassermann AM, Peltason L, Bajorath J: Computational analysis of multi-target structure-activity relationships to derive preference orders for chemical modifications toward target selectivity. ChemMedChem 2010; 5(6): 847–858. PubMed Abstract | Publisher Full Text\n\nLounkine E, Wawer M, Wassermann AM, et al.: SARANEA: a freely available program to mine structure-activity and structure-selectivity relationship information in compound data sets. J Chem Inf Model. 2010; 50(1): 68–78. PubMed Abstract | Publisher Full Text\n\nPeltason L, Iyer P, Bajorath J: Rationalizing three-dimensional activity landscapes and the influence of molecular representations on landscape topology and formation of activity cliffs. J Chem Inf Model. 2010; 50(6): 1021–1033. PubMed Abstract | Publisher Full Text\n\nWassermann AM, Bajorath J: Chemical substitutions that introduce activity cliffs across different compound classes and biological targets. J Chem Inf Model. 2010; 50(7): 1248–1256. PubMed Abstract | Publisher Full Text\n\nWawer M, Bajorath J: Similarity-potency trees: a method to search for SAR information in compound data sets and derive SAR rules. J Chem Inf Model. 2010; 50(8): 1395–1409. PubMed Abstract | Publisher Full Text\n\nVogt M, Stumpfe D, Geppert H, et al.: Scaffold hopping using two-dimensional fingerprints: true potential, black magic, or a hopeless endeavor? Guidelines for virtual screening. J Med Chem. 2010; 53(15): 5707–5715. PubMed Abstract | Publisher Full Text\n\nWawer M, Bajorath J: Extracting SAR information from a large collection of anti-malarial screening hits by NSG-SPT analysis. ACS Med Chem Lett. 2011; 2(3): 201–206. Publisher Full Text\n\nHu Y, Bajorath J: Combining horizontal and vertical substructure relationships in scaffold hierarchies for activity prediction. J Chem Inf Model. 2011; 51(2): 248–257. PubMed Abstract | Publisher Full Text\n\nDimova D, Wawer M, Wassermann AM, et al.: Design of multitarget activity landscapes that capture hierarchical activity cliff distributions. J Chem Inf Model. 2011; 51(2): 258–266. PubMed Abstract | Publisher Full Text\n\nWawer M, Bajorath J: Local structural changes, global data views: graphical substructure-activity relationship trailing. J Med Chem. 2011; 54(8): 2944–2951. PubMed Abstract | Publisher Full Text\n\nHeikamp K, Bajorath J: Large-scale similarity search profiling of ChEMBL compound data sets. J Chem Inf Model. 2011; 51(8): 1831–1839. PubMed Abstract | Publisher Full Text\n\nRipphausen P, Wassermann AM, Bajorath J: REPROVIS-DB: a benchmark system for ligand-based virtual screening derived from reproducible prospective applications. J Chem Inf Model. 2011; 51(10): 2467–2473. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Activity profile sequences: a concept to account for the progression of compound activity in target space and to extract SAR information from analogue series with multiple target annotations. ChemMedChem 2011; 6(12): 2150–2154. PubMed Abstract | Publisher Full Text\n\nLi R, Stumpfe D, Vogt M, et al.: Development of a method to consistently quantify the structural distance between scaffolds and to assess scaffold hopping potential. J Chem Inf Model. 2011; 51(10): 2507–2514. PubMed Abstract | Publisher Full Text\n\nStumpfe D, Bajorath J: Assessing the confidence level of public domain compound activity data and the impact of alternative potency measurements on SAR analysis. J Chem Inf Model. 2011; 51(12): 3131–3137. PubMed Abstract | Publisher Full Text\n\nGupta-Ostermann D, Hu Y, Bajorath J: Introducing the LASSO graph for compound data set representation and structure-activity relationship analysis. J Med Chem. 2012; 55(11): 5546–5553. PubMed Abstract | Publisher Full Text\n\nHu Y, Bajorath J: Extending the activity cliff concept: structural categorization of activity cliffs and systematic identification of different types of cliffs in the ChEMBL database. J Chem Inf Model. 2012; 52(7): 1806–1811. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "272",
"date": "20 Aug 2012",
"name": "Patrick Walters",
"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 fills a much-needed gap in the molecular modeling and cheminformatics arena. Far too often, authors of computational papers publish work that is difficult, if not impossible, to reproduce.In many cases, the structures and data used to develop and validate a new method are not included with the paper. When structures are included, they are typically supplied as images. Thus, the only way to reproduce the work is to go through a painstaking, often error prone, process of redrawing structures. If modeling and informatics are to advance, we need to do everything we can to promote reproducibility. We also need to have standard datasets that can be used to readily compare existing methods with new techniques. Datasets like DUD from the Shoichet group and MUV from the Baumann group provide a good start, but we need more. We also need more dialog and critical assessments of these datasets. The datasets provided with this paper will provide important tools for the development, comparison, and evaluation of new computational methods.In addition to releasing a large number of datasets, the authors have also provided software that allows those reading their papers to critically evaluate their group’s published methods on their own datasets. The release of this software also enables the reader to compare new methods with methods they may already be using. I applaud Hu and Bajorath for their efforts to promote open science and to provide datasets that will hopefully provide the basis for a large body of future work.",
"responses": []
},
{
"id": "273",
"date": "04 Sep 2012",
"name": "Michael K. Gilson",
