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https://f1000research.com/articles/2-175/v1	16 Aug 13	{ "type": "Case Report", "title": "Case Report: A rare cause of multiple organ dysfunction syndrome: Human Herpes Virus 6 infection", "authors": [ "Hakan Tekguc", "Nilufer Galip", "Ceyhun Dalkan", "Nazan Çobanoğlu", "Nerin Nadir Bahceciler", "Nilufer Galip", "Ceyhun Dalkan", "Nazan Çobanoğlu", "Nerin Nadir Bahceciler" ], "abstract": "Human herpes virus 6 (HHV-6) is a member of the β-herpes virus subfamily which targets mainly CD4 T cells and is a well-known cause of roseola infantum. Fever without roseola, encephalitis and hepatitis however are not uncommon after HHV-6 infection. More severe clinical cases are commonly observed in immune compromised patients. Case: An 11-month old girl, after a 24-hour fever, and with poor appetite was admitted into the hospital. Oral antibiotic treatment was initiated and she was discharged from the state hospital’s out-patient clinic two hours later. The following day, the patient continued to experience high fever, and hematemesis, and a tendency to sleep were added to her condition and she was once more admitted to the hospital. Lab results showed thrombocytopenia, alanine aminotransferase over 3000 U/L, INR was 2.5 and urea and creatinine were elevated at 75 mg/dl and 1.1 mg/dl, respectively. Due to persistent high fever and somnolence, a lumbar puncture was performed. The cerebrospinal fluid (CSF) was clear of any cells; protein and glucose were within normal range. However, test results were positive for HHV-6 DNA in the CSF, serum, and lymphocytes. Four organ dysfunctions including the central nervous-, hematologic-, renal- and hepatic systems, developed because of HHV-6 infection. Organ functions were normalized within one week of supportive treatment. HHV-6 is a benign virus that very rarely causes severe infection and hardly ever leads to a fatal infection. However, in our case, a healthy child, with a HHV- viral infection led to multiple organ dysfunction without any predisposing reason.", "keywords": [ "cerebrospinal fluid", "creatinine", "DNA" ], "content": "Introduction\n\nHuman herpes virus 6 (HHV-6) is a member of the β-herpesvirus subfamily of herpesviruses and targets mainly CD4 T cells1,2; however, it can infect many other cells of the hematologic, neurologic and hepatobiliary systems. HHV-6 infection is a well-known cause of roseola infantum (sixth disease); however, fevers without roseola, encephalitis and hepatitis are not uncommon1. Usually, primary infection occurs between 6 and 15 months of age and seropositivity is almost 100% by age 3. HHV-6 establishes latency in mononuclear cells and probably in entire body systems. HHV-6 can reactivate in immunocompromised patients3. Multiple organ dysfunction syndromes (MODS) define two or more organ failures because of systemic inflammatory response (Table 1), infections and sepsis are the most common reasons for MODS4.\n\n\nCase\n\nWritten informed consent for publication of clinical details was obtained from the legal guardian of the 11-month-old girl. The patient was admitted to the state hospital, with a 24-hour fever and poor appetite, and subsequently treated with amoxicillin/clavulinic acid with 40 mg/kg at a private clinic. She was diagnosed with upper respiratory tract infections and otitis media. The patient returned to the hospital the following day as the fever continued. She was restless and had one-time hematemesis. Vital signs were noted as normal, although she had a tendency to sleep. Initial investigation showed thrombocytopenia (normal hemoglobin and leukocyte count), and aspartate levels, alanine aminotransferase activity, prothrombin time, urea- and creatinine levels were all elevated (Table 2). Serum electrolytes, blood glucose, and calcium levels were normal. Ceftriaxone treatment, 100 mg/kg, was started on the first day of admission. On the following day, a lumbar puncture was performed because of persistent high fever and somnolence. Acyclovir (10 mg/kg every 8 hours), and teicoplanin (10 mg/kg/day) treatment were given, even though the cerebrospinal fluid was clear of cells, and the protein- and glucose levels were within the normal range. It was noticed that urine output was progressively decreasing (less than 0.5 cc/kg/h) and creatinine values were rising (maximum; 1.09 mg/dl).\n\nOn day four, the girl was transferred to the Near East University Pediatric Intensive Care Unit, due to dysfunction of four organ systems (the central nervous-, hematologic-, renal- and hepatic systems, Table 2)4. At her arrival, she was restless with a temperature of 36.4ºC, she had a tendency to sleep, her capillary refill time was prolonged, heart rate was 116/minute, and blood pressure was 100/55 mmHg. The patient had generalized edema but no hypotension, respiratory distress, hepatosplenomegaly, lymphadenopathy or rash was seen. Electroencephalography (EEG) was performed and showed bitemporal slow waves, but no electrical activity causing seizures was detected. During four nights of intensive care unit hospitalization, no fever was observed, but on the second day, a maculopapular rash started from the trunk and expanded to the whole body. The polymerase chain reaction test results on the first lumbar puncture were positive for HHV 6 DNA in the cerebrospinal fluid, serum and lymphocytes. Epstein Barr virus, cytomegalovirus, hepatitis A IgM results and bacterial cultures were all negative. The patient was diagnosed with HHV 6 infection and acyclovir treatment was continued for two weeks. Although ganciclovir or foscarnet would have been more appropriate for HHV 6 encephalitis and hepatitis treatment3, we had to use acyclovir because these other drugs were not available at our hospital at that time. One week after admission to Near East University Hospital, creatinine levels became normal, and were still normal 15 days later (0.44 mg/dl). Immunoglobulin and lymphocyte subset analysis was normal. The patient was discharged from the hospital without any sequelae and at a check-up three months later her laboratory parameters and developmental status were completely normal. In the following two years, the patient showed no sign of immune deficiency or another severe infection.\n\n\nDiscussion\n\nRoseola infantum is characterized by a sudden onset of fever, lasting for three to five days, followed by maculopapular rash, which may be transient or in fact may not appear at all1. HHV 6 usually causes roseola infantum but can also cause encephalitis and acute hepatitis2,5. In general, it is a benign virus that very rarely causes severe infection and hardly ever leads to a fatal infection6. However, our case showed MODS due to HHV 6 virus infection. The patient had a Glasgow Coma Scale of 11 and EEG abnormalities, a more than twofold increase in baseline creatinine, the INR was over 2 and ALT was twice the normal upper limit for her age. Besides hepatitis and encephalitis, our patient displayed renal insufficiency, which can not be explained by prerenal or postrenal reasons. The patient did not display any hypotension or dehydration, and renal ultrasonography did not show any pathology. The patient was diagnosed with intrinsic renal failure. We were unable to find out whether the exact reason for intrinsic renal pathology was the HHV 6 infection. In the current literature there are no reports of renal insufficiency in an otherwise healthy child, although it has been shown that in renal-transplanted patients HHV 6 can cause re-infection7 and allograft rejection8.\n\nWe used a polymerase chain reaction technique to detect HHV 6 DNA in the CSF, serum and lymphocytes. We checked for other viruses which can cause hepatitis and encephalitis like enterovirus, herpes simplex type 1 and 2. The test results for all these were negative. For an 11-month-old girl without an predisposing immune deficiency, sepsis or transplantation, we diagnosed her with primary infection9 after detecting HHV 6 DNA.\n\n\nConclusions\n\nHHV 6 is usually a benign virus which causes no or negligible organ dysfunction in a healthy child. It is known that when the patient is immunocompromised HHV 6 can cause organ dysfunction and encephalitis10,11. In addition; HHV 6 infection can cause organ rejection problems in transplant patients12,13. In the current literature, there are no other studies that report renal and hepatic insufficiency proceeding to MODS due to HHV-6 virus infection in a healthy child without any predisposing factors.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the legal guardian of the 11-month-old patient.", "appendix": "Author contributions\n\n\n\nHakan Tekguc was the primary doctor who followed the patient in the intensive care unit, and wrote the manuscript. Ceyhun Dalkan and Nilufer Galip were the other doctors following the patient at Near East University Hospital and helped writing the manuscript. Dr. Behceciler and Dr. Cobanoglu were two senior professors who supervised our team while following the patient in university hospital, also both reviewed the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgments\n\nThis article was based on a poster presented at 30th Annual Meeting of the European Society for Pediatric Infectious Diseases in Thessaloniki, Greece in 2012.\n\n\nReferences\n\nDebiasi RL, Tyler KL: Molecular methods for diagnosis of viral Encephalitis. Clin Microbiol Rev. 2004; 17(4): 903–925. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams MV: HHV-6: response to antiviral agents. In Human herpesvirus-6: epidemiology, molecular biology, and clinical pathology. Ablashi, DV Krueger, RF and Salahuddin, SZ (Eds), Elsevier Biomedical Press, Amsterdam, the Netherlands. p. 317. Publisher Full Text\n\nDe Bolle L, Manichanh C, Agut H, et al.: Human herpesvirus 6 DNA polymerase: enzymatic parameters, sensitivity to ganciclovir and determination of the role of the A961V mutation in HHV-6 ganciclovir resistance. Antiviral Res. 2004; 64(1): 17–25. PubMed Abstract \| Publisher Full Text\n\nGoldstein B, Giroir B, Randolph A: International pediatric sepsis consensus conference: Definitions for sepsis and organ dysfunction in pediatrics. Pediatr Crit Care Med. 2005; 6(1): 2–8. PubMed Abstract \| Publisher Full Text\n\nZerr DM, Meier AS, Selke SS, et al.: A population-based study of primary human herpesvirus 6 infection. N Engl J Med. 2005; 352(8): 768–776. PubMed Abstract \| Publisher Full Text\n\nLautenschlager I, Razonable RR: Human herpesvirus-6 infections in kidney, liver, lung, and heart transplantation: review. Transpl Int. 2012; 25(5): 493–502. PubMed Abstract \| Publisher Full Text\n\nDeborska-Materkowska D, Sadowska A, Matłosz B, et al.: [Human herpes virus 6 infection in renal transplant recipient--case report]. Przegl Epidemiol. 2006; 60(1): 141–146. PubMed Abstract\n\nAcott PD, Lee SH, Bitter-Suermann H, et al.: Infection concomitant with pediatric renal allograft rejection. Transplantation. 1996; 62(5): 689–691. PubMed Abstract\n\nKainth MK, Caserta MT: Molecular diagnostic tests for human herpesvirus 6. Pediatr Infect Dis J. 2011; 30(7): 604–605. PubMed Abstract \| Publisher Full Text\n\nWaruiru C, Slatter MA, Taylor C, et al.: Outcome of hematopoietic stem cell transplantation in severe combined immune deficiency with central nervous system viral infection. Pediatr Infect Dis J. 2007; 26(2): 129–133. PubMed Abstract \| Publisher Full Text\n\nMagalhães GS, Guardia AC, Sampaio AM, et al.: HHV-6: Clinical and Laboratory Investigations and Correlations With Encephalitis in Liver Transplant Recipients. Transplant Proc. 2013; 45(5): 1997–9. PubMed Abstract \| Publisher Full Text\n\nGuardia AC, Stucchi RS, Sampaio AM, et al.: Human herpesvirus 6 in donor biopsies associated with the incidence of clinical cytomegalovirus disease and hepatitis C virus recurrence. Int J Infect Dis. 2012; 16(2): e124–e129. PubMed Abstract \| Publisher Full Text\n\nSampaio AM, Guardia AC, Milan A, et al.: Co-infection and Clinical Impact of Human Herpesvirus 5 and 6 in Liver Transplantation. Transplant Proc. 2012; 44(8): 2455–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1655", "date": "24 Oct 2013", "name": "Esra Şevketoğlu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall I ‘Approve’ of this paper but I have the following comments:\n\nAll the patient's laboratory values (normal-abnormal) should be displayed in table 2.In the Case section the authors state that the capillary refill time was prolonged and that the patient had ‘generalised edema’; please state how these symptoms were treated.In the Discussion section the authors state that ‘The patient had a Glasgow Coma Scale of 11’; this should probably also be mention in the Case section.In the Discussion section the authors state that ‘ALT was twice the normal upper limit for her age’ yet this measurement is recorded as being >3000 in table 2.In the Discussion section the authors state that ‘We checked for other viruses which can cause hepatitis and encephalitis like enterovirus, herpes simplex type 1 and 2’ yet in the Case section different viruses are mentioned: 'Epstein Barr virus, cytomegalovirus, hepatitis A IgM results and bacterial cultures were all negative.'Finally the authors state that ‘The patient was diagnosed with intrinsic renal failure.’ but renal failure cannot be distinguished as intrinsic or extrinsic on the basis of this kind of laboratory data.", "responses": [] }, { "id": "3965", "date": "04 Mar 2014", "name": "Peter Medveczky", "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\nTekguc and colleagues describe an 11 month-old child hospitalized for high fever, thrombocytopenia, and possible multi-organ dysfunction. While it is possible that this is an unusual case of HHV-6 primary infection another obvious alternative is that the child has inherited or “chromosomally integrated” HHV-6 commonly referred to as ciHHV-6. Unfortunately this possibility was not considered in the differential diagnosis. The diagnosis of ciHHV-6 can be established by either quantitative PCR, or alternatively by PCR of hair follicles. Therefore the title “A rare cause of multiple organ dysfunction syndrome: Human Herpes Virus 6 infection” and the overall conclusions of the paper are not supported by the laboratory data.", "responses": [] } ]	1	https://f1000research.com/articles/2-175
https://f1000research.com/articles/2-171/v1	12 Aug 13	{ "type": "Review", "title": "Post-sphincterotomy bleeding: fully-covered metal stents for hemostasis", "authors": [ "Anthony T DeBenedet", "Grace H Elta", "Grace H Elta" ], "abstract": "Background/objectives: In endoscopic retrograde cholangiopancreatography, post-sphincterotomy bleeding (PSB) is a common complication of biliary sphincterotomy. Recently, the temporary placement of fully-covered metal stents (FCMS) into the biliary tree in order to achieve a tamponade effect has been described as an additional therapeutic option for PSB. The aim of this article is to review the literature on FCMS for hemostasis in PSB and update the treatment algorithm for this complication.Methods: A PubMed literature search was conducted using the search terms post-sphincterotomy, bleeding, and stent. 33 articles were reviewed, along with their references, and four were found to describe the use of FCMS for hemostasis in PSB.Results: A total of 21 patient cases were described in the four articles. All patients received FCMS for PSB hemostasis following the application and subsequent failure of traditional therapies (conventional pharmacologic injection, thermal or electrocoagulation, and mechanical therapy (balloon tamponade or endoclip)). Successful hemostasis was achieved in all patients through FCMS placement. No major complications were observed.\n\nConclusion: These 21 cases demonstrate that FCMS are a viable therapeutic option for PSB. It is reasonable to consider stent placement for patients in which traditional interventions fail in order to avoid the need for angiographic or surgical hemostasis.", "keywords": [ "In endoscopic retrograde cholangiopancreatography (ERCP)", "post-sphincterotomy bleeding (PSB) is a common complication of biliary sphincterotomy. The incidence varies from 1–48% depending on what definition is applied", "specifically whether bleeding during the procedure is included1", "intraprocedural bleeding is classified as immediate", "whereas bleeding that occurs after the procedure is considered delayed2–4. The accepted criteria for grading PSB are: mild", "which is a hemoglobin drop of less than 3 grams and no transfusion requirement", "moderate", "which is a transfusion requirement of 4 units or less and no angiographic intervention or surgery", "and severe", "which is a transfusion requirement of 5 units or more or intervention (angiographic or surgical)2." ], "content": "Introduction\n\nIn endoscopic retrograde cholangiopancreatography (ERCP), post-sphincterotomy bleeding (PSB) is a common complication of biliary sphincterotomy. The incidence varies from 1–48% depending on what definition is applied, specifically whether bleeding during the procedure is included1; intraprocedural bleeding is classified as immediate, whereas bleeding that occurs after the procedure is considered delayed2–4. The accepted criteria for grading PSB are: mild, which is a hemoglobin drop of less than 3 grams and no transfusion requirement; moderate, which is a transfusion requirement of 4 units or less and no angiographic intervention or surgery; and severe, which is a transfusion requirement of 5 units or more or intervention (angiographic or surgical)2.\n\nImmediate bleeding will often spontaneously stop without endoscopic intervention. But for immediate or delayed PSB that requires intervention, the endoscopic hemostasis options parallel those for bleeding elsewhere in the gastrointestinal tract: conventional pharmacologic injection (epinephrine); thermal or electrocoagulation; and mechanical therapy (balloon tamponade or endoclip). With the exception of pharmacologic injection, it can be challenging to effectively administer these various interventions through a side-viewing endoscope; however, endoscopic hemostasis is usually successful. When hemostasis is not achieved, angiographic embolization or surgery is required.\n\nRecently, the temporary placement of fully-covered metal stents (FCMS) into the biliary tree to achieve a tamponade effect has been described as an additional therapeutic option for PSB5–8. The aim of this article is to review the literature on FCMS for hemostasis in PSB and update the treatment algorithm for this complication1.\n\n\nLiterature review\n\nA PubMed literature search was conducted in July 2013 using the search terms post-sphincterotomy, bleeding, and stent. 33 articles were reviewed, along with their references, and four were found to describe the use of FCMS for hemostasis in PSB.\n\nIn 2010, Pisa and colleagues were the first to describe the technique in a two-patient case series that included one adult and one pediatric patient5. The adult initially received epinephrine injection and endoclip placement, but bleeding persisted. A plastic stent (10 Fr, 5 cm, OASIS, Wilson Cook Medical, Winston-Salem USA) was then placed, which also did not achieve hemostasis. Thus, a fully-covered metallic stent (4 cm in length and 1 cm in diameter, Wallstent, Boston Scientific, Natik USA) was placed. No further bleeding occurred. The stent was removed four weeks later without complication. The child received the same metal stent, after initially failing epinephrine injection and balloon tamponade. This stent was removed three weeks later without complication.\n\nShah and colleagues were next to describe the use of FCMS for hemostasis in PSB6. Their case series included five adult patients who failed various traditional therapies prior to stent placement (epinephrine injection, thermocoagulation, endoclip, and angiographic embolization). Each patient received a stent that was sized according to the patient’s anatomy (4–6 cm in length and 8–10 mm in diameter, Wallstent, Boston Scientific, Natik USA). The stents were placed so that the proximal end did not obstruct the cystic duct orifice and the distal end extended 1–2 cm beyond the biliary orifice. For each patient, stent removal was scheduled 2–4 weeks following stent placement. In three of the five patients, the stents were removed without complications; in two patients, the stents were not seen at the time of removal and were presumed to have migrated distally, as there was no fluoroscopic evidence of stent retention.\n\nItoi et al. published their case series of 11 patients in 20117. All patients had bile duct stones and had failed either mono- or combined therapy (hypertonic saline epinephrine injection, balloon tamponade, and/or endoclip placement) prior to stent placement. The FCMS were 6 cm in length and 10 mm in diameter (Covered Wallstent or Covered WallFlex, Boston Scientific Tokyo, Japan or Combistent, Taewoong, Seoul South Korea). Complete hemostasis was achieved in all patients who received stent placement, and stents were left in place for approximately one week. All were removed successfully without complication (one stent had migrated to the duodenum).\n\nFinally, Valats and colleagues described three patients with PSB who received various sized FCMS (Biliary Wallflex; Boston Scientific, Natick USA), which resulted in successful hemostasis8. These patients had also failed conventional endoscopic treatment prior to stent placement. No complications were observed. This case series also described several cases of common bile duct trauma causing hemobilia that were subsequently treated with FCMS.\n\n\nDiscussion\n\nThese 21 cases demonstrate that FCMS are a viable therapeutic option for PSB. However, there are several important issues that remain unanswered, including: which patients should be considered for stents, optimal stent size, timing of stent removal, the frequency of complications from stent placement, and whether stent placement is cost-effective.\n\nIt is reasonable to consider stent placement in patients who fail traditional interventions to avoid surgery or angiographic hemostasis. Whether stents should be used earlier in the hemostasis algorithm is unclear. It has been suggested that stenting could be considered prior to traditional interventions in patients with severe bleeding, a history of coagulopathy, and/or in patients who have required multiple endoscopic procedures for hemostasis9. This approach may also be both clinically and financially more effective.\n\nWith regard to stent size, the diameter should be large enough to avoid upstream migration into the bile duct or downstream displacement into the duodenum. Stent migration occurred in three of the 21 cases reviewed for this article, which is congruent with migration rates published for other conditions10–13. However, migration rates may actually be higher in the setting of PSB compared to biliary strictures because of the biliary sphincterotomy, which theoretically fosters a looser \"fit\" within the biliary tree. Itoi et al. suggested 10 mm as the ideal diameter7. Smaller diameter stents may become dislodged and may also not effectively achieve tamponade. The distal end of the stent should be beyond the biliary orifice to ensure effective tamponade, and the proximal end, if possible, should be distal to the cystic duct takeoff to theoretically reduce the risk of cholecystitis, although this risk is uncertain and may even be non-existent14,15.\n\nWhen placing covered stents in general, there is also a risk of pancreatic duct outflow obstruction which may potentially cause pancreatitis. This has been reported in up to 7% of patients in a previous study12. There were no reported cases of pancreatitis when placing stents for PSB in the cases reviewed for this article. It is possible that having a biliary sphincterotomy decreases the risk of pancreatitis in the setting of stent placement because there is less pressure from the stent on the pancreatic orifice16. Shah et al. suggest that stents should be removed within 2–4 weeks of placement6.\n\nFrom a cost perspective, there are no published cost-effectiveness analyses on FCMS for PSB. There are certainly costs associated with the placement and removal of FCMS. However, if stent placement prevents the need for angiographic embolization or surgery then there would hypothetically be cost-savings. Similarly, if stent placement is pursued as a first-step in patients who are high-risk for recurrent bleeding or complications, this could also be cost-saving7.\n\nIn conclusion, FCMS are a viable therapeutic option for the treatment of PSB that is refractory to traditional endoscopic interventions. The optimal patient population for this intervention, as well as the optimal stent size and removal time should be determined on a case-by-case basis until more data becomes available.", "appendix": "Author contributions\n\n\n\nDeBenedet: conception and design; analysis and interpretation of the data; drafting of the manuscript; critical revision of the manuscript for important intellectual content; final approval of the article. Elta: critical revision of the article for important intellectual content; final approval of the article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nFerreira LE, Baron TH: Post-Sphincterotomy Bleeding: Who, What, When, and How. Am J Gastroenterol. 2007; 102(12): 2850–2858. PubMed Abstract \| Publisher Full Text\n\nCotton PB, Lehman G, Vennes J, et al.: Endoscopic sphincterotomy complications and their management: An attempt at consensus. Gastrointest Endosc. 1991; 37(3): 383–93. PubMed Abstract \| Publisher Full Text\n\nFreeman ML, Nelson DB, Sherman S, et al.: Complications of endoscopic biliary sphincterotomy. N Engl J Med. 1996; 335(13): 909–18. PubMed Abstract \| Publisher Full Text\n\nFreeman ML: Understanding risk factors and avoiding complications with endoscopic retrograde cholangiopancreatography. Curr Gastroenterol Rep. 2003; 5(2): 145–53. PubMed Abstract \| Publisher Full Text\n\nDi Pisa M, Tarantino I, Barresi L, et al.: Placement of covered self-expandable metal biliary stent for the treatment of severe postsphincterotomy bleeding: outcomes of two cases. Gastroenterol Res Pract. 2010. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShah JN, Marson F, Binmoeller KF: Temporary self-expandable metal stent placement for treatment of post-sphincterotomy bleeding. Gastrointest Endosc. 2010; 72(6): 1274–78. PubMed Abstract \| Publisher Full Text\n\nItoi T, Yasuda I, Doi S, et al.: Endoscopic hemostasis using covered metallic stent placement for uncontrolled post-endoscopic sphincterotomy bleeding. Endoscopy. 2011; 43(4): 369–372. PubMed Abstract \| Publisher Full Text\n\nValats J, Funakoshi N, Bauret P, et al.: Covered self-expandable biliary stents for the treatment of bleeding after ERCP. Gastrointest Endosc. 2013; 78(1): 183–187. PubMed Abstract \| Publisher Full Text\n\nFerreira LE, Fatima J, Baron TH: Clinically significant delayed postsphincterotomybleeding: a twelve year single center experience. Mineva Gastroenterol Dietol. 2007; 53(3): 215–223. PubMed Abstract\n\nTraina M, Tarantino I, Barresi L, et al.: Efficacy and safety of fully covered self-expandable metallic stents in biliary complications after liver transplantation: a preliminary study. Liver Transpl. 2009; 15(11): 1493–8. PubMed Abstract \| Publisher Full Text\n\nCahen DL, Rauws EA, Gouma DJ, et al.: Removable fully covered self-expandable metal stents in the treatment of common bile duct stricture due to chronic pancreatitis: a case series. Endoscopy. 2008; 40(8): 697–700. PubMed Abstract \| Publisher Full Text\n\nMahajan A, Ho H, Sauer B, et al.: Temporary placement of fully covered self-expandable metal stents in benign biliary stricture: midterm evaluation (with video). Gastrointest Endosc. 2009; 70(2): 303–9. PubMed Abstract \| Publisher Full Text\n\nKahaleh M, Behm B, Clarke BW, et al.: Temporary placement of covered self-expandable metal stents in benign biliary strictures: a new paradigm (with video)? Gastrointest Endosc. 2008; 67(3): 446–54. PubMed Abstract \| Publisher Full Text\n\nFumex F, Coumaros D, Napoleon B, et al.: Similar performance but higher cholecystitis rate with covered biliary stents: results from a prospective multicenter evaluation. Endoscopy. 2006; 38(8): 787–92. PubMed Abstract \| Publisher Full Text\n\nTelford JJ, Carr-Locke DL, Baron TH, et al.: A randomized trial comparing uncovered and partially covered self-expandable metal stents in the palliation of distal malignant biliary obstruction. Gastrointest Endosc. 2010; 72(5): 907–14. PubMed Abstract \| Publisher Full Text\n\nSimmons DT, Petersen BT, Gostout CJ, et al.: Risk of pancreatitis following endoscopically placed large-bore plastic biliary stents with and without biliary sphincterotomy for management of postoperative bile leaks. Surg Endosc. 2008; 22(6): 1459–63. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1483", "date": "19 Sep 2013", "name": "Guido Costamagna", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article provides a precise review of a \"salvage\" technique for refractory post-sphincterotomy bleeding, the discussion underlines the pro and cons of the technique, and the article can be useful for the endoscopic community.", "responses": [] }, { "id": "1976", "date": "11 Nov 2013", "name": "David Carr-Locke", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSevere post-sphincterotomy bleeding during or after ERCP requiring intervention other than simple measures, such injection of 1:10,000 epinephrine, or that does not respond to first line attempts at hemostasis, is uncommon. This review, by DeBenedet and Elta, describes 21 patients from four articles where fully-covered self-expanding metallic stents were placed to provide tamponade to achieve successful hemostasis in 100% without complication. All stents were subsequently removed endoscopially or had passed spontaneously. This is a highly select group of patients but this option is a useful addition to the range of hemostatic modalities available for this difficult situation.", "responses": [] } ]	1	https://f1000research.com/articles/2-171
https://f1000research.com/articles/2-169/v1	09 Aug 13	{ "type": "Short Research Article", "title": "Anti-rhesus D prophylaxis in pregnant women is based on sialylated IgG antibodies", "authors": [ "André Winkler", "Markus Berger", "Marc Ehlers", "André Winkler", "Markus Berger" ], "abstract": "Red blood cells (RBCs) from a rhesus D (RhD)-positive fetus that reach the bloodstream of an RhD-negative pregnant woman during birth can induce a pathogenic antibody (Ab) response against the RhD-positive RBCs, leading to fetal hemolytic disease in subsequent pregnancies. To prevent a pathogenic immune reaction, the RhD-negative mother receives serum immunoglobulin G (IgG) containing polyclonal RhD-specific IgG Abs that is purified from healthy RhD-negative men immunized with RhD-positive RBCs. However, the protective mechanism of these polyclonal RhD-specific IgG Abs is unclear. It has become increasingly clear that the effector function of IgG Abs is regulated by the glycan pattern linked to the Fc region of IgG Abs. Non-fucosylated (afucosylated) IgG Abs have a higher affinity for activating Fc gamma receptors, and thus induce a stronger Ab-dependent cellular cytotoxicity (ADCC) reaction than do fucosylated IgG Abs. Agalactosylated and asialylated, autoantigen-specific serum IgG Abs correlate with pro-inflammatory immune responses and disease activity in patients with rheumatoid arthritis. In contrast, galactosylated and sialylated IgG Abs are immunosuppressive and inhibit in form of immune complexes (ICs) dendritic cell (DC) maturation and pro-inflammatory T and B cell immune responses in an antigen-specific manner. However, the galactosylation and sialylation levels of the protective polyclonal RhD-specific IgG Abs are unknown. Here, we purified RhD-specific IgG Abs from the approved commercial product Rhophylac® (CSL Behring) and found that these RhD-specific IgG Abs were even more galactosylated and sialylated than the total Rhophylac® IgG Abs. This result suggests that these galactosylated and sialylated polyclonal RhD-specific IgG Abs are immunosuppressive and induce tolerance against RhD, which would be in strong contrast to a low fucosylated, low galactosylated and low sialylated monoclonal RhD-specific IgG Ab developed to prevent fetal hemolytic disease that has recently passed a clinical phase II study.", "keywords": [ "Red blood cells (RBCs) from a rhesus D (RhD)-positive fetus that get in contact with immune cells of an RhD-negative pregnant woman during birth can induce a pathogenic antibody (Ab) response against the RhD-positive RBCs", "leading to fetal hemolytic disease in subsequent pregnancies with RhD-positive fetuses after transplacental passage. To prevent allo-immunization by RhD-positive fetal RBCs", "the RhD-negative mother receives one prenatal and one postnatal injection of serum immunoglobulin G (IgG) containing polyclonal RhD-specific IgG Abs that is purified from healthy RhD-negative men immunized with RhD-positive RBCs. Such a passive anti-RhD IgG Ab treatment i) induces a rapid clearance of RhD-positive RBCs from the bloodstream of the mother and ii) inhibits the development of pathogenic anti-RhD Abs by the mother1." ], "content": "Introduction\n\nRed blood cells (RBCs) from a rhesus D (RhD)-positive fetus that get in contact with immune cells of an RhD-negative pregnant woman during birth can induce a pathogenic antibody (Ab) response against the RhD-positive RBCs, leading to fetal hemolytic disease in subsequent pregnancies with RhD-positive fetuses after transplacental passage. To prevent allo-immunization by RhD-positive fetal RBCs, the RhD-negative mother receives one prenatal and one postnatal injection of serum immunoglobulin G (IgG) containing polyclonal RhD-specific IgG Abs that is purified from healthy RhD-negative men immunized with RhD-positive RBCs. Such a passive anti-RhD IgG Ab treatment i) induces a rapid clearance of RhD-positive RBCs from the bloodstream of the mother and ii) inhibits the development of pathogenic anti-RhD Abs by the mother1.\n\nHowever, the protective mechanism of passive anti-RhD treatment remains unclear. It is hypothesized that the clearance of RhD-positive RBCs is mediated through an Fcγ receptor (Fcγ R) IIIA-mediated Ab-dependent cellular cytotoxicity (ADCC) reaction and that rapid clearance prevents immunization1–4. To prevent immunization, it has also been suggested that polyclonal anti-RhD IgG Abs have to inhibit the activation of RhD-specific B cells through the co-ligation of the B cell receptor and an inhibitory receptor, such as the IgG inhibitory receptor FcγRIIB2,3. Furthermore, tolerance induction via antigen-presenting cells (APCs) has been posited to inhibit pro-inflammatory, RhD-specific T cell responses2,5,6. However, it is questionable whether FcγRIIIA crosslinking on APCs inhibits pro-inflammatory RhD-specific T cell responses but rather enforces pro-inflammatory T cell responses7. It is more likely that polyclonal anti-RhD IgG Abs target an inhibitory receptor (complex) on APCs to induce regulatory T cells and tolerance for inhibiting pro-inflammatory T cell and therewith also T cell-depemdent B cell responses.\n\nAttempts to substitute this polyclonal anti-RhD IgG prophylaxis with RhD-specific monoclonal IgG Abs have failed because the monoclonal RhD-specific IgG Abs were relatively unstable due to intramolecular rearrangements or did not clear RhD-positive RBCs as rapidly as the available polyclonal anti-RhD IgG Abs in in vitro assays or clinical trials and/or did not sufficiently inhibit allo-immunization in clinical trials1,4,8.\n\nIt has become increasingly clear that the effector function of IgG Abs is highly regulated by the Abs’ Fc N-linked glycosylation pattern (Figure 1A). Non-fucosylated (afucosylated) IgG Abs have a higher affinity for activating FcγRs, such as FcγRIIIA, and thus induce a stronger ADCC reaction than do fucosylated IgG Abs9,10. This finding is currently translated, for example, into tumor-specific Ab therapy.\n\n(A) The biantennary IgG Fc glycan core structure, which is coupled to Asn 297, consists of two N-acetyl-glucosamines (GlcNAc; dark blue) and three mannoses (Man), which can be further decorated with fucose; bisecting GlcNAc (light blue) and terminal GlcNAc (dark blue), galactose (G) and sialic acid (S). (B and C) The purified total and RhD-specific IgG samples were hydrolyzed with EndoS and analyzed by MALDI-TOF MS. The cleavage site of EndoS is indicated by an arrow in (A). (B) The bar graph indicate the frequency of all glycan structures with 0, 1 or 2 galactose (G) residues and 0, 1 or 2 sialic acid (S) residues. The mean values with the standard error of the mean (SEM) from independent experiments are shown. (C) The bar graphs separately indicate the frequency of G0, G1, G2 and S1 and S2 glycan structures from the bar graph in (B).\n\nAgalactosylated and asialylated (G0), autoantigen-specific serum IgG Abs correlate with pro-inflammatory immune responses and disease activity in patients with rheumatoid arthritis (RA)11–14. In contrast, pregnancy-induced and anti-tumor necrosis factor (TNF) therapy-induced remission in RA patients is associated with an increase in galactosylated and sialylated IgG Abs15,16. In this context, an anti-inflammatory role has been suggested for sialylated IgG Abs. Accordingly, the sialylated IgG subfraction of intravenous IgG (IVIG), which is purified from pooled human plasma from healthy donors and used to systemically treat autoimmunity in high doses (2 g/kg), exhibits anti-inflammatory activity17–20. We have recently shown that low doses of immune complexes (ICs) containing sialylated antigen-specific IgG Abs inhibit dendritic cell maturation and pro-inflammatory T and B cell immune responses in an antigen-specific manner21–23. ICs containing 15%, but not 5%, sialylated IgG Abs have further been sufficient to inhibit B cell activation in vitro23. Furthermore, it has recently been shown that ICs containing galactosylated, but not sialylated, IgG Abs are already sufficient to inhibit neutrophil activation24.\n\nThus, G0 IgG Abs enhance, whereas galactosylated and sialylated IgG Abs suppress, pro-inflammatory immune responses. Accordingly, ICs containing galactosylated and sialylated IgG Abs inhibit rather than induce an ADCC reaction by at least reduced binding affinity of galactosylated and sialylated IgG Abs to FcγRIIIA17,25 but also likely by active suppression mechanisms via inhibitory receptors on immune cells.\n\nHowever, based on the assumption that anti-RhD IgG Abs clear RhD-positive RBCs through an ADCC reaction, the different outcomes of monoclonal anti-RhD IgG Abs have been particularly attributed to different levels of fucose1,2,4,26–29. By contrast, the role of anti-RhD IgG galactosylation and sialylation has hardly been investigated.\n\nBased on the idea that anti-tumor as well as anti-RhD IgG Abs should induce a strong ADCC response by recruiting FcγRIIIA-expressing immune cells, the French biotechnology company Laboratoire Francais du Fractionnement et des Biotechnologies (LFB; Les Ulis, France) has generated a monoclonal anti-CD20 IgG1 Ab (ublituximab; LFB-R603) and a human monoclonal RhD-specific IgG1 Ab (roledumab, LFB-R59330) with low Fc fucosylation, low Fc galactosylation and low Fc sialylation based on their patent31. In the meantime the company has performed clinical phase I32 and II (NCT00952575; completed 2011) studies on roledumab33. The phase II study was designed to demonstrate the ability of roledumab to effectively eliminate exogenously administered RhD-positive RBCs from the circulation of an RhD-negative individual, thereby preventing RhD allo-immunization. The results have not been published yet.\n\nHowever, based on the findings described above regarding the effector functions of differentially glycosylated IgG Abs, it is questionable whether an anti-tumor IgG1 Ab and an anti-RhD IgG1 Ab should have the same Fc glycosylation. Whether low-galactosylated, low-sialylated RhD-specific IgG Abs can inhibit the induction of pathogenic immune reactions against RhD-positive fetal RBCs or rather enhance allo-immunization is also questionable. To identify the Fc galactosylation and sialylation of RhD-specific IgG Abs in a commercially available polyclonal anti-RhD IgG product, we purified RhD-specific IgG Abs from the approved product Rhophylac® (CSL Behring, King of Prussia, PA, USA) and analyzed the Abs’ Fc glycosylation.\n\n\nMethods\n\nTotal IgG from the commercial polyclonal anti-RhD IgG product Rhophylac® was purified using protein-G-sepharose (GE Healthcare, Fairfield, CT, USA). RhD-specific IgG Abs from the purified total IgG Abs of Rhophylac® were enriched using RhD-positive human erythrocytes. For this purpose, anonymous RhD-positive erythrocyte concentrates were obtained from the blood bank of the Charité - University Hospital Berlin. The erythrocyte concentrate was washed with 1 mM EDTA in PBS. Next, 20 ml of erythrocyte concentrate was diluted 1:1 with purified total IgG from Rhophylac® in PBS, which contained approximately 600 µg of RhD-specific IgG Abs as indicated by the company, and was incubated for 2h at 4°C. The erythrocytes were then washed five times with PBS. Subsequently, RhD-specific IgG Abs were eluted with 0.15 M glycine pH 3.0; neutralized with 1 M Tris/HCl pH 9.0 and dialyzed against PBS. Two independent RhD-specific IgG purifications (A and B) were done. Enrichment of the RhD-specific IgG Abs was verified by fluorescence-activated cell sorting (FACS) analysis. IgG Fc glycosylation was characterized through MALDI-TOF mass spectrometry (MS).\n\nEnrichment of the purified polyclonal RhD-specific IgG Abs was verified by FACS analysis (FACS Calibur with CellQuest Pro software, version 6.0 (BD Biosciences, Franklin Lakes, NJ, USA)). RhD-positive human RBCs were stained with 260 μg/ml of total IgG from Rhophylac® (blue), 2 μg/ml purified RhD-specific IgGs (black) or 2 μg/ml of total IgG from Rhophylac® (red) and an anti-human IgG APC-coupled secondary Ab (#550931; BD Biosciences) in cold PBS containing 0.5% bovine serum albumin (Sigma-Aldrich; St. Louis, MO, USA). The stained cells were gated on the main erythrocyte population in a FSC/SSC blot to exclude fragments and aggregates and analyzed in an anti-human IgG (APC) histogram. The data were analyzed with FlowJo 7.2.5 from Tree Star, Inc. Ashland, Oregon, USA. An overlay of different histograms, containing the analysis of the RhD-specific IgG Abs from purification A, is shown in Supplementary figure 1. The FCS files can be found in the data files below.\n\n\n\nThe purified total and RhD-specific IgG samples were hydrolyzed with recombinantly expressed endoglycosidase S (EndoS) from Streptococcus pyogenes, an enzyme that hydrolyzes N-glycans only from the Fc portion of the IgG Abs, to prevent the analysis of glycans from pontential contaminating RBC proteins34. The resulting N-glycans were purified through solid-phase extraction using reversed-phase C18 and graphitized carbon columns (Alltech, Deerfield, IL, USA), permethylated and further investigated by MALDI-TOF MS21. The spectra were recorded on an Ultraflex III mass spectrometer (Bruker Corporation, Billerica, MA, USA) equipped with a Smartbeam laser. Calibration was performed on a glucose ladder, and 2,5-dihydroxybenzoic acid (DHB) was used as the matrix. Spectra were recorded in reflector positive ionization mode and mass spectra from 3,000 laser shots were accumulated. The crude glycan analysis data of the two independent RhD-specific IgG Ab purifications A and B can be found in the data files below.\n\n\n\n\nResults and discussion\n\nTo assess the Fc galactosylation and sialylation of RhD-specific IgG Abs in a commercially available polyclonal anti-RhD IgG product, we purified RhD-specific IgG Abs from Rhophylac® and analyzed the Abs’ Fc glycosylation (Figure 1, Supplementary figure 1 and Supplementary figure 2 and the data files). We found that the purified polyclonal RhD-specific IgG Abs were even more galactosylated and sialylated than the total Rhophylac® IgG Abs (Figure 1, Supplementary figure 1 and Supplementary figure 2 and the data files), which are comparable to total IgG Abs in immunosuppressive IVIG. The injection of RhD-positive RBCs into RhD-negative men obviously induced no pathogenic immune response. Comparable results have recently been generated by analyzing allergen-specific human IgG Abs after successful allergen-specific immunotherapy for birch pollen allergy21. Together, these results further indicate that tolerance induction induces mainly galactosylated and sialylated IgG Abs that have the potential to inhibit pathogenic immune responses via ICs21–24.\n\nThese results strongly suggest that only galactosylated and sialylated, immunosuppressive monoclonal RhD-specific IgG Abs can be substituted for polyclonal anti-RhD IgG products to inhibit pathogenic allo-immunity in RhD-negative pregnant woman. Low fucosylated, low agalactosylated and low sialylated RhD-specific IgG Abs might not only enhance pro-inflammatory immune responses against RhD-positive fetal RBCs but also have the potential to attack RhD-positive fetal RBCs after transplacental passage.", "appendix": "Author contributions\n\n\n\nAW contributed to the experimental design and carried out the research. MB helped with the glycan analysis. ME contributed to the experimental design and prepared the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAW and ME were supported by the German Research Foundation (DFG: EH221-5 and SFB/TR654 project C9). In addition ME was a fellow of the Claussen-Simon-Foundation, and supported by the Max Planck Institute for Infection Biology, Berlin, Germany and by additional DFG grants (RTG1727 associated project 16, IRTG1911 project A4 and the cluster of excellence 306 “Inflammation at Interfaces” project K-TP2). MB was supported by the German Ministry of Research and Education (03IP511) and the Sonnefeld Foundation.\n\n\nAcknowledgements\n\nWe thank Mattias Collin for support with EndoS.\n\n\nSupplementary figures\n\nPurification of RhD-specific IgG Abs was verified by FACS analysis. RhD-positive human RBCs were stained with 260 µg/ml of total IgG from Rhophylac® (blue), 2 µg/ml purified RhD-specific IgGs (black; purification A) or 2 µg/ml of total IgG from Rhophylac® (red) and an anti-human IgG APC-coupled secondary Ab and analyzed by FACS analysis. The gray curve represents the unstained RBCs and the green curve represents the RBCs stained only with the secondary antibody. An overlay of different histograms, containing the analysis of the RhD-specific IgG Abs from purification A, is shown.\n\n(A) The biantennary IgG Fc glycan core structure, which is coupled to Asn 297 consists of two N-acetyl-glucosamines (GlcNAc; dark blue) and three mannoses (Man), which can be further decorated with fucose; bisecting GlcNAc (light blue) and terminal GlcNAc (dark blue), galactose (G, Gal) and sialic acid (N-acetylneuraminic acid (Neu5Ac), S, Sial). The cleavage site of EndoS is indicated by an arrow. (B) The molecular mass (m/z) of the possible Fc glycan structures (permethylated) released from Asn 297 upon EndoS treatment.\n\n\nReferences\n\nKumpel BM: Lessons learnt from many years of experience using anti-D in humans for prevention of RhD immunization and haemolytic disease of the fetus and newborn. Clin Exp Immunol. 2008; 154(1): 1–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumpel BM, Elson CJ: Mechanism of anti-D-mediated immune suppression--a paradox awaiting resolution? Trends Immunol. 2001; 22(1): 26–31. PubMed Abstract \| Publisher Full Text\n\nKumpel BM: In vivo studies of monoclonal anti-D and the mechanism of immune suppression. Transfus Clin Biol. 2002; 9(1): 9–14. PubMed Abstract \| Publisher Full Text\n\nKumpel BM: Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti-D immunoglobulin: more questions than answers. Vox Sang. 2007; 93(2): 99–111. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumpel BM, Rademacher TW, Rook GA, et al.: Galactosylation of human IgG monoclonal anti-D produced by EBV-transformed B-lymphoblastoid cell lines is dependent on culture method and affects Fc receptor-mediated functional activity. Hum Antibodies Hybridomas. 1994; 5(3–4): 143–151. PubMed Abstract\n\nKumpel BM, Wang Y, Griffiths HL, et al.: The biological activity of human monoclonal IgG anti-D is reduced by beta-galactosidase treatment. Hum Antibodies Hybridomas. 1995; 6(3): 82–88. PubMed Abstract\n\nSibéril S, de Romeuf C, Bihoreau N, et al.: Selection of a human anti-RhD monoclonal antibody for therapeutic use: impact of IgG glycosylation on activating and inhibitory Fc gamma R functions. Clin Immunol. 2006; 118(2–3): 170–179. PubMed Abstract \| Publisher Full Text\n\nBeliard R, Waegemans T, Notelet D, et al.: A human anti-D monoclonal antibody selected for enhanced FcgammaRIII engagement clears RhD+ autologous red cells in human volunteers as efficiently as polyclonal anti-D antibodies. Br J Haematol. 2008; 141(1): 109–119. PubMed Abstract \| Publisher Full Text\n\nPCT/FR2010/050376 (international publication number: WO/2010/100383) Anti-rhesus d monoclonal antibody. Reference Source\n\nUS 7931895 B2 patent “Monoclonal antibodies with enhanced ADCC function”; issued 2011. Reference Source\n\nYver A, Homery MC, Fuseau E, et al.: Pharmacokinetics and safety of roledumab, a novel human recombinant monoclonal anti-RhD antibody with an optimized Fc for improved engagement of FCγRIII, in healthy volunteers. Vox Sang. 2012; 103(3): 213–222. PubMed Abstract \| Publisher Full Text\n\nBeck A, Reichert JM: Marketing approval of mogamulizumab: a triumph for glyco-engineering. MAbs. 2012; 4(4): 419–425. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "1716", "date": "23 Sep 2013", "name": "Tracy McGaha", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article “Anti-rhesus D prophylaxis…” by Winkler, Berger, and Ehlers the authors present data demonstrating enrichment of galactosylation and sialylation in the anti Rh-D fraction of Rhophylac. This study is well written with adequate discussion of methods. Moreover, the report builds on exciting recent findings that the pattern of glycosylation is an important determinant factor in the effector function of IgG providing mechanistic control of FcGR interactions and tolerogenic v.s. inflammatory immunity.Their results would suggest the therapeutic value of anti-RhD therapy may lie in an active tolerogenic response rather than via clearance mechanisms, a theory which should be tested moving forward.The title is a bit misleading as it suggests experimental evidence contained within the report demonstrates anti-RhD activity is due to glycosylation, a theorized outcome that has yet to be demonstrated. A revision to reflect this fact may be appropriate.Otherwise, it is an exciting report pointing to a novel mechanism that may drive prophylactic anti-RhD therapy.", "responses": [ { "c_id": "589", "date": "23 Oct 2013", "name": "André Winkler", "role": "Author Response", "response": "Dear Tracy,many thanks for reviewing our manuscript.I agree that the title is a bit misleading. I would propose to slightly change it to: \"Anti-rhesus D prophylaxis in pregnant women is might based on sialylated IgG antibodies\"This statement is based on the fact that AntiD IgG are highly sialylated and the immunoregulatory properties of sialylated IgG has been shown previously by us and by others.Do you agree?" }, { "c_id": "594", "date": "28 Oct 2013", "name": "Tracy McGaha", "role": "Reviewer Response", "response": "Dear Dr Winkler,I think the proposed revision is appropriate and acceptable.Regards,Tracy McGaha." } ] }, { "id": "1718", "date": "27 Sep 2013", "name": "Mikael C. I. Karlsson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAnti-RhD therapy is one of the most successful antibody therapies ever invented and still we do not know how antibodies block Rh immunization. Recently, a novel level of regulation through alterations in glycosylation patterns has been started to be appreciated. This study is a good starting point to investigate if this mechanism of regulation by antibodies is involved. The glycosylation pattern found in the anti-RhD pool of antibodies suggests that an active tolerogenic pathway is involved and encourages further investigation.", "responses": [] } ]	1	https://f1000research.com/articles/2-169
https://f1000research.com/articles/2-168/v1	09 Aug 13	{ "type": "Opinion Article", "title": "Observational articles: a tool to reconstruct ecological history based on chronicling unusual events", "authors": [ "Ferdinando Boero" ], "abstract": "Natural history is based on observations, whereas modern ecology is mostly based on experiments aimed at testing hypotheses, either in the field or in a computer. Furthermore, experiments often reveal generalities that are taken as norms. Ecology, however, is a historical discipline and history is driven by both regularities (deriving from norms) and irregularities, or contingencies, which occur when norms are broken. If only norms occured, there would be no history. The current disregard for the importance of contingencies and anecdotes is preventing us from understanding ecological history. We need rules and norms, but we also need records about apparently irrelevant things that, in non-linear systems like ecological ones, might become the drivers of change and, thus, the determinants of history. The same arguments also hold in the field of evolutionary biology, with natural selection being the ecological driver of evolutionary change. It is important that scientists are able to publish potentially important observations, particularly those that are unrelated to their current projects that have no sufficient grounds to be framed into a classical eco-evolutionary paper, and could feasibly impact on the history of the systems in which they occurred. A report on any deviation from the norm would be welcome, from the disappearance of species to their sudden appearance in great quantities. Any event that an “expert eye” (i.e. the eye of a naturalist) might judge as potentially important is worth being reported.", "keywords": [ "Modern ecology started when Elton (1927)1 published his masterpiece “Animal Ecology” and wrote:" ], "content": "Introduction\n\nModern ecology started when Elton (1927)1 published his masterpiece “Animal Ecology” and wrote:\n\n\"Ecology is a new name for a very old subject. It simply means scientific natural history...the words ‘natural history’ bring up a rather clear vision of parties of naturalists going forth on excursion, prepared to swoop down on any rarity that will serve to swell the local list of species. It is a fact that natural history has fallen into disrepute...\"\n\nAt the onset of ecology, it was felt that the new science should be better formulized, with the identification of “rules” that were more than the simple “story telling” that was so deprecated by Elton (1927)1. And rightly so. But we have since started to perceive that these “rules” are rather flexible. As a matter of fact, this flexibility had already been perceived before Elton by the father of ecology, Charles Robert Darwin, who, in the Origin of Species (1859)2 wrote:\n\n\"Throw up a handful of feathers, and all must fall to the ground according to definite laws. But how simple is this problem compared to the action and reaction of the innumerable plants and animals which have determined, in the course of centuries, the proportional numbers and kinds of trees now growing on the old Indian ruins\".\n\nEighty-five years after Elton, the President of the American Society of Naturalists, Robert Ricklefs (2012)3, felt the need to stress the importance of natural history as a potential source of insight in ecology and evolutionary biology, and I took the chance to signal his article to the readers of F1000Prime4. I have also advocated the importance of natural history in previous publications (e.g. Boero and Bondorff 20075, Boero et al. 20086 and references therein).\n\nThere are no equations describing the succession of phases leading to a community and there is no unified model for this process. Connell and Slatyer (1977)7, for instance, identified three models, and each time one proves valid, the universality of the others is falsified (if we want to use Popperian logic in ecology)8. From an epistemological point of view, the three models (facilitation, inhibition, tolerance) exist but none of them are universal. Furthermore, in most cases the communities evolve through a blend of models, with a host of ad hoc explanations. The same is true in evolutionary biology, with neither gradual nor punctuated models being universal norms.\n\nEcology, just like evolutionary biology, is a historical discipline. What we see today is the result of a series of events that lead to other events. Sometimes, such as with gradual evolution, the pattern of events follows a gradual trend that might even be predictable but, at other times, there can be special events or contingencies that produce great changes in an abrupt fashion. The great changes, today, are called regime shifts, and they occur when tipping points are reached. Chaos theory, which describes non linear systems, teaches us that apparently negligible events can have a disproportionate bearing on the functioning of a given system. The negligibility of these events is due to the fact that, often, when they occur, their impact is very low. But, sometimes, the impact can be very big.\n\nWhen a regime shift, or an otherwise unexpected change, occurs we are surprised and it is difficult to determine what the drivers of change might have been. Sometimes, we might detect the triggering event a posteriori, but we often cannot, simply because we do not have the information available.\n\nSimply describing what we see is not considered very scientific nowadays and ‘descriptive science’ has become a derogatory term. We must have a hypothesis to test, or better, a controlled experiment that can be performed to identify ecological rules and laws. However, if “ecological rules” were followed by all systems, unexpected things would not happen, which is evidently not the case. Deviations from rules are the main determinants of history, but we cannot test something that is unexpected. As such, our quest to identify rules and regularities could be preventing us from understanding the history of these systems. Paradoxically, we aim at understanding historical systems while using ahistorical approaches! We need a means of reporting these contingencies so that we can better understand the historical trajectory of ecological systems.\n\nThe real world out there is like a gigantic jigsaw puzzle where many pieces are missing. Some pieces can be determined through planned projects that seek to test hypotheses, but if another piece is unexpectedly observed in the process, it might as well be collected rather than discarded.\n\n\nPublishing observation articles\n\nLong term series are the only way to follow ecological histories but, unfortunately, these are becoming increasingly rare since they do not serve to test specific hypotheses, even though they are crucial for understanding the history of ecological systems. If we are talking about global change, for instance, we must be able to compare the situation of today with that of yesterday, and identify when the main changes occurred, while linking them to possible drivers. But current research trends prevent us from recording this information since we have stopped storing observations in scientific publications. Sometimes, unusual events attract the attention of the media, such as jellyfish blooms, but are disregarded by scientists who are not directly interested in them.\n\nThe under-appreciated importance of natural history can also lead to the under-reporting of rare (but important) events, which in turn may lead to ecological patterns not being accepted by others in the research community. For instance, Condon et al. (2012)9 questioned shifts from fish-dominated to jellyfish-dominated oceans, considering the phenomenon “unsubstantiated” due to a lack of a substantial number of accounts published in the scientific literature, with reports of jellyfish bloom sightings more prevalent in the popular press. However, the problem is that a jellyfish bloom per se, does not have any interest for a scientific journal. It is considered just an observation, an anecdote with no hypothesis to test. As such, even if the frequency of jellyfish bloom sightings increases, the proof of this will be lacking in the scientific record. This is explicitly stated by Riisgård et al. (2012)10:\n\n\"The important former commercial fishing of plaice, cod, eel and flounder has been replaced by a countless number of commercially ‘useless’ jellyfish, but no monitoring data have systematically been collected in order to document and to understand the undesirable change within the ecosystem substituting fish with jellyfish as top-predators. Obviously, the basic problem is eutrophication, but knowledge about when, why and how jellyfish became a major pelagic group of key organisms remains largely unknown\".\n\nWe need to be able to collect apparently irrelevant information that might result, when assembled, in a goldmine of facts, which could help us to understand why certain ecological changes occur. Such observations should be written up and submitted to journals that accept submissions based on single observations, including Herpetology Notes (herpetology), Marine Biodiversity Records (marine biology) and F1000Research (all fields). All of these observations, once pooled together, might help build projects with hypotheses to test, whilst arranging observations into a chronological chain of events may help to determine how past contingencies may have led to present systems.\n\n\n‘Scientist science’, that’s what we want\n\nCitizen science, where scientists ask for the help of citizens to gather information, is a popular approach to collecting data in ecology. It is increasingly used as a means to gather information about species over a wide geographical area that scientists would be unable to cover by themselves. For instance, I have carried out a citizen science experiment on jellyfish that began in 2009 and have learnt much from it11,12. The database of this experiment is a collection of anecdotes but, once charted, provides a clear picture.\n\nDespite the advantages of citizen science, sometimes an “expert eye” can see a potentially important event that might pass unnoticed if observed by lay people. Ecologists and environmental scientists go into the field to carry out their projects and will naturally focus their attention on the variables that are relevant to their projects. However, they often see many other things that may be irrelevant to the fulfilment of their objectives, but that could be of ecological interest and it would be useful if this information was captured and disseminated. For instance, I may be on a cruise, and see a massive bloom of a species (e.g. a jellyfish, or a red tide); I may then go diving, and see that all the sponges are dead.\n\nPerhaps even negative observations can be worthy of publication. It may be the case that I used to see lots of specimens of species X when I started my career, but have not seen any in the last decade. Extinction is a negative result and one that is of ecological interest. If a notable vertebrate species such as a dolphin or a fish becomes extinct, then the International Union for Conservation of Nature may express some concern, but inconspicuous invertebrates often do not make these lists. Most biodiversity is made of inconspicuous stuff. Even a monstrosity might be worth reporting. Monstrous specimens might just be one-off freaks, but then if more and more such individuals are sighted at some specific location, or over a vast area, this could be an indicator that there is some problem in the environment.\n\nIn summary, I urge field scientists to take note of anything they see that is unusual and worth recording in their expert eyes. Take some pictures, write a report and submit it to a journal that accepts observation articles. Each observation that is published can be cited; it does not take much of your time and can enhance your CV. We need natural history, we need scientists who can use the most fantastic instrument in the known universe - the human eye. Darwin is our beacon. He collected an enormous number of apparently irrelevant observations during his voyage with the Beagle, continued to observe at home, and treasured the anecdotes that his correspondents sent him. With that mass of little observations, he assembled the theories of both evolution and ecology in one shot.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgements\n\nI thank the people at F1000Research for the enthusiasm they expressed when I proposed the idea of publishing observations.\n\n\nReferences\n\nElton CS: Animal Ecology. London: Sidgwick and Jackson, [ISBN: 978-0226206394] 1927. Reference Source\n\nDarwin CR: On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life. London: John Murray. 1859. Reference Source\n\nRicklefs RE: Naturalists, natural history, and the nature of biological diversity. Am Nat. 2012; 179(4): 423–35. PubMed Abstract \| Publisher Full Text\n\nBoero F: F1000Prime Recommendation of Ricklefs RE. Am Nat. 2012; 179(4): 423–35. Publisher Full Text\n\nBoero F, Bonsdorff E: A conceptual framework for marine biodiversity and ecosystem functioning. Mar Ecol Evol Persp. 2007; 28(Suppl 1): 134–145. Publisher Full Text\n\nBoero F, Bouillon J, Gravili C, et al.: Gelatinous plankton: irregularities rule the world (sometimes). Mar Ecol Progr Ser. 2008; 356: 299–310. Publisher Full Text\n\nConnell JH, Slatyer RO: Mechanisms of succession in natural communities and their role in community stability and organization. Am Nat. 1977; 111: 1119–1144. Publisher Full Text\n\nBoero F, Belmonte G, Bussotti S, et al.: From biodiversity and ecosystem functioning to the roots of ecological complexity. Ecological Complexity. 2004; 2: 101–109. Publisher Full Text\n\nCondon RH, Graham WM, Duarte CM, et al.: Questioning the Rise of Gelatinous Zooplankton in the World's Oceans. Bio Science. 2012b; 62(2): 160–169. Publisher Full Text\n\nRiisgård H, Andersen P, Hoffmann E: From Fish to Jellyfish in the Eutrophicated Limfjorden (Denmark). Estuaries and Coasts. 2012; 35(3): 701–713. Publisher Full Text\n\nBoero F: Recent innovations in marine biology. Mar Ecol. 2009; 30(Suppl s1): 1–12. Publisher Full Text\n\nBoero F: Review of jellyfish blooms in the Mediterranean and Black Sea. GFCM Studies and Reviews. 2013; 92: 53. Reference Source" }	[ { "id": "1417", "date": "14 Aug 2013", "name": "Joachim Mergeay", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFerdinando Boero's plea for documenting unusual events in 'life sciences' is a really important one, but there are some points of discussion. When we try to understand and predict ecological patterns and events, we use universalities, general rules in a probabilistic sense. Whereas ecologists and evolutionary biologists tend to search for generalities, in order to discover general but still probabilistic rules in the mass of data and variables influencing our dependent variables (all kinds of life-forms), we find it hard to acknowledge that these may be very weak at predicting the fate of individual systems. Ultimately, predictions about individual systems is what policy is interested in. Politicians don't want to hear about projections about the average response of a thousand parallel earth-like systems to climate change, they want us to predict the exact response of this planet and its biota to the human CO2-pump. But because we can only \"predict\" based on past observations - history - we tend to predict the response averaged over a large number of hypothetical systems that behaved according to rules of the past. Due to contingencies, this average predicted response may strongly differ from the actual response of the individual system we're actually interested in. And every time we are proven, a posteriori, wrong in our predictions, policy loses faith in soft - highly probabilistic - sciences like ecology and evolutionary biology. It is therefore tremendously important to be aware of the enormous role of contingencies in the historical system that is our world.To use an analogy from dispersal ecology: modelling a dispersal kernel is not that difficult, except for the shape and length of the tail; rare dispersal events over long distances (at the tail of the distribution) may have large influences (order-of-magnitude!) on the spread rate of organisms (e.g., Clark et al. 2003: Ecology 84:1979-88). So it's not enough to know the average response of organisms, outliers can be equally important. It is therefore also important to publish anecdotal facts, observations that may not be framed in a current hypothesis or are part of a large dataset, or which add to existing data. In the face of global change, the oddball of today may be the norm of tomorrow. Deviations from the norm, as Boero puts it, are just as key to understanding life, ecology and evolution, and how we function on this planet.As mentioned in the paper, citizen science can be a very powerful tool for this. I disagree, however, that we need less of that and more of similar scientist science. There are currently large observation databases based on what citizens observe on their favourite taxonomic groups. They enter their anecdotal data or complete datasets in repositories such as www.observado.org (worldwide), www.waarneming.nl (the Netherlands), www.observations.be (Belgium), http://data.nbn.org.uk/ (UK), and participate in long-term monitoring schemes and so on. Many other observations and datasets are published through www.GBIF.org and related data-portals such as http://bison.usgs.ornl.gov/. Moreover, the real taxonomic specialists are increasingly being found among citizens rather than among scientists. Partly, that's the result of science funding focusing less on natural history and taxonomy, but it's also the result of a world in which digital data and information is accessible much more than it was a decade (or two) ago. Add this availability of information to rapid interaction with peers from all over the world, including rapid dissemination of documentation and evidence, and easy cross-checks of observations (for example by photographic documentation), and becoming a citizen expert is much easier than a decade or two ago. The role of citizen science, also for documenting anecdotal facts, will only increase. Overall, this paper made me think about how to best deal with anecdotal data: Is the best way to deposit observational data through publication as a single remarkable fact, in a specialized journal willing to publish such data, or to deposit them in citizen-science like databases? Boero argues that once published in a journal, the data is available. But that doesn't necessarily make these observations and data very accessible.Boero argues that even writing anecdotal (typically low-impact) data-papers is still good for your career. But there are just 24 hours in a day. Many scientists have more than enough data in their drawers to publish, but lack time to publish them. How many zombie manuscripts remain unpublished forever? I have a dozen or so hoping to be published sooner or later, but I won’t bet money on them getting published soon. In the rat race for funding, we need high-impact papers, preferably many. So we make a selection of what to publish first, and we tend to aim for expected impact. Writing a high-impact paper does not require much more time than writing a low-impact paper and since time is limited, we focus on writing the former, and skip the scientifically less impacting descriptive anecdotal papers. It’s a real pity these papers and the data behind them don’t get published, but that’s the reality of how science and funding forces us to work. If there’s a fast way to publish anecdotal data in scientific journals, and which doesn't require a lot of writing effort, I think many would welcome this way of publishing.Next, is there really a shortage of journals wanting to publish anecdotal observations? Since the digital era of publication, the number of journals has sky-rocketed, and authors are continuously spammed with unsolicited requests to write papers for new journal X or Y. This said, it would be good to have dedicated journals for this purpose. At the very least, anecdotal data should be deposited in existing data repositories to make them truly accessible. The GBIF data portal or similar initiatives seems like a good initiative for this.", "responses": [] }, { "id": "1584", "date": "27 Aug 2013", "name": "John Chapman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paper is a good idea, and agree that “unusual” events and anecdotes (particularly when they are brief and to the point) are not published enough. The jellyfish blooms are a great example, but dead zones and massive invertebrate changes are also. The article is well organized and to the point. I think that quantitative and experimental ecologists have been vexed for the last 100 years or so, with the daunting problem of measuring anything in nature on the scale that nature occurs. We only have samples and can only measure a small fraction of interactions over very short periods. Anecdotes and periodic observations over time could help fill out the mosaic of ecological dynamics, but we must also admit that we can never see the whole picture.", "responses": [] } ]	1	https://f1000research.com/articles/2-168
https://f1000research.com/articles/2-167/v1	09 Aug 13	{ "type": "Research Article", "title": "Patient perspectives on the promptness and quality of care of road traffic incident victims in Peru: a cross-sectional, active surveillance study", "authors": [ "J Jaime Miranda", "Edmundo Rosales-Mayor", "D Alex Quistberg", "Ada Paca-Palao", "Camila Gianella", "Pablo Perel", "Luis Lopez", "Diego Luna", "Pablo Best", "Luis Huicho", "PIAT Working Group", "Edmundo Rosales-Mayor", "D Alex Quistberg", "Ada Paca-Palao", "Camila Gianella", "Pablo Perel", "Luis Lopez", "Diego Luna", "Pablo Best", "Luis Huicho" ], "abstract": "Background: Road injuries are the second-leading cause of disease and injury in the Andean region of South America. Adequate management of road traffic crash victims is important to prevent and reduce deaths and serious long-term injuries. Objective: To evaluate the promptness of health care services provided to those injured in road traffic incidents (RTIs) and the satisfaction with those services during the pre-hospital and hospital periods. Methods: We conducted a cross-sectional study with active surveillance to recruit participants in emergency departments at eight health care facilities in three Peruvian cities: a large metropolitan city (Lima) and two provincial cities (an urban center in the southern Andes and an urban center in the rainforest region), between August and September 2009. The main outcomes of interest were promptness of care, measured by time between injury and each service offered, as well as patient satisfaction measured by the Service Quality (SERVQUAL) survey. We explored the association between outcomes and city, type of health care facility (HCF), and type of provider. Results: We recruited 644 adults seeking care for RTIs. This active surveillance strategy yielded 34% more events than anticipated, suggesting under-reporting in traditional registries. Median response time between a RTI and any care at a HCF was 33 minutes overall and only 62% of participants received professional care during the initial “golden” hour after the RTI. After adjustment for various factors, there was strong evidence of higher global dissatisfaction levels among those receiving care at public HCFs compared to private ones (odds ratio (OR) 5.05, 95% confidence interval (CI) 1.88-13.54). This difference was not observed when provincial sites were compared to Lima (OR 1.41, 95% CI 0.42-4.70).\nConclusions: Response time to RTIs was adequate overall, though a large proportion of RTI victims could have received more prompt care. Overall, dissatisfaction was high, mainly at public institutions indicating much need for improvements in service provision.", "keywords": [ "Emergency Medical Services", "Developing Countries", "Accidents", "Traffic", "Delivery of Health Care", "Quality of Health Care" ], "content": "Introduction\n\nWorldwide, road traffic incidents (RTIs) constitute the primary cause of death due to injuries, the tenth leading cause of all deaths, and the ninth leading contributor to the global burden of disease1. Latin America and Peru are not exceptions to these statistics2,3. Adequate management after the occurrence of a traffic incident can decrease the probability of death and disability, limit the severity caused by injury and ensure that survivors are optimally reintegrated into the community4. In high-income countries, studies have shown that of those that die due to a RTI, 50% will die within minutes after the incident or on the way to the hospital, 15% die within the first 4 hours, and 35% die after 4 hours5. In low- and middle-income countries (LMIC), the majority of deaths occur among victims before arriving at a health care facility (HCF), indicating that many deaths and complications could be prevented via adequate initial management and response5–8. The experiences of developing countries with regards to evaluating the care of RTI victims are scarce and unsystematic9. Providing quality health care services to RTI victims is also important, ensuring that they are at the center of attention of all services offered10. Patients and users have a wide spectrum of cultures and attitudes, and both demand and hope for quality in all scientific, technical and humanistic aspects11,12.\n\nDetermining the response and quality of emergency services in LMICs is important for understanding how to strengthen these services in low-resource areas. One of the first steps to approach the problem of RTIs is to rely on solid information at a national level13, and RTIs have been considered a research priority for Peru14.\n\nTo evaluate how well the promptness of care complies with the recommendations for emergency care services during the pre-hospital and hospital periods as suggested by the US Department of Transportation (USDOT) and the National Highway Transportation Safety Administration (NHTSA) in the USA15, and how much satisfaction the victims have with respect to the care received. Additionally, we sought to assess the association between the city and type of HCF where the victim was cared for with the quality of attention received.\n\n\nMethods\n\nWe conducted a cross-sectional study over a period of four weeks, August-September 2009, in the Emergency Departments of 8 HCFs in Ayacucho (South Andes), Lima (Peru’s capital) and Pucallpa (rainforest region). These regions include urban areas with higher socioeconomic status and complex referral hospitals (Lima), and inner cities with a lower level of socioeconomic development (Ayacucho and Pucallpa)16. Both public (hospitals) and private (clinics), were selected on the basis of their closeness to each city’s arterial routes (highways and expressways) and the volume of RTI victims they received. The average number of potential eligible subjects receiving care at the selected facilities, for 2007–2008, ranged from 43 to 150 RTI victims per month. Based on these figures, we assumed that approximately 60 patients per site per month would be expected between August and September 2009.\n\nThe study population was adult victims of RTIs seeking health care for injuries at the selected HCFs, arriving on their own or carried by others (e.g. physician, firefighter, ambulance service, police, or other road user). We did not recruit anyone that presented in an altered state of consciousness or speech, since they were not in a condition to respond to the interview; therefore we excluded critically ill patients.\n\nThe protocol was reviewed and approved by the ethical committee of Peru’s Instituto Nacional de Salud and by ethics committees at the HCF if they had one. Written consent was obtained from each participant.\n\nActive surveillance was used to identify all possible participants by having research interviewers present 7 AM to 1 PM and 1 PM to 7 PM, Monday to Saturday. Subjects were interviewed without interrupting their care by staff. We visited patients at home if they had been admitted or discharged during the hours or days not covered by interviewers.\n\nWe assessed whether pre-hospital care was provided by untrained personnel (other road users, police, or municipal patrol guards also called “Serenazgo”) or by trained ones (firefighters and/or health professionals, assuming that firefighters receive first aid training). The promptness of attention was evaluated through an interviewer–administered, semi-structured questionnaire about waiting times before receiving care during the pre- and hospital periods.\n\nThe survey questionnaire was pilot-tested beforehand. There was sufficient evidence that there was adequate comprehension of the SERVQUAL instrument and its face validity. The modified Service Quality questionnaire (SERVQUAL)17,18 had a Cronbach alpha of 0.96, indicating good internal consistency and reliability of the instrument in the target population.\n\nWe defined the pre-hospital period as starting from the moment the incident occurred until the moment prior to arrival at the HCF, and hospital period as starting from arrival at the HCF onwards. Waiting times were self-reported by subjects. The quality of care during the pre-hospital period was evaluated on a 5-point Likert-type scale ranging from “Very Poor Care” to “Very Good Care”. To evaluate the pre-hospital quality of care for the purpose of analysis, we combined the categories of “Very poor care” and \"Poor care\".\n\nThe Service Quality (SERVQUAL) questionnaire was used to determine customer satisfaction17,18 with the HCF service received. SERVQUAL proposes a “discrepancy model” where the difference between expectations of what should be received versus perceptions of what was actually received at the point-of-care indicates the quality of service18–20. We used a modified SERVQUAL using 18 (out of 22) pairs of questions that have the most relevance to health care19, and that has been frequently used to evaluate quality of health care in Peru21–25. SERVQUAL evaluates satisfaction in five dimensions: i) tangibles: appearance of the physical facilities, equipment, staff, and communication materials; ii) reliability: delivering the service exactly as promised or as agreed; iii) responsiveness: willingness to quickly and immediately assist customers at a given opportunity; iv) assurance: courtesy and the ability to convey credibility without risks or prejudice when providing service; and v) empathy: willingness to put oneself in the place of another, to think first of the patient, and to serve according to specific characteristics and situations.\n\nIn addition to SERVQUAL, we examined other variables that demonstrate evidence of deficiencies in distinct processes of patient care, including the explanation of the diagnosis and the treatment of the victim using a structured questionnaire (see Data File). Finally, we asked the participants which primary aspect of the HCF where they attended should be improved.\n\nThe hospital user satisfaction score for each of the five SERVQUAL dimensions was determined by taking the difference between expectations and perceptions (very satisfied <0, moderately satisfied=0, somewhat or moderately dissatisfied >0 and ≤2, and extremely dissatisfied >2). The global score was calculated by taking the average scores of the 5 dimensions described above. Then, for analysis, satisfaction scores were dichotomized as satisfied (very satisfied and satisfied) and dissatisfied (somewhat or moderately dissatisfied and extremely dissatisfied).\n\nWe conducted a descriptive analysis of the frequencies and distribution of the variables and tested for statistically significant differences between groups using the Kruskal-Wallis test for outcomes with medians and χ2 tests for outcomes with frequencies. We used a multivariable logistic regression to evaluate the association between the degree of dissatisfaction with city type and facility type. For this analysis, we first developed models adjusted for age and sex (Model 1) and then adjusted for potential confounding variables defined a priori, such as education and employment (Model 2). Model 2 was also adjusted for the type of facility and type of city depending on the outcome of interest. We report odds ratios (OR) and 95% confidence intervals (95% CI). Statistical analysis was conducted with Stata 10 (STATA Corp, College Station, TX, USA).\n\n\nResults\n\nSix public HCFs, one in Ayacucho, two in Pucallpa and three in Lima; and two private HCFs, both in Lima, participated in the study. A total of 1064 RTI victims sought care in the eight emergency departments during the study period (Figure 1). Of them, 644 participated in the study: response rate 82%, 46% female, mean participant age 37.3 (SD 15.7 years), age range 18–88 years.\n\n* Victims admitted to emergency services of health care facilities (HCFs) included in the study. This includes all patients cared for in the emergency department and discharged with or without previous hospitalization. ** Altered states of consciousness and speech directly attributable to the road traffic incident (RTI; for example, encephalocranial traumas) or not (for example, alcohol intoxication, Alzheimer’s, dementia, senility, etc.) that affected the perception of the event and did not allow the respondent to answer the questionnaires.\n\nOf those eligible to participate but were not interviewed, 40% were female, mean age 39.4 (SD 15.7 years), age range 18–87 years. There were no significant differences between participants and non-participants in terms of gender or age. There were no significant differences between participants by cities with respect to education or employment.\n\nProfile of first responders after the RTI event. According to the victims, another road user was the first responder in 72% of the cases (Table 1). The median time to first response was 2 minutes (inter quartile range (IQR) 1–5) in all three cities, with no evidence of a difference in time (p=0.089). Other road users had the shortest median response time (Table 2) at 2 minutes (IQR 1–3) and firefighters (in Lima only) had the longest median time at 15 minutes (IQR 10–20).\n\nAll numbers are proportions (%) of total N for each column except where noted. Serenazgo: municipal patrol guards.\n\n* P-value calculated with Kruskal-Wallis test.\n\n*Serenazgo: municipal patrol guards.\n\n† No subjects were served by these providers.\n\n‡ Not applicable.\n\nN.B. The number is higher in the hospital period than the pre-hospital period as some participants could not remember details about the pre-hospital period.\n\nFirst response by trained personnel. With regards to trained personnel as first care providers, firefighters and ambulance services represented 4% of first responders in Lima (Figure 2) with a median response time of 10 minutes (IQR 5–20). There were no instances of trained personnel being the first responders in the other locations.\n\nThere were a total of 644 patients. Serenazgo: municipal patrol guards.\n\nCare received after first response. Only 21% of the respondents who were not treated by a trained first responder received further care from a trained provider before arriving at the HCF, though 2% did not respond to the question. The median additional time-to-trained-care by a trained provider, following a first response by any non-trained care provider, was 15 minutes (IQR 10–20). There was strong evidence of a difference in time-to-trained-care (p=0.0002) between cities with Lima at 15 minutes (IQR 10–20), Pucallpa at 10 minutes (IQR 6–10), and Ayacucho at 120 minutes (IQR 20–240).\n\nGlobal care during the pre-hospital period. Overall, 22% of the victims reported having received some sort of care by trained responders, be it first response or afterwards (27% in Lima, 7% in Pucallpa and 18% in Ayacucho). Taking together the entire pre-hospital period, 66% never received care by a qualified responder before arriving to a HCF (75% in Lima, 92% in Pucallpa and 74% in Ayacucho, p<0.001).\n\nObserved versus recommended standard pre-hospital response times. Taking into account the response time standards of the DOT and NHTSA for pre-hospital care, only 35% of RTI victims received care during the first 8 minutes or less. Only 10% of the victims arrived at the HCF within the response time standard of 38 minutes after the incident. Cities differed in meeting this standard: 13% in Lima, 4% in Pucallpa and 5% in Ayacucho; p<0.002. The median time from the occurrence of the RTI until arrival at the HCF was 30 minutes (IQR 15–45). When examining this time by city we found strong evidence of a difference between cities (p=0.0001); the median time in Lima was 30 minutes (IQR 20–45), 20 minutes (IQR 10–35) in Pucallpa, and 40 minutes in Ayacucho (IQR 20–270).\n\nProvision of care at HCF. At arrival at the HCF, the type of professionals involved in the provision of care varied dramatically between cities (Figure 3). For all victims, the median time of response-to-initial-care after arriving at a facility was 5 minutes (IQR 2–10). There was no evidence of difference in this time by city (p=0.1547) or by type of HCF (p=0.3327). When disaggregated by professions, the time of response-to-initial-care was different for doctors and medical students but not for nurses and nurse technicians (Table 2).\n\nThere were a total of 644 patients.\n\nWhen a physician was not the initial caregiver, the additional median waiting time to see a physician was 10 minutes (IQR 5–20). Reported times for Lima were 7 minutes (IQR 3–20), 10 minutes (IQR 5–20) in Pucallpa and 10 minutes (IQR 10–20) in Ayacucho, though these different times were not borderline statistically significant (p=0.0505).\n\nTotal elapsed time from the road traffic incident until intra-hospital care. The overall median time from the RTI until any care was received at a HCF was 33 minutes (IQR 22–65). In Lima the overall median time was 35 minutes (IQR 23–70), 25 minutes (IQR 17–42) in Pucallpa, and 52.5 minutes (IQR 27–275) in Ayacucho. Care at a HCF was received by 62% of the participants during the first “golden hour”. The proportion in Lima that received care in these first 60 minutes i.e. within the “golden hour” was 60%, 77% in Pucallpa, and 44% in Ayacucho (p<0.001).\n\nSatisfaction with care received during the pre-hospital period. Less than 10% of the victims reported that their first responder provided them with a poor care service. By type of first responder, 6% of the victims reported poor care by pedestrians, 7% by firefighters, 8% by the police, 8% by other ambulance services, and 9% by serenazgo.\n\nSatisfaction with care received during the intra-hospital period. The response rate to this survey component was 99%. In all cities and public HCFs we found a high proportion, around 75% or more, of victims that were dissatisfied. This was observed in each of the specific domains as well as in the global aggregate (Table 3). The highest proportions of satisfaction were at private HCFs, but only in the domains of tangibles, assurance, and empathy (Table 3).\n\n* P-value calculated with χ2 for trend.\n\nIn an adjusted multivariable analysis (Table 4) there were no evidence of an association between city and satisfaction, except in the domain of responsiveness. Compared to Lima, provincial HCFs showed more satisfaction in responsiveness (OR 0.32, 95% CI 0.15–0.68). Initially, the chances of lower satisfaction, in both global and specific domains, was 3 to 4-fold higher in the provinces compared to Lima, but these estimations became attenuated in the fully adjusted model (Table 4).\n\n* Provinces: Ayacucho y Pucallpa.\n\n** Model 1 adjusted for age and sex.\n\n¶ Model 2 adjusted for age, sex, education, employment, type of establishment, caregiver that provided care in the facility, time from the event to facility, and explanation of diagnosis to the patient.\n\nThere was strong evidence of an association between dissatisfaction and the type of HCF, which was observed in all domains of care provision. Compared to private services, participants who received care at public HCFs were 4 to 7 times more likely to report dissatisfaction. These estimates became stronger in fully adjusted models, and the magnitude of the odds estimates nearly doubled in the domains of responsiveness and empathy (Table 5).\n\n* Model 1 adjusted for age and sex.\n\n¶ Model 2 adjusted for age, sex, education, employment, city, caregiver that provided care in the facility, time from the event to facility, and explanation of diagnosis to the patient.\n\nNearly 20% of participants reported that their diagnosis was not explained to them: 12% in Lima, 37% in Pucallpa, and 26% in Ayacucho (p<0.001). The person explaining the diagnosis was usually a physician (95%). For the rest of the RTI victims, it was a nurse (3%) or medical student (2%). A lower proportion of subjects (16%) reported a lack of explanation about the treatment or procedures indicated. In Lima, 12% reported their treatment was not explained, 24% in Pucallpa, and 31% in Ayacucho (p<0.001).\n\nThe response rate to this question was 90%. The majority, 58% of RTI victims, reported that promptness of care was the top ranking HCF’s aspect that should improve. This was followed by conduct or behavior towards patients, and then by improvements in equipment and tools (Table 6). Most of the aspects highlighted for improvement were similar between public and private HFCs. While only a small difference (6% vs. 0%) the provision of more administrative and medical information ranked worse in private facilities compared to public ones.\n\n\n\n\nDiscussion\n\nBy taking advantage of active surveillance at HCFs that had a high volume of RTIs in three Peruvian cities, this study demonstrated notable deficiencies in the promptness and quality of care in the management of RTI victims. These include untrained first responders, a lengthy elapsed time until receiving attention by qualified caregivers, and above all, a marked dissatisfaction with the care received. Strong evidence of dissatisfaction was observed in public HCFs as compared with private institutions but not by geographical location, with the exception of responsiveness. Participants from Lima, the largest city in the country, were more likely to show dissatisfaction in terms of responsiveness as compared with other settings.\n\nThe implications of these findings are important for improving emergency services in these locations. Considering that other road users were usually the first responders and that the victims often had to wait until they arrived at a HCF to receive their first care from trained personnel, it is likely that most victims are not receiving adequate care in the pre-hospital period and so not meeting standards suggested by the USDOT and the NHTSA in the USA15. Ideally, the response time should be 8 minutes or less in at least 90% of all care. In our study, the median time from an RTI event to any form of care received at a HCF was 33 minutes. Indeed, 66% never received care by a qualified responder before arriving to a HCF. A previous study by the Assisted Emergency Transport System of ESSALUD in Lima, Peru by Lira et al.26 looked at the times from ambulance departure to arrival to an RTI event. They found that only 7/258 events (2.7%) met the target response time (defined as within 8 minutes in at least 90% of events).\n\nThe differences between the aforementioned international standards and those we observed could be due to incompletely developed service networks and assistance models in our study setting. In Peru these are comprised of a fragmented rendering of services and a disjointed reference and counter-reference system27. It is important to note that in Peru pre-hospital care is limited to the care and transport of patients to a HCF, thus the adequate conditions of opportunity (i.e. early treatment, ideally as close to the RTI as possible), quality, and appropriateness (i.e. the best possible care for a given scenario) are limited and not necessarily guaranteed.\n\nThough the reasons for the delayed responses were not an objective of this study, the findings were distinct enough in each context, i.e. city or type of HCF, that more research should be dedicated to understanding response time delays in the different locations and facility types. In relation to the notable delay observed in Ayacucho, one might speculate that the type of incidents and victims differ substantially than those in other cities. For example, these incidents could have occurred outside the city and/or could be related to highway incidents, which is suggested by the statistics from the police (Policía Nacional del Perú)28 and another study by our Program3. The factors associated with a variation in the quality of care of patients that suffered from a RTI were diverse and also intimately related to the problems faced by health care systems. These factors are variable and depend on the context of where care is offered-from any immediate care given after an incident, continuing through the specialized care during the hospital period, and also the rehabilitation of the victims.\n\nThe SERVQUAL instrument used in this study sheds light on remarkable statistics of global dissatisfaction, close to 90% in all cities and all types of HCFs. Thus, these findings increase our understanding of the most salient aspects of dissatisfaction with trauma care in Peru. One method to improve the quality of hospital care that has demonstrated cost-effectiveness is the implementation of improvement programs for trauma and emergency services laid out in the Guidelines for Essential Trauma Care, published by the WHO4. Their application to our health system has been insufficient at present29.\n\nOne of the strengths of the study was the active surveillance strategy we enacted. First, the coverage of the study (n=644) exceeded the volume of victims expected; as described in the methods approximately 60 patients per site per month were expected, 480 in all sites. Our active surveillance strategy yielded 34% more events than anticipated and unveils ongoing underreporting in traditional registries 30 . Second, the response rate obtained (82%) indicates that the methodology used was acceptable for fulfilling the objectives of the study. This strategy, therefore, constitutes a high-quality tool that could be replicated to evaluate RTIs, for the measurement of quality in other areas and as a tool linked to a continual evaluation process with the objective of improving the quality of services. Additionally, being able to carry out an active monitoring of victims of RTI that sought care at HCFs enabled us to obtain information that truly occurred to the said victims during the period of interest.\n\nSome limitations deserve consideration. The HCFs included in the study were restricted to larger institutions, specifically those with a high volume of RTI victims. Yet, knowledge generated by this study might inform other areas of Peru and other countries in the region with regards to gaps in management and quality of care in trauma services. Another limitation of this study is that all recorded times were self-reported by the injured or his/her family with a tendency to round the numbers. This could vary according to the seriousness of the injuries of the victim, and one could argue that there are alternative mechanisms of measurement of time that might avoid the potential biases of self-reporting. For example, a better way to estimate times in promptness of care is to use the exact date and time that an emergency call was registered and the moment that the ambulance leaves the scene. Given, however, that the majority, 66%, of those affected did not receive care from a qualified caregiver until arriving at a HCF, self-reporting was the most valid strategy to obtain the information of interest.\n\n\nConclusion\n\nIn conclusion, our study revealed important limitations of the health system that need to be strengthened if the objective of limiting death and disability of RTI victims is to be fulfilled. This was demonstrated by the low quality of care provided in terms of satisfaction with hospital care, especially outside Lima and in public facilities. Because of this, improving the capacity of care of RTI victims in the pre-hospital period with knowledge of first aid, particularly by other road users; reducing the wait time for care, particularly by trained caregivers; and improving the quality of hospital care are indispensable.", "appendix": "Author contributions\n\n\n\nJJM and LH obtained funding for the study. JJM, ERM and LH designed the study. JJM, ERM, DAQ and LH participated in the literature search. ERM reviewed and pilot tested the instruments. JJM, ERM, APP, LL, DL and LH carried out the research. JJM, ERM and LL analyzed the data. JJM, ERM and DAQ prepared the first draft of the manuscript. DAQ, CG, PP and PB provided critical input during the preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe Programa de Investigación en Accidentes de Tránsito (PIAT) and this work were funded by Peru’s Instituto Nacional de Salud. The sponsor had no role in the design, analysis, and interpretation of the literature, nor in the writing of the report and the decision to submit for publication.\n\n\nPIAT working group\n\nThe Programa de Investigación en Accidentes de Tránsito (PIAT) Working Group was hosted by Salud Sin Límites Perú in Lima, Peru. Principal Investigators: J Jaime Miranda, Luis Huicho; Coordinator: Ada Paca-Palao; Research Assistants: Luis López, Diego Luna, Edmundo Rosales; Associate Investigator: Pablo Best; PIAT Members: Miriam Egúsquiza, Camila Gianella, Claudia Lema, Esperanza Ludeña.\n\n\nAcknowledgements\n\nWe extend our thanks to all those involved with PIAT, the coordinators and field workers in each of the offices and all the participants in the study. We also thank all those from various phases that offered support: Eduardo Bedriñana (Salud Sin Límites Perú, Ayacucho, Peru), Lucie Ecker (Instituto de Investigación Nutricional, Lima, Peru), Fernando Llanos (Universidad Peruana Cayetano Heredia, Lima, Peru), Willy Lescano (US Naval Medical Research Center Detachment, Lima, Peru), David Moore (London School of Hygiene and Tropical Medicine, London, UK), Jorge Rey de Castro (Universidad Peruana Cayetano Heredia, Lima, Peru), Ian Roberts (London School of Hygiene and Tropical Medicine, London, UK), Paul Valdivia (Universidad Peruana Cayetano Heredia, Lima, Peru), Walter Valdivia (Ministerio de Economía y Finanzas, Lima, Peru).\n\n\nReferences\n\nMurray CJL, Lopez AD: The global burden of disease: a comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in 1990 and projected to 2020. Cambridge, Mass.: Published by the Harvard School of Public Health on behalf of the World Health Organization and the World Bank; 1996. Reference Source\n\nPerel P, Casas JP, Ortiz Z, et al.: Noncommunicable diseases and injuries in Latin America and the Caribbean: time for action. PLoS Med. 2006; 3(9): e344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiranda JJ, Huicho L, Lopez L, et al.: Incidencia, tendencia de los accidentes de tránsito en el Perú y factores de riesgo dependientes del peatón, vehículo y conductor [Informe Técnico]. Lima: Instituto Nacional de Salud, Salud Sin Límites Perú; 2009.\n\nMock C, Lormand JD, Goosen J, et al.: Guidelines for essential trauma care. London: World Health Organization, International Society of Surgery International Association for the Surgery of Trauma and Surgical Intensive Care; 2004. Reference Source\n\nMock CN, Jurkovich GJ, nii-Amon-Kotei D, et al.: Trauma mortality patterns in three nations at different economic levels: implications for global trauma system development. J Trauma. 1998; 44(5): 804–12; discussion 812–4. PubMed Abstract\n\nPapadopoulos IN, Bukis D, Karalas E, et al.: Preventable prehospital trauma deaths in a Hellenic urban health region: an audit of prehospital trauma care. J Trauma. 1996; 41(5): 864–9. PubMed Abstract\n\nHussain LM, Redmond AD: Are pre-hospital deaths from accidental injury preventable? BMJ. 1994; 308(6936): 1077–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGorman DF, Teanby DN, Sinha MP, et al.: Preventable deaths among major trauma patients in Mersey Region, North Wales and the Isle of Man. Injury. 1996; 27(3): 189–92. PubMed Abstract \| Publisher Full Text\n\nPeden M, Scurfield R, Sleet D, et al.: World report on road traffic injury prevention. Geneva: World Health Organization; 2004. Reference Source\n\nOrganización Panamericana de la Salud. Perfil del Sistema de Servicios de Salud de Perú.2001. Reference Source\n\nAranaz J: La calidad en sanitarios. Una propuesta general para los servicios clínicos.1998.\n\nGrapentine T: The history and future of service quality assessment: connecting customer needs and expectations to business processes. Marketing Research. 1998; 10(4): 5–20. Reference Source\n\nWorld Health Organization. A 5–year WHO strategy for road traffic injury prevention. Geneva: World Health Organization; 2001. Reference Source\n\nInstituto Nacional de Salud. Prioridades de investigación en salud en el Perú: análisis del proceso. Lima: INS; 2007. Reference Source\n\nDepartment of Transportation, National Highway Traffic Safety Administration. A Leadership Guide to Quality Improvement for Emergency Medical Services (EMS) Systems.1997. Reference Source\n\nPrograma de las Naciones Unidas para el Desarrollo. Cuadro de Índice de Desarrollo Humano Nacional. Lima; 2005.\n\nVan Dyke TP, Prybutok VR, Kappelman LA: Cautions on the Use of the SERVQUAL measure to assess the quality of information systems services. Decision Sciences. 1999; 30(3): 877–891. Publisher Full Text\n\nParasuraman A, Zeithaml VA, Berry LL: A conceptual model of service quality and its implications for future research. Journal of Marketing. 1985; 49(4): 41–50. Publisher Full Text\n\nBabakus E, Mangold WG: Adapting the SERVQUAL scale to hospital services: an empirical investigation. Health Serv Res. 1992; 26(6): 767–86. PubMed Abstract \| Free Full Text\n\nScardina SA: SERVQUAL: a tool for evaluating patient satisfaction with nursing care. J Nurs Care Qual. 1994; 8(2): 38–46. PubMed Abstract\n\nCasalino G: Calidad de servicio de la consulta externa de Medicina Interna de un hospital general de Lima mediante la encuesta Servqual. Rev Soc Peru Med Interna. 2008; 21(4): 143–52. Reference Source\n\nNúñez Z: Estudio de evaluación de la calidad de servicio de los consultorios externos del servicio de medicina del HNAL, Lima 2006 [Tesis de Maestría]. Lima: Universidad Peruana Cayetano Heredia; 2006.\n\nRicci V: Calidad de servicio percibida por los usuarios de la consulta externa de Medicina Interna del Hospital Nacional Hipólito Unanue, 2005 [Tesis de Maestría]. Lima: Universidad Peruana Cayetano Heredia; 2007.\n\nArteaga B: Calidad de servicio en consulta externa de medicina del Hospital Regional Docente de Trujillo desde la perspectiva del usuario externo [Tesis de Maestría]. Trujillo: Universidad Peruana Cayetano Heredia; 2004.\n\nCerna NB: Calidad de servicio, expresada en la satisfacción del usuario externo e interno del C.S. Baños del Inca [Tesis Doctoral]. Lima: Universidad Peruana Cayetano Heredia; 2002.\n\nVillavicencio ML: Tiempo de respuesta en el transportes primario de prioridades I y II en el servicio de STAE-EsSalud [Tesis Licenciatura]. Lima: Universidad Peruana Cayetano Heredia; 2003.\n\nMinisterio de Salud: Informe Técnico de la Comisión Multisectorial encargada de proponer los mecanismos que permitan consolidar un sistema nacional de salud.Resolución Suprema N° 002–2008–SA. Lima - Perú; 2008. Reference Source\n\nPolicía Nacional del Perú. Estadísticas Oficiales de la PNP.2008.\n\nRosales-Mayor E, Miranda JJ, Lema C, et al.: Resources and capacity of emergency trauma care services in Peru. Cad Saude Publica. 2011; 27(9): 1837–46. PubMed Abstract \| Publisher Full Text\n\nMiranda JJ, Paca-Palao A, Najarro L, et al.: Evaluación situacional, estructura, dinámica y monitoreo de los sistemas de información en accidentes de tránsito en el Perú – 2009. [Assessment of the structure, dynamics and monitoring of information systems for road traffic injuries in Peru – 2009]. Rev Peru Med Exp Salud Publica. 2010; 27(2): 273–87. Reference Source" }	[ { "id": "1412", "date": "30 Aug 2013", "name": "Andrej Michalsen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes the promptness and quality of care as perceived by approximately 640 road traffic incident victims at 8 healthcare facilities in Peru within 4 weeks. Both the promptness and the quality leave room for improvement because the average response time is far longer than internationally recommended and the quality of in-hospital services is considered low by those patients who took part in the surveillance. The abstract provides a reasonable summary of the article, except perhaps where promptness is rated as 'adequate overall'; a better description here might be \"adequate under the given circumstances\".The strength of the research is the clear methodology, which is comprehensively explained and followed. Without reliable data there will not be a sound basis for policy makers to decide on the allocation of scarce resources. Perhaps, the study period could have been longer than just four weeks to increase the validity of the results. Also, the goals for improving promptness of care lean especially towards U.S. standards without explaining their (alleged) superiority over standards in other parts of the world. The conclusions of the study appear balanced and justified. A replication of the study at more sites in Peru and beyond would be helpful to make policy makers aware of the importance of improvements in healthcare for injury victims as injury is one of the leading causes of morbidity and mortality worldwide.", "responses": [] }, { "id": "1692", "date": "05 Sep 2013", "name": "Naoto Morimura", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 is very important for assessing and recognizing the present status of trauma systems in the region. I believe an analysis of mortality however, for example an investigation of preventable trauma death by trauma scoring, will give this article greater support. In addition to the evaluation of access time to the hospital and patient’s satisfaction of this study, further study based on the outcome can enable the quality of the trauma management.", "responses": [] } ]	1	https://f1000research.com/articles/2-167
https://f1000research.com/articles/2-54/v1	18 Feb 13	{ "type": "Case Report", "title": "Case Report: Tuberculosis IRIS : a mediastinal problem", "authors": [ "Leonardo Valentin", "Andrew DiNardo", "Elizabeth Chiao", "Laila Woc-Colburn", "Arun Nachiappan", "Andrew DiNardo", "Elizabeth Chiao", "Laila Woc-Colburn", "Arun Nachiappan" ], "abstract": "We present a case of a 39-year-old male patient with Acquired Immune Deficiency Syndrome (AIDS) who developed Mycobacterium tuberculosis related Immune Reconstitution Inflammatory Syndrome (IRIS) after initiation of Highly Active Antiretroviral Therapy (HAART) treatment. The inflammatory response resulted in mediastinal necrotic lymphadenopathy and subsequent perforation of the esophageal wall.", "keywords": [ "Mycobacterium tuberculosis", "Immune Reconstitution Inflammatory Syndrome", "HAART" ], "content": "Presentation\n\nA 39 year-old man with a history of Acquired Immune Deficiency Syndrome (AIDS) presented to the emergency room with fever, productive cough, fatigue, diarrhea, and weight loss. Three weeks prior, he had been initiated on antiretroviral therapy (ART) with darunavir, ritonavir and combination tenofovir and emtricitabine. At that time, he had a CD4 count of 85 cells/μL (9%) and HIV-1 viral load of 336,950 RNA copies/mL. He was now febrile (41.0°C), with a heart rate of 100 beats/min and respiratory rate of 24/min. Physical examination revealed oral thrush and palpable cervical, supraclavicular and axillary lymphadenopathy. His laboratory evaluation was significant for a CD4 count of 28 cells/uL (10%), HIV-1 viral load of 3,410 RNA copies/mL, and hemoglobin of 6.6 g/dL. Chest radiograph on admission (not shown) demonstrated a 2.9 × 4.4 cm soft tissue mass in the anterior mediastinum.\n\nThe initial computed tomography (CT) scan of the chest showed low-attenuation, multiple mediastinal lesions, indicative of abscesses or necrotic lymphadenopathy (Figure 1), as well as esophageal discontinuity in the subcarinal region, a sign of esophageal fistula or perforation (Figure 2). Multiple cavitary lung nodules were also present (Figure 3).\n\n\nDiagnosis\n\nMediastinoscopy revealed purulent fluid drainage from necrotic lymph nodes. An esophagogastroduodenoscopy (EGD) demonstrated a 2 cm linear tear in the esophagus with proximal perforation at 29–31 cm level.\n\nThe mediastinal fluid was found to be 4+ acid-fast bacilli (AFB) smear positive. PCR of the mediastinal fluid was also positive for Mycobacterium tuberculosis (MTB) complex. In addition, the patient’s blood culture also grew MTB. The clinical presentation, recent initiation of ART, current 2-log decrease in viral load, and thoracic CT findings suggested a diagnosis of unmasking MTB immune reconstitution inflammatory syndrome (IRIS).\n\n\nDiscussion\n\nIRIS, previously known as immune restoration disease (IRD) and immune reconstitution syndrome (IRS) is a paradoxical deterioration in the clinical status of a patient after initiation of antiretroviral therapy1,2. The pathophysiology is related to the inflammation that occurs when the recovered immune system targets either live microorganisms or antigens from dead microorganisms3–7. Although recently proposed criteria for IRIS differ slightly, most criteria include the evidence of a recovered immune system along with a decrease in HIV viral load and/or increase in CD4 cell count. IRIS may occur as a paradoxical worsening of a known disease that has been under control with treatment, or an unmasking of a previously unsuspected disease4. Common pathogens associated with IRIS include tuberculous and non-tuberculous mycobacteria, cytomegalovirus, Pneumocystis jirovecii, JC virus, Cryptococcus neoformans, herpes simplex virus, hepatitis B virus, hepatitis C virus and Kaposi's sarcoma4,5. Non-infectious diseases such as sarcoidosis, Grave’s disease and thrombotic thrombocytopenic purpura have also been described8. While IRIS can occur acutely or up to 18 months after initiation of ART, most cases occur within the first two weeks to two months after initiation. IRIS is more likely to occur in the setting of high viral loads and low CD4 counts at the time of initiation of ART9–11.\n\nIn pre- and early Highly Active Antiretroviral Therapy (HAART) studies, the most common cause of lymphadenopathy (as seen on imaging) for an HIV patient with a CD4 count less than 50 cells/μL is mycobacterial infection12. Determining the presence of IRIS is not always straightforward; however, several key features help support correct diagnosis. The most common imaging feature in MTB-IRIS includes lymph node enlargement with central necrosis, most commonly located in the abdominal, axillary and mediastinal distributions13. The marked mediastinal lymphadenopathy in our patient is of particular interest, as this is common in patients with MTB-related IRIS14.\n\nThis patient initiated ART with a low CD4 count of 85 cells/uL (9%) and a high HIV viral load of 336,950 RNA copies/mL. After initiation of ART, his viral load decreased by 2 logs to 3,410 RNA copies/mL.\n\nThe exaggerated immune response to our patient’s mediastinal mycobacterial burden resulted in extension of inflammation from necrotic lymphadenopathy to the esophageal wall, which underwent necrosis and perforation. This resulted in a gas collection replacing the necrotic lymph nodes (Figure 1). Esophageal perforation can occur from extensive coughing and retching in an MTB patient, with or without an underlying infectious esophagitis.\n\n\nManagement\n\nAlthough definitive management of IRIS has not been established by carefully controlled studies, current management may include the addition of corticosteroids and, in severe cases, temporarily withholding ART. Future management may include evaluation for a combination of cytokines and inflammatory markers such as interleukin 7, interleukin 6 and/or C-reactive protein to predict who is at higher risk of developing IRIS, which can be assessed prior to initiation of ART15,16. Future therapies may include immunomodulatory medications (C-C chemokine receptor 5 inhibitors, TNF antagonists or interleukin 6 receptor inhibitors) to temper the vigorous immune reconstitution16.\n\nOur patient had a complicated hospitalization including recurrent pneumothoraces (Figure 4), empyema, and unmasking of cutaneous Kaposi sarcoma. The esophagus in an HIV patient is particularly vulnerable to pathology. Our case illustrates a mediastinal infectious process in which TB-IRIS was the etiology and causative factor for an esophageal perforation that further complicated the treatment of this patient with AIDS.\n\n(A) Multiple cavitary and non-cavitary lung nodules (same as Figure 3). (B) Hospital day 6: increased pulmonary tree-in-bud nodules and consolidations, new small right-sided pneumothorax (arrowhead), and new esophageal stent (arrow). (C) One month follow-up: nearly-resolved pulmonary opacities decreased tiny right pneumothorax, and removal of esophageal stent.\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\nLeonardo Valentin and Andrew DiNardo wrote the manuscript. Elizabeth Chiao, Laila Woc-Colburn, and Arun Nachiappan revised the manuscript. Arun Nachiappan selected the images. 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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nLeone S, Nicastri E, Giglio S, et al.: [Tuberculosis-associated immune reconstitution inflammatory syndrome]. Infez Med. 2008; 16(4): 193–199. PubMed Abstract\n\nShelburne SA 3rd, Hamill RJ, Rodriguez-Barradas MC, et al.: Immune reconstitution inflammatory syndrome: emergence of a unique syndrome during highly active antiretroviral therapy. Medicine (Baltimore). 2002; 81(3): 213–27. PubMed Abstract\n\nAchenbach CJ, Harrington RD, Dhanireddy S, et al.: Paradoxical immune reconstitution inflammatory syndrome in HIV-infected patients treated with combination antiretroviral therapy after AIDS-defining opportunistic infection. Clin Infect Dis. 2012; 54(3): 424–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarber DL, Andrade BB, Sereti I, et al.: Immune reconstitution inflammatory syndrome: the trouble with immunity when you had none. Nat Rev Microbiol. 2012; 10(2): 150–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBehrens GM, Meyer D, Stoll M, et al.: Immune Reconstitution Syndromes in Human Immuno-deficiency Virus Infection Following Effective Antiretroviral Therapy. Immunobiology. 2000; 202(2): 186–193. PubMed Abstract \| Publisher Full Text\n\nLetang E, Miró JM, Nhampossa T, et al.: Incidence and predictors of immune reconstitution inflammatory syndrome in a rural area of Mozambique. PloS One. 2011; 6(2): e16946. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh N, Perfect JR: Immune reconstitution syndrome associated with opportunistic mycoses. Lancet Infect Dis. 2007; 7(6): 395–401. PubMed Abstract \| Publisher Full Text\n\nMounzer K, DiNardo A, Goldstein K: Thrombotic thrombocytopenic purpura during immune reconstitution. AIDS. 2007; 21(18): 2559–60. PubMed Abstract \| Publisher Full Text\n\nElliott JH, Vohith K, Saramony S, et al.: Immunopathogenesis and diagnosis of tuberculosis and tuberculosis-associated immune reconstitution inflammatory syndrome during early antiretroviral therapy. J Infect Dis. 2009; 200(11): 1736–45. PubMed Abstract \| Publisher Full Text\n\nFrench MA, Lenzo N, John M, et al.: Immune restoration disease after the treatment of immunodeficient HIV-infected patients with highly active antiretroviral therapy. HIV Med. 2000; 1(2): 107–115. PubMed Abstract \| Publisher Full Text\n\nGrant PM, Komarow L, Andersen J, et al.: Risk Factor Analyses for Immune Reconstitution Inflammatory Syndrome in a Randomized Study of Early vs. Deferred ART during an Opportunistic Infection. PLoS One. 2010; 5(7): e11416. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLichtenberger JP 3rd, Sharma A, Zachary KC, et al.: What a differential a virus makes: a practical approach to thoracic imaging findings in the context of HIV infection--part 2, extrapulmonary findings, chronic lung disease, and immune reconstitution syndrome. AJR Am J Roentgenol. 2012; 198(6): 1305–12. PubMed Abstract \| Publisher Full Text\n\nRajeswaran G, Becker JL, Michailidis C, et al.: The radiology of IRIS (immune reconstitution inflammatory syndrome) in patients with mycobacterial tuberculosis and HIV co-infection: Appearances in 11 patients. Clin Radiol. 2006; 61(10): 833–43. PubMed Abstract \| Publisher Full Text\n\nBuckingham SJ, Haddow LJ, Shaw PJ, et al.: Immune reconstitution inflammatory syndrome in HIV-infected patients with mycobacterial infections starting highly active anti-retroviral therapy. Clin Radiol. 2004; 59(6): 505–13. PubMed Abstract \| Publisher Full Text\n\nAntonelli LR, Mahnke Y, Hodge JN, et al.: Elevated frequencies of highly activated CD4+ T cells in HIV+ patients developing immune reconstitution inflammatory syndrome. Blood. 2010; 116(19): 3818–3827. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSereti I, Rodger AJ, French M: Biomarkers in immune reconstitution inflammatory syndrome: signals from pathogenesis. Curr Opin HIV AIDS. 2010; 5(6): 504–510. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "836", "date": "14 Mar 2013", "name": "Eric Daar", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 somewhat novel manifestation of TB IRIS. The comments related to IRIS are relevant and of some value. Minor comments include:The discussion under management probably should at least raise the importance of continuing pathogen specific therapy and the potential role of nonsteroidal anti-inflammatory agents.Page 3, bottom of column 1, top of column 2 states “The esophagus in a HIV patient is particularly vulnerable to pathology.”\n\nThe use of the term “HIV Patient” is awkward and the overall statement is not supported by any data or citation.", "responses": [ { "c_id": "486", "date": "13 Jun 2013", "name": "Leonardo Valentin", "role": "Author Response", "response": "Thank you for your report Dr. Daar. Please find below the response to some of the comments: Case reports suggest NSAIDS may offer symptomatic relief, however randomized evidence for this is lacking. There is a BIII recommendation to use NSAIDS for TB- associated IRIS according to the updated IDSA/CDC guidelines.References:Murdoch DM. Incidence and risk factors for the immune reconstitution inflammatory syndrome in HIV patients in South Africa: a prospective study. AIDS. 2008;22(5):601Centers for Disease Control and Prevention. Guidelines for Prevention and Treatment of Opportunistic Infections in HIV-Infected Adults and Adolescents. MMWR 2009;58 (No. RR-4) April 10, 2009. Updated March 2013. Patients with very low CD4 counts commonly have esophageal pathology, most commonly due to Candida, CMV or HSV and more rarely due to HPV, mycobacteria, idiopathic ulceration, syphilis or H. ducreyi.Reference:Wilcox CM. Esophageal disease in acquired immunodeficiency syndrome: etiology, diagnosis, and management. Am J Med 1992 Apr 92 412-421." } ] }, { "id": "953", "date": "15 May 2013", "name": "Susana N. Asin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, about a mediastinal manifestation of unmasking Tuberculosis Immune Reconstitution Inflammatory Syndrome (IRIS). Specific remarks are:1- Since the optimal time to start ARV in the course of anti-tuberculosis treatment is unclear detailed comments about case management i.e. TB-treatment are encouraged. 2- The discussion should consider the fact that even though ARV decrease HIV-1 viral load, CD4 levels after three weeks of treatment were even lower (85 cells/ul compared to 28 cells/ul). Was this a clear indication of a recovering immune system? Should an increase in CD4 cells count have been expected?. 3-This report underscores the relevance of an inflammatory response targeting either live microorganisms or antigens from dead microorganisms as the pathophysiology of IRIS. Is there any evidence to support ARVs as a contributing factor to the on-going inflammatory response?", "responses": [ { "c_id": "488", "date": "16 Jun 2013", "name": "Leonardo Valentin", "role": "Author Response", "response": "Thank you for your review Dr. Asin. See below for our response:The patient had been inconsistently receiving anti-retroviral treatment since his diagnosis of HIV 10 years ago. Three weeks after having restarted antiretroviral therapy (ART) the patient presented to the emergency department with fever, productive cough, diarrhea, fatigue and weight loss. During his admission ART was continued, albeit with a brief interruption between hospital days 2 and 9, during his admission. Mediastinoscopy was performed on hospital day 3. The following day, PCR for TB was positive and the patient was started on four-drug anti-tuberculous therapy (ATT) consisting of Isoniazid, Rifabutin, Ethambutol, and Pyrazinamide. While the absolute CD4 count decreased, the percentage increased. We believe the absolute CD4 decrease was likely related to mycobacterial bone marrow invasion and subsequent inflammation causing pancytopenia & total leukocytosis. In addition CD4 increases are known to lag behind viral load decrease and are relatively delayed to the IRIS event. Some authors question the efficacy of CD4 rise as part of a proposed standardized IRIS diagnostic criteria. Reference: Achenbach, C. J. et al. 2012). Paradoxical immune reconstitution inflammatory syndrome in HIV-infected patients treated with combination antiretroviral therapy after AIDS-defining opportunistic infection. Clin Infect Dis. 54(3), 424–33. doi:10.1093/cid/cir8023.While the pathophysiology is unclear, the varied and multiple presentations of IRIS can provide some insight. There are probably some cases in which IRIS occurs due to neither dead nor live organisms, but an unbalanced immune system. Manifestations like thrombotic thrombocytopenic purpura, Grave’s thyroiditis, and other autoimmune conditions provide some examples of the non-infectious presentations.Reference: Mounzer K, DiNardo A, Goldstein K: Thrombotic thrombocytopenic purpura during immune reconstitution.AIDS. (2007); 21: 2559–60." } ] }, { "id": "998", "date": "12 Jun 2013", "name": "Paul Klenerman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 short case report and the images are striking.A couple of points:The actual therapy for this patient was not described in any detail.The timing of therapy for TB/HIV disease could be discussed.The evidence for use of anti-inflammatory agents could be discussed and referenced.It would help to expand on the apparent drop in CD4+ T cell count and anemia. What were the other blood indices and how did these recover over time?As a small point the 1st line of abstract should be “39 year-old”.", "responses": [] } ]	1	https://f1000research.com/articles/2-54
https://f1000research.com/articles/2-51/v1	15 Feb 13	{ "type": "Research Article", "title": "Cortical long-range interactions embed statistical knowledge of natural sensory input: a voltage-sensitive dye imaging study", "authors": [ "Selim Onat", "Dirk Jancke", "Peter König" ], "abstract": "How is contextual processing as demonstrated with simplified stimuli, cortically enacted in response to ecologically relevant complex and dynamic stimuli? Using voltage-sensitive dye imaging, we captured mesoscopic population dynamics across several square millimeters of cat primary visual cortex. By presenting natural movies locally through either one or two adjacent apertures, we show that simultaneous presentation leads to mutual facilitation of activity. These synergistic effects were most effective when both movie patches originated from the same natural movie, thus forming a coherent stimulus in which the inherent spatio-temporal structure of natural movies were preserved in accord with Gestalt principles of perceptual organization. These results suggest that natural sensory input triggers cooperative mechanisms that are imprinted into the cortical functional architecture as early as in primary visual cortex.", "keywords": [ "The early visual cortex comprises an extended and densely interwoven network", "acting on millisecond time scales1. Radially", "activity is rapidly distributed by local feedback loops2", "3. Tangentially", "long", "horizontal fibers enable neurons to sense regions beyond their receptive field borders4–7. Investigating the dynamics of this circuitry with simple parametric stimuli reveals well-defined selective response properties. In carnivores and primates", "where these neurons are further organized in overlaid maps8–11", "it is possible to satisfactorily predict changes in the layout of these maps in accord with the distribution of the stimulus energy across different visual features12", "but see13." ], "content": "Introduction\n\nThe early visual cortex comprises an extended and densely interwoven network, acting on millisecond time scales1. Radially, activity is rapidly distributed by local feedback loops2,3. Tangentially, long, horizontal fibers enable neurons to sense regions beyond their receptive field borders4–7. Investigating the dynamics of this circuitry with simple parametric stimuli reveals well-defined selective response properties. In carnivores and primates, where these neurons are further organized in overlaid maps8–11, it is possible to satisfactorily predict changes in the layout of these maps in accord with the distribution of the stimulus energy across different visual features12, but see13.\n\nHowever, these stimulus-response relationships can flexibly be modified by the specific connectivity patterns between distant neurons4,14,15. For example, a surrounding stimulus which is in itself not sufficient to drive cortical neurons above firing thresholds may exert strong contextual influences on local processing16–19. These integrative phenomena are conceived as the functional backbone of various Gestalt criteria of perceptual organization20 and naturally occurring visual tasks such as contour forming, figure-ground separation, object segmentation, or perceptual completion. Hence, local-to-local interactions are intrinsically tied to the integrative functionality of cortical operation. They can be conceptualized as biases originating from the cortical architecture that foster optimal coordination of large numbers of neurons in accord with the statistics of incoming signals21–23.\n\nToday, a large body of evidence indicates that the functional properties of neurons are specifically adapted to process signals that are of ecological relevance24–26. Neuronal stimulus-response properties exhibit higher sensitivity27, selectivity28 and reliability29,30 in response to visual features when these are presented within their natural sensory context. However, direct functional evidence showing that cortical connectivity mediating local-to-local coupling embeds empirical statistical knowledge of natural inputs is at best scarce and it is not clear whether these interactions studied with simple stimulus configurations extrapolate to complex dynamic conditions that mimic natural input.\n\nWe recorded cortical activity using voltage-sensitive dye imaging31 in response to locally presented natural movies recorded by cats in a natural habitat26. We characterized contextual effects by manipulating the spatiotemporal statistical regularities between two movie patches; we tested the hypothesis that cortical circuits responsible of contextual effects are functionally adapted to natural input statistics. Our results show that, under dynamic natural stimulation conditions, facilitatory interactions across distances beyond the classical receptive field characterize contextual effects, demonstrating that cortical circuits embed functional knowledge about the spatiotemporal relationships inherent in natural scenes.\n\n\nMaterials and methods\n\nStimulus acquisition and presentation hardware was the same as in a previous study26. Natural movies (see Figure 1A, for two example frames) were recorded at a sampling rate of 25 Hz by freely moving cats exploring a natural habitat. The recorded natural movies were presented locally, through a single or a pair of Gaussian apertures (referred to as patches in the text) for a duration of 2 s including the 200 ms prestimulus interval. We included in the analysis presented here only the initial 750 ms. Local patches were created by modulating the contrast of the movies as a function of space according to a two dimensional Gaussian function with full width at half maximum (FWHM) (~3–4°). The FWHM depended on the distance of the center point to the area centralis in individual experiments (~3–6°). The average luminance value of pixels overlapping with the two dimensional Gaussian mask were first subtracted and then multiplied pixel-wise with the Gaussian mask. This effectively reduced the luminance contrast from center to periphery. Following this step the average values were set back to the background luminance level, which was kept constant across the whole experiment.\n\n(A) Movie frames extracted from two different movies (marked orange/green) are shown. Prior to optical recordings retinotopy across the imaged cortical region was evaluated by hand mapping of receptive field locations (colored rectangles) at various penetration sites. This ensured that the distance between the mid-point of apertures was bigger than the range of classical receptive fields. (B) For each experiment, two base natural movies (marked orange/green) were used to create different stimulation conditions. Movies were presented through either one or two Gaussian masks (3° or 4° FWHM, see Materials and Methods) centered at positions A (top position) or B (bottom position), illustrated by white circles. According to the position label (A or B) and the index of the natural movies (Movie 1 or Movie 2), the following conditions were displayed: single (A1, B1, A2, B2), in which only one local patch was shown; coherent (A1B1, A2B2), in which two local patches belonged to the same full-field movie; and incoherent (A1B2 and A2B1), with local patches derived from different movies (see rightmost column).\n\nLocal conditions were indexed by two parameters: position on the screen (A or B) and movie index (1 or 2) specifying which full-field movies were to be masked. We used different movies in different experimental sessions to increase the generalizability of our results. By displaying either one or simultaneously two local movies, the conditions A1, B1, A1B1, and A2, B2, A2B2 were created (Figure 1B and Supplemental Movie S1). For conditions with a pair of patches, the distance between the centers of the two Gaussian apertures was equal to 2.5 FWHM. Local movies were corrected for mean luminance so that the average of the pixels within the Gaussian apertures was always equal to the brightness of the background in which they were embedded. However, we did not equalize the contrast within each aperture, as it is not possible to do so without introducing strong artifacts, particularly in cases where the local portion of a movie frame contains regions with homogenous brightness values belonging to object surfaces.\n\nPrior to optical recordings, the topographic mapping between the cortical surface and the visual field were scrutinized by means of several electrode penetrations, and local stimuli were positioned so that the upper movie patch matched the receptive field position of the simultaneously recorded multiunit activity. This ensured that the distance between the centers of the Gaussian masks extended beyond the borders of classical receptive fields.\n\nAnimals were initially anesthetized with ketamine (15 mg kg-1 intramuscularly (i.m.)) and xylazine (1 mg kg-1 i.m.), supplemented with atropine (0.05 mg kg-1 i.m.). After tracheotomy, animals were artificially respirated, continuously anaesthetized with 0.8–1.5% isoflurane in a 1:1 mixture of O2/N2O, and fed intravenously. Heart rate, intratracheal pressure, expired CO2, body temperature, and electroencephalograms (EEG) were monitored during the entire experiment. The skull was opened above the primary visual cortex and the dura was resected. Paralysis was induced and maintained by alcuronium dichloride (Alloferin®). Eyes were covered with zero-power contact lenses for protection. External lenses were used to focus the eyes on the screen. To control for eye drift, the position of the area centralis and receptive field positions were repeatedly measured. A stainless steel chamber was mounted and the cortex was stained for 2–3 hours with voltage-sensitive dye (RH-1691), and unbound dye was subsequently washed out with artificial cerebrospinal fluid. All surgical and experimental procedures were approved by the German Animal Care and Use Committee (AZ 9.93.2.10.32.07.032) in accordance with the Deutsche Tierschutzgesetz and NIH guidelines.\n\nOptical imaging was accomplished using an Imager 3001 (Optical Imaging Inc, Mountainside, NY) and a tandem lens macroscope32, 85 mm/1.2 toward camera and 50 mm/1.2 toward subject, attached to a CCD camera (DalStar, Dalsa, Colorado Springs). The camera was focused ~400 µm below the cortical surface. For detection of changes in fluorescence, the cortex was illuminated with light of 630 ± 10 nm wavelength and emitted light was high-pass filtered with a cutoff of 665 nm using a dichroic filter system. Cortical images were acquired at a frame rate of 220 Hz and covered regions of approximately 10×5 mm2 of primary visual cortex. The relevant retinotopic region of area 18 was captured (lower contralateral quadrant of the visual field), and parts of area 17 were also occasionally captured. For electrophysiological recordings, a custom-built device was used that allowed targeted penetrations at different locations without opening the sealed recording chamber.\n\nThe raw data was processed in two steps26. First, in order to remove differences in illumination across different pixels, divisive normalization was performed on all the recorded raw samples of a given pixel by its mean during prestimulus period. Second, heart-beat and respiration-related artifacts were removed by subtracting the average blank signal recorded in the absence of stimulation. These differences were later normalized by the blank signal in order to gain independence from the global activity level fluctuations occurring during the course of an experiment. As our recordings were synchronized with the heart-beat cycle of the animal, this blank subtraction step effectively removes these artifacts. Moreover, this method is preferred over the cocktail blank correction because our conditions were not composed of orthogonal stimuli. These steps were applied for each trial separately and the outcome was averaged across trials. The number of trials ranged from 25 to 35 for different experiments.\n\nWe computed spatial profiles by averaging the evoked data across the temporal dimension. These were fitted with a two dimensional Gaussian function of the form:\n\nG(x,y) = A exp((x – μx)2/σx + (x – μy)2/σy + (x – μx)(y – μy)/ρ)\n\nWhere A, σx, σy, μx, μy, and ρ represent the peak value, the horizontal (medio-lateral) and vertical (antero-posterior) spreads, the position of the Gaussian function (medio-lateral or antero-posterior), and the rotational parameter, respectively. We used lsqcurvefit function provided by Matlab (2007b, The MathWorks, Natick, MA) using a large-scale trust-region-reflective algorithm. Prior to optimization runs, initial parameters of the Gaussian function were roughly estimated using different heuristics for each experiment and condition separately. As the fitting function we used superposition of two Gaussian functions G1(x,y) + G2(x,y) centered roughly on separate activation spots. This was true even for single conditions that included only one single activity blob. We could therefore estimate the value of the indirect activation. We also constrained the value of different parameters to avoid fitting to irrelevant cortical activity; this was most useful in the single condition case, where there was only one single activity spot.\n\nFor the statistical evaluation of confidence intervals, we used the bootstrapping method with 1000 repetitions and an alpha value of 0.05. The confidence intervals are provided within square brackets following average values. We tested the significance of median activities using the Wilcoxon sign rank test; the p-values are provided within brackets.\n\n\nResults\n\nWe performed voltage-sensitive dye imaging (VSDI) in the primary visual cortex of anesthetized cats (n = 4). Multi-unit recordings complemented these measurements and provided information on receptive field properties and localization and spatial extent (see Materials and methods). In order to investigate long-range cortical interactions under ecologically relevant dynamic stimulation conditions, natural movies were presented locally by applying either one or two Gaussian masks (3–4° FWHM) to the original full-field movies (Figure 1A). Presentation of two movies (Movie 1 and Movie 2) viewed through apertures at two different positions (position A and B) creates a total of 8 different local stimulation conditions (Figure 1B, please see also Video S1): Single conditions (A1, B1, A2 and B2) consisted of isolated local movie patches that provided no contextual information (Figure 1B, first and second column). In contrast, coherent (A1B1 and A2B2) and incoherent (A1B2 and A2B1) conditions provided contextual information in the form of another distant movie-patch. Here, two movie-patches stemming from either the same (coherent) or different (incoherent) original natural movies were presented simultaneously at two locations that were larger than the typical classical receptive field sizes (Figure 1A, colored boxes representing receptive fields). Whereas coherent conditions leave the spatiotemporal characteristics of natural movies intact, incoherent stimulation eliminates naturally occurring correlations between apertures and induces an evident dissonance (please see Video S1).\n\nCortical responses to two different movies presented in single and coherent conditions for a duration of 750 ms are shown as space-time plots (Figure 2, top and bottom rows for Movie 1 and Movie 2, see also Video S2). Conditions are indicated on the left-most column. Upon localized stimulation by natural movie patches, activity emerged from baseline level with variable delays among conditions. The cortical dynamics induced by the individual stimuli show different temporal profiles and suggest that instantaneous activity levels were determined by the specific properties of each natural movie. Each single movie evoked well-separated spots of activity on the cortical surface indicating that the thalamic input was spatially well resolved at this stimulus configuration. Furthermore, we observed large differences in the activity levels between single and coherent conditions (note the difference in color scale). In this experiment, while the peak activity (maximum activity level across all pixels) during single conditions (bottom two rows in each panel) was 6.67×10-4 ΔF/F, coherent conditions (top row in each panel) led to a value of 7.86×10-4 ΔF/F, corresponding to an increase of 18%. As the direct input to the recorded cortical region was identical during single and coherent conditions, we attribute these differences in activity to the impact of long-range interactions on cortical dynamics under natural stimulation conditions.\n\n(A) Spatiotemporal activity patterns produced by locally presented natural movies is shown overlaid on the vascular image. Leftmost column schematically shows the corresponding stimulus condition and the movie used (orange and green). Each single frame represents the average activity across trials (n = 35) within 50 ms of non-overlapping segments of recorded data. The cortical representations of these stimuli were located along the antero-posterior axis, reflecting the vertical positioning of the movie patches in the visual field. Different spatial extents and peak values of cortical activation were observed along the presentation duration at different frames. Please note that different color-scales are used for different conditions in order to emphasize the detailed spatial structure. Upper color scale is clipped to 3.3 standard deviations. Only the most active pixels corresponding to highest 25th percentile are shown. For a video presentation please refer to the Video S2. Scale bar (shown in the first row in the frame zero) = 1mm.\n\nTo quantify long-range interactions we computed spatial profiles of activity levels under stimulation conditions with or without context (Figure 3, first and third rows). These maps represent the average activity of each single pixel across stimulation duration and demonstrate clearly the spatially restricted non-overlapping foci of activity. Note that different movies produced different amplitudes of activity. Thus, occasionally, incoherent conditions, incorporating a movie that drives the cortex more strongly, can appear more highly activated than in coherent conditions. Therefore, cross-wise comparisons (as in Figure 5B) between both incoherent conditions are needed to calculate the net interaction effects between coherent and incoherent movies.\n\nSpatial activity profiles (first and third rows) in response to two different natural movies (upper and lower panels) presented at various stimulation conditions with (third and fourth columns) or without (first and second columns) contextual information. Icons schematically represent the stimulation conditions. These are computed by time-averaging the data presented in Figure 2. Activity during no-context conditions was used to define a pair of interest regions per movie. This was done by selecting the pixels with highest activity located within the top 5th percentile (see contour lines). Within these regions of interest we derived direct (cyan), indirect (yellow) and coherent (black) and incoherent (magenta) activity levels. The observed activity profiles were parametrically fitted using a composite function involving two 2-D Gaussians (second and fourth rows). The goodness of fit values characterized by the correlation coefficient is shown for the whole data set in Figure 4D.\n\nThe precise shape of spatial activity profiles shown in Figure 3 varied considerably across different experiments. This is to be expected, as the location and extent of activity spots depend strongly on the recording conditions specific for each experimental session. It was therefore not straightforward to compare these spatial activity profiles across different experiments. We used a parametric approach in order to circumvent this problem and modeled spatial activity profiles recorded during different experiments using two-dimensional Gaussian functions with 6 free parameters. These parameters consisted of peak value (A), its horizontal and vertical position (μx and μy), horizontal and vertical spread (σx and σy) as well as a rotational parameter (ρ) (see Materials and methods). The modeled spatial activity profiles are presented together with the empirical data in Figure 3 (second and fourth rows, same colorbar). The correlation coefficient between these fits and the empirical data was on average 0.88 [0.82, 0.91] (average, [95% bootstrap confidence intervals], same convention in the following). For the whole data set, the distribution of correlation coefficients (Figure 4D) was negatively skewed and equal to 0.82 [0.78, 0.86] on average. Hence, compared to many thousands of pixels typically recorded in optical imaging, our parametric approach provided a major reduction in dimensionality without compromising the precise characterization of response patterns.\n\n(A) Modelized activity profiles averaged across all experiments (n=4) and movies in response to indirect, direct, coherent and incoherent stimulation types are depicted (first row, three dimensional depiction; second row, top view). Transparent orange plane (third and fourth columns) marks the peak level of the Gaussian function representing the responses to direct stimulation at the absence of contextual information (second column). We compared different parameters of the Gaussian fits across different stimulation types in order to characterize different contextual interactions (see red, green, blue arrows). (B–D) For each of the comparisons, peak (B) and joint spread values (C,D) of Gaussian fits are presented as scatter plots (see matching arrow and dot colors). Each experiment contributes 4 different points. The plus sign represents the mean. (D) The distribution of correlation coefficient between fits and the observed data for the whole dataset.\n\nWe computed 4 types of characteristic spatial activity profiles from 3 different stimulation conditions (Figure 4A, first row, three-dimensional depiction; second row, top view representation). From activity during single conditions we derived the characteristic activity profiles for direct (cyan border) and indirect (yellow border) stimulation types. Whereas the direct activity represents the baseline responses to a single movie-patch in the absence of any contextual stimuli, the indirect activity captures the influence of an isolated distant movie-patch. Similarly we computed the characteristic activity patterns in response to movie-patches presented either in coherent (dark gray border) or incoherent conditions (magenta border). In Figure 4A, we visualize these four characteristic activity profiles after normalizing separately peak and spread parameters by their corresponding values obtained during direct stimulation. This was done for each experiment separately and the median fitted values were computed subsequently (this was necessary in order to eliminate outliers that originated from the normalization procedure).\n\nWe observed major changes in the characteristic activity profiles that were reflected in peak and spread parameters (compare different columns in Figure 4A). Concerning the peak activity, the indirect effect (Figure 4A, first column, yellow borders) of a single movie-patch presented at a distant location was on average slightly excitatory; however, this was statistically not significant (sign-test = 0.8). However, it should be noted that we observed net-excitatory effects as frequently as suppressive effects with similar amplitudes at the distant non-stimulated locations. The occasional occurrence of suppression of net activity below baseline levels in the far periphery have been shown with VSDI when presenting local stimuli without contextual surround (see Figure 1 in33). During the two conditions where context was present (Figure 4A, third and fourth columns, magenta and dark gray borders), the peaks were higher than during stimulation without context (compare to second column, cyan border). Importantly, among those conditions where context was present, coherent context resulted in higher peak activity values (compare third and fourth columns).\n\nWe quantified the total facilitation effect by comparing the activity induced by direct and coherent stimulation (Figure 4A, see blue lines). This is depicted in (Figure 4B, blue dots) for each individual comparison (4 dots per experiments). In nearly all cases, the peak activity during coherent stimulation was higher than direct activity measured during single conditions. In 2 cases, a positive activation was observed only during coherent conditions. While direct drive evoked an average peak activity of 0.23×10-3 ΔF/F [0.16×10-3, 0.29×10-3 ΔF/F] (average, [95% bootstrap confidence intervals], same convention in the following) across experiments, a value of 0.34×10-3 ΔF/F [0.27×10-3, 0.39×10-3 ΔF/F] was observed during coherent input. The pairwise difference between peak values was equal to 0.11×10-3 ΔF/F [0.04×10-3, 0.14×10-3 ΔF/F], corresponding to an increase of 45.2% [19.8%, 62.3%] in peak value and this was significantly different than zero (p = 0.0011, pairwise t-test). We therefore conclude that contextual stimuli presented at distant locations have a substantial modulatory effect on local activity. We further compared the peak values between direct and incoherent stimulation conditions (not shown as a scatter plot). The presence of an incoherent context resulted in an increase of 24.5%; this increase was, however, not significantly different from zero (sign-test, p = 0.8; t-test, p = 0.08).\n\nTo what extent can the total facilitatory effect be accounted for by the indirect additive effect of the distant movie-patch? We compared the activity during coherent stimulation conditions to the predicted activity by the sum of direct and indirect responses (Figure 4A, green lines). We found a superadditive effect of contextual stimulation in nearly all comparisons (Figure 4B, green dots). The superadditive facilitatory effect quantified as the difference between coherent conditions and the sum of single and indirect activations corresponded to 41.9% [20.9% 76.2%] (signtest, p = 0.004; t-test, p = 0.009), hence only about 3.3% of the contextual effect was accounted for by linear interactions. This shows that long-range interactions result to a large extent from non-linear interactions between cortical sites.\n\nTo what extent are the non-linear contextual influences adapted to the statistical regularities of natural movies? The total facilitatory effect quantified above incorporates both the specific and unspecific influences originating from contextual stimulation. While the modulation of peak activity by an incoherent context can be attributed to the unspecific effect of the distant stimuli, any incremental effect of a coherent context can be attributed to the specific adaptation of these interactions to the statistics of natural movies. To evaluate the specificity of these interactions we compared the peak values between coherent and incoherent conditions (Figure 4A, red line; Figure 4B, red dots). We observed an increase of 20.7% [4.18% 38.12%] in the peak activity level, and this was found to be marginally significant (sign-test, p = 0.21; t-test, p = 0.053). However, compared to the 45.2% observed for total facilitation, this analysis shows that about 54.2% of the facilitation results from specific interactions. Therefore, the non-linear facilitatory effects were fully effective only when the two movie-patches complied with the statistical regularities specific to natural movies.\n\nIt is possible that long-range facilitation by the contextual sources of information are accompanied by a modification of the total spatial extent of cortical activity. For example, the presence of contextual information could result in a more tuned spatial activity profile leading to a decrease in spread parameters with the presence of context. Alternatively, contextual information could potentially cause a larger number of cortical neurons to be allocated. In order to test these different hypotheses, we evaluated the influence of context on the spatial extent of activated cortical space and compared the average spread parameters (σx and σy) between single and coherent conditions. The cortical activation extended larger surfaces during conditions of stimulation where context was present (Figure 4C). We found that the presence of a coherent context increased 10.9% [1.4, 29.2%] the joint spread parameter (Figure 4C, left panel). Considering each dimension separately we found that contextual information increased the spread parameter only along the direction of the context (along anterio-posterior axis). Consequently while an increase of 18.1% [5.3, 39.4%] in σy was observed, there was no significant change in σx [-1.9%, 24.0%]. This effect was further boosted by the presence of a coherent context. Comparing incoherent and coherent activations (Figure 4C, right panel), we found a similar result. Here again only the σy parameter was significantly different and an increase of 8.8% [0.5, 19.9%] was observed. Therefore, as in the case of peak activity modulations, coherent context had a stronger impact on the spread parameter compared to the case where the contextual information was absent or incoherent. We conclude that rather than a sharpening of the spatial profile, more cortical space is allocated when contextual information is present. Furthermore, the direction of this increase is biased towards the location of the contextual information.\n\nIn order to have a better grasp on the temporal unfolding of long-range interactions, we next characterized the time-course of the facilitatory effects. To this end we used the evoked activity values and limited the analysis to pixels that were most strongly driven by the movie patches. Based on the activity profiles during two single conditions (Figure 3, first and second rows), we defined two non-overlapping regions of interest for each movie condition by choosing those pixels that lay within the highest 5th percentile of activity (see contour lines). These most responsive pixels were typically located centrally with respect to the activity spot. For each of the afore-mentioned activation types (indirect, direct, incoherent and coherent) we computed the mean time-course of activity across all movies and experiments within these most strongly driven pixels (Figure 5A, same colors as in Figure 4A, and Figure 3). Please note that here experiments were conducted using different natural movies leading to the loss of a specific temporal profile. Samples of the time course of the facilitatory effect with mean activity significantly different than zero are depicted with filled circles (t-test).\n\n(A) The temporal evolution of activity in response to indirect (yellow), direct (cyan), coherent black) and incoherent (magenta) stimulation (left panel). Average activity within the regions of interest (depicted in Figure 3) is used. For each temporal window the median across experiments and movies is presented. Median values that are statistically different (sign-rank test, alpha = 0.05) from zero are depicted with filled circles. The pair-wise difference of amplitudes between coherent and single conditions (black line) as well as between coherent and incoherent (gray line) conditions is shown in the right panel. (B) The similarity between the time-courses of activity recorded at two distant regions of interest is evaluated for each movie separately under different conditions (2 dots per experiment). rcoherent measures the correlation between two time-courses of activity evoked by the simultaneous presentation of two local movie patches. rincoherent and rsingle were computed using the activity evoked by exactly the same input however presented at different conditions (see icons and arrows). The scatter plot depicts the comparison of rcoherent to rsingle and rincoherent.\n\nAs noted before, the indirect influence of the distant single movie-patch was slightly excitatory (Figure 5A, yellow line). However, contrary to the previous parametric analysis, which was not temporally resolved, we detected here a significant effect of the indirect input at ~100 ms (p = 0.04, see filled circle). This confirms that the indirect influence of a movie patch presented in isolation to its neighboring regions is of excitatory nature and occurs quickly.\n\nDuring direct stimulation in the absence of context (Figure 5A, cyan line), activity increased with stimulus onset and quickly reached a plateau at 100 ms, exactly where the indirect drive reached the significance level. At this point, the activity was 3.7-times stronger than the indirect drive. All samples following stimulus onset were statistically different from zero (p < 0.002). As expected, with the presence of a coherent context, the facilitatory interactions caused stronger activity levels throughout stimulus presentation. These were effective as early as 100 ms following stimulus onset (Figure 5A, left panel, cyan). We computed the pair-wise differences between coherent and single conditions and evaluated whether these deviated significantly from zero level (Figure 5A, right panel, black line). These facilitatory effects quickly followed after stimulus onset and reached significance around 300 ms (p < 0.049). The presence of an incoherent context had a smaller impact on activity levels (left panel, magenta line) and consequently the time-course of activity was similar to conditions where no context was present. The difference between coherent and incoherent conditions (right panel, gray line) computed over the stimulus presentation was positive throughout the stimulus presentation and reached the significance levels at two time frames (p < 0.042, see filled circles). The time-resolved analysis presented here complements the parameteric approach. We conclude that the interactions between different cortical locations occur quickly following stimulus onset and they persist across the stimulus presentation.\n\nDuring single conditions, the activity within the region of interest is mainly determined by direct sub-cortical input and, therefore, the bottom-up characteristics of the input stream are presumably the sole determinants of the precise time-course. Additionally, as natural movies contain non-zero correlations across long distances, it is expected that activity profiles at locations A and B exhibit certain amount of similarity that would lead into correlations in the activity time-courses. We quantified these similarities at locations A and B during two single conditions by measuring the correlation coefficient (Figure 5B, schematic representation). The correlations, rsingle, between the time-courses of activity recorded in both locations were never negative. Temporal resolution of the time-courses in this analysis was 200 Hz in order to capture its detailed structure. We observed an average correlation of rsingle = 0.57 [0.47, 0.67], suggesting that low-level characteristics of movies were to a large extent common to both locations. How do the lateral interactions, which are effective during simultaneous presentation of two movie patches, influence the precise time-course of activity? To answer this question, we computed rcoherent by quantifying the correlation between activities evoked by two simultaneously presented movie patches (Figure 5B, see arrows). All correlation values were higher than corresponding rsingle values (Figure 5, red dots). rcoherent was equal to 0.84 [0.74, 0.89], resulting in an increase of 46.9%. This result suggests that long-range interactions increase the similarity of the activity time-course. In accord with this conclusion, we observed that an incoherent movie-patch presented simultaneously had a detrimental effect on the correlation values. Consequently, rincoherent was 30% smaller than rcoherent and equal to 0.58, ([0.38, 0.74], Figure 5B, black dots). This result suggests that long-range interactions, in addition to their facilitatory effects, lead to an increase in the similarity of the time-course.\n\n\n\nMovie 1. Natural movies and stimulus conditions. Two different natural movies and derived local stimulation conditions are shown as they were used during one of the experiments. To facilitate inspection, movies are shown at half of the speed (12 Hz) used during the experiments. The right-hand color labels (red/blue) code for condition identity. The monitor covered a visual field of approximately 30×40 degrees.\n\n\n\nMovie 2. Activation patterns during locally presented natural images. Evoked optical activity during local stimulation with a single or a pair of natural movie patches. First and third rows show full-field natural movies (left) and derived local conditions. Second and fourth rows depict cortical activity patterns measured with VSDI. The scale bar represents 1 mm.\n\n\n\n\nDiscussion\n\nWe used VSDI to investigate long-range cortical interactions during processing of natural images in the primary visual cortex at the mesoscopic population level. By using \"keyhole-like\" presentations of the original natural movies through either one or two distant Gaussian masks, we quantified the effect of surrounding stimulation on local activity. We provide evidence that contextual integrative mechanisms are indeed operative under natural stimulus conditions. We show that under these conditions the horizontal cortical network34–37 forms the basis for synergistic interactions across several millimeters of cortex. Contextual stimulation led to a net facilitatory effect compared to the case when the movies were shown in isolation. An important attribute of these interactions was their sensitivity to the intrinsic spatiotemporal regularities of natural movies38,39. Moreover these contextual interactions led to an increased similarity of the population dynamics across long-range cortical distances.\n\nContextual processing has been investigated extensively both experimentally at the single neuronal level40 and in recent modeling approaches41. A large variety of facilitatory and/or inhibitory contextual effects have been observed, however the final outcome crucially depends on the precise configuration of the parametrized stimulus used to stimulate center and surround regions. While the surround effect was found to be mainly inhibitory35,42,43 and spatially asymmetrically organized44, the precise nature of the effect depends on the contrast of contextual stimuli relative to the contrast threshold of the recorded neuron45–47.\n\nAn important cornerstone of long-range facilitation is its dependence on the precise spatial configuration of the surrounding context48. It has been shown that facilitatory effects increase proportionally with the congruency of the contextual stimuli with respect to the center stimulus18,49. Using static stimuli, such coherence is generally controlled parametrically by changing the orientation difference between center and surround patches17,18,50,51. Since we here used natural movies recorded by cats that freely explored a natural habitat, our stimuli were complex and contained simultaneous multiple features. The head and body movements of the cats added to this complexity as the recorded visual stimuli contained motion cues that were correlated across large visual distances. In order to control the coherency of the stimuli between the apertures, we adopted a non-parametric method by exploiting the unique spatiotemporal characteristics of each original movie.\n\nWhen the same movie was presented through both apertures they were perceptually grouped without effort, and appeared to belong to a single scenery. On the other hand, when two differing movies were used, the content within both apertures appeared to be immediately incompatible (please see Video S1). There are a number of factors that determine coherence between patches taken from the same movie. First, stimulus motion was similar between the two distant apertures. This was due to the body- and head-motions of the recording cat, which induced large and equal motion fields across the visual scene captured by the camera. It has been earlier noted that such temporal phase relationships across distant regions are perceptually salient and enable object segmentation even in the absence of any spatial information52. Second, natural images tend to possess large spatial correlations because of the dominance of low spatial frequencies in their spectrum21. Moreover auto-correlations of orientations may cover large portions of visual field reaching up to 8 degrees53. Therefore, our stimulus paradigm can be conceived as a dynamic illustration of Gestalt criteria of good continuation.\n\nThere are different idealized mechanisms, each based on different anatomical substrates, which could mediate the observed facilitatory long-range interactions. Overlapping feed-forward thalamo-cortical input could be an explanation for increased cortical drive during stimulation with adjacent movie patches. However, there are a number of counter-arguments against this explanation. First, cortical locations driven most strongly by the individual movies were separated by distances larger than the anatomical spread of direct thalamo-cortical projections5,15,54,55. This was in accord with relatively smaller spatial extents of mapped receptive fields. Second, the activity at the distant location during stimulation with one single movie patch was only minimal and reached significant levels about 50 ms later in comparison to directly stimulated locations. Third, and most decisively, the total drive to the recorded cortical area was constant across the two coherent and incoherent conditions. Only the order of the presentation being different, it is not possible to account for facilitatory interactions in a purely feed-forward scheme.\n\nRather, the dense network of horizontal connections linking distant neurons across several millimeters is a likely candidate for the observed long-range effects. It has been shown that unmyelinated intralaminar connections contribute to subthreshold responses evoked from distant stimuli placed outside of the classical receptive fields both with intracellular5 and combined extracellular recordings and VSDI in cat7. Furthermore, the selective intracortical connectivity pattern of these tangential connections linking neurons with similar feature selectivity is well-suited to mediate the specific enhancement of activity levels dependent on the stimulus coherence. However, we cannot exclude that feedback signals originating from higher visual areas with larger receptive field sizes than in primary visual cortex could add to these interactions56,57. Back-propagating waves of activity have been shown to be initiated in further downstream cortical areas as early as ~100 ms after stimulus onset58,59. Thus, these connections act fast60,61 and are likely to mediate surround modulations spanning considerable distances in visual space, while lateral intra-laminar connectivity may account for modulations within shorter distances62.\n\nWe observed that, compared to incoherent stimulation, stimulation with coherent pairs of natural stimulus patches led to stronger facilitatory effects. Since the total input analyzed across coherent and incoherent stimulation was identical across the recorded cortical area, these results cannot be solely explained by the local properties of movie patches. Rather, this facilitation necessarily reflects the outcome of an integrative phenomenon sensitive to the content of both local movie patches when presented simultaneously. Therefore, we suggest that the functional architecture of early visual cortical circuits may have empirically internalized the typical contextual relationships21–23 found in dynamic natural visual scenes.", "appendix": "Author contributions\n\n\n\nSO, DJ, PK: Conceived and designed the experiments, analyzed the data, wrote the manuscript, contributed materials and analysis tools. SO, PK: Stimulus recording and preparation. DJ: performed the experiments.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work has been financed by the Bundesministerium für Bildung und Forschung (BMBF), the Deutsche Forschungsgemeinschaft (DFG) SFB-874 (TP A2 Eysel, Jancke), the Bernstein Group for Computational Neuroscience Bochum, the German-Israeli Project Cooperation (JA 945/3-1, SL 185/1-1), the International Graduate School of Neuroscience (IGSN) Ruhr-University Bochum, Germany and the EU commission (FP7-ICT-270212, eSMCs).\n\n\nAcknowledgements\n\nWe thank Markus Swierczek for contribution to data acquisition; Stefan Dobers and the mechanical shop of the Ruhr-University Bochum for excellent technical support; Cliodhna Quigley and Benedict Ng for comments on the manuscript; Agnieszka Grabska and Alper Açık for helpful discussions; Jörg Conradt, Gudrun Möller, Rodrigo Salazar for their valuable help in natural movie recording.\n\n\nReferences\n\nCallaway EM: Local circuits in primary visual cortex of the macaque monkey. 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J Neurosci. 2002; 22(19): 8633–8646. PubMed Abstract" }	[ { "id": "784", "date": "19 Feb 2013", "name": "Marcello Rosa", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 seem to only deal with linear addition of activation. We are not sure that this is a reasonable assumption, without more information about how optical activity scales with spiking rate. Thus, supra-linear summation may not be that surprising. i.e. the presence of one stimulus patch might lift the optical activity to close to threshold, so a second patch then seems to create a lot of activation, but it's just because there was a \"hidden\" threshold. Figure 5 is pretty complicated. We had a hard time understanding it. Maybe it needs to be broken into smaller, more digestible bits of information and have a clearer legend.This paper will be more useful if they also present data using gratings as stimuli.", "responses": [ { "c_id": "516", "date": "02 Aug 2013", "name": "Selim Onat", "role": "Author Response", "response": "Point by point response to Dr Rosa: This is an important point. The relationship between the VSD imaging signal and spiking activity can indeed be complex. For instance, a close relationship between spike rate and the derivative of the VSD response rather than its magnitude has been proposed 69. This might especially apply to the rising phase of the membrane potential after stimulus onset 6,70. Combined VSD and Calcium-sensitive dye imaging suggests that the relationship between spiking activity and the amplitude of the VSD response depends also on stimulus intensity 71. However, Chen et al., 72 found that these relationships are well-captured by a power function with an exponent of ~4 [similarly as for the relationship between average membrane potential and spike rates in single V1 neurons 73,74, indicating that the threshold for observing significant spiking activity is about 30–40% of the maximal VSD response. Amplitudes evoked by our natural movies were on average well-within this range as compared to full-field moving gratings that we used as a standard control for maximal stimulation. Moreover, we compared the fitted peak amplitudes and this ensures that only pixels with highest amplitude at the center of the movie representations entered our analysis. Therefore, we are confident that subthreshold activity was excluded and that we describe the effect of long-range connectivity on postsynaptic spiking activity. We added this information to Discussion (page 10, before the last paragraph) Dividing the Figure 5 might make it more difficult to understand given that the data that is shown on the scatter plots are summarized on the top panels, which represents the average observations. Interestingly, using LFP as a population measure of postsynaptic activity, a recent study showed that superimposed gratings (i.e. plaids) lead to responses that were not predicted by activity in response to their single components (Bartolo et al., 2011). Thus, a simple parametric manipulation (addition) of most simple stimuli like gratings can lead to drastic changes in neuronal population response behavior limiting explanatory power. If this is the case, the use of gratings may be a weak predictor with respect to complex stimulation, in particular with respect to natural scenes. Please also see above for our reply to Dr Contreras who had a similar concern.Reference numbers refer to the reference list in the manuscript" } ] }, { "id": "811", "date": "06 Mar 2013", "name": "Diego Contreras", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhat is the point of using “ecological” stimuli if the response is then reduced to 6 parameters? How is that different from using drifting gratings? The authors should either provide the comparison with simpler stimuli or better justify their choices. The main point here is that the larger summation of two stimuli from the same movie versus two stimuli from different movies may be simply due to objects that span the two masks generating a similarity in the orientation and spatial frequency in the two stimuli, which, in turn, would lead to very well-known orientation-specific effects of contextual stimuli. How is presenting another stimulus “contextual”? the stimuli where either one or two stimuli of identical size. It would be easier to read if the authors used only one terminology, such as single, coherent and incoherent. How was a 2D Gaussian fit to the response of one of the stimulus while presenting both? The response is merged, doesn't this affect the fitting procedure?What was learned from the multi-unit recordings?Figure 2: It is pretty but mostly useless without quantification, the authors should show values of dF/F or contour plots of the significant response (3 or 5 SDs above baseline noise) so that the movies can be properly compared. It is remarkable that the responses to the same mask in movie 1 and 2 are so different, are they really statistically different? Is the VSD signal capable of discriminating the differences between the two movies? Looking at the movies it seems that they have similar spectrotemporal composition.Where were the values presented on top of page 5 taken from? What area? What time?Figure 4:What is “modelized”? Does figure 4A not represent data?Minor Points:Page 4, 2nd column: Dissonance? What is “superadditive”? Does it mean supralinear? Very difficult to understand, mainly because the term is used in combinations such as “superadditive facilitation”", "responses": [ { "c_id": "519", "date": "09 Aug 2013", "name": "Selim Onat", "role": "Author Response", "response": "Point by point response to Dr Contreras: These are important questions (please also see our response to question 3 from Dr Rosa below). Clearly, there exist a remarkable number of studies (references in main text) about contextual effects using local grating patches of certain orientations and spatial frequencies.In our work we follow instead the hypothesis that the low-level sensory regions (if not the whole nervous system) are wired to optimally process inputs that are most likely to be received from the sensory organs. Our recent report 26 suggests that the cortical state (i.e. both the dynamic range and mean activity) is qualitatively different when stimulated with gratings as compared to ecological stimuli. Thus, response behavior to natural input can deviate significantly from predictions based on simple parameterized stimuli, probably due to the extensive spatial information in natural images 22, 29.These observations may place the burden of justification on the use of simple stimuli, and not of natural stimuli as used in the present report. Many intra-areal long-range facilitation phenomena in the early visual system have been shown to occur in response to local presentations of simple stimuli. The evidence showing that these are also functional under more natural stimulation conditions that are more complex and also more ecological is not clear. For instance, both the extent and the impact of long-range connections may critically depend on the correlation structure across neuronal populations (Lindén et al., 2011), which in turn emerges from the specific spatio-temporal correlations of visual input. Therefore, in this manuscript, we aimed to demonstrate that long-range interactions are also functional and effective under more naturalistic stimulation conditions, which were so far not investigated. We provide this evidence in this manuscript and we have added a paragraph to the discussion section (paragraph 4) of our manuscript.Finally, the choice of a low number of parameters to characterize the response is data driven. We demonstrate that our analysis captures the larger part of variance of the response as obtained by voltage sensitive dye imaging (VSDI) and is therefore a valid procedure. A comparison of the dimensionality of stimulus and response space has to take into account the non-linearity of processing in the visual system. Our use of “contextual” is inspired by usage of the terms classical receptive field and non-classical receptive field/context in the visual sciences: Here one visual stimulus elicits a reliable response at a certain location. The other stimulus does not elicit detectable activity at that location, but as our data demonstrate modulates the response to the former stimulus. The term “context” is intended to denote that dependence. That is, we consider this neighboring information as a contextual input as it doesn’t directly influence the processing at the distant center. In all cases, including single stimulation conditions, there were two 2D Gaussians that were combined to fit the observed responses. The parameters were estimated simultaneously. Using two Gaussians in all stimulation conditions ensured that the activation at the distant locations did not influence the fit. Most of the time, the activity spots were clearly distant and well-isolated from each other, therefore possible cross-contributions were negligible. Furthermore the center position of each single Gaussian fit was constrained around the location of the peak responses at locations A and B. This helped avoiding complications where Gaussians could overfit noise. We now included this information in the manuscript (Methods, page 8, last paragraph). Electrical recordings were performed to allow for rapid retinotopic hand mapping of the imaged area before VSDI. Electrical recordings parallel to VSDI were done to verify occurrence of evoked spiking activity at those cortical locations where high levels of the dye signal was observed, i.e. around the center representation of one of the local movie patches. Note that a single recording site was used. Thus, analysis of reciprocal interactions at two locations simultaneously, as resolved by VSDI, was not feasible. Analysis of the electrical recordings related to particular stimulus features will be subject of a following report. The colorbar depicts x10-4 dF/F values. This is now added to the legends of Figures 2 to 4. Pixels with an activity smaller than 1.5x10-4 dF/F are not shown. This corresponds to the 75th percentile of the activity distribution, as noted on the figure legend. Responses of each single movie are depicted with their own colormap so that the precise detailed structure of the evoked responses can be clearly seen for each single condition. Using a common color scale would basically eliminate the possibility to clearly see responses to movies that evoked weaker responses. Furthermore, the colorbar helps comparison of amplitudes across different movies and positions. VSDI can clearly distinguish these differences. In Onat et al.13, we have shown that VSDI can distinguish diverse stimulus related components in response to drifting gratings including actual position, i.e. the retinotopic trajectory, of the grating stripes. In the current manuscript, in Figure 2, it is also possible to see that the same movie during double or single conditions leads to similar temporal activity profiles (as analyzed in Figure 5). These are peak values computed across the entire stimulation duration and all recorded pixels. Figure 4A (top row) presents the spatial activity profiles observed on the cortical surface that are reconstructed using the Gaussian parameters (mu, sigma, amp) averaged across ROIs and cats. Please notice that averaging the raw recorded data across cats and ROIs (A and B) is not possible. This is due to the large differences in the shape of the activity profiles across different ROIs and cats, therefore we reconstructed these activity profiles using the fitted parameters. Each individual data point that contributes to these spatial profiles are depicted in Figure 4B. We replaced the word “modelized” with “modeled”. You might view this analysis technique as a low dimensional parametric description of the experimental data. Minor PointsWe used the term dissonance to describe the perceptual quality of movie patches originating from different movies when shown simultaneously. We used the term super-additive, to indicate a response that is more than the sum of the two individual components when these individual components are presented simultaneously. The meaning is essentially the same as supralinear, although in the literature super-additive is used more often. Reference numbers refer to the reference list in the manuscript" } ] } ]	1	https://f1000research.com/articles/2-51