"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 takes a valuable step in the direction of improved scientific communication by making compound data sets and software, which have been created by the authors in the course of their research over a number of years, available to the scientific community in machine-readable format.This step dramatically lower barriers for reproducing, using, and building upon this research and sets an important standard for other authors who write papers about digital collections and software.Currently, the downloads are provided at the authors’ institutional web-site. Given the possibility that this URL may change, it may be helpful to assign a stable digital object identifier (DOI) to the entire collection of 30 data sets and programs; or potentially to assign a separate DOI to each data set and program, so that they can be referenced and accessed in a more fine-grained manner.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/1-11
|
https://f1000research.com/articles/1-9/v1
|
08 Aug 12
|
{
"type": "Research Article",
"title": "Exploring the membrane topology of prohormone convertase 1 in AtT20 Cells: in situ analysis by immunofluorescence microscopy",
"authors": [
"Niamh X Cawley",
"Meera Sridhar",
"Hong Hong",
"Peng Loh",
"Meera Sridhar",
"Hong Hong",
"Peng Loh"
],
"abstract": "Prohormone convertase 1 (PC1) was previously characterized as a partially transmembrane protein in purified chromaffin granules of bovine adrenal medulla1. This was challenged with experiments on transfected PC1 in COS1 cells, a non-endocrine cell line2. To address this issue, we undertook to analyze its extraction properties in vitro and its immunocytochemical localization in situ in AtT20 cells, an endocrine cell line that expresses PC1. Most of the 87 kDa form of PC1 was resistant to carbonate extraction suggesting that it had properties of a transmembrane protein. Under semi-permeabilized conditions whereby only the plasma membrane was permeabilized, the carboxy-terminus of PC1 was specifically immunostained whereas the amino-terminus was not. These results indicate that the amino-terminus of PC1 was within the lumen of the Golgi and granules, and some of the C-terminus was exposed to the cytosol. Thus, endogenous PC1 can assume a transmembrane orientation in situ in AtT20 cells.",
"keywords": [
"The proprotein convertases (PCs) belong to a family of endoproteinases that cleave proproteins specifically at basic residue cleavage sites3. The first mammalian member of this group",
"furin",
"was identified by sequence homology4",
"5 to the yeast prohormone convertase",
"Kex2",
"which was the first eukaryotic enzyme to be described as a prohormone convertase6",
"7. Both Kex2 and furin are transmembrane proteins5",
"7–9. Other mammalian enzymes",
"homologous to furin were subsequently cloned",
"of which PC1 (also described as PC3 or SPC3) and PC2 were found to be exclusively expressed in (neuro)endocrine tissue10–14",
"suggesting their function to be specific for the maturation of peptide hormones and neuropeptides. Both enzymes do not contain classical amino acid sequences that would predict them to have a transmembrane domain."
],
"content": "1. Introduction\n\nThe proprotein convertases (PCs) belong to a family of endoproteinases that cleave proproteins specifically at basic residue cleavage sites3. The first mammalian member of this group, furin, was identified by sequence homology4,5 to the yeast prohormone convertase, Kex2, which was the first eukaryotic enzyme to be described as a prohormone convertase6,7. Both Kex2 and furin are transmembrane proteins5,7–9. Other mammalian enzymes, homologous to furin were subsequently cloned, of which PC1 (also described as PC3 or SPC3) and PC2 were found to be exclusively expressed in (neuro)endocrine tissue10–14, suggesting their function to be specific for the maturation of peptide hormones and neuropeptides. Both enzymes do not contain classical amino acid sequences that would predict them to have a transmembrane domain.\n\nPC1 is expressed as a pre-pro-protein of ~92 kDa in mass. After removal of the signal peptide, the pro-protein undergoes autocatalytic conversion in the ER to an 87 kDa form15–18. This form of PC1 can subsequently be converted to a 64–66 kDa form19,20 which is the predominant form found in dense-core granules of the bovine pituitary21. The conversion from 87 kDa to 64 kDa is the result of the removal of the carboxyl terminus in a late compartment of the secretory pathway22. We and others have investigated the function of the C-terminus of PC1, since it does not appear to be involved in catalysis per se and is distinct from the P-domain of PC1 which is involved in the stability and pH and calcium dependence of PC1 activity23. Initial studies revealed that the C-terminus of PC1 is involved in the efficient trafficking of PC1 to the regulated secretory pathway (RSP)24–26, giving rise to the identification of three alpha-helical amphipathic sequences important in this function. Recent studies by Dikeakos et al.27,28, have characterized by NMR the extreme C-terminal sequence and identified important residues within the sequence involved in binding to membrane patches which were important for sorting.\n\nPreviously, we showed by immuno-labeling and classical extraction studies, that in intact purified bovine chromaffin granules, PC1 behaved in part like a transmembrane protein1. We identified, immunologically and functionally, the putative transmembrane (TM) sequence (aa617–638), and showed that when fused with the soluble extracellular domain of the IL2 receptor alpha-subunit (Tac), it could direct this protein to secretory granules of the RSP1. Indeed, deletion studies identified this sequence as necessary for PC1 sorting to the RSP29. We speculated about how PC1 might assume a TM orientation and the consequences of having a cytosolic domain. However, Stettler et al. provided evidence that transfected PC1 is not synthesized as a TM protein in COS1 cells30. In our current study we address in a model endocrine cell line, AtT20 cells, which expresses PC1 endogenously, whether PC1 has properties consistent with that of a TM protein.