https://f1000research.com/articles/2-120/v1	02 May 13	{ "type": "Method Article", "title": "Microbiopsy engineered for minimally invasive and suture-free sub-millimetre skin sampling", "authors": [ "Lynlee L Lin", "Tarl W Prow", "Anthony P Raphael", "Robert L Harrold III", "Clare A Primiero", "Alexander B Ansaldo", "H Peter Soyer", "Lynlee L Lin", "Anthony P Raphael", "Robert L Harrold III", "Clare A Primiero", "Alexander B Ansaldo", "H Peter Soyer" ], "abstract": "We describe the development of a sub-millimetre skin punch biopsy device for painless and suture-free skin sampling for molecular diagnosis and research. Conventional skin punch biopsies range from 2-4 mm in diameter. Local anaesthesia is required and sutures are usually used to close the wound. Our microbiopsy is 0.50 mm wide and 0.20 mm thick. The microbiopsy device is fabricated from three stacked medical grade stainless steel plates tapered to a point and contains a chamber within the centre plate to collect the skin sample. We observed that the application of this device resulted in a 0.21 ± 0.04 mm wide puncture site in volunteer skin using reflectance confocal microscopy. Histological sections from microbiopsied skin revealed 0.22 ± 0.12 mm wide and 0.26 ± 0.09 mm deep puncture sites. Longitudinal observation in microbiopsied volunteers showed that the wound closed within 1 day and was not visible after 7 days. Reflectance confocal microscope images from these same sites showed the formation of a tiny crust that resolved by 3 weeks and was completely undetectable by the naked eye. The design parameters of the device were optimised for molecular analysis using sampled DNA mass as the primary end point in volunteer studies. Finally, total RNA was characterized. The optimised device extracted 5.9 ± 3.4 ng DNA and 9.0 ± 10.1 ng RNA. We foresee that minimally invasive molecular sampling will play an increasingly significant role in diagnostic dermatology and skin research.", "keywords": [ "skin punch biopsy", "microbiopsy", "neoplasm", "diagnostic tool" ], "content": "Introduction\n\nSkin biopsy is one of the most essential techniques in dermatology for accurate diagnosis of neoplastic or inflammatory skin diseases through histopathological assessment. This technique is performed under local anaesthetic by trained medical personnel, normally a dermatologist, to remove a skin sample 2–4 mm in diameter (Figure 1) that is then preferably sent to a dermatopathologist for histopathological diagnosis. Pathological examination using skin biopsies often complements and/or confirms diagnosis of common neoplastic or inflammatory skin diseases1. This technique is usually performed using a punch biopsy tool or a scalpel. The wound is then either sutured or left to heal.\n\nA conventional biopsy punch is shown on the left, an 18 gauge syringe needle in the centre and the inner chamber of our microbiopsy device on the right Panel (a). Our microbiopsy device chamber is 0.15 mm in width with an outer width of 0.25 mm. The top row of Panel (b) contains a conventional 3 mm biopsy site and tissue, whereas the bottom panels show microbiopsied skin and tissue.\n\nAn excisional biopsy, a common alternative in neoplastic skin conditions, is performed when the entire tumour is needed for histopathological examination, or when melanoma is suspected. This depends on the size and location of the lesion and can be performed using a larger 4–6 mm diameter punch biopsy, a deep shave biopsy, or with a fusiform excision2. These biopsy techniques coupled with histopathology are able to provide accurate diagnosis of the disease occurrence and progression, however the downsides of these techniques are that they require local anaesthesia and sutures in addition to the time required to carry out the procedure. Furthermore, samples are generally fixed with formalin that hinders molecular analysis. Molecular fingerprinting of skin disease has the potential to dramatically improve diagnostic sensitivity and open the door for personalised medicine3–5. To date, there have been no reports on biomarker profiling of lesional samples in a prospective study due to the lack of suitable technologies to perform multiple sampling over time. This is due to the iatrogenic issue of cutting out the lesion through biopsy precluding further study of that lesion.\n\nThis problem exists in many medical disciplines and has led to the evolution of experimental diagnostic devices towards miniaturised versions of their predecessors. This miniaturisation trend is enabled by several factors including improved micro-manufacturing tolerances, decreasing costs and increasing availability. One of the earliest micro-devices developed to obtain biopsy samples was patented by Krulevitch et al.6. They patented microbiopsy/precision cutting devices fabricated by conventional machining, silicon micromachining, precision machining and injection moulding. Over the years, there have been many similar patents that report a variety of such microbiopsy devices. The unifying theme is that these patents all describe micro-biopsy devices for breast or intestinal tissue sampling7–10 and importantly none were engineered for skin or skin lesions.\n\nOur group has developed a new microbiopsy platform technology that enables the collection of tiny pieces of skin using a micro-medical device that is minimally invasive and does not require local anaesthesia (IP Australia Appl. Num. 2012901490, filed on 16/04/2012). There are situations when conventional biopsies are not appropriate. Skin disease may present with multiple lesions and/or in a cosmetically sensitive area. Certain areas such as the lower leg may have problems with wound healing and so biopsies are avoided. The microbiopsy device fills a void in dermatology where conventional biopsies are not feasible or could do more harm than good. We have observed that microbiopsy sampling does not interfere with downstream histopathological diagnosis11. The data presented herein describe the fabrication, optimisation and early steps toward the validation of a novel microbiopsy device for use in vivo.\n\n\nMaterials and methods\n\nThe microbiopsy devices used in our studies were fabricated by laser cutting plates of stainless steel. The in-house fabrication of microbiopsy devices involves laser cutting of two-dimensional designs on 0.05 mm thick medical grade stainless steel (304, Mastercut Technologies, Australia) at 95% power, 30% frequency and speed of 0.9 using a 20 W laser marking system (LaserPro S290, GCC, Taiwan). All of the 2D microbiopsy components were assembled into 3D devices, and sterilized using a glass bead sterilizer (Steri Inotech 350, Sigma-Aldrich, USA) at 250°C for 30 seconds or submerged in 70% ethanol for 1 min and air-dried prior to use. The microbiopsy device was then fitted into a spring-loaded applicator.\n\nA bench top scanning electron microscope (SEM) (JCM-6000 Neoscope, JEOL, USA) was used to acquire high-resolution images of the microbiopsy device. The roughness amplitude of the microbiopsy chamber was obtained by measuring the average distances of the edge to a regression-fitted straight line using MatLab (Mathworks, Australia).\n\nMicrobiopsy samples were either obtained in vivo from healthy volunteers (HREC/12/QPAH/082, Metro South Human Research Ethics Committee, Centres for Health Research, Princess Alexandra Hospital) or ex vivo from excised actinic keratosis (AK) lesions. The skin of a healthy volunteer was swabbed with alcohol and the microbiopsy applied. Conventional biopsies e.g. punch (Figure 1b) or shave biopsies, were performed with informed consent from patients (HREC/08/QPAH/207 for healthy skin or HREC/11/QPAH/477 for AK lesions) prior to being microbiopsied. Samples were collected in sterile RNase and DNase free 1.5 ml microcentrifuge tubes containing either RNALater® (Life Technologies, USA) or pH 7.0 phosphate buffered saline (PBS) on ice within 20 minutes after tissue removal from patients. All samples placed in RNALater® were kept overnight at 4°C and then stored at -80°C.\n\nVolunteer pain scores were evaluated with Metro South Human Research Ethics Committee, Princess Alexandra Hospital approval (HREC/12/QPAH/082). Each of the 20 volunteers (Table 1) was presented with an assessment form to grade his/her expected pain score based on a numerical rating 10-point Likert scale, 0 as having no pain and 10 as pain as bad as they can imagine before the start of the experiment. Each microbiopsy application was performed one minute apart and the pain score recorded immediately after application. Five minutes after the final microbiopsy application, the volunteers were asked to rate the level of pain for each microbiopsy site.\n\nVolunteers for microbiopsy classified by age, ethnic group, gender and Fitzpatrick skin type.\n\nMicrobiopsy sites were visualized with a reflectance confocal microscope (RCM, Vivascope 1500® Multilaser, Lucid, Inc., USA) in volunteers or in excised skin (HREC/12/QPAH/217). RCM was also used to perform in vivo examination of the skin after microbiopsy. RCM images were collected through the microscope head, integrated with a water immersion objective, at a near-infrared wavelength of 785 nm. Blocks consisting of 7 × 7 mm mosaics of stitched RCM images and 2 µm to 200 µm vertical z-stacks were acquired at the microbiopsy sampling site. Dermatoscopic images were collected with a Canon Power Shot G10 digital camera (Canon, Japan) and a dermatoscope attachment (Dermlite©, 3Gen, USA).\n\nMicrobiopsied tissue was embedded (Optimal cutting temperature compound, Sakura Finetek, USA) and cryosectioned (CM1850, Leica Microsystems Pty Ltd, Australia). The 10 µm thick sections were fixed with 100% cold methanol for 10 minutes, air dried and stained with haematoxylin and eosin (Varistain Gemini ES, Thermo Fisher Scientific Inc., USA) to visualize the microbiopsy sites.\n\nThe microbiopsy extracted tissue was incubated with DRAQ5 (BioStatus Limited, UK) at 10 µM in PBS for 30 minutes at room temperature. Confocal microscopy images were taken with a Zeiss 510 META confocal microscope (Carl Zeiss Microscopy GmbH, Germany). A 3D projection was then created from the z-stacks (2 μm steps) of confocal images using Imaris (Bitplane Scientific Software, Switzerland). ImageJ (NIH, USA) was used to estimate the nuclei in microbiopsy samples.\n\nDNA was extracted from the microbiopsy samples using QIAamp DNA Micro purification kits (QIAGEN GmbH, Germany) using a protocol modified to accommodate the unique microbiopsy sample. The microbiopsy was removed from the applicator housing after application. The presence of a tissue sample was quickly confirmed by opening the microbiopsy device and visualizing with a stereo microscope (Carl Zeiss Microscopy GmbH, Germany). The opened device was then immersed in 180 μl lysis buffer (Buffer ATL, QIAGEN GmbH, Germany) in the presence of proteinase K (20 μl) in a 1.5 ml tube. Fifteen seconds of pulse-vortexing was applied to ensure that the sample was well-mixed. The sample was then placed in a thermomixer (Eppendorf, Australia) at 56°C with shaking at 800 rpm overnight. The tube containing the device was briefly centrifuged for 10 seconds to remove the solution adhering to the cap of the tube before transferring the lysate into a new tube. The tube containing the device was centrifuged again at 6000 g for 30 seconds to remove any lysate adhering to the device. The lysates were combined and the remaining procedure followed the manufacturer supplied instructions.\n\nThe modified sample lysing processing described in DNA isolation above was also used with the Arcturus® PicoPure® RNA Isolation Kit (Life Technologies, USA) to obtain total RNA from microbiopsy samples. RNA isolation of lesional samples was performed using RNeasy Mini Kit (QIAGEN GmbH, Germany) according to the RNeasy Mini Handbook (version 2010).\n\nA quantitative fluorometer-based assay (Qubit® 2.0, Life Technologies, USA) was used to determine the concentration of DNA and RNA with the protocol provided by the manufacturer.\n\nAgilent RNA 6000 Nano and Pico kits (Agilent Technologies, USA) were used to determine the RNA integrity number (RIN) of lesional and microbiopsy samples after RNA isolation. The supplied protocol was followed.\n\nIdentical amounts of total RNA (14 ng) for both lesional and microbiopsy samples were subjected to whole transcriptome amplification. The procedure was carried out using the supplied instructions (QuantiTect Whole Transcriptome Kit, QIAGEN, Australia). The cDNA obtained from the amplification process was used as a template in a polymerase chain reaction (PCR) reaction using human beta actin primers (forward: ATC TGG CAC ACC TTC TAC AAT GA; reverse: CGT CAT ACT CCT GCT TGC TGA TCC AC) (Integrated DNA Technologies, NSW, Australia). There was an initial denaturation for 2 minutes at 98°C, followed by 30 cycles of 98°C for 30s (denaturation), 67°C for 30s (annealing), and 72°C for 1 minute (extension). The cDNA and PCR products were run on a 1% agarose gel (Bio-rad Laboratories, Inc., Australia) and visualized with RedSafe (ChemBio, UK). HyperLadder™ 1 kb (HyperLadder I) (Bioline, UK) was used to determine the mass of cDNA and human beta actin amplicons.\n\nStatistical analysis was performed using PRISM 6 for Windows (GraphPad Software, Inc., USA). Channel width, velocity and roughness amplitude data were presented as mean ± SD. Pain score for channel width and velocity were presented as minimum to maximum box-and-whiskers plot. One-way ANOVA combined with a Tukey’s multiple comparison post-test was performed to determine the statistical significance.\n\n\nResults\n\nOur microbiopsy device has a chamber volumetric size of 0.003 mm3 that is more than 6000 times smaller than a conventional 3 mm punch biopsy and more than 5 times smaller than an 18 GA syringe needle (Figure 1a). Figure 1b demonstrates the size differences between a conventional 3 mm punch biopsy (top panels) and our microbiopsy (bottom panels).\n\nWe conducted a volunteer study in 20 healthy individuals to determine the optimal channel width and application velocity for this device. Extracted DNA mass was chosen to be the primary indicator for sample-to-sample comparisons. Interestingly, we observed tissue collection (4.5 ± 1.5 ng DNA) around the rough edges of a microbiopsy device without a chamber (Figure 2a–b, channel width of 0 mm). After applying the microbiopsy, the device was opened up and visualized under a dissecting microscope. Successful collection was achieved when tissue was evident within or around the device and unsuccessful if no tissue was present. Tissue was collected from all volunteers (n=20) when a 0.15 mm channel width microbiopsy device was used. Only 13 successful collections were achieved from 20 applications when a 0.20 mm channel width microbiopsy device was used. This indicated that the collection rate decreased from 100% to 65% when channel width was increased by 0.05 mm. The device without a channel (0 mm channel width) captured tissue around the edges of the tapered plates in all replicates (n=20). This experiment showed that a channel width of 0.15 mm obtained the highest average amount of DNA (5.9 ± 3.4 ng), which was significantly higher than 0.25 and 0.30 mm channel widths (p < 0.0001). There was no significant difference in the total DNA extracted between 0–0.20 mm channel widths (Figure 2a).\n\nChannel width and velocity were varied to optimise the microbiopsy device configuration. Total DNA was used as a surrogate for sample size. Panel (a) shows the DNA extracted from device with varying channel width and that the maximum amount of DNA was collected with 0.15 mm channel width. Panel (b) displays high resolution scanning electron microscopic images showing different channel widths of the microbiopsy device. Panel (c) shows the level of pain volunteers reported when different channel width microbiopsies were used. Panel (d) shows the varying velocity applied and that the maximum amount of DNA was collected when the device was applied at 16.6 m/s. Panel (e) shows the level of pain volunteers reported when application velocity was varied.\n\nMicrobiopsy application velocity was evaluated by using defined applicator springs to achieve velocities between 0–20.2 m/s (Figure 2d). Only negligible amounts of DNA were recovered when the device was applied at less than 9.2 m/s. However, there was a 7.5-fold increase (0.8 ± 0.8 to 6.0 ± 3.0 ng) in DNA recovered when the application velocity was increased from 9.2 m/s to 16.6 m/s (p < 0.0001). An additional increase to 20.2 m/s increase in application velocity did not result in significant increases in DNA collection.\n\nThere was no change in the level of pain reported when microbiopsies with varying channel widths were applied (Figure 2c). The level of pain significantly increased and variation increased when microbiopsies were applied at increasing velocities (Figure 2e). All of the volunteers had a pain score of 0 at 5 minutes after the final microbiopsy application (data not shown). The volunteers scored pain between 0 to 10 with an average score of 1.5 ± 1.1, when the 0.15 mm channel width microbiopsy was applied at 20.2 m/s.\n\nWe observed DNA collection without a centre chamber (Figure 2a, 4.5 ± 1.4 ng from channel width 0 mm) and hypothesized that surface roughness could be key to successful sample collection. We compared microbiopsy devices with varying roughness amplitudes (RA) and observed an increasing trend in total DNA extracted with increasing RA (Figure 3a) (n=20 for 5.36 RA and n=5 for 0.92 and 1.32). The SEM images of the inner chamber edge were used to measure the RA. These measurements ranged from relatively smooth (RA 0.92) to rough (RA 5.36) (Figure 3b, side view).\n\nPanel (a) shows that microbiopsy devices with higher roughness amplitude channels are capable of collecting more DNA. Panel (b) contains high resolution scanning electron microscopic images showing different channel widths and roughness of the microbiopsy device.\n\nRCM was used to visualise the microbiopsy application site. RCM images revealed a microbiopsy site similar to the size of a small hair follicle in the dermal papillary post microbiopsy application (Figure 4a and 4b). The inset shows that microbiopsy application resulted in a puncture site that was approximately 0.10 × 0.50 mm in dimension (Figure 4b). Microbiopsy samples were stained with DRAQ5 (BioStatus Limited, UK) to highlight the nuclei. Confocal images were used to generate a 3D model of the microbiopsy sample. The usual skin strata were apparent in this model. We estimated that there were 1634 nuclei in Figure 4c. We observed 0.22 ± 0.12 mm wide and 0.26 ± 0.09 mm deep puncture sites in excised abdominal skin from 10 histological sections (Figure 4d).\n\nPanels (a) and (b) are reflectance confocal microscopy mosaics of a microbiopsy site, see the hair follicles featured in the centre and on the right hand side of the images for size comparison (bar indicates 0.5 mm in a and b). Panel (c) shows a 63x magnification, 3D rendering of the microbiopsy tissue with a nuclear counter stain (orange) derived from a confocal microscopy z-stack of the sample within the microbiopsy device. The stratum corneum (SC), viable epidermis (VE), dermal-epidermal junction (DEJ) and superficial dermis (DER) are labeled. This microbiopsy contained an estimated 1634 nuclei. Haematoxylin and eosin stained section of human skin after microbiopsy application shows a 0.10 mm wide and 0.25 mm deep puncture Panel (d). * indicates the site of microbiopsy application.\n\nOne of our goals was to compare AK lesions to microbiopsy samples since many of these lesions can be present in patients but only a few progress12 to squamous cell carcinoma and the molecular mechanism behind this transition is a focus of intense research. Microbiopsy samples were taken from freshly excised AKs. The RNA from these matched samples had comparable quality with an average RIN difference of -0.85 ± 0.85 for n=4. Representative Bioanalyzer (Agilent Technologies, USA) results for the conventional biopsy (RIN 6.50) and microbiopsy (RIN 5.10) are shown in Figure 5, left panel. The results showed that there are similarities and differences in the RNA bands, which may be due to the large amount of tissue sampled with conventional biopsy and the relatively small number of cells sampled with the microbiopsy.\n\nThe left panel shows the Bioanalyzer readout from RNA isolated from a conventional actinic keratosis (AK) shave biopsy next to microbiopsy obtained RNA from the same AK lesion. The right panel compares the RNA quality of a shave biopsy and normal skin microbiopsy that were subjected to total transcriptome amplification to generate cDNA. The cDNA was then used as a template for a PCR reaction containing actin specific primers with an expected product at 800 bp.\n\nThe small sample size is an inherent limitation of the microbiopsy device. To mitigate this, a pilot experiment was conducted where the microbiopsy sample was subjected to whole transcriptome amplification for RNA analysis. We included a skin sample with the same amount of starting material (14 ng) as an amplification control in the experiment. We observed a 2000-fold increase in cDNA from both samples based on total RNA and cDNA measurements. The cDNAs were of comparable quality and quantity (Figure 5, Transcriptome Amplification to cDNA). Subsequently, we used PCR to amplify human beta-actin mRNA. We observed 2 identical bands at 800 bp with comparable PCR product quality both lanes (Figure 5, Actin PCR).\n\nImmediately after microbiopsy application we observed local erythema that resolved within 24 hours. The tiny excision site from the microbiopsy healed quickly and was invisible to the naked eye after 24 hours (Figure 6). We used dermoscopy and RCM to monitor the wound healing process at regular intervals (Figure 6, center and right columns).\n\nThe left column shows dermoscopic images of the microbiopsy site over time. The middle and right column are mosaics and at 30x magnification reflectance confocal microscopy (RCM) images, respectively, of the microbiopsy site.\n\n\nDiscussion and conclusion\n\nIn this proof of concept study, we have shown that an arrangement of stacked plates to form a 3D microbiopsy device is capable of providing molecular samples from normal and diseased skin. DNA extraction from human skin is critical for stratifying lesions in terms of mutational status. This type of characterization is becoming more and more relevant as targeted signalling inhibitors are being added to the therapeutic arsenal. Recent developments in molecular inhibitors for skin cancer, e.g. Vemurafenib13 and Trametinib14,15, are driving a new need for molecular diagnostic information that cannot be gathered through morphologic analysis. Molecular biomarkers have been used to detect cancer, but are now being developed to determine which molecular therapeutic will be the most effective. Melanoma clinical biology research has resulted in genomic data identifying oncogenes that can be targeted by protein kinase inhibitors e.g. BRAF and MEK16. Diagnostic assays to detect BRAF and NRAS mutations are now being used to stratify skin cancer lesions17 . The microbiopsy device has the potential to help screen multiple lesions for specific mutations, and to be important in discovery based research.\n\nYancovitz et al. describe intra- and inter-tumour heterogeneity of melanoma in the context of BRAFV600E mutations18. Hematoxylin and eosin stained sections of primary and metastatic lesions were subjected to laser capture microdissection to isolate the lesions of interest. They captured 30–300 cells for each lesion of interest. This is less than 5 times that captured with the microbiopsy device. The authors used the same DNA extraction kit as we did to isolate lesional DNA for analysis. These samples were then used to detect BRAFV600E tumour heterogeneity. Based on our analysis, we estimate that Yancovitz et al. isolated <1 ng of DNA in this study and were able to amplify BRAF exon 15 using conventional PCR followed by mutation analysis. We found that we can reproducibly isolate 5 ng of DNA from the superficial skin and in another publication show the use of the microbiopsy device in dysplastic nevi11. This work sets the stage for future microbiopsy studies focused on BRAF mutational analysis in nevi in situ. The addition of RNA analysis to mutational studies would give additional insights into gene expression profiles of these lesions.\n\nBerglund et al. (2007) reported that their optimised methodology yielded an average 1.4 ± 0.4 µg of consistently high quality RNA (8.4–8.9 RIN) from 3 mm skin punch biopsies19. Another group reported that they had isolated an average of 1.5 µg of RNA with an average RIN value of 8.1 from half of a 4 mm punch biopsy skin sample (n=97)20. The average total RNA yielded from the microbiopsy device is 9.0 ± 10.1 ng (n=5). Even though we isolated far less RNA with the microbiopsy device, our proof of concept study supports the hypothesis that this limitation can be addressed using commercial amplification kits and PCR (Figure 5) for investigating the molecular basis of skin disease.\n\nDiscovering non-melanoma skin cancer biomarkers and potential therapeutic targets is an emerging area of research. For example, Dang et al. (2006) focused on the genetic changes that occur in AK to squamous cell carcinoma (SCC) for prospective development of new diagnostic tools and therapeutic approaches. They reported that 7 out of 14 genes in 10 AK, 10 SCC and 20 normal skin samples related to cell adhesion, communication, metabolism and respiration were significantly dysregulated in AK and SCC (p < 0.05)21. The microbiopsy device has the potential to help facilitate longitudinal studies within a single lesion.\n\nApplication of the relatively small microbiopsy device does not require local anaesthesia or sutures and therefore no set up for a minor clinical procedure is necessary. Observations from our volunteer study suggest that collecting multiple microbiopsies within in a short period is feasible. We envision that microbiopsy devices will become a routine clinical and research device in dermatology allowing the dermatologist to obtain targeted lesion data for molecular stratification.\n\n\nConsent\n\nWritten informed consent for publication of their clinical images was obtained from the patients (HREC/11/QPAH/477 and/or HREC/12/QPAH/082, Metro South Human Research Ethics Committee, Centres for Health Research, Princess Alexandra Hospital).", "appendix": "Author contributions\n\n\n\nLLL, HPS and TWP contributed to the experimental design and preparation of the manuscript. LLL, APR, CAP, RLH and ABA carried out the research. APR contributed to the design of experiments. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project was supported by Epiderm (Boronia Park, NSW 2111, Australia) and NHMRC Practitioner Fellowship (APP1020148) (HPS).\n\n\nAcknowledgements\n\nWe thank Drs. Sudipta Sinnya and Jean-Marie Tan for patient recruitment and clinical assistance. We thank Dr. Roy Hall for providing the human actin primers and Dr. Yin Xiang Setoh for assisting in the PCR experiment. We thank Dr. Rick Sturm for his valuable input on the molecular characterization of this device.\n\n\nReferences\n\nSina B, Kao GF, Deng AC, et al.: Skin biopsy for inflammatory and common neoplastic skin diseases: optimum time, best location and preferred techniques. A critical review. J Cutan Pathol. 2009; 36(5): 505–510. PubMed Abstract \| Publisher Full Text\n\nPickett H: Shave and punch biopsy for skin lesions. Am Fam Physician. 2011; 84(9): 995–1002. PubMed Abstract\n\nUgurel S, Utikal J, Becker JC: Tumor biomarkers in melanoma. Cancer Control. 2009; 16(3): 219–224. PubMed Abstract\n\nShivers S, Jakub J, Pendas S, et al.: Molecular staging for patients with malignant melanoma. Expert Rev Anticancer Ther. 2007; 7(11): 1665–1674. PubMed Abstract \| Publisher Full Text\n\nGreinert R: Skin cancer: new markers for better prevention. Pathobiology. 2009; 76(2): 64–81. PubMed Abstract \| Publisher Full Text\n\nKrulevitch PA, Lee AP, Northrup MA, et al.: Microbiopsy/Precision cutting devices. In: Edited by Patent US. United States: The Regents of the University of California; 1999. Reference Source\n\nByun S, Lim JM, Paik SJ, et al.: Barbed micro-spikes for micro-scale biopsy. J Micromech Microeng. 2005; 15(6): 1279–1284. Publisher Full Text\n\nCho D, Park SK, Lee AR, et al.: Catheter capable of being equipped with micro biospy tool. In. Edited by Patent US; 2011. Reference Source\n\nCosnier ML, Martin F, Bouamrani A, et al.: A minimally invasive microdevice for molecular sampling and analysis. IEEE Trans Biomed Eng. 2009; 56(12): 2898–2904. PubMed Abstract \| Publisher Full Text\n\nPflueger DR: Micro-invasive breast biopsy device. In. Edited by Patent US; 2004. Reference Source\n\nBanan P, Lin LL, Lambie D, et al.: Effects of Ex Vivo Skin Microbiopsy on histopathological diagnosis in melanocytic skin lesions. JAMA Dermatol. 2013; 149(9): 1107–9. PubMed Abstract \| Publisher Full Text\n\nFuchs A, Marmur E: The kinetics of skin cancer: progression of actinic keratosis to squamous cell carcinoma. Dermatol Surg. 2007; 33(9): 1099–1101. PubMed Abstract \| Publisher Full Text\n\nChapman PB, Hauschild A, Robert C, et al.: Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011; 364(26): 2507–2516. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlaherty KT, Infante JR, Daud A, et al.: Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med. 2012; 367(18): 1694–1703. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlaherty KT, Robert C, Hersey P, et al.: Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med. 2012; 367(2): 107–114. PubMed Abstract \| Publisher Full Text\n\nRomano E, Schwartz GK, Chapman PB, et al.: Treatment implications of the emerging molecular classification system for melanoma. Lancet Oncol. 2011; 12(9): 913–922. PubMed Abstract \| Publisher Full Text\n\nJin SA, Chun SM, Choi YD, et al.: BRAF mutations and KIT aberrations and their clinicopathological correlation in 202 Korean melanomas. J Invest Dermatol. 2013; 133(2): 579–582. PubMed Abstract \| Publisher Full Text\n\nYancovitz M, Litterman A, Yoon J, et al.: Intra- and inter-tumor heterogeneity of BRAF(V600E) mutations in primary and metastatic melanoma. PLoS One. 2012; 7(1): e29336. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerglund SR, Schwietert CW, Jones AA, et al.: Optimized methodology for sequential extraction of RNA and protein from small human skin biopsies. J Invest Dermatol. 2007; 127(2): 349–353. PubMed Abstract \| Publisher Full Text\n\nBruning O, Rodenburg W, Radonic T, et al.: RNA isolation for transcriptomics of human and mouse small skin biopsies. BMC Res Notes. 2011; 4: 438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDang C, Gottschling M, Manning K, et al.: Identification of dysregulated genes in cutaneous squamous cell carcinoma. Oncol Rep. 2006; 16(3): 513–519. PubMed Abstract\n\nFitzpatrick TB: The validity and practicality of sun-reactive skin types I through VI. Arch Dermatol. 1988; 124(6): 869–871. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1009", "date": "19 Jun 2013", "name": "Jack L. Arbiser", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, article content, conclusions and data are all highly appropriate. I am particularly impressed with the ability to obtain intact DNA and RNA from this technique. Given the almost total lack of scarring, I predict that this method will be highly used and this paper will be highly cited in the field. I look forward to utilizing it in my own practice. The ease of wound healing makes it particularly attractive", "responses": [] }, { "id": "1011", "date": "19 Jun 2013", "name": "Marco Ardigò", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, innovative and well conducted study about the use of microinvasive (“quasi” on invasive) tissue sampling for confocal-molecular correlation performed with the new method of the sub-millimeter punch. Title and abstract are appropriate.Conclusions are justified on the basis of results of the study.In conclusion, the study really increases the chances of routine applications of micro-punches for tissue sampling for targeting molecular analysis in skin diseases.", "responses": [] }, { "id": "1017", "date": "21 Jun 2013", "name": "Neil Rajan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, describing the development and characterisation of a device to take small biopsies from skin lesions. Attractively, it appears to allow for sampling of tissue with minimal discomfort. The extracted sample can be processed through DNA and RNA extraction protocols. The authors describe how this device may influence future changes in molecular diagnostics in skin tumours. The paper is well written. There are a few minor revisions that should be made for it to be acceptable for indexing. •\n\nTitle and Abstract: The word painless in the second line should be removed as this is not substantiated by the data provided. The volunteers expressed that pain was experienced at the time of the biopsy, indicated in the score in Table 1. •\n\nArticle content:Improved wound healing at high risk sitesPage 3 of 11 – “Certain areas such as the lower leg may have problems with wound healing and so biopsies are avoided. The microbiopsy device fills a void in dermatology where conventional biopsies are not feasible or could do more harm than good.” – This needs to be substantiated by data showing that the device resulted in fewer lower leg complications for this statement to stand. Otherwise it should be removed.Comparison to 4mm punch biopsiesThe disadvantages of the punch biopsy approach (anaethesia, suturing) are highlighted. Whilst the thrust of the article is the extraction of tissue for molecular analysis at a nucleic acid level, for a balanced view, it should be pointed out that punch biopsies allow for histological assessment of the tissue sampled, whilst the microbiopsy device does not. It may also be worth considering future studies where pain scores are assessed in patients who volunteer to have both a punch biopsy and a microbiopsy undertaken, to compare the difference between the 2 approaches.RNA qualityIt has to be acknowledged that whilst the RNA extracted was amplified to cDNA, the starting RNA integrity number of the sample published (5.5) would not be considered suitable for whole transcriptome approaches, such as RNA –seq and microarray profiling, where ideally a RIN of >8 is desirable. DNA qualityIt would be good to see an indication of the DNA quality and integrity from tissue extracted with this device, using either a bioanalyzer approach or an agarose gel.•\n\nConclusions: The conclusions are sensible, balanced and justified on the basis of the results of the study.", "responses": [] } ]	1	https://f1000research.com/articles/2-120
https://f1000research.com/articles/2-5/v1	10 Jan 13	{ "type": "Research Article", "title": "De novo genetic variation revealed in somatic sectors of single Arabidopsis plants", "authors": [ "Marianne T Hopkins", "Aaron M Khalid", "Pei-Chun Chang", "Karen C Vanderhoek", "Dulcie Lai", "Meghan D Doerr", "Susan J Lolle", "Marianne T Hopkins", "Aaron M Khalid", "Pei-Chun Chang", "Karen C Vanderhoek", "Dulcie Lai", "Meghan D Doerr" ], "abstract": "Concern over the tremendous loss of genetic diversity among many of our most important crops has prompted major efforts to preserve seed stocks derived from cultivated species and their wild relatives. Arabidopsis thaliana propagates mainly by self-fertilizing, and therefore, like many crop plants, theoretically has a limited potential for producing genetically diverse offspring. Despite this, inbreeding has persisted in Arabidopsis for over a million years suggesting that some underlying adaptive mechanism buffers the deleterious consequences of this reproductive strategy. Using presence-absence molecular markers we demonstrate that single Arabidopsis plants can have multiple genotypes. Sequence analyses reveal single nucleotide changes, loss of sequences and, surprisingly, acquisition of unique genomic insertions. Estimates based on quantitative analyses suggest that these genetically discordant sectors are very small but can have a complex genetic makeup. In ruling out more trivial explanations for these data, our findings raise the possibility that intrinsic drivers of genetic variation are responsible for the targeted sequence changes we detect. Given the evolutionary advantage afforded to populations with greater genetic diversity, we hypothesize that organisms that primarily self-fertilize or propagate clonally counteract the genetic cost of such reproductive strategies by leveraging a cryptic reserve of extra-genomic information.", "keywords": [ "Arabidopsis", "genome instability", "genetic heterogeneity", "mosaicism", "inbreeding" ], "content": "Introduction\n\nPlants live in ever changing environments and must adapt using strategies that fundamentally differ from those employed by animals. Developmental plasticity is at the core of those strategies allowing plants to modify their growth and the organs they produce in response to different environmental signals. This type of open-ended modular development enhances survival because damaged or diseased units can readily be discarded without compromising viability. Furthermore, because plants are constrained to sessile life styles, a modular growth habit affords greater versatility allowing phenotypic and genetic variation between modules to be used to the plant’s advantage, aiding adaption to pathogen life cycles1 or to longer-term environmental perturbations such as climate change. As a consequence of this profound developmental versatility, even individuals composed of cell populations derived from different plant species are viable and can coordinate the growth and development of chimeric organs2. In an elegant paper published in 1981, Whitham and Slobodchikoff3 proposed that mosaicism offers a unique adaptive advantage for plants by allowing introduction of genetic variants into the gene pool either through vegetative propagation or through sexual reproduction. They further propose that mutations arising somatically have a greater probability of being incorporated into the gene pool than mutations that arise in the gametes3 precisely because germ line cells are derived from somatic tissues that arise late in the developmental history of the plant4,5.\n\nThe relatively frequent occurrence of mosaics among various plant species has been extensively utilized in the development of novel ornamentals and for the selection and maintenance of desirable traits in many cultivated crops. Any desirable cultivars that have arisen in this manner have been maintained through vegetative propagation and, to date, are responsible for a significant fraction of agriculturally important perennial plants. On the other hand, desirable traits in many important annual crops, such as rice, soybean, maize and wheat, have been introduced through classical genetic manipulations using directed breeding strategies. Once generated, annuals with good agronomic performance are usually maintained by inbreeding.\n\nIn recent years, concern has grown over the presumed loss of genetic diversity resulting from the application of modern horticultural and breeding practices. Therefore, the benefit of excellent performance may come with a significant cost6,7. However, recent and surprising results suggest that even highly inbred species harbor unanticipated sources of intrinsic genetic variation. For example, highly inbred soybean cultivars have been shown to manifest significant phenotypic and genetic variation in the absence of sexual manipulation8–10. Such high intrinsic genetic variation has also been demonstrated for a number of other crop plants11.\n\nIn the natural world, inbreeding occurs in many highly successful flowering plant species including wild relatives of Arabidopsis thaliana12. Therefore, in nature species that are highly inbred have persisted despite their predicted reduction in genetic diversity. Why would such inbreeding strategies be successful and what are the implications from an adaptive perspective? One possibility put forward by Barrett13 is that such populations are very successful in their particular niches and benefit from producing large numbers of genetically identical offspring. Nevertheless, selection should favor plant species that can co-evolve on time scales reflecting particular environmental challenges such as fluctuations and variations in pathogen populations. In keeping with this view, it has been shown that sequence variation in 20 diverse strains of Arabidopsis is highly non-random. In gene families mediating biotic interactions, such as those implicated in pathogen defense, variation far exceeds that seen in families involved in basic biological processes14.\n\nThe underlying mechanisms driving phenotypic variation in highly inbred lines, whether domesticated or wild, have often been inferred and have had limited experimental verification. Nevertheless, relatively simple molecular approaches have provided insight into some of the genomic events coinciding with visible changes in phenotype. In flax, for example, molecular assays have demonstrated that heritable phenotypic changes induced by environmental shifts are accompanied by reproducible changes in genomic DNA including changes in total DNA content, non-random changes in DNA sequences or sequence rearrangements15–18. In soybean, reproducible non-random DNA sequence changes induced by in vitro culturing of root explants have also been demonstrated using restriction fragment length polymorphic markers19. Genomic changes manifesting similar hallmarks of biased sequence alterations have also been described for banana20 and in rice hybrids21.\n\nIn the work described by Roth et al.19 soybean root explants were shown to repeatedly give rise to particular alleles that were absent in the donor plants but had previously been found and characterized in other varieties of cultivated soybean. To account for the appearance of these particular allelic variants the authors proposed that these organisms had evolved \"internal generators of genetic variation\" that mediated genome changes through some type of recombination process. In 2005, Lolle and colleagues22 described a genome-wide phenomenon in Arabidopsis hothead (hth) mutants that was very reminiscent of that described by Roth et al.19. Based on the nature and genome-wide locations of the sequence changes detected, it was proposed that a template-directed process mediated these changes and that these cryptic but stable extra-genomic templates themselves had persisted since at least the grandparental generation. Not surprisingly, this proposal met with considerable skepticism and numerous alternative explanations for these data have since been published23–28.\n\nIn this study we have employed presence-absence molecular markers to test for non-Mendelian inheritance and found that Arabidopsis plants can inherit novel insertion sequences that were absent in their immediate parents. Furthermore, we show that discordant DNA-based marker profiles can be found between tissues isolated from different parts of an individual plant. These experiments demonstrate that individual plants spontaneously produce somatic sectors and are genetic mosaics. Since genetic variation can occur in the same plant in the absence of sexual reproduction, we propose that these novel insertion sequences must originate from cryptic reserves intrinsic to the host plant itself. The data presented support the original contention that a previously unknown template-directed mechanism exists22 and raise the encouraging possibility that other inbreeding species, including crop plants, may also harbor a cryptic reserve of genetic variation.\n\n\nMethods\n\nAll genetic stocks of Arabidopsis thaliana used for these experiments have been described previously29. Arabidopsis seeds derived from these stocks were sown onto moistened potting mix (1:1 mixture of LC1:LG3 Sungro Sunshine potting mixes, Sungro Horticulture, Seba Beach, AB) and vernalized at 4°C for 2–5 days. Plants were maintained in growth chambers (Econoair AC60, Ecological Chambers Inc., Winnipeg, MB; GC8-VH/GCB-B, Environmental Growth Chambers, Chagrin Falls, Ohio; Conviron PGW36/E15, Controlled Environments Ltd., Winnipeg, MB) and illuminated with a mixture of incandescent and fluorescent lights (140–170μmol m-2 sec-1 at pot level) with a 24-hour photoperiod. Growth chambers were maintained at 20 ± 4°C at 40–60% relative humidity. Plants were grown in flats or in 3- or 6-inch pots and watered as needed. Seeds used for seedling root-shoot comparison were surface sterilized using bleach and plated on agar medium containing half strength MS basal salts (Sigma, St. Louis, USA). Seedlings were harvested approximately 5 days post-germination. Hybrid lines were generated between Landsberg and Columbia accessions by manual pollination and all crosses were done reciprocally. F2 seed was obtained from self-fertilized F1 plants. Individual F2 plants were reared in plastic tubes (Johnston Industrial Plastics, Ontario, Canada) and F3 seed collected from each F2 plant individually. Tissue samples were collected from individual F2 and F3 plants, and genotypic profiles were determined using insertion-deletion polymorphic molecular markers (see Figure 1).\n\nNine of the markers are intergenic (*). Marker names reflect clone designations. The size of the insertion sequence is indicated in base pairs (bp). The relative location of HOTHEAD (HTH) is shown at the bottom of chromosome 1.\n\nExperimental set ups were replicated twice and the net out-crossing frequencies determined. Herbicide-resistant transgenic Arabidopsis pollen donors previously transformed with the pCB302 mini binary vector only30 and mutant test plants were grown in a 1:1 ratio and arranged in randomized positions (www.random.org). Out-crossing frequencies were also compared to plants under the same conditions but reared within plastic tubes. Progeny were sprayed with glufosinate (40 micrograms ml-1 active ingredient: WipeOut, Nu-Gro IP Inc., Ontario) to test for herbicide resistance and resistant plants tested for segregation of hth mutant progeny plants.\n\nFor DNA extraction, rosette or cauline leaf tissue was collected and DNA extracted according to the method of Edwards et al.31. Samples not processed immediately were stored at -20°C. Sixteen sets of DNA oligonucleotide primers were designed to amplify approximately 150–300bp genomic regions by polymerase chain reactions (PCR), each containing one 45–94bp marker which is present in the Columbia but absent in the Landsberg accession (Table 1). PCR amplicon products were size separated by agarose gel electrophoresis.\n\nExpected amplicon product sizes for the Columbia and Landsberg accessions are shown in adjacent columns.\n\nPortions of genomic DNA were PCR amplified and sequenced directly or products cloned into standard pGEM TA vectors (Promega). Amplified or cloned PCR products were sequenced at the Centre for Applied Genomics (http://www.tcag.ca/, Toronto, Ontario). Sequence alignments were generated using CLC Sequence Viewer 6.4 software (CLC bioA/S; www.clcbio.com).\n\nQuantitative PCR was performed on a Bio-Rad Real-Time thermal cycler CFX96 attached to a computer running CFX Manager. SsoFast EvaGreen Supermix (Bio-Rad) was used according to manufacturer’s instructions. A series of primers either flanking or internal to the insertion sequences were used to generate control and experimental amplicons. The positive control was a PCR product amplified from the Columbia accession, spanning the indel sequence of interest by ~700–900bp. The positive control was gel purified and used to generate a standard curve for conversion of C(t) value to copy number of the insertion sequence and the external reference sequence. External reference primers immediately flanked the indel markers. Insertion sequences were detected using one external reference primer paired with a primer homologous to sequences within the insertion itself. Primer sequences and amplicon product sizes are listed in Table 2. The colors indicated in the first column (insertion-deletion marker) correspond to the colors used for the qPCR-generated bar graphs.\n\nPrimer positions, left and right primer sequences and expected amplicon sizes are indicated for each marker. Colors correspond to those used in Figure 5B and Figure 6B.\n\n\nResults\n\nHomozygous hth mutant Arabidopsis plants were previously shown to give rise to wild type (wt) progeny at relatively high frequencies22,29. Although an intrinsic mechanism was proposed22, cross-pollination with neighboring plants was subsequently put forward as the more likely explanation for the appearance of these wt revertant offspring26,27. To test the susceptibility of hth plants to out-crossing under our growth conditions, experiments were conducted using a pollen donor harboring a dominant gene conferring resistance to the herbicide glufosinate. Herbicide-resistant transgenic lines were grown together with hth and eceriferum-10 (cer-10)32 floral fusion mutants and wt Landsberg plants. These analyses confirmed that the majority of hth mutant plants did not cross-pollinate. However, when cross-pollination was detected, frequencies varied considerably between individual hth mutant plants. Mutants with floral fusion phenotypes were predisposed to higher pollen capture than wild type plants (0.02–0.43% for hth-4, 8 and 10 mutants, 0.89% for cer10 mutants, 0.01% for wt plants). In addition, factors such as donor-recipient proximity, the severity of the floral fusion phenotype, growth chamber airflow patterns and plant handling influenced the propensity to cross-pollinate. Nevertheless, growing hth mutant F2 plants in the complete absence of HTH pollen donors did not eliminate wt progeny from F3 progeny pools and, on average, 1.53% of F3 progeny were phenotypically wt for HTH despite being derived from self-fertilized homozygous F2 hth mutant parent plants (2/133 hth-4, 2/131 hth-8 and 2/127 hth-10 gave rise to wt F3 progeny). Under our laboratory conditions, out-crossing could not be completely eliminated within hth mutant populations if mutants were grown together with wt plants, even if every hth mutant plant was shielded in transparent plastic tubes.\n\nWhile conducting segregation analyses and scoring offspring for herbicide resistance, a single hth mutant plant with a large phenotypically wt floral sector was identified (Figure 2). Sampling of shoot tissues confirmed that phenotype corresponded to genotype and that both mutant hth-4 and wt HTH alleles could be detected in tissue derived from this large wt sector (Figure 2B).\n\n(A) Two mutant branches (white boxes) flank a phenotypically wt flower branch (magenta box). Examples of normal wt (HTH/HTH) and mutant (hth/hth) flowers are shown on the right. (B) DNA was extracted from tissue samples and allele-specific PCR-based molecular markers used to determine genotype. The wt branch scored as heterozygous (hth-4/HTH-4), while mutant branches scored as homozygous for the hth-4 allele.\n\nThe identification of this sectored individual provided the first phenotypic evidence that hth plants were capable of producing somatic sectors. This finding suggested that perhaps some of the wt revertants originally found among hth mutant progeny might have arisen from genetically heterozygous sectors on the parent plant22. Since well over 300,000 mutant plants were screened in the course of our out-crossing experiments and only one plant with a very large phenotypically wt sector found such as that shown in Figure 2B, we reasoned that if sectoring does occur, the vast majority of sectors would be too small to result in a visible phenotype. This possibility prompted us to test whether novel genotypes could be detected in tissue samples obtained from single hth plants.\n\nFor these experiments we chose to focus exclusively on molecular markers consisting of genomic DNA sequence tracts between 45–94 nucleotides in length that are either present or absent in the Columbia and Landsberg Arabidopsis accessions (insertion-deletion polymorphic markers or indels; Figure 1). In choosing to use indel markers we reasoned that deletions would be recalcitrant to enzyme repair or modification and therefore would help differentiate between enzyme-based mechanisms such as the one put forth by Comai and Cartwright24 and a template-directed mechanism like the one previously proposed22. Hybrid lines were constructed between Columbia and Landsberg accessions and descendants used as experimental material. For all of the indel markers used in this study, Columbia is homozygous for the insertion.\n\nInitially, F3 seed progeny derived from hybrid F2 parent lines with known indel marker profiles were screened to test whether or not these markers were stable. All F2 parent plants were reared in plastic tubes. When marker profiles were compared between hth-4 parent plants and their F3 adult offspring, 2.16% [6/277] deviated from the expected profile. This frequency is approximately 5 times higher than baseline rates (0.02–0.43%) seen in outcrossing experiments described above. When F3 progeny were assayed as seedlings, similar frequencies were seen, with 2.5% [15/600] of the F3 seedlings showing discordant marker profiles. Altogether 600 seedlings were tested using a total of 30 seedlings per F2 plant (eleven hth-4, five hth-7, two hth-8 and two hth-10 F2 plants). Of the 15 F3 seedlings that tested positive for at least one non-parental marker, 7 had acquired insertions.\n\nTo test whether the observed genetic discordance between parent and offspring was due to sectoring, multiple tissue samples were collected from individual adult plants and indel marker profiles compared between these different samples. Molecular analyses confirmed that some tissue samples taken from individual hth mutant plants had novel marker profiles. For the plant shown in Figure 3A, seven out of eight samples scored as expected and were homozygous for the Landsberg deletion marker, however, one sample produced two amplicon products, one of which co-migrated with the Landsberg deletion allele and a larger amplicon that co-migrated with Columbia insertion allele.\n\n(A) DNA was extracted from multiple tissue samples and PCR-amplified using F8D20 primers. A novel PCR amplicon product corresponding in size to the insertion allele (C) was detected in hth-7 tissue sample 3 (arrow). (B) Sterile seeds were sown onto petri plates (top left) and 5-day old seedlings cut at the root-shoot junction (illustrated in the top right panel) and genotyped individually. DNA extracted from shoot (S) and root (R) samples derived from individual hth-3 or wt seedlings were PCR-amplified using F12K11 and F4C21 primers, respectively. Samples were loaded in pairs (indicated by horizontal bars). Novel amplicon bands were detected in five seedling samples (arrows) that correspond in size to the insertion allele (C). In one hth-3 sample, both organs (S, R) had a novel band, while a novel amplicon was detected only in the root in a second sample. In three cases, DNA extracted from wt seedlings gave rise to novel bands corresponding in size to the insertion allele (C) (arrows, S). In both cases, the parent plant was homozygous for the deletion allele (L) at the corresponding marker. Heterozygote (H), no DNA control sample (ND).\n\nTo test whether sectors could be detected earlier in development, the molecular genotype of shoots and roots of single seedlings grown under sterile conditions were compared to one another. On the assumption that wild type plants would not produce sectors, identical tests were also conducted on wt hybrid lines as negative controls. In the majority of cases, as expected, there was a perfect correspondence between the molecular profiles of root and shoot. However, in some cases, individual seedlings were found to have molecular signatures that differed between the two organ systems (10/44 hth-3; 1/50 hth-4; 9/76 hth-7; Figure 3B). Surprisingly, wt hybrid seedlings also showed novel genotypes when roots and shoots from the same seedling were compared (10/184 wt hybrids; Figure 3B).\n\nA subset of amplicon samples were subjected to DNA sequence analyses in order to determine their molecular features. Sequence analyses of DNA clones derived from individuals where the non-parental amplicon co-migrated with the smaller deletion allele showed identity with the Landsberg deletion marker (Figure 4). In two instances, polymorphisms immediately upstream of the deletion were also detected (Figure 4A). As indicated, the Landsberg accession differs from Columbia at these exact three nucleotides. DNA sequence analysis of novel amplicons that co-migrated with the larger insertion allele showed that this seedling shoot had acquired a 54-nucleotide insertion that shares identity with the Columbia reference genome (Figure 4B). This same insertion was absent in the F2 parent plant. These particular seedlings descended from the same wt hybrid parent plant as the F3 progeny whose profiles are shown in Figure 3B.\n\n(A) The F2 hth-3 parent (F2 hth) shares sequence identity with 2 of 3 DNA clones isolated from this single hth-3 seedling (F3 R2 and F3 S1). DNA sequence data obtained from a root clone (F3 R1) shares identity with the Landsberg sequence (Ler), including 3 flanking sequence polymorphisms (arrows) and a corresponding 85 base-pair deletion. The Columbia reference sequence (Col) is shown on the top line of the alignment. (B) The HTH wt hybrid parent (F2 wt) shares sequence identity with 2 of 3 DNA clones isolated from this single seedling (F3 S2 and F3 R1). DNA sequence data obtained from one shoot clone (F3 S1), however, reveals a 54 base-pair insertion sequence (junctions shown by arrows) and shares identity with the Columbia reference sequence (Col).\n\nTo obtain an estimate of sector size, tissue samples were subjected to quantitative assays where the copy number of a genomic reference sequence immediately flanking the marker of interest was compared to the copy number of a sequence internal to that particular insertion marker (Figure 5 and Figure 6). Hybrid plants verified to be homozygous for a deletion at specific indel markers were subjected to the quantitative assays. The quantitative polymerase chain reaction (qPCR) data reveal two remarkable findings. First, the majority of tissue samples collected from individual hth mutant plants tested positive for the presence of at least one insertion marker (Figure 5). In addition, multiple insertion sequences could be detected in many of the tissue samples tested (Figure 5B). In most instances the copy number of any given insertion sequence, relative to the reference, was very low (less than one copy per 1000). Second, wt hybrid plants also showed evidence of sectors with novel genotypes (Figure 6). Only two out of four wt plants tested, however, showed evidence of novel insertions.\n\n(A) DNA was extracted from branches 1–7 of this hth-7 mutant plant and amplified using qPCR or standard PCR reactions. (B) Graphical representation of qPCR results using four different indel markers (F6D8 (red), F15H11 (yellow), T14G11 (blue), and T6H20 (green)). Colored bars show the number of insertion sequences per 1000 copies of the reference sequence (lines indicate standard error of the mean, n=3). All 7 samples showed novel insertion sequences. (C) Standard PCR-amplification using T6H20 primers showed amplicons that corresponded exclusively to the deletion allele (L). Primer positions (arrows) relative to the T6H20 indel (green box) are depicted to the right of the gel image. (D) Pooled amplicon product from T6H20 reference primers demonstrate that this region was amplified equally in all samples, as was the positive control (+). The reference sequence is upstream of the T6H20 insertion marker, as depicted on the right. (E) Quantitative PCR using a primer anchored within the T6H20 indel gave rise to amplicons that corresponded in size to the positive control (+). No product was amplified from sample six. T6H20 indel (green box), Columbia (C), Landsberg (L), heterozygote (H), no DNA control sample (ND).\n\n\n\n\n\n\n\n\n\n\n\n(A) DNA was extracted from branches 1–5 of two wt hybrid plants (9B and 10B) and amplified using qPCR. (B) Graphical representation of qPCR results using three different indel markers ((F15H11 (yellow), T14G11 (blue), and MGI19 (pink)). Colored bars show the number of insertion sequences per 1000 copies of the reference sequence (lines indicate standard error of the mean, n=3). Novel insertion sequences could be detected in all 10 samples.\n\n\n\n\n\n\n\n\nDiscussion\n\nBy employing classical genetic approaches in conjunction with low and high-resolution molecular methods, we show that one Arabidopsis plant can have multiple genotypes. We have found instances of intra-organismal variation in different genetic backgrounds, in plants reared in different growth chambers, at different developmental stages and under sterile growth conditions. Furthermore, the incidence of sectoring and genetic discordance appears to be in some way conditioned by the hth mutant background as we found a consistently higher frequency of genetic discordance within single hth plants as compared to HTH wt plants. This was also true for shoot and root systems compared between aseptically grown seedlings and for tissue samples taken from adult plants and subjected to qPCR. Of critical importance, in showing that single Arabidopsis plants are genetic mosaics, experimental error due to cross-pollination and seed contamination can be completely discounted. To the best of our knowledge, this is the first report that documents the spontaneous but targeted appearance of unique genomic insertions at multiple discreet loci in single plants.\n\nOnly two other cases of spontaneous genomic insertions have been reported in plants that similarly could not be explained by any previously known mechanism. In both cases the insertion was non-random and targeted a specific locus. In the case of flax, the insertion sequence was 5.7 kilobase pairs in size16 while in rice the insertion was comparatively small, being only 34 base pairs in size33. Our data suggest that these reported cases of spontaneous genomic insertion events, like the sequence changes reported here, occur by a process intrinsic to the plant. As before, we propose the possibility that Arabidopsis plants harbor a cryptic store of sequence templates that can overwrite the parentally contributed genomes by a template-directed mechanism22.\n\nIf intrinsic drivers of genetic variation exist in inbreeding plant species, have additional incidents of cryptic genetic variation been documented in other systems? We believe that in soybean and cauliflower such events have indeed been reported and presented as cases of enigmatic phenotypic variation8,9,34. In other studies, molecular data have been featured. Again in flax, for example, molecular assays have demonstrated that heritable phenotypic changes induced by environmental shifts are accompanied by reproducible locus-specific copy number changes in genomic DNA16–18. In soybean, reproducible non-random changes in restriction length polymorphic markers induced by in vitro35,36 culturing of root explants have also been documented19. Genomic changes manifesting similar hallmarks of biased sequence alterations have also been described in rice21,33 and corn37 hybrids, as well as in Arabidopsis38–40.\n\nIn long-lived arborescent plants, intra-organism genetic variation has been demonstrated in a variety of systems3,36,41. The fitness benefits have also been validated using models that test whether the production of genetically divergent modules is an effective strategy for achieving adaptive co-evolution with organisms that feed on or infect the plant35,42,43. Models testing fitness benefits of module-level selection show that this is an effective strategy for achieving adaptive co-evolution between long-lived trees and short-lived herbivores when individual tree branches diverge genetically35. Furthermore, this held true across a range of assumptions, even when reproduction was predominantly asexual. However, the fitness benefits were only fully realized for sufficiently long-lived trees that experienced strong selection35. This fitness paradox is not exclusive to plants but also is relevant to organisms outside of the plant kingdom that have remained evolutionarily robust even though reproduction is predominantly asexual43.\n\nFor a short-lived organism such as Arabidopsis, what adaptive value would within-organism genetic variation have? One possibility is that this heterogeneity offsets the predicted decline in genetic variation that should result from inbreeding. Plant development is open-ended and reiterative, allowing for the continuous output of repetitive units or modules that function to support the growth and reproduction of the individual. When combined with developmental plasticity and the absence of a sequestered germ line, modular development may actually drive plants toward becoming genetically heterogeneous41,43–45. As posited by Whitham and Slobodchikoff3, somatic sector formation permits the introduction of genetic variants into the gene pool either through vegetative propagation or through sexual reproduction. As these authors point out, germ line cells are derived from somatic tissues that arise late in the developmental history of the plant and therefore somatic mutations are more likely to introduce genetic variation than mutations that arise in the gametes3,4,46. By expanding the window of tolerance for genetic variation, plants may be afforded a better adaptive strategy given lifestyle constraints. The versatility of modular development combined with tolerance for genetic variation may allow plants to adapt at rates tailored to pathogen life cycles1 or to relatively expanded time scales, such as those affecting climate change. Even though self-fertilization is thought to have evolved approximately one million years ago12, Arabidopsis plants have not suffered the consequential genetic erosion but have continued to thrive.\n\nIn addition to benefiting from a natural tendency toward genetic heterogeneity, the plant genome itself is thought to buffer the cost of having limited genetic diversity. In wild relatives of Arabidopsis the genome is thought to be highly dynamic and to respond to changes in environmental conditions or other extrinsic factors42,47. Genome responses include elevated rates of homologous recombination that persist for multiple generations48, changes in copy number49 and modulation of epigenetic gene regulation50. Pervasive genetic buffering46,51 ensures that phenotypes with potentially deleterious consequences are attenuated. In addition to the genome responses listed above, our findings suggest that an intrinsic source of genetic variation can be leveraged to enhance the diversity in genetic output achieved by Arabidopsis plants.\n\nIn considering alternate template-dependent mechanisms, such as gene conversion or homologous recombination, none can account for the de novo appearance of unique sequence insertions. Nevertheless, it is possible that the insertion or deletion of small DNA sequence tracts, as described here, could reflect the activity of transposable elements52,53. However, numerous lines of evidence argue against this possibility. For instance, when novel amplicons were detected, they co-migrated with their corresponding insertion or deletion allele and did not show size heterogeneity, as would have been expected for transposon-driven excision or insertion events. Sequence data confirm that deletion events reproducibly eliminate a fixed length of sequence while insertion events reproducibly introduce a fixed sequence tract and both events repeatedly target precise genomic sites. Insertion and deletion events do not appear to produce obvious junction sites with altered nucleotides. Similarly, insertion events introduce sequences that share identity with the Columbia reference genome and do not appear to be chimeric gene or genome fragments. Furthermore, transposable element-mediated events cannot account for the fact that these insertion sequences appear to be generated de novo since no comparable conserved region of homology exists elsewhere in the host genome, as demonstrated by our qPCR data. Lastly, as determined by DNA database searches, none of the indel markers used in this study share significant sequence homology with annotated Arabidopsis transposable elements.\n\nIf the genome of an intensely studied model organism such as Arabidopsis is subject to modification by the template-directed mechanism we propose, why has this phenomenon not been described previously? Our research shows that target choice and methodological approach are critical in differentiating these genomic events from other processes that also modify DNA sequences. Based on our findings, the only genomic targets that are truly diagnostic of this phenomenon are deletions. To the best of our knowledge, deletion alleles have been used in genetic studies precisely because they are known to be stable and not to revert but have not been used to study phenomena related to epigenetic inheritance. There is no generalized precedent for genetic instability of deletions and assuming otherwise would go against an established biological paradigm. Polymorphic molecular markers such as single nucleotides, simple sequence repeats, or insertions that are subject to alterations by other processes will not provide sufficient resolution to differentiate mechanism, even though they are also likely targets for this process. In particular, our findings may explain why genome sequencing efforts have failed to register these sequence deviations or, if detected, why they may have been attributed to sequencing error and eliminated during curation. One possibility that immediately emerges from this prediction is that raw sequence data contained in existing genome database archives may already contain evidence of extra-genomic sequence information, revealed by features such as highly biased loci-specific \"errors\".\n\nCollectively, our genetic and molecular data show that many, and perhaps most, insertion events occur somatically in both seedlings and adult plants. Sectoring may therefore be a constitutive process that takes place throughout development but may be limited such that, at any given time, only a few cells host these genetic changes. Importantly, this may explain why sequence changes seen in revertant hth progeny have rarely been found to affect both alleles. Although sexual transmission of non-parental markers clearly does occur22, the fact that we have not found HTH/HTH progeny among seed-derived offspring suggests that sectors populating the gamete forming lineages are unstable or very rare. The qPCR data are consistent with this supposition. However, it is also possible that mechanistic differences exist between somatic and germ line tissues or that insertion events remain dynamic, limiting sexually transmitted changes to those that stabilize. It is also possible that certain genetic backgrounds condition this process as suggested by the greater number of events detected in hth mutants.\n\nIn addition to validating our genetic and molecular data, the qPCR results extend those findings and suggest that the genetic make up of individuals can be surprisingly complex. Our data show that each plant can produce multiple discreet sectors, at many different growing points and each with unique marker profiles. This finding implies that sectoring may be a relatively common occurrence, even in wt genetic backgrounds. Since the adult plants used for these experiments were left largely intact and only a small proportion of the plant sampled, many more sectors may have been present than quantified. As such, it is possible that our current census underestimates the frequency with which these smaller islands of genetic variation arise. Although sectors are more readily detected using qPCR, this method cannot distinguish, for example, between copy number variation within a small cluster of cells versus multiple cells that remain strictly diploid and are clonally related. Similarly, it is not possible to distinguish whether one sector hosts the full complement of genetic sequence changes, whether independent events occur in multiple discreet sectors, or if sectors overlap. Visualization of sectors in living tissue or tissue sections should help distinguish between these possibilities.\n\nIn addition to models demonstrating the fitness benefits of module-level selection35, computational models provide surprisingly strong support for an ancestrally based \"error-correcting\" mechanism such as the one we propose to exist in Arabidopsis plants54. In these constrained-optimization simulations, the evolutionary benefit of \"genetic repair\" strategies was compared between populations that access repair templates derived either from parents, grandparents or great-grandparents. Interestingly, a grandparent- or great grandparent-based genetic repair strategy is strongly favored over parental repair strategies. Furthermore, simulation results show that using a randomly selected template consistently gave superior results to those achieved using templates from the fittest parent or grandparent. From a biological perspective, such a strategy has considerable merit. Retaining a cache of templates derived from grandparental lineages would guarantee greater allele diversity precisely because the reservoir of allele variants would be deeper and allele redundancy would be less likely to occur. Random selection of templates would be the most parsimonious strategy to affect genome repair, again because it would promote diversity across alleles and between individuals. Since only those individuals that survived in previous generations would contribute to these cached templates, represented alleles would be biased to those that have proven robust under a spectrum of selective pressures.\n\nIn summary, the research presented here brings to light five striking findings. First, individual Arabidopsis plants are capable of producing somatic sectors during the course of normal vegetative development. Second, those sectors can have distinct and unique marker profiles and can differ in single nucleotide composition, can acquire small DNA insertions or can experience DNA sequence loss. Third, the de novo appearance of genomic insertions supports our original contention that cryptic sequence templates drive some of these changes22. Fourth, this phenomenon can be detected in wt genetic backgrounds raising the possibility that many Arabidopsis lab strains may be genetic mosaics. Finally, this process is genome-wide, impacting all 5 chromosomes, whether or not the target loci reside within genes or between genes.\n\nOur data expand on the ideas put forth by Whitham and Slobodchikoff3 and suggest that sector formation, even in a short-lived organism like Arabidopsis, may be a normal part of development and, furthermore, that the formation of sectors serves to capture novel genetic variation, irrespective of the source of that variation. Models testing the benefit of within organism genetic heterogeneity suggest that the average fitness of the population increases if some individuals within that population are genetic mosaics35. As our data show, not all individuals in the populations we tested showed evidence of genetically distinct sectors but for those individuals that did, the number of sectors varied greatly. Our findings raise the possibility that inbreeding plants and, perhaps other organisms that predominantly propagate asexually, may sequester cryptic sources of genetic variation that can be harnessed to promote greater genetic diversity.", "appendix": "Author contributions\n\n\n\nSJL conceived the study. MTH, AMK, PCC KCV, DL, MDD and SJL carried out the research. MTH, AMK, PCC and SJL contributed to the design of the experiments. SJL prepared the manuscript. All authors were involved in the revision of the 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\nSJL gratefully acknowledges funding from the Natural Sciences and Engineering Research Council of Canada (NSERC: RGPIN-341446) and the University of Waterloo (UW). MTH, PCC, and DL each were supported by NSERC Fellowships. AMK was supported by an Ontario Graduate Scholarship.\n\n\nAcknowledgements\n\nWe thank K. Cuddington, H. Engelhardt, D. Enstone, S. Goggi, B. Moffatt, D. O’Donoghue, R. Palmer, D. Rose and J. Witt for insightful discussions and for sharing their perspectives and expertise. We thank L. Hoyles and J. Waite for helpful discussions and technical assistance. 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Publisher Full Text" }	[ { "id": "729", "date": "30 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\nThis paper describes the occurrence of somatic sectors in hth mutant genotypes, with the frequency of rare visible sectors at a specific locus being 1/300,000. However, if there is a selective advantage during development, such sectors might be higher and could also produce clonal selected events and easily be identified by PCR.The data provide adequate support against contamination by outcrossing as a possible explanation of the results.Minor comment: should change the word 'vernalized' to 'stratified' in the Methods section, sentence beginning \"Arabidopsis seeds derived from these stocks...\"", "responses": [] }, { "id": "745", "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\nThere is nothing 'wrong' with the paper, although this research is still quite controversial. I would still like to see the mRNA and DNA from hth individual plants to be sequenced and compared. Also, if genetic recombination mechanism is in play and if mRNA needs to be converted to cDNA for integration, one could show that some of the mutants involved in recombination or in reverse transcription would be impaired in the process of acquisition of new alleles.All the experiments were done correctly, but as I said the story is far from completion.", "responses": [] }, { "id": "746", "date": "31 Jan 2013", "name": "David Oppenheimer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 current manuscript, Lolle and colleagues show that plants can spontaneously produce mosaic sectors, and they show that insertion sequences can arise de-novo. Importantly, growing the hth plants in the absence of pollen donors did not eliminate the appearance of HTH plants in the next generation. It is hard to come up with a model for how one could get 1.5% wt plants unless the researchers were covered in wt pollen while tending to the isolated hth plants. It would be useful for the authors to remind the reader that the hth mutants are in the Ler background.The authors should state the degree to which they tried to eliminate PCR contamination (setting up reactions in UV-sterilized hoods, positive displacement pipets, etc.).Some important questions/comments:What are the allele-specific molecular markers used to genotype the plant shown in Figure 1B? Is the marker used one of the Col/Ler indel markers? What tissues do the individual lanes represent (individual flowers, stem tissue, etc.)? On Page 8, the authors reared the plants in plastic cones, which they conceded does not eliminate outcrossing. Although the frequency of deviation from the expected profile is significantly higher than that due to outcrossing, it would have been better to grow the plants in isolation. This will remain an important criticism, as outcrossing frequencies rely on many variables, and it is possible (albeit difficult) to completely eliminate the possibility of outcrossing.On page 8, the authors should clarify the experimental details to make it easy for the readers to follow. The authors should state (if I am following it correctly) that: homozygous Ler hth mutants were crossed with Col wt plants to generate the F1 plants. F1 plants were grown and allowed to set seed. The F2 plants were screened for hth mutants which were saved for F3 analysis. All the markers listed in Figure 2 were used to genotype the individual F2 plants. The individual F2 plants chosen for further study showed the homozygous Ler indel pattern. F3 seedlings were analyzed to address whether or not the marker patterns were stable.The key results in Figure 3 cannot be explained by outcrossing, period. Page 8, Change \"Markers are discordance…\" to \"Markers are discordant…\"Page 9, ‘targeted appearance of unique genomic insertions’: it might be better to say ‘re-appearance of the allele from a previous generation’.General:Since HTH has not been cloned yet, this adds an additional layer of complexity to the story. Nonetheless, molecular data were completely absent when transposable elements were discovered, so the identity of HTH is not crucial to the story of de novo genomic changes other than to further understand the nature of the reversion events.The key aspects of the Peng and Mercier papers were that neither lab was able to find revertants when the plants were grown in isolation. These authors then concluded that there was no non-mendelian mechanism at work, and therefore the rest of the arguments were moot. However, the inability to reproduce the phenomenon does not mean that the phenomenon does not exist; it only means that the Peng and Mercier labs were not able to reproduce the appropriate growth conditions to see the effect. This is not unexpected given that an uncharacterized stress response may be at play here leading to the reversion phenotype.In my own work with particular Arabidopsis mutants that are mostly sterile, we do see revertant sectors, albeit rarely. With one particular mutant, a single inflorescence showed many full seed pods even though the other inflorescences on the same plant showed completely empty pods. Clearly this is not seed or pollen contamination. We have yet to follow up on this rare observation, mainly because it is hard to study rare phenomena.", "responses": [] } ]	1	https://f1000research.com/articles/2-5
https://f1000research.com/articles/2-164/v1	30 Jul 13	{ "type": "Short Research Article", "title": "The nature and prevalence of chronic pain in homeless persons: an observational study", "authors": [ "Rebecca Fisher", "Judith Ewing", "Alice Garrett", "E Katherine Harrison", "Kimberly KT Lwin", "Daniel W Wheeler", "Rebecca Fisher", "Judith Ewing", "Alice Garrett", "E Katherine Harrison", "Kimberly KT Lwin" ], "abstract": "Background: Homeless people are known to suffer disproportionately with health problems that reduce physical functioning and quality of life, and shorten life expectancy. They suffer from a wide range of diseases that are known to be painful, but little information is available about the nature and prevalence of chronic pain in this vulnerable group. This study aimed to estimate the prevalence of chronic pain among homeless people, and to examine its location, effect on activities of daily living, and relationship with alcohol and drugs.Methods: We conducted face-to-face interviews with users of homeless shelters in four major cities in the United Kingdom, in the winters of 2009-11. Participants completed the Brief Pain Inventory, Short Form McGill Pain questionnaire, Leeds Assessment of Neuropathic Symptoms and Signs, and detailed their intake of prescribed and unprescribed medications and alcohol. We also recorded each participant’s reasons for homelessness, and whether they slept rough or in shelters.Findings: Of 168 shelter users approached, 150 (89.3%) participated: 93 participants (63%) reported experiencing pain lasting longer than three months; the mean duration of pain experienced was 82.2 months. The lower limbs were most frequently affected. Opioids appeared to afford a degree of analgesia for some, but whilst many reported symptoms suggestive of neuropathic pain, very few were taking anti-neuropathic drugs.Interpretation: The prevalence of chronic pain in the homeless appears to be substantially higher than the general population, is poorly controlled, and adversely affects general activity, walking and sleeping. It is hard to discern whether chronic pain is a cause or effect of homelessness, or both. Pain is a symptom, but in this challenging group it might not always be possible to treat the underlying cause. Exploring the diagnosis and treatment of neuropathic pain may offer a means of improving the quality of these vulnerable people’s lives.", "keywords": [ "Homeless", "chronic pain" ], "content": "Introduction\n\nHomeless people suffer disproportionately from health problems, to such an extent that the life expectancy of a person sleeping on the streets is 42–52 years1–4. Their lifestyle predisposes them to various potentially painful and unpleasant complaints (including dental caries, trench foot, infectious diseases, peripheral neuropathy and depression), but such conditions often go untreated due to multiple barriers to care5–8. Inadequate living conditions, frequent exposure to the elements, and violence, substance misuse, and poor nutrition combine with inadequate access to healthcare to exacerbate health problems4,9–11.\n\nIn England, the Housing Act 1996 defines a person as being homeless if there is no accommodation that they are entitled to occupy; or they have accommodation but it is not reasonable for them to continue to occupy it12. Although the United Kingdom (UK) government has reported an overall annual decline in homelessness as defined by this legislation since 2004, the most recent data show that this trend has reversed. In the second quarter of 2012, local authorities in England accepted 12,960 applicants as being homeless, and Rough Sleeping England estimated that 1,768 people were ‘sleeping rough’ on the streets13,14. As government figures do not include people who satisfy the legal definition of homelessness but who have not applied to be classified as such, it is likely that true incidence of homelessness is substantially higher. Crisis, a charity representing the interests of single homeless people in the UK, estimates that there are around 400,000 ‘hidden homeless’ in England, Wales and Scotland3.\n\nLarge epidemiological studies in Australia, Europe and Scotland, using a definition of chronic pain as ‘pain that persists beyond normal tissue healing time, which is assumed to be three months’15 have found a prevalence ranging from 17% to 50% in the general population16–18. Given the increased prevalence of health problems in homeless people4–8, we hypothesised that they may also experience more pain than the general population.\n\nTo date there have been no studies primarily addressing the prevalence of pain in homeless persons, although in a study from Canada, almost 52% of randomly selected homeless individuals fulfilled the criteria for chronic pain19. Pain has featured as a secondary topic in studies of oral health, end of life care, and the measurement of pain in the context of homelessness5,20,21. Pain is a symptom, and, although it would be preferable to treat the underlying causes, barriers to care and the presence of potentially irreversible disease mean that it is important to gain insights into the quantity and character of pain that homeless people experience, its impact on function, and the strategies they use to manage it. This study addresses pain as a primary focus, as better characterisation of pain may pave the way for targeted intervention.\n\nWe used standard questionnaires to estimate the prevalence, duration and nature of pain in homeless people, and examine its character, location, severity, impact on activities of daily living, and the strategies used for pain relief.\n\n\nMethods\n\nWe conducted a pilot questionnaire survey of users at two homeless shelters in Cambridge, UK, over 3 months in late 2009 and early 2010. Five of the authors acted as interviewers (RFRF, JCE, AG, EKH and KKTL), having received training in the use of the Brief Pain Inventory (BPI) and short form McGill (SF-MPQ) questionnaires from DWW22–25. Introductions to shelter users were facilitated by shelter staff when required. Written consent was obtained from all participants.\n\nParticipants in our pilot study completed the BPI and the SF-MPQ questionnaires face-to-face during a single interview. Interviews followed a standardised format, with questions read aloud to avoid issues with literacy. Participants reporting pain for more than 3 months were asked to rate its average and its greatest intensities during the past 24 hours on an 11-point numeric rating scale (NRS 0–10, where 0 represents no pain and 10 equates to the worst pain imaginable), to mark primary pain location on whole-body diagrams, and provide a list of their current prescribed and unprescribed medications. Each participant was asked to detail their alcohol use in the past 24 hours (converted into number of units), and their recreational drug use (by drug used and amount).\n\nHaving collected and analysed these pilot data, we extended the study to include more centres, recruiting homeless adults from five shelters in Oxford, Belfast, and London. We also recruited from day centres that aim to help homeless people ‘sleeping rough’ on the streets. In total, eight institutions were approached, but one declined to participate as they dealt mostly with homeless adolescents in whom they did not consider pain to be a problem. Participants from these additional centres were asked further questions concerning the main reason for their homelessness if possible, which was categorised into drug or alcohol misuse, mental health problems, family or relationship breakdown, financial difficulties, and leaving prison or the armed forces. These participants were also asked their duration of pain, how long they had been homeless, whether they predominantly slept rough on the streets, or under a roof in night shelters or hostels, for example.\n\nAs our pilot data suggested a high prevalence of neuropathic pain, these participants were also asked to complete the Leeds Assessment of Neuropathic Symptoms and Signs questionnaire and examination (LANSS)26, to improve the precision with we could detect the prevalence of neuropathic pain27.\n\nUsing the BPI, short form McGill and LANSS questionnaires together provides a comprehensive analysis of an individual’s pain that we thought would be easy to use and appropriate for the homeless population; cover current treatments for pain and their perceived efficacy rated on a percentage scale of pain relief and assess the extent to which pain interferes with daily functions28.\n\nAll shelter users present at that shelter on the day of interviewing were offered the chance to participate. Those that declined to participate or who were unable to give informed consent due to language difficulties or intoxication were excluded from the study. Double enrolment was avoided as participants’ names and dates of birth were known to interviewers. No incentive was offered for participation.\n\nIt was calculated that 150 participants would need to be chosen to provide a 95% confidence interval of ±8% for the prevalence of chronic pain. Data were entered into a spreadsheet and statistical analysis performed with GraphPad Prism version 4.0b (GraphPad Software, CA). Continuous data are expressed as the mean, with range and 95% confidence intervals (95% CI) where appropriate and non-continuous data are expressed as the median with the interquartile range (IQR). Groups were compared using non-parametric statistical tests: the Komolgorov-Smirnov test was used to examine data for normal distribution; where data were not distributed normally, the Mann-Whitney test was used. Relationships between continuous data were examined using linear regression, the gradient of the best-fit slope with 95% CI and r2 values are reported. Statistical significance was indicated by a p value of less than 0.05.\n\nThe study was approved by the University of Cambridge Psychology research ethics committee (approval references 2009.73 and 2011.41).\n\n\nResults\n\nOne hundred and sixty-eight homeless people were invited to participate in the study: 150 (89.3%) completed the interviews, but 14 declined to participate, two were too intoxicated and two did not speak English. Of those completing the study, 134 (89.3%) were male and 16 (10.7%) were female; their mean age was 42.2 years (range 23–73 years, 95% CI 40.1–44.5 years).\n\nFifty-one participants (34.0%) reported sleeping rough on the streets, 95 (63.3%) used night shelters, and four (2.7%) were sleeping with friends or in squats. The mean duration of homelessness was 58.5 months (range 0.1–384.0 months, 95% CI 50.4–66.5 months).\n\nThe most common cause of homelessness in the 111 participants who were asked was breakdown of family relationships, cited by 44 (39.6%) participants. Further causes were: financial difficulties (29.6%); drug or alcohol misuse (17.4%); leaving prison or the armed services (11.9%); and health problems (10.9%). Twelve participants (8%) reported multiple causes of homelessness; hence the sum of percentages exceeds 100%.\n\nOf the 150 shelter users interviewed, 107 reported experiencing pain in the past 24 hours (71.3%, 95% CI 67.5%–74.8%). The reported mean duration of pain experienced was 78.3 months (range 0.1–480.0 months, 95% CI 66.4–90.2); 89 of 107 (83.2%) had experienced pain for more than three months suggesting an overall prevalence of 59.3% (95% CI 55.1%–63.0%). The worst, least and average pain intensities experienced are summarised in Table 1.\n\nThe lower limbs were the most common site of pain, with 51.4% of participants reporting pain in this area (Figure 1). Further areas affected were: abdomen, pelvis or back (36.9%); chest, arms and shoulders (25.2%); and head or neck (15.3%). Thirty-one participants (27.9%) reported more than one affected area; hence the sum of percentages exceeds 100%.\n\nGeneral activity was the domain most affected by pain, with the majority (85.6%) of individuals with pain reporting that this aspect of their life was affected, the mean extent of this interference being 5.6 on an NRS of 0–10 (Table 2). Relationships appeared to be the activity least affected by pain, with just over half (54.1%) of participants stating that their pain had an impact on relationships, and an average NRS score of 3.5. There was no statistical difference between the average pain experienced by those who slept outdoors and those who slept under a roof (p = 0.114).\n\nFifty-nine of the 107 participants reporting chronic pain (55.1%) were taking over-the-counter and prescribed analgesics. Paracetamol (acetaminophen) was most popular, followed by non-steroidal anti-inflammatory drugs and opioids such as codeine, dihydrocodeine and morphine. Polypharmacy was common: 25 of the 59 participants (42.4%) taking over-the-counter and prescribed analgesics took more than one agent. None of the participants who reported no pain took over-the-counter or prescribed analgesics. Only four participants reported taking anti-neuropathic drugs: two were taking gabapentin, one dosulepin, and one amitriptyline. Other therapies were also used: three participants had access to physiotherapy; and a handful used complementary techniques such as acupuncture, osteopathy, and aromatherapy.\n\nThirty-three participants (22.1%) admitted to using illegal drugs such as heroin, methadone, crack cocaine, cannabis, and non-prescribed diazepam; one participant declined to answer these questions. One participant reporting severe pain used cannabis for analgesia, and two who reported no pain said they did so because it was adequately treated by illegal methadone, heroin or diazepam. For those taking opioid analgesia, the mean reduction in pain intensity with their chosen regime was 46.3% (range 0%–100%, 95% CI 38.9%–53.8%). The average pain reported by those taking opioids, whether prescribed or unprescribed, was significantly lower than those who did not (p < 0.0001, Figure 2).\n\nScatter plot to show the severity of average pain reported by homeless people who either take prescribed opioids, non-prescribed opioids, or cannabis; or those who take over the counter non-opioid analgesics or no drugs. Bars indicate means. Data compared using Mann-Whitney test.\n\nThe majority (59.3%) of the 150 participants had not consumed alcohol in the previous 24 hours. Of the 61 (40.7%) who did, the mean number of units consumed was 19.1 (range 1.0–94.0, 95% CI 17.3–21.1). One participant declined to answer questions about alcohol. There was no apparent relationship between alcohol consumption and pain intensity: the number of units consumed by those reporting pain in the previous 24 hours was not significantly different (mean consumption 9.0 units versus 12.0 units, p = 0.149), nor was there a relationship between the quantity of alcohol consumed and pain experienced (Figure 3).\n\nA) Scatter plot showing the amount of alcohol consumed by homeless people reporting versus those who did not. Bars indicate means. B) Linear regression plot showing lack of relationship between quantity of alcohol consumed and intensity of pain reported. The unbroken line represents the best-fit slope and the broken lines the 95% confidence intervals (best-fit slope 0.579, 95% CI -0.06–2.24, r2=0.034).\n\nOf the 107 participants who reported pain, 90 completed the SF-MPQ questionnaire but only 59 completed the LANSS, as fewer participants wished to undergo the limited physical examination required completing this questionnaire. The median sensory pain score of SF-MPQ was 10 (IQR 5–16), the median affective pain score was 4 (IQR 0–7), and the median total combined component score was 14 (IQR 7–23). Table 3 shows a comparison of our data with that reported in clinical trials that used the SF-MPQ to assess the analgesic effect of anti-convulsant drugs in neuropathic pain29–31. Data shown from these studies are the mean scores of baseline data from the patients with neuropathic pain in the placebo arm of the trials. For ease of comparison this table contains our mean data, although arguably SF-MPQ is not a continuous scale. Fewer participants completed the LANSS scale: the median score was 6 (IQR 1–12); ten of those who did (16.1%) reported a score of 12 or more, highly suggestive of neuropathic pain26. Therefore the LANSS data suggest a prevalence of neuropathic pain of 16.9%, and the SF-MPQ suggest that it is of a greater intensity than volunteers diagnosed with neuropathic pain entering clinical trials.\n\n\nDiscussion\n\nPain is a substantial problem in homeless shelter users: 71.3% reported acute pain, and 59.3% fulfilled the criteria for chronic pain, the mean duration of which exceeded 6 years. The prevalence of chronic pain in our participants is substantially higher than that reported in several large population studies16–18. It also appears that a substantial component of the pain experienced is neuropathic in nature: 16.9% of participants who completed the LANSS scored 12 or more, which compares unfavourably with 8% in the general population32.\n\nIt is difficult to ascertain whether pain is a potential cause or effect of homelessness in these individuals. Many factors associated with homelessness predispose to pain, and homeless people experience numerous barriers to health care, such as psychiatric illness, substance misuse, and the lack of a fixed address for correspondence33,34.\n\nLower limb pain was the most common location for pain in our study population. This is likely to be a result of lifestyle factors, such as ill-fitting shoes, long periods of standing, poor foot care, or sensory neuropathy. Many respondents commented that a lack of accessible facilities during the day resulted in a great deal of time spent on their feet. Abdominal pain was also common, which could result from opioid-induced constipation or alcohol-related diseases such as chronic pancreatitis, gastritis and peptic ulceration. The extent of the pain that shelter users experience exerts a considerable impact on their daily activities, with general activity and walking most affected. The severity of the pain reported by those with neuropathic symptoms is comparable to volunteers diagnosed with neuropathic pain enrolled into the placebo arms of three recent large trials of anti-neuropathic drugs29–31. The pain is also poorly controlled: just over half of those with chronic pain were using analgesia but the mean reduction in pain was only 46.3%, which compares unfavourably with the primary outcome measure of many trials of analgesic drugs, namely a 50% reduction in pain on a visual analogue scale35. Inadequate pain control may result from reluctance to seek help with pain or investigation of underlying disease, for example due to a lack of awareness of help available, psychiatric illness, or low expectations of health professionals. Concerning treatment, physicians may be reluctant to prescribe opioids to individuals with a history of substance misuse, and non-steroidal anti-inflammatory drugs for those for those with a history of alcohol misuse19.\n\nOur findings corroborate previous studies that found a high prevalence of alcohol and substance misuse amongst homeless people, although it should be stressed that the majority of homeless people do not misuse alcohol or take illegal drugs4,19. We found no association between pain and alcohol use, but cannot exclude the possibilities that alcohol is being used to self-medicate, or that pain might arise as the result of alcohol-associated disease. Those using opioids, either prescribed, bought over the counter or obtained illegally, reported lower average pain intensity than those who did not. Many participants admitted obtaining opioids illegally specifically and solely for the purpose of pain relief. A small number also used illegally obtained cannabis and benzodiazepines for analgesia.\n\nOur study has strengths and limitations. The time that our interviewers spent in the shelters and with homeless people meant they built up a rapport with a large number of homeless people, which is reflected in the high response rate. We found our participants pleasant, engaging and coherent; most defied our preconceived ideas about homeless people and did not fulfil the usual stereotypes. All interviewers were medical students, a particular advantage as participants reported speaking more freely and truthfully than perhaps they would with doctors. Previously, difficulties have been reported when measuring pain in the context of homelessness, perhaps due to participants’ lack of literacy skills21. We chose questionnaires that are widely accepted as being valid in adults and read them through with our participants. We did not experience substantial problems with vague answers or engagement, and do not believe that introducing and validating new pain questionnaires specifically for homeless people is worthwhile as long as this paradigm is used. We reduced bias as far as possible by including a range of shelters and rough sleeping outreach services in our capital and provincial cities. Rough sleepers are some of the most difficult homeless people to engage, but a large proportion of our participants regularly slept outdoors. Therefore, we believe that our study population is as representative of the homeless population as practicable, and our findings could be generalised to other developed countries.\n\nMost of the limitations of our study result from our desire not to overburden our participants with too many questionnaires and potentially intrusive personal questions. For example, we did not perform diagnostic interviews to collect data on psychiatric and physical disorders. The pain questionnaires used are independent of diagnosis and pathophysiology; they serve merely to quantify the amount of pain that a person is experiencing. This somewhat limits our ability to comment on causation of pain, and as some potential participants were intoxicated the possibility of inaccurate self-reporting of drug and alcohol use must also be considered. Although we collected high quality data from a relatively large number of homeless people, our study did not have sufficient power to detect differences between men and women, different age groups, or those with different social or economic reasons for homelessness.\n\nExisting literature about pain among homeless persons is very limited, and a formal prevalence rate has not previously been reported. We found that almost two thirds of homeless people that we interviewed experienced chronic pain. Meeting the medical needs of homeless people is extremely difficult, so treating the underlying cause of the pain might not be possible. Our findings should raise awareness amongst healthcare professionals that chronic pain is common in homeless people and is generally inadequately controlled, and therefore requires thorough investigation and treatment. The drugs that our participants took to alleviate pain, either prescribed or unprescribed, afforded pain relief of less than 50%. Further studies are needed to establish the causes of pain in homeless persons, but it is notable that a large proportion reported symptoms and signs suggestive of chronic neuropathic pain. The observation that very few took anti-neuropathic drugs suggests that there may be opportunities to treat pain in this challenging but vulnerable population more effectively.", "appendix": "Author contributions\n\n\n\nRFRF, JCE, KKTL, AG, EKH and DWW were all involved with study conception and design, analysis and interpretation of data, drafting the article and revising it critically for important intellectual content, and final approval of the version to be published. DWW did not collect data. DWW is the guarantor.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe study was funded by the University Division of Anaesthesia and the School of Clinical Medicine at the University of Cambridge, UK. The sponsors played no role in 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\nAcknowledgements\n\nWe are grateful for the assistance of staff and guests at shelters for homeless people in Cambridge, Oxford, Belfast, and London, although to ensure the confidentiality of our participants we cannot identify them.\n\n\nReferences\n\nWright JD: Poor people, poor health: the health status of the homeless. J Soc Issues. 1990; 46(4): 49–64. Publisher Full Text\n\nNordentoft M, Wandall-Holm N: 10 year follow up study of mortality among users of hostels for homeless people in Copenhagen. BMJ. 2003; 327(7406): 81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrisis: Homelessness policy watch: homelessness kills.2008. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "1780", "date": "04 Oct 2013", "name": "Benjamin Henwood", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written and well conceived study that focuses on an important topic: the nature and prevalence of pain among individuals experiencing homelessness. This is an obvious and overlooked area of research that represents an important target of clinical intervention. The methods are clear, appropriate, and appear well executed. The findings confirm what one might expect: there are high rates of chronic pain among the homeless. They also add insight into the type of pain individuals experience including high rates of neuropathic pain. The notion that these individuals use drugs to self-medicate the effects of pain seems only partially supported, suggesting that pain is an important consideration, but only one of many in terms of understanding substance use in homeless populations. High rates of the use of acetaminophen raise questions about potential liver damage and toxicity, and it would be worth considering homeless individuals' knowledge on this topic, which is perhaps a point that could be raised in the discussion. One final suggestion is to consider whether and how providing housing would help address or alleviate pain among this population.", "responses": [] }, { "id": "2090", "date": "25 Oct 2013", "name": "Lara Carson Weinstein", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 researched article on an important and little studied topic. I have a few comments for the authors to consider:I would find it helpful to include a brief description of the questionnaires used in the study, perhaps with a comment on their reliability and validity.In the discussion about lower limb pain you may want to mention trauma as a likely source in this population.The relatively low percentage of women in the sample limits the generalisability to women.", "responses": [ { "c_id": "597", "date": "28 Oct 2013", "name": "Rebecca Fisher", "role": "Author Response", "response": "The questionnaires used in the study are well validated and were chosen by the study team in part for ease of use with our study population, and also to provide comprehensive coverage of both neuropathic and non-neuropathic pain. There is little work on how to best measure pain in the context of homelessness (see reference 21, Matter et al), and we felt a combination of these questionnaires would allow a comprehensive assessment of pain, that was not overly burdensome to participants. References 22-28 provide further information on the questionnaires used in this study.We agree that trauma could have been added as a cause of lower limb pain in this population. Indeed several participants anecdotally mentioned abdominal stab wounds as contributing to their pain, and this could have been discussed further.We agree that the low percentage of women in the study limits its generalisability. In our study the small number of female participants reflected the overwhelmingly male occupancy of the night shelters at which participants were recruited. It would be interesting to expand the study to shelters catering only for women." } ] } ]	1	https://f1000research.com/articles/2-164