\n\n\n2. Materials and methods\n\nAtT20 and COS7 cells were grown in high glucose DMEM containing 10% fetal bovine serum, 1X penicillin/streptomycin and 50 μg/ml normocin (Invitrogen, San Diego, CA) in an incubator maintained at 37°C and 5% CO2. For sodium carbonate extraction, AtT20 cells were rinsed 3 times with ice cold phosphate buffered saline (PBS), scraped and collected in PBS, sedimented by centrifugation (3,000 rpm for 3 min) and resuspended in a smaller volume of PBS. The cell suspension was dispensed into equal aliquots and stored at -30°C until used.\n\nA frozen aliquot of AtT20 cells was thawed on ice. One hundred μl of the cell suspension was saved immediately and 100 μl were placed into two airfuge tubes for centrifugation at 24 psi for 10 min, i.e. >100,000 × g (Airfuge, Beckman, Fullerton, CA). To block the non-specific binding of proteins to the plastic, the airfuge tubes were previously incubated on ice with 10% BSA for 30 min, after which they were rinsed 3 times with PBS. After centrifugation of the samples, the supernatants were removed and saved, and the pellets were resuspended in an additional 100 μl PBS each. The samples were centrifuged again and the resulting supernatants were combined with their original supernatants. To collect and save the pellet from the PBS extraction, the pellet from one tube was resuspended with 100 μl PBS and saved. The empty tube was rinsed once with 100 μl PBS and this was combined with the resuspended pellet. The other pellet was resuspended by vigorous pipetting with 100 μl of 0.1 M sodium carbonate, pH 11.5, and allowed to incubate on ice for 30 min. The sample was then centrifuged at 24 psi for 10 min and the resulting supernatant saved. The tube was rinsed carefully with 100 μl of fresh carbonate solution and this was added to the supernatant. The pellet was collected in PBS in the same way as was done for the first pellet. Thirty μl 1 M Tris/Cl, pH 7.4, 90 μl 4X SDS sample buffer and 36 μl 10X sample reducing agent were added to each of the extracted samples (containing 200 μl). To the original 100 μl aliquot of the starting material, half these volumes were added to maintain equivalent dilutions. Ten μl of the starting material and 20 μl of each extracted samples were analyzed by Western blot after SDS-PAGE through a 4–12% NuPAGE gel using the Bis-Tris buffer system and electro-blotting to nitrocellulose. Three transmembrane proteins; transferrin receptor, synaptotagmin 1 and aquaporin 1, and 3 non-transmembrane proteins; chromogranin A (β-granin), p115 and Grasp65 were analyzed. The blots were also probed for PC1 (N-terminal specific, ABR Inc., Golden, CO) in order to determine its pattern of extraction and to compare it to the patterns of the known transmembrane and non-transmembrane proteins listed above. Visualization of the proteins was by detection of secondary antibodies labeled with fluorophores that emit in the infra-red region of the spectrum using the Odyssey Infrared Imaging System (Licor Biosciences).\n\nFor the detection of the C-terminus of PC1, a new antibody was generated against the peptide DSEDSLYSDYVDVFYN, which is present within the C-terminus of PC1 (amino acid numbering D714-N729). A cysteine residue was incorporated at the amino terminal to facilitate the coupling of the peptide to keyhole limpet hemocyanin (KLH). The synthesis of the peptide, coupling to KLH and generation of immune sera was performed under contract by Covance Research (Princeton, NJ). The antibodies were immunopurified as follows. Three mg of peptide (without KLH), dissolved in DMSO, were coupled to Affi-Gel 15 beads (Biorad, Hercules, CA) according to the manufacturer’s protocol. The beads were loaded into a column (~1.5 ml bed volume) and prepared for affinity chromatography. Five ml of the PC1 C-terminal antiserum (#5450) were mixed with 5 ml PBS containing 0.1% Tween 20 (PBST) and added to the column. The flow through sample was re-applied 3 times after which the column was washed with 30 ml PBST. The bound antibodies were eluted with 0.9 ml aliquots of 0.1 M Glycine, pH 2.9 into eppendorf tubes containing 100 μl 1 M Tris/Cl buffer, pH 7.5. Analysis by SDS-PAGE under reducing conditions and Coomassie Blue staining of the eluted fractions verified the presence of 50 kDa and 25 kDa immunoglobulin bands at an apparent purity estimated at >95% (data not shown). The purified IgGs were pooled and concentrated by centrifugation through 50 kDa molecular mass cutoff membranes (Pall Filtron, Northborough, MA). The buffer was also replaced with PBS/0.1% sodium azide by diafiltration through the same membranes. The resultant sample of immuno-purified IgGs (165 μg/ml) was stored at 4°C. These IgGs were used for the immunoprecipitation (IP) and immunocytochemistry (ICC) experiments.\n\nTo demonstrate the specificity of the new C-terminal PC1 purified IgGs, an immunoprecipitation was performed using radio-labeled proteins from AtT20 cells. The cells were labeled for 24 h with a mixture of [35S]-Met/[35S]-Cys (100 μCi/ml). Following this, the cells were rinsed 3 times with ice cold PBS and then harvested in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 2 mM EDTA (TNE buffer) containing 0.1% Triton X-100 and freshly prepared phenyl-methylsulfonyl fluoride (PMSF, 1 mM). The homogenate was centrifuged at 13,000 rpm for 10 min to sediment insoluble cellular material and a second extraction was performed on this pellet. The supernatants from both extractions were combined and incubated at 60°C for 20 min after addition of SDS to a final concentration of 0.1%. Following centrifugation of the sample (13,000 rpm, 10 min), Triton X-100 was added to the supernatant to a final concentration of 1%. The sample was pre-cleared by addition of protein A-sepharose beads (50 μl of a 50% slurry, 30 min, 4°C). After centrifugation to remove the beads, the supernatant was incubated with 1 μg of PC1 C-terminal immuno-purified IgGs or 1 μg of PC1 N-terminal immuno-purified IgGs for 18 h at 4°C. Antibody:antigen complexes were precipitated with 30 μl of protein-A sepharose beads and were washed extensively. The beads were resuspended in 1X tris-glycine SDS sample buffer containing β-mercaptoethanol, boiled for 10 min to elute the proteins and then analyzed by autoradiography after SDS-PAGE and electroblotting onto PVDF membrane. An additional gel was run later for the analysis of the IP supernatant. In that case, a 4–12% NuPAGE gel was used and the proteins transferred to nitrocellulose for autoradiography.\n\nIt was demonstrated previously in purified bovine adrenal chromaffin granules that some PC1 could adopt a transmembrane orientation1. This suggested that some of the C-terminus of PC1 is localized on the cytosolic side of secretory granules in the cell. In order to investigate this, in situ, we employed a procedure that has previously been characterized31 and one which we had been investigating independently. This procedure utilizes the observation that fixation of cells with para-formaldehyde (PFA) in PBS selectively permeabilizes the plasma membrane and allows access by immunoglobulins to the cytosolic space. Thus, cytosolic epitopes would be accessible for immunofluorescence microscopy in PFA/PBS fixed cells. On the other hand, proteins within the lumen of organelles, such as those found within the secretory pathway would not be accessible31. Using this procedure it is therefore possible to demonstrate the topology of a transmembrane protein within cells in situ if domain specific antibodies were available.\n\nAtT20 cells were grown in two-chambered glass slides, rinsed 3 times with room temperature (RT) PBS and then fixed in 2% PFA/PBS for 30 min at RT. One set of chambers was permeabilized by 0.25% Triton X-100/PBS for 5 min at RT while the other set received only PBS. After blocking with 1% bovine serum albumen (BSA)/PBS for 2 h at RT, the cells were then incubated for 16–20 h at 4°C with primary antiserum diluted as indicated in 1% BSA/PBS. Primary antibodies used were as follows; mouse anti-transferrin receptor (1:1,000, cytoplasmic epitope) (Invitrogen, Carlsbad, CA), mouse anti-p115 (1:1,000) (BD Biosciences, San Jose CA), rabbit anti-GRASP65 (1:2,000) (Proteus Biosciences, Ramona, CA), mouse anti-ACTH (1:1,000) (Abcam, Cambridge, MA), rabbit anti-PC1 (10 μg/ml, N-terminal specific) (Affinity Bioreagents Inc., Golden, CO) and rabbit anti-chromogranin A (1:10,000)32. The rabbit anti-PC1 (C-terminal specific) immuno-purified antibody, which was generated in our laboratory (see above), was used at a concentration of 1.6 μg/ml. This antibody was also used in combination with the mouse anti-p115 in a double labeling experiment. To demonstrate specificity, the C-terminal specific PC1 purified antibodies were pre-absorbed with the immunogenic peptide (1 μg/ml) and also used. After extensive washing with PBS, primary antibodies were detected with Alexa dye-conjugated secondary antibodies; goat anti-rabbit-568 (1:1,000) or goat anti-mouse-488 (1:1,000) from Molecular Probes (Invitrogen, Carlsbad, CA). All pictures were captured on an LSM 510 inverted scanning confocal microscope in the NICHD Microscopy and Imaging Core facility. For each antigen, power settings were optimized for the Triton X-100 (TX-100) treated cells until a clear, strong picture was obtained. These settings were then used to detect the same antigen in the non TX-100 treated cells, so that a direct comparison could be made between the staining intensities of the same antigen under the two conditions.\n\nProhormone convertase 1 cDNA29, encoded in the mammalian expression vector, pcDNA3.1, was transfected into COS7 cells using Lipofectamine 2000 according to the manufacturer (Invitrogen). Forty-eight h after transfection, the cells were processed for ICC under TX-100 treated and untreated conditions and images captured as described above.\n\n\n3. Results\n\nThree transmembrane proteins; transferrin receptor, aquaporin 1 and synaptotagmin 1, were studied as a set of control proteins for the classical procedure of alkaline sodium carbonate extraction. All 3 of these proteins were predominantly recovered in the sodium carbonate pellet indicative of their resistance to extraction by this procedure (Figure 1). Three non-transmembrane proteins were also studied as another set of control proteins for this procedure. These proteins, β-actin, chromogranin A (or β-granin when processed) and p115, a protein peripherally associated with the cytoplasmic side of the Golgi33, were recovered in the PBS supernatant (Figure 1). Residual levels of these proteins that were recovered in the PBS pellet were subsequently extracted in the sodium carbonate supernatant, demonstrating, as non-transmembrane proteins, their susceptibility to extraction by this procedure. These 6 proteins were established as positive and negative controls for the carbonate extraction procedure of AtT20 cells and all volumes were maintained equivalently so that a direct relative quantification of the proteins recovered at each step could be obtained when compared to the starting material. The distribution of PC1 was also analyzed using an N-terminal specific PC1 antibody. It was found that the 64 kDa form of PC1 was predominantly recovered in the PBS supernatant whereas the 87 kDa form was recovered in the PBS pellet. The 87 kDa form was subsequently recovered predominantly in the sodium carbonate pellet along with a small amount of the 64 kDa form (Figure 1).\n\nAtT20 cells were subjected to extraction by PBS followed by 0.1 M sodium carbonate, pH 11.5. Equivalent volumes from each step were analyzed by Western blot. Three TM proteins (transferrin receptor (TfR), synaptotagmin 1 (Syt-1) and aquaporin 1 (AQP-1) and 3 non-TM proteins (β-actin, β-granin and p115) were analyzed as controls. The 3 TM proteins were recovered in the sodium carbonate pellet while the 3 non-TM proteins were predominantly recovered in the PBS supernantant. The distribution of PC1 was also analyzed. The 64 kDa form was predominantly recovered in the PBS supernatant while the 87 kDa form (and a small amount of the 64 kDa form) was predominantly recovered in the carbonate pellet. This suggested that the 87 kDa form and some of the 64 kDa form of PC1 have properties consistent with a TM protein. T, total; S, supernatant; P, pellet.