https://f1000research.com/articles/2-142/v1	19 Jun 13	{ "type": "Research Article", "title": "Identification and molecular characterization of a Chlamydomonas reinhardtii mutant that shows a light intensity dependent progressive chlorophyll deficiency", "authors": [ "Phillip B Grovenstein", "Darryel A Wilson", "Kathryn D Lankford", "Kelsey A Gaston", "Surangi Perera", "Mautusi Mitra", "Phillip B Grovenstein", "Darryel A Wilson", "Kathryn D Lankford", "Kelsey A Gaston", "Surangi Perera" ], "abstract": "The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms. These tetrapyrroles are synthesized via a common branched pathway that involves mainly nuclear encoded enzymes. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4) protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.", "keywords": [ "Chlamydomonas reinhardtii", "algae", "Chlorophyll", "Gun4", "6F14", "light sensitivity" ], "content": "Introduction\n\nChlamydomonas reinhardtii is a green micro-alga that can grow heterotrophically in the dark by metabolizing exogenous acetate. It possesses a photosynthetic apparatus very similar to higher plants, has a short and simple haplontic life cycle, can synthesize Chl both light dependently and light independently (unlike most angiosperms) and its genome has been sequenced1. In addition, well developed molecular tools exist for genetic manipulations of its genome. All these traits make this alga an elegant system for dissecting photosynthesis and chloroplast biogenesis2,3.\n\nChl, heme, siroheme, cobalamin, heme d1 and factor F430 are major tetrapyrroles that are involved in wide variety of essential life processes in all living organisms. Chl and heme are synthesized via a common branched pathway4,5 (outlined in Figure 1). Photosynthetic eukaryotes synthesize 5-aminolevulinic acid (ALA) from glutamine (Glu) bound to tRNAGlu through the C5 pathway consisting of two steps catalyzed by glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase4,5. ALA is subsequently converted in six steps to PPIX, the last common precursor for both Chl and heme biosynthesis4,5. Insertion of Fe2+ into PPIX by ferrochelatase (FeChel) leads to heme. Insertion of Mg2+ in PPIX by the heterotrimeric MgChel (comprised of three subunits: CHLD, CHLH and CHLI6) leads to MgPPIX, the first biosynthetic intermediate in the Chl branch6. MgPPIX is converted to Pchlide via three enzymatic steps. The reduction of Pchlide to form chlorophyllide (Chlide) can occur by two different mechanisms. One mechanism is catalyzed by the strictly light dependent enzyme NADPH:Pchlide oxidoreductase (LPOR) and occurs in all photosynthetic organisms; it is the only mechanism of Chl formation in angiosperms7–10. The second mechanism is catalyzed by the light independent NADPH:Pchlide oxidoreductase (LiPOR) and is present in anoxygenic bacteria, alga, ferns and gymnosperms11–20. The Chlide a undergoes a phytylation reaction, catalyzed by Chl synthase (CS), resulting in the formation of Chl a. In vascular plants and green algae a portion of the Chlide a is converted to Chlide b by Chlide a oxygenase (CAO) prior to phytylation21–24. Chl a is converted to Chl b by CAO via formation of 7-hydroxymethyl chlorophyll a (HCA) and Chl b can be converted back to Chl a via HCA by chlorophyll b reductase (CBR) and 7-hydroxymethyl chlorophyll a reductase (HCAR)25. This inter-conversion of Chl a and Chl b, referred to as the “chlorophyll cycle”, plays an important role in greening, acclimation to light and senescence25.\n\nLight regulated steps are in red. Dashed arrows denote multiple enzymatic steps and green arrows point to steps that are positively regulated by the GUN4 protein, respectively. Tetrapyrrole intermediates and enzymes are shown in black and bold black type, respectively. Readers are advised to look in the text for full names of tetrapyrrole intermediates and enzymes, which are abbreviated in this figure.\n\nStringent control of tetrapyrrole biosynthesis is especially essential for oxygenic photosynthetic organisms that are often prone to oxidative stress. Free Chl, heme and their immediate precursors are highly photo-toxic molecules and generate reactive oxygen species (ROS) under aerobic conditions26. Hence most of the cellular Chls are usually bound to the light harvesting complex (LHC) and other photosystem (PS) proteins. Chl is made in the plastid. Most of these Chl binding proteins and enzymes of the tetrapyrroles biosynthetic pathways are encoded by the nuclear genes5. Hence a tight coordination of biosynthesis of Chl with its apoprotein is necessary27. Chl and heme biosynthesis in plants is under transcriptional, translational and post-translational control at multi level and is accomplished by a complex regulatory network among the chloroplasts, mitochondria and nucleus, that is not well understood28–30.\n\nOne of the major research interests of our laboratory is to identify components that play a role in the regulation of Chl biosynthesis under different irradiance conditions. We have generated a random DNA insertional Chlamydomonas mutant library and have screened it to isolate twenty one mutants that are either defective in Chl biosynthesis and/or are incapable of photo-autotrophic growth under different irradiance conditions. One of the isolated mutants (6F14) is a light sensitive mutant which shows a light intensity dependent progressive photo-bleaching and is incapable of photosynthesis under low light intensities (90–100 µmol m-2 s-1). Molecular analyses revealed that 6F14 is defective in the GUN4 (genome uncoupled 4) gene which codes for a protein that stimulates MgChel activity. 6F14 is the second gun4 mutant to be identified in Chlamydomonas31. Transformation of 6F14 with a functional copy of the GUN4 gene restored the wild type phenotype. Western analyses show that the two isolated gun4 complements are over-expressing the GUN4 protein. Chl analyses show that these gun4 complements have 50–60% more Chl than that of the wild type strain. In this study, we present our molecular data on the identification of the mutation locus in 6F14 and its complementation.\n\n\nMaterials and methods\n\nChlamydomonas strains 4A+ (a gift from Dr. Krishna Niyogi (UC, Berkeley), gun4 and gun4 complements (both generated by our laboratory) were grown either in Tris-Acetate Phosphate (TAP) heterotrophic media or in Sueoka’s High Salt (HS) photo-autotrophic media. TAP and HS liquid media and agar plates were prepared in the lab using reagents from Fisher Scientific (Pittsburgh, PA) according to the protocol given in Gorman and Levine (1965)32 and Sueoka (1960)33, respectively. The 4A+ strain and gun4 complements were maintained on TAP agar plates and TAP + zeocin (Sigma, St. Louis, MO) plates, respectively under dim light intensities (10–15 µmol photons m-2 s-1) at 25°C. The final zeocin concentration was 15 µg/ml. The gun4 mutant (6F14) was maintained in the dim light or in the dark on TAP 1.5% agar plates containing 10 µg/ml of paromomycin (Sigma, St. Louis, MO). Liquid algal cultures used for RNA and genomic DNA extractions and protein analyses were grown in 100 ml flasks on the New Brunswick Scientific Excella E5 platform shaker (Enfield, CT) in TAP media at 150 rpm in the dim light.\n\nThe purified pBC1plasmid from the DH5α Escherichia coli-pBC1 clone (obtained from Dr. Krishna Niyogi’s laboratory at UC, Berkeley) was used for random DNA insertional mutagenesis. This plasmid contains two antibiotic resistance genes: APHVIII and AmpR (Figure 2). APHVIII confers resistance against the antibiotic paromomycin and was used as a selection marker for screening of Chlamydomonas transformants. AmpR was used as a selection marker for screening of E. coli clones harboring the pBC1 plasmid. E. coli was grown in 1 l of Luria Bertani (LB) broth containing 1% tryptone, 0.5% of yeast extract, 1% NaCl and ampicillin (final concentration of ampicillin:100 µg/ml). LB media was prepared in the laboratory using reagents purchased from Fisher (Pittsburgh, PA). Ampicillin was purchased from Fisher (Pittsburgh, PA). The culture was incubated at 37°C overnight. Plasmid purification from E. coli cells was facilitated by a Qiagen plasmid mega kit according to the protocol given in the technical manual (Qiagen, Valencia, CA). Once purified from E. coli, the circular pBC1 vector was linearized with the restriction enzyme KpnI (NEB, Beverly, MA) according to the protocol given in the technical manual. The linearized DNA was purified using a QIAEX II gel extraction kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. All agarose DNA gel electrophoresis was visualized by BioRad Molecular Imager Gel Doc XR+ (BioRad, Hercules, CA). Transformation of parental strain 4A+ by the linearized pBC1 vector was performed utilizing the glass bead transformation technique described by Kindle et al. (1989)34 and Dent et al. (2005)2. Transformants were plated onto fresh TAP agar plates containing 10 µg/ml paromomycin (TAP+P) in the dark. Single colonies of mutants were picked and transferred onto fresh TAP+P plates using a numbered grid layout. Screening of photosynthetic and pigment deficient mutants was done by visual inspection and monitoring of growth under different light intensities in heterotrophic, mixotrophic and photo-autotrophic conditions2.\n\nThe cleavage site of Kpn1 restriction enzyme, used for linearization of the vector is shown. APHVIII is under the control of combo promoters which consist of the promoter of the gene encoding the small subunit of Rubisco (RbcS2) and the gene encoding the heat shock protein 70A (Hsp70A). pBC1 is a phagemid and its F1 origin (F1 ori) and pUC origin (pUC ori) are shown. The size of the plasmid is 4763 bp.\n\n4A+, gun4 complements and gun4 were grown in TAP liquid media in the dim light to a cell density of about 5 × 106 cells/ml of the culture. Genomic DNA was purified using a phenol-chloroform extraction method35. RNA extraction was facilitated by TRIzol reagent from Invitrogen (Carlsbad, CA) following the protocol in the technical manual. DNA and RNA concentrations were measured using a Nanodrop 1000 spectrophotometer from Thermo Fisher Scientific (Wilmington, DE). DNase treatment was performed using Ambion’s TURBO DNA-free kit from Invitrogen (Carlsbad, CA) following the protocol in the technical manual to remove genomic DNA from the RNA preparation. Generation of cDNA was performed using Life Technologies Superscript III First-Strand Synthesis System from Invitrogen (Carlsbad, CA) following the protocol in the technical manual.\n\nTAIL (Thermal Asymmetric InterLaced) PCR was implemented, following the protocol of Dent et al. (2005)2. HotStar Taq Plus DNA polymerase kit reagents (Qiagen, Valencia, CA) were used for PCR. The PCR reaction mixture consisted of 1 × PCR buffer, 200 µM of each dNTP, 1 × Q-solution, 2.5 units of HotStar Taq Plus DNA polymerase, 60 pmoles of the random degenerate primer RD1 and 5 pmol of the APHVIII specific primer. Primers were ordered from IDT (Skokie, IL; Table 1). Degenerate primer RD1 has an average Tm of 51°C while the three APHVIII specific primers used had Tm ranging from 58°C to 64°C. PCR cycling programs were created using the program given in Dent et al. (2005)2. TAIL1 PCR product was diluted 10-fold and 2 µl of the diluted TAIL1 PCR product was used for TAIL2 PCR reactions. The TAIL2 PCR product was gel purified using a QIAEX II gel extraction kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. Purified TAIL2 PCR product was sequenced at the UC, Berkeley DNA Sequencing Facility (Berkeley, CA). All primer sequences are shown in Table 1.\n\nThese primers were used to generate the data in Figure 7 and Figure 8.\n\nPrimers were designed based on genomic DNA sequences available in the Chlamydomonas genome database in Phytozome. Amplifications of genomic DNA and cDNA were executed using the MJ Research PTC-200 Peltier Thermal Cycler (Watertown, MA). HotStar Taq Plus DNA polymerase kit (Qiagen, Valencia, CA) was used for PCR following the cycling conditions given in the Qiagen protocol booklet. Annealing temperature was between 55 and 60°C depending on the Tm of the primers. Extension time was varied according to the size of the PCR product amplified. Final extension was set at 72°C for ten minutes. All genomic and reverse transcription PCR products were amplified for a total of thirty-five cycles. A 50–150 ng sample of genomic DNA or cDNA were used for PCR reactions. For semi-quantitative RT-PCR reactions, 3 µg of total RNA was converted into cDNA and then 150 ng of cDNA templates were used for RT-PCR. Sequences of primers used for genomic and RT-PCR are shown in Table 2–Table 4.\n\nThese primers were used for GUN4 (Cre05.g246800) genomic DNA PCR on 6F14 and 4A+ and also for DNA sequencing to generate the data in Figure 8 and Figure 9.\n\nThese primers were used to generate the data in Figure 10 and Figure 11.\n\nThese primers were used to generate the data in Figure 12. The gene loci numbers in Phytozome for the three neighboring genes of GUN4 on chromosome 5 and the control actin gene on chromosome 13 are: HYP1 [Cre05.g246750], HYP2 [g5195] and SOXE [Cre05.g246900] and Actin (Cre13.g603700), respectively.\n\nThe pDBle vector (obtained from Dr. Saul Purton, University College London, UK) was double-digested with restriction enzymes EcoRI and NdeI (NEB, Beverly, MA) according to the protocol given in the technical manual. The GUN4 gene was amplified using primers given in Table 5. Ligation of the double digested (NdeI and EcoRI digested) GUN4 gene and the NdeI/EcoRI double-digested pDBle vector was done using the T4 ligase and 1 mM ATP (NEB, Beverly, MA). Chemically competent (CaCl2 treated) E. coli cells were used for transformation. After transformation, E. coli cells were plated on LB+ampicillin (final concentration of ampicillin:100 µg/ml) plates and incubated at 37°C overnight. Single colonies were picked the next day and plasmids were isolated from these clones. Isolated plasmids were double-digested with EcoRI and NdeI to verify the cloning of the GUN4 gene. The GUN4-pDBle construct from the selected clone was sequenced by the UC, Berkeley DNA Sequencing Facility (Berkeley, CA). Chromas Lite (http://technelysium.com.au/) and BLAST were used to analyze DNA sequences.\n\nThese primers were used in the experiments that generated the data in Figure 13 and Figure 16 and were also used for GUN4 gene amplification for cloning.\n\nComplementation of the gun4 was performed utilizing the glass bead transformation technique described by Kindle et al. 198934. 2 µg of the linearized GUN4-pDBle was used to complement 6F14. Transformed cells were plated onto fresh TAP plates containing 15 µg/ml zeocin (Z) and placed in the dark at 25°C. Single colonies were picked and transferred onto fresh TAP+Z plates using a numbered grid template for screening of potential gun4 complements. Screening of gun4 complements was done by monitoring the Chl content and growth of complement strains either on TAP or HS plates under medium light (300 µmol photons m-2 s-1) in the presence or absence of antibiotics zeocin and paromomycin.\n\nChlamydomonas cells from different strains grown in TAP in the dim light were harvested, washed twice with fresh medium and resuspended in TEN buffer (10 mM Tris-HCl, 10 mM EDTA and 150 mM NaCl; pH 8). Gel lanes were loaded with an equal amount of Chl (4 µg Chl). Resuspended cell suspension was mixed in a 1:1 ratio with the sample solubilization buffer SDS-urea buffer (150 mM Tris-HCl, pH 6.8; 7% w/v SDS; 10% w/v glycerol; 2 M urea; bromophenol blue and 10% β-mercaptoethanol) and were incubated at room temperature for about thirty minutes, with intermittent vortexing. The sample solubilization buffer was prepared according to the protocol of Smith et al. (1990)36 using reagents from Fisher (Pittsburgh, PA). After incubation, the solubilized protein samples were vortexed and spun at a maximum speed of 20,000 g in a 1.5 ml eppendorf tube (USA Scientific, Ocala, FL) for five minutes at 4°C. The soluble fraction was loaded on a “any kD™ Mini-PROTEAN® TGX™ Precast Gel” (BioRad, Hercules, CA) and SDS-PAGE analysis was performed according to Laemmli (1970)37 using a Page Ruler prestained molecular weight protein ladder (Fermentas, Glen Burnie, Maryland) at a constant current of 80 V for 2 hours. Gels were stained with colloidal Coomassie Gel Code blue stain reagent (Thermo Fisher Scientific, Rockford, IL) for protein visualization.\n\nElectrophoretic transfer of the SDS-PAGE resolved proteins onto an Immobilon P–PVDF membrane (Millipore, Billerica, MA) was carried out for 2 hours at a constant current of 400 mA in the transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol). The GUN4 polyclonal antibody was raised in rabbit against the full length Chlamydomonas GUN4 mature protein that lacks the first 45 amino acids corresponding to the predicted chloroplast transit peptide31. This antibody was generated by Dr. Roberto Bassi’s laboratory (University of Verona, Italy) and was provided to us by Dr. Krishna Niyogi (UC, Berkeley). GUN4 primary antibodies were diluted to a ratio of 1:1000 before being used as a primary probe. The secondary antibodies used for Western blotting were conjugated to horseradish peroxidase (Pierce protein research product, Thermo Fisher Scientific, Rockford, IL) and diluted to a ratio of 1:20,000 with the antibody buffer. Western blots were developed by using the Supersignal West Pico chemiluminescent substrate kit (Pierce protein research product, Thermo Fisher Scientific, Rockford, IL).\n\nCell density (number of cells per ml of the culture) was calculated by counting the cells using a Neubauer ultraplane hemacytometer (Hausser Scientific, Horsham, PA). Pigments from intact cells were extracted in 80% acetone and cell debris was removed by centrifugation at 10,000 g for 5 minutes. The absorbance of the supernatant was measured with a Beckman Coulter DU 730 Life Science UV/Vis spectrophotometer (Brea, CA). Chl a and b concentrations were determined by Arnon (1949)38 equations, with corrections as described by Melis et al. (1987)39.\n\n\nResults\n\nMutant 6F14 was generated by random insertional mutagenesis of the C. reinhardtii wild type strain 4A+ (137c genetic background). 6F14 was identified as a slightly Chl deficient paromomycin resistant mutant on TAP+P plate in the dark (Figure 3).\n\nThis figure shows the phenotypic difference of 6F14 compared to the parental strain, 4A+ on heterotrophic agar media (TAP) plates under two different growth conditions: dark + paromomycin (P) and dark.\n\nGrowth analyses in heterotrophic and photo-autotrophic liquid media revealed that 6F14 is light sensitive and shows progressive photo-bleaching with increase in light intensities (Figure 4 and Figure 5). In mixotrophic conditions under 10–15 µmol photons m-2 s-1, 6F14 possesses 58% less Chl/cell than 4A+. At 40–50 µmol photons m-2 s-1, 6F14 has 72% less Chl/cell than the wild type. At 75–80 µmol photons m-2 s-1, 6F14 possesses 99% less Chl/cell than the wild type. At 75–80 µmol photons m-2 s-1, 6F14 starts to photo-bleach and turns yellow; it dies at light intensities 100–120 µmol photons m-2 s-1 in TAP (Figure 4).\n\nDark adapted cells of 6F14 and 4A+ were shifted to different light intensities in this experiment. Light conditions and strains are labeled above the culture flasks. The cell density (cells/ml) and nmol chlorophyll (Chl) per cell are shown below the culture flasks in red and black numbers, respectively. For each light condition, experiments were performed on three biological replicates of each strain. Statistical error (±SD) was ≤ 10%.\n\nDark adapted cells of 6F14 and 4A+ were shifted to different light intensities in this experiment. The mean cell density (cells/ml) and the Chlorophyll (Chl) content (nmol Chl per cell) are shown below the culture flasks in red and black numbers, respectively. For each light condition, experiments were performed on three biological replicates of each strain. Statistical error (±SD) was ≤ 10%.\n\nFigure 5 shows photo-autotrophic cultures of 6F14 and 4A+. 6F14 has the ability to grow photo-autotrophically in HS media in dim light (10–15 µmol of photons m-2 s-1). However, the mutant grows extremely slowly in comparison to the wild type. When grown at 10–15 µmol photons m-2 s-1 in HS media, 6F14 possesses 60% less Chl/cell than the wild type. At 40–50 µmol photons m-2 s-1 in HS media, 6F14 has 79% less Chl/cell than 4A+, and at 75–80 µmol photons m-2 s-1 in HS media, 6F14 possesses 83% less Chl/cell than the wild type. At 100–120 µmol photons m-2 s-1 in HS media, 6F14 fails to survive (Figure 5).\n\nFigure 6 demonstrates that when dim light adapted 6F14 was shifted to 40–50 µmol photons m-2 s-1 there was no significant change in Chl/cell content (Figure 4). Dark adapted 6F14 showed a 50% reduction in Chl/cell when moved to 40–50 µmol photons m-2 s-1. When dim light adapted 6F14 was shifted to 75–80 µmol photons m-2 s-1, it showed a 98% reduction in Chl/cell while the dark adapted 6F14 failed to survive under 75–80 µmol photons m-2 s-1. Taken together, the results shown in Figure 4 and Figure 6 show that dark adapted 6F14 is more sensitive to the magnitude of light intensity changes in the environment than the dim light adapted 6F14 (Figure 6).\n\n6F14 was adapted to dim light (10–15 µmol photons m-2 s-1) or dark for one week in TAP media. Dark and dim light adapted cultures were then shifted to 40–50 or 75–80 µmol photons m-2 s-1. The mean cell density (cells/ml) and the Chlorophyll (Chl) content (nmol Chl per cell) are shown below the culture flasks in red and black numbers, respectively. For each light condition, experiments were performed on three biological replicates of 6F14. Statistical error (±SD) was ≤ 10%. The average Chl content in the dim light and dark adapted 6F14 was 1.7 × 10-6 and 2.18 × 10-6 nmol/cell.\n\nThe linearized pBC1 plasmid was used to generate 6F14 (Figure 2). To find the insertion of the APHVIII end of the plasmid in 6F14, TAIL PCR method was employed. Figure 7A shows the position of the vector specific TAIL PCR primers and also shows the arbitrary position of the random degenerate primer. A 2.9 kb DNA product from TAIL2 PCR was purified from the agarose gel (Figure 7B, Table 1). This purified DNA product was used for further PCR using internal primers specific to the 3´ UnTranslated Region (UTR) of the APHVIII gene. The PCR results confirmed that the 2.9 kb DNA product contains the 3´ UTR of the APHVIII gene (Figure 7C). Sequencing of the 2.9 kb TAIL2 PCR product revealed that the APHVIII end of the plasmid has been inserted 344 bp away from the GUN4 gene (Cre05.g246800) on chromosome 5. The GUN4 locus was cleaved at least at two places (Figure 8). The first cleavage was about 781 bp away from the 5′ end of the GUN4 gene and the second cleavage was 1131 bp away from the 3´ end of the GUN4 gene. These cleavages were followed by the inversion of the cleaved genomic DNA which then ligated to the 3´ UTR of the GUN4 gene (Figure 8). Plasmid insertion also led to an addition of 29 bp at the APHVIII end of the plasmid. An addition of 45 bp was found at the breakage point in the 3´ UTR of the GUN4 gene (Figure 8).\n\n(A) A diagram showing a truncated pBC1 illustrating the APHVIII end of the linearized pBC1 vector. Primers used for PCR and DNA sequencing are shown by numbered black arrows. Thermal Asymmetric InterLaced1 (TAIL1) PCR was performed using primers 4R and RD1 (a random degenerate primer). (B) TAIL2 PCR was performed using primers 3R and RD1. In lane 1, 10-fold diluted TAIL1 PCR product was used for TAIL2 PCR; Lane 2 is a zero DNA control lane. The 2.9 kb TAIL2 PCR product used for DNA sequencing is highlighted in the red box. Initial DNA sequencing was performed using vector specific primers 2R and 3R (Table 1). (C) Gel purified DNA product (2.9 kb) from TAIL2 PCR was used to verify if the product is specific to the APHVIII gene. PCR primer names are labeled on the top of the gel. PCR product size is labeled. F and R stand for forward and reverse primers, respectively. All primer sequences are shown in Table 1. ST stands for 1 kb plus ladder (Invitrogen, Carlsbad, CA). DNA samples were run on a 1% agarose gel.\n\n(A) A schematic genomic map showing a 3525 bp genomic DNA region spanning the GUN4 locus. The number at the bottom of the map denotes distance between respective points on the genomic DNA. The two GUN4 exons are represented by white block arrows. Grey boxes in GUN4 denote UnTranslated Regions (UTRs). The two break points are shown by the dashed pink and green lines. (B) A schematic diagram showing the rearrangement of the GUN4 locus after the insertion of the plasmid. The big and the small tan boxes, denote addition of 45 and 29 bp, respectively. The genomic DNA sequence obtained by sequencing the 2.9 kb Thermal Asymmetric InterLaced2 (TAIL2) PCR product is highlighted in red. The bold black small arrow indicates insertion point of the pBC1 plasmid. DNA sequencing was performed using GUN4 specific primers 2R, 7F and 7R, 12R and 14R. F and R stand for forward and reverse primers, respectively. Primer sequences are shown in Table 2.\n\nFurther genomic DNA PCR analyses with GUN4 specific primers confirmed that the 3´ part of the GUN4 first exon and the 5′ part of the GUN4 second exon were deleted or displaced (Figure 9). We also used primers specific to the genomic region upstream of the GUN4 gene and primers specific to the 3´ UTR of a hypothetical gene, HYP2, (g5195) located downstream of GUN4 to see the extent of deletion on either side of the GUN4 gene. Our PCR analyses show that a 1.354 kb genomic DNA region, located upstream of GUN4 was deleted/displaced. Additionally, there is a deletion of approximately 526 bp in the 3´ UTR of the downstream HYP2 gene (Figure 10 and Figure 11). Taken together the data show that plasmid insertion in the 6F14 genome has rearranged the GUN4 locus and has affected a part of the 3´ UTR of the HYP2 gene. We do not yet know the exact location of the pUC ori end of the plasmid in the 6F14 genome (Figure 11).\n\n(A) A schematic of the GUN4 gene. Small black arrows denote GUN4 specific primers. The two GUN4 exons are represented by white block arrows. Grey boxes in GUN4 denote UnTranslated Regions (UTRs). (B), (C) and (D) DNA gels showing DNA products obtained from genomic DNA PCR using GUN4 specific primers. Lanes 1, 2 and 3 are the zero DNA controls, 6F14 and 4A+, respectively. PCR primer names are labeled on the top of the gel. 14F/3R gives a 517 bp product while 3F/8R gives a 942 bp product. 8F/7R gives a 449 bp product. 7F/12R gives a 313 bp product. 12F/10R gives a 606 bp product and 12F/11R gives a 623 bp product. F and R stand for forward and reverse primers, respectively. Primer sequences are shown in Table 2.\n\n(A) A schematic diagram showing GUN4 (white arrow) and its two neighboring genes HYP1 (black arrow) and HYP2 (grey arrow). Black numbers at the bottom denote distances between respective genes. The two red highlighted regions and corresponding numbers show the distance between primer ACF7 and the start of the GUN4 gene and the distance between the primer H5F and the end of the HYP2 gene, respectively. Primers used for PCR are labeled. (B) A DNA gel showing the genomic DNA amplified using GUN4 upstream region specific primers. Lanes 1, 3 and 5: 6F14; Lanes 2, 4 and 6: 4A+. (C) A DNA gel showing the genomic DNA amplified using primers spanning the 3´ UTR region of HYP2 gene. Lanes 1, 3 and 5: 6F14; Lanes 2, 4 and 6: 4A+. Product size of ACF6/ACR7: 937 bp (this primer set also produces a nonspecific 550 bp product); product size of ACF7/ACR11: 244 bp; product size of H3F/H6R: 667 bp; product size of H4F/H6R: 391 bp; product size of H5F/H6R: 278 bp. F and R stand for forward and reverse primers, respectively. Primer sequences are shown in Table 3.\n\n(A) A schematic genomic map showing an 8.064 kb genomic DNA region spanning the GUN4 locus on chromosome 5. The numbers at the bottom of the map denote distances between respective points on the genomic DNA. The red highlighted region and number show the distance between primer ACF7 and the start of the GUN4 gene and the distance between the primer H5F and the end of the HYP2 gene, respectively. The two GUN4 exons are represented by white block arrows. The tan arrow and the black block arrow, denotes a part of HYP1 3´ UTR and HYP2 gene, respectively. (B) An updated schematic diagram showing the rearrangement of the GUN4 locus based on PCR analyses and DNA sequencing. Two break points in the genome are denoted by green and pink dashed lines. The big and the small grey boxes, denote addition of 45 and 29 bp, respectively. The small black arrows denote primers that were used for genomic PCRs in Figure 9 and Figure 10. Red dashed lines denote possible deletions. The small red arrow indicates the point of insertion of the pBC1 plasmid. The black numbers at the bottom of the map denote distances between respective points on the genomic DNA. The red highlighted region and the corresponding number show the distance between the end of the primer H5F and the stop codon of the HYP2 gene.\n\nTranscript levels of GUN4 and the neighboring genes (HYP1 [Cre05.g246750]; HYP2 [g5195] and SOXE [Cre05.g246900]) were checked using semi-quantitative RT-PCR using GUN4, HYP1, HYP2 and SOXE specific primers, respectively (Figure 12). Reduced levels of HYP1 and HYP2 transcripts were observed in 6F14 compared to that in the wild type (Figure 12). GUN4 transcript is missing in 6F14 as expected (Figure 12). The transcript level of SOXE, the second gene downstream of GUN4, was not affected. Cre05.g246750 and g5195 are genes in the Chlamydomonas database coding for hypothetical proteins. We have named these genes as HYP1 and HYP2 arbitrarily for our study. The SOXE gene codes for sulfocyanin, a blue copper protein. Readers are requested to identify GUN4 and its neighboring genes by the gene locus number (Cre or the g number) in the Phytozome database.\n\n(A) A schematic map of a 21.497 kb genomic region spanning the GUN4 locus on chromosome 5. HYP1 and HYP2 are genes located upstream and downstream of the GUN4 gene, respectively coding for hypothetical proteins. Sulfocyanin (SOXE) codes for a blue copper protein. The top black number denotes size of a gene (bp) while the bottom black number denotes distance between genes (bp). (B) Semi-quantitative RT-PCR results. Lanes: 1, 3, 5, 7, 9 denote 4A+ cDNA products. Lanes 2, 4, 6, 8, 10 denote gun4 cDNA products. Primer sequences are shown in Table 4. All primers span an intron. Actin was used as a control. Actin genomic product size: 527 bp; Actin cDNA product size: 305 bp. HYP1 genomic product size 726 bp; HYP1 cDNA product size: 459 bp. GUN4 genomic product size: 942 bp; GUN4 cDNA product size 775 bp. HYP2 genomic product size: 797 bp; HYP2 cDNA product size: 184bp. SOXE genomic product size: 517 bp; SOXE cDNA product size: 119 bp.\n\nWe will be referring to 6F14 as gun4 from here onward. As gun4 specifically lacks a functional GUN4 gene, we cloned the GUN4 gene in the pDBle vector to transform 6F14 (Figure 13, Table 5). The trans GUN4 expression is driven by the constitutive PsaD promoter in the GUN4-pDBle construct. pDBle has two Ble genes that confer resistance to the antibiotic zeocin. Figure 14 shows growth phenotypes of two gun4 complements (gun4-19 and gun4-27), 6F14 and 4A+. gun4 complements are not light sensitive and are able to grow and photosynthesize under medium light intensities (300 µmol photons m-2 s-1) without photo-bleaching (Figure 14). As gun4 complements harbor the Ble gene (from the pDBle vector) and APHVIII gene (derived from the parental strain 6F14), they can grow both on zeocin and paromomycin media plates unlike 6F14 and 4A+ (Figure 14).\n\nNdeI/EcoRI double digested GUN4 gene (956 bp) was cloned into the NdeI/EcoRI double digested pDBle plasmid. Primers used for amplification of the GUN4 gene are shown in Table 5. GUN4 expression is driven by the constitutive PsaD promoter. NdeI and EcoRI restriction sites are labeled. pDBle contains two copies of BleR genes driven by the Rubisco (RbcS2) promoter. The size of the GUN4-pDBle construct is 7653 bp. The black arrow and the white arrow, denotes GUN4 and BleR genes, respectively. Grey and tan boxes denote UnTranslated Regions (UTRs).\n\ngun4-19 and gun4-27 complements, 6F14 and 4A+ were grown for a week under six different growth conditions: TAP + Z (zeocin) in the dark, TAP in the dark, TAP + P (paromomycin) low light (LL; 50 µmol photons m-2 s-1), TAP LL, TAP medium light (ML; 300 µmol photons m-2 s-1) and HS ML.\n\nChl analyses show that under heterotrophic conditions both gun4 complements have 65–68% more Chl than that of the wild type cells (Figure 15). Under photo-autotrophic conditions gun4 complement cells possess 50–60% more Chl than that of the wild type cells (Figure 15). Figure 16A shows a schematic figure of the trans GUN4 gene used for complementation. PCR analyses using the genomic DNA show that the gun4 complements possess the functional trans GUN4 gene (Figure 16B, 16C and 16D). Figure 17A shows a stained protein gel that was loaded on equal Chl basis. Western analyses of the two gun4 complements with a Chlamydomonas GUN4 specific antibody show that the GUN4 protein is absent in the gun4 mutant but present in the gun4 complements (Figure 17B). Western analyses also show that the two gun4 complements have higher levels of the GUN4 protein compared to that of the wild type (Figure 17B).\n\nLight intensity is labeled above the culture flask. Growth media is labeled to the left of the culture flask. The mean cell density (cells/ml) and the Chlorophyll (Chl) content (nmol Chl per cell) are shown below the culture flasks in red and black numbers, respectively. For each light condition and growth condition, experiments were performed on three biological replicates of each strain. Statistical error (±SD) was ≤ 10%.\n\n(A) A schematic diagram of the GUN4-pDBle construct. Primers used for PCR are shown on the map. (B) Genomic DNA PCR analyses with GUN4 cloning primers (product size: 969 bp). Lane 1: gun4; Lane 2: 4A+; Lane 3: gun4-19; Lane 4: gun4-27. (C) RT-PCR analyses with GUN4 cloning primers (product size: 802 bp). Lane 1: gun4; Lane 2: 4A+; Lane 3: gun4-19; Lane 4: gun4-27. (D) Genomic PCR analyses using a PsaD 5′ UTR specific forward primer with a GUN4 cloning reverse primer (product size: 976 bp). Lane 1: gun4; Lane 2: 4A+; Lane 3: gun4-19; Lane 4: gun4-27. Primer sequences are shown in Table 5.\n\n(A) A stained protein gel. Lanes 1, 2, 3 and 4 represent gun4, gun4-19, 4A+ and gun4-27, respectively. PS denotes prestained molecular weight protein ladder. Total cell extract of different strains were loaded on equal Chlorophyll (Chl) basis (4 µg of Chl). (B) Western analyses using a GUN4 antibody generated against the Chlamydomonas mature full length GUN4 protein. Lanes 1, 2, 3 and 4 represent gun4, gun4-19, 4A+ and gun4-27, respectively. GUN4 (24kDa) protein detected by the antibody is labeled. * denotes a 25kDa protein detected non-specifically by the GUN4 antibody.\n\n\nDiscussion\n\nPlastid development and gene expressions are largely under nuclear “anterograde” control40. Additionally, chloroplast functional and developmental states can regulate expression of nuclear genes encoding chloroplast localized proteins via retrograde signaling40. The first evidence for the involvement of Chl biosynthetic precursors in retrograde signaling came from the work in Chlamydomonas41. In Arabidopsis MgPPIX was hypothesized to be a retrograde signal from the chloroplast to the nucleus on the basis of data obtained with mutants that are defective in the norflurazon (NF) induced down-regulation of transcription of light harvesting complex protein B (LHCB)[gun (genomes uncoupled) phenotype]40,42. Six gun mutants are known; five of which directly influence tetrapyrrole biosynthesis (gun2-gun6)43,44. The gun4 mutation is localized to a porphyrin binding protein GUN4. GUN4 enhances the sensitivity of MgChel to Mg2+ at physiologically low Mg2+ concentration31,45. Cyanobacterial and higher plant GUN4 directly interacts with the CHLH subunit of MgChel and binds PPIX and MgPPIX, the substrate and the reaction product of the MgChel30,46–50. Although GUN4 is not an essential component of the MgChel complex, the presence of GUN4 markedly improves the enzyme activity in vitro by increasing the apparent substrate-binding capacity of CHLH for PPIX, particularly under low Mg2+ concentrations45,51,52. It is proposed that GUN4 upon porphyrin binding, stabilizes interactions between the catalytic subunit of MgChel and the chloroplast membranes, the site of Chl biosynthesis46,47. This enables MgChel to interact with enzyme complexes involved in the further downstream steps in the pathway46,47. Apart from its role in substrate channeling into the Chl synthesizing branch of tetrapyrrole biosynthesis, GUN4 has also been implicated in providing photo-protection under increasing light intensities30,46,47. The porphyrin binding property of GUN4 has been implicated in ROS attenuation but conclusive experimental support is lacking47. In higher plants, GUN4 has been implicated as an essential component in a post-translational feedback regulation mechanism that modulates ALA biosynthesis in response to enzymatic activities of the Mg branch of tetrapyrrole biosynthesis as well as to the accumulating Mg porphyrin levels30 (Figure 1).\n\n6F14 is the second gun4 mutant to be identified in C. reinhardtii. The first C. reinhardtii gun4 mutant was identified and characterized in 2012 by Formighieri et al.31. In this gun4 mutant, 184 bp of the second exon of the GUN4 gene was deleted. In 6F14 the plasmid insertion outside the GUN4 gene has caused a genetic rearrangement of the GUN4 gene that prevented gene expression (Figure 11). Transcripts of GUN4 and the neighboring genes of GUN4 in 6F14 were checked by performing semi-quantitative reverse transcription PCR. In 6F14, the transcript level of the first downstream hypothetical (HYP2) gene was lower than that in the wild type (Figure 12). The plasmid insertion in 6F14 has led to a deletion of part of the 3´ UTR region of the HYP2 gene (270 bp away from the stop codon of the coding region of HYP2; Figure 11). The 3´ UTR is usually responsible for the stability of the transcript. Hence the nature of the deletion in HYP2 explains the decrease in transcript levels of HYP2. GUN4 and the upstream gene, HYP1, are separated from each other by 2.784 kb (Figure 10). There is a possible deletion/genetic rearrangement in the 5′ genomic region upstream of the GUN4 which does not extend into the HYP1 gene (Figure 10 and Figure 11). Although the transcription of HYP1 was not hampered, the HYP1 transcript level is lower in our gun4 compared to that in the wild type (Figure 12). Based on the RT-PCR analyses, it is speculated there might be some uncharacterized downstream regulatory sequences present in the 2.784 kb region that might regulate HYP1 transcription. In future, quantitative real time-PCR experiments can be used to accurately quantify transcript levels of HYP1 and HYP2 in 6F14.\n\nThe photosensitive phenotype of our gun4 mutant resembles that of the earlier identified Chlamydomonas gun4 mutant. Over-accumulation of photo-excitable PPIX leads to photo-oxidative damage to the cells in presence of light and oxygen4,26,53. The light sensitivity of 6F14 is most probably due to an over-accumulation of the PPIX which occurs due to the inactivity of MgChel enzyme as has been shown by Formighieri et al. (2012)31 in their gun4 mutant. Future HPLC (High Performance Liquid Chromatography) analyses of steady state tetrapyrrole intermediates in 6F14 will confirm this hypothesis. Formighieri et al. (2012)31 explored four light conditions (dark, 6-, 50-, and 500 µmol photons m-2 s-1) and showed that the gun4 Chlamydomonas mutant dies under high light (500 µmol photons m-2 s-1). These researchers did not explore or clarify the maximum light irradiance condition that can be tolerated by the Chlamydomonas gun4 mutant in heterotrophic and photosynthetic growth conditions. In this study, we found that 6F14 photo-bleached at 75–80 µmol photons m-2 s-1 and could not tolerate light intensity above 100 µmol photons m-2 s-1 (Figure 4 and Figure 5). The earlier identified C. reinhardtii gun4 was able to grow in continuous light slightly better than in photoperiodic shifts31. In Arabidopsis, the gun4 mutant was seen to exhibit significant improved growth in continuous light compared to periodic shifts in light30. In this study, 6F14 and the wild type were adapted to dark or dim light and then shifted to two different light irradiances (40–50 µmol photons m-2 s-1 and 75–80 µmol photons m-2 s-1). Cultures exposed to light shifts showed a significant reduction in the Chl content than those grown under a constant light intensity (Figure 4 and Figure 6). Additionally, dark adapted 6F14 showed a significant reduction in the Chl content compared to the dim light adapted 6F14, when cells were shifted to similar light intensities (Figure 6). These results show that 6F14 is very sensitive to the magnitude of light intensity fluctuations in the environment unlike the earlier reported Chlamydomonas gun4 mutant31. Our light shift experimental results support the findings in cyanobacterial and Arabidopsis gun4 mutants30,48–50.\n\nBy spectrophotometric analysis we have shown that in the dark 6F14 possesses almost similar Chl content like that in the wild type (Figure 4). This phenotype is very different from that of the previously identified Chlamydomonas gun4 mutant which possesses 50% of the wild type level of Chl/cell31 in the dark. Variation in Chl/cell in the dark between the two C. reinhardtii gun4 mutants could possibly be due to a variation of the parental strain’s ability to synthesize Chl in the dark. The parental strain used by Formighieri et al. (2012)31 was cw15mt-. However, the 50% decrease in Chl seen in the previously described Chlamydomonas gun4 mutant was determined through HPLC analyses. Hence the discrepancy in Chl content in the two gun4 mutants could be due to the sensitivity of the HPLC method compared to that of the spectrophotometric method used for Chl assays.\n\nInterestingly, it has been shown by Formighieri et al. (2012)31 that Chlamydomonas gun4 complements expressing more GUN4 protein grow better under high light and that there is no correlation between the accumulation of PPIX and the ability to grow better under high light31. However these gun4 complements were not over-expressers of the GUN4 protein compared to the wild type strain cw15, used in their experiments31. Our two gun4 complements (gun4-19 and gun4-27) are over-expressing the GUN4 protein compared to the wild type strain 4A+ in the dim light (Figure 17B). These two gun4 complements open up new avenues to test if GUN4 has a distinct photo-protective role that is independent from the PPIX-induced GUN4 photo-protective role proposed by several researchers46,47. Comparative growth studies, quantitative measurements of GUN4 transcripts by Real Time PCR, GUN4 protein levels by Western analyses and PPIX content by HPLC analyses of the high light-(500 µmol photons m-2 s-1) and dim light-(15–20 µmol photons m-2 s-1) adapted gun4 complements and the wild type strain will help to confirm if GUN4 has a distinct photo-protective role that is independent of tetrapyrrole metabolism.\n\nTaken together our work reconfirms the results of other researchers who have studied GUN4 in other photosynthetic organisms30,31,46,47,49,50. Although loss of GUN4 caused a perturbation in Chl biosynthesis in our gun4 mutant, the effect is not as dramatic as it is in Arabidopsis, where the loss of GUN4 results in a nearly albino mutant51. The earlier identified Chlamydomonas gun4 mutant phenotypically resembles our gun4 mutant31. Therefore it seems that in C. reinhardtii Chl biosynthesis is less dependent on the GUN4 function. One explanation for this difference in the mutant phenotype could be that Chlamydomonas is capable of synthesizing Chl in the dark unlike the angiosperms. GUN4 binds PPIX and acts at the branch point in the tetrapyrrole biosynthetic pathway where PPIX is diverted to heme and Chl biosynthesis. Hence, although GUN4 has a conserved physiological role in all oxygenic photosynthetic organisms, it might have a somewhat different role in different evolutionary groups depending on the channelization of PPIX into the heme and Chl branch in the pathway.", "appendix": "Author contributions\n\n\n\nMM and PG conceived the study, designed the experiments and took the lead role in preparing the manuscript. KG generated the mutant 6F14. PG did the PCR analyses, isolated genomic DNA, performed growth and Chl analyses of different strains, prepared cDNA, cloned the GUN4 gene and complemented the mutant 6F14. KG and SP maintained all the strains and were involved in the mutagenesis experiment. KL and DW isolated genomic DNA and performed PCRs. MM performed the TAIL-PCR analyses, extracted TAIL-PCR product from agarose gels, prepared DNA samples for sequencing and analyzed the DNA sequencing data and also performed the protein and Western analyses. All authors were involved in the revision of the manuscript draft 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 project was supported by several grants awarded to Dr. Mautusi Mitra. These are: the start-up grant of the University of West Georgia (UWG), the Faculty Research Grant by the UWG College of Science and Mathematics, the Internal Development Grant by the UWG office of Research and Sponsored Project, the Research Incentive grant by the UWG College of Science and Mathematics, the UWG Student Research Assistance Program (SRAP) grant and the UWise-BOR-STEM II grant from UWG.\n\n\nAcknowledgements\n\nWe would like to thank Dr. Krishna K. Niyogi (UC, Berkeley) for providing the 4A+ strain and the pBC1 plasmid that were used for mutagenesis and the GUN4 antibody for Western analyses. We are grateful to Dr. Saul Purton (University College London, UK) for providing the complementation vector pDBle. We are grateful to Dr. Bernhard Grimm and Dr. Pawel Brzezowski (Humboldt University, Berlin, Germany) for providing us the SOXE gene specific primers for RT-PCR analyses. 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PCC6803 and plants. J Plant Physiol. 2011; 168(12): 1380–1386. PubMed Abstract \| Publisher Full Text\n\nSobotka R, Dühring U, Komenda J, et al.: Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes. J Biol Chem. 2008; 283(38): 25794–25802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilde A, Mikolajczyk S, Alawady A, et al.: The gun4 gene is essential for cyanobacterial porphyrin metabolism. FEBS Lett. 2004; 571(1–3): 119–123. PubMed Abstract \| Publisher Full Text\n\nLarkin RM, Alonso JM, Ecker JR, et al.: GUN4, a regulator of chlorophyll synthesis and intracellular signaling. Science. 2003; 299(5608): 902–906. PubMed Abstract \| Publisher Full Text\n\nVerdecia MA, Larkin RM, Ferrer JL, et al.: Structure of the Mg-chelatase cofactor GUN4 reveals a novel hand-shaped fold for porphyrin binding. PLoS Biol. 2005; 3(5): e151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTanaka R, Tanaka A: Tetrapyrrole biosynthesis in higher plants. Annu Rev Plant Biol. 2007; 58: 321–346. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1047", "date": "09 Jul 2013", "name": "Kittisak Yokthongwattana", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting paper by Mitra et al. presents results that show another allele of the gun4 mutant in Chlamydomonas. Although this is not the first article to report on gun4 mutant, it is important as it does provides extra phenotypic studies that show both consistent results and novel investigations. I do have some minor concerns that the authors should consider to improve the manuscript:1. The title is somewhat misleading as the progressive Chl deficiency is just one of the phenotypes of this mutant; please consider properly revising it.2. Why did the authors need to put up so many figures in one article? Some of the figures are not really necessary and can be put attached online as supplementary figures. With too many figures, the article can become very boring to read.3. The authors should put some more emphasis on the interesting findings like the genetic rearrangement of the gun4 gene in the mutant, for example.4. Unless the authors made further characterization on the two HYP genes regarding their sequences and deduced amino acid and possible functions, they can be omitted from the paper as they would confuse the reader.", "responses": [] }, { "id": "1048", "date": "09 Jul 2013", "name": "EonSeon Jin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents data regarding phenotype of isolated mutants (6F14), identification of the mutation locus in 6F14 and its complementation.The results revealed that 6F14 is defective in the GUN4 (genome uncoupled 4) gene which regulate MgChel activity. Therefore, the transformation of 6F14 with a GUN4 gene restored the wild type phenotype with over-expressing the GUN4 protein. The authors concluded that 6F14 is the second gun4 mutant identified in Chlamydomonas. In that sense, the study of 6F14 (gun4 II) is not novel. However, the authors have shown the difference of mutation locus of gun4 I (earlier identified) and gun4 II (in this study) and also the differences of light sensitivity and chlorophyll content which is very interesting. I do have some suggested revisions:1. If the authors compare all physiological responses for two gun4 mutants and their parental wild types in the same culture condition, it would explain more clearly the differences between the gun4 II and gun4 I mutants. 2. Authors may need to propose the future experiment with this new gun4 mutant (6F14) in the discussion section of the article. 3. I noticed that the introduction part of your abstract (up to 5 lines) is almost identical with your recent paper “Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis” paper. I would recommend you to revise the introduction part of this abstract.", "responses": [] } ]	1	https://f1000research.com/articles/2-142
https://f1000research.com/articles/2-138/v1	10 Jun 13	{ "type": "Research Article", "title": "Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis", "authors": [ "Phillip B Grovenstein", "Darryel A Wilson", "Cameron G Lennox", "Katherine P Smith", "Alisha A Contractor", "Jonathan L Mincey", "Kathryn D Lankford", "Jacqueline M Smith", "Tashana C Haye", "Mautusi Mitra", "Phillip B Grovenstein", "Darryel A Wilson", "Cameron G Lennox", "Katherine P Smith", "Alisha A Contractor", "Jonathan L Mincey", "Kathryn D Lankford", "Jacqueline M Smith", "Tashana C Haye" ], "abstract": "The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis and chloroplast to nucleus retrograde signaling in Chlamydomonas, which has never been studied before.", "keywords": [ "The green micro-alga Chlamydomonas reinhardtii possesses a photosynthetic apparatus very similar to that of higher plants", "can grow photo-autotrophically and heterotrophically (it can metabolize exogenous acetate as a carbon source) and possesses a completely sequenced genome1. These attributes make it an elegant model organism to study oxygenic photosynthesis and chloroplast biogenesis2", "3. In photosynthetic organisms", "tetrapyrroles like Chl and heme are essential for energy metabolism (i.e. photosynthesis and respiration). Biosynthesis of Chl and heme occur via a common branched pathway that involves both nuclear- and chloroplast-encoded enzymes in most photosynthetic organisms4. In photosynthetic eukaryotes", "5-aminolevulinic acid (ALA) is synthesized from glutamine through glutamyl-tRNA5. Conversion of ALA through several steps yields protoporphyrin IX (PPIX)", "the last common precursor for both heme and Chl biosynthesis5. Ferrochelatase inserts iron in the center of PPIX thus committing it to the heme branch of the pathway. Insertion of Mg2+ in PPIX by MgChel leads to Mgproto", "the first biosynthetic intermediate in the Chl branch6. Magnesium chelatase has three subunits", "which are CHLD", "CHLH and CHLI7. The ATP-dependent catalytic mechanism of the heterotrimeric MgChel complex includes at least two steps7–9: an activation step", "followed by the Mg2+ insertion8. Activation of MgChel with ATP involves CHLD and CHLI while CHLH is required for the chelation step10. CHLI belongs to the AAA+ family of ATPases. Plants have two isozymes of CHLI1 (CHLI1 and CHLI2) which are 70%–81% identical in protein sequences10. Although the functional role of CHLI1 is well characterized", "that of CHLI2 is not. Most of the data on CHLI comes from studies on Arabidopsis thaliana chli mutants and the functional significance of CHLI1 and CHLI2 has not been studied in green algae10–16. In Arabidopsis CHLI2 plays a limited role in Chl biosynthesis because of its lower expression level compared to that of CHLI112–15. In Arabidopsis the CHLI2 protein amount is lower than that of CHLI1. When overexpressed", "CHLI2 can fully rescue an Arabidopsis chli1chli2 double mutant12." ], "content": "Introduction\n\nThe green micro-alga Chlamydomonas reinhardtii possesses a photosynthetic apparatus very similar to that of higher plants, can grow photo-autotrophically and heterotrophically (it can metabolize exogenous acetate as a carbon source) and possesses a completely sequenced genome1. These attributes make it an elegant model organism to study oxygenic photosynthesis and chloroplast biogenesis2,3. In photosynthetic organisms, tetrapyrroles like Chl and heme are essential for energy metabolism (i.e. photosynthesis and respiration). Biosynthesis of Chl and heme occur via a common branched pathway that involves both nuclear- and chloroplast-encoded enzymes in most photosynthetic organisms4. In photosynthetic eukaryotes, 5-aminolevulinic acid (ALA) is synthesized from glutamine through glutamyl-tRNA5. Conversion of ALA through several steps yields protoporphyrin IX (PPIX), the last common precursor for both heme and Chl biosynthesis5. Ferrochelatase inserts iron in the center of PPIX thus committing it to the heme branch of the pathway. Insertion of Mg2+ in PPIX by MgChel leads to Mgproto, the first biosynthetic intermediate in the Chl branch6. Magnesium chelatase has three subunits, which are CHLD, CHLH and CHLI7. The ATP-dependent catalytic mechanism of the heterotrimeric MgChel complex includes at least two steps7–9: an activation step, followed by the Mg2+ insertion8. Activation of MgChel with ATP involves CHLD and CHLI while CHLH is required for the chelation step10. CHLI belongs to the AAA+ family of ATPases. Plants have two isozymes of CHLI1 (CHLI1 and CHLI2) which are 70%–81% identical in protein sequences10. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. Most of the data on CHLI comes from studies on Arabidopsis thaliana chli mutants and the functional significance of CHLI1 and CHLI2 has not been studied in green algae10–16. In Arabidopsis CHLI2 plays a limited role in Chl biosynthesis because of its lower expression level compared to that of CHLI112–15. In Arabidopsis the CHLI2 protein amount is lower than that of CHLI1. When overexpressed, CHLI2 can fully rescue an Arabidopsis chli1chli2 double mutant12.\n\nWe have isolated the first chli1 mutant of Chlamydomonas reinhardtii (5A7) which possesses an intact CHLI2 gene. Transformation of 5A7 with a functional copy of the CHLI1 gene restored Chl biosynthesis. Western analyses show that the CHLI2 protein level is lower than that of CHLI1 in the wild type strain and CHLI2 protein is barely detectable in the mutant strain. In this study, we present our molecular data on the identification of the mutation locus in 5A7 and its complementation.\n\n\nMaterials and methods\n\nChlamydomonas strains 4A+ (a gift from Dr. Krishna Niyogi (UC, Berkeley), 5A7/chli1 (generated by our laboratory) and chli1 complements (generated by our laboratory) were grown either in Tris-Acetate Phosphate (TAP) heterotrophic media or in Sueoka’s High Salt (HS) photo-autotrophic media. TAP and HS liquid media and agar plates were prepared in the lab using reagents from Fisher Scientific (Pittsburg, PA) according to the protocol given in Gorman and Levine (1965)17 and Sueoka (1960)18, respectively. The 4A+ strain and chli1 complements were maintained on TAP agar plates and TAP+zeocin (Sigma, St. Louis, MO) plates, respectively under dim light intensities (10–15 µmol photons m-2s-1) at 25°C. The final zeocin concentration was 15 µg/ml). The chli1 mutant (5A7) was maintained in the dark on TAP 1.5% agar plates containing 10 µg/ml of paromomycin (Sigma, St. Louis, MO). Liquid algal cultures used for RNA and genomic DNA extractions and protein analyses were grown in 100 ml flasks on the New Brunswick Scientific Excella E5 platform shaker (Enfield, CT) at 150 rpm in the dark or in the dim light.\n\nThe purified pBC1 plasmid from the DH5α Escherichia coli clone harboring the pBC1 plasmid (obtained from Dr. Krishna Niyogi’s laboratory at UC, Berkeley) was used for random DNA insertional mutagenesis. This plasmid contains two antibiotic resistance genes: APHVIII and AmpR (Figure 1). APHVIII confers resistance against the antibiotic paromomycin (Sigma, St. Louis MO) and was used as a selection marker for screening of Chlamydomonas transformants. AmpR was used as a selection marker for screening of E. coli clones harboring the pBC1 plasmid. E.coli was grown in 1 l of Luria Bertani (LB) broth containing 1% tryptone, 0.5% of yeast extract, 1% NaCl and ampicillin [final concentration of ampicillin:100 µg/ml]. LB reagent was prepared in the laboratory using reagents purchased from Fisher (Pittsburgh, PA). Ampicillin was purchased from Fisher (Pittsburgh, PA). The culture was incubated at 37°C overnight. Plasmid purification from E. coli cells was facilitated by Qiagen plasmid mega kit according to the protocol given in the technical manual (Qiagen, Valencia, CA). Once purified from E. coli, the circular pBC1 vector was linearized with the restriction enzyme Kpn1 (NEB, Beverly, MA) according to the protocol given in the technical manual. The linearized DNA was purified using a QIAEX II gel extraction kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. All agarose DNA gel electrophoresis was visualized by BioRad Molecular Imager Gel Doc XR+ (BioRad, Hercules, CA). Transformation of parental strain 4A+ by the linearized pBC1 vector was performed utilizing the glass bead transformation technique described by Kindle et al. (1989)19 and Dent et al. (2005)2. Transformants were plated onto fresh TAP agar plates containing 10 µg/ml paromomycin (TAP+P) in the dark. Single colonies of mutants were picked and transferred onto fresh TAP+P plates using a numbered grid layout. Screening of photosynthetic and pigment deficient mutants was done by visual inspection and monitoring of growth under different light intensities in heterotrophic, mixotrophic and photo-autotrophic conditions2.\n\nThe cleavage site of the Kpn1 restriction enzyme, used for linearization of the vector is shown. APHVIII is under the control of combo promoters consisting of the promoter of the gene encoding the small subunit of Rubisco (RbcS2) and the promoter of the gene encoding the heat shock protein 70A (Hsp70A). pBC1 is a phagemid and its F1 origin (F1 ori) and pUC origin (pUC ori) are shown. The size of the plasmid is 4763 bp.\n\n4A+, chli1 complements and 5A7/chli1 were grown in TAP liquid media in the dark to a cell density of about 5 × 106 cells/ml of the culture. Genomic DNA was purified using a phenol-chloroform extraction method19. RNA extraction was facilitated by TRIzol reagent from Invitrogen (Carlsbad, CA) following the protocol in the technical manual. DNA and RNA concentrations were measured using a Nanodrop 1000 spectrophotometer from Thermo Fisher Scientific (Wilmington, DE). DNase treatment was performed using Ambion’s TURBO DNA-free kit from Invitrogen (Carlsbad, CA) following the protocol in the technical manual to remove genomic DNA from the RNA preparation. Generation of cDNA was performed using Life Technologies Superscript III First-Strand Synthesis System from Invitrogen (Carlsbad, CA) following the protocol in the technical manual.\n\nTAIL (Thermal Asymmetric InterLaced) PCR was implemented, following the protocol of Dent et al. (2005)2. This protocol was implemented with one modification of utilizing a non-degenerate primer (AD2) derived from the original degenerate primer (AD) for TAIL PCR as this non-degenerate primer was giving us optimum yield without generating excess nonspecific products. HotStar Taq Plus DNA polymerase kit reagents (Qiagen, Valencia, CA) were used for PCR. The PCR reaction mixture consisted of 1× PCR buffer, 200 µM of each dNTP, 1× Q-solution, 2.5 units of HotStar Taq Plus DNA polymerase, 60 pmoles of the non-degenerate primer AD2 and 5 pmol of the APHVIII specific primer. Primers were ordered from IDT (Skokie, IL; Table 1). The non-degenerate primer AD2 has a Tm of 46°C while the three APHVIII specific primers used had Tm ranging from 58°C to 64°C. PCR cycling programs were created using the program given in Dent et al. (2005)2. TAIL1 PCR product was diluted 10 and 25-fold and 2 μl of the diluted TAIL1 PCR product was used for TAIL2 PCR reactions. The TAIL2 PCR product was gel purified using QIAEX II gel extraction kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. The purified TAIL2 PCR product was sequenced at the UC, Berkeley DNA Sequencing Facility (Berkeley, CA).\n\nThese primers were used to generate the data in Figure 3 and Figure 4.\n\nPrimers were designed based on genomic DNA sequences available in the Chlamydomonas genome database in Phytozome. Amplifications of genomic DNA and cDNA were executed using MJ Research PTC-200 Peltier Thermal Cycler (Watertown, MA). HotStar Taq Plus DNA polymerase kit (Qiagen, Valencia, CA) was used for PCR following the cycling conditions given in the Qiagen protocol booklet. Annealing temperature was between 55 and 60°C depending on the Tm of the primers. Extension time was varied according to the size of the PCR product amplified. Final extension was set at 72°C for ten minutes. All genomic and reverse transcription PCR products were amplified for a total of thirty-five cycles. 50–150 ng of genomic DNA or cDNA templates were used for PCR reactions. For semi-quantitative RT-PCR using CHLI1 and CHLI2 gene specific primers, 3 μg of total RNA was converted into cDNA and then 150 ng of cDNA templates were used for RT-PCR. Sequences of primers used for genomic and RT-PCR are shown in Table 2, Table 3 and Table 4.\n\nThese primers were used to generate the data in Figure 6A. The gene loci in Phytozome (http://www.phytozome.net/) are: CHLI1 (Cre06.g306300), UP1 (Cre06.g306250), UP2 (Cre06.g306200), UP3 (Cre06.g306150) and UP4 (Cre06.g306100).\n\nThese primers were used to generate the data in Figure 6B and 6C. The gene loci in Phytozome (http://www.phytozome.net/) are: CHLI1 (Cre06.g306300), FDX3 (Cre06.g306350), AMT (g7098), UP5 (Cre06.g306450) and UP6 (Cre06.g306500) and Actin (Cre13.g603700).\n\nThese primers were used in the experiments that generated the data in Figure 7 and Figure 10 and also used for CHLI1 cDNA amplification for cloning.\n\nThe pDBle vector (obtained from Dr. Saul Purton, University College London, UK) was double-digested with restriction enzymes EcoR1 and Nde1 (NEB, Beverly, MA) according to the protocol given in the technical manual. The CHLI1 cDNA template was amplified using primers given in Table 4. Ligation of the double digested (NdeI and EcoR1 digested) CHLI1 cDNA and the NdeI/EcoRI double-digested pDBle vector was done using the T4 ligase and 1 mM ATP (NEB, Beverly, MA). Chemically competent (CaCl2 treated) E. coli cells were used for transformation. After transformation, E. coli cells were plated on LB+ampicillin (100 µg/ml) plates and incubated at 37°C overnight. Single colonies were picked the next day and plasmids were isolated from these clones. Isolated plasmids were double-digested with EcoR1 and Nde1 to verify the cloning of the CHLI1 cDNA. The CHLI1-pDBle construct from the selected clone was sequenced by the UC, Berkeley DNA Sequencing Facility (Berkeley, CA). Chromas Lite (http://technelysium.com.au/) and BLAST were used to analyze DNA sequences.\n\nComplementation of the chli1 was performed utilizing the glass bead transformation technique described by Kindle et al. 198920. 2 µg of the linearized CHLI1-pDBle was used to complement chli1. Transformed cells were plated onto fresh TAP plates containing 15 µg/ml zeocin (Z) and placed in the dark at 25°C. Single colonies were picked and transferred onto fresh TAP+Z plates using a numbered grid template for screening of potential chli1 complements. Screening of chli1 complements was done by visual inspection of green coloration and monitoring growth of light adapted complement strain cells either on TAP in the dark or in the dim light or HS plates under medium light (300 μmol photons m-2s-1).\n\nChlamydomonas cells from different strains grown in TAP in the dark were harvested, washed twice with fresh medium and resuspended in TEN buffer (10 mM Tris-HCl, 10 mM EDTA and 150 mM NaCl; pH 8). Protein concentrations of samples were determined by the method of Lowry et al. (1951)21 with bovine serum albumin as standard. Gel lanes were either loaded with an equal amount of Chl (4 μg Chl) or with 40 μg of protein. Resuspended cell suspension was mixed in a 1:1 ratio with the sample solubilization buffer SDS-urea buffer (150 mM Tris-HCl, pH 6.8; 7% w/v SDS; 10% w/v glycerol; 2 M urea, bromophenol blue and 10% β-mercaptoethanol) and were incubated at room temperature for about thirty minutes, with intermittent vortexing. The sample solubilization buffer was prepared according to the protocol of Smith et al. (1990)22 using reagents from Fisher (Pittsburgh, PA). After incubation, the solubilized protein samples were vortexed and spun at a maximum speed of 20,000 g in a microcentrifuge for five minutes at 4°C. The soluble fraction was loaded on a \"any kD™ Mini-PROTEAN® TGX™ Precast Gel\" (BioRad, Hercules, CA) and SDS-PAGE analysis was performed according to Laemmli (1970)23 using a Page Ruler prestained or unstained protein ladder (Fermentas, Glen Burnie, Maryland) at a constant current of 80 V for 2 hours. Gels were stained with colloidal Coomassie Gel code blue stain reagent (Thermo Fisher Scientific, Rockford, IL) for protein visualization.\n\nElectrophoretic transfer of the SDS-PAGE resolved proteins onto an Immobilon P–PVDF membrane (Millipore, Billerica, MA) was carried out for 2 hours at a constant current of 400 mA in the transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol). The CHLI1 polyclonal antibody was raised in rabbit against the full length Arabidopsis thaliana CHLI1 mature protein that lacks the predicted transit peptide24. This antibody is a gift from Dr. Robert Larkin (Michigan State University). CHLI1 primary antibodies were diluted to a ratio of 1:2,000 before being used as a primary probe. The secondary antibodies used for Western blotting were conjugated to horseradish peroxidase (Pierce protein research product, Thermo Fisher Scientific, Rockford, IL) and diluted to a ratio of 1:20,000 with the antibody buffer. Western blots were developed by using the Supersignal West Pico chemiluminescent substrate kit (Pierce protein research product, Thermo Fisher Scientific, Rockford, IL).\n\nCell density (number of cells per ml of the culture) was calculated by counting the cells using a Neubauer ultraplane hemacytometer (Hausser Scientific, Horsham, PA). Pigments from intact cells were extracted in 80% acetone and cell debris was removed by centrifugation at 10,000 g for 5 minutes. The absorbance of the supernatant was measured with a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer (Brea, CA). Chl a and b concentrations were determined by Arnon (1949)25 equations, with corrections as described by Melis et al. (1987)26.\n\n\nResults\n\nMutant 5A7 was generated by random insertional mutagenesis of the Chlamydomonas reinhardtii wild type strain 4A+ (137c genetic background). 5A7 lacks detectable chlorophyll, appears yellowish-brown in color and grows only under heterotrophic conditions in the dark or in the dim light in the presence of acetate in the growth media (Figure 1). It is incapable of photosynthesis and is sensitive to light intensities higher than 20 μmol photons m-2s-1 (Figure 2).\n\n(A) This figure shows the phenotypic difference of 5A7 compared to the parental strain, 4A+ on heterotrophic/mixotrophic agar media (TAP) plates under five different light conditions: dark + paromomycin (P), dark, very dim light (VDL, 2–4 μmol photons m-2s-1), dim light A (DLA, 10–15 μmol photons m-2s-1) and dim light B (DLB, 20–25 μmol photons m-2s-1). (B) This figure shows the growth phenotype of 5A7 in liquid photo-autotrophic media (HS) under dim light (DL = 10–15 μmol photons m-2s-1).\n\nThe linearized plasmid pBC1 was used to generate 5A7 (Figure 1). To find the insertion of the APHVIII end of the plasmid in 5A7, a modified TAIL (Thermal Asymmetric InterLaced) PCR method was used. Figure 3A shows the position of the vector specific TAIL PCR primers and also shows the arbitrary position of the random non-degenerate primer. A 850 bp DNA product from TAIL2 PCR was purified from the agarose gel (Figure 3B, Table 1). This purified DNA product was used for PCR using internal primers specific to the 3´UTR (UnTranslated Region) of the APHVIII gene. The PCR results confirmed that the 850 bp DNA product contains the 3´UTR of the APHVIII gene (Figure 3C). Sequencing of the 850 bp TAIL2 PCR product revealed that the APHVIII end of the plasmid has been inserted in the fourth exon of a hypothetical gene which we have named as UP6 (Figure 4). UP6 (Cre06.g306500) is located on chromosome 6.\n\n(A) A diagram showing a truncated pBC1 illustrating the APHVIII end of the linearized pBC1 vector. Primers used for PCR are shown by numbered black arrows. Thermal Asymmetric InterLaced 1 (TAIL1) PCR was performed using primer 4R and AD2. (B) TAIL2 PCR was performed using primer 3R and AD2. Lanes 1 and 4 are zero DNA lanes; in lane 2, a 10-fold diluted TAIL1 PCR product was used for TAIL2 PCR; in lane 5, a 25-fold diluted TAIL1 PCR product was used for TAIL2 PCR; lanes 3 and 6 are blank lanes. The 850 bp product used for DNA sequencing is highlighted. (C) Gel purified DNA product (850 bp) from the TAIL2 PCR was used to verify if the product was specific to the APHVIII gene. F and R stand for forward and reverse primers, respectively. AD2 is a non-degenerate primer. PCR primer names are labeled on the top of the gel. In lanes A and B, where triple primers were used for PCR, PCR products are labeled by the corresponding primer combinations that gave rise to the specific product. PCR product sizes are shown beside the primer combinations. All primer sequences are shown in Table 1. ST stands for 1 kb plus ladder (Invitrogen, Carlsbad, CA). DNA samples were run on a 1% agarose gel.\n\nPrimer 2R (Table 1), specific to the 3´UTR of the APHVIII gene was used for sequencing the 850 bp Thermal Asymmetric InterLaced 2 (TAIL2) PCR product. 3´UTR sequence of APHVIII is in bold black, extra nucleotide additions are in bold blue. The flanking Chlamydomonas UP6 genomic sequence is denoted in red. The APHVIII end of the plasmid has been inserted after the eighth nucleotide in the fourth exon of UP6 gene.\n\nFigure 5 shows a schematic map of the UP6 locus with its eight neighboring genes UP4 (Cre06.g306100), UP3 (Cre06.g306150), UP1 (Cre06.g306250), UP2 (Cre06.g306200) CHLI1 (Cre06.g306300), FDX3 (Cre06.g306350), AMT (g7098) and UP5 (Cre06.g306450). It is to be noted that we have named all of these genes arbitrarily for our study except for the CHLI1 and FDX3 genes, which were annotated in the Chlamydomonas genome database. Readers are requested to identify these unknown genes by the gene locus number (Cre or g number) in the Phytozome database. PCR analyses with the genomic DNA of 4A+ and 5A7 were performed using primers specific to four neighboring genes upstream of the CHLI1 (including UP6) and four neighboring genes downstream of the CHLI1 locus (Table 2 and Table 3; Figure 5A and 5B). PCR analyses revealed that all eight genes neighboring the CHLI1 locus were deleted or displaced from their native location (Figure 5A and 5B). UP5 primers gave nonspecific multiple products in 5A7 (Figure 5B). The first two exons of UP6 are present in the 5A7 genome as the UP6 primers spanning the first and the second exon, gave similar genomic DNA PCR product of the expected size as in the 4A+ lane (Figure 5B). Reverse transcription (RT-PCR) analyses using the same UP6 primers on 5A7 and 4A+ cDNA did not yield a PCR product in 5A7 unlike that in 4A+ (Figure 5C; Table 3). This shows that the insertion of the plasmid in the fourth exon of UP6 in 5A7 has hampered the transcription of the UP6 gene.\n\nThe map shows a 35.7 kb genomic DNA region that harbors the UP6 and eight genes located upstream of it. Each arrow represents a gene. The name of the gene is given on top of the arrow. The black numbers on the top of arrows denote sizes of genes (bp) while black numbers below denote distances in between genes (bp).\n\n(A) PCR using the genomic DNA of 5A7 and 4A+ with primers specific to CHLI1 and four neighboring genes (UP1, UP2, UP3, UP4) downstream of the CHLI1 gene. The sizes of the genomic DNA PCR products for CHLI1, UP1, UP2, UP3 and UP4 are, 459, 100, 342, 550 and 672 (bp), respectively. Odd numbered lanes denote 5A7; even numbered lanes denote 4A+; ST denotes 1 kb plus DNA ladder. (B) PCR using the genomic DNA of 5A7 and 4A+ with primers specific to CHLI1 and four neighboring genes (FDX3, AMT, UP5 and UP6) upstream of the CHLI1 gene. The sizes of the genomic DNA PCR products for CHLI1, FDX3, AMT, UP5 and UP6 are, 459, 90, 369, 379 and 369 (bp), respectively. Odd numbered lanes denote 5A7; even numbered lanes denote 4A+; M denotes 50 bp DNA ladder (NEB, Beverly, MA). (C) PCR and RT-PCR with UP6 gene specific primers using the 5A7 and 4A+ genomic DNA and cDNA. Actin was used as a control. Actin genomic and cDNA product sizes are 527 and 305 (bp), respectively. Odd numbered lanes denote genomic DNA PCR products; even numbered lanes denote cDNA products. All primers used spanned an intron. M denotes 50 bp DNA ladder. All DNA samples were run on a 1.8% agarose gel. Gene names are given at the bottom of the gel. Primer sequences are shown in Table 2 and Table 3.\n\nTaken together the data shows that at least a 35,715 bp genomic region has been deleted and or/displaced when the plasmid got inserted in the 5A7 genome. Except for the CHLI1 gene, the functions of the remaining eight genes (including UP6) are not known. We do not yet know the exact location of the pUC origin (pUC ori) end of the plasmid (Figure 1) in the 5A7 genome.\n\nAs CHLI plays a role in Chl biosynthesis, we checked for the presence/absence of the CHLI1 and CHLI2 in 5A7. RT-PCR results show that CHLI1 transcript is absent and CHLI2 transcript is present in 5A7 (Figure 7A, Table 4). Figure 7B shows the presence of the CHLI2 gene in 5A7.\n\n(A) Semi-quantitative RT-PCR analyses of 5A7 and 4A+ using CHLI1 and CHLI2 primers. (B) PCR analyses using 5A7 and 4A+ genomic DNA with CHLI2 gene specific primers. Odd numbered lanes denote 5A7; even numbered lanes denote 4A+. PCR product sizes (bp) are labeled. ST denotes 1 kb plus DNA ladder. All DNA samples were run on a 1.8% agarose gel. Gene names are given at the top of the gel. Primer sequences are shown in Table 4.\n\nWe will be referring to strain 5A7 as chli1 from here onward. As our chli1 lacks Chl and CHLI1 is involved in Chl biosynthesis, we cloned the CHLI1 cDNA in the pDBle vector to transform 5A7 (Figure 8, Table 4). CHLI1 expression is driven by the constitutive PsaD promoter in the CHLI1-pDBle construct (Figure 8). pDBle has two Ble genes that confer resistance to the antibiotic zeocin. Figure 9 shows growth phenotypes of two chli1 complements (chli1-7 and chli1-8); 5A7 and 4A+. chli1 complements are able to synthesize Chl, are not light sensitive and are capable of photosynthesis (Figure 9). As the chli1 complements harbor the Ble gene (from the pDBle vector) and APHVIII gene (derived from the parental strain 5A7), they can grow both on zeocin and paromomycin media plates unlike chli1 and 4A+ (Figure 9).\n\nNdeI/EcoR1 double digested CHLI1 cDNA (1260 bp) was cloned into the NdeI/EcoRI double digested pDBle plasmid. Primers used for amplification of CHLI1 cDNA are shown in Table 4. CHLI1 expression is driven by the constitutive PsaD promoter. NdeI and EcoRI restriction sites are labeled. pDBle contains two copies of BleR genes driven by the Rubisco (RbcS2) promoter. The size of the CHLI1-pDBle construct is 7957 bp. Black arrow and white arrow denotes CHLI1 cDNA and BleR gene, respectively. Grey boxes denote UnTranslated regions (UTR).\n\nchli1-7 and chli1-8 complements were grown with 5A7 and 4A+ under five growth conditions: TAP+Z (zeocin) in the dark, TAP in dim light (DL) (15 µmol photons m-2s-1), TAP+P (paromomycin) in DL, TAP in medium light ML (300 µmol photons m-2s-1) and HS in ML.\n\nChl analyses show that both chli1 complements are about 33–46% Chl deficient. chli1complements have a similar Chl a/b ratio as that of the wild type (Table 5, Data File below). Figure 10A and 10B show a schematic figure of the native Chlamydomonas CHLI1 gene and the trans CHLI1 gene used for complementation, respectively. PCR analyses using the genomic DNA show that the chli1 complements have the trans CHLI1 gene (Figure 10C and 10D). In Figure 10D the genomic DNA PCR product sizes in the two complement lanes are smaller than that in the 4A+ lane as we have cloned the CHLI1 cDNA for complementation. The Chlamydomonas CHLI1 protein has about 71% sequence identity to the Arabidopsis CHLI1 protein. Figure 11A shows a stained protein gel. The two chli1 complements and the 4A+ were loaded on an equal Chl basis in each lane in the protein gel (Figure 11A). As 5A7 lacks Chl, the maximum amount of protein (40 µg) that can be loaded in a mini protein gel, was used (Figure 11A). Light harvesting complex proteins (LHCs) can barely be detected in the chli1 mutant (Figure 11A).\n\nChlorophyll analyses were done on three biological replicates for each strain. Strains were grown mixotrophically in TAP under 15–20 µmol photons m-2s-1. Mean values are shown in the table. Statistical error (± SD) was ≤10% of the values shown. ND: not detected.\n\n(A) A schematic of the native CHLI1 gene. The tan bars denote UnTranslated Regions (UTRs), the white arrows represent exons and the black lines denote introns. (B) A schematic of the CHLI1-pDBle complementation vector containing the CHLI1 cDNA. PsaD promoter, 5´UTR, 3´UTR and CHLI1 specific primers are labeled. (C) Genomic DNA PCR using a PsaD 5´UTR specific primer and a CHLI1 specific primer. Product size: 1272 bp. Lane 1: 5A7; Lane 2: 4A+; Lane 3: chli1-7; Lane 4: chli1-8. ST represents 1 kb plus DNA ladder. (D) Genomic DNA PCR using CHLI1 specific primers. Genomic DNA product size: 459 bp; cDNA product size: 249 bp. Lanes 3 and 4 show smaller PCR products compared to that in lane 2 as cDNA was used for complementation. Lane 1: 5A7; Lane 2: 4A+; Lane 3: chli1-7; Lane 4: chli1-8. ST represents 1 kb plus DNA ladder. All primer sequences are shown in Table 4.\n\n(A) A stained protein gel. Lanes 1, 2, 3 and 4 represent chli1-8, chli1-7, 4A+ and 5A7, respectively. Light harvesting complex (LHC) protein bands are labeled. PS and US denote pre stained and unstained molecular weight protein ladders, respectively. Total cell extract of different strains were loaded on equal Chl basis (4 µg of Chl) in lanes 1, 2 and 3. In lane 4, 40 µg of protein (the maximum amount of protein that can be loaded on a mini protein gel) was loaded as 5A7 lacks Chl. (B) Western analyses using a CHLI1 antibody generated against the Arabidopsis CHLI1 protein. Lanes 1, 2, 3 and 4 represent chli1-8, chli1-7, 4A+ and 5A7, respectively. CHLI1 (40 kDa) and CHLI2 (42 kDa) proteins detected by the antibody are labeled.\n\n\n\nWestern analyses of the two chli1 complements with a CHLI1 antibody show that the CHLI1 protein is absent in the chli1 mutant but present in the chli1 complements (Figure 11B). Western analyses also show that the Arabidopsis CHLI1 antibody detects both the CHLI1 (40 kDa) and CHLI2 (42 kDa) protein in Chlamydomonas as the Chlamydomonas CHLI2 has about 62% sequence identity to the Arabidopsis CHLI1 (Figure 11B). In the wild type the CHLI2 protein amount is much lower than that of CHLI1. As the chli1 complements are Chl deficient compared to the wild type, the two complement lanes show higher amount of protein loadings (Figure 11A). Although more protein was loaded in the 5A7 lane in the protein gel compared to that in the 4A+ and the chli1 complement lanes, the CHLI2 protein was barely detectable in 5A7 (Figure 11B).\n\n\nDiscussion\n\n5A7 is the first chli1 mutant to be identified in C. reinhardtii and in green algae. CHLI1 deletion has affected Chl biosynthesis and photosynthetic growth in the chli1 mutant (Figure 2). Over-accumulation of photo-excitable PPIX leads to photo-oxidative damage to the cells in presence of light and oxygen4–6. The light sensitivity of the chli1 is most probably due to an over-accumulation of PPIX which occurs due to the inactivity of MgChel enzyme which converts PPIX to MgPPIX. Future HPLC (High Performance Liquid Chromatography) analyses of steady state tetrapyrrole intermediates will confirm this hypothesis.\n\nBased on the current molecular analyses, our chli1 mutant has a deletion of at least nine genes (including the CHLI1 gene). Currently we are investigating the exact insertion point of the pUC ori end of the plasmid in the chli1 genome (Figure 1). This will provide us with a precise estimate of the number of gene deletions in chli1. Although complementation of chli1 with the CHLI1 gene restored Chl biosynthesis, tolerance to high light levels and photo-autotrophic growth, chli1 complements are still Chl deficient to some extent (Table 5). This is probably due to a lower expression of the CHLI1 protein in these chli1 complements (Figure 11). Semi-quantitative RT-PCR shows that the CHLI2 transcript level in chli1 is much lower than that in the wild type strain (Figure 7). Western analyses show that the CHLI2 protein level is severely reduced in the chli1 mutant (Figure 11). Real Time PCR analyses can be used to confirm whether the reduction in the CHLI2 protein level is due to a low abundance of the CHLI2 transcript. Additionally, the roles of any of the other missing eight genes in Chl biosynthesis cannot be ruled out as currently the functions of these genes are unknown.\n\nIn Arabidopsis, it has been shown that CHLI2 does play a limited role in Chl biosynthesis in the absence of CHLI112–15. 5A7 possesses an intact CHLI2 gene but the CHLI2 protein is barely detectable in the mutant. This raises two questions:\n\n1) Is the low abundance of the CHLI2 protein a general effect or is it a specific effect of the CHLI1 mutation?\n\n2) Is the total absence of Chl in strain 5A7 due to the specific absence of CHLI1 or due to the absence of both CHLI1 and the near absence of CHLI2 protein?\n\nThe first question can be addressed by performing Western analyses of 5A7 with antibodies raised against any non-photosynthetic and/or photosynthetic protein. If the low abundance of the CHLI2 protein is due to a general effect of the mutation, there will be an overall reduction of different cellular proteins. The second question can be addressed by overexpressing CHLI2 in 5A7 to see if Chl biosynthesis can occur in the absence of CHLI1 or by silencing CHLI2 in the wild type strain using RNA interference or micro RNA based techniques.\n\nNorflurazon (NF) causes photo-oxidative damage to the chloroplast by inhibiting carotenoid biosynthesis.24,27–31. In Arabidopsis MgPPIX is hypothesized to be a retrograde signal from the chloroplast to the nucleus on the basis of data obtained with mutants that are defective in the NF, induced down-regulation of the transcription of the light harvesting complex protein B(LHCB) expression [gun (genomes uncoupled) phenotype]27,28. In Arabidopsis, there are controversies regarding whether chli1 mutants are gun mutants12,29–31. Our chli1 lacks detectable LHC proteins on the stained gel (Figure 11A). LHC transcript and protein analyses in NF treated and untreated cells and HPLC based analyses of steady state tetrapyrroles in chli1 will help to confirm if it is a \"gun\" mutant. In summary, in the future our chli1 mutant can be used to clarify the functional role of CHLI1 and CHLI2 in Chl biosynthesis as well as their roles in retrograde signaling in C. reinhardtii.", "appendix": "Author contributions\n\n\n\nMM and PG conceived the study, designed the experiments and took the lead role in preparing the manuscript. DW generated the mutant 5A7 and isolated genomic DNA from the strains. PG did the PCR analyses, isolated genomic DNA, performed growth and Chl analyses of different strains, prepared cDNA, cloned the CHLI1 gene and complemented the mutant 5A7. JS maintained all the strains and was involved in the mutagenesis experiment. MM, PG, CL, KS, JM, KL, TH and AC maintained cultures, performed the TAIL-PCR analyses, extracted TAIL-PCR product from agarose gels, prepared DNA samples for sequencing and analyzed the DNA sequencing data. MM performed the protein and Western analyses. All authors were involved in the revision of the manuscript draft 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 project was supported by several grants awarded to Dr. Mautusi Mitra. These are: the start-up grant of the University of West Georgia (UWG), the Faculty Research Grant by the UWG College of Science and Mathematics, the Internal Development Grant by the UWG office of Research and Sponsored Project, the Research Incentive grant by the UWG College of Science and Mathematics, the UWG Student Research Assistance Program (SRAP) grant and the UWise-BOR-STEM II grant from UWG.\n\n\nAcknowledgements\n\nWe would like to thank Dr. Krishna K. Niyogi (UC, Berkeley) for providing the 4A+ strain and the pBC1 plasmid that were used for mutagenesis, Dr. Saul Purton (University College London, UK) for providing the complementation vector pDBle and Dr. Robert Larkin (Michigan State University) for giving us the Arabidopsis CHLI antibodies. We are grateful to Dr. Bernhard Grimm and Dr. Pawel Brzezowski (Humboldt University, Berlin, Germany) for providing us the FDX3, UP1, UP2, and AMT gene specific primers for PCR analyses. We would also like to thank Dr. Leos Kral (University of West Georgia) for allowing us to use his nano spectrophotometer and Dr. Anastasios Melis (UC, Berkeley) for allowing us to perform the protein and Western analyses at his laboratory.\n\n\nReferences\n\nMerchant SS, Prochnik SE, Vallon O, et al.: The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science. 2007; 318(5848): 245–250. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "1014", "date": "20 Jun 2013", "name": "David Oppenheimer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describe the isolation and characterization of a mutant of Chlamydomonas reinhardtii deficient in chlorophyll biosynthesis. The authors screened a population of Chalamydomonas that was transformed with an insertional mutagenesis vector. They found a chlorophyll deficient mutant that only grew under heterotrophic conditions. The authors provided convincing evidence that this mutant lacked the CHLI1 gene as well as several flanking genes. The CHLI1 gene encodes a subunit of the enzyme responsible for inserting Mg2+ into protoporphyrin IX. Interestingly, the chli1 mutant also showed decreased expression of a homolog of CHLI1 and CHLI2. The authors also rescued the chli1 mutant phenotype using a wildtype CHLI1 cDNA transgene. This work is important because it is the first report of a chli1 mutant from a green alga. This mutant provides a starting point for additional genetic and biochemical analyses of chlorophyll biosynthesis in Chlamydomonas.\n\nSuggested revisions:The authors should change their mutant name to chli1-1, since it is the first mutant allele isolated. This sets a precedent for naming new alleles as they are discovered. Instead of calling the rescued transformants that contain the wildtype CHLI1 cDNA \"complements\", the authors should consider calling them \"rescued transformants\". Some geneticists adhere to a more strict use of the term \"complementation\" and its derivatives where complementation is done by crossing two mutants, and not by genetic transformation of a mutant using a wildtype gene.", "responses": [] }, { "id": "1023", "date": "24 Jun 2013", "name": "Tatsuru Masuda", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 have successfully identified a Chlamydomonas mutant deficient in chlorophyll biosynthesis using insertional mutagenesis. Through molecular genetic analyses, the authors demonstrated that the deficiency of the CHLI1 gene, which encodes a subunit of Mg-chelatase, caused the chlorophyll-deficient phenotype. Although Chlamydomonas has another homologous CHLI2 gene, this mutant failed to accumulate chlorophylls, suggesting the limited function of CHLI2 on chlorophyll biosynthesis. To clarify whether the reduced expression of CHLI2 caused chlorophyll deficiency, overexpression of CHLI2 in the mutant should give further insights into the functionality of CHLI2. In addition, it is desirable to determine the levels of chlorophyll intermediates that are correlated to the photosensitivity of the mutant. Suggested revisions:Considering the distinct regulation of chlorophyll biosynthesis and nuclear gene expression between Chlamydomonas and Arabidopsis, I am not sure whether this mutant is useful for analysis of the retrograde signaling. Actually, a number of previous papers suggested distinct roles of tetrapyrroles on nuclear gene expression in Chlamydomonas (Kropat et al. 1997, Kropat et al. 2000, Chekounova et al. 2001 and Vos et al. 2011). In this sense, the last paragraph of the Discussion section should be largely revised including already known mechanisms in Chlamydomonas cells and not be so focused on Arabidopsis.", "responses": [] }, { "id": "1027", "date": "26 Jun 2013", "name": "Ruby A. Ynalvez", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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. In addition, the abstract represents a suitable summary of this interesting work. The authors were able to successfully isolate a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis. Using molecular biology techniques they have provided sufficient evidence that the novel mutant is missing a CHLI1 gene and having an intact CHLI2 gene. Their results also provided evidence that CHLI1 in C. reinhardtii is important in chlorophyll biosynthesis and that the presence of its homolog CHLI2 is not sufficient to make up for the function of CHLI1 in chlorophyll biosynthesis. The growth phenotype analysis of the rescued transformants has shown clearly the role of CHLI1 in chlorophyll biosynthesis. It would be interesting to know what percentage of rescued transformants was in their complementation analysis.\n\nOverall this is an interesting article. More importantly, this work reports the first chli1 mutant to be identified in C. reinhardtii, as well as in green algae. The chli1 mutant will open up new avenues to further explore the functional roles of CHLI1 and CHLI2 in chlorophyll biosynthesis in particular in C. reinhardtii. Suggested revisions: On page 6, sentence 2 “5A7 lacks detectable chlorophyll, appears yellowish-brown in color and grows only under heterotrophic conditions in the dark or in the dim light in the presence of acetate in the growth media (Figure 1).” Figure 1 should be Figure 2. On page 6, sentence 3 “It is incapable of photosynthesis and is sensitive to light intensities higher than 20 μmol photons m-2s-1 (Figure 2),” ---can be more clearly referred to as Figure 2A instead of Figure 2 only.\n\nOn page 8, Figures 5A, 5B and 5C mentioned in text actually refers to Figures 6A, 6B and 6C.", "responses": [] } ]	1	https://f1000research.com/articles/2-138
https://f1000research.com/articles/2-163/v1	29 Jul 13	{ "type": "Commentary", "title": "“Don’t forget the migrants”: exploring preparedness and response strategies to combat the potential spread of MERS-CoV virus through migrant workers in Sri Lanka", "authors": [ "Kolitha Wickramage", "Sharika Peiris", "Suneth B Agampodi", "Kolitha Wickramage", "Sharika Peiris" ], "abstract": "From September 2012 to July 2013, 81 laboratory-confirmed cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV), including 45 deaths (a case fatality ratio of 55%) have been reported from eight countries. Human-to-human transmission is now confirmed showing potential for another pandemic of zoonotic disease, with an extremely high mortality rate. Effective surveillance strategies are required in countries with a high influx of migrants from the Middle East to mitigate the probable importation of MERS-CoV. We discuss here the risk of MERS-CoV in major labor sending countries and list the probable strategies for control and prevention of MERS-CoV using Sri Lanka as an example. It is conservatively estimated that 10% of Sri Lanka’s population work as international labor migrants (1.8 to 2 million workers), with 93% residing in the Middle East. An average of 720 workers depart each day, with the majority of these workers (71%) departing to the Kingdom of Saudi Arabia (the country with 81.5% of total MERS-CoV cases). We also describe other inbound migration categories such as tourists and resident visa holders relevant to the context of preparedness and planning. The importance of partnerships between public health authorities at national and regional levels with labor migration networks to establish institutional and/or policy mechanisms are highlighted for ensuring effective preparedness and response planning. Strategies that can be taken by public health authorities working in both labor sending and labor receiving counties are also described. The strategies described here may be useful for other labor sending country contexts in Asia with a high frequency and volume of migrant workers to and from the Gulf region.", "keywords": [ "coronavirus", "Middle East", "Sri Lanka", "South Asia", "preparedness", "planning", "migrants" ], "content": "Introduction\n\nThe global health community is experiencing one of the deadliest coronavirus outbreaks that has been reported in recent times. The first case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection was reported in September 2012 from the Kingdom of Saudi Arabia (KSA)1. Since then, 81 laboratory-confirmed cases of infection with 45 deaths were reported by eight countries, of which 66 (81.5%) were from the KSA2 (Table 1). Even though France, Germany, Italy, Tunisia and the United Kingdom have also reported laboratory-confirmed cases, these patients had been either transferred to these countries from hospitals in the Middle East for specialist care or had returned from the Middle East and subsequently became ill. Hitherto, there have been no cases reported in Asia.\n\nCoronaviruses have long been known to cause widespread human infections such as the common cold and global pandemics such as severe acute respiratory syndrome (SARS)3. MERS-CoV has not been identified previously among humans4, thus knowledge about the natural history of the disease is still limited. The clinical syndrome of MERS-CoV is primarily a respiratory disease including fever, cough and shortness of breath, resembling SARS. More than half of cases develop life threatening complications, such as respiratory failure5,6, acute respiratory distress syndrome (ARDS)6–8, renal failure4–6,8, and consumptive coagulopathy8. Studies of clusters of cases suggest that the spread may occur by both large and small aerosols and possibly via the faecal-oral route9. The pathogenesis of MERS-CoV is not fully understood. It appears to cause respiratory problems by attacking and infecting the cells in the nasopharynx; laboratory studies show that the virus has the ability to cause profound apoptosis of human bronchial epithelial cells10. All confirmed cases have had respiratory disease and most have developed pneumonia11. Complications during the course of illness have included severe pneumonia with respiratory failure requiring mechanical ventilations, ARDS with multi-organ failure, renal failure requiring dialysis, consumptive coagulopathy and pericarditis11. Hitherto, 45 out of 81 cases (55%) have died as a result of infection (Table 1). The rapid transmission and high attack rate in hospital settings have raised concerns about the risk of health care associated transmission of this virus12.\n\nAlthough the transmission of the disease is still not as rapid as seen during the SARS epidemic in 200313, human to human transmission of MRES-CoV has now been established5. Given the high case fatality rate compared to previous coronavirus pandemics, continued risk assessment, surveillance, and preparedness measures by all countries are required to minimize the impact of a probable global pandemic of MERS. The WHO encourages “all Member States to continue their surveillance for severe acute respiratory infections (SARI) and to carefully review any unusual pattern”2.\n\nThe annual Hajj pilgrimage, attended by 3 million pilgrims from all over the globe, has been identified as a potential threat for major spread14. A recent study has shown evidence of rapid acquisition of respiratory viruses among pilgrims during their stay during the Hajj in the KSA, most notably rhinovirus14,15. The authors highlight the potential of spreading these infections in the pilgrims' home countries upon their return. Memish and colleagues also suggest a ‘high degree of clinical vigilance’ required for the possibility of MERS-CoV infection in patients with respiratory infections who have visited the Middle East in the preceding 10 days6. Despite these concerns, the WHO does not recommend changing travel plans for Hajj or Umrah because of MERS-CoV. However, at a recent meeting organized by the WHO in Cairo (June, 2013), public health officials specifically emphasized the importance of preparedness and response at Hajj and contexts of mass gatherings ‘as a priority action’, with Member States of WHO agreeing to develop specific plans for MERS-CoV16. No emphasis at this meeting or in peer-reviewed literature has been made in relation to the large volumes and frequent travel patterns of international labor migrant workers to the Middle Eastern countries, especially from Asia17.\n\n\nInternational labor migrants in the Middle Eastern region\n\nLabor migration from Asia to the Middle East involves the movement of contractual workers from many ‘labor sending' nations such as the Philippines, India, Sri Lanka and Indonesia, to ‘labor receiving' ones, mainly within the Middle Eastern region18. Estimates of total migrant workers by the International Labor Organization for 2010 were 105.5 million, 30 million of which were from within Asia19. It is estimated that there is a net annual outflow of two million migrant workers from the ‘top five’ South Asian labor sending countries of Sri Lanka, India, Bangladesh, Nepal and Pakistan20 (Table 2). Unregistered ‘irregular’ migrant workers also contribute to this outflow of contractual workers from Asia, although estimates are difficult to assess due to the clandestine nature of their travel. It is important to highlight that remittance from labor migrants contribute significantly to the economic growth of most developing countries in Asia. The Sri Lankan economy is highly dependent on foreign exchange earnings from its migrant workforce, with remittance from workers in Middle Eastern countries alone contributing 58.9% of all total foreign exchange earned in 201121.\n\n\nPreparedness measures and screening strategies relevant to Sri Lanka\n\nAlthough the WHO has not yet issued a travel health warning for any country, nor recommended conducting on-arrival screenings at ports of entry, the infectious nature of MERS-CoV means that there is a risk of contracting the disease through infected individuals who have visited the Middle East in the preceding 10 to 14 days. Health authorities in some countries in the region have already begun making advanced arrangements for the diagnostic test kits developed by the CDC for MERS-CoV to be made available to National Reference Laboratories16. Ensuring guidance for health care professionals regarding case definition, diagnosis and management for MERS-CoV infection, and establishing an active surveillance system for ‘influenza-like’ illnesses in hospitals are essential steps for surveillance. Elaborating pandemic preparedness and response measures are not the focus of this current paper since these have already been well described and indeed established in Sri Lanka through previous efforts against SARS and H1N122. Rather, this article will focus on understanding the importance of the large volumes of migration categories and their dynamics, which may yield more specific and targeted public health and screening interventions for MERS-CoV.\n\n\nInbound migration categories to Sri Lanka from the Middle East region\n\nInbound migration refers to the flow of persons traveling into a country23. We identify five major inbound migrant flows from the Middle East to Sri Lanka with the potential of introducing MERS-CoV (Figure 1).\n\nThis figure shows the different categories of inbound travelers arriving at the Bandaranaike International Airport, Sri Lanka.\n\nKSA, Qatar, Kuwait, UAE and Jordan are the major destination (labor receiving) countries, encompassing 85% of Sri Lanka’s total international labor migrant force (1.8 to 2 million workers in 2011)21. Each day, around 720 migrant workers leave Sri Lanka to the Middle East as labor migrants through Bandaranaike International airport24. Over 93% of the 262,960 labor migrants were employed in Middle Eastern countries in the year 2011 (Table 2). Female participation in foreign employment is 48.3% of the total departures during the same year, and 85% of them worked as domestic housemaid25. The recent evidence of virus spreading within family clusters may be a significant factor in determining household transmission6.\n\nData on patterns of returning migrant workers are not available since there is no registry of returning workers. However, inflow is expected to be greater than outflow considering both the cyclical nature of labor migration (where a worker usually returns to the country for a short period before departing again - a cycle which can last 10 years or more), and the large stock total of formally registered workers from Sri Lanka.\n\nEvery year, Muslims from all over the world converge in KSA to take part in the annual Hajj (pilgrimage). KSA hosted 2.5 million pilgrims in 2009 amidst the H1N1 pandemic27. In 2013, the Hajj is expected to fall between the 13–18 October. A quota system operates to limit the number of people from each country visiting Mecca each year based on the number of Muslims in each country. The Sri Lankan quota for 2013 is currently set at 2,80028.\n\nA residence visa is a permit for non-Sri Lankan citizens to obtain residence facilities for purposes of long stays, work and study. The numbers of both residency visa holders and tourists visiting Sri Lanka from the Middle East, disaggregated by country of residence, are shown in Table 3. Both KSA and the UAE remain the primary source countries of migrants within this inbound category.\n\n*Others: Yemen, Cyprus, Iraq, Palestine and Syria.\n\nIf a highly conservative estimate on the number of labor migrants returning from the Middle East is placed at 220,000 persons per year, then based on data from the five major categories of migrant flows presented here, an estimated 280,901 persons will travel from the Middle East to Sri Lanka. This number does not account for the number of returning Sri Lankan tourists and irregular migrants from the Middle Eastern region. Based on the fact that 71% of the current caseload of Sri Lankan migrant workers depart for the KSA, it is expected that the majority of inbound migrants will be traveling from the same country.\n\n\nPreparedness and response strategies for labor migrants\n\nIt is important to note that the following recommendations are suggested as a way of enhancing, not substituting, existing frameworks on pandemic disaster preparedness and response. There are currently no established guidelines for MERS-CoV established at country level, unlike in other settings29.\n\nA number of prevention and screening strategies for migrant workers are presented here, classified according to the three phases of migration: ‘pre-departure’ (departing Sri Lanka), ‘at destination’ (time spent in the Gulf States) and ‘upon-arrival’ (arrival back in Sri Lanka). Each stage in the migration cycle offers unique opportunities for public health action/intervention based on enabling mechanisms and capacities harnessed in routine migrant worker pathways (Figure 2). These may be useful in refining into other country contexts.\n\nPotential places for interventions (intervention space) in relation to labor migration.\n\nA. Strategies at the 'pre-departure’ phase. The majority of labor receiving countries require pre-departure health assessment as a pre-requisite for a work visa. Migrant workers to Gulf State countries are expected to undertake a mandatory pre-departure medical examination in Sri Lanka to ensure their ‘fitness to travel’ and fulfillment of health assessment criteria set by the recipient country. Health care workers could provide health information on MERS-CoV to potential migrant workers during the medical examination. The Gulf-Approved Medical Centers Association (GAMCA) has a network of 13 private medical centers in Sri Lanka, which are accredited to conduct health assessments of Sri Lankan migrant workers prior to departure to the GAMCA countries KSA, Kuwait, Bahrain, Qatar, UAE and Oman. As a preparedness measure, medical staff at these health assessment centers can be trained with up-to date information on MERS-CoV and be encouraged to disseminate language specific information-exchange communication (IEC) materials on signs, symptoms and preventative actions for the migrant worker30.\n\nB. Strategies at the ‘destination’ phase. Sri Lankan embassies and diplomatic missions at destination countries could disseminate public health service messages in relation to MERS-CoV in Singhalese/Tamil languages via embassy welfare programs, social networks and through ethno-specific radio programs. It is vital that local health authorities and employers provide access for migrant workers to seek primary health care and that they are supported with specialized/referral care within the health system in the Gulf States. The importance of health accessibility, irrespective of visa status, for migrant workers to primary and specialized health care facilities in these destination countries also needs to be emphasized through state-to-state and regional advocacy mechanisms. It is recommended that public health authorities and global bodies such as the WHO and the International Organization for Migration utilize the support of existing inter-regional and trans-national migrant worker networks such as the members of the ‘Colombo process’ and ‘Abu-Dhabi process’ in order to promote effective public health messages and strategies31.\n\nC. Strategies at the ‘on-arrival’ phase. The Sri Lanka Bureau of Foreign Employment (SLFBE) which provides policy direction and regulation of labor migrants has a dedicated 24-hour administrative desk at Sri Lanka’s Bandaranaike International Airport, to manage grievances from returning migrant workers. A worker welfare center to house migrant workers in need of support managed by the SLFBE is also available near the airport. Currently there are no medical personnel attached to the SLFBE services for on-arrival phase. It is recommended that the Ministry of Health make arrangements to establish a coordination mechanism with the SLFBE and with airport health authorities, which currently have no linkage to migrant worker programs. A rotating roster of trained health professionals allocated at the health center at the airport could ensure each returning worker completes the rapid symptom checklist (see assessment algorithm in Figure 3). The algorithm was developed after augmenting the guidance frameworks for MERS-CoV created by the public health authorities in Canada29 and the CDC32. It is important for port health authorities to also build effective partnerships and protocols with immigration control officers at ‘on arrival counters’. This will ensure referral of travelers returning from the Middle East where cases of MERS-CoV have been reported to the health screening desk. Leaflets advising travelers of symptoms of the influenza-like illness could also be distributed at the immigration counter to arriving passengers.\n\n1Immigration Counter Referral for those migrant workers returning from Arabian peninsula and neighboring Middle East countries (Saudi Arabia, Qatar, Jordan, United Arab Emirates, Bahrain, Iran, Iraq, Israel, Kuwait, Lebanon, Oman, Palestinian Territories, Yemen, Syria). Referral to Airport health unit may also be directed from the SLFBE migrant worker arrival desk.\n\n2Acute Respiratory Infection (ARI): Any new onset acute respiratory infection that could potentially be spread by the droplet route (either upper or lower respiratory tract), which presents with symptoms of a new or worsening cough or shortness of breath and often fever (>38°Celsius).\n\n3This token will identify the migrant worker as a susceptible person for MERS-CoV.\n\nManaging risk communication also forms a vital strategy for any form of public health preparedness and response. Studies have shown that when responding to a novel infectious disease outbreak, policy and planning decisions can limit the ability to control the outbreak and result in unintended consequences including lack of public confidence33. Communication of risk to target populations needs to be carefully planned to avert maladaptive behaviors due to fear and defensive avoidance (the motivated resistance to the message, such as denial or minimization of the threat34). Individuals may defensively avoid a message by being inattentive to the communication (e.g., looking away from the message), or by suppressing any thoughts about the threat over the long term. Mitigating such threats through targeted communication strategies to migrant workers and other categories such as those described above may be useful35. The strategies outlined above do not warrant large scale ‘national level’ awareness campaigns, which may exacerbate anxiety and induce maladaptive rather than positive health seeking behaviors36.\n\n\nConclusion\n\nIt has been one year since MERS-CoV was discovered, yet many questions remain unanswered about its pathogenesis, host reservoirs and transmission dynamics. What is clear from global health authorities is that countries need to plan for preparedness and response planning29. We recommend partnerships between public health authorities, at national and regional levels, with the labor migration industry and migrant worker networks in establishing both institutional and policy mechanisms to ensure effective preparedness and response planning in response to a potential MERS-COV threat through labor migrants from South Asia.", "appendix": "Author contributions\n\n\n\nKW conceived the paper and drafted the first version of the manuscript. SP contributed in the concept and manuscript preparation. SBA revised and edited and finalized the manuscript for submission.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBermingham A, Chand MA, Brown CS, et al.: Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September 2012. Euro Surveill. 2012; 17(40): 20290. PubMed Abstract\n\nWorld Health Organization. Global Alert and Response (GAR) Middle East respiratory syndrome coronavirus (MERS-CoV) - update.2013. Reference Source\n\nWeiss SR, Navas-Martin S: Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev. 2005; 69(4): 635–664. 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N Engl J Med. 2013. PubMed Abstract \| Publisher Full Text\n\nPeiris JS, Chu CM, Cheng VC, et al.: Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet. 2003; 361(9371): 1767–1772. PubMed Abstract \| Publisher Full Text\n\nBenkouiten S, Charrel R, Belhouchat K, et al.: Circulation of respiratory viruses among pilgrims during the 2012 Hajj pilgrimage. Clin Infect Dis. 2013. PubMed Abstract \| Publisher Full Text\n\nAl-Tawfiq JA, Zumla A, Memish ZA: Respiratory tract infections during the annual Hajj: potential risks and mitigation strategies. Curr Opin Pulm Med. 2013; 19(3): 192–197. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization. Regional Office for the Eastern Mediterranean. Public health officials agree priority actions to detect and control MERS-CoV.2013. Reference Source\n\nInternational Organization for Migration. Middle East. 2013. Reference Source\n\nKaur A: International labour migration dynamics and inequality in Southeast Asia, in Migration and Inequality T. Bastia, Editor. 2013, Routledge: Oxon.63–92. Reference Source\n\nGabriel C, Pellerin H: Governing International Labour Migration: Current. Issues, Challenges and Dilemmas. Routledge/RIPE Studies in Global Political Economy, ed. I.B. Jacqueline Best, Paul Langley, Anna Leander. Routledge: Oxon. 2008; 26. Reference Source\n\nBaruah N: Trends and Outlook for Labour Migration in Asia, in The 3rd ADBI-OECD-ILO Roundtable on Labour Migration in Asia. Assessing Labour Market Requirements for Foreign Workers and Developing Policies for Regional Skills Mobility. ; Bangkok, Thailand. 2013. Reference Source\n\nSri Lanka Bureau of Foreign Employment Annual statistics report on foreign employment, Sri Lanka Bureau of Foreign Employment: Battaramulla. 2011. Reference Source\n\nEpidemiology Unit. Information on Influenza.2013. Reference Source\n\nMinistry of Health, National Migration Health Policy (Draft).2013; Colombo.\n\nSri Lanka Bureau of Foreign Employment, Annual statistics report on foreign employment. Sri Lanka Bureau of Foreign Employment: Battaramulla. 2010p. 5. Reference Source\n\nSri Lanka Bureau of Foreign Employment, Annual statistics report on foreign employment. Sri Lanka Bureau of Foreign Employment: Battaramulla. 2010p. 19–20. Reference Source\n\nAsian Development Bank Institute and Organization for Economic Coorporation and Development, Managing Migration to Support Inclusive and Sustainable Growth Tokyo: Asian Development Bank Institute 56. 2013Reference Source\n\nOmar SR, Afifi RM: A Mass Gathering Experience at the 2009 Pilgrimage in Makkah, Saudi Arabia, During the 2009 Novel Influenza A (H1N1) Pandemic. Journal of American Science. 2013; 9(4): 563–571. Reference Source\n\nNo increase in Sri Lanka Haj quota, in Daily Mirror. Wijaya Newspaper ltd: Colombo 2013. Reference Source\n\nProvincial Infectious Diseases Advisory Committee, Tools for Preparedness: Triage, Screening and Patient Management for MERS-CoV Infections in Acute Care Settings. O.A.f.H.P.a. Promotion, Editor. Queen’s Printer for Ontario: Ontario 2013; 1–9. Reference Source\n\nCenter for Disease Control. MERS-Frequently Asked Questions. 2013. Reference Source\n\nInternational Organization for Migration. Regional Consultative Process (RCP) on the management of overseas employment and contractual labor for countries of origin in Asia.2013. Reference Source\n\nCenter for Disease Control. Interim Infection Prevention and Control Recommendations for Hospitalized Patients with Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Middle East Respiratory Syndrome 2013. Reference Source\n\nRosella LC, Wilson K, Crowcroft NS, et al.: Pandemic H1N1 in Canada and the use of evidence in developing public health policies – A policy analysis. Soc Sci Med. 2013; 83: 1–9. PubMed Abstract \| Publisher Full Text\n\nWitte K, Allen M: A meta-analysis of fear appeals: implications for effective public health campaigns. Health Educ Behav. 2000; 27(5): 591–615. PubMed Abstract \| Publisher Full Text\n\nWitte K, Allen M: A Meta-Analysis of Fear Appeals: Implications for Effective Public Health Campaigns. Health Educ Behav. 2000; 27(5): 591–615. PubMed Abstract \| Publisher Full Text\n\nSmith RD: Responding to global infectious disease outbreaks: lessons from SARS on the role of risk perception, communication and management. Soc Sci Med. 2006; 63(12): 3113–3123. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1251", "date": "06 Aug 2013", "name": "Kayvon Modjarrad", "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 current manuscript, submitted by Wickramage et al., comments on the potentially important role migrant workers may play in the evolving MERS-CoV epidemic. The authors focus most of their discussion on laborers traveling between Sri Lanka and countries bordering the Persian Gulf where most MERS cases have been identified. The authors raise a couple of important concerns: 1) Migrant laborers could serve as a major vehicle for the international spread of the MERS coronavirus; 2) MERS-specific surveillance strategies are needed, particularly for Southeast Asian countries that receive large numbers of returning laborers from the Middle East.\n\nThese points can be presented, however, with much greater clarity and brevity. The authors occasionally use hyperbole and repetition to make misleading assertions. For example, \"the extremely high\" mortality rate they cite has been falling as more cases are identified and will likely continue along this trend as milder cases are discovered. Claims of \"rapid transmission\" and a \"high attack rate\" are made without any epidemiologic basis or support from literature. Throughout the commentary, the authors also make redundant, disorganized, and extra-contextual statements about MERS clinical features, labor sending/receiving nations, the Hajj pilgrimage, and strategies for controlling a potential MERS epidemic. The only way this manuscript can be salvaged is if it is refashioned as a brief communication of no more than a few hundred words with one figure or table. The manuscript should be narrowly focused on migrant workers between Gulf States and Southeast Asia and the threat they may pose to the spread of the virus. The additional commentary on screening and surveillance adds nothing to the existing WHO report and essentially boils down to education, awareness, and appropriate referral. The following are specific points of concern:Instead of presenting the number of cases and deaths in each country in tabular format, the authors should provide a link to WHO’s running tally as the authors numbers will be (as they are now) inaccurate and out-of-date. Remove alarmist statements such as “extremely high mortality rate”, “deadliest coronavirus outbreaks” and platitudes such as “the infectious nature of MERS-CoV means that there is a risk of contracting the disease through infected individuals”. I’m confused by the last sentence of the introduction’s first paragraph: “there have been no cases reported in Asia.” The second paragraph of the introduction repeats the clinical features of MERS. This should be corrected. The Hajj pilgrimage is discussed briefly in the introduction, dropped, and then brought up again without any context later in the manuscript. The same is true regarding labor migration patterns and surveillance strategies. There is no real coherent flow to any of these topics.Figure 1 and 2 - no information to what is stated in the text. The terms created for the different phases of labor migration cycle make no sense. To the best of my understanding, “pre-departure” means departure and “upon-arrival” means return. These terms are superfluous and are more jargon than they are informative. Figure 3 is unclear. Is this for anybody returning from a Gulf State or only those returning with symptoms? The first diamond makes this unclear.", "responses": [] }, { "id": "1250", "date": "19 Aug 2013", "name": "Christian Gericke", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWell written paper the spreading potential of MERS-CoV through migration in Sri Lanka.", "responses": [] }, { "id": "2374", "date": "11 Nov 2013", "name": "Maria van Kerkhove", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 edit that I would recomend for this article; Table 1 is not necessary and can be removed.", "responses": [] } ]	1	https://f1000research.com/articles/2-163
https://f1000research.com/articles/2-162/v1	29 Jul 13	{ "type": "Method Article", "title": "A toolkit for transgenesis at the ROSA26 locus by recombinase-mediated cassette exchange", "authors": [ "Kristina Williams", "Xiaoyun Zhao", "Wallace S Chick", "Kristina Williams", "Xiaoyun Zhao" ], "abstract": "We describe a toolkit to perform transgenesis at the ROSA26 locus by recombinase-mediated cassette exchange that will eliminate the inherent problem of random insertion via traditional pronuclear injection. A recombination site-tagged embryonic stem (ES) cell line and two cloning vectors were constructed to facilitate the generation of targeted ES cells with the transgene of interest at ROSA26. The experimental procedure is simple and efficient, and can be readily adapted in many laboratories for rapid generation of transgenic mice.", "keywords": [ "Recombinase mediated cassette exchange", "RMCE", "ROSA26", "ES cells", "Knock in", "transgenic mouse", "gene targeting", "GFP" ], "content": "Introduction\n\nTransgenic mice created by pronuclear injection of a transgene have been a valuable resource for studying gene effects in a whole animal context, albeit there are several pitfalls of this technique. A well-known drawback was the randomness of transgene insertion where an endogenous gene or important regulatory element might be disrupted, resulting in a phenotype independent of the transgene of interest. Usually, multiple founders had to be tested and characterized before a clean transgenic mouse line could be established. To circumvent the inherent defect of this type of passive transgenesis, several approaches have been developed1 that allow transgenes to be inserted at defined genomic loci, such as ROSA26. These active forms of transgenesis can be achieved by utilizing a site-specific recombination system2 to facilitate the knock-in of the gene of interest. We have developed a toolkit (a ROSA26-tagged mouse embryonic stem (ES) cell line and two exchange vectors) for efficient gene knock-in at the ROSA26 locus in mouse ES cells by recombinase-mediated cassette exchange (RMCE). Standard microinjection of the knock-in ES cells would subsequently produce a chimera and the knock-in mice expressing the gene of interest, constitutively or conditionally, depending on which exchange vector is used.\n\n\nMethod\n\nFirst, we generated a mouse ES cell line, designated as R26FNF3-1F1 (1F1), in which the ROSA26 allele was targeted with a FRT and F3 flanked phosphoglycerate kinase (PGK) promoter and neomycin (Neo) selection cassette by homologous recombination in EC7.1 ES cells (Figure 1A), a hybrid strain with a background of C57BL/6 and 129X1/SvJ3. A targeting construct was made by the subcloning of a 5.8 kb BamHI fragment from the BAC clone RP23-401D9; a FRT-PGK-neo-F3 cassette amplified from PL451 (a gift from NCI Frederick) was inserted into the XbaI site so that the left and right homologous arms were both 2.9 kb in length. The PGK-neo marker was cloned in an opposite direction with respect to the ROSA26 locus. EC7.1 ES cells (5 x 106)3 were electroporated with 20 µg of linearized targeting construct and a total of 192 clones were screened for the homologous recombination event by long-range PCR. The PCR products were digested with specific enzymes (EcoRV for the left arm and ScaI for the right arm) to confirm the identities. A total of 7 targeted clones were recovered; clone R26FNF3-1F1 was established and was used in the subsequent experiments.\n\n(A) Targeting the ROSA26 allele. The ROSA26 allele was targeted with a FRT and F3 flanked PGK promoter and neomycin selection cassette by homologous recombination in EC7.1 embryonic stem (ES) cells. Clones were screened for the homologous recombination event by long-range PCR. The PCR products were digested with specific enzymes (EcoRV for the left arm and ScaI for the right arm) to confirm the identities. (B) Cloning vectors for the generation of the recombination cassette. pF-WLPLF3: a transgene could be cloned at the Apal or BamHI site in a way that the downstream WPRE-pA will allow proper transcription. The floxed PGK-puro selection marker could be removed from the targeted allele after recombination if so desired. pFLSL-WF3: a transgene could be cloned at the NotI site which is downstream of a floxed-STOP sequence (a tandem array of four SV40 polyA signals). The transgene will express once the STOP sequence is deleted by Cre-recombinase.\n\nTo facilitate the cloning of the transgene, we made two exchange vectors; one for constitutive expression (pF-WLPLF3), and another for conditional expression, mediated by the cre/loxP system (pFLSL-WF3) (Figure 1B). A woodchuck hepatitis post-transcriptional regulatory element (WPRE) was also incorporated to enhance transgene expression. Both vectors have the recombination sites FRT and F3 flanking the transgene cassette, and in the case of pFLSL-WF3, a floxed-STOP cassette was inserted upstream of the transgene to prevent expression until a cre-mediated deletion of the STOP cassette had taken place, allowing spatial and temporal control of expression4,5.\n\nThe 1F1 ES cells were cultured with primary mouse embryonic fibroblasts (PMEF) (Millipore, Billerica, MA) as feeders in KnockOut DMEM (Life Technologies, Carlsbad, CA) supplemented with 15% FBS (Tissue Culture Biologicals, Los Alamitos, CA) 1,000 U/ml LIF (ESGRO) (Millipore, Billerica, MA), 100 µM non-essential amino acids (Life Technologies, Carlsbad, CA), 2 mM GlutaMAX (Life Technologies, Carlsbad, CA), 55 µM 2-mercaptoethanol (Life Technologies, Carlsbad, CA), and 25 U Pen/Strep (Life Technologies, Carlsbad, CA), at 37°C and 5% CO2. They were passaged one-tenth when confluent, typically every two days. To illustrate the efficiency of RMCE in knocking-in and expressing a green fluorescent protein (GFP) marker, we transfected 5 x 106 1F1 ES cells by electroporation with 15 µg of pF-GFP-WLPLF3 and 15 µg of pCAG-Flpe6 (Addgene plasmid 13787). Electroporation was carried out in a 4 mm cuvette on the Gene Pulser Xcell (Bio-Rad, Hercules, CA) at 250 volts and 500 µF capacitance. The transformants were plated onto three 100 mm dishes containing DR4 mouse fibroblasts as feeders (Applied Stem Cell, Menlo Park, CA); puromycin (1 µg/ml) (Sigma-Aldrich, St. Louis, MO) was applied 24 hr post-plating. Resistant clones were allowed to grow for seven days after which a total of 96 individual colonies were hand picked, trypsinized, and cultured in a 96-well plate. We made replica plates when the colonies were confluent so that one set of cells was cryo-preserved in 10% DMSO (Sigma-Aldrich, St. Louis, MO) at -80°C, one set was subjected to a G418 (250 µg/ml) (Life Technologies, Carlsbad, CA) sensitivity test, and another set of cells was lysed to isolate genomic DNA for genotyping analysis7. From the 96 clones tested, we found 8 clones that were G418 sensitive (Figure 2B), indicating loss of the original neo cassette, through the exchange of the transgene cassette (Figure 2A). The clones that were puromycin resistant but still retained G418 resistance should be clones that had random integration of the transgene cassette without displacing the neo cassette at the ROSA26 locus. The exchange was further confirmed by PCR analysis (Figure 2C). Briefly, a pair of primers annealing to the upstream sequence of the ROSA26 locus and the GFP (arrows indicated in Figure 2A) were employed to amplify the cassette exchange product. We then analyzed GFP expression of the knock-in as well as a randomly picked background clone (PuroR G418R). By using fluorescent microscopy on a Axiovert 200M microscope (Zeiss, Jena, Germany) on live ES cells, we confirmed two of the selected knock-in clones (B7 and B12) exhibited GFP expression while the background clone D5 did not (Figure 2D), suggesting that the knock-in GFP marker at the ROSA26 locus was functionally expressed.\n\n(A) Schematic of RMCE. Circular plasmids of the exchange vector pF-GFP-WLPLF3 and the pCAG-Flp expression vector were transfected into 1F1 embryonic stem (ES) cells. Recombinants were selected for puromycin resistance; each individual clone was then seeded onto the well of a 96-well plate. (B) G418 sensitivity test. The black wells represent those ES cell clones that were killed by G418 (PuroR G418S), indicating the loss of the originally tagged neo marker. (C) Genotyping analysis for the RMCE event. ES cells were analyzed by PCR (the primers are represented by the arrows in Figure 2A) for the correct integration of GFP into the ROSA26 allele. (D) Expression of GFP. Knock-in ES cell clone B7 is expressing GFP as observed live under a fluorescent microscope. The background clone D5 (PuroR G418R) that has a random integration does not have GFP expressed.\n\n\nSummary\n\nIn summary, we present here a protocol and the associated reagents required for a very efficient gene knock-in at the ROSA26 locus in mouse ES cells using recombinase-mediated cassette exchange. Two cloning vectors are described for the generation of a transgene exchange cassette in a single cloning step. The timeline from transfection of ES cells to the isolation of recombinant clones is approximately two to three weeks. We observed that the recombination rate of RMCE was quite high so that recombinants could be isolated easily. For the GFP transgene we observed an 8% recombination rate. Ninety-six clones of two other RMCE transgenesis (Pik3r1 and NpHR-eYFP) generated by the same approach were screened (by PCR and the G418 sensitivity test) as described above, yielding 12% and 31% recombination rates, respectively, suggesting that no more than a single 96-well plate of clones need to be harvested and analyzed. Moreover, the screening of recombinants was straightforward and easy to interpret. Unlike gene targeting by homologous recombination, the PCR genotyping for RMCE events in these experiments was very efficient because of the small size of amplicons (~500 bp). As a side note, we observed that PCR was optimal when using DMSO (2.5%) and a reduced Mg2+ concentration (1 mM) in the PCR buffer, an observation we encountered whenever we attempted to amplify at the ROSA26 locus. In parallel, the G418 sensitivity test served as confirmatory evidence of gene exchange. Overall, we found that the experimental procedure is simple to perform and exhibits high efficiency. It could easily be adopted by other laboratories. The 1F1 ES cells were derived from a F1 hybrid strain with a background of C57BL/6 and 129X1/SvJ. Our experience with these hybrid ES cells is that they contribute to the germline efficiently, even after a variety of in vitro manipulations3,8. The FRT-neo-F3 cassette was targeted to the C57BL/6 allele into which the transgene will be exchanged; mice derived could then be backcrossed to attain a pure C57BL/6 background which, depending on the intended mouse study, might be desirable.", "appendix": "Author contributions\n\n\n\nKristina Williams: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing. Xiaoyun Zhao: Collection and assembly of data, manuscript writing. Wallace Chick: Conception and design, financial support, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is supported by the National institute of Health (NIH), Rocky Mountain Neurological Disorders core grant P30 NS048154. This paper is subject to the NIH Public Access Policy.\n\n\nAcknowledgements\n\nWe would like to thank Ms Irene Choi for her proofreading of the manuscript.\n\n\nReferences\n\nShinohara ET, Kaminski JM, Segal DJ, et al.: Active integration: new strategies for transgenesis. Transgenic Res. 2007; 16(3): 333–9. PubMed Abstract \| Publisher Full Text\n\nAraki K, Araki M, Yamamura K: Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites. Nucleic Acids Res. 2002; 30(19): e103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChick WS, Mentzer SE, Carpenter DA, et al.: X-ray-induced deletion complexes in embryonic stem cells on mouse chromosome 15. Mamm Genome. 2005; 16(9): 661–671. PubMed Abstract \| Publisher Full Text\n\nKwan KM: Conditional alleles in mice: Practical considerations for tissue-specific knockouts. Genesis. 2002; 32: 49–62. PubMed Abstract \| Publisher Full Text\n\nHayashi S, McMahon AP: Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol. 2002; 244(2): 305–18. PubMed Abstract \| Publisher Full Text\n\nMatsuda T, Cepko CL: Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci U S A. 2007; 104(3): 1027–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamírez-Solis R, Rivera-Pérez J, Wallace JD, et al.: Genomic DNA microextraction: a method to screen numerous samples. Anal Biochem. 1992; 201(2): 331–335. PubMed Abstract \| Publisher Full Text\n\nChick WS, Drechsel DA, Hammond W, et al.: Transmission of mutant phenotypes from ES cells to adult mice. Mamm Genome. 2009; 20: 734–740. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "1263", "date": "08 Aug 2013", "name": "Jennifer Clancy", "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 describes a method to insert a recombination cassette specifically into the ROSA26 allele. However I find it hard to establish how novel this method is and how much better it is then existing protocols from the work outlined here. Also, the results did not summarise the efficiency and specificity of this system well (which would have been nice to see in the figures) and I was unclear as to whether the pCAG-Flpe plasmid was a control or a comparison plasmid (and there were unclear results from this work). Additionally, only using examples to show the outcomes instead of statistics does not demonstrate how good the method is, only some of the potential outcomes. Also, what is the FRT-neo-F3 cassette mentioned in the last line?I am not an expert in transgenic mouse generation, but I do know that the ROSA locus has been used for some time. What is the essential advancement with this method?Also, please label panel 2C so meaning can be derived from it.I think some work needs to be done to enhance the clarity of the objectives, outcomes and any novelty in this article. Also, perhaps some better schematics of the various cassettes mentioned in the text would help the reader follow what they are being used for.", "responses": [] }, { "id": "1850", "date": "03 Oct 2013", "name": "Juergen Bode", "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\nOver all, the method described in this article does not seem to provide any benefits over previously published methods. Several details of the procedure are hard to understand since they are not clearly described either in the text or the figure legends. We also have concerns that the quality of the figures is below the standard we would expect. Specifically we have the following comments/queries:\n\nWhy was the targeting construct inserted in the opposite orientation? Was this perhaps to circumvent the promoter effects from the ROSA26 intrinsic promoter? This is not clearly stated in the article and should be explained.Why was Flpe been used and not Flpo (which has been known to be superior for several years)?Fig. 1b: The text is hard to follow. Which transgenes were cloned into the constructs? It would be helpful if the authors could provide a schema depicting the cloning strategy.Fig.2 is of poor quality overall. 2C: This PCR picture is inacceptable (as there are no annotations of size). Something has been written on the gel picture but it is hard to read as it is far too small.The ES cells used for RMCE were not characterized with regard to pluripotency and checked for karyotypic aberrations. Including these points in the article would be useful.We had major problems understanding the loxP site insertions in the vector. The positions are not clearly described in the text. Why is the stop site 4 x pA?The manuscript does not mention that the promoter driving the transgenes is the endogenous one. This should be added and explained.Our most serious concern is that very similar approaches (ROSA26 targeted by Flp-RMCE using F and F3 sites) have already been published, at least three times 1,2,3. We would have expected these papers to have been cited in the article and any advantages in the authors’ method to be discussed in relation to these previously described approaches.", "responses": [] }, { "id": "2313", "date": "05 Nov 2013", "name": "Lieven Haenebalcke", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a method to generate transgenic mouse embryonic stem cells by knocking in a constitutive or conditional expression vector to the ROSA26 locus by a recombinase-mediated cassette exchange (RMCE) approach. Although the authors already state in their abstract that this can be a simple and efficient procedure to generate transgenic mice in many laboratories, this is not proven by this article. Additionally and as already mentioned by the other two reviewers, this story clearly lacks novelty and does not refer to the most recent publications describing similar and even more efficient ROSA26-based RMCE technologies.Major comments: The introduction is not up to date as it does not refer to the most recent reports on RMCE-based transgenic technologies. More importantly, it does not describe how this study is of added value to the field or how it distinguishes itself from these other reports (see reference list reviewer Juergen Bode).It is not clear why one particular RMCE-compatible ES cell clone was chosen over the others. It would be nice to see Southern blot confirmation of correct and single transgene integration, karyotyping data, pluripotency analysis and ability of these ES cells to establish transgenic animals after in vitro manipulation.A more schematic overview of the constructs, RMCE-strategy and expression mechanism should be given to enhance the clarity of this method for less-experienced readers, e.g. include the splice acceptor which is required to obtain endogenous ROSA26 promoter-based expression of the RMCE-inserted transgene.As already mentioned by the other two reviewers, Figure 2C is not clear at all. Additionally, the PCR strategy should also be backed-up by Southern blot analysis to give an idea on possible secondary, random integrations. Separate targetings should be performed to get a real, statistically significant estimate on the efficiency of this RMCE approach.Figure 2D: Did all correctly targeted ES cell clones express the same levels of GFP and was this expression equal in all cells of one particular clone? In other words, how robust and wide spread is this ROSA26-based system?No experimental data is given for the pFLSL-WF3 conditional vector system (Cre/loxP). Was this vector tested? Please include data. If it did not work, the authors cannot state they have also developed a conditional systemSummary: the authors state this is a very efficient system for gene knock-in at the ROSA26 locus, both constitutive and conditional. However, this approach still requires traditional cloning of the cDNA of interest into the targeting vectors and the picking and screening of approximately 100 individual ES cell clones per construct. It is not clear to me how this method is superior to a very similar but previously published ROSA26-based approach, which even makes use of homologous recombination instead of RMCE (Nyabi et al. Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells Nucleic Acids Research; 37(7), 2009), especially because no data is provided to prove that the method described here can generate transgenic animals.In general, I am convinced that there are other, more reliable, tested and efficient systems available to generate such ROSA26-based transgenic ES cells and animals than the RMCE approach described here.", "responses": [] } ]	1	https://f1000research.com/articles/2-162
https://f1000research.com/articles/2-144/v1	27 Jun 13	{ "type": "Commentary", "title": "High-resolution view of compound promiscuity", "authors": [ "Ye Hu", "Jürgen Bajorath", "Ye Hu" ], "abstract": "Compound promiscuity is defined as the ability of a small molecule to specifically interact with multiple biological targets. So-defined promiscuity is relevant for drug discovery because it provides the molecular basis of polypharmacology, which is increasingly implicated in the therapeutic efficacy of drugs. Recent studies have analyzed different aspects of compound promiscuity on the basis of currently available activity data. In this commentary, we present take-home messages from these studies augmented with new results to generate a detailed picture of compound promiscuity that might serve as a reference for further discussions and research activities.", "keywords": [ "polypharmacology", "compound promiscuity", "drug efficiacy" ], "content": "Introduction\n\nPolypharmacology is an emerging theme in drug discovery1,2. It is generally accepted that drugs often elicit their therapeutic effects through interactions with different targets and the ensuing modulation of multiple signaling pathways. In some therapeutic areas such as oncology, polypharmacology is heavily exploited, for example, through the use of promiscuous ATP site-directed protein kinase inhibitors3. In other areas, such as the treatment of infectious or chronic inflammatory diseases, achieving a high degree of target selectivity of drug candidates plays a major role.\n\nThe study of drug polypharmacology has become an important topic in pharmaceutical research4,5, especially focusing on combined computational and experimental analysis5. On the basis of drug-target networks, it was estimated early on that a drug interacts on average with approximately two targets4. More recent estimates from computational data analysis suggest that drugs might bind on average to two to seven targets, depending on the primary target families, and that more than 50% of current drugs might interact with more than five targets6.\n\nCompound promiscuity as defined herein is the origin of polypharmacology. Promiscuity analysis can be extended from drugs to bioactive compounds through computational mining of currently available activity data. The results of activity data analysis are generally affected by data incompleteness7. This potential influence can only be eliminated by reaching the ultimate (and probably elusive) goal of chemogenomics8, i.e., testing all compounds against all targets. In the presence of data incompleteness, compound promiscuity rates are likely underestimated. However, it is not certain that further increasing amounts of assay data will indeed significantly alter the currently emerging view of compound promiscuity (vide infra).\n\nRecent studies have generated a differentiated picture of compound promiscuity. The interested reader is also referred to comprehensive reviews of compound promiscuity analysis9 and polypharmacology6. In this commentary, we summarize key messages from recent promiscuity analysis in a compact format. It is hoped that this summary might be helpful as a reference for further studies.\n\n\nKey results of compound promiscuity analysis\n\nPublic data sources for compound promiscuity analysis discussed herein have been ChEMBL10, the major repository of compound activity data from medicinal chemistry (currently in May 2013 containing 1,295,510 compounds with a total of 11,420,351 activity annotations), the PubChem BioAssay database11, the major repository of screening data (with more than 3300 confirmatory assays), and DrugBank12, which currently contains 1518 approved and 5080 experimental drugs.\n\nIt is important to note that collecting all activity annotations for a compound reported in the literature including, for example, reporter gene or other cell-based assays is at best providing a measure of assay promiscuity, but not of specific interactions with different targets9. Therefore, it is generally required to apply data confidence criteria such as the presence of well-defined activity measurements or evidence for direct ligand-target interactions9 (as provided in ChEMBL as activity data filters).\n\nWhen monitoring the growth of compound activity data in ChEMBL over a period of more than two years from its original release (January 2010) to release 13 (May 2012), a significant increase in the number of promiscuous compounds was detected13. However, by quantifying compound-based target relationships, it was determined that the increase in compounds with activity against targets from different families was largely due to (assay-dependent) IC50 measurements, rather than (assay-independent) equilibrium constants (Ki values)13. IC50 values are easier to determine than Ki values and provide the readout of most primary biochemical assays (except single-point screening assays), which might at least in part rationalize greater target coverage and the IC50-dependent increase in compound promiscuity across different families. However, it can also not be excluded that apparent promiscuity in different assays is higher on the basis of IC50 measurements, given their assay dependence (and often limited accuracy). Regardless, the type of activity measurements that are taken into account influences the outcome of promiscuity analysis. Thus, clear specification of activity measurements and data selection criteria are required.\n\nThe subset of compounds with available Ki measurements from ChEMBL release 13 was further investigated. On the basis of Ki measurements, approximately 62% of all compounds were only annotated with a single target, ~36% with two or more targets from the same family, and only ~2% of all active compounds with multiple targets from different families14. A promiscuous bioactive compound was found to interact on average with two to three targets.\n\nOne might anticipate that the degree of compound promiscuity would be particularly high in screening assays (even if frequent hitters and other non-specific compounds are excluded). Therefore, 1085 confirmatory bioassays from PubChem were systematically analyzed. It was found that ~77% of all confirmed active compounds were tested in more than 50 different assays15. Thus, these active PubChem compounds provided a sound basis for promiscuity assessment. These results were in part surprising. An active PubChem compound displayed a ~50% probability to interact with two or more targets. The probability to interact with more than five targets was only ~8%. On average, a PubChem screening hit was active against 2.5 targets. For comparison, compounds from the IC50- and Ki-based subsets of ChEMBL release 14 (August 2012) interacted on average with 1.4 and 1.7 targets, respectively15. The comparably low ratios observed for both compound subsets indicated that IC50 measurements did not systematically increase promiscuity rates (vide supra). The analysis of active compounds from PubChem confirmatory assays provided an upper level estimate of promiscuity, which was not significantly higher than that for ChEMBL compounds.\n\nDetailed analysis of compound activity data from ChEMBL release 14 (August 2012) has made it possible to derive a promiscuity profile that is most characteristic of bioactive compounds from medicinal chemistry sources. The majority of currently available promiscuous compounds is active in the sub-µM range against two to five targets from the same family and displays potency differences against these targets within one or two orders of magnitude16. An important aspect of this representative profile is that promiscuity does not imply low potency. Furthermore, compounds that are highly potent against a (primary) target and weakly potent against others are not frequently found16.\n\n\nUp-to-date promiscuity rates\n\nIn Table 1, current average promiscuity rates are summarized for compounds from ChEMBL, PubChem, and DrugBank. For promiscuity assessment of drugs, all targets reported in DrugBank were considered.\n\nThe average number of targets is reported for compounds from ChEMBL release 14 (divided into Ki and IC50 value-based subsets), approved or experimental drugs from DrugBank 3.0, and active compounds from PubChem confirmatory bioassays. Corresponding statistics are provided in italics for promiscuous compounds (having two or more target annotations). For compounds from ChEMBL, only high-confidence activity annotations were taken into account (i.e., explicit activity measurements with the highest confidence level of direct ligand-target interactions). For calculations on drugs, all DrugBank target categories were taken into account.\n\nIf all compounds with single or multiple target annotations are analyzed, ChEMBL compounds interact on average with one to two targets and PubChem compounds with two to three. However, approved drugs have on average close to six targets. In contrast, the degree of promiscuity of experimental drugs is considerably lower, with less than two targets per drug candidate. If only promiscuous compounds or drugs are taken into account (i.e., if compounds with single target annotations are excluded), promiscuity rates only slightly increase by about one target per compound, the exception being experimental drugs whose average number of targets increases from 1.8 to 4.7.\n\nIn Table 2, the probability of promiscuity is reported for compounds from different sources (calculated from target distributions of compounds). For a ChEMBL compound with available IC50 and Ki measurements, the current probability of activity against two or more targets is ~25% and ~38%, respectively (if both IC50 and Ki measurements were available for a compound, they were separately considered). However, for activity against more than five targets, the probabilities are reduced to only ~1%. Similar observations are made for confirmed PubChem screening hits (providing an upper-limit promiscuity assessment for bioactive compounds, vide supra). In this case, the probability of activity against two or more, or against more than five targets is ~51% and ~8%, respectively. Furthermore, the probability of promiscuity of approved drugs from DrugBank is ~84% and the probability to interact with more than five targets still ~37%. For experimental drugs, the corresponding probabilities are much lower, with only ~24% and ~3%, respectively.\n\nFor different compound categories and activity measurements, the probability of a compound to be active against two or more targets or more than five targets is reported.\n\n\nConclusions\n\nHerein, we have provided a detailed and up-to-date view of compound promiscuity, the molecular basis of polypharmacology. For active compounds from medicinal chemistry and biological screening sources, the degree of promiscuity is lower than for drugs. There is a notable increase in promiscuity from bioactive compounds over drug candidates to approved drugs. The exploration of possible reasons for this apparent \"promiscuity enrichment\" along the drug discovery pathway should provide interesting opportunities for future research. On the basis of currently available high-confidence activity data, promiscuity of bioactive compounds is limited (and very low across different target families). However, if compounds are promiscuous, they typically bind to their targets with relatively high potency. Given the overall low degree of promiscuity of bioactive compounds including screening hits in the presence of nearly exponential data growth in recent years, it remains an open question if future chemogenomics efforts might substantially change the current picture of compound promiscuity (vide supra). The majority of available bioactive compounds have single target annotations and we believe it is unlikely that most of them will display a high degree of currently undiscovered promiscuity. Hence, we would also conclude that the target specificity paradigm that has long dominated small molecule discovery efforts should continue to play a major role, despite emerging \"anti-reductionism\" and the increasing focus on phenotypic readouts.", "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 were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nPaolini GV, Shapland RH, van Hoorn WP, et al.: Global mapping of pharmacological space. Nat Biotechnol. 2006; 24(7): 805–815. PubMed Abstract \| Publisher Full Text\n\nBoran AD, Iyengar R: Systems approaches to polypharmacology and drug discovery. Curr Opin Drug Discov Devel. 2010; 13(3): 297–309. PubMed Abstract \| Free Full Text\n\nKnight ZA, Lin H, Shokat KM: Targeting the cancer kinome through polypharmacology. Nat Rev Cancer. 2010; 10(2): 130–137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYildirim MA, Goh KI, Cusick ME, et al.: Drug-target network. Nat Biotechnol. 2007; 25(10): 1119–1126. PubMed Abstract \| Publisher Full Text\n\nLounkine E, Keiser MJ, Whitebread S, et al.: Large-scale prediction and testing of drug activity on side-effect targets. Nature. 2012; 486(7403): 361–367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJalencas X, Mestres J: On the origins of drug polypharmacology. Med Chem Comm. 2013; 4(1): 80–87. Publisher Full Text\n\nMestres J, Gregori-Puigjané E, Valverde S, et al.: Data completeness--the Achilles heel of drug-target networks. Nat Biotechnol. 2008; 26(9): 983–984. PubMed Abstract \| Publisher Full Text\n\nRognan D: Chemogenomic approaches to rational drug design. Br J Pharmacol. 2007; 152(1): 38–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu Y, Bajorath J: Compound promiscuity: what can we learn from current data? Drug Discov Today. 2013; 18(13–14): 644–650. PubMed Abstract \| Publisher 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\nWang Y, Xiao J, Suzek TO, et al.: PubChem’s bioassay database. Nucleic Acids Res. 2012; 40(Database issue): D400–D412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnox C, Law V, Jewison T, et al.: DrugBank 3.0: a comprehensive resource for ‘omics’ research on drugs. Nucleic Acids Res. 2011; 39(Database issue): D1035–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu Y, Bajorath J: Growth of ligand-target interaction data in ChEMBL is associated with increasing and activity measurement-dependent compound promiscuity. J Chem Inf Model. 2012; 52(10): 2550–2558. PubMed Abstract \| Publisher Full Text\n\nHu Y, Bajorath J: How promiscuous are pharmaceutically relevant compounds? A data-driven assessment. AAPS J. 2013; 15(1): 104–111. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu Y, Bajorath J: What is the likelihood of an active compound to be promiscuous? Systematic assessment of compound promiscuity on the basis of PubChem confirmatory bioassay data. AAPS J. 2013; 15(3): 808–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu Y, Bajorath J: Promiscuity profiles of bioactive compounds: potency range and difference distributions and the relation to target numbers and families, in revision (the reference status will be updated as soon as possible). Med Chem Commun. 2013; 4: 1196–1201. Publisher Full Text" }	[ { "id": "1036", "date": "01 Jul 2013", "name": "Stefan A Laufer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nExcellent work. My field (“the kinase community”) will benefit a lot form this commentary as compound promiscuity is an issue.", "responses": [] }, { "id": "1052", "date": "09 Jul 2013", "name": "Hans Matter", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting manuscript presents a view on compound promiscuity based on in-vitro data and the number of potential targets per compound in public databases such as ChEMBL, PubChem and DrugBank. In particular the authors investigate and challenge the notion that most compounds today in lead findings are active on a large multitude of biological data. The title is appropriate for this contribution and the abstract sufficiently summarizes this study. The conclusions are balanced and justified on the basis of the data analysis; this is therefore an essential view on the number of targets.It is an interesting observation from this study that DrugBank annotated drugs appear to interact with a higher number of molecular targets compared to early phase compounds in ChEMBL or PubChem. Any interpretation of this finding should be treated with caution, but it is tempting to discuss from a partially historical view as DrugBank may be enriched with older drugs that would have been subjected to less strict requirements for in-vitro selectivity than in today’s drug discovery. In addition during and after approval, drugs may have been tested in more profiling assays as is the case with earlier screening-type substances.Following the authors, this interesting argument also supports target-specific drug discovery paradigms used in past years. However, working with public databases leads to many caveats, all of which have been pointed out earlier, e.g. incompleteness of the data matrix and differences of data from different sources. It might be interesting for future investigations to cross-check this conclusion for compounds targeting families like kinases or GPCRs. Due to the challenges of inherent selectivity in those families one could expect a larger percentage of promiscuous compounds. The same discrimination might possibly be true for smaller versus larger compounds.", "responses": [] }, { "id": "1067", "date": "16 Jul 2013", "name": "Jeremy L. 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\n\nHu and Bajorath’s update on compound promiscuity in public compound bioactivity databases is timely with the increasing cognizance of polypharmacology and its role in the efficacy and safety of drugs. The article aims to raise reader awareness and present new questions to be answered, and so the title, abstract, and content are appropriate. All data are freely available for download for primary sources mentioned.The conclusions are fair and unbiased.Additional questions do arise from this survey. First, does the average number of targets per compound differ from the median (or do highly promiscuous compounds skew the average?).Second, is it reasonable to begin distinguishing promiscuous from privileged compounds? For example, by incorporating target class information, staurosporine might be viewed differently from quercetin, where the former represents a highly privileged scaffold among kinases and the latter displays IC50 values against an abundance of target types. Third, the drug discovery field needs to understand if the \"promiscuity enrichment\" that occurs between the screening hits phase to the marketed drug phase largely reflects the depth of bioactivity data coverage for drugs, as drugs are highly profiled globally. The hit rates of drugs and medchem compounds across the same set of assays and targets would be needed to definitively conclude that drugs are more promiscuous. However, the apparent increased promiscuity of drugs supports the growing resurgence of phenotypic screening, the impetus for exploring compound combinations in the context of multiple genotypes, and begs the question of how medchem optimization of multiple targets should be attacked.", "responses": [] } ]	1	https://f1000research.com/articles/2-144
https://f1000research.com/articles/2-161/v1	26 Jul 13	{ "type": "Short Research Article", "title": "Photodynamic therapy with new sublingual sensitiser Photosoft®E4 for cancer: a case series", "authors": [ "Karin Ried", "Avni Sali", "Michelle Wang", "Brian Meade", "Donald Murphy", "Avni Sali", "Michelle Wang", "Brian Meade", "Donald Murphy" ], "abstract": "Background: An increasing number of patients seek complementary therapies for cancer treatment, the leading cause of death in the developed world. Photodynamic therapy (PDT), the combination of light and a photosensitiser agent, has provided some promising results in cancer therapy. New photosensitiser agents are continuously being developed to improve tolerability and effectiveness. There is a need to objectively evaluate clinical data from PDT patients.Methods: Here we report a case series using the new sublingually administered, chlorophyll-based photosensitiser Photosoft®E4 and an external laser light in a group of ten adult cancer patients not undergoing other concurrent therapies. PDT was administered for three treatment cycles with an average of 14 light treatments per patient, consisting of agent administration and laser treatment on alternate days over 3 months. Safety, tolerability and effectiveness on tumour palliation were monitored. Results: Patients in this study presented with a variety of cancer types and stages; half of the patients had breast cancer, and 40% had metastases. We found Photosoft®E4 to be safe and highly tolerable. However, overall disease status was not improved in our group of patients. Conclusions: Future research is required to determine the bioavailability of Photosoft®E4 and its uptake in tumour tissue, pharmacokinetics and dosing regimen, as well as the best mode of light delivery for the in vivo sensitiser activation.", "keywords": [ "Cancer is the leading cause of death in the Western world", "accounting for 13% of all deaths worldwide", "and about three in ten deaths in Australia1", "2. It is estimated that 800", "000 Australians (3.3% ) were living with cancer in 2007", "and an additional 100", "000–125", "000 are expected to be diagnosed every year", "contributing 19% of the total disease burden in 20123." ], "content": "Introduction\n\nCancer is the leading cause of death in the Western world, accounting for 13% of all deaths worldwide, and about three in ten deaths in Australia1,2. It is estimated that 800,000 Australians (3.3% ) were living with cancer in 2007, and an additional 100,000–125,000 are expected to be diagnosed every year, contributing 19% of the total disease burden in 20123.\n\nAn increasing number of patients seek complementary and unconventional cancer therapies. While in 1998 an average of 31.4% of patients in Western countries sought complementary and alternative medical (CAM) therapies4, three-quarters of the patients in a cancer centre in the United States used at least one CAM modality in 2005, and the majority of those (58%) were initiated after cancer diagnosis5.\n\nThe main CAM modalities sought in addition to conventional cancer therapies include special diets, dietary supplements and herbs, psychotherapy, movement therapy, mind-body therapies and spirituality based interventions4–7. More recently, photodynamic therapy (PDT) has become an acceptable adjunct cancer therapy, providing some promising results. A systematic review of the effect of PDT - alone or in conjunction with conventional therapy - on patients with non-small cell lung carcinoma, showed a response rate of 30.8–84.8% on mainly inoperable disease, with a 5-year survival rate of 43.4–72%, a considerable improvement to the survival rate of 15–35% for inoperable and metastasised cancers using conventional treatment alone8. Other types of cancers treated with PDT such as head and neck cancer showed an 89% 5-year survival rate for superficial cancers, compared with 75% using conventional therapies alone1,9,10.\n\nPDT combines a photosensitiser and light to cause selective damage to the target tissue10–13. Laser light of a specific wavelength of maximum absorbance matched to the photosensitiser is directly toxic to the cell and also interacts with local intracellular oxygen molecules to activate singlet oxygen, which in turn destroys the target tissue14. Cancer cells accumulate a higher concentration of the photosensitiser than surrounding healthy tissue, and therefore are a selective target for light treatment10–13.\n\nA range of photosensitisers are available for malignant tumours. Early drugs were based on haemato-porphyrin derivatives, while newer drugs based on porphyrin, chlorin or chlorophyll derivates have been developed to reduce the phototoxic effects of the early photosensitisers15. Pharmacokinetic in vitro studies on chlorophyll-based photosensitisers have demonstrated uptake in tumour cells with a range of clearance times16, and the safety of similar chlorophyll-based photosensitisers, such as Radachlorin® or Sonoflora/Sonnemed® has been demonstrated in animal studies and high tolerability has been observed in small human trials17–19.\n\nWhile most photosensitisers are administered intravenously, the new chlorophyll-based sensitiser Photosoft®E4 was developed to be taken sublingually20. A similar chlorophyll-based and sublingually administered photosensitiser Sonoflora® was found to be safe and effective in the sonodynamic treatment (a combination of light and ultrasound) of breast cancer patients19.\n\nAs patients are increasingly seeking and accessing information on complementary treatment options for their cancer treatment, there is a need for the objective evaluation and dissemination of current clinical data on available therapies such as PDT, to help with decision making21.\n\nRecently, a new generation photosensitiser has become available in Australia, Photosoft®E4. While there is extensive clinical experience with this sublingual photosensitiser in China where there have been several promising case study reports22, concerns have been expressed about the effectiveness of the modified technology by others23,24.\n\nFor these reasons, there is a need to evaluate clinical experiences with PDT in other countries, including Australia. Here we report on the safety and tolerability of Photosoft®E4 and gauge the effect of the PDT treatment in a group of cancer patients not undergoing any concurrent therapies.\n\n\nMethods\n\nData for this case series report was collected at the National Institute of Integrative Medicine, Melbourne between January and November 2012. The aims of this pilot study were to determine the safety and tolerability of sublingual administration of a new chlorophyll-based photosensitiser and external laser treatment, and to ascertain the effectiveness of PDT and tumour palliation.\n\nTen patients (8 women, 2 men) were enrolled (mean age 56.6 years, range 35–81 years), representative of the common clinical population. Inclusion criteria were: adults with primary or metastatic cancer, no concomitant other therapies during PDT treatment (including chemo- or radiotherapy, sublingual or intravenous medical therapy, as per patient’s request), ability to take medication sublingually, compliance with study protocol, and informed written consent. Patients were excluded if tumours were close to large blood vessels, or invading the trachea, if patients were pregnant or lactating, or with mental impairment. Participating patients were treated with PDT free of charge.\n\nThe pilot study was approved by the Human Research Ethics Committee at the National Institute of Integrative Medicine. Clinical Trial Notification (CTN) numbers for equipment and the photosensitiser were obtained from the Therapeutic Goods Administration Australia.\n\nThe protocol was based on the protocol used at clinics in China at the time of the start of our trial22. The photosensitiser PhotoSoft®E4 (Chlorin-e6-Chlorophyllin-A-Zinc-complex, The Cho group, supplied by Sekhsaria Chemicals Lid, CTN 050/2012), was administered sublingually at a dose of 5mg/kg body weight.\n\nA multi-frequency laser model PDT630II (The Cho group, Guilin Xingda Photoelectricity Medical Equipment Co Ltd, CTN 062/2012) emitting blue, red and infra-red laser light with peak wavelengths at 460 nm, 660 nm, and 870 nm with a total light-dose of 45 J/cm2 and a fluence-rate of 25 mW/cm2 was used to irradiate each tumour site for 30 min (Figure 1). After the external focused laser, all patients were treated in a whole-body LED-light-bed model NGPDT (The The Cho group, Guilin Xingda Photoelectricity Medical Equipment Co Ltd) emitting the same wavelengths as the external laser for 30 min to target any circulating tumour cells (Figure 2).\n\nThe treatment comprised of three cycles, with each cycle consisting of 4–5 laser treatments on alternate days over the course of nine days, followed by a 4–5 week break. The photosensitiser was taken sublingually 18–26 hours prior to each laser treatment. In total, patients who completed the three treatment cycles received 12–15 light treatments (mean=14) over three months.\n\nSafety and tolerability were assessed during and after each treatment session by the treating physician. A semi-structured questionnaire assessed whether patients had any unusual sensations, including gastrointestinal symptoms, feeling of tiredness or malaise, pain, or elevated temperature between treatment sessions (see Supplementary material).\n\nThe appearance and size of the primary tumour site and the presence and extent of any metastases were assessed clinically and by Computer-Tomography (CT), Magnetic-Resonance-Imaging (MRI), or ultrasound scan before baseline and at three months after completion of treatment.\n\n\nResults\n\nHalf of the patients (n=5) had breast cancer, three of which had declined surgery and were without metastases, and two had local reoccurrence and/or metastases after mastectomy (Table 1). Other types of cancer were basal salivary (n=1), neck (n=1), both of whom had no prior mainstream cancer treatment, and stage-IV cervical (n=2) and stage-IV pancreatic cancer (n=1) who had all undergone surgery and/or chemotherapy and radiotherapy previously (Table 1).\n\nMainstream treatment, including surgery, chemotherapy, radiotherapy.\n\n* 1 cycle consisted of 4-5 laser treatment sessions.\n\nCP = Clinical Photoimage; CT = Computer Tomography; F = Female; M= Male; MRI= Magnetic Resonance Imaging; U= Ultrasound; x = times laser treatment.\n\nAll breast cancer patients completed the full course of 3x4 treatments (n=5); other patients underwent 1–2 cycles of treatment before withdrawal due to disease progression (n=3) or uptake of other therapies (n=2).\n\nPatients were treated with a 30 min external laser per tumour site plus 30 min exposure in the light bed, ranging from 1–2 hour-sessions for primary non-metastatic cancers (n=5), and 2–4 hour-sessions for patients with metastasis (n=5).\n\nAll patients tolerated the sublingual administration of the photosensitiser well and did not report any side effects other than unpleasant taste (n=10). Laser treatment caused localised sensations such as tingling in some patients (n=5). No phototoxic reactions were experienced. Some patients (n=3) reported localised tingling of variable intensity at the tumour site 1–2 weeks after treatment, lasting between 30 min to overnight.\n\nOne patient with a superficial breast tumour showed some localised reduction of tumour size (Table 1, ID4). Diagnostic imaging by CT, MRI or ultrasound scans showed evidence of disease progression in all patients after three months regardless of the type or stage of cancer (Table 1). However, one patient (ID1, breast cancer) who continued with PDT treatment reported clinically stable disease at six months after study completion (tumour-size 3.6 cm3).\n\n\nDiscussion\n\nIn our study, the photosensitiser PhotoSoft®E4 taken sublingually at a dose of 5 mg/kg up to 15 times over three months was tolerated well by all cancer patients and no phototoxic effects were observed. The high tolerability of the photosensitiser is in line with international reports from clinical centres using similar agents19. However, photodynamic therapy of the sublingually administered photosensitiser with external laser light did not improve the overall disease status in our group of patients.\n\nTo date, most photosensitisers are administered intravenously, and the pharmacokinetics of sublingually administered photosensitizing agents has not been studied extensively. However, one study found that biodistribution and pharmacokinetics of another photosensitiser 5-aminolaevulinic-acid (ALA) in the human gastrointestinal tract and urinary bladder was comparable if administered sublingually or intravenously25. In addition, sublingual administration of Photosoft®E4 was shown to be taken up selectively by tumour cells in prostate cancer patients26. However, more research is needed on the bio-distribution, uptake in different types of tumour tissue27, and pharmacokinetics of the photosensitiser PhotoSoft®E4. Pharmacokinetic data would also ascertain the optimal time interval between drug intake and light administration28. In our study, we used a 24-hour-interval, which is in line with intervals used in studies with early photosensitisers such as Photofrin®, as well as the chlorophyll-based photosensitiser Sonoflora®19,29,30. Interestingly, a fractionated drug-dose (the administration of the photosensitiser at multiple time intervals before light activation) ranging between 15 minutes to four hours prior to light therapy has resulted in superior therapeutic effects for a variety of chlorophyll-based photosensitisers11,29,31. Fractionated drug-dosing has been shown to target tumours by different mechanisms, involving either vascular damage with a short-interval laser treatment or direct tumour-cell apoptosis with a long-interval laser treatment29. Furthermore, sonodynamic therapy has provided promising results with sublingually administered chlorophyll-based photosensitisers similar to PhotoSoft®E419.\n\nOur study had a few limitations, including the small number of patients, and the diverse range of cancer types and stages. However, patients were representative of the common clinical population seeking alternative options for their cancer treatment. In addition, the number of patients was sufficient to elucidate the potential effect of the treatment following the protocol as outlined.\n\nA limitation of the protocol was that we used external laser equipment emitting wavelengths between 460–870 nm. Light penetration through skin or muscle increases with wavelength, and is estimated to reach with 37% density between 2.5 and 9 mm of tissue32,33. Interstitial administration of laser light facilitates penetration and reach of deeper tumours34, but may be too invasive for most patients.\n\n\nConclusions\n\nThis clinical study indicates the safety and high tolerability of the photosensitiser PhotoSoft®E4, if taken sublingually at a dosage of 5mg/kg up to 15 times within a three month period. However, the protocol for the photodynamic therapy used in this trial as well as in clinical settings by others previously provided no benefit to treatment-related outcomes. More research is required to determine the bioavailability and uptake in tumour tissue, pharmacokinetics and dosing regimen, as well as the best mode of light delivery for the activation of the in vivo sensitiser.", "appendix": "Author contributions\n\n\n\nAS conceptualised the study, secured support, and developed study design with contributions from all authors. MW was responsible for enrolment of patients, application of PDT treatment, and data collection. MW and KR analysed and interpreted data. KR prepared the manuscript with feedback from all co-authors. All authors approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nVillage Roadshow provided financial support for the study.\n\n\nAcknowledgements\n\nWe thank The Cho group for loaning the laser equipment, and all patients for their contribution. We are grateful to Wolfgang Marx for feedback on the manuscript.\n\n\nSupplementary material\n\n\n\n\nReferences\n\nCancer Council Australia. Facts and figures. Cancer in Australia. 2013. Reference Source\n\nWorld Health Organisation Cancer. Factsheet No. 297 2013. Reference Source\n\nAIHW Cancer survival and prevalence in Australia: period estimates from 1982 to 2010 Australian Institute of Health and Welfare. Reference Source\n\nErnst E, Cassileth BR: The prevalence of complementary/alternative medicine in cancer: a systematic review. Cancer. 1998; 83: 777–82. PubMed Abstract\n\nPerlman A, Lontok O, Huhmann M, et al.: Prevalence and correlates of postdiagnosis initiation of complementary and alternative medicine among patients at a comprehensive cancer center. J Oncol Pract. 2013; 9: 34–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRichardson MA, Sanders T, Palmer JL, et al.: Complementary/alternative medicine use in a comprehensive cancer center and the implications for oncology. J Clin Oncol. 2000; 18: 2505–14. PubMed Abstract\n\nYates JS, Mustian KM, Morrow GR, et al.: Prevalence of complementary and alternative medicine use in cancer patients during treatment. Support Care Cancer. 2005; 13: 806–11. PubMed Abstract \| Publisher Full Text\n\nMaziak DE, Markman BR, MacKay JA, et al.: Photodynamic therapy in nonsmall cell lung cancer: a systematic review. Ann Thorac Surg. 2004; 77: 1484–91. PubMed Abstract \| Publisher Full Text\n\nFeyh J: Photodynamic treatment for cancers of the head and neck. J Photochem Photobiol B. 1996; 36: 175–7. PubMed Abstract \| Publisher Full Text\n\nHuang Z: A review of progress in clinical photodynamic therapy. Technol Cancer Res Treat. 2005; 4: 283–93. PubMed Abstract \| Free Full Text\n\nDolmans DE, Fukumura D, Jain RK: Photodynamic therapy for cancer. Nat Rev Cancer. 2003; 3: 380–7. PubMed Abstract \| Publisher Full Text\n\nBrown SB, Brown EA, Walker I: The present and future role of photodynamic therapy in cancer treatment. Lancet Oncol. 2004; 5: 497–508. PubMed Abstract \| Publisher Full Text\n\nAgostinis P, Berg K, Cengel KA, et al.: Photodynamic therapy of cancer: an update. CA Cancer J Clin. 2011; 61: 250–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllison RR, Moghissi K: Photodynamic Therapy (PDT): PDT Mechanisms. Clin Endosc. 2013; 46: 24–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu DY: Research and development of photodynamic therapy photosensitizers in China. Photodiagnosis Photodyn Therapy. 2007; 4(1): 13–25. Publisher Full Text\n\nDouillard S, Lhommeau I, Olivier D, et al.: In vitro evaluation of Radachlorin sensitizer for photodynamic therapy. J Photochem Photobiol B. 2010; 98: 128–37. PubMed Abstract \| Publisher Full Text\n\nKochneva EV, Filonenko EV, Vakulovskaya EG, et al.: Photosensitizer Radachlorin®: Skin cancer PDT phase II clinical trials. Photodiagnosis Photodyn Ther. 2010; 7: 258–67. PubMed Abstract \| Publisher Full Text\n\nLewis TJ: Toxicity and cytopathogenic properties toward human melanoma cells of activated cancer therapeutics in zebra fish. Integr Cancer Ther. 2010; 9: 84–92. PubMed Abstract \| Publisher Full Text\n\nWang X, Zhang W, Xu Z, et al.: Sonodynamic and photodynamic therapy in advanced breast carcinoma: A report of 3 cases. Integr Cancer Ther. 2009; 8: 283–7. PubMed Abstract \| Publisher Full Text\n\nCho Group Next Generation PDT. http\n\nBalneaves LG, Weeks L, Seely D: Patient decision-making about complementary and alternative medicine in cancer management: context and process. Curr Oncol. 2008; 15(Suppl 2): s94–s100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNext Generation PDT. Clinical Studies. Reference Source\n\nBown SG: How mainstream medicine sees photodynamic therapy in the United Kingdom. J Natl Compr Canc Netw. 2012; 10(Suppl 2): S69–74. PubMed Abstract\n\nMoseley H, Eljamel S, Moghissi K: Concerns around so-called ‘next generation’ PDT and sonodynamic therapy.2013. Reference Source\n\nLoh C, MacRobert AJ, Bedwell J, et al.: Oral versus intravenous administration of 5–aminolaevulinic acid for photodynamic therapy. Br J Cancer. 1993; 68: 41–51. PubMed Abstract \| Free Full Text\n\nMurphy D: Prostate cancer treated by Sono- and photodynamic therapy (Abstract 89). The 66th Australian and New Zealand Urological Nurses Society (ANZUNS) Annual Scientific Meeting. Melbourne, Victoria, Australia 2013. Reference Source\n\nYoshida T, Tokashiki R, Ito H, et al.: Therapeutic effects of a new photosensitizer for photodynamic therapy of early head and neck cancer in relation to tissue concentration. Auris Nasus Larynx. 2008; 35: 545–51. PubMed Abstract \| Publisher Full Text\n\nCastano AP, Demidova TN, Hamblin MR: Mechanisms in photodynamic therapy: part one—photosensitizers, photochemistry and cellular localization. Photodiagnosis Photodyn Ther. 2004; 1: 279–93. Publisher Full Text\n\nDolmans DE, Kadambi A, Hill JS, et al.: Targeting tumor vasculature and cancer cells in orthotopic breast tumor by fractionated photosensitizer dosing photodynamic therapy. Cancer Res. 2002; 62: 4289–94. PubMed Abstract\n\nDougherty TJ: An update on photodynamic therapy applications. J Clin Laser Med Surg. 2002; 20: 3–7. PubMed Abstract\n\nTogashi H, Uehara M, Ikeda H, et al.: Fractionated photodynamic therapy for a human oral squamous cell carcinoma xenograft. Oral oncol. 2006; 42: 526–32. PubMed Abstract \| Publisher Full Text\n\nDougherty TJ, Marcus SL: Photodynamic therapy. Eur J Cancer. 1992; 28A: 1734–42. PubMed Abstract\n\nKolari P: Penetration of unfocused laser light into the skin. Arch Dermatol Res. 1985; 277: 342–4. PubMed Abstract\n\nChen Q, Huang Z, Luck D, et al.: Preclinical Studies in Normal Canine Prostate of a Novel Palladium Bacteriopheophorbide (WST09) Photosensitizer for Photodynamic Therapy of Prostate Cancer. Photochem Photobiol. 2002; 76: 438–45. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1947", "date": "03 Oct 2013", "name": "Michael Hamblin", "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 reports a case series of 10 adult cancer patients treated with a procedure involving sublingual administration of a “photosensitizer” called PhotosoftE4 and illumination of tumors with a laser and the whole body with a “LED bed”. Not surprisingly there was no real therapeutic effect.Using a sub-optimal PDT regimen such as that described in this report and describing it as “complementary and alternative medicine, CAM” is analogous to snipping at a tumor with nail scissors and calling it “CAM surgery”, or using a diagnostic X-ray machine and calling it “CAM radiotherapy”. Of course PDT performed with this bizarre methodology didn’t work; the question is more why would one think it possibly could?PDT treatments for cancer using chlorophyll-based photosensitizers (PS) such as HPPH, ce6 or TOOKAD work perfectly well when the PS is injected IV 1. If the investigators in this study were intent on only using an orally delivered PS they could have used oral 5-aminolevulinic acid (ALA) 2. While this has some problems in terms of side effects such as blood pressure changes, it has been shown to work. Putting this type of ce6 preparation under the tongue would not be expected to lead to any appreciable systemic absorption and therefore no therapeutic effect. The assertion that a whole-body illumination procedure could eradicate “circulating tumor cells” is frankly laughable.PDT is a perfectly respectable but not yet mainstream medical procedure and I am concerned that flawed studies such as this one could affect the reputation of this potentially useful therapeutic technique and offer false hope for desperate cancer patients.", "responses": [ { "c_id": "572", "date": "08 Oct 2013", "name": "Karin Ried", "role": "Author Response", "response": "In this trial we tested reproducibility of a protocol previously used by others, and are the first, to our knowledge, to have objectively evaluated and reported on the safety (primary outcome) and effectiveness (secondary outcome). We agree with the referee that our findings suggest that the PDT regime used in this trial is sub-optimal. Therefore we consider it important to have published our findings to inform patients and practice, and to counteract publication bias and indeed false hope. Our article discusses potential improvements of PDT to be considered for future applications." } ] }, { "id": "2164", "date": "23 Oct 2013", "name": "David Kessel", "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 not clear what information this report is designed to convey. PDT is not 'alternative' therapy, having been approved by the FDA for some indications and showing evidence for effective cancer control. The approach described here seems to be based on a random trial & error method with no consideration for pharmacokinetics, biodistribution or dosimetry. There has been some enthusiasm for publication of negative reports so as to tell future investigators what not to do, so in that sense, this report qualifies.", "responses": [] } ]	1	https://f1000research.com/articles/2-161