\n\nUnder steady state conditions, 2 forms of PC1 are found in AtT20 cells, an 87 kDa form and a 64 kDa form, both of which have an identical N-terminus. Immunoprecipitation by the N-terminal specific IgGs resulted in the capture of both these forms (Figure 2, N-term). When the C-terminal specific IgGs were used, one major band was captured consistent with being the 87 kDa PC1 form as it co-migrated with the 87 kDa form captured by the N-terminal specific IgGs (Figure 2, C-term). A faint band, with an apparent molecular mass of ~20 kDa based on the molecular mass standards (SeeBlue Plus 2, Invitrogen) was also seen. This band was considered likely to be the processed carboxyl terminus of PC1 since it was only present in the C-terminal specific IP lane and it has the same molecular mass as a previously expressed form of the C-terminal domain of mouse PC134. Western blot analysis of a subsequent IP of unlabeled AtT20 cells (both carried out with the PC1 C-terminal IgGs), failed to show such a PC1 immunoreactive protein (data not shown), indicating that it’s levels were too low for Western blot detection (compared to the radiolabeled band) or was possibly a protein that co-immunoprecipitated with the 87 kDa form of PC1.\n\nTo demonstrate the specificity of the immuno-purified PC1 C-terminal specific IgGs, an immunoprecipitation (IP) was performed on radiolabeled AtT20 cells. From a multitude of labeled proteins (Sup lane), one major band at 87 kDa was immunoprecipitated with these IgGs (C-term lane). As a control, immunoprecipitation with N-terminal specific PC1 IgGs yielded the two expected bands of 87 kDa and 64 kDa PC1 (N-term lane). A faint band at ~20 kDa was deemed non-specific as it was not immuno-reactive with the C-terminal specific IgGs in a Western blot of a subsequent IP of unlabeled AtT20 cell lysate (data not shown).\n\nTo demonstrate that PFA fixation selectively permeabilizes the plasma membrane, we performed ICC on TX-100 treated and non-treated AtT20 cells and analyzed the staining pattern of 6 endogenous proteins; 3 with epitopes localized in the cytosol and 3 proteins localized within the lumen of organelles belonging to the regulated secretory pathway (RSP) which includes the Golgi and secretory granules. For all 3 lumenal proteins, CgA, ACTH and the N-terminus of PC1, strong staining of the Golgi and tips of the processes were observed only in the TX-100 treated cells consistent with their presence in the RSP (Figure 3A-C). In the absence of TX-100, no staining of these proteins could be detected (Figure 3D-F), demonstrating the requirement for the detergent to expose these proteins to the antibodies by permeabilizing the membranes of the organelles. For the 3 proteins with cytosolic epitopes, p115, Grasp 65 and transferrin receptor, strong immuno-staining was observed whether TX-100 was used or not (Figure 4). Thus PFA fixation allows accessibility of the IgGs to the cytosol, where they can bind their antigens, but not to the lumen of the organelles of the RSP.\n\nAtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. Three luminal proteins belonging to the RSP (Chromogranin A, ACTH and the N-terminus of PC1) were stained by indirect immunofluorescence. For all three proteins, the Golgi (arrows) and the tips of the processes (arrow heads) were specifically stained when Triton X-100 was used (A–C). No staining was seen when Triton X-100 was not used (D–F). This staining pattern is consistent with the presence of these proteins within the organelles of the RSP and demonstrates that PFA fixation does not cause an access of the antibodies to these compartments. Bar 20 μm\n\nAtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. Three cytosolic proteins (p115, Grasp65 and N-terminus of the transferrin receptor) were stained by indirect immunofluorescence. For p115 (A, D) and Grasp65 (B, E), staining of the Golgi area was observed and for the transferrin receptor (C, F), staining of the plasma membrane/endosomes were observed, whether the cells were treated with Triton X-100 or not. This demonstrated that the antibodies had access to the cytosol even with only PFA fixation. Bar 20 μm\n\nUsing this procedure with the C-terminal specific immuno-purified PC1 antibodies, a pattern of staining for PC1 was observed in the TX-100 treated cells that was similar to that of CgA, ACTH and the N-terminal specific PC1 antibodies, i.e. strong staining of the Golgi and a punctate pattern in the processes (Figure 5, top panel, PC1). The staining pattern exhibited by these purified antibodies is consistent with the localization of PC1 in the Golgi, as evidenced by its colocalization with p115 (Figure 5, top panel, p115 and Merged) and secretory granules of the RSP. No staining could be seen when immuno-purified C-terminal PC1 IgGs were used that had been pre-absorbed by the antigenic peptide (Figure 5, Absorption control). In the absence of TX-100, however, while staining with the N-terminal specific PC1 antibodies was negative (Figure 3F); staining of the Golgi and processes was observed in the untreated cells with the C-terminal specific IgGs (Figure 5, lower panel, PC1). This pattern of staining indicated that the C-terminus of PC1 is present in the cytosol and the N-terminus of PC1 is in the lumen of the Golgi and secretory granules, indicating that at least some PC1 is in a transmembrane orientation in situ and supports the results of the extraction experiments (Figure 1).