https://f1000research.com/articles/2-160/v1	24 Jul 13	{ "type": "Study Protocol", "title": "Systematic assessment of benefits and risks: study protocol for a multi-criteria decision analysis using the Analytic Hierarchy Process for comparative effectiveness research", "authors": [ "Nisa M Maruthur", "Susan Joy", "James Dolan", "Jodi B Segal", "Hasan M Shihab", "Sonal Singh", "Nisa M Maruthur", "Susan Joy", "James Dolan", "Jodi B Segal", "Hasan M Shihab" ], "abstract": "Background: Regulatory decision-making involves assessment of risks and benefits of medications at the time of approval or when relevant safety concerns arise with a medication. The Analytic Hierarchy Process (AHP) facilitates decision-making in complex situations involving tradeoffs by considering risks and benefits of alternatives. The AHP allows a more structured method of synthesizing and understanding evidence in the context of importance assigned to outcomes. Our objective is to evaluate the use of an AHP in a simulated committee setting selecting oral medications for type 2 diabetes. Methods: This study protocol describes the AHP in five sequential steps using a small group of diabetes experts representing various clinical disciplines. The first step will involve defining the goal of the decision and developing the AHP model. In the next step, we will collect information about how well alternatives are expected to fulfill the decision criteria. In the third step, we will compare the ability of the alternatives to fulfill the criteria and judge the importance of eight criteria relative to the decision goal of the optimal medication choice for type 2 diabetes. We will use pairwise comparisons to sequentially compare the pairs of alternative options regarding their ability to fulfill the criteria. In the fourth step, the scales created in the third step will be combined to create a summary score indicating how well the alternatives met the decision goal. The resulting scores will be expressed as percentages and will indicate the alternative medications' relative abilities to fulfill the decision goal. The fifth step will consist of sensitivity analyses to explore the effects of changing the estimates. We will also conduct a cognitive interview and process evaluation. Discussion: Multi-criteria decision analysis using the AHP will aid, support and enhance the ability of decision makers to make evidence-based informed decisions consistent with their values and preferences.", "keywords": [ "Decision-making", "Analytical Hierarchical Process", "Multi-criteria decision analysis", "Type 2 Diabetes" ], "content": "Background\n\nRegulatory decision-making involves assessment of the risks and benefits of medications at the time of approval or when relevant safety concerns arise with a medication. The objectives of regulatory decisions are to determine whether a particular drug is safe and effective for use in the population at a specific dose for a particular indication. With increasing pressure on government agencies to improve transparency, the Food and Drug Administration (FDA) is making significant changes to make its processes and decisions more transparent to industry stakeholders and consumers (http://www.fda.gov/AboutFDA/Transparency/TransparencyInitiative/default.htm). This has included initiatives to improve the transparency and consistency of risk-benefit assessments (http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/UCM296379.pdf). Recently, the FDA issued draft guidance on a structured qualitative benefit-risk framework, with several aspects of the decision making being quantitative (http://www.fda.gov/downloads/forindustry/userfees/prescriptiondruguserfee/ucm329758).\n\nThe Institute of Medicine report on the ethics of post-marketing safety studies (http://www.iom.edu/Reports/2012/Ethical-and-Scientific-Issues-in-Studying-the-Safety-of-Approved-Drugs.aspx) recommended that the FDA consider systematic assessment of benefit and risk during its regulatory advisory committee meetings. It recommended evaluation of the Multi-criteria decision analysis (MCDA) as one approach to group decision making during regulatory advisory committee meetings. MCDA methods are designed to help people make good decisions by helping them better understand the available information, assess their decision preferences and priorities, and enhance communication among involved stakeholders1–3. A MCDA using the Analytic Hierarchy Process (AHP; see Methods for a description) allows us to make better decisions in complex situations involving tradeoffs by explicitly considering the risks and benefits of alternatives4.\n\nThe AHP is equipped to address a wide range of decisions that involve both quantitative data and additional, less-tangible input from stakeholders5–8. This is highly relevant to comparative effectiveness research as the comparison of alternative drugs or interventions is paramount. These complex situations may include tradeoffs between imperfect options, a mix of objective data and subjective options, and uncertain future outcomes9.\n\nDecisions always involve evaluative judgments; MCDA helps this process by making these judgments explicit, systematic and transparent1. It also allows input from multiple stakeholders who may assign different preference weightings to the various risks and benefits. It has been used in other governmental organizational decision making10–12. Type 2 diabetes is a priority condition for comparative effectiveness research as it is a condition with multiple treatment options with multiple potential benefits and harms13,14.\n\nAs a part of the Johns Hopkins FDA Partnership in Applied Comparative Effectiveness we plan to conduct a MCDA using the AHP to determine the optimal choice of oral medications for type 2 diabetes in a simulated advisory committee setting.\n\n\nMethods\n\nWe will invite a small group of at least eight diabetes experts from clinical (primary care, endocrinology, and pharmacy), research (epidemiology and clinical trials), operations (pharmacy and therapeutics), and public health disciplines (department of public health) related to diabetes treatment to participate. Recruitment methods will include invitations extended by email with attachment of an approved document (Appendix A). This project received ethical approval from the Institutional Review Board of Johns Hopkins University (Approval Number: NA_00048562).\n\nThe study intervention will be a four part structured interview consisting of a) overview of current medications and options for type 2 diabetes; b) a MCDA using the AHP; c) a cognitive interview; d) evaluation of the AHP-based priority assessment procedure.\n\nWe will use the most updated version of the regulatory label for the treatment options for type 2 diabetes. When data on outcomes is not available, we will use data from our Agency for Healthcare Research and Quality (AHRQ) comparative effectiveness review15.\n\nThe first step in AHP analysis involves defining the goal of the decision, the alternatives being considered, and the criteria to determine how well the alternatives can be expected to meet the goal5–8. These are organized into a hierarchical decision model with the goal at the top, the alternatives at the bottom, and the criteria in between (Figure 1). In the second step, information about how well the alternatives can be expected to fulfill the decision criteria is collected. The third step consists of two parts: a) comparing the ability of the alternatives to fulfill the prespecified criteria, and b) judging the importance of the criteria relative to the decision goal. Pairs of alternative options are sequentially compared regarding their ability to fulfill the criteria using pairwise comparisons7.\n\nThe first step in Analytic Hierarchy Process analysis consists of defining the goal of the decision, the alternatives being considered, and the criteria to determine how well the alternatives can be expected to meet the goal. As seen above, these are organized into a hierarchical decision model with the goal at the top, alternatives at the bottom, and criteria in between.\n\nA combined normalized ratio scale summarizes the results of the direct and indirect comparisons. Priorities for alternatives are compared using ratios; relative differences of 1.1 are considered significant according to standard AHP criteria10. A ratio, or relative difference, of 1.1 between two alternatives implies a 10% multiplicative difference with respect to how the alternatives meet a given objective at the next level above in the hierarchy. To measure the quality of AHP, we will measure the internal consistency of the judgments within a set of pairwise comparisons using a measure called the consistency ratio. A consistency ratio of 0 indicates perfect consistency. By convention, consistency ratios < 0.15 were considered acceptable7.\n\nSeparate judgements are made for various decision perspectives. After the pairwise comparisons of alternatives, the pairwise comparison methods are used to determine the priorities of the criteria relative to the decision goal7.\n\nIn the fourth step, the scales created in step three are combined to create a summary score indicating how well the alternatives can be expected to meet the decision goal7.\n\nThis is similar to estimating a weighted average by multiplying the scores indicating how well the alternative options meet the decision goal by the priorities assigned to the criteria and adding the results7. The priority of a given alternative with respect to meeting an objective at the next level up in the hierarchy is obtained by summing the products of the alternative weight and each objective weight at the level below in the hierarchy. The pair wise ratings will be transformed into relative weights by calculation of the right principal eigenvector of the relevant matrix (e.g., matrix of the pairwise comparisons between objectives at one level of the hierarchy). Expert Choice, uses the matrix multiplication method, considered to be accurate16, for this calculation. The resulting scores are commonly expressed as percentages and indicate the alternative medications' relative abilities to fulfill the decision goal.\n\nThe fifth step consists of sensitivity analyses to explore the effects of changing the estimates or judgements used in the original analysis.\n\nAll AHP analyses will be conducted using Expert Choice 11.5 standard program. We will use the ideal synthesis mode with which rank is preserved in the case of addition or removal of an 'irrelevant' alternative17. In the ideal mode, the priorities of alternatives or options at a given level of the hierarchy are divided by the priority of the highest-scoring alternative or objective (the “ideal”), and the results are weighted10.\n\nIn our example, this means that the two identical highest-priority alternatives will both receive the same weight, and it will not make any difference to the weights of the other alternatives if both or only one of the irrelevant alternatives are included. In the distributive mode, priorities are divided by the sum of priorities to give a normalized weight and this allows for rank reversal10. We will examine our results for robustness using the distributive mode.\n\nWe will also conduct a cognitive interview and process evaluation to evaluate the user perspectives on the process. The cognitive interview will consist of an investigator accompanying the participants as they complete the AHP task and asking a range of probing questions. Questioning will be guided by a checklist to ensure that all important aspects of the hierarchy, the choice tasks, and the instrument itself were functioning as intended. Qualitative feedback will be entered into NVivo (http://www.qsrinternational.com/products_nvivo.aspx), coded and analyzed to identify recurring themes. Once all participants have completed the AHP, they will be invited back for a group session. At this session group results will be presented and discussed and the participants will be asked to complete an evaluation form (Appendix B) regarding their experience with, and confidence in the AHP method. The operationalization of the AHP for our study is described below.\n\nThe decision goal will be placed at the top of the hierarchy. The level of the hierarchy below the decision goal will include the general objectives. More specific objectives will be placed below the general objectives with each lower level consisting of more specific objectives. General objectives and more specific objectives at a given level of the hierarchy will be comparable. We will aim for seven or fewer objectives on any given level of the hierarchy, and objectives will be expressed positively. The pharmacologic alternatives will be placed at the lowest level of the hierarchy. The decision context, model content, and structure of the hierarchy will be validated for content and refined through in-person group sessions with experts. The panel of experts will consist of clinical experts (e.g., internists, endocrinologists, and pharmacists) and research experts (e.g., epidemiologists and clinical trialists). The decision context and hierarchical model will be presented to a panel of experts on at least two occasions. Experts will be invited to these validation sessions informally in-person or by e-mail. Sessions will be 60 to 90 minutes in duration. After each session, the expert feedback obtained will be synthesized and incorporated into a revised version of the model and decision context. During each expert session, a member of the study team will present an overview of the use of the AHP in decision-making. A list of three to five open-ended questions will be distributed in hard copy to the group for feedback on the model (Supplementary Table 1).\n\nThe decision context will be ranking medication for type 2 diabetes. The clinical scenario presented will be an adult patient with moderately uncontrolled type 2 diabetes (glycated hemoglobin-7–9 g/dl). The stated decision goal will be to rank the options for type 2 diabetes treatment. Two criteria will be defined as determining the best treatment of type 2 diabetes: 1) to maximize benefits via glucose reduction; 2) to minimize medication adverse effects. The criteria for maximizing benefits will be focused on reducing glycated hemoglobin; the criteria for minimizing medication adverse events will be sub-divided into two sub criteria: minimizing non-serious harm and minimizing serious harm. The serious harms include severe hypoglycemia, congestive heart failure, acute pancreatitis, and bladder cancer. The non-serious harms include fractures, weight gain and gastrointestinal symptoms. The five medication options that will be considered are metformin, sulfonylureas, exenatide, sitagliptin and pioglitazone.\n\nWe will use several sources of data for the decision-analysis. The treatment-specific probabilities or mean differences for objectives at the lowest level of the hierarchy will be identified from the FDA label or medical review documents available for each drug as available on the FDA website. In the absence of quantitative data on the specific objectives, we will substitute data from a Comparative Effectiveness Review of diabetes medications developed under contract from AHRQ18.\n\nThese treatment-specific quantitative data (“evidence matrix”) will be presented relative to either the comparator identified in the decision context, or to placebo/usual care (see section on, “Presentation of objectives”).\n\nThe data from the evidence matrix will be formatted to create a visual representation which facilitates interpretation. Formats for presentation will be considered based on Dolan et al.19 and will include display of risks on a plot with 2 axes; bar charts displaying risks; flow charts displaying risks; and pictograms that depict probabilities by displaying a box that contains 100 items, some of which are filled. These formats will be compared and discussed among the study team, and the final presentation format selected by the study team will be used to display evidence on objectives during AHP sessions. See Supplementary Figure 1 for the different visual representations to be considered.\n\nComparisons among alternative drugs relative to criteria: We will compare the alternative drugs’ ability to achieve the decision goal (i.e. best treatment for type 2 diabetes) by making comparisons among the alternative drugs with regard to fulfilling each criterion. This will be conducted using standard AHP pairwise comparisons among the alternatives for each of the benefit and risk criteria defined in the previous step using the 9-point ratio scale. This “bottom-up approach” will be used to account for the potential consideration when one key assumption for AHP – that the higher level of elements in the hierarchy are independent of lower level elements – may not hold.\n\nComparisons among the criteria: The same pairwise comparison method will be used to determine the priorities of each of the criteria relative to the decision goal (i.e. safe and effective medication for type 2 diabetes).\n\nWe will use the standard AHP weighting to combine the results of the judgments made in step three to determine the relative abilities of the medications to meet our stated goals for the decision context. Relative differences > 1.1 will be considered significant.\n\nWe will explore the impact of different judgments’ on the relative importance of the criteria varying their priorities from 0 (no importance) to 1 (most important) and recalculating their alternative scores. We will invite input on the relative importance of the criteria from various stakeholders and conduct additional sensitivity analysis to determine their impact on the decision goals.\n\nDelivery of the AHP web based instrument. The AHP instrument will be delivered as a series of questions in an online version of Expert Choice. The first screen, or “welcome screen”, will present a comprehensive description of the decision context, instructions as to how pair-wise comparisons should be judged using a ratio scale, and instructions for navigating through the experiment. Each subsequent screen will contain a set of pair-wise questions designed to elicit each respondent’s opinion on the relative weight of each objective or alternative in terms of meeting the overall goal or relevant objective.\n\nAlternatives will be presented first, then specific objectives, moving from the bottom of the hierarchy up to the decision goal. A bottom-up order of presentation was considered more appropriate than a top-down order because expert respondents will have underlying knowledge about details that could influence decisions at higher levels in the hierarchy in an uncontrolled manner. For pair-wise comparisons of objectives and sub-objectives, participants will see the text: “Which of the two objectives below is more important?” For pair-wise comparisons of alternatives, participants will see the text: “Which of the two alternatives below is more preferable?” Rating of alternatives is necessary in order to transfer the evidence into subjective comparisons of importance on the ratio scale. The Expert Choice software translates all judgments to a ratio scale, regardless of whether they are entered using a verbal, graphical, or numerical scale.\n\nThe maximum number of pair-wise comparisons will be presented in order to give the best possible accuracy. Participants will be able to click on information icons to display evidence-based information (described above in “Presentation of objectives”) on how each alternative meets the relevant objective. The instructions on the welcome screen and questions will be drafted according to survey methodology best practice and will be tested and redrafted as necessary by the study team. Participants will be able to see their individual intermediate results throughout the process and their overall results when the process is completed. The option for participants to see their inconsistency ratios will be turned off because presentation of the inconsistency ratio may encourage participants to aim for consistency over accuracy. At the end of the process, participants will see a “thank you” screen which will provide a brief overview of the analysis process and group session as well as contact details for the principal investigator and ethics committee. Analysis will be conducted using the ideal mode, and various sensitivity analyses will be conducted for the group session.\n\nParticipants will be invited to participate in one individual session and one group session. Trained interviewers who are co-investigators on the project will schedule one-on-one appointments with respondents in which they will complete the instrument and probe questions. Interviews will take approximately 90 minutes. Once all pretests are completed, individual and group results will be analyzed and presented to respondents in a group debrief session lasting approximately 60 minutes. This will provide respondents with further opportunity to comment on the face validity of the approach and to raise any additional concerns about the instrument.\n\nThe individual interviews will involve an experienced investigator working one-on-one with each respondent to work through the AHP task and conduct the cognitive debriefing concurrently. Interviews will be scheduled at quiet locations and times convenient to the respondent and will take approximately 90 minutes to complete. Respondents will not be remunerated for their participation. Respondents will not be asked to provide any personal health information, which helps minimize the risk of breaches of confidentiality.\n\nAt the end, participants will be invited back for a group session. The objectives of the group session are: 1) to present the group results from the AHP and any significant findings from the cognitive debriefing; 2) to assess the extent to which respondents agree with the experiment’s findings; and 3) to assess whether they find the method sufficiently trustworthy to consider using it in other decision making contexts.\n\nThe group session will be held in a secure location. It will take approximately two hours, and respondents will not receive remuneration. The agenda for the group session will include presentation of overall results followed by results at each level of the hierarchy. For each trade-off in the hierarchy, presentation of results will be accompanied by discussion of any outlying results, with respondents encouraged to discuss reasons for differing views. Where there is significant heterogeneity in responses, as measured by the standard deviation of the priority weights, or where respondents do not agree with results, investigators will show the impact on results of alterations to responses and/or the evidence matrix. The session will conclude with a short poll on the extent to which respondents agree with the findings and trust the method.\n\nCognitive interviewing will be conducted alongside the individual interviews. The cognitive debriefing protocol will be developed based on standard methodology building on methods used for cognitive debriefing of conjoint analysis instruments. The protocol will involve prospective and retrospective probing, as well as assessing information volunteered by participants to assess the AHP instrument according to the following checklist shown in Table 1. Interviews will be recorded and transcribed, if agreed to by respondents, to facilitate analysis of the cognitive interviewing information. Analysis of the cognitive interview will involve assessing the extent to which the scale performed adequately on each of the checklist items. Major themes or concerns about the instrument will be elicited from the transcripts and presented through representative quotes.\n\nThe study team will comprise of co-investigators with methodologic expertise in preference assessment methods, decision analysis, evaluation of pharmacotherapeutic published and unpublished literature, epidemiology, and clinical care of type 2 diabetes. The study team will meet for at least 45 minutes each week to discuss and refine the decision context and model development. One member of the team will take primary responsibility for development of the decision context and model based on study team discussions and feedback during validation sessions.\n\nThe protocol was approved by The Johns Hopkins Medicine Institutional Review Board. Risks to human subjects are minimal because the tasks involve opinion research, with no medical interventions or collection of personal health information. Participants will be provided with information about the study at the beginning of the individual sessions and asked for their consent to participate. They will be informed that they do not have to answer all questions and that they can withdraw from the study at any time without penalty.\n\n\nDiscussion\n\nWe will provide the FDA with a new and innovative decision making method to ensure that potentially life-saving treatments are available to patients while protecting public health. The results from this study are likely to benefit patients and regulatory decision-makers in making judgments about the risks and benefits of drugs and devices.\n\nOur study will provide a demonstration of the development and conduct of an AHP in the context of benefit-risk analysis that might occur in a committee setting. Using the AHP can aid, support, and enhance the understanding of decision-making processes. Cognitive interviewing will provide detailed qualitative data that could be used to improve the validity, clarity, and usability of the instrument.\n\nOur protocol has several strengths. First, the AHP will be developed in an iterative manner that incorporates expert feedback at several steps in the process. While all participants will be experts in diabetes, each group will include some people who are new to AHP. This structure ensures that all technical considerations are addressed but also that the instrument will be as accessible as possible for people using an AHP for the first time. Second, this study will include formal process evaluations at several steps in the process, which gives structured insight into users’ perspectives on the performance of the instrument and confidence in the method. Third, we will evaluate the results of the cognitive interview to ensure confidence that the process is valid.\n\nThis will be the first study to use the AHP to rank alternatives for treatment of type 2 diabetes. The findings will be compared to other applications in the healthcare literature where the AHP has been used in a committee-style decision-making process20,21. The study will determine to what extent the optimal choice of treatment for type 2 diabetes depends on the performance of the alternatives on various benefit and harm outcomes, and the importance assigned to these outcomes.\n\nThis study will provide information on a novel comprehensive approach using the AHP to systematically address the risk-benefits in a transparent patient-centered evidence-based manner.", "appendix": "Author contributions\n\n\n\nSS, NM, SJ, JD, JS conceived the study idea and design. SS, NM, SJ, JS, JD and HS participated in writing and revising the manuscript. JS secured funding for the study. 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\nThis work was funded by a grant contract number HHSF2232010000072C, entitled, \"Partnership in Applied Comparative Effectiveness Science,\" sponsored by the Food and Drug Administration, Department of Health and Human Services.\n\n\nSupplementary figure\n\nA) The flow chart shows the occurrence gastrointestinal (GI) side effects by diabetes medication pair. B) Bar chart shows excess number of patients per 100 with GI side effects. SU-Sulfonylureas; MET-Metformin; Pio-Pioglitazone.\n\n\nReferences\n\nBelton V, Stewart T: Multiple Criteria Decision Analysis: An Integrated Approach. United Kingdom: Kluwer Academic Publishers. 2002. Reference Source\n\nFigueira J, Greco S, Ehrgott M: Multiple Criteria Decision Analysis: State of the Art Surveys. New York: Springer 2005. Reference Source\n\nDolan JG, Boohaker E, Allison J, et al.: Patients' preferences and priorities regarding colorectal cancer screening. Med Decis Making. 2013; 33(1): 59–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMussen F, Salek S, Walker S: A quantitative approach to benefit-risk assessment of medicines - part 1: The development of a new model using multi-criteria decision analysis. Pharmacoepidemiol Drug Saf. 2007; 16(Suppl 1): S2–S15. PubMed Abstract \| Publisher Full Text\n\nDolan JG, Isselhardt BJ Jr, Cappuccio JD: The analytic hierarchy process in medical decision making: A tutorial. Med Decis Making. 1989; 9(1): 40–50. PubMed Abstract \| Publisher Full Text\n\nSaaty T: How to make a decision: The analytic hierarchy process. Interfaces. 1994; 24(6): 19–43. Publisher Full Text\n\nSingh S, Dolan JG, Centor RM: Optimal management of adults with pharyngitis--a multi-criteria decision analysis. BMC Med Inform Decis Mak. 2006; 6: 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDolan JG: Shared decision-making--transferring research into practice: The analytic hierarchy process (AHP). Patient Educ Couns. 2008; 73(3): 418–425. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDolan JG: Multi-criteria clinical decision support: A primer on the use of multiple criteria decision making methods to promote evidence-based, patient-centered healthcare. Patient. 2010; 3(4): 229–248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaaty T: Fundamentals of Decision Making and Priority Theory with the Analytic Hierarchy Process. Vol VI of the AHP Series. Pittsburgh, PA: RWS Publications. 2006. Reference Source\n\nGolden BL, Wasil EA, Harker PT: The Analytic Hierarchy Process: Applications and Studies. Berlin: Springer-Verlag. 1989. Reference Source\n\nLiberatore MJ, Nydick RL: The analytic hierarchy process in medical and health care decision making: A literature review. European Journal of Operational Research. 2008; 189(1): 194–207. Publisher Full Text\n\nSingh S, Loke YK, Furberg CD: Thiazolidinediones and heart failure: A teleo-analysis. Diabetes Care. 2007; 30(8): 2148–2153. PubMed Abstract \| Publisher Full Text\n\nSingh S, Loke YK, Furberg CD: Long-term risk of cardiovascular events with rosiglitazone: A meta-analysis. JAMA. 2007; 298(10): 1189–1195. PubMed Abstract \| Publisher Full Text\n\nBennett WL, Wilson LM, Bolen S, et al.: Oral diabetes medications for adults with type 2 diabetes: An update. Agency for Healthcare Research and Quality (US); Rockville (MD). 2011. PubMed Abstract\n\nSaaty TL, Vargas LG: Models, Methods, Concepts & Applications of the Analytic Hierarchy Process. Second ed. New York: Springer. International Series in Operations Research & Management Science 2012; 175. Publisher Full Text\n\nForman EH: Ideal and distributed synthesis modes for the analytical hierarchy process. The International Federation of Operations Research, Lisbon, Portugal. 1993.\n\nBennett WL, Maruthur NM, Singh S, et al.: Comparative effectiveness and safety of medications for type 2 diabetes: An update including new drugs and 2-drug combinations. Ann Intern Med. 2011; 154(9): 602–613. PubMed Abstract \| Publisher Full Text\n\nDolan JG, Qian F, Veazie PJ: How well do commonly used data presentation formats support comparative effectiveness evaluations? Med Decis Making. 2012; 32(6): 840–850. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrishnan JA, Lindenauer PK, Au DH, et al.: Stakeholder priorities for comparative effectiveness research in chronic obstructive pulmonary disease: A workshop report. Am J Respir Crit Care Med. 2013; 187(3): 320–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPecchia L, Martin JL, Ragozzino A, et al.: User needs elicitation via analytic hierarchy process (AHP). A case study on a computed tomography (CT) scanner. BMC Med Inform Decis Mak. 2013; 13: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "1261", "date": "30 Jul 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\n\nThe article sets out to evaluate the Analytic Hierarchy Process (AHP) in a simulated committee setting for type 2 diabetes. AHP presents a new way of decision-making about drugs and devices. AHP is potentially valuable to patients and regulators as it can help weigh the risks and benefits.", "responses": [] }, { "id": "2193", "date": "28 Oct 2013", "name": "Faith Michael Uzoka", "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 paper addresses a very important multi-criteria decision analysis technique (AHP), applied to an area where it is greatly needed - drug prescription. The authors present a proposal that utilizes the AHP in drug benefit-risk analysis, with the intent of submitting the research findings for consideration by the FDA. The authors made efforts to elaborate on the AHP methodology. However, in the process of doing so, too many (at times unnecessary and confusing) details were presented:\n\nFigure 1: The AHP hierarchy is unclear. The decision box at the top is not indicative of the hierarchy structure associated with AHP and should be replaced by a goal box (rectangle). The diagram also presents the drugs (decision alternatives) as the lowest level of the hierarchy; that is, a sub level of all other levels. The authors need to present them as decision alternatives and not as a lower level hierarchy.The second to last paragraph of page 4 is unclear. What do the authors mean by “irrelevant alternative”? How does the distributive mode of Expert Choice help “examine….results for robustness”?Step 1, page 5: The authors state that “more specific objectives will be placed below the general objectives…” Do they mean criteria? Why should they aim for seven or fewer objectives (criteria)?Step 2, page 5, final paragraph: The sentence “Formats for ….based on Dolan et al…box containing 100 items…” is unclear. What is the substance of Dolan et al? What does the entire sentence mean? The authors need to a better explanation in this paragraph. Also Supplementary Figure A is unclear and not properly explained.Step 3, page 5: this is very confusing. The authors initially indicated that they would carry out a pair-wise comparison of the decision variables to determine the priority ratings of the variables yet now, in addition, the authors want to carry out a pair-wise comparison of alternatives. The alternatives are supposed to be evaluated against the global priorities. In addition I have the following questions about the methodology of the study:Why do the authors spend so much effort on multiple interviews and sessions with physicians, only to test the model with one patient (page 5, step one, second paragraph). A medical experiment of this nature (that could lead to far reaching decisions), should be tested using an adequate amount of data theerby increasing the generalisability of the model.If this paper is a proposal paper (as I understand it to be), the authors do not need to discuss implementation issues such as the looks and prompts by Expert Choice.Have the authors considered other concomitants that could affect the benefits and harms associated with a given drug? For exampple age, gender, race, and other genetic factors could contribute to the benefits and risks associated with a given drug. A fuzzy-cognitive map technique would likely produce a better result than AHP in the sense that it is a better tool for modelling cause and effect relationships. In all, the idea of the study is very good. The authors need to better decide on the appropriate technique for modelling drug risk-benefit analysis, and clearly describe the technique.", "responses": [] }, { "id": "2259", "date": "10 Dec 2013", "name": "Saravana Kumar", "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 research study which aims to use an Analytic Hierarchy Process (AHP) in a simulated committee setting for selecting oral medications for type 2 diabetes. The study protocol is well presented and “ticks” many important methodological constructs.However, there are areas which can be further strengthened:At times, the information presented is not clear and lacks justification. For example, in page 5, the authors state that the “these treatment-specific quantitative data (“evidence-matrix”) will be presented relative to either the comparator...”. What is the “evidence-matrix” and how will it be presented?Similarly, in step 4, the authors state that they will “…use the standard AHP weighting to combine the results and judgements made in step three to determine relative abilities of the medications to meet our stated goals for decision contexts”. How will the authors “combine” the “results and judgements”? What process will they use?Also, what does “meet our stated goals for decision contexts” actually refer to? The manuscript is littered with similar statements creating ambiguity and confusion. Simple sentences with clearer explanations may be required. Sampling – This may be buried in the manuscript, but what sampling framework will be used to identify the diabetes experts? Will it be purpose or theoretical sampling? Convenience sampling should be avoided if possible due to issues of bias. Qualitative data analysis – How will the data collected through interviews and group sessions be analysed and reported? Will it be via content or thematic analysis? Given that this research also collects qualitative data, it is important to recognise and address rigour and trustworthiness in the research methodology.Overall, this is an interesting and worthwhile study which could be strengthened with additional clarity and specific focus on key methodological issues. I wish the authors the best of luck with their research.", "responses": [] } ]	1	https://f1000research.com/articles/2-160
https://f1000research.com/articles/2-52/v1	15 Feb 13	{ "type": "Review", "title": "Use of botulinum toxin in musculoskeletal pain", "authors": [ "Jasvinder A Singh" ], "abstract": "Chronic musculoskeletal pain is a common cause of chronic pain, which is associated with a total cost of $635 billion per year in the U.S. Emerging evidence suggests an anti-nociceptive action of botulinum toxin, independent of its muscle paralyzing action. This review provides a summary of data from both non-randomized and randomized clinical studies of botulinum toxin in back pain and various osteoarticular conditions, including osteoarthritis, tennis elbow, low back pain and hand pain. Three randomized controlled trials (RCTs) of small sizes provide evidence of short-term efficacy of a single intra-articular injection of 100 units of botulinum toxin A (BoNT/A) for the relief of pain and the improvement of both function and quality of life in patients with chronic joint pain due to arthritis. Three RCTs studied intramuscular BoNT/A for tennis elbow with one showing a significant improvement in pain relief compared with placebo, another one showing no difference from placebo, and the third finding that pain and function improvement with BoNT/A injection were similar to those obtained with surgical release. One RCT of intramuscular BoNT/A for low back pain found improvement in pain and function compared to placebo. Single RCTs using local injections of BoNT in patients with either temporomandibular joint (TMJ) pain or plantar fasciitis found superior efficacy compared to placebo. One RCT of intramuscular BoNT/B in patients with hand pain and carpal tunnel syndrome found improvement in pain in both BoNT/B and placebo groups, but no significant difference between groups. Most evidence is based on small studies, but the use of BoNT is supported by a single, and sometimes up to three, RCTs for several chronic musculoskeletal pain conditions. This indicates that botulinum toxin may be a promising potential new treatment for chronic refractory musculoskeletal pain. Well-designed large clinical trials are needed.", "keywords": [ "musculoskeletal pain", "arthritis", "botulinum toxin", "intra-articular", "pain", "function" ], "content": "Introduction\n\nPain is a major public health problem. Pain was recognized as a major challenge by the Institute of Medicine (IOM) in their recent report1. This report highlighted that persistent pain affects 100 million Americans with a cost of $635 billion in direct costs and lost wages. Musculoskeletal pain is the most common cause of persistent pain and is most often due to arthritis, back pain and/or local musculoskeletal conditions (such as tendinitis, bursitis, sprains), and (less commonly) injuries. In a European survey of pain prevalence in the general population, moderate to severe pain lasting 6-months or longer was reported by 19% respondents, of whom 40% (~8% of all respondents) had joint pain2. Arthritis affects nearly 50 million adults in the U.S., and is projected to increase in prevalence to 67 million adults, or 25% of those aged 18 years and older, by the year 20303. Arthritis is the leading cause of disability in adults ≥18 years in the U.S.4, the third leading cause of work limitation in the U.S.5, is associated with 992,100 annual hospitalizations6 and 44 million annual outpatient visits7. In 2003, the total cost of arthritis was $128 billion in the U.S., including $81 billion in direct medical costs and $47 billion in indirect costs (lost earnings)8. Back pain is a leading cause of persistent pain and is the fifth-most-common reason for physician visits9,10. Back pain was responsible for 50% of all chronic pain problems in a European survey2. In a U.S. survey, each year 15% of American adults report frequent back pain or pain lasting more than two weeks11. The annual costs of lower back pain in the U.S. exceed $100 billion12,13. Treatment of chronic musculoskeletal pain secondary to joint pain and/or back pain constitutes a major challenge.\n\nAll joint structures except articular cartilage are innervated with articular nerves that contain A-delta, A-beta and C-fibers14–17. In normal healthy individuals, these nociceptors have a high threshold for excitation in response to mechanical and thermal stimuli. Normal activities such as walking, stair climbing and sports (and palpation of the joint) do not cross this threshold for nociceptor activation and therefore these activities are not associated with pain or unpleasant sensations. However, joint injury or inflammation is associated with decrease in the excitation threshold of these nociceptors. This leads to enhanced responses to both innocuous and noxious mechanical, chemical and thermal stimuli. This phenomenon is called peripheral sensitization18. Chronic joint inflammation is also associated with hyperexcitability of spinal nociceptive neurons i.e., central sensitization19,20. A variety of mediators can sensitize joint nerves and nociceptors to mechanical stimuli including bradykinin, serotonin, substance P, prostaglandin E2, prostaglandin I2 and neuropeptide Y17. Another contributor to joint pain and inflammation is neurogenic inflammation, which involves the release of neuropeptides from nerve terminals in response to inflammation, the release of neuropeptides from postganglionic sympathetic nerves due to sympathetic reflexes and the release of neuropeptides due to cytokine or neuropeptide-stimulation of local inflammatory cells21. It is likely that these processes underlie the clinical observed signs and symptoms of pain and mechanical hyperalgesia in inflamed joints. Recent reviews describe the processes of sensitization and neurogenic inflammation and summarize our current understating of joint pain and the various mechanisms contributing to it17,18. Activation of glial cells, other immune cells and cytokines also contribute to the generation of pain22–27. The role of pain pathways, pain receptors and various contributors to both inflammatory and neuropathic pain has been summarized in recent studies and reviews28–33.\n\nCommonly used approaches to treat musculoskeletal pain include oral therapies, intra-articular therapies, topical treatments and physical therapy. The main challenges to the use of therapies for refractory musculoskeletal pain include their limited efficacy and the risk of toxicity. Use of common oral therapies such as non-steroidal anti-inflammatory drugs (NSAIDs), opioid medications (narcotics) and analgesics (acetaminophen etc.), which are effective for some patients with chronic musculoskeletal pain, is associated with a significant risk of side effects, especially in the elderly34–36. Common side effects include: peptic ulcer disease with its complications (bleeding and perforation), renal failure and liver toxicity in patients using NSAIDs; sedation, confusion, constipation and falls in those taking narcotics; and liver toxicity in patients taking acetaminophen. Both intra-articular therapies (including the use of corticosteroids and hyaluronic acid) as well as topical preparations such as capsaicin and lidocaine have limited efficacy in patients with osteoarthritis (OA) and/or joint pain37–41. Physical therapy is effective42, but feasibility remains an issue as some patients are unable to adhere to physical therapy regimens due to personal preference and/or comorbidities, the need for transportation for frequent visits and the inconvenience of frequent travel and the time commitment involved. Thus, at present, limited effective and safe therapeutic options are available for refractory musculoskeletal pain. Therefore, new therapeutic options for treatment of musculoskeletal pain are needed.\n\nBotulinum toxin (BoNT) is one of the most potent neurotoxins and consists of a 50 kDa light chain and a 100 kDa heavy chain linked with a disulfide bond. It exists in seven serotypes, A through G. Botulinum toxin has been shown to interfere with the expression of various neuropeptides such as substance P and calcitonin gene-related protein (CGRP), which are key mediators of neurogenic inflammation43. In an animal model, botulinum toxin A (BoNT/A; Onabotulinumtoxina) injections into the rat paw reduced formalin-induced paw edema, tissue glutamate release and spinal cord electrical excitations44. BoNT/A inhibited stimulated substance P-release45,46 and CGRP-release45,47,48 in models of acute and chronic inflammation. In vitro studies showed that BoNT/A inhibited stimulated CGRP release from rat trigeminal ganglia49 and capsaicin-stimulated substance P-release from embryonic rat dorsal root ganglia neurons50. In particular, BoNTs have been shown to inhibit cytokines, neuropeptides and other inflammatory mediators that play an important role in the pathophysiology of both rheumatoid arthritis and rat adjuvant arthritis (an animal model that is similar to human rheumatoid arthritis)51–54. Thus, there is pre-clinical evidence suggesting an anti-nociceptive mechanism of action of botulinum toxin. Various clinical studies support the anti-nociceptive action of botulinum toxin55–57, while some studies have found no such evidence58–60.\n\nThe objective of this systematic review was to synthesize data from both randomized and non-randomized trials to assess the safety and efficacy of botulinum toxin in osteoarticular pain.\n\nIn this evidence-based literature review, I performed a focused systematic review of published non-randomized (prospective and retrospective cohort studies and case series) and randomized studies of botulinum toxin for arthritis and musculoskeletal pain by performing two PubMed searches: one using the terms \"botulinum toxin\" and \"musculoskeletal pain\" and another using the terms \"botulinum toxin\" and \"arthritis\". In addition to summarizing the key studies identified from this search, I also reviewed the bibliography of key reviews to identify any studies that may have been missed using the search strategy, including our recent review of the use of botulinum toxin in osteoarticular pain61. Inclusion criteria included the use of botulinum toxin, randomized or non-randomized study that included adults with musculoskeletal pain and reported clinical outcomes. Studies of myofascial pain syndromes were not included in this review, since they have been the focus of previous reviews62,63. I summarized case reports only when they provided new evidence for a particular use of botulinum toxin. In the sections below, I summarize data that provides initial evidence for the anti-nociceptive action of botulinum toxin, followed by existing evidence related to the use of botulinum toxin in various musculoskeletal pain conditions including refractory arthritis.\n\n\nResults\n\nIn this section, I summarize the data from non-randomized studies followed by data from randomized controlled trials (RCTs) for the efficacy of botulinum toxin’s efficacy in refractory shoulder joint pain (1 randomized study) and refractory knee joint pain (2 randomized studies) (Table 1).\n\nRCT, randomized controlled trial; BoNT, botulinum toxin; PL, placebo; SQ, subcutaneous; VAS, Visual Analog Scale; SD, standard deviation; IA, intra-articular.\n\nThere are three non-randomized studies that provide evidence for the efficacy of BoNT in cases of refractory joint pain64–66. In a retrospective study, Mahowald et al. reported the results of intra-articular injections of botulinum toxin type A (IA-BoNT/A) in 15 shoulder and lower extremity joints in 11 patients experiencing refractory pain64. All patients were receiving analgesics/NSAIDs and none had experienced relief with intra-articular corticosteroids. Patients received 25–100 units of IA-BoNT/A. There were 9 men and 2 women with an age range of 42–82 years. Joint pain was measured on a 0–10 numeric rating scale (NRS). Pain relief began within 2 weeks in most patients. A single intra-articular injection of botulinum toxin was associated with a clinically and statistically significant decrease in pain severity and improvement in function compared to baseline: lower extremity and shoulder pain decreased from 7 to 2.7 and 8.2 to 2.4, respectively; shoulder flexion improved from 68 to 113 degrees and shoulder abduction improved from 50 to 74 degrees, respectively. Timed stand tests (the time taken to stand up ten times from a sitting position) improved from 36 to 23 seconds. The duration of relief/improvement lasted 3–10 months. None of the patients reported any adverse events and no motor-sensory deficits were found on lower extremity examination after the IA-BoNT/A injection.\n\nAnother report described the long-term follow-up of these 11 patients with 15 treated joints, of which ten joints were re-injected with 30–150 units of BoNT/A intra-articularly (Botox, Allergan) at the patient’s request, when pain returned65. Nine of the ten re-injections were associated with pain reduction, as seen with the first injection. Pain severity decreased from 6.6 (SD, 1.2) to 3.3 (SD, 2.7) on a 0–10 NRS, which was statistically significant (p=0.003). Pain relief lasted 3–17 months. None of the patients reported any local or systemic adverse events related to BoNT/A. One patient experienced increased joint swelling with no increase in joint pain 3 weeks after BoNT/A injection. Another patient experienced a continued increase in joint pain after BoNT/A injection, which was relieved with a subsequent BoNT/A injection.\n\nIn another case series, 11 adults with refractory pain underwent injection of botulinum toxin type A (25–100 units; Botox, Allergan) or type B (5,000 units; Myobloc, Solstice Neurosciences) into the sacroiliac, cervical/lumbar facet or sternoclavicular joints, C-2 roots and lumbar disc66. This case series comprised 9 women and 2 men with a mean age of 48 years (SD, 10 years; range, 32–68 years). Median pain scores decreased significantly after BoNT injections, with a median decrease of 3 on a 0–10 NRS pain scale (range for pain NRS reduction, 0–5; p=0.008). Three patients experienced no change in pain severity after botulinum toxin injections, while eight experienced a decrease. No patients reported any increase in pain severity after BoNT injection. Pain reduction began within 3–5 days. Five patients received repeat injections. There was no evidence of decreasing efficacy of pain relief with repeated BoNT injections; in fact, the duration of pain relief increased with each successive treatment for 4 of the 5 patients with multiple treatments. All patients that experienced pain reduction with BoNT injections also noted improved function in activities of daily life and the range of motion in their joints. The median duration of pain relief with BoNT injections was 1.6 months longer than that seen with previous corticosteroid injections. There were no adverse events reported by the patients.\n\nSingh et al. performed a double-blind RCT of BoNT/A (Allergan, Inc.) into glenohumeral (shoulder) joints of patients with refractory, chronic shoulder joint pain due to osteoarthritis or rheumatoid arthritis of the shoulder joint, who had all failed conservative management67 (Table 1). Patients were randomized to one of the two treatment groups - a single injection of 100 units of botulinum toxin type A (IA-BoNT/A) reconstituted in 1cc of saline plus 1 ml of 2% lidocaine (treatment group; n=21) or a single injection of IA-saline plus 1 ml of 2% lidocaine (placebo group; n=22). Patients were enrolled in the study if they had chronic knee pain of 4.5 or more on a 0–10 NRS pain scale for at least 6-months, evidence of radiographic OA, had a failed response to oral analgesics/anti-inflammatory drugs and IA-corticosteroid injections, and were not candidates for shoulder joint replacement surgery. Patients were excluded if they were currently using aminoglycoside antibiotics, curare-like agents, or other agents that might interfere with neuromuscular function; had shoulder joint malignancy, a prosthetic shoulder joint or planned shoulder joint surgery in the next 6 months; prior botulinum toxin injection into the index shoulder joint; had disorders of neuromuscular function including myasthenia gravis, Eaton-Lambert syndrome, amyotrophic lateral sclerosis; known allergy/sensitivity to study medications; a history of recent or ongoing alcohol or drug abuse, uncontrolled systemic disease; or were pregnant, breast feeding or planning pregnancy during the study period. The study had >80% power to detect 1.5 unit difference in pain NRS scale between intervention and placebo.\n\nAt a 1-month follow-up or later, if patients did not experience any reduction in pain severity (due to inefficacy of the treatment), they were given the option to drop out of the randomized phase of the study, and request the second unblinded injection with 100 units of IA-BoNT/A, which led to the beginning of an open-label phase. The published paper reported the results from the 1-month double-blind portion of the study67.\n\n36 patients with 43 painful shoulder joints were randomized. Mean age was >70 years, 95% were men, >85% had OA as the underlying diagnosis, mean shoulder pain duration was 8–11 years, comorbidity index was high, at least 50% had been treated each with narcotics or non-steroidal anti-inflammatory drugs (NSAIDs), and all had failed previous IA-corticosteroid injections.\n\nReductions in shoulder pain severity were significantly greater in the IA BoNT/A group compared to the IA placebo group at 1 month (-2.4 vs. -0.8 unit reductions NRS respectively, p=0.014; primary outcome). 61% of patients in the IA BoNT/A group experienced clinically meaningful pain relief (defined as a 30% or 2-point reduction previously68,69) at 1 month compared with 36% in the placebo group (p=0.22).\n\nSeveral secondary outcomes were significantly better in the IA-BoNT/A versus the IA-placebo group. Quality-of-life improvements were significantly greater in the BoNT/A group versus the placebo group for five of the eight short form-36 (SF-36) subscales at 1-month, namely bodily pain, physical role functioning, vitality, emotional role functioning and mental health (p-values ranging from 0.04 to 0.001). Improvements in quality of life exceeded the clinically meaningful threshold of 10-points for all 5 SF-36 subscales in IA-BoNT/A group versus as opposed to only 1 subscale in the IA-placebo group. McGill affective pain dimension improved significantly more in BoNT/A compared to placebo at 1-month (p=0.047). Other secondary measures showed a trend in improvements, but were not statistically significantly different between treatment groups. The Shoulder Pain and Disability Index (SPADI) showed a trend towards greater improvement in BoNT/A versus placebo group (p=0.0826). Treatment-failure after 1 month, defined as a drop-out from the blinded phase due to inefficacy and request for an active treatment injection, was 2.5-times higher in the placebo vs. the BoNT/A group; 45% (10/22) vs. 19% (4/21; p=0.128) respectively. 61% in BoNT/A group experienced clinically meaningful pain relief at 1 month (a 2-point or 30% reduction in pain severity on the visual analogue pain scale (VAS)) compared to 36% in the placebo group (p=0.22).\n\nThe change in pain severity was influenced by baseline SPADI disability. In those patients with a higher SPADI disability score (>61.3), the decrease in VAS pain ratings at 1 month was significantly greater in the BoNT/A group (a 3.6 reduction) compared with the placebo (0.5; p=0.015). On the other hand, in patients with lower SPADI disability score (≤61.3), no significant difference was observed in VAS pain severity reduction between BoNT/A (a 1.9 reduction) and placebo groups (a 1.6 reduction; p=0.73).\n\nOverall adverse events were similar in IA-BoNT/A and IA-placebo groups. No significant difference in serious adverse events was noted. Common adverse events such as dry mouth, flu-like symptoms, dizziness etc. were not statistically significantly different between treatment groups in this small study, powered for efficacy, but not safety outcomes.\n\nComparison of IA-BoNT/A to IA-corticosteroid. Boon et al. compared IA-BoNT/A to IA-corticosteroids in patients with radiographic and clinical knee OA in an RCT70 (Table 1). In a single-center, prospective double-blind RCT, patients were randomized to one of three treatments - a single injection of 100 units (2 standard doses) of IA-BoNT/A (20 patients), 200 units (20 patients) or an IA-corticosteroid injection with 40 mg of methylprednisolone acetate (20 patients), into the painful knee joint. Patients were eligible for the study if they were adults ≥40 years that had both symptomatic knee OA with knee pain severity of ≥6 on a 10-point NRS scale that interfered with function most days of the week and Kellgren grade II or III tibiofemoral knee OA identified by plain radiographs. Patients were excluded if they had Kellgren grade I or IV tibiofemoral knee OA, inflammatory arthritis (such as rheumatoid arthritis, gout or pseudogout), reconstructive surgery on the affected knee, body mass index >35 kg/m2, recent IA-corticosteroid or hyaluronic acid injection (last 3-months), a clinically unstable medical or psychiatric condition or history of neuromuscular disease, aminoglycoside or curare-like agents use or severe peripheral neuropathy.\n\nThe primary outcome was VAS (0–10) pain scores at 8-weeks post-injection (rated using a 10cm line). Secondary outcomes included lower extremity pain and function assessment using the Western Ontario McMaster Universities Arthritis Index (WOMAC), quality of life assessment using the SF-36, patient global assessment and time taken for a 40-meter self-paced walk (in seconds). The study was powered to detect a 2 cm reduction in a VAS pain rating within each group compared to baseline scores. Non-responders were allowed to request a second injection at 8-weeks during face-to-face clinic visit after the blinded injection. Additional follow-up assessments were done at 12 and 26 weeks using mailed questionnaires.\n\nBaseline characteristics were similar in the three groups. Patients had a mean age of 61–64 years, body mass index 28–31 kg/m2, symptom duration ranging from 6–10 years and 50–65% were women. Patients had tried multiple modalities for knee pain relief in the past, including: 70–80% had tried IA-corticosteroid injections, 35–50% had tried IA-hyaluronic acid injections, 55–65% had tried acetaminophen, 75–90% had tried corticosteroids and 45–80% had tried physical therapy. All 60 patients (35 women and 25 men) completed the 8-week follow-up.\n\nVAS pain scores decreased from 6.4±1.8cm to 5.4±2.3cm (p=0.15; 22% reduction) in the IA-corticosteroid group. In the 100-unit IA-BoNT/A (low dose) group, pain severity decreased significantly from 6.6±1.9cm to 4.5±2.2cm (p=0.01; 28% reduction); and in the 200 units (high-dose) IA-BoNT/A group decreased from 6.6±1.4cm to 5.9±2.4cm (p=0.15; 26% reduction). A 2cm reduction in VAS pain scores, which is considered a clinically meaningful reduction in pain68,69, was reported by 42% in the IA-corticosteroid group, 60% in 100-unit IA-BoNT/A group and 26% in the 200-unit IA-BoNT/A group (p=0.10 for comparison between three groups; differences between 100 and 200 units not significant). Secondary outcomes including WOMAC pain scores, function and stiffness and self-paced 40 meter walk time improved significantly within each of the three groups at 8-weeks, compared to baseline (p<0.05 for each; Table 1). However, there were no significant differences for WOMAC scores between groups at 8-weeks. SF-36 scales did not improve significantly in any group, except in the mental health subscale score at 8-weeks in the IA-BoNT/A 200 unit group (Table 1). At the 8-week follow-up 65% in the IA-corticosteroid group, 75% in 100-unit IA-BoNT/A group and 70% in 200-unit IA-BoNT/A group said that they would have the treatment again. Seventeen patients withdrew from the study at the 8-week follow-up requesting re-injection and an additional 11 patients did not complete the 6-month follow-up questionnaire. For the patients remaining in the trial, the reduction in VAS pain was sustained at 12-weeks for both 100- and 200-unit IA-BoNT/A and at 12 and 26 weeks in the 100-unit IA-BoNT/A group, but not in the IA-corticosteroid group. No deaths, anaphylactic reaction or septic arthritis were reported in any group, strength testing did not reveal any significant changes at any time-point in treatment groups and none of the common adverse events were significantly different between groups in this small study, powered for efficacy, but not safety assessments.\n\nComparison of IA-BoNT/A to IA-placebo. In a recent review article, Mahowald et al. summarized the results of an RCT comparing a single injection of 100 units of BoNT/A reconstituted in 1 ml of saline plus 1 ml of 2% lidocaine (treatment group) to a single injection of IA-saline plus 1 ml of 2% lidocaine (placebo group) in patients with refractory knee pain due to knee OA71 (Table 1). Patients were enrolled in the study if they had chronic knee pain of 4.5 or more on the NRS pain scale for at least 3 months, evidence of radiographic OA, had received no benefit from oral analgesics/anti-inflammatory drugs, IA-corticosteroid or IA-hyaluronic acid injections, and were not candidates for joint replacement surgery. Exclusion criteria were the same as those stated in the shoulder RCT described in the section earlier72. We considered the patient’s report of knee pain relief 5–10 minutes after the injection and/or aspiration of joint fluid as a surrogate for an accurate intra-articular placement of the needle.\n\nForty-two patients were randomized to either a IA-BoNT/A or IA-placebo group. At 1-month, McGill pain scores decreased significantly in both the IA-BoNT/A (4.7, p=0.048) and IA-placebo groups (5.6, p=0.02). However, at 3 months the decrease in pain was significant only in the IA-BoNT/A (4.2, p=0.002), but not in the placebo group (4.6, p=0.09).\n\nDue to a differential treatment effect evident on scatter plots, patients were stratified by baseline NRS pain scale scores into moderate (4.5–6.9) or severe (7 or higher) pain for exploratory analyses. In the severe knee pain group, significant changes in McGill sensory, affective and total pain scores were noted in the IA-BoNT/A, but not in the placebo group (Table 1).\n\nIn summary, three RCTs and three non-randomized studies indicate that a single intra-articular injection of botulinum toxin is associated with a clinically meaningful reduction of pain and an improvement of function in patients with refractory joint pain due to osteoarthritis or other underlying arthritic-conditions. However, larger studies are needed to assess the most effective dose, the long-term safety of intra-articular injections and to define as to which patients might be the best candidates for this treatment option.\n\n\nTennis elbow\n\nIn an open-label case series of 14 patients with \"treatment-resistant\" tennis elbow, Moore and colleagues injected 20–40 units of BoNT/A into the extensor digitorum communis and found >50% pain relief in 9/14 and complete pain relief in 4/14 patients during the 6–8 month follow-up73. Pain relief began by 2 weeks in 10 patients, 3 weeks in one and after 1 month in two patients.\n\nHayton et al. studied 40 patients with refractory tennis elbow pain with a duration of over 6 months, who had experienced no pain relief from ≥1 corticosteroid injections and a full course of physiotherapy74 (Table 2). Patients were randomized to either 50 units of botulinum toxin type A (Allergan; n=19) or normal saline placebo (2 ml; n=21) injected 5 cm distal to the area of maximal tenderness at the lateral epicondyle74 (Table 2). There were no statistically significant differences in pain between the botulinum toxin and placebo groups at 3 months after the injection (difference of 1.1 between groups, p=0.54), grip strength (difference of 0.57 kg between groups, p=0.90), or quality of life measured by the Short Form-12 physical (difference of 6.24 points between groups, p=0.16) and mental (difference of 4.26 points between groups, p=0.42) component summary scores. Twelve of the 18 patients in the BoNT group had a transient extensors lag of the long finger at 1-weeks that disappeared 3-months after the injection; none reported this in the placebo group. The 1-point difference in pain scores between the BoNT and placebo group in this study is similar to that noted in studies of joint pain67,70,75, however, the larger standard deviation likely led to this difference being non-significant in this case.\n\nRCT, randomized controlled trial; BoNT, botulinum toxin; PL, placebo; SQ, subcutaneous; VAS, Visual Analog Scale; SD, standard deviation.\n\nIn another study by Wong et al. 60 patients experiencing tennis elbow pain for 3 months or longer were randomized to either a single injection of 60 units of botulinum toxin A (Dysport; Ipsen) or saline placebo injections into soft tissue and muscle 1 cm from the lateral epicondyle76 (Table 2). Patients were treatment-naive with no prior local injections. Patients were followed for 12 weeks in a double-blinded multicenter study. The mean age was 45 years, 49 were women and symptom duration was between 12 and 19 months in the two groups. Pain on a VAS scale (0–100mm) decreased significantly more in the active treatment group (65.5mm at baseline to 25.3mm at week 4 and 23.5mm at week 12) than placebo (66.2mm at baseline to 50.5mm at week 4 and 43.5mm at week 12). The differences between groups were statistically significant at both week 4 (p<0.001) and week 12 (p=0.006). Mild weakness in finger extension at 4 weeks was seen in 10 patients in the BoNT/A group versus 6 patients in the saline group. Four patients in the Botulinum group had paresis of the fingers at 4 weeks (in one patient this persisted to week 12) compared to none in the placebo group, but grip strength was similar in both groups at the two time points.\n\nAnother randomized study compared 1 to 2 injections of 30–40 units of Botulinum toxin type A (Allergan Inc.) into the wrist extensor with the surgical release of the extensor origin of the extensor carpi radialis brevis tendon77 (Table 2). Forty patients with refractory chronic tennis elbow pain (average duration of symptoms ~10 months) were randomized, 20 to each treatment. The mean age of the patients was 43 years, average symptom duration was 11 months (range 6–48 months), and 21 were women. They found no differences between the groups with regards to pain and grip strength up to 2 years of follow-up, while minor differences were noted at shorter follow-up periods. At 1 year, 65% of patients in the Botulinum toxin group and 75% in the operative group had good to excellent results (based on a validated composite scale with pain and patient satisfaction items). Limitations of the study included the lack of description of outcomes and the lack of a priori designation of outcomes as primary versus secondary.\n\nIn summary, results from three RCTs of patients with tennis elbow indicate that botulinum toxin may be effective (one of the three RCTs were positive; two RCTs were negative but had a small sample size), in at least some patients with tennis elbow. A small sample and short follow up period are limitations of these studies. These studies differed in patient population (patients with non-refractory vs. refractory disease), duration of disease (>3 months vs. >6 months vs. 10 months), site of injection (1cm vs. 5cm from the lateral epicondyle or direct into the wrist extensor) and the dosage of preparation used (60 units of Dysport vs. 50 units of Botox vs. 30–40 unit injections of Botox) in the three RCTs respectively.\n\n\nTemporomandibular joint (TMJ) pain\n\nIn an open-label study of 41 patients with painful hyperactivity of the masticatory muscles, 200 units of BoNT/A (Dysport) was injected intramuscularly, with 8 conducted under electromyographic guidance)78. The patients in this study had not gained pain relief from conservative treatment after 3 to 12 months of symptom duration. In 29 cases, only one treatment was administered. The majority of the injections were administered intraorally. Patients were observed for up to 12 months. 80% of patients reported an improvement in pain. The mean pain severity decreased from 6.4 to 3.5 on the 0–10cm VAS scale. Thirteen patients experienced a \"major improvement\" as evident by the disappearance of pain. Relief lasted 3–12 months during the observation period for most individuals; only 7 patients requested re-injection. One patient experienced temporary speech impairment and swallowing difficulty post-injection, which had completely reversed after 2–5 weeks.\n\nIn an open-label study, 46 patients who had experienced TMJ pain for a median duration of 96 months were injected with 50 units of botulinum toxin A in each masseter muscle and 25 units in each temporalis muscles under electromyographic guidance79. Patients were followed up to 8 weeks. The mean age was 41 years and 39 were women. Comparing pre-injection to 8-week post-injection assessments, a significant improvement was noted in the pain VAS from a median of 8 to 5, functional disability index scores dropped from 5.3 to 3.9mm, tenderness to palpation reduced from 15.5 to 6mm and jaw opening extent improved from 29.5 to 34.5 mm (p<0.05 for assessments). No subjects reported worsening of their condition or any side effects after the injection.\n\nIn another study, 15 patients with temporomandibular disorder received 150 units of BoNT/A with 50 units in each masseter and 25 units in each temporalis muscle bilaterally80. The mean age was 39 years and 13 were women with a mean symptom duration of 10 years. Compared to pre-injection, at 8 weeks post-injection, a significant improvement was noted in patients; mean pain VAS scores dropped from 7.3 to 4, functional disability index scores reduced from 5.5 to 3.1, tenderness to palpation scores improved from 17 to 7.6 and jaw opening extent increase from 27 to 34mm (p<0.05 for assessments).\n\nIn a single-blinded randomized study, 90 patients with chronic facial pain caused by hyperactivity of the masticatory muscles, received intramuscular injections of 35 mouse units (based on the amount required for a lethal dose in mice) of botulinum toxin A (Allergan; n=60) or placebo (n=30) in both masticatory muscle81 (Table 3). The patients in the study had experienced no relief from conservative treatment after 3 to 34 months of use. A significantly greater reduction in 0–10 VAS pain scores was noted in the Botulinum toxin (a 3.2 unit decrease) compared with the placebo group (a 0.4 unit decrease) (p<0.01) was found at a follow-up conducted between 1–3 months. A greater proportion of patients in the BoNT/A group had ≥2-point improvement in VAS pain scores during the follow-up. One patient experienced temporary paralysis of facial expression muscles and swallowing difficulty post-injection, which resolved after 4 weeks. No speech impairment or systemic botulism was reported.\n\nRCT, randomized controlled trial; NRS, Numeric Rating Scale; VAS, Visual analog scale; WHYMPI, West Haven-Yale Multidimensional Pain Inventory.\n\nIn summary, intramuscular injections of botulinum toxin seem to induce short-term pain relief in cases of temporomandibular disease. The evidence for this is based on non-randomized studies and one single blind study where the nature of the blinding (patient- or physician- blinded) as well as the time-point for outcome measurement were not described. The results of this study are also limited by the short follow-up duration.\n\n\nLow back pain\n\nNey et al. reported the results of injection of 200–500 units of botulinum toxin A intramuscularly in up to 4–5 trigger points per each side of the back from L2 to S1 in 60 patients with chronic low back pain in an open-label prospective study82. The mean age was 47 years, mean disease duration was of 9 years, 18 were women and 62% had concurrent radicular pain. A positive response was defined by the occurrence of 2 out of the following 3 criteria: a ≥50% improvement in pain VAS scores, an improvement of 2 or more grades in the pain and functional subsets of the Oswestry Low Back Pain Questionnaire, and a ≥30% increase in the number of pain-free days from baseline. 58% of patients responded positively at 2 months and of these 17% still reported improvement at 4 months of follow-up. Most patients with improvements at 2 months reported that the beneficial effects weaned off by 4 months. 18 of the 19 patients that received re-injection reported beneficial response at a 2 month follow up. Two patients reported a mild, flu-like reaction lasting 3–5 days. None of the patients reported any muscle weakness.\n\nFoster and colleagues compared 200 units of Botulinum toxin A (Allergan) injected intramuscular paravertebrally from L1-5/L2-S1 (n=15) to placebo (n=16) in 31 patients with chronic back pain of ≥6 month duration83 (Table 3). 40 units/site were injected at five lumbar paravertebral levels on the side of maximum discomfort. The mean age was 46 years, 16 were women and the mean pain duration was 6 years (with a range of 0.5–30 years). The number of individuals experiencing greater than 50% reduction in VAS pain scores from baseline was significantly higher in the botulinum toxin group compared with placebo: 73% vs. 25% at 3 weeks (p=0.012), and 60% vs. 13% at 8-weeks (p=0.009). The Oswestry Low Back Pain Questionnaire score improved significantly more patients in patients receiving Botox (67%) compared with those receiving placebo (19%, p=0.011). No patient reported worsening of pain or function after the BoNT injection, but two patients reported worsening of pain after placebo injections.\n\nIn summary, intramuscular injections of botulinum toxin in patients with chronic back pain seems to provide short-term benefits of pain reduction. This is based on one non-randomized study and one randomized study. More evidence is needed.\n\n\nPlantar fasciitis\n\nPlaczek et al. reported a case series of 9 patients with chronic plantar fasciitis, who were injected with 200 units of botulinum toxin A (Dysport) in the plantar fascia84. Patients were followed up to one year after the injection. Statistically significant pain relief began two weeks after the injection (from 4.2 to 1.9 on a 0–10 VAS pain scale, p=0.012) and persisted for the 52 weeks of follow up (from 4.2 to 0.4, p=0.043). Muscle weakness or systemic effects were not seen.\n\nBabcock and colleagues compared 70 units of Botulinum toxin A injected into the plantar fascia at two sites per foot to a placebo in 27 patients (43 feet in total) who had experienced chronic refractory plantar fasciitis for 6 months or more, and who had failed to respond to conventional therapies except surgery or extracorporeal shock therapy85 (Table 3). In this study, 22 feet were randomized to BoNT/A and 21 to placebo. The median age was 44 years and 18 patients (12 with bilateral and six with unilateral plantar fascia) were women. Of the sixteen bilateral patients (male and female), 12 improved on all measures in the BoNT/A-treated foot and only one improved in the placebo-treated foot. Both pain and pain relief were measured on separate 0–10cm VAS scales. Compared to the placebo group, the Botulinum toxin group had significantly improved VAS pain relief scores (4.75cm vs. 0.6cm at 3 weeks and 4.95cm vs. 1.2cm at 8 weeks, p<0.005 for both), significantly lower VAS pain scores (2.7cm vs. 4.7cm at 3 weeks and 1.6cm vs. 4.4cm at 8 weeks, p<0.005 for both), significantly better foot function as assessed by the Maryland Foot Score (100-point scale) (72 vs. 49 at 3 weeks and 81 vs. 54 at 8 weeks, p<0.001 for both) and less muscle tenderness at plantar fascia insertion as assessed by a pressure algometry response (2.7 vs. 1.8 at 3 weeks and 2.8 vs. 1.8 at 8 weeks, p<0.003 for both). No complications were reported.\n\n\nHand pain and carpal tunnel syndrome\n\nBreuer and colleagues randomized 20 patients with carpal tunnel syndrome with associated hand pain to receive electromyographically guided placebo or botulinum toxin B injections in three hypothenar muscles anatomically attached to the carpal tunnel86 (Table 3). The dose used in 18 of the 20 patients due to modified protocol was 2,500 units, since the first two patients reported weakness and stiffness of the fourth and fifth fingers with higher doses of 5000 and 7500 units. During the 13-week trial, significant decreases in pain outcomes and improvements in function were noted in both placebo and the BoNT/A groups compared to the baseline, but there were no significant differences between the two groups. The unblinding of the first two patients and variation in the BoNT/A dose during the trial make interpretation of this study’s findings difficult.\n\n\nAnterior knee pain\n\nOne non-randomized study provides evidence for the use of botulinum toxin in cases of anterior knee pain. Singer et al. injected 300 to 500 units of botulinum toxin A (Dysport) into the vastus lateralis muscle in 8 women with chronic anterior knee pain of more than 6 months duration who had failed to respond to conservative management, e.g. patello-femoral bracing or taping87 (Table 3). The injection was followed by a 12-week home exercise program to strengthen the vastus medialis. The mean age was 29 years and mean symptom duration was 5 years (range, 1–19 years). Patients reported a decrease in knee pain (individual changes were shown in the article, not means), an increase in function with an improvement in the mean force production in the affected limb from 22.7 kg to 24.3 kg) and an improvement in the mean time taken to ascend and descend a flight of 11 stairs from 12 to 10 seconds. These improvements were maintained at a 24-week follow up.\n\n\nHip pain\n\nOne non-randomized study provides evidence for the use of botulinum toxin in hip pain. A total of 400 units of Botulinum toxin type A (Dysport) were injected into the adductor longus and the adductor magnus muscles in 39 patients with hip osteoarthritis88. The mean age was 68 years (age range 41–82 years). The Harris Hip Score, a commonly used measure of hip function, increased significantly after 2, 4 and 12 weeks after the BoNT/A injection (p<0.0001). There was a significant decrease in pain 2, 4 and 12 weeks after the BoNT/A injection (p<0.001).\n\n\nSummary and conclusions\n\nIn this review, I have summarized studies of the use of botulinum toxin for musculoskeletal pain. Most studies used either an intra-articular or intramuscular route of administration in patients with refractory pain, who had not responded to multiple other treatment interventions. Pre-clinical laboratory evidence supports an independent anti-nociceptive mechanism of action of botulinum toxin. While there are several osteoarticular conditions for which botulinum toxin has been studied in an RCT compared to placebo, for several conditions such as low back pain, plantar fasciitis, temporomandibular disorder and carpal tunnel syndrome, the evidence is based on non-randomized data and only a single RCT. For other conditions, such as osteoarthritis of the knee/shoulder and tennis elbow data in support of its use from several RCTs and non-randomized studies was available. Most RCTs included in this review had several limitations. Most studies were single center, had a small sample size, short follow-up and in some cases, non-standardized injection techniques. The evidence for an anti-nociceptive action of botulinum toxin in osteoarticular pain is growing. Side effects seem to be mild, and in cases of muscle weakness, reversible; however, data from larger samples needs to be generated. Evidence from larger multicenter studies of longer duration that test various doses, regimens and routes of administration of botulinum toxin are needed to better define its role in management of osteoarticular pain.", "appendix": "Competing interests\n\n\n\nThere are no financial competing interests related directly to this study. JAS has research and travel grants from Takeda, Savient, and Allergan; and consultant fees from URL pharmaceuticals, Savient, Takeda, Ardea, Allergan and Novartis. The views expressed in this article are those of the author and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government.\n\n\nGrant information\n\n\n\n\nReferences\n\nInstitute of Medicine: Relieving Pain in America: A Blueprint for Transforming prevention, care, Education and Research. Washington, D.C.: The National Academies Press; 2011. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "826", "date": "11 Mar 2013", "name": "Robert Gerwin", "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 reviews the use of botulimum toxin in the treatment of pain, rather than in the treatment of muscle contraction. This is a new area of potentially great interest and therefore the review is both timely and presents a new use of botulinum toxin to be considered. The drawback in the article is that it is a systematic review, not a metaanalysis and not a critical review. As such, it could be shortened by summarizing the studies with less detail. The greater detail in which the cases are described does not add a great detail, since they are simply recapitulations of the study, and do not tell tell us if the study is credible or not. I suggest that the author either be more concise in the summaries, or be more critical in assessing the studies. Moreover, the author includes both intra-articular injections and intramuscular injections. The intramuscular injections may have complex effects, including; analgesic effects, effects from blocking acetylcholine release from motor nerve endings, and the elimination of muscle myofascial trigger points. However, the review is incomplete. For example, he discusses low back pain with injections into the lumbar paraspinal musculature, but only gives Ney and Forster as two references. He could have included the work of Gul et al and of DeAndres et al. The title and abstract are appropriate. My comments regarding the nature of his review are given above. The conclusion is accurate and justified. There are no experimental data to discuss, since this is a review article. In summary, the topic is timely, the review summarizes the data from some but not all relevant studies and in that sense is not complete, and the summary does not address the credibility of the articles cited, and that is a weakness.", "responses": [ { "c_id": "467", "date": "21 May 2013", "name": "Jasvinder Singh", "role": "Author Response", "response": "We thank the reviewers, who provided great insights. The revisions made in response to their comments have improved the quality and clarity of this paper. Here are our responses to the comments from reviewer #2. Reviewer comments Response to reviewers Robert Gerwin, Pain & Rehabilitation Medicine, Bethesda, MD, USA Status and Report: 11 Mar 2013 This article reviews the use of botulimum toxin in the treatment of pain, rather than in the treatment of muscle contraction. This is a new area of potentially great interest and therefore the review is both timely and presents a new use of botulinum toxin to be considered. Thank you. The drawback in the article is that it is a systematic review, not a metaanalysis and not a critical review. As such, it could be shortened by summarizing the studies with less detail. The greater detail in which the cases are described does not add a great detail, since they are simply recapitulations of the study, and do not tell tell us if the study is credible or not. I suggest that the author either be more concise in the summaries, or be more critical in assessing the studies. We agree and have listed this as a limitation of our review. We have added an overall limitation section for our review. We have also added a section of comments related to study strengths and weaknesses of each indication to provide a more critical assessment of each study. “Summary and Study limitations: plantar fasciitis In summary, the evidence is based on one non-randomized study and one RCT. Pain and function both improved short-term in botulinum toxin compared to placebo at 3- and 8-week follow-up in RCT and pain improved up to 1-year follow-up in non-randomized study. While the data seems promising, more RCTs are needed and evidence of efficacy at longer-term is needed to assess whether botulinum toxin can provide a longer-term relief in patients with plantar fasciitis.” “Limitations of this review This was a focused review of randomized and non-randomized studies, not a systematic review or a meta-analysis. Therefore findings must be interpreted with caution. In particular, the summary section for various musculoskeletal pain disorders highlights the limitation of studies included. In particular, evidence for the efficacy of botulinum toxin in anterior knee pain and hip pain was based on non-randomized study data only (one study for each), and is subject to bias. The intramuscular injections may have complex effects, including the analgesic effects, effects from blocking acetylcholine release from motor nerve endings, and the elimination of muscle myofascial trigger points. On the other hand, intraarticular injections may work primarily by their effect on the analgesic pathways. “ Moreover, the author includes both intra-articular injections and intramuscular injections. The intramuscular injections may have complex effects, including; analgesic effects, effects from blocking acetylcholine release from motor nerve endings, and the elimination of muscle myofascial trigger points. We agree that intramuscular and intra-articular injections likely have different mechanisms of action. Both types of injections are commonly used in the treatment of MSK pain, the focus of our paper. We mention the important point brought up by the reviewer in the discussion of results. However, the review is incomplete. For example, he discusses low back pain with injections into the lumbar paraspinal musculature, but only gives Ney and Forster as two references. He could have included the work of Gul et al and of DeAndres et al. We have now added DeAndres et al. and Lew et al. to our review. We are sorry but could not find Gul et al. The title and abstract are appropriate. Thank you. My comments regarding the nature of his review are given above. The conclusion is accurate and justified. There are no experimental data to discuss, since this is a review article. Thank you. In summary, the topic is timely, the review summarizes the data from some but not all relevant studies and in that sense is not complete, and the summary does not address the credibility of the articles cited, and that is a weakness. We have now included the additional studies pointed out by the reviewer. We have also added statements related to the credibility of each studies for each condition (please see responses to reviewer comments in the sections above and the changes in the revised article)." } ] }, { "id": "824", "date": "12 Mar 2013", "name": "Julia Funk", "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 manuscript is a decent, very detailed, and almost complete review. The title and abstract are appropriate for the content of the article and the conclusions are sensible and justified.However, I do have two significant reservations. Unfortunately, for a complete review of the published data concerning the topic 'botulinum toxin and pain' we found two RCTs missing: Lin YC (2010) Am J Phys Med Rehabil, Placzek R (2007) JBJS Am.Furthermore, there have been at least 3 review articles with the same topic in 2012 (Hameed F (2012), Cheng OT (2012), Sim WS (2011)), hence by reading this manuscript, little information may be new to the informed reader.", "responses": [ { "c_id": "466", "date": "21 May 2013", "name": "Jasvinder Singh", "role": "Author Response", "response": "We thank the reviewers, who provided great insights. The revisions made in response to their comments have improved the quality and clarity of this paper. Here are our responses to the comments from reviewer #1. Reviewer comments Response to reviewers Reviewer#1: Julia Funk, Centrum für Muskuloskeletale Chirurgie, Orthopädische Universitätsklinik der Charité, Berlin, Germany Status and Report: 12 Mar 2013 The manuscript is a decent, very detailed, and almost complete review. The title and abstract are appropriate for the content of the article and the conclusions are sensible and justified. Thank you. However, I do have two significant reservations. Unfortunately, for a complete review of the published data concerning the topic 'botulinum toxin and pain' we found two RCTs missing: Lin YC (2010) Am J Phys Med Rehabil, Placzek R (2007) JBJS Am. We thank the reviewer for pointing us to the two missing studies, which have now been included in the review. Furthermore, there have been at least 3 review articles with the same topic in 2012 (Hameed F (2012), Cheng OT (2012), Sim WS (2011)), hence by reading this manuscript, little information may be new to the informed reader. We thank the reviewer for pointing out these pertinent reviews. We refer to them in our paper and discuss how our review adds to what’s already been published. Both Hameed and Cheng et al. were focused on osteoarthritis (OA) treatments only. Our review is more comprehensive in that it includes muscucloskeletal (MSK) pain, not just knee OA or OA. Sim et al. provides a very brief overview of a few studies of botulinum toxin for any indication, without providing much detail about key studies. Thus, we believe our review adds to the already published reviews, and provides a complete perspective on the potential role of botulinum toxin in the treatment of MSK pain. “Recent reviews have summarized the injectable treatments for osteoarthritis (64), injectable treatments for knee osteoarthritis (65) or use of neurotoxin for the treatment of painful disorders (66), In this review, we provide a review of evidence regarding Botulinum toxin for the treatment of musculoskeletal pain conditions.”" } ] } ]	1	https://f1000research.com/articles/2-52
https://f1000research.com/articles/2-159/v1	17 Jul 13	{ "type": "Short Research Article", "title": "Do children with mental disorders have higher prevalence of hypovitaminosis D?", "authors": [ "Mini Zhang", "Keith Cheng", "Robert Rope", "Elizabeth Martin", "Ajit Jetmalani", "Keith Cheng", "Robert Rope", "Elizabeth Martin", "Ajit Jetmalani" ], "abstract": "Inadequate vitamin D level is associated with various adverse medical outcomes. There is a growing concern that insufficient vitamin D may play a role in the development of psychiatric symptoms. This study aims to answer the question: do children with mental disorders have a higher prevalence of hypovitaminosis D? A retrospective chart review examined 25 hydroxyvitamin D (25(OH)D) levels in youth ages 7 to 17 (n=67) at two Oregon psychiatric residential facilities. Vitamin D deficiency is defined as <20 ng/ml and insufficiency as <30 ng/ml. Diagnoses were organized into six categories. 25(OH)D levels were compared across genders and diagnostic groups using a two-sample t-test and ANOVA, respectively. Statistical differences in prevalence across diagnostic categories were calculated using a Pearson chi-square test. Using the data from Saintonge’s NHANES III study on healthy US children for comparison, 21% of our cohorts were found to be vitamin D deficient and 64% insufficient, in contrast to 14% and 48%, respectively. While our results are not statistically significant, mainly because of small sample size, the overall mean 25(OH)D level in our cohort was insufficient (27.59 ± 9.35 ng/ml), compared to a sufficient mean value of 32.1 ng/ml in the general population. No statistical significant difference was found in the prevalence across diagnostic categories. This study found that children with psychiatric disorders might have a higher prevalence of hypovitaminosis D than the general pediatric population. Although a causal relationship between hypovitaminosis D and psychiatric disorders cannot be derived based on the study design, our study provides initial descriptive data on the prevalence of hypovitaminosis D in children with psychiatric disorders, which has not been previously reported to our knowledge. Prospective studies with a larger sample size and controlled variables would allow more precise analysis of the relationship between hypovitaminosis D and childhood mental disorders.", "keywords": [ "Vitamin D", "mental illness", "psychoses", "autism", "mood", "insufficiency", "deficiency", "25(OH)D", "paediatric" ], "content": "Introduction\n\nAn inadequate vitamin D level is increasingly being linked to diverse disease states. Beyond its importance in endocrine and bone health, there is a growing concern that vitamin D insufficiency may affect brain function and mental health.\n\nStudies have linked hypovitaminosis D to various psychiatric disorders such as depressed mood1 and schizophrenia2,3. In children, emerging evidence suggests that vitamin D plays a role in brain development4–6. A Finnish study found that vitamin D supplementation in infancy reduced the risk of schizophrenia later in life among males7. A Swedish study found that patients diagnosed with schizophrenia and autism had the lowest 25-hydroxyvitamin D (25(OH)D) levels among psychiatric diagnoses, and proposed that low 25(OH)D may not only be a predisposing developmental factor, but may also affect mental health in adulthood3. Therefore, prevention of hypovitaminosis D in early life may be associated with reduced risk of developing certain psychiatric disorders.\n\nThe optimal level of vitamin D is controversial. The cutoff serum levels range from 20 to 50 ng/ml8. The American Academy of Pediatrics (AAP) recommends a minimal level of 20 ng/ml in children. Using the same cut-off value, the US Institute of Medicine (IOM) reports that the majority of North Americans have sufficient vitamin D required for bone health8. However, some subgroups, particularly those who are older, those living in institutions, or those with darker skin, may be at increased risk for hypovitaminosis D8.\n\nVitamin D insufficiency in the general population was estimated to range from 1% to 78% among different studies9. Using the Third National Health and Nutrient Examination Survey (NHANES III, 1988–94) data, Saintonge et al. estimated that 14% of healthy children had 25(OH)D levels below 20 ng/ml, and 48% had levels below 30 ng/ml10. The prevalence of hypovitaminosis D among children with mental disorders remains unclear.\n\nThis study attempts to ascertain whether children with mental illness have a higher prevalence of hypovitaminosis D and whether there is a difference in prevalence across various disorders.\n\n\nMethods\n\nA retrospective chart review was conducted at two residential psychiatric treatment programs in Oregon, USA, (latitude 45°N). There were 67 patients aged from 7 to 17 years, whose serum 25(OH)D levels were measured between October 2009 and 2010. Patients had one to four co-morbid psychiatric diagnoses. There were no exclusion criteria. Given the retrospective nature and lack of identifiable health data used in the study, no institutional review board approval was needed.\n\nDeficiency was defined as <20 ng/ml, based on the AAP recommended value. Insufficiency was defined as <30 ng/ml, by the local laboratory standard used in Oregon. For the 14 patients who had multiple 25(OH)D levels recorded, we used the lowest 25(OH)D level in our analyses. We felt this method was justified clinically if any period with this degree of hypovitaminosis D during childhood is correlated to developmental differences. The diagnoses were organized into six categories shown in Table 1. For patients with multiple diagnoses, their 25(OH)D level was counted individually in each diagnostic category to calculate the mean and prevalence.\n\n25(OH)D levels were compared across genders and diagnostic groups using a two-sample t-test and ANOVA, respectively. Statistical differences in prevalence across diagnostic groups were calculated using a Pearson chi-square test. The analysis was performed using STATA IC (version 11) from StataCorp LP, College Station, Texas.\n\n\nResults\n\nA total of 67 patients with 168 diagnoses were included in this study. Using the NHANES III study10 for comparison, 21% of our cohort were vitamin D deficient (<20 ng/ml), compared to 14% reported in the general US population. If a cut-off value of 30 ng/ml was used, 64% of the children in our study population were classified as being insufficient, compared to 48% of healthy US children.\n\nThe overall mean 25(OH)D level in our study cohort was 27.59 ± 9.35 ng/ml (i.e. insufficient), compared to a mean value of 32.1 ng/ml (i.e. sufficient) in the general US population10. Females in our study (n=29) had a mean level of 27.4 ± 9.1 ng/ml, comparable to the mean of 27.74 ± 9.66 ng/ml amongst males (n=38). The gender difference was non-significant (p=0.89). The mean 25(OH)D levels by diagnostic category are shown in Table 2. No statistical significant differences could be concluded in the mean level (p=0.80) across diagnostic categories.\n\nThe prevalence of patients with hypovitaminosis D across diagnostic groups using both cutoff values are shown in Figure 1. There was no statistical significance found in the prevalence across diagnostic groups, perhaps due to the small sample size. It is interesting to note that psychotic disorder had the highest prevalence of deficiency and insufficiency among specific diagnostic groups: 43% and 71%, respectively.\n\nValues are given as percentages. See Table 1 for a list of ‘Other Disorders’.\n\n\nDiscussion\n\nBeyond its importance in endocrine function, there is growing awareness of the role that vitamin D plays in brain function. However, the prevalence of hypovitaminosis D in children with mental illnesses is uncertain. This study found that children with serious psychiatric disorders may have a higher prevalence of hypovitaminosis D and a lower mean 25(OH)D level, compared to the general US population10. Although no statistical significance can be concluded, it is noteworthy that psychotic disorders had the highest prevalence of hypovitaminosis D among the specific diagnostic categories, which supports previous studies2,3,7. We suggest using a cut-off value of 20 ng/ml for clinical interventions, as recommended by the AAP and IOM10. Clinicians should discuss the costs and benefits of treatment with patients when levels are between 20 and 30 ng/ml.\n\nThe primary limitation of this study was its small sample size. Due to its retrospective design, we were limited by the availability of 25(OH)D studies without specific clinical indications. Other limitations include not controlling for the length of inpatient stay, ethnicity, age, nutritional status, sun exposure, or skin pigmentation. For the patients with multiple diagnoses, their 25(OH)D was counted in each diagnostic category, which might overestimate the prevalence of hypovitaminosis D. The study utilized the lowest level of 25(OH)D for patients with multiple measurements, which might result in lower mean level and higher prevalence. Although a causal relationship between hypovitaminosis D and psychiatric disorders cannot be derived based on the study design, our study provides important initial descriptive data on the prevalence of hypovitaminosis D in a pediatric population with psychiatric disorders which has not, to our knowledge, been previously reported.\n\n\nConclusion\n\nAs research continues on the impact of vitamin D in medicine, its implication for psychiatric disorders may be clarified. While no robust statistical conclusions can be made mainly due to small sample size, this study provides initial data suggesting that children with mental illnesses might have lower vitamin D levels and a higher prevalence of hypovitaminosis D than the general population.\n\nGiven the high prevalence of hypovitaminosis D and its profound impact on overall health, clinicians should have a higher suspicion of hypovitaminosis D in the pediatric psychiatric population.\n\nImportant future steps include the design of a larger prospective study with more controlled variables would allow more precise analysis to establish the prevalence of hypovitaminosis D, as well as to infer any correlation between hypovitaminosis D and childhood mental illness. Preventative and ameliorative measures might subsequently be instigated to assess causation and affect the development and treatment of certain mental disorders.", "appendix": "Author contributions\n\nMZ and KC conceived the study. MZ designed the study, compiled the data, and prepared the first and final drafts of the manuscript. KC provided the data source, provided expert guidance, revised the manuscripts, and oversaw the study. RR contributed to the study design and manuscript preparation. EM provided statistical analysis of the data and contributed to revision of the manuscript. AJ provided revisions 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\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declare that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nSpecial thanks to Dr. Eric Turner for guidance in the submission and publication process.\n\n\nReferences\n\nGanji V, Milone C, Cody MM, et al:Serum vitamin D concentrations are related to depression in young adult US population: the Third National Health and Nutrition Examination Survey. Int Arch Med. 2010; 3: 29.\n\nMcGrath JJ, Eyles DW, Pedersen CB, et al:Neonatal vitamin D status and risk of schizophrenia: a population-based case-control study. Arch Gen Psychiatry. 2010; 67: 889–94.\n\nHumble MB, Gustafsson S, Bejerot S: Low serum levels of 25-hydroxyvitamin D (25-OHD) among psychiatric out-patients in Sweden: Relations with season, age, ethnic origin and psychiatric diagnosis. J Steroid Biochem Mol Biol. 2010; 121: 467–70.\n\nEyles D, Burne T, McGrath J: Vitamin D in fetal brain development. Semin Cell Dev Biol. 2011; 22: 629–36.\n\nKesby JP, Eyles DW, Burne TH, et al:The effects of vitamin D on brain development and adult brain function. Mol Cell Endocrinol. 2011; 347: 121–7.\n\nGrant WB, Soles CM: Epidemiologic evidence supporting the role of maternal vitamin D deficiency as a risk factor for the development of infantile autism. Dermatoendocrinol. 2009; 1: 223–8.\n\nMcGrath J, Saari K, Hakko H, et al:Vitamin D supplementation during the first year of life and risk of schizophrenia: a Finnish birth cohort study. Schizophr Res. 2004; 67: 237–45.\n\nInstitute of Medicine of the National Academies. Implications and Special Concerns. In A. Catharine Ross, Christine L. Taylor, Ann L. Yaktine, and Heather B. Del Valle, ed. DRI Dietary Reference Intakes for Calcium Vitamin D. 1st ed. Washington, DC: National Academies Press, 2011: 487.\n\nRovner AJ, O'Brien KO: Hypovitaminosis D among healthy children in the United States: a review of the current evidence. Arch Pediatr Adolesc Med. 2008; 162: 513–9.\n\nSaintonge S, Bang H, Gerber LM: Implications of a new definition of vitamin D deficiency in a multiracial US adolescent population: the National Health and Nutrition Examination Survey III. Pediatrics. 2009; 123: 797803." }	[ { "id": "1188", "date": "23 Jul 2013", "name": "Shailesh Jain", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title of the article is appropriate to the content of the article. The abstract is able to summarize the key elements of the research including the brief overview of the study design, statistical methods used and the key conclusion. The article is well constructed and clear in its presentation. It is succinct, direct, to the point and conveys clear message.Study design has been conducted, properly measured and collected appropriately. Due diligence has been made as pointed out regarding privacy of the patients. The ethnicity of IRB involvement is also clearly pointed out.The study design and methodology has been clearly delineated. The methodology and the steps described in this study can easily be replicated in future studies.The data are presented in an easy to understand and informative way which a busy reader can easily grasp the key findings.The study presentation has included all the data that are necessary to understand the key findings and helpful for future studies.Limitations are adequately described though the fact that other possible explanations, such as medical conditions, are not taken into account. Though the data ‘look’ okay, I would recommend a review by a statistician.", "responses": [ { "c_id": "508", "date": "25 Jul 2013", "name": "Mini Zhang", "role": "Author Response", "response": "Dear Dr. Jain, Thank you very much for your nice words and comments! We appreciate your time and expertise in reviewing our article. Please feel free to contact me if you have further comments or questions. Sincerely, Mini Zhang, MD" } ] }, { "id": "1117", "date": "25 Jul 2013", "name": "Linda Mayes", "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\nWhile the report offers an interesting hypothesis, the sample is under-powered and the findings are not significant. There are also many biases in why vitamin D levels were or were not measured in psychiatric samples. The authors note limitation of sample size. I would suggest increasing sample size and being more targeted in the definition of psychopathology in future case control design.", "responses": [ { "c_id": "511", "date": "29 Jul 2013", "name": "Mini Zhang", "role": "Author Response", "response": "Dear Dr. Mayes, Thank you for your comments and suggestions. We are aware of several limitations of our study including the sample size and the method of vitamin D measurement, as we stated in our paper. We agree that a bigger sample size and better design with controlled variables, and a prospective study will significantly increase the power and validity of our study. This study is the initial step, and we hope we would be able to pursue this a step further in the future. Thank you again for your time and expert opinion. Regards, Mini Zhang, MD" } ] }, { "id": "1112", "date": "31 Jul 2013", "name": "Hans-Peter Volz", "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 should clearly state whether they included all patients of this age group or whether the included patients represent a sub-sample; if yes, according to which inclusion criteria?Since patients with multiple diagnoses have been counted in each diagnostic category they fulfilled, a second analysis would be helpful in which every patient is counted only once.Was there any medication given that could influence vitamin D-Levels?If the authors can meet these points of criticism, the paper is well suited to be approved", "responses": [] } ]	1	https://f1000research.com/articles/2-159