\n\nAtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. PC1 was stained with C-terminal specific immunopurifed IgGs by indirect immunofluorescence. In the Triton X-100 treated cells, the staining pattern of the C-terminus of PC1 was similar to that of the N-terminus of PC1 described in Figure 3, in that Golgi staining (arrows) was observed (top panel). In the Triton X-100 untreated cells, however, a reduced but similar staining pattern was observed to that of the Triton X-100 treated cells (lower panel). This demonstrates that some of the C-terminus of PC1 was localized in the cytosol. Bar 20 μm.\n\nThe topology of PC1 transfected into non-endocrine COS7 cells was assessed also by this procedure. After fixation by PFA, the C-terminus of PC1 was strongly stained by the C-terminus specific purified IgGs only after permeabilization with TX-100 (Figure 6A). This result indicated that PC1 did not assume a TM orientation in COS7 cells consistent with the results of Stettler et al. in COS1 cells30.\n\nCOS7 cells, expressing transfected full length PC1, were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. PC1 was stained with C-terminal specific immunopurifed IgGs by indirect immunofluorescence. In the Triton X-100 treated cells, a strong staining pattern of the C-terminus of PC1 was observed in the transfected cells consistent with a distribution in the reticular network of the ER and in the Golgi. (A) Only a low level of background staining was observed for the TX-100 untreated cells (B). Bar 20 μM.\n\n\n4. Discussion\n\nProhormone convertase 1 (PC1) is sorted to the regulated secretory pathway (RSP) of (neuro)endocrine cells where it functions to cleave prohormones and proneuropeptides into smaller peptides that ultimately function in important biological processes. How PC1 is sorted to the RSP has been actively studied and several proposals have been put forward. A commonality among these ideas is the belief that association of the C-terminal tail of PC1 with components of the trans-Golgi network (TGN) membrane, where sorting to the RSP is believed to be initiated, must occur, although binding via the prodomain has also been implicated35. In light of the extraction and/or binding studies by Hill et al.21, Arnaoutova et al.1 and Jutras et al.25, it is considered that such binding or membrane association is quite strong. Without evidence of amino acid sequences that predict a classical transmembrane (TM) domain similar to furin and Kex2, and with the previously identified membrane binding amphipathic α-helices within the C-terminus25, it is reasonable to expect that binding would be to the luminal side of the TGN membrane in a non-TM manner and that such binding would be necessary for sorting to the RSP.\n\nHowever, we have previously studied the membrane association properties of carboxypeptidase E (CPE) that contains an amphipathic α-helix at its C-terminus36,37 and demonstrated that it can assume a transmembrane topology in a lipid raft dependent manner. Indeed, live cell imaging demonstrated a role of its cytoplasmic tail in peptide hormone granule transport via interaction with dynactin and the microtubule dependent motor proteins, kinesin and cytoplasmic dynein38,39. Hence, based on these and other observations of the novel TM behavior of CPE and PC2 via their C-termini37,40, we speculated that lipid raft-association and TM orientation of PC1 might occur through a similar C-terminal domain that was identified in PC1. Thus, in our previous work we tested this idea principally in intact purified, bovine adrenal medulla chromaffin granules and showed the presence of the C-terminus of PC1 on the outside of these granules. In such a case the cytosolic C-terminus would be quite long (~114 aa). To explain how this might occur, we speculated that, since insertion through the membrane in the Golgi or post-Golgi compartment would be energetically unlikely, PC1 might be synthesized as a TM protein1.\n\nOur speculations, however, have been challenged by Stettler et al. who showed by various methods that transfected PC1 is not synthesized as a TM protein in COS1 cells30. The results of Stettler et al. were important for two reasons. One, it provided information about the initial synthesis of PC1, albeit in a non-endocrine cell, and two; it questioned whether PC1 was a TM protein at all, because the data by Arnaoutova et al. which demonstrated it to be a TM protein in intact purified granules, was discounted by Stettler et al. simply as the result of contamination. While this is a valid point, we do not consider it to be probable because, had there been even a small amount of contamination, chromogranin A, the most abundant protein in chromaffin granules (47% of soluble granule content, 7% of total adrenal medulla content41, would, like PC1, also have been biotinylated when the purified granules were used in the biotinylation experiment. The observation that PC1 was biotinylated and CgA was not provided very strong evidence that PC1 was there in a transmembrane orientation rather than by contamination from ruptured organelles.\n\nRegardless of this explanation and to readdress the issue of PC1 topology, we undertook to analyze endogenously expressed PC1 in a model endocrine cell line where both forms are known to exist at steady state. Initial extractions by sodium carbonate, pH 11.5, a classical procedure for the characterization of TM proteins initially described by Fujiki et al.42, suggested that the 87 kDa form (and some of the 64 kDa form) of PC1 had properties of a TM protein because its partitioning behavior was similar to three known TM proteins (Figure 1). These results are consistent with the previously published data on PC1 from bovine chromaffin granules1 and indicated to us that PC1 in AtT20 cells had similar properties to PC1 found in chromaffin cells from bovine adrenal medulla. While resistance to alkaline sodium carbonate extraction is not definitive proof of TM orientation, it is considered to be strong evidence for such a conclusion. To investigate this further we studied PC1 topology in AtT20 cells under steady state conditions in situ by immunofluorescence confocal microscopy. This simple but powerful procedure is based on the observation that fixing cells for immunocytochemistry (ICC) with para-formaldehyde (PFA) in PBS, permeabilizes the plasma membrane sufficiently to allow immunoglobulins (IgGs) into the cytosol31. However, since PFA/PBS does not have the same effect on membranes of internal organelles, we can determine the topology of organellar proteins if domain specific IgGs are available.\n\nTo demonstrate the validity of this technique in AtT20 cells, we performed ICC on PFA fixed cells with and without detergent permeabilization and analyzed the staining of 3 known cytosolic proteins (Grasp65, p115 and the N-terminus of the transferrin receptor) and 3 known luminal proteins belonging to the regulated secretory pathway (RSP) (ACTH, CgA and the N-terminus of PC1). As expected, the RSP proteins were only stained when the cells were permeabilized with the detergent, Triton X-100 (TX-100) demonstrating the integrity of the membranes of the internal organelles (Figure 3). The cytosolic proteins were stained whether TX-100 was used or not (Figure 4) demonstrating that the antibodies had access to the cytosol even in the absence of detergent treatment. Staining of the C-terminus of PC1 with our immuno-purified IgGs gave a similar pattern of staining to that of the PC1 N-terminal specific IgGs when performed on TX-100 treated cells. In the absence of TX-100, however, reduced but specific staining with the C-terminal specific IgGs was also observed indicating that some of the C-terminus of PC1 was present in the cytosol (Figure 5). Golgi staining of the C-terminus was observed (as demonstrated by its colocalization with p115) as well as punctate staining in the processes, indicative of granules, suggests that PC1 (or some of it) can be in a TM orientation in the Golgi and granules.\n\nHow this happens is currently unknown. However, assuming that the results observed by Stettler et al. in the COS1 cells are similar for the synthesis of endogenous PC1 in classical (neuro)endocrine tissue/cell lines, we are now directed to re-consider the possibility that an insertion event might be taking place after synthesis in the ER. Although the sequence identified as the TM domain (aa619–638)1,29,43 does not have classical TM characteristics, it is conceivable that one or several factors that are still unknown may facilitate or stabilize PC1 in such an insertion/orientation, thus reducing the free energy necessary for such an event. An example of a \"helper\" protein exists for the diphtheria toxin where insertion into model membranes as a TM protein only occurs in the presence of molten globule-like proteins44. In addition, while it is known that C-terminal tail-anchored proteins (TA proteins, e.g. cytochrome b5) are TM proteins, the mechanism by which membrane insertion of these cytosolic proteins occurs is still unknown as it is independent of the Sec61 translocon45.\n\nIt is probable that not all of the PC1 becomes TM, in which case an equilibrium may exist between luminal/peripheral and TM partitioning and that this equilibrium may depend on levels of endogenous factors as indicated above. Indeed a more intense signal of the PC1 C-terminal signal in the granules was observed in the TX-100 treated cells (Figure 5, top panel versus lower panel) indicating the presence of the C-terminus within the lumen of the granules presumably as a cleaved product. The concept of \"helper\" proteins is also supported by our observations that when we transfected PC1 into COS7 cells (Figure 6) or PC12 cells (a model neuroendocrine cell line) (data not shown) and performed the ICC experiment we could only observe specific PC1 C-terminal staining when TX-100 was used indicating that in transfected cells the PC1 did not measurably adopt a TM orientation. This suggested to us that TM insertion is a saturable process that appears to require components that are limiting in (neuro)endocrine cells or not present in non-endocrine cells. Studies are underway to identify such components.",
"appendix": "Author contributions\n\n\n\nNXC devised and planned the experiments. MS, HL and NXC carried out the experiments. NXC and YPL interpreted the results and NXC wrote the paper. YPL supported the project.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests declared.\n\n\nGrant information\n\nThis research is supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH.\n\n\nAcknowledgements\n\nMicroscopy imaging was performed at the Microscopy & Imaging Core (National Institute of Child health and Development, NIH) with the assistance of Dr. Vincent Schram and Mr. Chip Dye.\n\n\nReferences\n\nArnaoutova I, Smith AM, Coates LC, et al.: The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein. Biochemistry. 2003; 42(35): 10445–10455. 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PubMed Abstract | Publisher Full Text\n\nO'Connor DT, Frigon RP: Chromogranin A, the major catecholamine storage vesicle soluble protein. Multiple size forms, subcellular storage, and regional distribution in chromaffin and nervous tissue elucidated by radioimmunoassay. J Biol Chem. 1984; 259(5): 3237–3247. PubMed Abstract\n\nFujiki Y, Hubbard AL, Fowler S, et al.: Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982; 93(1): 97–102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArnaoutova I, Jackson CL, Al-Awar OS, et al.: Recycling of Raft-associated prohormone sorting receptor carboxypeptidase E requires interaction with ARF6. Mol Biol Cell. 2003; 14(11): 4448–4457. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRen J, Kachel K, Kim H, et al.: Interaction of diphtheria toxin T domain with molten globule-like proteins and its implications for translocation. 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}
|
[
{
"id": "267",
"date": "13 Aug 2012",