https://f1000research.com/articles/2-157/v1	16 Jul 13	{ "type": "Short Research Article", "title": "Maintenance of empathy levels among first and final year medical students: a cross sectional study", "authors": [ "Areeb Sohail Bangash", "Nisreen Feroz Ali", "Abdul Haseeb Shehzad", "Sobia Haqqi", "Nisreen Feroz Ali", "Abdul Haseeb Shehzad", "Sobia Haqqi" ], "abstract": "Objectives: The purpose of this study was to quantify the levels of empathy amongst medical students in the first year and final year of the medical curriculum at a medical university in Karachi, Pakistan.\nMethods: A cross-sectional study, comprising of participating students in their first year and final year of the medical curriculum at Ziauddin University Medical College, was carried out, using the Empathy Quotient (EQ) scale consisting of 60 questions through a self-administered questionnaire. The results were collected anonymously over a time period of six months from a sample of 171 participants. Results: According to our analysis, we found 82.67% of fifth year students and 80.21% of first years showing average or above average levels of empathy. Female mean scores were 42±9.60 while males were 38.7±9.358 (P=0.03). No association was found between empathy and age of the participants (p=0.77). Conclusion: We found no significant difference in the levels of empathy between the first and fifth year medical students. However, it was shown that females exhibited higher levels of empathy than males.", "keywords": [ "Carl Rogers", "one of the founders of the humanist school of psychology", "states that empathy is:" ], "content": "Introduction\n\nCarl Rogers, one of the founders of the humanist school of psychology, states that empathy is:\n\n\"To sense the patient’s private world as if it were your own but without ever losing the \"as if\" quality…..to accurately sense the feelings and personal meanings the patient is experiencing and communicating…\"1\n\nEmpathy plays a crucial role in the physician-patient therapeutic relationship2. Patient satisfaction and their compliance with treatment improve when their physician understands them better3. In a recent survey, patients of physicians who had scored high in empathy, reported better disease control and prognosis, in comparison to patients of physicians with low empathy scores4,5.\n\nEmpathy is as important for medical students as it is for physicians. Interestingly, there seems to be a decline in empathy levels during medical training as reported by Tavakol et al. and Pederson et al6,7. Of the many reasons given to explain this phenomenon, it was reported that, as they progressed through medical school, a number of medical students were starting to become more cynical about life in academia and the medical profession.\n\nThe purpose of this study was to quantify empathy levels amongst medical students in the first and the final year of their medical curriculum at a medical university in Karachi, Pakistan.\n\n\nMaterials and methods\n\nWritten consent was obtained from each participant. The ethical review committee at Ziauddin University approved this study and the consent procedure.\n\nThe study participants included 171 out of total 195 medical students from the first and fifth year curriculum of the Medical College at Ziauddin University in Karachi, Pakistan, in 2012. There were a total of 96 students in the first year and 75 students in the fifth year. There were 107 female and 64 male students in the population studied. Students who were not present during the administration of the questionnaire (N=18) and incomplete forms (N=6) were not included in the study. Ziauddin Medical College follows a five-year medical school curriculum with the first three years focused on preclinical study, with limited patient contact, followed by two years of clinical study. The teaching language at the institute is English.\n\nTwo cohorts completed the Empathy Quotient Scale questionnaire anonymously, one comprised of first year medical students, the other of fifth year students. The study population included all students who provided complete information in the questionnaire. A list of total students for both the cohorts was extracted from the admissions department at Ziauddin University.\n\nThe medical students completed the Baron-Cohen and Wheelwright Empathy Quotient Scale (EQ), which is a psychometrically validated and reliable instrument for measuring the components of empathy8,9. The questionnaire used was in its original language (English) and format. The EQ consists of 60 questions, divided into two types: 40 questions judge the empathy of the participant (see Table 1). The remainder are filer questions, which are included to distract the participant from the constant exposure to empathy questions, thus acting as a control.\n\nEach question had the option of four different responses (strongly agree, slightly agree, slightly disagree and strongly disagree). The \"strongly agree\" response carried two points while \"slightly agree\" response scored one point, on the following questions: 1, 6, 19, 22, 25, 26, 35, 36, 37, 38, 41, 42, 43, 44, 52, 54, 55, 57, 58, 59, and 60.\n\nThe \"strongly disagree\" response carried two points while \"slightly agree\" response score one point, on the following questions: 4, 8, 10, 11, 12, 14, 15, 18, 21, 27, 28, 29, 32, 34, 39, 46, 48, 49, and 508.\n\nThe participants were asked to provide their age and gender before filling out the EQ. Gender was included because it has been previously reported that female medical students and physicians tend to have more empathy compared to their male counterparts10,11. As people age they become mature and develop concern for others, which correlates with a gain in empathy hence we included age as a variable12. The age range for first year students was 17–22 years and for fifth year students was 21–26 years.\n\nOne author distributed the self-administered questionnaires among the medical students between December 2011 and February 2012. Participation was voluntary and students were assured the responses were confidential. Informed consent was obtained from participants after explaining the purpose of the survey to them. The surveys were conducted immediately after a scheduled lecture, where attendance was compulsory for all medical students within the same cohort. Each student was given ample time to fill the questionnaire; students who were in a hurry were allowed to take the questionnaire and complete it at their own convenience and return it.\n\nParticipants who failed to complete or return the administered survey were defined as a non-responder. Baron-Cohen and Wheelwright define a score of 33 or more, out of a total of 80, as adequate empathy levels8. Individuals who scored below 33 were defined as \"class 1\", they had a lower than average ability to understand and respond to other people’s feelings. Students who scored 33 or more were divided into 3 categories according to their scores. A score between 33–52 was defined as \"class 2\", where participants showed an average ability to understand and respond to others feelings. A score between 53–63 was defined as \"class 3\", where the students showed an above average ability to understand and respond to another person’s feelings. A score between 64–80 was defined as \"class 4\", where the responder showed a very high ability to understand and respond to the feelings of others.\n\nAll computation was done using SPSS (version 20). For numeric data mean and standard deviation was used. For categorical data, we used percentages and frequencies. An independent t-test was applied to assess differences between the means of gender and the year of study. The measure of association between age and score was calculated using coefficient of correlation. A chi-square test was applied between the score cohorts, year of study and age group. A P-value lower than 0.05 was deemed significant.\n\n\nResults\n\nOf the 177 surveys distributed, 171 were completed and returned. The responders represent 88% of the total body of students in first and fifth year of Ziauddin Medical College.\n\nFigure 1 displays the distribution of EQ scores in the two groups. A comparative analysis revealed that the mean scores for fifth year students (40.85 ± 9.833) were almost equivalent to the first year students (40.70 ± 9.497).\n\nEQ scores were not significantly different between year groups.\n\nOur analysis of the EQ score for first years revealed that 67.7% of the students had class 2 scores while the remaining 19.8% and 12.5% students scored within classes 1 and 3, respectively. Similarly, the majority of fifth years had class 2 scores (73.3%) while the remaining students obtained class 1, 3 and 4 scores (18.7%, 6.7% and 1.3%), respectively. However, a non-significant association was found between the year of study with EQ scores (P=0.401), as shown in Table 2.\n\nWe found no association between age and EQ scores (P=0.77).\n\nTable 3 shows the empathy score classification according to gender in both years. According to the data, females had a higher mean score (85%) compared to their male counter parts (75%) when both year groups were combined. These results were also consistent with our findings between sexes within each year group. Female mean scores were 42 ± 9.60 while males were 38.7 ± 9.358 (P=0.03).\n\n\nDiscussion\n\nTo the best of our knowledge, our study is one of the first to assess empathy amongst medical students in Pakistan. Our results display novel findings, with adequate empathy levels in both first year and final year medical students. Contrary to various studies, which state that empathy decreases as the level of medical education increases5,6, in our study students in their final year had adequate empathy levels similar to students in first year. This is in support of results obtained in previous Japanese and Korean studies13,14. Morling and Lamoreaux15 have reported that Asians have more ‘collectivistic and less individualistic social cultures’ than Westerners, and a possible parallel can be drawn between our results and those seen in other culturally similar Asian countries such as Japan and Korea.\n\nThe empathy scores recorded amongst senior medical students in our study could also be a result of cohort effects. The emphasis placed during medical training o medical ethics, a considerate attitude towards the patient’s wellbeing and a concentration on a patient centered approach may increase empathy. Furthermore, after the second year of medical school, there is constant patient exposure where students are required to learn history-taking skills and regular examinations that build the student’s professional attitude and approach to gaining patient cooperation, factors that may enhance student empathy. Subjects such as behavioral sciences and medical ethics are taught in the third year of medicine; integrating behavioral sciences at the undergraduate levels can help doctors-in-training to have a better understanding of behavioral issues in clinical settings later on16. Hence, the training obtained throughout medical school may persuade students to implement a sympathetic manner in their interpersonal relationships with patients. It is also worth noting that in Pakistan a great deal of emphasis is placed on apt history taking, focused clinical examinations and particularly minimal lab tests to arrive at a diagnosis. The majority of the population is underprivileged and cannot afford the expense of treatment let alone costly diagnostic tests. Hence, the lack of ‘computer based diagnostic and therapeutic technology’17 enables physicians and the students shadowing them to rely mainly on good patient interaction and clinical expertise.\n\nIn contrast, the literature often shows a positive association of empathy with respect to age. The younger year groups may be less exposed to clinical situations and may hold more idealistic views when starting out in their medical education. Stressors such as academic performance, long work hours18–26, lack of sleep and subsequent increases in responsibility with age are all contributory factors to a decrease in empathy12,27,28. It has been suggested by Rosenfield and Jones29 that a high emphasis is placed on the student’s ability to objectively assess the patient and to maintain a professional and neutral approach. This may all contribute to a decrease in empathy with over the course of medical training.\n\nThrough our results, it was also found that females are more empathic than males in both year groups, a finding that is consistent with many international studies30–33.\n\nWomen show a greater understanding of the emotional support that the patient may need and generally tend to give a higher significance to developing inter-personal relationships with patients34–37, whereas men tend to assign greater significance to authority, independence and control38.\n\nMany reasons have been cited for greater empathy levels in females, including the suggestion that women have evolved to be more gentle and compassionate towards their offspring than their male counterparts30, and hence demonstrate better communication skills and a higher level of understanding towards their offspring. A possible connection can be drawn here, as offspring and patients both require care.\n\nThere were several limitations to our research. Due to the cross-sectional nature of our study, we were only able to examine empathy levels in year 1 and year 5. It would be of interest to measure how empathy varies each year throughout the five years of medical school using a prospective longitudinal study design. Secondly, it may also be that the candidates own self-perception influences his/her choices while filling out the questionnaires and this may vary from the actual behavior that is implemented in their everyday interactions. Thirdly, our study only focuses on students attending a private medical school in Pakistan. The results cannot be generalized for all medical colleges and a greater coverage of different medical schools and a larger study population are needed to validate our results further. It is also important to note that other characteristics such as cultural backgrounds and specialty preferences can have an impact on empathy levels; previous research has shown that students opting for ‘people oriented specialties’ show higher empathy levels than those who prefer ‘technology oriented specialties’17.\n\nThe strengths of our study include an appropriate sample size, which represents 88% of the sample population. Our research is the first of its kind in Pakistan and assesses multiple variables such as age, gender and year of education.\n\n\nRecommendations\n\nIt would be valuable to carry out a prospective study where students are followed annually from the beginning of first year until graduation, to give a true representation of change in empathy levels. Other variables such as cultural backgrounds and specialty preferences should be noted.\n\n\nConclusion\n\nMedicine is a field where interpersonal skills and concern for others are of key importance, for it is a field whose very core is based on service to humanity. It is thus essential that empathy should be nurtured in medical students rather than eroded away with time and clinical exposure. It was surprising to see that a large majority of both first years and fifth years maintained adequate empathy levels despite no significant emphasis placed on the matter throughout the medical curriculum. Gender differences were significant and further research needs to be carried out to determine reasons for this. Our research paves the way for further research to be carried out with Pakistani medical students, which may further justify our results and identify additional influencing variables.", "appendix": "Author contributions\n\n\n\nAreeb Sohail conceived the study and design, collected, analyzed and interpreted the data and drafted the article. Nisreen Ali and Abdul Haseeb contributed to the design of the study, collected and analyzed data and drafted the article. Sobia Haqqi supervised, interpreted the data and drafted the article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to thank all the students from batch 13 of Ziauddin Medical College for their voluntary participation in this study. Special thanks to Dr. Farhana Zafar and Dr. Nosheen Zehra, for their assistance during the data collection and for their endless support throughout the study.\n\n\nReferences\n\nRogers CR: Empathic: An unappreciated way of being. Couns Psychol. 1975; 5: 2–10. Publisher Full Text\n\nHemmerdinger JM, Stoddart SD, Lilford RJ: A systematic review of tests of empathy in medicine. BMC Med Educ. 2007; 7: 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPederson C: Presence as a nursing intervention with hospitalized children. Matern Child Nurs J. 1993; 21(3): 75–81. PubMed Abstract\n\nHojat M, Louis DZ, Markham FW, et al.: Physicians' empathy and clinical outcomes for diabetic patients. Acad Med. 2011; 86(3): 359–64. PubMed Abstract \| Publisher Full Text\n\nTavakol S, Dennick R, Tavakol M: Medical students' understanding of empathy: a phenomenological study. Med Educ. 2012; 46(3): 306–16. PubMed Abstract \| Publisher Full Text\n\nPedersen R: Empirical research on empathy in medicine-A critical review. Patient Educ Couns. 2009; 76(3): 307–22. PubMed Abstract \| Publisher Full Text\n\nPedersen R: Empathy development in medical education–a critical review. Med Teach. 2010; 32(7): 593–600. PubMed Abstract \| Publisher Full Text\n\nBaron-Cohen S, Wheelwright S: The empathy quotient: an investigation of adults with asperger syndrome or high functioning autism, and normal sex differences. J Autism Dev Disord. 2004; 34: 163–175. PubMed Abstract \| Publisher Full Text\n\nLawrence EJ, Shaw P, Baker D, et al.: Measuring empathy: reliability and validity of the Empathy Quotient. Psychol Med. 2004; 34: 911–919. PubMed Abstract \| Publisher Full Text\n\nHojat M, Mangione S, Nasca TJ, et al.: The Jefferson Scale of Physician Empathy: development and preliminary psychometric data. Educ Psychol Meas. 2001; 61: 349–65. Publisher Full Text\n\nHojat M, Gonnella JS, Mangione S, et al.: Empathy in medical students as related to academic performance, clinical competence and gender. Med Educ. 2002; 36: 522–7. PubMed Abstract \| Publisher Full Text\n\nDavis MH: Measuring individual differences in empathy; evidence for a multidimensional approach. J Pers Soc Psychol. 1983; 44: 113–26. Publisher Full Text\n\nKataoka HU, Koide N, Ochi K, et al.: Measurement of empathy among japanese medical students: psychometrics and score differences by gender and level of medical education. Acad Med. 2009; 84(9): 1192–1197. PubMed Abstract \| Publisher Full Text\n\nRoh MS, Hahm BJ, Lee DH, et al.: Evaluation of empathy among korean medical students: a cross-sectional study using the korean version of the jefferson scale of physician empathy. Teach Learn Med. 2010; 22(3): 167–171. PubMed Abstract \| Publisher Full Text\n\nMorling B, Lamoreaux M: Measuring culture outside the head: a meta-analysis of individualism-collectivism in cultural products. Pers Soc Psychol Rev. 2008; 12: 199–221. PubMed Abstract \| Publisher Full Text\n\nHumayun A, Herbert M: Towards behavioral sciences in undergraduate training: a core curriculum. J Pak Med Assoc. 2011; 61(8): 800–7. PubMed Abstract\n\nHojat M, Vergare MJ, Maxwell K, et al.: The devil is in the third year: a longitudinal study of erosion of empathy in medical school. Acad Med. 2009; 84: 1182–1191. PubMed Abstract \| Publisher Full Text\n\nWesterman GH, Grandy TG, Ocanto RA, et al.: Perceived sources of stress in the dental school environment. J Dent Educ. 1993; 57(3): 225–31. PubMed Abstract\n\nSanders AE, Lushington K: Effect of perceived stress on student performance in dental school. J Dent Educ. 2002; 66(1): 75–81. PubMed Abstract\n\nPolychronopoulou A, Divaris K: Perceived sources of stress among Greek dental students. J Dent Educ. 2005; 69(6): 687–92. PubMed Abstract\n\nRajab LD: Perceived sources of stress among dental students at the University of Jordan. J Dent Educ. 2001; 65(3): 232–41. PubMed Abstract\n\nBurk DT, Bender DJ: Use and perceived effectiveness of student support services in a first-year dental student population. J Dent Educ. 2005; 69(10): 1148–60. PubMed Abstract\n\nYap AU, Bhole S, Teo CS: A cross-cultural comparison of perceived sources of stress in the dental school environment. J Dent Educ. 1996; 60(5): 459–64. PubMed Abstract\n\nte Brake H, Bloemendal E, Hoogstraten J: Gender differences in burnout among Dutch dentists. Community Dent Oral Epidemiol. 2003; 31: 321–7. PubMed Abstract \| Publisher Full Text\n\nAcharya S: Factors affecting stress among Indian dental students. J Dent Educ. 2003; 67(10): 1140–1148. PubMed Abstract\n\nDahlin M, Joneborg N, Runseson B: Stress and depression among medical students: a cross-sectional study. Med Educ. 2005; 39: 594–604. PubMed Abstract \| Publisher Full Text\n\nSpencer J: Editorial: decline in empathy in medical education: how can we stop the rot? Med Educ. 2004; 38: 916–8. PubMed Abstract \| Publisher Full Text\n\nAktekin M, Karaman T, Senol YY, et al.: Anxiety, depression, and stressful life events among medical students: a prospective study in Antalya, Turkey. Med Educ. 2001; 35: 12–7. PubMed Abstract \| Publisher Full Text\n\nRosenfield PJ, Jones L: Striking a balance: training medical students to provide empathetic care. Med Educ. 2004; 38: 927–33. PubMed Abstract \| Publisher Full Text\n\nHojat M, Gonnella JS, Nasca TJ, et al.: Physician empathy: Definition, components, measurement and relationship to gender and specialty. Am J Psychiatry. 2002; 159: 1563–1569. PubMed Abstract \| Publisher Full Text\n\nHojat M, Mangione S, Nasca TJ, et al.: The Jefferson Scale of Physician Empathy: Development and preliminary psychometric data. Educ Psychol Meas. 2001; 61: 349–365. Publisher Full Text\n\nHojat M, Gonnella JS, Nasca TJ, et al.: The Jefferson Scale of Physician Empathy: Further psychometric data and differences by gender and specialty at item level. Acad Med. 2002; 77(10 suppl): S58–60. PubMed Abstract\n\nHojat M, Zuckerman M, Magee M, et al.: Empathy in medical students as related to specialty interest, personality, and perceptions of mother and father. Pers Individ Dif. 2005; 39: 1205–1215. Publisher Full Text\n\nChen D, Lew R, Hershman W: A cross-sectional measurement of medical student empathy. J Gen Intern Med. 2007; 22(10): 1434–1438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAustin EJ, Evans P, Magnus B, et al.: A preliminary study of empathy, emotional intelligence and examination performance in MBChB students. Med Educ. 2007; 41: 684–689. PubMed Abstract \| Publisher Full Text\n\nNewton BW, Barber L, Clardy J, et al.: Is there hardening of the heart during medical school? Acad Med. 2008; 83(3): 244–249. PubMed Abstract \| Publisher Full Text\n\nHojat M, Gonnella JS, Mangione S, et al.: Empathy in medical students as related to academic performance, clinical competence and gender. Med Educ. 2002; 36: 522–527. PubMed Abstract \| Publisher Full Text\n\nDi Lillo M, Cicchetti A, Lo Scalzo A, et al.: The Jefferson Scale of Physician Empathy: preliminary psychometrics and group comparisons in Italian physicians. Acad Med. 2009; 84(9): 1198–1202. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1107", "date": "19 Jul 2013", "name": "Kevin J Black", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 surveyed first- and fifth-year medical students in a private medical school in Pakistan with the goal of quantifying empathy according to a standard questionnaire. The hypothesis was that medical students' empathy declines during training. A high survey return rate was achieved (88%). Women scored higher on empathy than men, which fits the results of other studies. However, there was no significant difference in empathy scores between beginning and senior medical students whether viewed as a mean or as a distribution between typical, low, and high score categories. The authors discuss possible explanations for their results appropriately and do not go beyond the data. They note the primary limitations: the cross-sectional approach and the focus on one school. The authors are impressed that ~80% of the students scored in the normal to above normal empathy ranges. By contrast, I was surprised that 19-20% of the students scored in the below-normal range. After all, these are people entering a profession devoted to taking care of others! I am not aware of whether other studies of empathy among physicians or trainees show higher or lower rates of empathy than in the general population, but if so, a mention of those results in Discussion would add interest.Overall, this is an interesting tidbit of information about medical training, presented clearly and without exaggeration.", "responses": [] }, { "id": "1095", "date": "25 Jul 2013", "name": "Pooria Sarrami Foroushani", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article has an appropriate title, and the abstract provides adequate information about the study. The paper is well written, containing clear and balanced data about the study and related literature. Bangash et al have used a standard questionnaire to compare empathy level of first and five year medical students. They had a high response rate and have found that empathy level remains at the high level of 80%. The authors have referred to the limitation of their methodology. They have argued that this finding is different from Westerners’ studies and is similar to the results of researches in other Asian countries, such as Japan and Korea. This is an interesting topic for further explorations. As Bangash et al have also recommended, it will be interesting to undertake prospective longitudinal studies and also to explore to the relationship between self-reported empathy with actual interactions with patients.", "responses": [] }, { "id": "1094", "date": "29 Jul 2013", "name": "Iman Hegazi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the manuscript with great interest. Empathy will remain an area of high importance for medical education and, despite being extensively studied, its research should still be encouraged world-wide.This is well-conducted research and the authors have succeeded in explaining the data clearly and concisely. The abstract is concise and provides an adequate summary of the article. The article is clearly written and well constructed.I recommend only a few minor changes:I believe that the current title may be somewhat misleading as the word “maintenance” implies a more longitudinal study, or a study across all cohorts from first to final year, whereas this research is more of a comparative cross-sectional study between first and final year medical students. I recommend that a more appropriate title be used.The methodological rigor is adequate with only a few pointers:Since the EQ test was not included in the article, I do not see any need for paragraphs 5 and 6 in the “Materials and methods” section. These paragraphs did not help explain the EQ and those who are interested in understanding the EQ test can easily refer to the reference. Similarly, table 1 does not add any information and can be removed.In the statistical analyses section, it was mentioned that the t-test and chi-square test were used. The values of these tests along with the p-values should be included in the text and in tables 2 and 3.The discussion and conclusion appear to be sensible and balanced. It would have been interesting to tell us about the selection process that medical students have to go through before being accepted in this private medical school.Overall, this is a well-written and interesting article.", "responses": [] } ]	1	https://f1000research.com/articles/2-157
https://f1000research.com/articles/2-8/v1	10 Jan 13	{ "type": "Research Article", "title": "I. Embryonal vasculature formation recapitulated in transgenic mammary tumor spheroids implanted pseudo-orthotopicly into mouse dorsal skin fold: the organoblasts concept", "authors": [ "Halina Witkiewicz", "Phil Oh", "Jan E Schnitzer", "Phil Oh", "Jan E Schnitzer" ], "abstract": "Inadequate understanding of cancer biology is a problem. This work focused on cellular mechanisms of tumor vascularization. According to earlier studies, the tumor vasculature derives from host endothelial cells (angiogenesis) or their precursors of bone marrow origin circulating in the blood (neo-vasculogenesis) unlike in embryos. In this study, we observed the neo-vasculature form in multiple ways from local precursor cells. Recapitulation of primitive as well as advanced embryonal stages of vasculature formation followed co-implantation of avascular (in vitro cultured) N202 breast tumor spheroids and homologous tissue grafts into mouse dorsal skin chambers. Ultrastructural and immunocytochemical analysis of tissue sections exposed the interactions between the tumor and the graft tissue stem cells. It revealed details of vasculature morphogenesis not seen before in either tumors or embryos. A gradual increase in complexity of the vascular morphogenesis at the tumor site reflected a range of steps in ontogenic evolution of the differentiating cells. Malignant- and surgical injury repair-related tissue growth prompted local cells to initiate extramedullar erythropoiesis and vascular patterning. The new findings included: interdependence between the extramedullar hematopoiesis and assembly of new vessels (both from the locally differentiating precursors); nucleo-cytoplasmic conversion (karyolysis) as the mechanism of erythroblast enucleation; the role of megakaryocytes and platelets in vascular pattern formation before emergence of endothelial cells; lineage relationships between hematopoietic and endothelial cells; the role of extracellular calmyrin in tissue morphogenesis; and calmyrite, a new ultrastructural entity associated with anaerobic energy metabolism. The central role of the extramedullar erythropoiesis in the formation of new vasculature (blood and vessels) emerged here as part of the tissue building process including the lymphatic system and nerves, and suggests a cellular mechanism for instigating variable properties of endothelial surfaces in different organs. Those findings are consistent with the organoblasts concept, previously discussed in a study on childhood tumors, and have implications for tissue definition.", "keywords": [ "“The significant problems we face cannot be solved at the same level of thinking we were at when we created them”." ], "content": "\n\n“The significant problems we face cannot be solved at the same level of thinking we were at when we created them”.\n\n– Albert Einstein\n\n\nIntroduction\n\nTumor vascular biology research has been beleaguered with multiple controversial experimental data and unanswered questions despite the technological progress and new concepts in network biology. The importance of understanding interactions among individual elements of a system has only recently gained popularity. Much of the information on mapping of mammalian interactome networks waits to be experimentally validated, although advancements have been made in small model organisms (yeast, Saccharomyces cerevisiae; fruit fly, Drosphila melanogaster; and worm, Caenorhabditis elegans)1. Richness and redundancy enabling the natural selection of evolving biological systems make their analysis difficult and call for new methods and ways of thinking2,3. Unsolved problems include lineage relationship between endothelial stem cells (SCs) and hematopoietic stem cells (HSCs)4,5; the role of platelets in embryonal development6,7; lineages of immune cells8, tolerance of tumors by the host immune system9 and reprogramming of energy metabolism by tumors5,9. The question of biological significance of the cellular heterogeneity within tumors also remains unanswered.\n\nTraditionally vasculogenesis and hematopoiesis were subjects of independent studies and some fundamental issues have remained unclear in each case. The origin of the precursors of endothelial cells (ECs) has created controversy in understanding tumor growth related vasculogenesis, whereas the illusive origin of HSCs had been a daunting problem in understanding embryonal development. As opposed to de novo vasculature formation during embryogenesis (vasculogenesis), tumor vasculature has been thought to emerge by expansion of the host vasculature through postnatal sprouting of endothelial cells (angiogenesis). Therefore, it was proposed that inhibiting angiogenesis would stop tumor growth10. When that conceptually simple objective turned out to be impossible to reach, a notion of neo-vasculogenesis was born, suggesting that tumor vessels form from circulating bone marrow-derived endothelial precursors rather than from mature ECs11. However, the views on tumor neo-angiogenesis have remained polarized3,12 and we have yet to stop tumor growth.\n\nDescriptions of the two main types of erythropoiesis, i.e. primitive and definitive, has kept changing over the years. Extremely large nucleated cells and smaller enucleated ones, distinguished first, were later referred to as nucleated and anuclear, respectively13,14. Each wave of erythropoiesis was reported to generate more potent progenitors (progenitors of more numerous cell types)15. Lately, five distinct classes of hematopoietic cells in murine conceptus emerged from studies on their activity in transplantation or clonogenic assays in vitro: primitive; pro-definitive (myeloid progenitors); meso-definitive (lymphoid-myeloid progenitors); meta-definitive (neonatal repopulating HSCs); and adult-definitive (adult repopulating HSCs). Maturation of primitive, nucleated precursors from yolk sac in circulation has also been reported16. These classes of cells appeared to be generated independently of each other in distinct hematopoietic sites17. In other words, the origins of HSCs seemed to be multiple. Conclusions on the embryonic location of HSCs capable of long-term repopulation depended on the assays used to test them. Those from yolk sac repopulated neonate recipients better than adults, whereas those from aorta-gonad-mesonephros (AGM) repopulated adult recipients better than neonates18. Evidently, the successful repopulation requires a certain level of developmental compatibility. After birth, bone marrow (medulla ossea) was proposed to be the proper niche where the formation of blood elements would continue for the lifetime as needed. Therefore, extramedullar hematopoiesis observed in liver19,20, adipose tissue21, cerebellar hemagioblastoma22, meningioma23, or subdural hematoma24 was perceived as unusual. However, in our study, it appeared essential for tumor growth and for regeneration of normal tissues.\n\nThe debate over the mechanism leading to the elimination of the nucleus from maturing erythroblast during erythropoiesis, expulsion of nucleus or karyolysis, dated back to the late nineteenth century25. The current consensus on achieving enucleation by nuclear extrusion came from the ultrastructural studies published in 1967 that addressed the controversy by looking at either peripheral blood from dogs intentionally made anemic26 or at erythroid clones grown in the spleen of irradiated mice transplanted with syngeneic marrow cells25. In normal peripheral blood or non-manipulated spleen, the enucleation event was too rare to study by transmission electron microscopy (TEM). Ultrastructural analysis of a developing embryo are not available in the literature, even for an organism as well studied as the mouse. The Edinburgh Mouse Atlas Project offers no TEM images (http://www.emouseatlas.org/emap/home.html). In our model, the enucleation event was frequent enough to reveal its mechanism and we found no evidence for nuclear extrusion. Instead, we observed several variants of generating erythrocytes by nucleo-cytoplasmic conversion. Thus, depending on the model, the same method could generate conflicting results. Interestingly, the analysis of chromosomal topology showed that at certain stages of erythroblast differentiation, two chromosomes were unidentifiable27. Below, we use the term “erythrosome” as a synonym for “erythrocyte” because the latter is not a cell.\n\nSimilarities and differences in the antigenic composition of tumors and embryonal tissues, as well as in patterns of isozymes distribution, have been studied for years28,29. Reappearance in tumors of embryonic and fetal antigens, as well as isozymes, has been shown to be a common phenomenon, yet similar alterations have sometimes been observed in non-neoplastic tissues under dietary and hormonal influences29,30. In cases of carcinoembryonic antigen (CEA), the concept ultimately did not apply, contradicting the antigen’s name, as CEA-cross-reactive antigens were located in normal human tissues including blood31. Also, “patterned vascular channels without EC”, i.e. vascular mimicry, were observed in embryos as well as in growing tumors32,33. The analogy between the two types of growing tissues was further enforced by showing that tumor cells could be epigenetically reprogrammed into normal cell types34,35. Here, we present an unprecedented collection of original images documenting the full range of growing dynamic complexity of the tumor-induced vasculature formation, reminiscent of the embryonal one.\n\nThe hallmark of the classic definition of adult TSCs is the potential of the cells to self-renew and differentiate36. The 2002 amendment to that definition37 emphasized additional aspects of the concept, namely: a shift from the cellular view to a system view including self-organizing processes, stemness as a capability rather than as a cellular property, dependence on the growth environment, within-tissue plasticity, and the functionality of the TSCs and the tissue. The stemness as an attribute of a system, rather than of a particular cell lineage, questioned the usefulness of presumed protein markers for identifying stem cells (SCs)37–39. With regard to both embryonal and cancer SCs (ESCs and CSCs, respectively) the views remain opposing. On the one hand, neither ESCs nor CSCs fulfill all those criteria40; on the other hand, both do but with certain qualifications. In the case of the ESCs, self-renewal and differentiation is likely to occur sequentially for two reasons. (1) The redundancy during development could serve as an evolutionary safety measure; in the context of the early embryo, that could mean generating a certain number of identical cells before their differentiation begins41. (2) Heterogeneity with respect to cell cycle activity was detected even in such an iconic cell lineage as the HSCs of adults42. In case of the CSCs, their role in tumorigenesis appeared to depend on microenvironment as discussed below and in reference43.\n\nThe goal of our study was to visualize the cellular mechanisms of vasculature formation associated with the growth of malignant tissue. We carried out the ultrastructural in situ analysis of initially avascular breast tumor spheroids co-implanted with homologous tissue graft, into the mouse dorsal skin folds44,45. A complementary study on such spheroids implanted without the graft is the subject of a separate report43. In contrast to the reductionism that dominated the ultrastructural studies of the second half of the last century, we analyzed tissue sections instead of isolated tissue components. The high resolution was necessary to investigate distinct physiology and functions of individual cells whereas the tissue context unveiled interactions among the cells. The results revealed interdependence between erythrogenesis and vasculogenesis localized at the site of tissue growth or repair and other new qualities of the emerging system. The data illustrated a great variability and increasing complexity of the vasculature morphogenesis, i.e., formation of blood and vessels, as both were evolving from primitive forms while enabling the tissue building process. Like in the embryo17, the new tumor vasculature was developing independently of the existing one. New erythrosomes and other blood elements formed extramedullary, and new vessels were assembling around them before initiation of the blood flow occurred. Vessel “sprouts” progressed toward other vessels not away from them. By suggesting local tissue cells as progenitors of the new vasculature, those observations related to the concept of tissue stem cells (TSCs).\n\nThe results of the morphological analysis of the vasculature formation in our model were consistent with the amended functional definition of the TSCs concept. They are not in harmony with the common understanding of the cell lineages as hierarchical and irreversible progress of differentiation. Moreover, the new findings suggest a hypothesis that the adult vasculature formation represents a partial recapitulation of the embryonal organogenesis involving coordinated activity of some cells capable to form all tissue types of a particular organ de novo. Such a perspective is not entirely new. The term “organoblast” was used in 1997 in reference to pediatric salivary gland tumors that had morphological characteristics of the organ with maturation arrested at a primitive state of development46. Later, mouse mammary SCs in the form of in vitro grown mammospheres (“organoids”) were shown to regenerate morphologically normal mammary epithelial ducts after implantation into mammary fat pads surgically cleared of the endogenous epithelium47.\n\nOur results also demonstrate the potential of immunocytochemistry to reveal further molecular-level details of the cellular interactions as they relate to regional tissue growth. This approach is suitable to validate concepts derived from proteogenomics as well as effects of pharmacological treatments. It is far more relevant to medical problems than in vitro studies and cheaper than conducting clinical trials.\n\n\nMaterials and methods\n\nThe study was performed according to protocols approved by 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\nThree recipient mice of the 5 used in the two accompanying articles were assessed in this report. 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, N202.1A48 was stably transfected to express GFP under histone H2B promoter49. The two cell lines, N202.1A parental and N202.1A+H2B-GFP (obtained from Drs. J. Lustgarten and P. Borgström) 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) as described earlier45. The tumors were incubated in the chambers for one or three weeks (Table 1). Their final size was about 1–3 mm in diameter. The blood circulation in tumor vessels was monitored in vivo by light microscopy before disassembling the chambers50.\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 perpendicularly 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 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, using a Leica Ultracut UCT ultramicrotome, and stained with lead citrate51 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 epitops (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 over night, mounted on metal pins and frozen in liquid nitrogen, as described by Tokuyasu52. Frozen tissues were cut into 70 nm sections, using 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 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 set of images included in the Supplement.\n\n\nResults\n\nMultiple steps of cellular differentiation and the growing complexity of the vasculature formation process were observed in tissue samples harvested at only two different time points (5 and 21 days after implantation) due to local variability resulting from lack of synchronization in vivo. The emergence of increasingly more specialized phenotypes of individual cells characterized those steps, signifying progress in the cellular differentiation. Multiple forms of erythropoiesis reflected the multiple levels of cell maturation.\n\nFive days after implantation, in hyaline membrane between the tumor surface and the glass chamber wall, the earliest event seen was migration of megakaryocytes and erythroblasts and their products, platelets and primitive erythrosomes respectively, along with the bipotent precursors of both cell types7,53–55 (Figure 1 & Figure S1). The cells seemed loosely bound together by fibrous (non-collagen) secretion, likely proteoglycans. They appeared to be migrating in columns and differentiating simultaneously, as if scouting the hypoxic avascular space under the glass and establishing a blueprint for vascular pattern. The presence of a composite dense body, characteristic of platelets56, in an extra-cellular matrix (ECM) producer indicated that the ECM building cell was related to megakaryocyte (Figure 1[E & F]). At the same time on the tumor surface, the two cell types had formed a primitive vascular pattern in the absence of ECs (Figure 2 & Figure S2). A crystalline inclusion resembling an Auer body57 was found in a small structure of such nature (Figure 2 [C]). In both cases, the cells represented two principal functional attributes of the vasculature. The first attribute, the metabolism-related functions of transporting gases (O2 & CO2) and of generating ATP anaerobicly58, were associated with erythrosomes. The second one, the structure-organizing functions, were represented initially by pro-megakaryocytes in the form of secreted elements of the extracellular matrix (ECM), possibly carcinoembryonic antigen (CEA)31 and later, by mature megakaryocytes and platelets. That way, megakaryocytes were in a position to receive additional nourishment (ATP) from the erythrosomes as their mitochondria suffered from some hypoxic distress ([F] in Figure 1 & Figure S1). ECs were absent. By shaping the vascular pattern, megakaryocytes presented themselves as a cell type involved in early vasculature morphogenesis, in addition to the known later role of platelets in mending vascular injuries by their aggregation and the thrombus formation. At higher magnification, the emerging erythrosomes revealed nucleo-cytoplasmic conversion as the mechanism of enucleation in single erythroblasts as well as in clusters of them (Figure 3 & Figure S3).\n\nThe pattern-forming heterogeneous population of the cells migrating in the hyaline membrane consisted of (a) those with large nuclei occasionally containing double nucleoli and with asymmetrically distributed, irregularly shaped, large cytoplasm and satellite platelets i.e. megakaryocytes, and (b) those with atypical, lobular, heterogeneously dark nuclei and dark cytoplasmic granules in the absence of mitochondria, usually accompanied by erythrosomes, i.e. most likely erythroblasts [A]. The four elements, cellular and sub-cellular (erythrosomes and platelets), appeared evenly dispersed among each other like in the narrow (about capillary short dimension) column in [B] or the megakaryocytes that displayed a tendency to migrate to the periphery of the larger cluster and surround the other elements [C]. Those poorly differentiated cells were probably bipotent, megakaryocyte and erythroid, progenitors [C]; compare with Figure 8 in55. The cellular and sub-cellular elements appeared to be held together with extracellular fibrous components, possibly proteoglycans and little collagen (the insert in [C]). The resulting red pattern appeared as a vascular network under low magnification of dissecting microscope. The parallel arrangement of extracellular, short, non-collagen fibers suggested mobility for the cells secreting them [D]. The extra-cellular matrix (ECM) producer [E] located to the right of the erythroblast [F] contained a composite dense body (arrows in [E & F]) characteristic of platelets (Figure 111-1, E in56).\n\nPrimitive vascular pattern in the tumor-harboring solid tissue below the hyaline membrane consisted of megakaryocytes and platelets engaged in direct and tight contact with one another and with erythrosomes and erythroblasts. There were no ECM-filled spaces left between them nor was there an additional cellular layer covering those elements. The elongated branching structure in [A] appeared kept together by megakaryocytes and platelets (N.B. the line in the middle of the image is a scratch caused by an imperfection of the knife-edge during cutting the section). In the two circled regions of that structure, incomplete separation of platelets from megakaryocytes provided ultrastructural evidence for their identity and for an ongoing process of generating platelets at that location. One region was the bifurcation area at the base of the right almost vertical branch, enlarged in [B] and the other was the tip of the lower left branch (enlargement not shown). Evidently, megakaryocytes were capable of organizing erythrosomes into branching primitive vascular structure before ECs emerged to provide protective walls of a vessel. In a smaller elongated structure of similar nature, although without platelets in the section plane, one of the cells contained an Auer body [C], previously described in neutrophils from cases of leukemia and defined as azurophilic, large, elongated granules (Figure 64-10 in57). The upper part of that cell contained two erythrosomes; whereas what remained of the nucleus had a different texture compared with that of the nucleus in the second cell of that complex. The prominent dark granules of the latter resembled those in the cell next to the megakaryocyte from the tip of the lower branching structure in [A]. If the irregularly shaped seemingly empty space next to those granules was a prototype of a lumen then both those cells could have potentially been angioblasts.\n\nThe single cells undergoing structural remodeling in the nucleus and cytoplasm [A & B] represented at slightly different stages of erythropoiesis. Conceivably, a few such cells, when gathered into a cluster, could fill up a larger space with erythrosomes creating a cobble stone pattern, also called ‘erythropoietic lake’ or myeloplaxes [C]. Platelets and megakaryocyte were recognizable at the periphery of the cluster.\n\nThe immunohistochemical staining detecting CD34 confirmed the erythropoietic lineage identity of the cells by analyzing frozen counterparts of the samples shown in Figure 1, Figure 2, Figure 3 & Figure S1, Figure S2, Figure S3 (Mouse 5, and some from Mouse 3). The CD34 label was present in mononuclear and giant polyploidal erythroblasts (Figure 4 & Figure S4 [A–F] & [G–I]) respectively. Both types were at the early stage of nucleo-cytoplasmic conversion but in the giant cells, the mitochondria were still intact and they were oversized. The few giant cells belonged to a larger cluster of such cells, most likely representing cells undergoing growth and endoreduplication before converting into giant erythroschizonts that would later break into erythroschizomers. Together those features suggested very efficient production of large number of erythrosomes. In 1909, Maximow interpreted the emergence of such extremely large primitive nucleated red cells found in mammalian embryos as a response to relative hypoxia of the fetus and the need for rapid generation of a large amount of blood13. The extramedullar erythropoiesis induced by hypoxia of growing tumors resembled fetal erythropoiesis in that regard.\n\nClusters of the gold label were found on the cell surface, in the nucleus, cytoplasm and mitochondria of cells morphologically fitting the description of erythroblasts [A, B, D & E] and on extracellular collagen fibers produced by them [B & C]. A fragment of cell with mixed phenotype is shown in [F]. The cluster of CD34-labeled giant erythroblasts (potentially leading to myeloplaxes) contained one cell with a double nucleus (right side in [H]). Two regions of [H] are enlarged in [G & I], left and right respectively, to show oversized mitochondria, some converting into peroxisomes [G]. That cluster of cells was bigger than the one shown earlier (Figure 3C), and the presence of mitochondria despite clear commitment to erythrogenesis suggested that here the process was more efficient. Arrows point out some clusters of Au grains.\n\nDetecting the green fluorescent protein (GFP) label of either the graft or the tumor spheroids helped to address the question of the source of hemato-vasculogenic cells in the tumor microenvironment. In Mouse 5, the graft tissue originated from the donor mouse that carried the GFP marker under a ubiquitin promoter. Thus the GFP label found in erythroblasts, erythrosomes, platelets and ECs indicated participation of the graft cells in building of the tumor vasculature (Figure 5 & Figure S5), although unlabeled cells with similar morphology were also present. Cells with ambivalent morphology, like the one in Figure 5 & Figure S5 [F], provided a hint that ECs were evolving from erythroblasts.\n\nGFP-specific label was present in erythroblasts [A], primitive erythrosomes like the one separating from the cell that made it (left lower corner in [B]), platelets [C] and in EC [D]. Morphologically interesting cells, erythroblast [E] and perishing hemangioblast [F], stained with nonspecific antibody are shown at low magnification (they had no gold grains). The erythroblast was entirely consumed by a successful incremental conversion into at least three erythropoietic vesicles [E] and the cell with ambivalent phenotype [F], possibly hemangioblast, contained three internal vesicles resembling erythrosomes and two of comparable size that appeared to be failing as erythrosomes (partially electron lucent ones).\n\nWe wondered if a mechanism similar to the one controlling hemostasis could play a role in establishing the vascular pattern before forming the endothelium. A protein best known for its involvement in platelet aggregation and thrombus stabilization, calmyrin, also known as calcium and integrin-binding protein (CIB1), was therefore detected next. Consequently, a new role for calmyrin in tissue morphogenesis emerged from such analysis of the hyaline membrane where the cells were mostly separated from one another. At low magnification, occasionally triplets of cells were seen within a single field; each one with different features that suggested that they were related but currently committing to become either megakaryocytes, erythroblasts or ECs ([A, B & C] in Figure 6 & Figure S6) and (Figure 7 & Figure S7). The calmyrin-specific label was located in all three morphologically distinct cells and in the fibers connecting the differentiating ECs with another cell and in the ECM (Figure 6 & Figure S6 [C] & [D] respectively), as well as in large, possibly polyploidal cells that contained internal vesicles similar to those in the cells of regular size (Figure 6 & Figure S6 [D & G]). Those vesicles were either becoming erythrosomes or losing their electron-dense content. The cells were perishing in the process but at the same time, they were changing the microenvironment by leaving traces of their presence in the ECM as developmental clues for other cells. All that suggested that the differentiation process was happening there by means of trial and error, i.e. cytoevolution. Calmyrin seemed to be instrumental in building this tissue canvas. Overall distribution of the protein inside the cells was compartmentalized ([E, F & I] in Figure 6 & Figure S6) but within each compartment, the calmyrin label was dispersed randomly. Inside the primitive erythrosomes of the hyaline membrane, the label was also dispersed (with rare exceptions), unlike in those from the solid tissue closer to the tumor.\n\nThe cells shown in the upper row [A, B & C] were located in the hyaline membrane 5 µm (or less) apart from one another (Figure 7 & Figure S7) suggesting their relatively recent derivation from a common precursor. The first, irregularly shaped cell with platelet-resembling attachment was probably a megakaryocyte [A]. The second one, had four large vesicles but only one of them was similar in size and electron density to a cell-free erythrosome (right upper corner in [A] and elswhere), whereas two other vesicles were much lighter with the exception of small patches at the periphery, and the fourth one was probably an incompletely converted nucleus [B]. The third cell had two large vesicles and both appeared to represent different stages of degradation of the material they were made of [C]. The outer membrane of that cell was at one side connected to another cell via calmyrin-containing extracellular fibers (insert in [C]). Both cells with the large vesicles made impression of cells with mixed phenotypes with some inclinations to specialize in either producing erythrosomes [B] or forming a lumen [C]. Fields shown in [D–I] were in the same section; they represented what seemed to be left after internal remodeling of polyploidal cells or multi-cellular clusters [D & G]. There were cell-free erythrosomes as well as empty vesicles, some broken [H]. Clearly, cells were perishing because of such restructuring but they were also changing the microenvironment. Insert in [D] shows calmyrin-specific label on fibers of ECM. [E, F & I] are enlarged regions from [D] and [H], respectively.\n\nCompare with Figure 6 [A, B & C]. The insert shows calmyrin-containing extracellular fibers connecting the adjacent cells.\n\nIn those more differentiated cells beneath the hyaline membrane, the calmyrin-specific label formed clusters over an area of about caveolar size, i.e. 70 nm in diameter (Figure 8 & Figure S8) revealing a new ultrastructural element, the calmyrite. The sub-cellular location of the calmyrites in mitochondria, peroxisomes and erythrosomes, suggested that all three of those organelles were homing previously unreported ultrastructural elements related to energy metabolism. In addition to cell types shown in Figure 8 & Figure S8 [A-H], calmyrites were seen in megakaryocytes, platelets, granulocytes, plasma cells, smooth muscle and rat normal EC(s). The included rat lung section (Figure 8 [I]) demonstrated that calmyrites existed in normal differentiated cells together with mitochondria and were not a product of necrotic process, but rather representing a backup metabolic pathway rendering robustness to the cellular metabolism.\n\nUneven distribution of the abundant calmyrin-specific label in erythrosomes from the solid tissue under the hyaline membrane was striking [A, B, C & D]. No such clusters were seen extracellularly [E]. The gold particles appeared arranged with certain symmetry in clusters of about 6 to 12, as if the protein became a part of distinct para-crystalline ultrastructures that in less differentiated regions were not formed ([E] and Figure 6 & Figure S6). Such gold grain clusters were also present in ECs [F] and tumor cells [G] where small dark mitochondria suggested hypoxic distress. In a giant cell the uneven distribution of scattered calmyrin-specific label and heterogeneity of electron density indicated an ongoing process of erythrogenic conversion [H]. One calmyrite in a section from normal rat lung is shown in [I]. Those ultrastructural para-crystalline elements visualized by calmyrin-specific label are being described here for the first time and referred to as calmyrites.\n\nThree weeks after implantation, some cells kept converting into the erythrosomes and others kept providing structural support to them but in a more complex way. Their phenotypes kept changing (Figure 9 & Figure S9). Some cells were producing large amounts of collagen as if converting their entire cytoplasm into masses of collagen while their nuclei were converting into erythroschizosomes (Figure 9 & Figure S9 [C]) leading to a new tissue canvas in the form of fields of collagen with erythrosomes scattered in there. Such vessel-free erythrosomes described earlier in a similar context were believed to be extravasated erythrocytes. Eosinophils, adipocytes and dendritic cells were occasionally present in that environment as well. Erythrosomes and cells providing structural support to them seemed to have been chemotacticly attracted to each other regardless of the current level of their differentiation. Moreover, segments of new vasculature with increasing structural complexity were assembling around emerging erythrosomes into capillaries and vessels of variable size simultaneously. Occasionally, megakaryocyte-resembling cells were positioning themselves like pericytes, i.e. abluminally, close to a forming vessel. The one in Figure 9 & Figure S9 [B] might have been a megakaryocyte evolving into ECs or pericyte. Still greater complexity of the vasculature morphogenesis was evident in the second mouse with tumor grown for three weeks. D epending on the location in the section, the more primitive forms of the vasculature were present as well. Collagen came across as an important factor for building not only vasculature but also tumor tissue (Figure 10 & Figure S10). Signs of hypoxic stress were in the form of relatively small darkening or necrotic mitochondria (loosing their internal structure) and dilated cisternae of endoplasmic reticulum (ER) partially depleted of ribosomes and resembling vesiculo-vacuolar organelles (VVOs)59, (Figure 10 & Figure S10 [D]).\n\nThree weeks after implantation, erythroblasts and megakaryocytes were accompanied by capillaries [A]. The close proximity of those three cell types suggested their recent differentiation from common precursors (HSCs) at that location [A]. Also evident were forming vessels larger than capillaries, i.e. nucleated cells extending themselves around groups of erythrosomes before completing the enclosure [B]. The snail shaped cell in the upper left corner of [B] had asymmetrically distributed cytoplasm (a feature of a megakaryocyte) flattened against the abluminal surface of the forming vessel like a podosome, and nucleus partially divided by a radial sinus or invagination of nuclear membrane, suggestive of a potential for stretching. Cells closer to the erythrosomes also had such features. A cell undergoing mitosis is in the lower right corner of the image. Some cells, with ambivalent phenotype that had not specialized into either erythrosome or EC, made collagen in exuberant amounts, comparable to the size of their entire cytoplasm (instead of a few fibers like in Mouse 5) and the nucleus seemed to be converting into an erythrosome on its own [C]. However, because those proficient collagen makers were also perishing in the process of tissue building, the result was an area filled with scattered erythrosomes amidst massive amounts of collagen like in the left lower corner in [D]. Capillaries and larger vessels were seen side by side [D].\n\nThe primitive vessels were embedded in copious amounts of collagen [A]. The wall of such vessels typically was polarized; it had caveolae on the luminal surface and collagen abluminally [B]. Collagen-making cell and tumor edges, accommodating each other’s curvatures, revealed interaction between the two [C]. The tumor seemed to benefit from collagen support provided by the nonmalignant neighbor as well as appearingto have acquired the ability to synthesize some by itself. Arrows in [B, C & D] point towards intracellular bundles of collagen fibers; [D] is the enlarged collagen-containing region of [C]. Dilated cisternae of endoplasmic reticulum depleted of ribosomes to a variable degree resembled vesiculo-vacuolar organelles (VVOs)59 [D].\n\nArteries and veins were formed independently, each vessel type from individual, differentiated cells. The level of differentiation varied. The two shown arteries assembling from differentiated cells of variable type (including chromaffin cells) would have likely joined eventually had the tissue been harvested later (Figure 11, Figure 12, Figure 13 & Figure S11, Figure S12, Figure S13).\n\nForming lumen of the emerging vasculature fragment shown in [A] was occupied by free erythrosomes and platelets. That “vasculature piece” was not a vessel yet because it included blood elements that were being generated simultaneously with the vessel wall. The wall was incomplete at the time of image capturing. Structural differences along the circumference of the future lumen included a gap on the left side. Such lack of continuity and the irregular forms of erythrosomes also indicated that the forming vessel was not yet a part of the circulation. In that region, cells other than endothelial cells were recognizable, namely chromaffin cells and megakaryocytes at the outer side although their products (platelets) accompanied the erythrosomes in the lumen. The right side of the vessel wall was structurally the most advanced; it consisted of ECs uncharacteristically non-flattened. The shape of their nuclei and corrugation of the basement membrane between those cells and supporting pericyte suggested a potential for physical expansion. Such wavy basement membrane likely contained elastic fibers. The image in [B] could have been from a similar region cut in a perpendicular plane with regard to that in [A]; nuclei of the centrally located ECs had their radial sinuses oriented towards the basement membrane. That too could facilitate the expansion of the vessel. ECs in the middle part of the main image [A] were flattened; in the upper side one could see an erythrosome passing through. The spatial relationship between structures shown in [A] and in Figure 13 is demonstrated in [C].\n\nThe images were from a section parallel to the one shown in Figure 11, i.e. from another region of the same vessel. Arrows point to two chromaffin cells in [A]; erythrosome, platelets and maturing platelets “ante portem”, i.e. facing opening in the endothelium, are shown in [B,C & D], respectively.\n\nA second forming complex vessel had a corrugated basement membrane at both ends of the elongated structure suggesting that the one in Figure 11 might not have been cut exactly parallel to its long axis. A round cell surrounded by a collagen-free region at the lower end suggested growth in that direction which happened to be pointing towards the first artery Figure 11 [C].\n\nMoreover, developing nerves accompanied some forming vessels (Figure 14, Figure S14, and Figure 15 & Figure S15 [D & E]). One displayed a few nerve fibers at early stages of myelination and a lack of mature synapses despite the presence of multiple nerve boutons with synaptic vesicles. Next to it, an equivalent number of cells committed to the erythropoietic pathway generated a giant erythro-schizosome, although perhaps normally there would have been a cluster of erythrosomes (“erythropoietic lake”) in a forming vessel. Whether this particular one was going to be successful in producing normal erythrosomes or was just a part of the ongoing cytoevolution was not clear. The cells that surrounded the developing nerve did not necessarily derive from the ectoderm. They ended up on the surface of neuroplax but had much in common with endothelium, morphologically and functionally – namely, a flattened shape, plenty of caveolae to control the microenvironment of the developing neurons and Schwann cells. Caveolae were shown elsewhere to contain ATP-ase that could be processing ATP secreted by the giant erythroschizont right next to it60–63. Such concerted differentiation resulting in different functions would have been unlikely to happen without coordinated regulation of the vasculature and nerves.\n\nSeveral nerve fibers were engulfed by Schwann cells on the right but progressing myelination could be recognized only on the one in the center with three mitochondria in the section plane (left arrow). Multiple nerve boutons with synaptic vesicles (right arrow) were present but mature synapses were absent. Surrounding the nerve fibers was one to three-layer neurothelium with abundant caveolae (insert in the right lower corner). Collagen fibers located internally were thinner (20–40 nm) than those lining the external surface of the neurothelium (60–100 nm). A giant erythroschizosome was located next to the forming nerve and it had similar dimensions (right lower corner behind the insert).\n\nIn summary, the observed variants of erythrogenesis included nucleo-cytoplasmic conversion and nuclear conversion with collagen production continuing in the cytoplasm. Erythroblasts were either entirely devoted to erythrogenesis or had ambiguous phenotype, including electron lucent (failing) vesicles, collagen synthesis and later also presence of caveolae. Erythrogenic conversion affected entire cells or syncytium and later generated schizomers (myeloplaxes, cobble stone areas, erythrocytic lakes) or the cellular remodeling occurred incrementally. Erythroblasts had aerobic or anaerobic metabolism as indicated by the presence or absence of mitochondria. They were polynuclear or mononuclear and of giant or average cell size. The erythrosomal membrane was or was not undergoing remodeling inside the erythroblast before release of the organelle. Some cells were perishing without succeeding in production of either proper erythrosomes or a lumen despite initiating those processes. Evidently, the cellular differentiation was happening by means of natural selection (cytoevolution). Cells perishing in the process were contributing to building tissue framework available for subsequent remodeling. Erythropoiesis appeared to be essential at every step of evolving cellular mechanisms of vasculature formation. Fusion of the neo-vasculature with the pre-existing one would be the final step needed to allow the newly created portion of tumor tissue, already equipped with its own vasculature (a network of blood-containing vessels of variable size), to grow beyond the limit set by the diffusion range of metabolites. That event would be equivalent to the first heartbeat in a developing embryo.\n\n\nDiscussion\n\nJudging by the activities of cells evolving in our new experimentally created environment, we retrospectively assumed the presence of their precursors, in agreement with the functional TSCs definition. The question was which cells participated in the formation of the tumor vasculature and how did the vasculature form? At the outset, the components of the tumor niche (tumor spheroid, graft and local host tissues) did not have an ongoing vasculature formation. In both variants of the model, with and without homologous tissue graft, the new qualities emerged after the implantation (this report and the accompanying article43, respectively). New tissues formed locally and integrated into the host animal. Some of the graft cells formed the new vasculature for the pseudo-orthotopically implanted tumor, whereas in the absence of the graft, some of the tumor cells eventually evolved to do the same. In both types of the tumor niche (pseudo-orthotopic and ectopic) some local cells differentiated to form vasculature at the site of the tissue growth. Regardless of the origin (lineage) of those cells, one could think of them as SCs - based on the definition of such cells37.\n\nThe amorphous continuously growing tumor mass was out of touch with tissue morphogenesis-controlling mechanisms but depended on the ability of the tumor cells to induce formation of vasculature either by CSCs or by local TSCs. Thus, tumors were subjected to selective pressure because those incapable of either inducing local nonmalignant SCs to form tumor-supporting vasculature or of generating their own vasculature43 could not grow. The ability of the tumor to generate its own vasculature in the ectopic environment implied a conditional existence of CSCs, i.e. evident in one in vivo environment but not in the other (and not in vitro). Any single cell of the spheroid population could potentially establish new tumors in the orthotopic environment. However, large numbers of those cells would be necessary in a variety of ectopic environments, whether encountered by metastatic tumors or created experimentally43. One should not be surprised at finding HSCs markers among CSCs and ESCs because some of the features perceived in the literature as defining the stemness might be attributed to tissue growth, shared by malignant and non-malignant tissues. Our results showed that growing tissue sectors were capable of generating their own vasculature (blood and vessels) independently of the existing circulatory system and therefore recapitulated the embryonal vasculogenesis. However, the newly created blood-containing vascular segments did eventually fuse with the host vasculature whereas the embryonal vasculature does not fuse with the maternal one.\n\nThe vascular “sprouts” grew towards existing vessels, not away from them (Figure 11 & Figure S11 [A, C] and Figure 13 & Figure S13). Yet the new tumor-supporting vasculature observed here originated from the graft, not the tumor. To understand the paradox, one needs to keep in mind that the implanted components, tumor spheroid and graft, were related; both originated from the breast. When implanted separately, each behaved differently. The graft (injured lactating breast tissue) first produced new normal mammary epithelial ducts (results not shown) whereas the tumor initially used some of its own cells to feed the others, a fraction of which evolved into megakaryocytes and ECs43. However, when implanted together, breast tumor and breast tissue reconstituted the primary tumor in its native environment, as intended. Self-organizing potentials of the two influenced each other and a new dynamic balance emerged because of such interaction. At the same time, the host was responding to the injury inflicted by the surgical procedure associated with the implantation. That enabled the comparison of the tumor and the scar tissue vasculature formation side-by-side. The involvement of non-malignant TSCs in the formation of the tumor vasculature suggested that normal and malignant tissues share the cellular mechanism of vasculature formation. The developing nerves accompanying the forming vessels also indicated that those vessels were normal, yet they were as close to a muscle as to the tumor (Figure 11 & Figure S11). The growing undersupplied region likely initiated the process because without new substrates, the growth would be energetically prohibitive, regardless of the genetic makeup. The oxygen diffusion range and the maturity level of cells in the microenvironment shaped the initial vascular pattern. The presence of calmyrites in non-malignant cells suggested that anaerobic metabolism was not unique for tumors either but could function in normal cells as an alternative, less-efficient pathway functional during mitosis and therefore more frequently used by tumors43. Morphologically, the tumor vascular pattern appeared abnormal because it never matured due to perpetual tumor growth64. Vascular tortuosity had been noticed to be a feature of young tissues65,66, presumably in anticipation of growth. The blood flow would help to establish the hierarchical pattern for optimal functionality.\n\nTraditionally, the ontogenetic tree symbolizing cellular differentiation pathways, for instance hematopoiesis, consisted of arrows arranged in certain sequence and often branching but always pointing in the same direction, therefore signifying progress and irreversibility. Such a concept of phenotypic changes does not reflect great flexibility in terms of the functions of differentiated cells for which compelling evidence has already been accumulated, including in vitro-induced pluripotent SCs67,68, and is fast growing as the stem cell research prospers. The reliance of erythroblast and EC differentiation on natural selection, i.e. differentiation of HSCs by cytoevolution illustrated here for the first time, suggested “broken lines” for lineages (the dead ends and some stage skipping). The process was characterized by great variability of forms and perishing of nonfunctional ones that nevertheless must have contributed to the progress in differentiation of others by changing the microenvironment. The dead ends were demonstrated also in vitro69. Another interesting point related to cell lineages that emerged here as examples of the cell types thought of as mature that could trans-differentiate into other phenotypes. Megakaryocytes could become pericytes or ECs, depending on circumstances, and collagen-producers could become ECs. To trans-differentiate might be more practical than to migrate long distances and deal with the forms that are no longer useful. The requirement for continuous regulation to maintain a differentiated phenotype was postulated previously70. A network of arrows pointing in different directions, including reverse, would represent the actual cellular differentiation pathways better than the traditional tree. “A good analogy likens cell types to metabolites in metabolic networks”40.\n\nMegakaryocytes are known to develop from bipotent erythroid/megakaryocytic progenitor7,53,71,72. However, their morphogenetic role in establishing vascular pattern in the absence of ECs is new (Figure 1, Figure 2, Figure 11 & Figure S1, Figure S2, Figure S11). Addressing one of the unanswered questions in vascular biology6,7 it demystifies the presence of platelets in the early embryo and demonstrates that megakaryocytes and erythroblasts develop before ECs. Together they are capable of forming primitive vascular patterns without ECs. Given the chemical nature of CEA, (200 kDa glycoprotein containing about 50% carbohydrate) and its presence in tumors and in pregnancy, one could suspect that the presumed proteoglycans of ECM (Figure 1 & Figure S1) indeed contain CEA31. The second dichotomy in the pathway corroborated the controversial derivation of ECs from hemangioblasts73. At a certain phase in vasculature morphogenesis, the three cell types co-localized as triplets and acted in concert. However, as the assembly of vessels progressed, megakaryocytes started positioning themselves abluminally suggesting their ability to trans-differentiate into pericytes (Figure 9 & Figure S9).\n\nThe presence of eosinophils (Figure 15 & Figure S15), neutrophils (Figure 1 & Figure S1), dendritic and plasma cells (not shown) in that environment, although relatively rare, suggests that they too differentiate locally and assist tumor growth. That possibility deserves further studies because, if confirmed, it would explain why the host immune system could tolerate the tumors. If primary tumors are similar enough to the tissue of origin to prompt local TSCs for vasculature formation, one could hardly rationalize the need for its rejection by the host. If, however, the tumor loses the tissue-specific characteristics, it would become alienated and dormant until an angiogenic switch occurred43,74, which would not necessarily be immunogenic (e.g. medical implants are non-immunogenic). In further support of this theory, the lymphopoietic potential of mouse embryonic HSCs cells was indeed observed in vitro and in vivo17,75.\n\n[A & B]: Erythrosome labeled with ubiquitin-detecting antibody, internalized by non-labeled macrophage (Mouse 5). [C]: Eosinophil (Mouse 3). [D]: Myelinated nerve fiber engulfed by Schwann cell (Mouse 2). [E]: Forming vessel located between muscle and nerves (Mouse 2). [F]: Empty vein or lymphatic vessel with an internal valve (Mouse 2).\n\nThe central role of the extramedullar erythrogenesis at every level of the dynamic complexity of vasculature formation was understandable because the ongoing tissue growth requires energy and nutrients. The complex issue of the origin and maturation of HSCs in developing embryos and their potential migration among AGM, yolk sac, umbilical artery, vitellin artery, placenta and fetal liver76 was echoed in the model used here. Without the guidance from the mature bone marrow niche, the erythrogenesis was recapitulating the embryonal stages. Calmyrites contributed a new criterion for grading the maturity level of erythropoiesis.\n\nOur observations offered new understanding of vasculature morphogenesis in relation to tissue morphogenesis. For example, plasticity of cells became well recognized but often perceived as “puzzling, surprising, intriguing, provoking”, etc. as single TSC types or even clones gave rise to multiple phenotypes unexpectedly. Epithelium evolved from bone marrow progenitors77, hematopoietic cells from endothelial or neuronal cells75,78,79, blood from brain80 or muscle81,82, muscle from HSCs82,83, and HSCs from stromal-vascular fraction of adipose tissue21,84, causing the confusion. The common presence of the hematopoietic component in those heterogeneous differentiated populations derived from TSCs was striking. It suggests that progenitor cells committed to a specific tissue type also have hematoblastic potential. One can anticipate more reports on finding erythropoietic cells during procedures intended to isolate tissue-specific cells. That reasoning does not contradict trans-differentiation of cell types other than those involved in erythropoiesis. For example, neuronal cells were shown to change their phenotype to endothelial cels when co-cultured with (ECs)85. Historically, the HSC lineage has shaped the traditional belief in terminal differentiation but, as discussed below, that lineage turned out to be a special case rather than the archetype from which to extrapolate to other tissues. Yet supposedly all connective tissues (and possibly others) derive from HSCs and therefore, transplantation of HSCs rather than mesenchymal SCs would be the choice therapy for genetic deficiencies of connective tissues, making HSCs a panacea for multiple related illnesses86. However, our results suggest the opposite. All tissues, at all stages of life, might have hemato-lympho-vaso-neuro-adipo-poietic potentials as long as they have the potential to grow or to repair themselves. However, execution of that potential would depend on morphogenesis-controlling factors and the availability of proper substrates. Various types of hematopoiesis (primitive, definitive and subclasses of those) reflect the differentiation levels of the tissues harboring the process. In an emergency, even endothelium could become hemogenic87.\n\nUnderstanding the mechanisms regulating tissue morphogenesis could have therapeutic implications. For example, the observation that collagen is essential for the process could be useful. Assuming that ECM of tumor tissue might be unprotected with tissue inhibitors of metalloproteinases (TIMPs), as was shown to be the case in embryo88, one could envision pharmacological intervention with proteolytic enzymes.\n\nThe classic functional definition of the adult TSCs was amended in 2002 to include its flexible and regulated tissue self-organization capability based on cell-cell and cell-environment interactions37. The amended definition allowed for the assumption that TSCs have tissue specificity and the ability to form vasculature de novo when needed. Conceptually one could view TSCs as histo-hematoblasts, as we did initially. Just like TSCs and SCs in general, the histo-hematoblasts was a concept, not a clone37. Which cells would produce a new piece of tissue or its vasculature did not have to be predetermined during embryogenesis but later, when and where the growth was to take place. The location of tissue growth must be governed by organogenesis-controlling factors like BMP489–91. An example demonstrating the spatial correlation between hematopoiesis and regions of tissue growth is available for the mouse embryo92. Simply put, when traced back along their ontogenetic lineage, cells engaged in building new vasculature for the growing part of a tissue would not all turn out to belong to a single HSC clone. Such multi-origin does not imply their universal applicability as transplants; as mentioned above, the developmental compatibility was shown to matter to some extent18. Another practical aspect that follows is that a strategy to stop the vasculature formation in tumors should not target specific precursor cells but rather the process itself, regardless of the lineage of cells executing the tumor supporting function. To think of SCs as “some cells” might be helpful. Due to the self-organizing potential of tissues in higher organisms, at any phase of growth and development or life of an individual, some cells in the growing region of an organ might be capable of addressing the basic metabolic needs locally, and of establishing communication with the rest of the organism. Which cells of a given organ they are, may be neither predetermined nor critical but dictated by morphogenesis-regulating factors (malfunctioning in tumors) and local energy metabolism requirements (applicable to tumors).\n\nThe concept of histo-hematoblasts was rooted in the fundamental role of neo-hematopoiesis in tissue morphogenesis, namely, fueling metabolism of any tissue mass exceeding diffusion range of oxygen (100 – 200 µm93,94; 150 µm64) and presumably of nutrients. Neo-hematopoiesis appeared to be the core of the process, both figuratively speaking and literally. Emerging erythrosomes, known for their ability to secrete ATP58, formed the central point to which other cells were chemotacticly attracted and around which endothelium evolved eventually with ATP-ase located in caveolae60–63. What triggered the local (extramedullar) vasculature formation was the localized incremental tissue growth. Any tissue growth, (developmental, repair-related or malignant) would require new vasculature; whereas, proliferation of cells replacing those that perish while fulfilling their physiological functions would not. Examples are intestinal and skin epithelia, hair follicles and secretory cells of some glands (as discussed in87. One could think of TSCs as “kits” containing potentials for all necessary elements of the tissue to be built. Chromaffin cells participated in assembling the new artery (Figure 11, Figure S11 & Figure 12, Figure S12) and cells from the immune system were present in that environment too. Developing nerves paralleled the forming vessels of similar dimensions (Figure 15, Figure S15 [E] and Figure 4 in43). GATA-2 transcription factor required for hematopoiesis was also reported to be involved in the maintenance of the pool of ventral neuronal progenitors92. Moreover, existence of common neural and vascular patterning tools (signaling molecules like ephrins, netrins, slits, semaphorins and neuropilins) was in fact pointed out earlier95,96. The correlation of vasculature formation with the local development of nerves, mentioned above and elsewhere97–101, called for modifying the concept of histo-hematoblasts to the organoblasts concept, to cover the observed phenomena more adequately.\n\nThe organoblasts (Organo-Bs) is designed to be a generic term for such organ-committed precursor cells. The plural of the term is deliberate here, like in the case of SCs, because those cells neither belong to a unique lineage nor are committed to a specific tissue type within an organ. In particular cases they would be for example: Nephro-Bs (kidneys), Hepato-Bs (liver), Osteo-Bs (bone), Dermo-Bs (skin), Pulmo-Bs (lungs), Entero-Bs (gut), Sarco-Bs (muscle), Spleno-Bs (spleen), Thymo-Bs (thymus), Ovario-Bs (ovary), Testo-Bs or Orchio-Bs (testes), Cerebro-Bs (brain) and Cardio-Bs (heart) and so on. Hemangio-Bs and Neuro-Bs would derive from the organoblasts of each organ independently to form vasculature, lymphatics and peripheral nerves. Accordingly, in the formation of any new tissue (or in the growing segment of an existing tissue), some of the growing cells would become hematoblasts or neuroblasts to connect them to the rest of the given organ in the system. Naturally, such a mechanism of vasculature morphogenesis would result in organ-specific variability in the endothelium. That would be consistent with published reports on the presence of tissue-specific markers on endothelium in particular organs, i.e. functional variability of endothelium in different organs102–107 as well as ultrastructural differences. Brain vessels provided the most prominent example108. The current definition of TSCs implies their potential to provide differentiated cells specific for a given tissue. The difference between TSCs and organoblasts (or organ SCs) is the ability of the latter to give rise to cells of specific tissue parenchyma as well as all the accessory cells necessary to maintain system homeostasis. All those cells would be locally differentiating from the organoblasts at the site of incremental tissue growth.\n\nFrom the evolutionary perspective, flexibility is more valuable than rigid perfection of the complex systems. Phylogenetic analysis applied to mouse cell lineages have indicated that extensive cellular mixing homogenized the lineages in the embryo before gastrulation41. Therefore, up to that stage of development, the cells were functionally equivalent. Consequently, dissimilar lineages participated in morphogenesis of particular tissues during organogenesis. Such redundancy combined with cell plasticity and the natural selection at the cellular level might have been compensating for the high number of mutations happening at every mitosis. C. elegans can afford to lose an entire individual because one organism can produce ~1000 fertilized eggs, whereas mammals are too complex to take care of mutations that way41.\n\nThe tissue definition based on the organoblasts concept would consist of two parts: (1) organ location (liver, heart, brain, stomach, pancreas, bone, striated muscle, ovary etc.), and (2) specific function, either unique for that organ, or system integrating (nerves, vasculature and lymphatics), or providing structural integrity. Tissues related to the latter two functions could be similar in different organs but not identical, as known for the endothelial surfaces. [Examples: brain grey matter, brain white matter, brain meninges, brain vessel; gut epithelium, gut smooth muscle, gut vessel; heart artery, heart vein, heart muscle; liver parenchyma, liver vessel; lung epithelium, lung endothelium, lung pulmonary pleura, lung costal pleura; lymph node capsule, lymph node nodule, lymph node hilum; peripheral nerve dendrites, peripheral nerve Schwan cells, peripheral nerve neurothelium; auricular cartilage, bronchial cartilage, articular cartilage, growth plate cartilage; bone marrow, bone cancellous, bone trabecular, bone periosteum, bone capillary; tumor parenchyma, tumor vessel, etc.]. There is more to organ development than differentiation of specific cell types. Dramatic qualitative changes of the tissue fabric in growing organisms occur by tissue remodeling; for example, bone morphogenesis involves formation of properly shaped cartilage first and its subsequent replacement with calcified tissue. In our model, free erythrosomes scattered in abundant collagen formed a scaffold that could support morphogenesis of the malignant tissue or scar.\n\nThe position of vasculature as a specific type of connective tissue equal in rank to other differentiated tissues, i.e. created during embryogenesis, deserves to be reassessed. Our results suggest extending the acceptance of the multiple origins of HSC precursors in the embryo to any tissue growth (malignant or reconstructive), also after birth. The vasculature formation (blood and vessels) is unique because it must be a part of any tissue growth, whether developmental, pathological or reconstructive and regardless of age, not just during embryogenesis. The novelty here was that not only vessels but also blood formed de novo at the location of growing tissue, not just in bone marrow and spleen. That could presumably occur in any organ. Under stress, a fraction of any tissue could feed the rest of the population if that meant survival of some cells. How the conversion of living cells into a meal could happen would depend on what kind of cells they were and on the microenvironment. Hematopoiesis appeared to have fundamental but accessory role in all tissues. That is why following HSC lineage was not a trivial issue. The implications go beyond cell lineages; they extend to organogenesis.", "appendix": "Author contributions\n\n\n\nJES conceived the study and participated in interpretation of the 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\nThis work was supported by National Institutes of Health Grants (to J. E. Schnitzer): R01CA119378, PO1CA104898 and R01CA083989.\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 materials\n\nThe pattern-forming heterogeneous population of the cells migrating in the hyaline membrane consisted of (a) those with large nuclei occasionally containing double nucleoli and with asymmetrically distributed, irregularly shaped, large cytoplasm and satellite platelets i.e., megakaryocytes (purple), and (b) those with atypical, lobular, heterogeneously dark nuclei and dark cytoplasmic granules in the absence of mitochondria, usually accompanied by erythrosomes (red), i.e. most likely erythroblasts (light red) [A]. The four elements, cellular and sub-cellular (erythrosomes and platelets), appeared evenly dispersed among each other like in the narrow (about capillary short dimension) column in [B] or the megakaryocytes that displayed a tendency to migrate to the periphery of the larger cluster and surround the other elements [C]. Those poorly differentiated cells were probably bipotent, megakaryocyte and erythroid, progenitors (brown) [C]; compare with Figure 8 in55. The cellular and sub-cellular elements appeared to be held together with extracellular fibrous components, possibly proteoglycans and little collagen (the insert in [C]). The resulting red pattern appeared as a vascular network under low magnification of dissecting microscope. The parallel arrangement of extracellular, short, non-collagen fibers suggested mobility for the cells secreting them [D]. The extra-cellular matrix (ECM) producer [E] located to the right of the erythroblast [F] contained a composite dense body (arrows in [E & F]) characteristic of platelets (see Figure 111-1, E in56).