"name": "Nabil Seidah",
"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": "268",
"date": "16 Aug 2012",
"name": "David R Cool",
"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": "269",
"date": "29 Aug 2012",
"name": "Regina Kuliawat",
"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\nProhormone convertases (PCs) are clearly a critical component for the normal function of secretory granules and the mechanism by which PCs are efficiently targeted to the correct compartment needs to be well understood.The carboxy (C) terminus of PC1/3 is important for efficient trafficking to granules and interaction of the C-terminal sequence with membranes appears to be important for sorting. In neuroendocrine cells, processing of the 87 kDa ER to the 64-66 kDa form of PC1/3 occurs in a late compartment of the secretory pathway and involves the removal of the C terminus. Based on the observations that in chromaffin granules PC1/3 may adopt a transmembrane orientation whereas in non-endocrine COS-1 cells, ER localized PC1/3 did not behave as a membrane spanning protein, Cawley and colleagues generated an antibody specific to the C terminus. In this report, the authors use biochemical and imaging approaches to characterize the behavior of the C terminus of PC1/3 in both endocrine (AtT20) and non-endocrine (COS7) cell lines to determine if the presence of the regulated secretory pathway influences the topology of PC1/3.Figure 1. AtT20 cells were lysed by freeze thawing and upon centrifugation and Western blot detection using the N-terminus directed antibody, proportionately more of the 87 kDa form is recovered in the membrane associated pellet. Alkaline carbonate extraction of the pellet fraction retrieves predominantly the proform. Overall, comparison of carbonate extraction profiles of PC1/3 to bone fide membrane or cytosolic proteins suggests that the unprocessed form of PC1/3 exhibits properties resembling those of a membrane protein.Figure 2. To examine the relationship between PC1/3 maturation and epitope recognition by the N-and C-directed antibodies, radiolabeled PC1/3 was evaluated. Prolonged labeling of cells is expected to load the more slowly turning over pools with radioactive PC1/3. Thus, 24 h metabolically labeled cells should have accumulated both the 87kDa and the processed 64 kDa forms preferentially within late compartments of the secretory pathway. The immunoprecipitation experiments demonstrate that both antibodies are capable of detecting PC1/3 but why, using the same cell lysate do the N-term and C-term antibodies differ so substantially in their recovery of the 87 kDa? In addition, PC1/3 does not have the traditional transmembrane segment and is thought to fully translocate into the ER lumen. Thus, a more careful pulse-chase analysis including sequential immunoprecipitations would have improved on the existing antibody characterization.Generally speaking, antibodies that recognize only denatured epitopes do not work well for immunofluorescence. Since the epitope recognition by the antibody is important for the analysis, the authors should provide an explanation why the rather elaborate heating/solubilization protocol was required to prepare the cell lysates for immunoprecipitation. Nevertheless, The C-and N-terminus directed antibodies show a distinct pattern of recognition in metabolically labeled cells, with C-term antibody capturing the immature forms of PC1/3.Figures 3-5. For immunocytochemical analysis, treatment with Triton permeabilizes plasma membrane as well as intracellular membranes and allows antibodies to bind to epitopes oriented towards both cytosol and lumen. Tested in this section of the report is the underlying assumption that paraformaldehyde fixation permeabilizes the plasma membrane leaving intracellular membranes intact and antibodies cannot gain access to the lumen of organelles. Antibodies raised against the N-terminal domain of PC1/3 (oriented towards the lumen) is expected to immunostain PC1/3 only in cells treated with Triton X-100, whereas antibodies raised against the C-terminal domain (if oriented towards cytoplasm) will stain PC1/3 in the presence or absence of Triton X-100. Indeed, PFA fixation is sufficient for access by the C-terminal PC1/3 antibody as well as the antibodies to cytosolic proteins, whereas lumenal proteins cannot be detected–antibody to N terminus of PC1/3 falls into this latter category. The reduced but similar pattern of C-PC1/3 antibody staining obtained in the absence or presence of detergent permeabilization is taken as evidence for the membrane spanning topology of PC1/3.To strengthen the conclusion and to exclude the possibility that some of the staining reflects damaged, hence leaky or ruptured organelles, controls to demonstrate organelle integrity are needed. Co-localization experiments shown in Figure 5, should have also characterized the appropriate lumenal markers. Protease protection is an additional method for assaying cytosolic protein domains and a fluorescence-based technique involving protease protection (Lorenz H, Hailey DW, & Lippincott-Schwartz J, 2008) would have been an additional test to use in support of PC1/3 trans-membrane orientation.Figure 6. Finally, in COS7 cells, the C terminus of PC1/3, clearly detectable in perinuclear regions when detergent is present, is not available to antibody binding in the absence of detergent. This is in agreement with the report from Stettler and colleagues (although the reference appears to be missing) and raises the intriguing possibility that ‘helper’ proteins, specific to cells with a regulated secretory pathway, may potentiate the insertion into membranes. Here as well, the work would have benefited from a more extensive characterization of ER localized PC1/3.",
"responses": []
}
] | 1
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https://f1000research.com/articles/1-9
|
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