\n\nPrimitive vascular pattern in the tumor-harboring solid tissue below the hyaline membrane consisted of megakaryocytes and platelets (purple) engaged in direct and tight contact with one another and with erythrosomes and erythroblasts (red). There were no ECM-filled spaces left between them nor was there an additional cellular layer covering those elements. The elongated branching structure in [A] appeared kept together by megakaryocytes and platelets (N.B. the cutting imperfection mention in Figure 2 has been removed here). In the two circled regions of that structure, incomplete separation of platelets from megakaryocytes provided ultrastructural evidence for their identity and for an ongoing process of generating platelets at that location. One region was the bifurcation area at the base of the right almost vertical branch, enlarged in [B] and the other was the tip of the lower left branch (enlargement not shown). Evidently, megakaryocytes were capable of organizing erythrosomes into branching primitive vascular structure before ECs emerged to provide protective walls of a vessel. In a smaller elongated structure of similar nature, although without platelets in the section plane, one of the cells contained an Auer body [C], previously described in neutrophils from cases of leukemia and defined as azurophilic, large, elongated granules (Figure 64-10 in57). The upper part of that cell contained two erythrosomes; whereas what remained of the nucleus had a different texture compared with that of the nucleus in the second cell of that complex. The prominent dark granules of the latter resembled those in the cell next to the megakaryocyte from the tip of the lower branching structure in [A]. If the irregularly shaped seemingly empty space next to those granules was a prototype of a lumen then both those cells could have potentially been angioblasts.\n\nThe single cells undergoing structural remodeling in the nucleus and cytoplasm represented at slightly different stages of erythropoiesis [A & B]. Conceivably, a few such cells (red), when gathered into a cluster, could fill up a larger space with erythrosomes creating a cobble stone pattern, also called ‘erythropoietic lake’ or myeloplaxes [C]. Platelets and megakaryocyte (purple) were recognizable at the periphery of the cluster.\n\nClusters of the gold label were found on the cell surface, in the nucleus, cytoplasm and mitochondria of cells morphologically fitting the description of erythroblasts [A, B, D & E] and on extracellular collagen fibers produced by them [B & C]. A fragment of cell with mixed phenotype is shown in [F]. The cluster of CD34-labeled giant erythroblasts (red), potentially leading to myeloplaxes, contained one cell with a double nucleus (right side in [H]). Two regions of [H] are enlarged in [G & I], left and right respectively, to show oversized mitochondria (orange), some converting into peroxisomes (dark & orange) [G]. That cluster of cells was bigger than the one shown earlier (Figure S3C), and the presence of mitochondria despite clear commitment to erythropoiesis suggested that here the process was more efficient. Arrows point out some clusters of Au grains.\n\nGFP-specific label was present in erythroblasts (red) [A], primitive erythrosomes (red) like the one separating from the cell that made it (left lower corner in [B]), platelets (purple) [C] and in EC (green) [D]. Morphologically interesting cells, erythroblast [E] and perishing hemangioblast [F] stained with nonspecific antibody are shown at low magnification (they had no gold grains). The erythroblast was entirely consumed by a successful incremental conversion into at least three erythrogenic vesicles (red) [E] and the cell with ambivalent phenotype [F], possibly hemangioblast, contained three internal vesicles resembling erythrosomes (red) and two of comparable size that appeared to be failing as erythrosomes (green) (partially electron lucent ones).\n\nThe cells shown in the upper row [A, B & C] were located in the hyaline membrane 5 µm (or less) apart from one another (Figure S14b) suggesting their relatively recent derivation from a common precursor. The first, irregularly shaped cell with platelet-resembling attachment was probably a megakaryocyte (purple) [A]. The second one (mixed shades of red), had four large vesicles but only one of them was similar in size and electron density to a cell-free erythrosome (right upper corner in [A]), whereas two other vesicles (yellow) were much lighter with the exception of small patches at the periphery, and the fourth one was probably an incompletely converted nucleus [B]. The third cell (green) had two large vesicles (red and pink) and both appeared to represent different stages of degradation of the material they were made of [C]. The outer membrane of that cell was at one side connected to another cell via calmyrin-containing extracellular fibers (insert in [C]). Both cells with the large vesicles made impression of cells with mixed phenotypes with some inclinations to specialize in either producing erythrosomes [B] or forming a lumen [C]. Fields shown in [D & I] were in the same section; they represented what seemed to be left after internal remodeling of polyploidal cells or multi-cellular clusters [D & G]. There were cell-free erythrosomes as well as empty vesicles, some broken [H]. Clearly, cells were perishing because of such restructuring but they were also changing the microenvironment. Insert in [D] shows calmyrin-specific label on fibers of ECM. [E, F & I] are enlarged regions from [D] and [H], respectively.\n\nCompare with Figure S4 [A, B & C]. The insert shows calmyrin-containing extracellular fibers connecting the adjacent cells.\n\nUneven distribution of the abundant calmyrin-specific label in erythrosomes (red) from the solid tissue under the hyaline membrane was striking [A, B, C & D]. No such clusters were seen extracellularly [E]. The gold particles appeared arranged with certain symmetry in clusters of about 6 to 12, as if the protein became a part of distinct para-crystalline ultrastructures that in less differentiated regions were not formed ([E] and Figure S4). Such gold grain clusters were also present in ECs (green) [F] and tumor cells (brown) [G] where small dark mitochondria suggested hypoxic distress. In a giant cell, the uneven distribution of scattered calmyrin-specific label and heterogeneity of electron density indicated an ongoing process of erythrogenic conversion [H]. One calmyrite in a section from normal rat lung is shown in [I]. Those ultrastructural para-crystalline elements visualized by calmyrin-specific label are being described here for the first time and referred to as calmyrites.\n\nThree weeks after implantation, erythroblasts (red) and megakaryocytes (purple) were accompanied by capillaries (green) [A]. The close proximity of those three cell types suggested their recent differentiation from common precursors (HSCs) at that location [A]. Also evident were forming vessels larger than capillaries, i.e. nucleated cells extending themselves around groups of erythrosomes before completing the enclosure (green) [B]. The snail shaped cell (purple) in the upper left corner of [B] had asymmetrically distributed cytoplasm (a feature of a megakaryocyte) flattened against the abluminal surface of the forming vessel like a podosome, and nucleus partially divided by a radial sinus or invagination of nuclear membrane, suggestive of a potential for stretching. Cells closer to the erythrosomes also had such features. A cell undergoing mitosis (bright yellow) is in the lower right corner of the image. Some cells, with ambivalent phenotype that had not specialized into either erythrosome or EC, made collagen in exuberant amounts, comparable to the size of their entire cytoplasm (yellow) instead of a few fibers like in Mouse 5, and the nucleus (red) seemed to be converting into an erythrosome on its own [C]. However, because those proficient collagen makers were also perishing in the process of tissue building, the result was an area filled with scattered erythrosomes amidst massive amounts of collagen like in the left lower corner in [D]. Capillaries and larger vessels were seen side by side [D].\n\nThe primitive vessels were embedded in copious amounts of collagen [A]. The wall of such vessels (green) typically was polarized; it had caveolae on the luminal surface and collagen abluminally [B]. Collagen-making cell (yellow) and tumor edges (brown), accommodating each other’s curvatures, revealed interaction between the two [C]. The tumor seemed to benefit from collagen support provided by the nonmalignant neighbor as well as appearing to have acquired the ability to synthesize some by itself. Arrows in [B, C & D] point towards intracellular bundles of collagen fibers; [D] is the enlarged collagen-containing region of [C]. Dilated cisternae of endoplasmic reticulum depleted of ribosomes to a variable degree resembled vesiculo-vacuolar organelles (VVOs)59 [D].\n\nForming lumen of the emerging vasculature fragment shown in [A] was occupied by free erythrosomes (red) and platelets (purple). That “vasculature piece” was not a vessel yet because it included blood elements that were being generated simultaneously with the vessel wall. The wall was incomplete at the time of image capturing. Structural differences along the circumference of the future lumen included a gap on the left side. Such lack of continuity and the irregular forms of erythrosomes also indicated that the forming vessel was not yet a part of the circulation. In that region, cells other than endothelial cells were recognizable, namely nerve and chromaffin cells (dark and light blue, respectively) and megakaryocytes (purple) at the outer side; although their products, platelets (purple), accompanied the erythrosomes in the lumen. The right side of the vessel wall was structurally the most advanced; it consisted of ECs (green) uncharacteristically non-flattened. The shape of their nuclei and corrugation of the basement membrane between those cells and supporting pericyte suggested a potential for physical expansion. Such wavy basement membrane likely contained elastic fibers. The image in [B] could have been from a similar region cut in a perpendicular plane with regard to that in [A]; nuclei of the centrally located ECs had their radial sinuses oriented towards the basement membrane. That too could facilitate the expansion of the vessel. ECs in the middle part of the main image [A] were flattened; in the upper side one could see an erythrosome passing through. The spatial relationship between the structures shown in [A] and in Figure S10 is demonstrated in [C].\n\nThe images were from a section parallel to the one shown in Figure S8, i.e., from another region of the same vessel. Arrows point to two chromaffin cells (blue) in [A]; erythrosome (red), platelets (purple) and maturing platelets “ante portem” i.e. facing the opening in the endothelium (green), are shown in [B,C & D], respectively.\n\nA second forming complex vessel had a corrugated basement membrane at both ends of the elongated structure suggesting that the one in Figure S8 might have been cut not exactly parallel to its long axis. A round cell (bright yellow) surrounded by a collagen-free region at the lower end suggested growth in that direction which happened to be pointing towards the first artery Figure S8 [C].\n\nSeveral nerve fibers (light blue) were engulfed by Schwann cells (dark blue) on the right but progressing myelination could be recognized only on the one in the center with three mitochondria in the section plane (left arrow). Multiple nerve boutons with synaptic vesicles (right arrow) were present but mature synapses were absent. Surrounding the nerve fibers was one to three-layer neurothelium (emerald green) with abundant caveolae (insert in the right lower corner). Collagen fibers located internally were thinner (20 – 40 nm) than those lining the external surface of the neurothelium (60 – 100 nm). 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "704", "date": "14 Jan 2013", "name": "Louise Purton", "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 rely heavily on their own identification of ultrastructure components of cells without any confirmation of the identity of the cells by immunostaining. Furthermore, all of the immunostaining they do show is questionable and likely non-specific staining (in the materials and methods the appropriate species-matched isotype control is not listed, the one that is listed is of incorrect species). While megakaryocytes and erythrocytes are easily morphologically identifiable, I disagree with their interpretation of the data at 5 days, when they state that no endothelial cells are visible. My opinion is that an EC is visible in Figure 1C at the bottom (they claim this is a megakaryocyte). The authors are strongly advised to read papers by Leon Weiss, who did excellent TEM studies of mouse bone marrow and is the expert in this field. Without confirmation of the different cell types in this study with specific immunostaining their conclusions are not valid. Larger numbers of mice at each time point are also required to show reproducibility of findings.", "responses": [ { "c_id": "432", "date": "10 Apr 2013", "name": "Halina Witkiewicz", "role": "Author Response", "response": "Identifying cells The referee’s notion that we rely heavily on cellular morphology (at the ultrastructural level) is accurate. To that experimental approach we devoted the opening section of the Discussion in the accompanying article (III) because we believe it is the strength of our study. The referee’s generic skepticism with regard to usefulness of morphological features for identifying cell phenotypes is inconsistent with her other statements. On one hand she states that “Without confirmation of the different cell types … with specific immunostaining… conclusions are not valid” and on the other that “… megakaryocytes and erythrocytes are easily morphologically identifiable…” and goes on to identify a cell in Figure 2 as endothelial (EC) - without confirmation with the specific immunostaining. However, the cell in question does not fulfill the criteria that we used for the EC phenotype, namely: a flattened shape, polarized cell membrane with abundant caveolae (mostly on luminal surface) and attachment to collagen or basement membrane (on abluminal surface), collagen synthesis and occasionally seen Weibel-Palade bodies (page 10 of the accompanying article III). Moreover, in vivo mature -thelial cells of any kind (endo-, epi- or meso-) have tight contacts with one another. That is how ECs can be identified also in absence of erythrosomes (in sections of tissues fixed by perfusion). In contrast, the hallmarks of megakaryocyte are: large nuclei often containing double nucleoli, large asymmetrically distributed irregularly shaped cytoplasm and satellite platelets (Figure 1 [A] and 2 [B]). The referee does not specify criteria by which the cell in Figure 2 was in her opinion an EC and not a megakaryocyte. Antibodies The referee questions specificity of the antibodies that we used: GFP-specific rabbit polyclonal IgG (ab290) from Abcam; calmyrin-specific rabbit polyclonal IgG (11823-1-AP) from ProteinTech Group, Inc. and CD34-specific monoclonal IgG2a (sc-18917) from Santa Cruz. All those vendors validated the specificity of the antibodies in ways presented in their datasheets. Abcam and Santa Cruz also provided references to published work by authors who used the antibodies successfully.Nevertheless, each experimental setting requires controls addressing issues specific for that setting. Out of 150 publications that used the GFP-specific antibody only one had the species matched isotype control and out of 15 that used the CD34-specific antibody also only one had it. Both reported flow cytometry studies. For in situ analysis the best controls are the internal ones, demonstrating variable pattern of staining that correlates with particular ultrastructures as expected based on the experimental design. We showed those. The pattern of GFP-specific immunostaining depended on the promoter controlling expression of the inserted GFP coding sequence. In case of the ubiquitin promoter the label was ubiquitous, including erythrosomes, whereas in case of the histone H2B promoter the label was associated with chromatin, regardless of the cell cycle phase (inter-phase nucleus or mitotic chromosomes), although not in all cells because neither the host nor the graft donor mouse contained the GFP. Therefore the erythrosome labeled with anti-ubiquitin antibody and internalized by unlabeled macrophage must have been of the graft origin (Figure 15 [A, B]). Similarly, the unlabeled fibroblast next to the labeled dividing tumor cell must have been of the host origin (Figure 2 [F] in the accompanying article II). Thus the same antibody produced different staining patterns in sections from differently handled host mice. Both were expected and easy to explain. The variable distribution of calmyrin-associated Au grains correlated with the level of morphological complexity in the evolving phenotypes of HSC lineage (Figure 6 EFI & Figure 8). In emerging erythrosomes they were randomly dispersed initially and in more mature forms they appeared in distinct clusters, the calmyrites, whereas the CD34 label vanished from the nuclear area at the start of the nucleo-cytoplasmic conversion (Figure 4 [F]) and was not found in erythrosomes. None of the three antibodies were essential in identifying particular phenotypes. CD34, thought of as a primitive stem cell marker, was present in various differentiated cells of HSC lineage as well, and the distinct clusters of Au particles attributed to calmyrites were present in even greater variety of differentiated cell types, including nonmalignant rat epithelium. Such clusters were not seen extracellularly. Finally, both the anti-GFP and anti-calmyrin antibodies were rabbit polyclonal IgGs and therefore, represented the species matched isotype controls for each other. Numbers Our goal was accomplished by generating unprecedented photographic documentation of spontaneous inter-cellular relations leading to the vasculature formation. The animals were not subjected to any kind of treatment except implantation of the tumor. The nature of the study was exploratory and observational therefore the results were qualitative. The images represented raw data, i.e. a direct demonstration of the phenomena that had actually occurred in vivo. The conventional thinking in terms of comparing treated and control groups over a period of time to measure an anticipated effect and to evaluate its statistical significance was not applicable here. The time progression was not critical either, in contrast to the embryonal growth and development, because the tumor never matures. In our model the cells were responding to locally variable microenvironment therefore they were not functionally synchronized. Consequently a range of different stages of vasculature morphogenesis could be found simultaneously within single sections. To extend that range we did have two different times of incubating tumors in the skin fold chambers: one and three weeks. Indeed, different yet overlapping sets of options were found. However, in tumors grown for three weeks without the graft we found the earliest stages of vasculature morphogenesis (Figure 1) repeated in necrotic areas that were being repopulated. Thus the range of options was well covered. To illustrate our conclusions we selected representative images after careful analysis of many (several hundreds per mouse at various magnifications). We stopped the analysis when the findings became redundant and we understood the observed phenomena. In the literature the number of studied animals tends to be inversely proportional to complexity of assays. For example, one study using ab290 involved animal groups of variable size: n = 18 for flow cytometry and n = 6 for histology (PM:15883207). For electron microscopy based studies it is common to use samples from small number of individuals (PM:20439620). In all five mice that we used (in this and the accompanying articles) the fundamental cellular mechanism of initiating the vasculature formation turned out to be the same. Thus, the number of animals was adequate for the goal of this particular study. Increasing the number of sacrificed animals above the necessary minimum, just for the number sake, would be superfluous and against the animal welfare rules." } ] }, { "id": "926", "date": "08 May 2013", "name": "Anita E. Bandrowski", "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 paper entitled 'I. Embryonal vasculature formation recapitulated in transgenic mammary tumour spheroids implanted pseudo-orthotopicly into mouse dorsal skin fold: the organoblasts concept' by Witkiewicz, Oh and Schnitzer presents a novel and interesting view of vasculature development in solid tumours. The notion that tumour cells become organized into a pseudo organ, complete with its' own division of labour is compelling. The methods and results reported herein show a significant meticulous scholarship in this group. The work is of a high quality and the paper is generally well written.The reporting of methods is refreshingly complete, but a few things should be clarified. In a recent announcement of Nature's policies on methods reporting (see http://www.nature.com/authors/policies/checklist.pdf) several items are pointed out as critical, which the authors address; they provide stock numbers for animals, catalogue numbers for significant reagents, the order of procedures and most of the other characteristics specified as significant. The parts of the methods that must be clarified are: The n's could be made explicit throughout the paper; and the reasons that incubation in addition to graft tissue differed between figures 1-8 vs 9+ should be clarified by the authors before final approval.As the first reviewer correctly points out, the interpretation of ultra-structural data can be an art, and it is my opinion that the only way to get a good sense of these figures is to bring the community in, allowing them to see the full size images and manipulate them. There are projects that collect images that are full size and allow the community to view and manipulate the data sets, comparing images from different data sets and deploying automated tools to align them (the cell: an image library cellimagelibrary.org or the Cell Centered DataBase ccdb.ucsd.edu). While this step is not required, it may be a good way to increase the visibility of the data and allow multiple parties to weigh in on interpretation.The conclusions presented in the study are coherent with the data, but at odds with a prevailing theory. The authors bring up the point that \"the views on tumour neo-angiogenesis have remained polarized and we have yet to stop tumour growth\" in no uncertain terms bringing ire from the community. The statement may not be completely fair, but given the lack of ability of independent researchers to reproduce most significant cancer studies (Nature 483, 531–533) it may not be unfounded and again it may generate controversy, spurring others to repeat the work presented here. If the work stands the test of time and the ire of the community, the implications will be profound.A set of minor points are found below.RT is not defined as acronym, seems to be room temperature, but should be statedPerhaps a table of acronyms found in the paper would help in readability?Please rewrite this sentence to clarify your point;'The presence of a composite dense body, characteristic of platelets56, in an extra-cellular matrix (ECM) producer indicated that the ECM building cell was related to megakaryocyte (Figure 1[E & F]).'", "responses": [ { "c_id": "492", "date": "03 Jul 2013", "name": "Halina Witkiewicz", "role": "Author Response", "response": "This and the two accompanying articles report results of basic research on tumor biology. The rules regarding ‘n’s and controls required for pre-clinical pharmacological studies do not apply here because we did not test any treatment or hypothesis for a mechanism of action of a potential new drug. The article describes phenomena that in vivo occur simultaneously, side by side, because unlike in embryo, the tumors keep growing but never mature. Total 5 mice were used to make observations that were unprecedented and consistently corrected the erroneous belief lasting since 1967 regarding the mechanism of erythrocyte enucleation by nuclear expulsion (Skutelsky & Danon, J Cell Biol (1967); Simpson & Kling, J Cell Biol (1967) ). In those two publications, the numbers of mice or dogs used for the ultrastructural analysis in situ were not mentioned. For our purposes, the numbers of animals with pseudo-orthotopicly and ectopicly implanted tumor spheroids (n=3 and n=2, respectively) appear adequate. A link to the image library will be provided when the images are deposited. Other critical comments made by the referee have been addressed in the amended version of the article." } ] }, { "id": "972", "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: “I. Embryonal vasculature formation recapitulated in transgenic mammary tumor spheroids implanted pseudo-orthotopicly into mouse dorsal skin fold: the organoblasts concept” is appropriate for the content of the article. The abstract represents a suitable summary of the work.The methods are well documented, except there is little information about the chamber that was implanted – that portion needs to be expanded (e.g. explain the chamber design, dimensions, material, the pore sizes). How the tumor spheroids were implanted into the chamber (e.g. injected by needle). Using the results section, I only assume that the chamber was made from glass and covered by a hyaline membrane. The article is well constructed and is bringing a different opinion about angiogenesis in a tumor microenvironment. I appreciate that the authors moved their observation from in vitro to in vivo studies and emphasize the fact that the processes in the tumor microenvironment are very dynamic.I appreciate that the authors studied cell morphology during their analyses rather than relying solely on immuno-markers.If the authors prefer to use the original black and white microphotographs, then I suggest adding arrows, arrowheads and other symbols to the figures, figure legends, and text to indicate exactly which cells are being discussed – e.g. megakaryocytes or erythroblast in figure 1A. This type of correction should be carried on to all other figures. Or the authors may switch the B/W Figure 1 with a color Figure S1 (there are the different cell types indicated by the colors). In figure 5 and S5, I suggest adding arrows pointing at GFP positive structures. In figure 10A and S10A, change the scale unit ‘µM’ to ‘µm’.The discussion seems longer than needed – I suggest that it should be cut to focus on the current results.", "responses": [ { "c_id": "491", "date": "03 Jul 2013", "name": "Halina Witkiewicz", "role": "Author Response", "response": "The scale bar unit in Figures 10 [A] & S10 [A] has been corrected. The details of setting up the chambers have been described in references 44, 45 & 50." } ] } ]	1	https://f1000research.com/articles/2-8
https://f1000research.com/articles/2-152/v1	09 Jul 13	{ "type": "Short Research Article", "title": "Why tyrosine kinase inhibitor resistance is common in advanced gastrointestinal stromal tumors", "authors": [ "Cristian Tomasetti", "George D Demetri", "Giovanni Parmigiani", "George D Demetri", "Giovanni Parmigiani" ], "abstract": "Background: Most patients with advanced gastrointestinal stromal tumors (GIST) develop drug resistance to tyrosine kinase inhibitors (TKIs) within two years of starting therapy, whereas most chronic myeloid leukemia (CML) patients in chronic phase still exhibit disease control after a decade on therapy. This article aims to explain this divergence in long term outcomes.Methods and results: By combining clinical and experimental observations with mathematical formulas we estimate that, in advanced GIST, the genetic changes responsible for resistance are generally already present at disease detection.Conclusion: This result has relevant clinical implications by providing support for the exploration of combination therapies.", "keywords": [ "Gastrointestinal stromal tumors (GIST) are sarcomas arising in the muscle wall of the gastrointestinal tract. The majority of GISTs are driven by activating mutations in the receptor tyrosine kinases KIT or PDGFR-α", "whose aberrant signaling induces uncontrolled proliferation and decreased apoptosis1. Because of the small number of driving genetic mutations", "GISTs represent a paradigm for kinase-driven solid tumors", "and offer one of the best models to shed light on fundamental questions about cancer", "providing a critical understanding of more genomically complex solid tumors." ], "content": "Introduction\n\nGastrointestinal stromal tumors (GIST) are sarcomas arising in the muscle wall of the gastrointestinal tract. The majority of GISTs are driven by activating mutations in the receptor tyrosine kinases KIT or PDGFR-α, whose aberrant signaling induces uncontrolled proliferation and decreased apoptosis1. Because of the small number of driving genetic mutations, GISTs represent a paradigm for kinase-driven solid tumors, and offer one of the best models to shed light on fundamental questions about cancer, providing a critical understanding of more genomically complex solid tumors.\n\nTyrosine kinase inhibitors (TKIs), such as imatinib and sunitinib, block the aberrant activation of KIT, leading to major clinical benefits of objective response and durable disease control. Imatinib represents the standard first-line therapy for this disease when it is surgically incurable. However, imatinib does not cure advanced GIST2. Moreover, while the large majority of patients treated with imatinib do not show signs of primary resistance (disease progression within the first six months of therapy), secondary resistance to imatinib emerges in at least half the patients after two years of therapy and in more than 80% of patients after seven years1,3,4.\n\nImatinib was originally introduced to treat chronic myeloid leukemia (CML), because of its specificity for the TK domain in the bcr-abl translocation gene, which characterizes the disease. Imatinib represents the standard first-line therapy and has led to dramatic improvements in outcomes for CML patients in the chronic phase: at six years the estimated event-free survival is 83%, and an estimated 93% of treated patients are free from progression5. Importantly, secondary resistance is far less frequent in CML5.\n\nIn this article we consider two fundamental, clinically relevant, but still unanswered questions: Why do patients with advanced GIST often relapse and does secondary resistance originate before or during TKI treatment?\n\n\nMaterials and methods\n\nIn order to obtain estimates for the probability of at least one drug resistant GIST cell being present at the time the tumor reaches a given diameter, and before the introduction of the TKI, we used formula (5) in Tomasetti et al.6:\n\n\n\nThis formula estimates the probability of having resistant mutants PR in a tumor of size M, (number of cells), and is derived by counting the number of divisions required for the tumor to reach that size. It is assumed that, at each cell division, there is a small probability u that one of the daughter cells is hit by a mutation known to induce drug resistance. The parameter l and d are the birth and death rates for the cell population, while a and b are the probabilities for the cells’ mode’s of division. This mathematical result is based on standard assumptions and has been successfully used to predict the development of acquired resistance to targeted epidermal growth factor receptor (EGFR) blockade in colorectal cancer7. In order to apply this formula we used parameter estimates available in the literature, as follows.\n\nThe somatic point mutation rate u can be estimated to be between 10-9 and 10-8 per base per cell division8,9. There are many known point mutations causing resistance to imatinib10. To obtain a conservative bound, we assumed a value of 10 point mutations in our calculations, though the actual number could be higher11. Overall, the probability of a point mutation causing drug resistance in GIST should then be at least 10-8 per cell division. It has been estimated that 10-9 cancer cells are present per cm3 of tumoral mass12. As our goal was to generate an unfavorable scenario to our hypothesis, and since stromal tissue and other types of cells may be present, we halved this amount. Long-term drug resistance requires secondary mutations to be present in cells that are long-lived or able to self-renew13. KIT is known to have anti-apoptotic activity, thus contributing to cell lifespan14. Alternatively, resistance could arise from mutations in rare cells possessing stem cell attributes. Interstitial cells of Cajal progenitors are a potential candidate15. Their frequency is estimated to be 6.2 × 10-3 of all cells16. To be conservative, we only considered the stem cell compartment and set all other parameters of the main formula (5) in Tomasetti et al.6 to zero.\n\nUsing these results and parameters values, we can derive the lower bound of Figure 1 for the probability of at least one drug resistant GIST cell being present at the time the tumor reaches a given diameter, and before the introduction of the TKI. Because we selected parameters to generate an unfavorable scenario to our hypothesis, we expected the actual curve to be above the curve of Figure 1. The calculation and the figure were obtained using the freely available R software (version 2.15.3)17.\n\n\nResults\n\nWe combined experimental data with our mathematical formulas6 that recently have been successfully used to predict the development of acquired resistance to targeted EGFR blockade in colorectal cancer7. By so doing, we have obtained the relationship shown in Figure 1 between the tumor diameter at detection, and the probability that the tumor already harbors a resistant mutant at that time (see Materials and methods for the derivation of Figure 1). For example, for GISTs with diameters of 2 and 6 cm we estimate this probability to be equal to 0.12 and 0.97, respectively.\n\nWe now bring these elements to bear in interpreting recent clinical observations. The size of a GIST at presentation may vary between 1 and >40 cm in diameter1. For example, in a clinical trial (NCT00237185) of 147 patients with unresectable or metastatic GIST expressing KIT and treated with imatinib, 75% of the patients had a GIST whose diameter was larger than 7 cm at treatment18. Based on Figure 1, we would expect the large majority of these patients to have resistant mutant cells already present by the time of GIST detection and therefore to develop resistance during therapy. In fact, only about 20% of the patients in this clinical trial were still progression-free after five and a half years from the start of imatinib treatment18.\n\nThe combination of experimental and clinical data with our mathematical estimates implies then that secondary resistance in patients with advanced GIST is to be expected and is due to the large size of the tumor at detection time. That is, mathematical estimates indicate that mutations responsible for secondary resistance are already present before the start of the treatment in patients with large tumor sizes, a key factor in explaining the high relapse of advanced GIST patients to TKI.\n\n\nDiscussion\n\nIt has been recently suggested that secondary resistance may be the result of the treatment itself rather than pretreatment mutations4. It is worth considering this issue further. Secondary mutations are not usually found at detection using conventional Sanger sequencing techniques4. However, these techniques are not sensitive for rare events. Because imatinib has not yet selected for resistant mutants, they may remain extremely rare, and thus are likely missed by the assays used for mutation analysis. Thus, the fact that mutants are rarely found at detection does not contradict the possibility that the point mutations did occur before treatment. Using our mathematical formulas again4, we estimate that if resistance is present in a GIST of 2 cm in diameter, only approximately 1 out of 108 tumor cells will be drug resistant. Most importantly, while resistance may also originate during treatment, this does not exclude the fact that random point mutations can occur during the pre-treatment phase at each cell division. We have shown that the estimated number of resistant mutants produced before the start of the treatment is sufficient to explain the observed clinical data. This logic also holds for the development of tertiary resistant clones that emerge within, on average, six months of starting second-line therapy with sunitinib following failure of imatinib19. Thus, our calculations, while not addressing the problem of primary resistance, are able to explain secondary resistance in advanced GIST.\n\nOur prediction that patients with smaller GISTs at detection will have a smaller probability of progression has been already supported by the clinical correlation of tumor size at presentation with improved outcomes on imatinib18. With sufficient data, it may also be possible to observe a relation between tumor diameter at detection and probability of resistance that is similar in shape to Figure 1, since a resistant clone originating before therapy should eventually grow to a detectable size. Lastly, in the future, with next-generation DNA sequencing techniques, it may become possible to detect the presence of secondary mutations that represent a very small fraction of cells at initial presentation.\n\nOur results, if confirmed, would have important clinical implications. A therapy using a combination of TKIs (for example by adding a new agent with novel spectrum activity to imatinib) would select only for those cells which have been hit by two point mutations, each causing resistance to one of the drugs. We estimate that such a probability is very small for realistic values of the GIST diameter (not considering cross resistance). Therefore, combination therapy could drastically reduce the development of drug resistance caused by point mutations. Also, earlier detection of GIST offers the potential to provide measurable improvements in outcome, as long as it can result in a sufficiently large reduction in the average diameter at detection.", "appendix": "Author contributions\n\n\n\nCT and GP conceived the idea. CT, GDD, and GP designed and performed research, and wrote the manuscript. CT contributed the mathematical analysis.\n\n\nCompeting interests\n\n\n\nCT and GP have no competing interests to disclose. GDD discloses the following: Novartis. Consultant, <$10k per annum. Honorarium <$10k per annum. Research support to Dana Farber for specific clinical trial agreements in our sarcoma unit, >$200,000 per year. Research support to Dana-Farber for specific clinical trial agreements in our sarcoma unit, >$200,000 per year. Pfizer. Consultant, <$10k per annum. Honorarium <$10k per annum. Research support to Dana-Farber for specific clinical trial agreements in our sarcoma unit, >$200,000 per year. Infinity Pharmaceuticals. Consultant, <$10k per annum. Research support to Dana-Farber for specific clinical trial agreements in our sarcoma unit, >$200,000 per year. Regulatory presentation support, uncompensated. Glaxo Smith Kline. Consultant, <$10k per annum. Kolltan Pharmaceuticals. Chair, Scientific Advisory Board. Chair, Medical Advisory Board. Consultant. Equity (minor stake, non-public).\n\n\nGrant information\n\nThe work of CT was supported in part by the National Institute of Health under Grant T32 CA009337. GDD was partially funded by DFHCC SPORE (NCI Grant) for GIST research, as well as Ludwig Center for Cancer Research at Dana-Farber/Harvard Cancer Center. GP was supported in part by the NIH/NCI grant 5P30 CA006516-46.\n\n\nReferences\n\nCorless CL, Heinrich MC: Molecular pathobiology of gastrointestinal stromal sarcomas. Annu Rev Pathol. 2008; 3: 557–86. PubMed Abstract \| Publisher Full Text\n\nLe Cesne A, Ray-Coquard I, Bui BN, et al.: Discontinuation of imatinib in patients with advanced gastrointestinal stromal tumours after 3 years of treatment: an open-label multicentre randomised phase 3 trial. Lancet Oncol. 2010; 11(10): 942–9. PubMed Abstract \| Publisher Full Text\n\nAntonescu CR, Besmer P, Guo T, et al.: Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation. Clin Cancer Res. 2005; 11(11): 4182–90. PubMed Abstract \| Publisher Full Text\n\nAntonescu CR: The GIST paradigm: lessons for other kinase-driven cancers. J Pathol. 2011; 223(2): 251–61. PubMed Abstract \| Publisher Full Text\n\nHochhaus A, O'Brien SG, Guilhot F, et al.: Six-year follow-up of patients receiving imatinib for the first-line treatment of chronic myeloid leukemia. Leukemia. 2009; 23(6): 1054–61. PubMed Abstract \| Publisher Full Text\n\nTomasetti C, Levy D: Role of symmetric and asymmetric division of stem cells in developing drug resistance. Proc Natl Acad Sci U S A. 2010; 107(39): 16766–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiaz LA Jr, Williams RT, Wu J, et al.: The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature. 2012; 486(7404): 537–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAraten DJ, Golde DW, Zhang RH, et al.: A quantitative measurement of the human somatic mutation rate. Cancer Res. 2005; 65(18): 8111–7. PubMed Abstract \| Publisher Full Text\n\nDrake JW, Charlesworth B, Charlesworth D, et al.: Rates of spontaneous mutation. Genetics. 1998; 148(4): 1667–86. PubMed Abstract \| Free Full Text\n\nHeinrich MC, Corless CL, Blanke CD, et al.: Molecular correlates of imatinib resistance in gastrointestinal stromal tumors. J Clin Oncol. 2006; 24(29): 4764–74. PubMed Abstract \| Publisher Full Text\n\nWang WL, Conley A, Reynoso D, et al.: Mechanisms of resistance to imatinib and sunitinib in gastrointestinal stromal tumor. Cancer Chemother Pharmacol. 2011; 67(Suppl 1): S15–24. PubMed Abstract \| Publisher Full Text\n\nKlein M, Bartoszynski R: Estimation of growth and metastatic rates of primary breast cancer. In Mathematical Population Dynamics. (eds. Arino, O., Axelrod, D.E. & Kimmel, M.) (Marcel Dekker, New York, 1991).\n\nTomasetti C: A new hypothesis: imatinib affects leukemic stem cells in the same way it affects all other leukemic cells. Blood Cancer J. 2011; 1(5): e19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRossi F, Ehlers I, Agosti V, et al.: Oncogenic Kit signaling and therapeutic intervention in a mouse model of gastrointestinal stromal tumor. Proc Natl Acad Sci U S A. 2006; 103(34): 12843–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBardsley MR, Horváth VJ, Asuzu DT, et al.: Kitlow stem cells cause resistance to Kit/platelet-derived growth factor alpha inhibitors in murine gastrointestinal stromal tumors. Gastroenterology. 2010; 139(3): 942–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLorincz A, Redelman D, Horváth VJ, et al.: Progenitors of interstitial cells of cajal in the postnatal murine stomach. Gastroenterology. 2008; 134(4): 1083–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nR_Core_Team. R: A Language and Environment for Statistical Computing. 2.15.3 edn (R Foundation for Statistical Computing, Vienna, Austria, 2013). Reference Source\n\nBlanke CD, Demetri GD, von Mehren M, et al.: Long-term results from a randomized phase II trial of standard- versus higher-dose imatinib mesylate for patients with unresectable or metastatic gastrointestinal stromal tumors expressing KIT. J Clin Oncol. 2008; 26(4): 620–5. PubMed Abstract \| Publisher Full Text\n\nDemetri GD, van Oosterom AT, Garrett CR, et al.: Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet. 2006; 368(9544): 1329–38. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1140", "date": "18 Jul 2013", "name": "Shiro Urayama", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 brief report depicting a mathematical model based estimation of presence of chemoresistant cells in gastrointestinal stromal tumor (GIST). The model incorporates the notion of the presence of rare cells possessing stem cell attributes (cancer stem cells) among heterogeneous cells within such mass, contributing to the chemotherapeutic resistance. On the basis of genetic point mutation as a source for the resistance, the probability formula for existence of resistant cells at a tumor size was constructed. Despite some constraints, the article presents reasonable assumptions providing for the lower bound of the probability. Based on this model, many of the clinical index GIST mass has significant likelihood of containing a tyrosine kinase inhibitor resistant cell at the time of the discovery. If future clinical findings support the assumptions made, the model would demonstrate a rational for combination therapeutics.Specific points:Title and Abstract: AppropriateArticle content: Methods and approaches are described appropriately and references made to the main formula utilizedConclusions: AppropriateData: Appropriate as includedMinor item: Materials and Methods second paragraph, 5th sentence should have “109 cancer cells” instead of “10-9 cancer cells are present per cm3 of tumor mass”.", "responses": [ { "c_id": "613", "date": "11 Nov 2013", "name": "Cristian Tomasetti", "role": "Author Response", "response": "We thank the referee. It should read \"estimated that 109 cells\"." } ] }, { "id": "1134", "date": "18 Jul 2013", "name": "Gerald W. Prager", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTomasetti et al. were combining clinical and experimental observations with mathematical formulas to estimate that in advanced GIST, the genetic changes responsible for resistance are already present from the beginning of treatment. The model is of highest interest, especially in respect of second and third line treatment with targeted therapies. However, prospective validation is required before this model can be incorporated in combination drug trials.", "responses": [] } ]	1	https://f1000research.com/articles/2-152
https://f1000research.com/articles/2-151/v1	09 Jul 13	{ "type": "Research Article", "title": "A histological and functional study on hippocampal formation of normal and diabetic rats", "authors": [ "Shaimaa N Amin", "Sandra M Younan", "Mira F Youssef", "Laila A Rashed", "Ibrahim Mohamady", "Sandra M Younan", "Mira F Youssef", "Laila A Rashed", "Ibrahim Mohamady" ], "abstract": "Background: The hippocampus is a key brain area for many forms of learning and memory and is particularly sensitive to changes in glucose homeostasis.Aim of the work: To investigate in experimentally induced type 1 and 2 diabetes mellitus in rat model the effect of diabetes mellitus on cognitive functions and related markers of hippocampal synaptic plasticity, and the possible impact of blocking N-methyl-D-aspartic acid (NMDA) receptors by memantine.Materials and methods: Seven rat groups were included: non-diabetic control and non-diabetic receiving memantine; type-1 diabetic groups - untreated, treated with insulin alone and treated with insulin and memantine; and type 2 diabetic groups - untreated and memantine treated. Cognitive functions were assessed by the Morris Water Maze and passive avoidance test. Biochemical analysis was done for serum glucose, serum insulin and insulin resistance. Routine histological examination was done, together with immunohistochemistry for detection of the hippocampal learning and memory plasticity marker, namely activity regulated cytoskeletal-associated protein (Arc), and the astrocytes reactivity marker, namely glial fibrillary acidic protein (GFAP). Results: Both type 1 and 2 untreated diabetic groups showed significantly impaired cognitive performance compared to the non-diabetic group. Treating the type 1 diabetic group with insulin alone significantly improved cognitive performance, but significantly decreased GFAP and Arc compared to the untreated type 1 group. In addition, the type 2 diabetic groups showed a significant decrease in hippocampus GFAP and Arc compared to the non-diabetic groups. Blocking NMDA receptors by memantine significantly increased cognitive performance, GFAP and Arc in the type 1 insulin-memantine group compared to the type 1-insulin group and significantly increased Arc in the type 2-memantine group compared to the untreated type 2 diabetic group. The non-diabetic group receiving memantine was, however, significantly adversely affected.Conclusion: Cognitive functions are impaired in both types of diabetes mellitus and can be improved by blockage of NMDA receptors which may spark a future therapeutic role for these receptors in diabetes-associated cognitive dysfunction.", "keywords": [ "Many organ systems are adversely affected by diabetes", "including the brain. Patients with either type 1 or type 2 diabetes are more prone to cognitive dysfunction", "including impaired memory and learning as well as Alzheimer’s disease", "compared to age-matched non-diabetic subjects1. Cellular mechanisms that explain how diabetes negatively influences brain functioning are still not well understood", "and the most appropriatemethods to diagnose and treat cognitive dysfunctionin diabetes have not yet been defined." ], "content": "Introduction\n\nMany organ systems are adversely affected by diabetes, including the brain. Patients with either type 1 or type 2 diabetes are more prone to cognitive dysfunction, including impaired memory and learning as well as Alzheimer’s disease, compared to age-matched non-diabetic subjects1. Cellular mechanisms that explain how diabetes negatively influences brain functioning are still not well understood, and the most appropriatemethods to diagnose and treat cognitive dysfunctionin diabetes have not yet been defined.\n\nThe hippocampal formation (formed of the hippocampus proper, the dentate gyrus and the subiculum) is a key brain area for many forms of learning and memory, and is particularly sensitive to changes in glucose homeostasis. Analyses of behavioral performance and hippocampal synaptic plasticity in experimental models of diabetes have yielded inconsistent findings. While some studies suggested that water maze performance and passive avoidance tests as measures of synaptic plasticity were reduced2, others reported that these measures were unaffected3.\n\nAstrocytes are proving critical for the survival of neurons in the central nervous system (CNS), playing a role in energy metabolism, maintenance of the blood-brain barrier, vascular reactivity, regulation of extracellular glutamate levels and protection from reactive oxygen species4,5. These cells react to neuronal damage resulting from physical or chemical insults by over expression of the glial fibrillary acidic protein (GFAP). This protects CNS cells through the uptake of excitotoxic glutamate, the production of the anti-oxidant glutathione and the neuroprotective adenosine6–8, the degradation of amyloid-beta peptides and by limiting the spread of inflammatory cells9,10. Alterations in astrocytes activity were associated with diabetes-related disturbances in the brain and levels of GFAP have been under debate11,12.\n\nChanges in synaptic strength can occur within minutes of stimulation. For these changes to represent memory, they must persist for days and months. It is suggested that for the activity regulated and cytoskeletal associated protein (Arc), an immediate early gene may serve an important role in the transition to long-lasting forms of potentiation and hippocampal-dependent learning, memory consolidation and synaptic plasticity13. N-Methyl-D-Aspartate receptors (NMDARs) are ionotropic glutamate receptors found in the CNS and it is thought that the flow of Ca2+ through these receptors can cause both long-term potentiation (LTP) and long-term depression (LTD) vital for memory and learning14. However, overstimulation of these receptors causes neurodegeneration and excitotoxicity15.\n\nSince the impairment of synaptic plasticity in streptozotocin (STZ)-induced diabetic rats was linked to an inappropriate level of NMDA receptor stimulation required for the induction phase of long-term-potentiation16, this study aimed to investigate the effect of type 1 and type 2 diabetes on cognitive functions and hippocampal astrocyte reactivity and synaptic plasticity markers, and the impact of partial NMDARs blocking on these parameters.\n\n\nMaterials and methods\n\nThe study protocol was approved by Physiology department committee, scientific committee and Faculty committee of Kasr Al Ainy Faculty of Medicine.\n\nAnimal experiments were performed in the Physiology Department, Kasr Al Aini Faculty of Medicine. A total of 42 male albino rats 5–6 months old obtained from Kasr Al Ainy animal house, weighing 200–250 g constituted the animal model in this study. Rats were housed each in a cage (Suzhou Suhang Technology Equipment Co., Ltd.) in a constant temperature-(22–24°C) and light-controlled room on an alternating 12:12 h light-dark cycle and had free access to food and water. Rats were fed a standard commercial pellet diet (Harlan Teklad) except groups for type 2 diabetes which were fed high fat diet (HFD) obtained by mixing 35 g of lard per 100 g of rat chow.\n\nRats were divided into the following groups (n=6/group):\n\nType 1 diabetes was induced in rats fed on standard diet (6.5% Kcal fat) by single intraperitoneal (i.p.) injection of 65 mg/kg STZ (Biomedicals, LLC, France) dissolved in 1 ml cold citrate buffer concentration 0.1 mol PH4.8 (life technology; USA)17. Type 2 diabetes was induced by feeding the rats high fat diet (HFD: 58% Kcal fat) for a period of 2 weeks followed by i.p. injection with a single lowdose of STZ 45 mg/kg. Both the low dose of STZ and the high fat diet are essential elements to induce type 2 diabetes with insulin resistance18. Rats were maintained on their respective diets till the end of the study. The non-diabetic group received the i.p injection of 1 ml citrate buffer. The diagnosis of diabetes mellitus was confirmed by measuring blood glucose levels (using spectrophotometer-Beckman; USA) one week after STZ injection.\n\nGroups treated with insulin received (1 U/100 g) of commercial insulin (Mixtard 30/70; Novo Nordisk) once/day subcutaneous (S.C.) in the evening before the rat activity phase19.\n\nMemantine was used in the form of commercial tablets: memantine hydrochloride (Ebixa, Lundbeck, A/S, Denmark 10 mg/tablet). Tablets were crushed, dissolved in water and the calculated dose (30 mg/kg/day) was given orally by gavage feeding for 3 weeks20. Memantine treatment was started 4 weeks after induction of diabetes in corresponding groups i.e. after diagnosis of cognitive dysfunction.\n\nThese tests were performed 4 weeks after induction of diabetes to allow time for development of the diabetic-associated behavioural changes21 and then repeated at the end of the study to assess the effect of the different treatment protocols.\n\nThe passive avoidance test is generally regarded as a measure of long-term memory, and was performed according to methods described previously22. An illuminated compartment (base side; 15.5×4.5 cm, floor side; 15.5×10 cm, height 8.5 cm, 20 W) connected to a dark compartment (base side; 15.5×4.5 cm, floor side; 15.5×10 cm, height 8.5 cm) through a guillotine door was used. On habituation day, the rat was placed in the illuminated compartment and allowed to explore freely for 30 s, then the door was raised and once the rat entered the dark compartment with four paws, the door was closed and the latency to enter was recorded (from the time the door was lifted). On the following day, one learning trial was given by repeating the steps of the habituation trial and 3 seconds after the door was closed, an unavoidable scrambled electric foot shock (0.5 mA for 2 s) was delivered through the grid floor of the dark compartment and the rat was removed 30 s later to its home cage. Retention of the passive avoidance response (task) was tested 24 h later by placing the animal on the lighted compartment and measuring the latency in re-entering the dark compartment; increased escape latency to dark compartment is a good index of long-term memory.\n\nThe spatial learning and memory of rats was tested according to the method of R. Morris23. A Morris water maze with a submerged platform and a video tracking system (ANY-maze™ Video Tracking System; version 4.72-Stoelting Co.) were used. The Morris water maze consisted of a circular tank, (diameter: 120 cm, height: 30 cm) filled to a depth of 24 cm. The water temperature was 26°C and a 10 cm clear circular platform was submerged 1 cm below the water level in the northwest quadrant of the maze.\n\nCue discrimination. Using the protocol seen in24 and25. A visible platform test was performed to exclude drug or experimental manipulation-induced changes in visual acuity. The video tracker system was not used and only a stop watch was used in this test. Habituation to the pool was done by permitting the rats to swim freely for 30 seconds and giving them four trials (from four different directions) to climb to the platform that had been extended 1 cm above the water level. The rats then had 15 trials of cue training in 3 block intervals, each including five trials; the intervals (intertrials and interblock) were approximately 10 minutes. During this stage we didn't provide the rats with cues except for the platform.\n\nSpatial discrimination. During spatial discrimination, the hidden platform was placed 1.5 cm below the water level changing the area of the pool from that used during cue discrimination training. We added powered milk to make the pool water opaque, rendering the platform ‘invisible’. The platform location had been fixed relative to the distal cues. Rats had trained in eighteen trials in the form of six blocks (three trials per block) and the intertrial intervals were about 10 minutes and after every trial we stirred the water to avoid the effect of odor trails as unwanted cues. Rats were allowed to start swimming in each trial from one of four locations (north, south, east, and west); the choice of the location was random for each rat and each trial. The rat should escape to the platform within 60 seconds and if that didn't occur we guided them gently toward the hidden platform where they remained for 10 seconds. The rats were dried with a towel and returned to their cage after every trial. The parameters recorded in these training blocks were: latency to reach the platform, distance traveled in the maze till reaching the platform and proximity (% of time spent within the quadrant where the platform was placed).\n\nProbe trial. In the probe trial (the immediate probe trial) we removed the platform from the swimming pool and allowed the rat to swim for 60 seconds. The probe trial was given after the fifth training block and the rats then had the sixth block of training that was not included in cognitive assessment. In order to assess 24-hour retention, rats were given another probe trial 24 hours later (the platform was removed from the pool).\n\nAt the end of experimental period rats were anesthetized with ether, blood samples were collected from retro-orbital venous sinus, fasting serum glucose was measured using oxidase-peroxidase method26 and fasting serum insulin was analyzed using enzyme-linked immunosorbent assay (ELISA) (DRG diagnostics, Germany) according to the manufacturer’s instructions. To estimate insulin resistance, the homeostasis model assessment for insulin resistance (HOMA-IR: insulin resistance index) was calculated27. HOMA-IR is an indirect method for the assessment of insulin resistance. It depends on relationship between fasting plasma glucose and insulin based on a mathematical model. HOMA-IR was calculated using the following equation28:\n\nHOMA-IR = Fasting glucose (mg/dl) × Fasting insulin (uU/ml)/450\n\nBrain sectioning and staining. The rats were anesthetized with ether followed by quick cervical dislocation. After death confirmation by lack of pulse, breathing, corneal reflex and response to firm toe pinch, inability to hear respiratory sounds and heartbeat by use of a stethoscope then we performed decapitation followed by harvesting of brain tissues which were placed in 10% formaldhyde for 2 hours29. The brains were removed and placed in a new formaldehyde solution for 24 hours before being dehydrated using ethanol (70% for 24 h, 90% for 1 h and 100% for 1 h) then cleaned in xylene and embedded in paraffin. Coronal sections were cut with a microtome (Leica RM 2025, Germany) at 5 µm thicknesses, mounted on glass slides and stained with the routine hematoxylin and eosin technique30. Examination of slides, photography and morphometric studies were done at Histology Department, Kasr El-Aini Faculty of Medicine.\n\nImmunohistochemical techniques. Serial brain sections cut at 5 µm thickness were mounted on positively charged glass slides (SuperFrost Plus® slides, MENZEL-GLÄSER) for immunohistochemical staining using primary antibodies: glial fibrillary acidic protein (GFAP) (ThermoScientific, USA, ready to use, 7 ml), and activity regulated-cytoskeletal associated protein (Arc) (Lifespan Biosciences, Seattle, WA-µl diluted 1:500) together with a secondary \"Ultravision detection system\" (ThermoScientific, USA, catalog no TP-015-HD). Sections were deparaffinised and hydrated in graded descending concentrations of alcohol, then incubated with hydrogen peroxide blocking solution (3%; ThermoScientific, USA) for 15 mins. Incubation was done in humid chambers at room temperature, and slides were continuously kept wet starting from this step onwards. Slides were washed twice in phosphate buffer (0.15 mol/L NaCl, pH 7.0; Dako; Denmark) incubated with pepsin digestive enzyme-one packet dissolved in 500 ml of 0.2 N Hcl (Dako; Denmark) and washed 4 times in buffer. Ultra V block (ThermoScientific, USA) was applied and incubated for 5 mins. Primary antibodies were then applied on the serial sections and each was incubated for 30 mins. Sections were then washed and biotinylated goat antipolyvalent antibody (secondary antibody) was applied for 10 mins, washed, then followed by streptavidin peroxidase for 10 mins and washed after. To develop color reaction, one drop of DAB Plus chromogen was added to 2 ml of DAB Plus substrate, mixed and applied on tissues for 5–15 mins. Sections were then counterstained with Mayer’s hematoxylin. A coverslip was applied using mounting media. A positive reaction appeared as brown color31.\n\nQuantitative morphometric study. Ten high-power fields were measured in each of the serial sections in the different studied groups. Area % was measured for GFAP and Arc. The data were obtained by using Leica QWin 500 image analyzer computer system (England). The image analyzer consists of an Olympus microscope, a colored video camera, colored monitor and a hard disc of a Leica IBM personal computer connected to the microscope and controlled by Leica QWin 500 software. Data were statistically described in terms of mean and standard deviation (mean±SD) for area %. Photography was performed using a Panasonic wv. GP 210 camera connected to the Olympus microscope (U-CMAD3-Japan) and Olympus camera for measurments (C3040-ADU, Japan) connected to the computer.\n\n\nStatistics\n\nThe results were analyzed using SPSS computer software package, version 16 (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA). Data were presented as mean±SD. Comparison of quantitative variables between the studied groups was done using analysis of variance (ANOVA) test with the Bonferroni post Hoc test or Kruskal Wallis test with Wilcoxon signed rank test depending on the result of the Shapiro-Wilk test for normality of distribution which determined if data was parametric or non-parametric. Results were considered statistically significant at p≤0.0532.\n\n\nResults\n\nAs revealed from Table 2, administration of memantine to non-diabetic rats significantly increased serum glucose compared to non-diabetic group. No statistical difference was observed in serum insulin and HOMA-IR between both groups.\n\n: significant compared to non-diabetic group.\n\n+: significant compared to non-diabetic-memantine group.\n\n#: significant compared to untreated type 1 diabetic group.\n\n@: significant compared to type-1 DM-insulin group.\n\n$: significant compared to untreated type 2 DM group.\n\nAs expected, induction of type 1 diabetes mellitus significantly increased serum glucose level and HOMA-IR, and significantly decreased serum insulin in the untreated type 1 DM group (p<0.05) compared to non-diabetic group.\n\nThe type 1 DM group treated with insulin or with insulin-memantine still had significantly increased serum glucose and HOMA-IR compared to the non-diabetic group and non-diabetic-memantine group, respectively. The type 1 insulin treated group showed a significantly decreased serum glucose with a significantly increased serum insulin and HOMA-IR compared to the untreated type 1 diabetic group (Table 2, p<0.05). The type 1 DM group treated with both insulin and memantine showed a significantly decreased serum glucose (p<0.05) with no statistical difference in the serum insulin and insulin resistance index compared to type 1 DM group treated with insulin alone (p>0.05).\n\nInduction of type 2 diabetes significantly increased serum glucose and insulin levels as well as HOMA-IR in untreated type 2 DM group compared to the non-diabetic group. Type 2 DM group treated with memantine showed a significant decrease in serum glucose and insulin and HOMA-IR compared to untreated type 2 DM group, although these parameters were still significantly increased compared to the non-diabetic-memantine group (Table 2, p<0.05). These results indicate that while memantine can improve serum glucose in both types of diabetes mellitus, it has no beneficial effect in non-diabetic rats.\n\nResults indicate that both types of diabetes induced deficiency in learning and spatial memory in rats during the passive avoidance and Morris water maze tests. Untreated type 1 and 2 diabetic groups compared to the non-diabetic group exhibited a significant decrease in the escape latency to the dark compartment in the passive avoidance test (Figure 1A&B, p<0.05), a significant increase in the escape latency and the travelled distance to hidden platform (Figure 2A&B and Figure 3A&B, p<0.05) and a significant decrease in proximity as a measure of the % time spent within 40 cm of the platform (Figure 4A&B, p<0.05) in all Morris maze training blocks. Also, a significant decrease was observed in the proximity of both the untreated diabetic groups during immediate and 24 h probe trials compared to the non-diabetic group (Figure 5A&B, p<0.05).\n\nLatency to enter the dark compartment in type 1 diabetic (A) and type 2 diabetic (B) subgroups compared to non-diabetic subgroups. : significant compared to non-diabetic group, +: significant compared to non-diabetic-memantine group, #: significant compared to untreated type 1 diabetic group, @: significant compared to type 1-insulin group, $: significant compared to untreated type 2 diabetic group at p<0.05.\n\nA: escape latency to hidden platform in type 1 diabetic subgroups compared to non-diabetic groups. B: escape latency to hidden platform in type 2 subgroups compared to non-diabetic groups. Data are presented as mean±SD (n=6/group).\n\nA: distance travelled to hidden platform by type 1 diabetic subgroups compared to non-diabetic groups. B: distance travelled to hidden platform by type 2 subgroups compared to non-diabetic groups. Data are presented as mean±SD (n=6/group).\n\nProximity (% time spent within 40 cm of the platform) in type 1 (A) and in type 2 (B) diabetic subgroups compared to non-diabetic groups. Data are presented as mean±SD (n=6/group).\n\nProximity (% time spent within 40 cm to where the platform was previously present) in type 1 (A) and in type 2 (B) diabetic subgroups compared to non-diabetic group during immediate (open bars) and 24 h (filled bars) probe trials. : significant compared to non-diabetic group, +: significant compared to non-diabetic-memantine group, #: significant compared to untreated type 1 diabetic group, @: significant compared to type 1-insulin group, $: significant compared to untreated type 2 diabetic group at p<0.05. Data are presented as mean±SD (n=6/group).\n\nThe type 1 diabetic group treated with insulin also showed a significant impairment in all performed tests compared to the non-diabetic group (p<0.05), and a partial improvement of the cognitive functions compared to the untreated type 1 diabetic group. Compared to the non-diabetic group, the insulin-treated group showed a significant decrease in the escape latency to the dark compartment in the passive avoidance test (Figure 1A, p<0.05), a significant increase in the escape latency and the travelled distance to the hidden platform (Figure 2A and Figure 3A, p<0.05), and a significant decrease in proximity observed during training and during immediate and 24 h probe trials (Figure 4A and Figure 5A, p<0.05) of the Morris water maze.\n\nCompared to the untreated type 1 diabetic group, the insulin-treated type 1 group showed a significant increase in the escape latency to the dark compartment of the passive avoidance test (Figure 1A), a significant decrease in the escape latency (Figure 2A) and distance travelled (Figure 3A) in all blocks of training and a significant increase in proximity only during the 1st and 5th blocks of training and immediate probe trial during the Morris water maze test (Figure 4A and Figure 5A, p<0.05).\n\nTreating the type 1 diabetic group with both insulin and memantine had a better impact on learning and spatial memory compared to insulin treatment alone. A significant increase was observed in the escape latency to the dark compartment of the passive avoidance test (Figure 1A) compared to the non-diabetic memantine- and insulin-treated type 1 groups. Also a significant decrease in escape latency to the hidden platform (Figure 2A) was revealed compared to the non-diabetic-memantine group in all training blocks and in the 4th and 5th training blocks compared to the type 1 diabetic-insulin group. Distance travelled in the Morris water maze test by the type 1-insulin-memantine group was significantly decreased in all blocks compared to the type 1 DM-insulin group (Figure 3A) and in the 1st, 4th and 5th block of training compared to the non-diabetic-memantine group indicating a beneficial effect of memantine over the insulin treatment alone.\n\nProximity in the type 1 insulin-memantine group was significantly increased in all blocks of training (Figure 4A) and in immediate and 24 h probe trials (Figure 5A) compared to that of the type 1 diabetic-insulin group. However, this was still significantly decreased in training blocks 4 and 5 and in the immediate and 24 h probe trials compared to non-diabetic-memantine group (Figure 4A and Figure 5A, p<0.05).\n\nTreating the type 2 diabetic group with memantine significantly increased escape latency to the dark compartment of the passive avoidance test (Figure 1A) compared to the non-diabetic-memantine and untreated type 2 diabetic groups. Also, it significantly decreased the escape latency to the hidden platform (Figure 2B) compared to the untreated type 2 diabetic group in all Morris water maze training blocks, although this still significantly increased compared to non-diabetic-memantine in the 1st 4 blocks and only significantly decreased in the 5th training block.\n\nDistance travelled in the Morris water maze was significantly decreased in the type 2-memantine group compared to untreated type 2 group in all training blocks, and to the non-diabetic-memantine group in the first three blocks with a statistically insignificant difference in the last two blocks, which indicated a positive effect of memantine on learning and spatial memory.\n\nProximity was significantly increased in the type 2 memantine group in the last four training blocks and in immediate and 24 h probe trials compared to the untreated type 2 DM. Moreover; proximity was significantly increased in the last three training blocks and the immediate probe trial in the type 2 memantine group compared to the non-diabetic-memantine group. These results suggest that memantine may consolidate the long-term memory in type 2 diabetes.\n\nAdministration of memantine to the non-diabetic group had less beneficial effects with a significant decrease in the escape latency to the dark compartment in the passive avoidance test (Figure 1) and a significant increase in the escape latency and distance travelled to the hidden platform during all training blocks of the Morris water maze test compared to the non-diabetic group (Figure 2 and Figure 3, p<0.05). Proximity was also significantly reduced in all training blocks and in the immediate and 24 h probe trials compared to the non-diabetic group (Figure 4 and Figure 5, p<0.05), which suggest that memantine has no role in learning and memory consolidation in the non-diabetic state.\n\n\nHistological results\n\nHistological examination of hematoxylin and eosin stained sections revealed the characteristic areas of hippocampal formation. These are the hippocampus proper, dentate gyrus and subiculum (Figure 6).\n\nDentate gyrus (DG) is seen surrounding CA4 by its upper & lower limbs. Note lateral ventricle (LV) related to CA1 & CA2. M denotes molecular layer inside concavity of CA and of DG. (H & E ×40).\n\nThe hippocampus proper is formed of Cornu Ammonis CA1 and CA2 formed of zone of small pyramidal cells, CA3 and CA4 formed of zone of large pyramidal cells. CA4 projects into concavity of dentate gyrus that is formed of small granule cells. Subiculum is outward continuation of CA1 region. Areas in between compact zones of cells comprise the molecular layer which consists of neuronal processes/(axons and dendrites), glial cells, and scattered nerve cells (Figure 7).\n\n(a): Section from non-diabetic control showing 5–6 compact layers of small pyramidal cells of CA1 region, most with vesicular nuclei; (b): shows few layers of large pyramidal cells in CA3 region, also with vesicular nuclei (arrows). Molecular layer (ML) shows many glial cells () among neuronal processes. (c): shows layers of compact granular cells with dark nuclei in dentate gyrus G. Molecular layer shows glial cells () as well as pyramidal cells (↑) (H & E ×400).\n\nSections from non-diabetic groups receiving memantine alone were shown to be markedly affected by the treatment showing decreased thickness of the small pyramidal cell layers, decreased thickness and areas of cell loss of the large pyramidal layers, with retraction of processes and vacuolation of granular cells. The molecular layer exhibited enlarged pyramidal cells (Figure 8).\n\nSection from non-diabetic group receiving memantine alone showing: (a): decreased thickness of layer of small pyramidal cells of CA1 to reach 2 layers in some areas (↑); and (b): more marked affection of large pyramidal cells of CA3 where areas are devoid of cells (↑). Cells have vesicular nuclei. (c): granular cells show marked retraction of processes with vacuolations, and molecular layer (ML) shows enlarged neurons (n) and enlarged glial cells (). (H & E ×400).\n\nThe type 1 DM group that was left untreated showed marked changes in all regions in the form of disorganization and cell loss of small pyramidal cells, some of which had pale nuclei while others were dark. There was also marked shrinkage in the size of the large pyramidal cells, the outer layer was more affected, with darkened nuclei. The granular layer also showed marked vacuolation, while the molecular layer exhibited enlarged neurons and excess glial cells (Figure 9).\n\nThe untreated type 1 diabetic group shows (a): disorganization and areas of cell loss of small pyramidal cells; some having pale nuclei and others dark. Note also clumping of neuronal processes. (b): marked shrinkage in size of large pyramidal cells, affecting outer layer more, with darkened nuclei (↑). (c): Granular cell layers also showed marked vacuolations. Molecular layer (ML) shows marked enlargement of neurons (n) and of glial cells ().\n\nTreatment with insulin alone caused improvement in the form of preservation of small pyramidal cells and markedly decreased apoptosis of large cells. Vacuolations, however, persisted in granular cells together with overall marked disorganization (Figure 10).\n\nThe type 1 diabetic group treated with insulin only shows (a): preservation of small pyramidal cells; but with (b): marked apoptosis of large pyramidal cells (↑) and (c): marked disorganization, vacuolation (V) and decreased population of granular cells. Molecular layer (M) mostly shows normal cells & fibres. (H & E ×400).\n\nAddition of memantine to insulin therapy caused an additional mild improvement in the preservation of the cells, but with persistence of clumping of neuronal processes and widened capillaries in many fields (Figure 11).\n\nThe type 1 group treated with insulin and memantine shows also (a): preservation of small pyramidal cells of CA1 while; (b): some of large pyramidal cells of CA3 show apoptosis (↑) with some clumping of neuronal fibrils (f) (c): Granular cells show less vacuolation, & molecular layer shows normal size of cells, with widened capillaries (c). (H & E ×400).\n\nThe type 2 DM group left untreated showed changes very similar to the type 1 untreated group. These included darkened small pyramidal cells mainly in deep part (inner layer nearer to the molecular layer), disorganization of layers and many apoptotic large cells, together with vacuolations and clumped processes, but with no significant change in the sizes of glial cells (Figure 12). Treatment with memantine, however, caused a significant improvement in the form of fewer apoptotic cells, less marked shrinkage, but with persistence of clumped processes and dilated vessels (Figure 13).\n\nThese include: (a): many darkened nuclei of small pyramidal layer (arrowhead) mainly in deep layer, with vacuolation (v) and clumping of processes. (b): layer of large pyramidal cells shows disorganization with many apoptotic cells (↑). (c): granular layer shows some cell loss (arrowhead) and increased glial cells with no change in their size () while (d) molecular layer shows apoptotic cells & (H & E ×400).\n\nThe type 2 diabetic group treated with memantine alone shows some protective effect for the drug in the form of: (a): preservation of small pyramidal cells except for deepest layer (↑) with clumping of neuronal fibrils (f); but with (b): shrinkage and darkening of many large pyramidal cells and (c): clumping & disorganization of granular cells with dilated vessels (v) and normal glial cells () in molecular layer. (H & E ×400).\n\nAs revealed in Figure 14, there was a highly significant increase in area % of GFAP staining of the non-diabetic-memantine group when compared to normal controls (p<0.001). The untreated type 1 DM group also showed a very highly significant increase. Insulin treatment alone, however, caused a highly significant decrease in the level of staining that was just slightly better with the addition of memantine. The untreated type 2 DM group and memantine treated group both showed very low levels of staining, with a highly significant decrease when compared to control (Figure 14).\n\n: significant compared to non-diabetic group, +: significant compared to non-diabetic-memantine group, #: significant compared to untreated type 1 diabetic group, @: significant compared to type 1-insulin group, $: significant compared to untreated type 2 diabetic group at p<0.05. Data are presented as mean±SD (n=6/group).\n\nThere was no statistically significant difference in area % distribution of Arc between control, non-diabetic-memantine and untreated type 1 DM groups. The type 1 DM group receiving insulin had significantly decreased levels of staining as compared to controls. The type 1 DM group receiving insulin and memantine had significantly elevated levels when compared to the previous group, and were more similar to controls. The type 2 DM group exhibited a significantly lower level than the control group, which was also improved by the use of memantine (Figure 14).\n\nTreating the type 2 diabetic group with memantine significantly increased Arc compared to the untreated type 2 diabetic group (Figure 14, p<0.05) and significantly decreased GFAP compared to the non-diabetic-memantine group. Administration of memantine to the non-diabetic group only significantly increased hippocampal GFAP expression compared to the non-diabetic group without affecting the hippocampal Arc.\n\n\nImmunohistochemical stains\n\nImmunohistochemical staining for GFAP showed its normal distribution in the control group as a mild positive reaction in glial cells of molecular layers. The non-diabetic group receiving memantine showed marked elevation in levels of immunostaining, becoming dense and more widespread in the enlarged glial cells. The type 1 DM untreated group also exhibited markedly high levels of reaction. However, this decreased to less than normal levels with insulin treatment alone, and was nearer to normal level with combined insulin and memantine therapy. The type 2 DM untreated group had a very low level of staining, which was not improved with memantine therapy (Figures 15a–15g).\n\n(a) Non-diabetic control group. (b) Non-diabetic memantine group. (c) Type 1 DM untreated. (d) Type 1 DM + insulin. (e) Type 1 DM + insulin + memantine. (f) Type 2 DM untreated. (g) Type 2 DM + memantine. Note increased staining in b and c.\n\nImmunohistochemical staining for Arc in the control group showed mild to moderate staining homogenously filling neuronal cell bodies, and widespread in neuronal processes. The level and distribution of staining was mostly not altered with memantine alone, or with type 1 DM alone. However, the type 1 DM group receiving insulin showed a significantly decreased level and disruption of normal homogenous patterns of staining in cell bodies. That returned to a near normal pattern and level with the addition of memantine therapy. The type 2 DM group exhibited only a mild level of widespread immunostaining that was significantly improved to a near-normal pattern with memantine therapy (Figures 16a–16g).\n\n(a) Non-diabetic Control group. (b) Non-diabetic memantine group. (c) Type 1 DM untreated. (d) Type 1 DM + insulin. (e) Type 1 DM + insulin + memantine. (f) Type 2 DM untreated. (g) Type 2 DM + memantine. Note patchy disorganized staining in d, and to a lesser extent in e.\n\n\nDiscussion\n\nDiabetes is associated with several adverse effects on the brain, some of which may result primarily from direct consequences of chronic hyperglycemia. In this work, both type 1 and 2 untreated diabetic groups showed significantly impaired learning and spatial memory in rats during the passive avoidance and Morris water maze tests compared to the non-diabetic group in agreement with previous studies1,33.\n\nInsulin treatment of type 1 diabetic group after development of cognitive dysfunction partially improved cognitive functions compared to the untreated type 1 group; however, all tested cognitive functions were still impaired compared to the non-diabetic group. These results strongly support a previous study suggesting that intervention with insulin failed to reverse water maze learning and partially affected long-term potentiation, unlike insulin treatment commenced at the onset of diabetes34.\n\nCognitive dysfunction and impaired synaptic plasticity in both types of diabetes have been linked to hyperglycemia, insulin deficiency and/or insulin resistance and altered insulin signaling35,36, hypophyseal-pituitary axis hyperactivity and elevated glucocorticoid levels37. Insulin diminishes hypothalamic-pituitary-adrenal-axis activity38 and modulates neurotransmitter levels39, thus promoting physiologic processes critical for memory.\n\nHyperglycemia increases the NMDA receptor-mediated calcium entry into the neurons and may induce neuronal excitotoxicity through an activation cascade ending by the release of ROS40. Memantine, an uncompetitive NMDA receptor antagonist, significantly improved serum glucose and insulin levels as well as HOMA-IR in type 1 and type 2 diabetic groups compared to the insulin-treated type 1 and untreated type 2 group, respectively. It also significantly improved all tested cognitive functions in the type 1 DM group (with the exception of the escape latency to hidden platform in the first three training blocks) compared to the type 1 group treated with insulin alone and in the type 2 DM group compared to the untreated type 2 diabetic group which indicates a positive effect of blocking NMDA receptors on memory and synaptic plasticity in diabetes. A similar dose of memantine has been shown to improve cognitive function and to slow cognitive and functional decline in Alzheimer disease transgenic rat models20.\n\nThe importance of maintaining normal synaptic NMDA signaling was demonstrated by Papadia et al.41. Memantine cannot act or accumulate in the NMDA channel when the channel is open for several milliseconds as occurs during normal synaptic activity. Instead, memantine only inhibits the prolonged influx of Ca2+ ions and blocks abnormal glutamate excitatory signals42.\n\nHippocampal formation has long been known to be responsible for learning and memory. These processes involve distinct, and interlinked, groups of efferent systems for episodic memory, affective and social learning, and sensory processing and integration43,44, all of which could be affected by chronic diseases like diabetes.\n\nExamination of normal and diabetic hippocampus sections revealed marked effects of diabetes in the form of cell death in several areas, with disruption of normal layer organization. This was associated by clumping of neuronal processes (appearing as excess eosinophilia) which is indicative of damage to neurons45. These changes were markedly improved by insulin therapy and by insulin and memantine, and were less improved by memantine therapy alone.\n\nThe reaction of astrocytes may be the earliest response of the brain tissue to an altered glucose metabolism46. In this work, GFAP was significantly decreased in the untreated type 2 diabetic group compared to the non-diabetic group, in agreement with several previous studies12,47. However, GFAP was significantly elevated in the untreated type 1 diabetic group compared to non-diabetics, supported by the findings of Baydas et al.11. These findings showed that untreated type 1 diabetes induced glial hyperactivity with increased GFAP in the rats' hippocampus. Baydas and colleagues attributed this to the effect of reactive oxygen species especially because oxidative stress occurs earlier in type 1 diabetes48.\n\nMoreover; Lebed et al.46, assessed GFAP 3 and 7 days after STZ injection using immunohistochemistry and demonstrated that the reduced GFAP-positive cell count was found on day 3 when these cells were significantly smaller and less arborized with respect to the control. This tendency reversed on day 7 when more numerous GFAP-positive cells grew in size and became more ramified.\n\nInsulin in the type 1 group significantly decreased hippocampal GFAP compared to untreated type 1 and non-diabetic groups. These results agree with those of Lechuga-Sancho et al.49, but contradict those of Coleman et al.50. Meanwhile, memantine significantly increased GFAP expression compared to the insulin-treated group, although it was still significantly lower than that of the non-diabetic-memantine group, and GFAP was changed insignificantly in the type 2 diabetic group compared to the untreated type 2 group.\n\nThe significant decrease in GFAP expression found in the current study in diabetic groups treated with memantine agrees with previous work by Miguel-Hidalgo et al.51, who studied the neuroprotective effect of memantine against β-amyloid-induced neurodegeneration and concluded that memantine-treated animals had significant reductions in GFAP immunostaining as compared with vehicle-treated animals. Decreases in GFAP expression are associated with detrimental conditions in the CNS52, several of which occur in GFAP knock-out mice and in diabetic individuals. Also; associated proliferation of astrocytic cells in the damaged area of hippocampus (increased GFAP immunostaining) is a common end result of damage to neurons in the CNS53. In support of the cognitive dysfunction, Arc was significantly decreased in the untreated type 2 DM group compared to control. However; Arc expression was insignificantly changed in the untreated type 1 diabetic group compared to the control group. Both synaptic plasticity and memory are impaired in the absence of Arc13, and inhibition of Arc protein in rats impaired maintenance of LTP beyond 4 hours54.\n\nWhile insulin in the type 1 group significantly decreased hippocampal Arc compared to the untreated and non-diabetic groups, memantine significantly increased its expression in the type 1 group compared to the insulin treated group, and in the type 2 diabetic group compared to untreated type 2 group making Arc expression nearer however still not identical to control group. Rosi et al.55 found that memantine restored Arc in hippocampus CA3 cells that was disrupted by chronic neuroinflammation. Rial et al.56 demonstrated that Arc reduces the number of glutamate receptors, leading to a decrease in α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) mediated synaptic currents, consistent with a role in the homeostatic regulation of synaptic strength.\n\nIn the present study, although Arc was insignificantly changed in the untreated type 1 diabetic group compared to the non-diabetic group, the type 2 diabetic groups showed a significant decrease in Arc expression in untreated type 2 compared to control. Mateos et al.57, found decreased expression of Arc in the cerebral cortex and hippocampus of HFD-fed animals and linked this finding to HFD supplied to type 2 diabetic rats.\n\nSince desensitization of insulin receptors and impaired insulin signaling are common features of diabetes58, memantine may qualify as potential future option to combat cognitive impairments and dementia in diabetes.\n\nThe positive effects of memantine were absent in the non-diabetic group. Memantine significantly impaired all tested cognitive performances and significantly increased serum glucose. However, it did significantly increase hippocampal GFAP compared to the non-diabetic group, indicating that the beneficial effects of memantine are only evident in the disease state.\n\nIn conclusion, both types of diabetes are associated with cognitive dysfunction. Insulin in type 1 DM and memantine in both diabetic types could improve these parameters. Memantine had an advantage to be able to improve astrocytic reactivity in type 1 DM and Arc expression in both types of diabetes, which may spark a future therapeutic role of the NMDARs antagonists in diabetes-associated cognitive dysfunction.", "appendix": "Author contributions\n\n\n\nSNA wrote the work protocol, performed cognitive tests, statistical analysis and conducted the writing and revision of the manuscript. SMY shared in protocol writing, writing of the manuscript and supervised the work. MFY produced the histological examination slides, performed the photography and morphometric studies and helped with writing of the manuscript. LAR performed the biochemical measurements. IM was the senior supervisor for the whole work and the group leader.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was partially funded by Cairo University by supplying the kits for insulin measurement (ELISA), the primary and secondary antibodies used for immunostaining.\n\n\nReferences\n\nArvanitakis Z, Wilson RS, Bienias JL, et al.: Diabetes mellitus and risk of Alzheimer disease and decline in cognitive function. Arch Neurol. 2004; 61(5): 661–666. PubMed Abstract \| Publisher Full Text\n\nReagan LP: Insulin signaling effects on memory and mood. Curr Opin Pharmacol. 2007; 7(6): 633–637. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelanger A, Lavoie N, Trudeau F, et al.: Preserved LTP and water maze learning in hyperglycaemic-hyperinsulinemic ZDF rats. Physiol Behav. 2004; 83(3): 483–494. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiguel-Hidalgo JJ, Alvarez XA, Cacabelos R, et al.: Neuroprotection by memantine against neurodegeneration induced by beta-amyloid(1–40). Brain Res. 2002; 958(1): 210–221. PubMed Abstract \| Publisher Full Text\n\nPekny M, Pekna M: Astrocyte intermediate filaments in CNS pathologies and regeneration. J Pathol. 2004; 204(4): 428–437. PubMed Abstract \| Publisher Full Text\n\nStevens A, Lowe JS, Young B: Nervous system, in Wheater's Basic Histopathology, a colour atlas and text, fourth edition. Churchill Livingstone, Elsevier Science Limited, Edinburgh, London, New York, 2003; 269–270. Reference Source\n\nGuzowski JF, Lyford GL, Stevenson GD, et al.: Inhibition of activity-dependent arc protein expression in the rat hippocampus impairs the maintenance of long-term potentiation and the consolidation of long-term memory. J Neurosci. 2000; 20(11): 3993–4001. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "1063", "date": "16 Jul 2013", "name": "Carlos Telleria", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents experiments that are very well described. The title is appropriate for the content of the manuscript and the conclusions are appropriate to the results obtained and not overstated. Materials and methods are clearly described so as the experiments can be replicated. The study could be strengthened by adding early on in the manuscript (e.g. introduction) more information about memantine, which is the NMDAR blocker used in the study.", "responses": [] }, { "id": "1065", "date": "16 Jul 2013", "name": "Zhenjie Zhang", "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 by Amin et al. accessed the effects of memantine (NMDA receptor blocker marketed as an Alzheimer’s disease medication) on hippocampal histology and functions in diabetes model rats. The authors presented interesting data. Both of their functional and histological results showed severe impairment in pathological groups, and memantine partially alleviated these impairments in some cases. However, in the non-diabetic group treated with memantine, impairments similar to the pathological groups were observed. Thus, the potential benefit by memantine was only evident in diabetic conditions. While there are interesting observations in the study, it also raises concerns regarding the experiment design and data interpretation. Major concerns: While the authors claimed memantine effectively improved nearly all quantitative test scores in both type I and type II diabetes groups, some improvement data upon the type I group were actually lacking. The authors only compared the type I diabetic rats treated with insulin to type I diabetic rats treated with both insulin and memantine, but did not compare untreated type I diabetic rats to memantine treated type I diabetic rats. Thus, based on the presented data, it is very difficult to access the effects of memantine alone on type I biabetic hippocampal function and histology.\n\nWhile the paper concludes that memantine significantly improved the type I diabetic pathology, the data presented showed only very marginal improvements by memantine on top of insulin (especially Figure 2, 4, 5). These differences were statistically significant only because of the extremely small standard deviations in the data.\n\nMinor concerns: Some wordings describing effects need to be changed. Specifically, the last paragraph of page 7 says “Administration of memantine to the non-diabetic group had less beneficial effects…” In fact, that treatment severely impaired all functions tested, instead of just being “less beneficial”. The SDs in many of the graphs are very small. It would be a lot more informative if statistical significance levels of p<0.01 and p<0.001 were included in addition to the mere p<0.05. This would help the readers to appreciate the statistical strength of the conclusions.", "responses": [] }, { "id": "1109", "date": "17 Jul 2013", "name": "Louiza Belkacemi", "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 sought to investigate the effect of diabetes mellitus on cognitive functions and related markers of hippocampal synaptic plasticity, and the potential impact of blocking N-methyl-D-aspartic acid (NMDA) receptors by memantine in type 1 and 2 diabetes mellitus in rat models. Their results showed that both types of diabetes negatively impact cognitive functions. The study is well designed and easy to follow but further clarifications are needed on the following points. METHODSExperimental design and animalsExplain why the albino rat was specifically chosen for their research.Explain how the 6 rats per group were chosen. Was power of analysis performed? Induction of diabetesFor the type 1 diabetes induced rats, when the single intraperitoneal (i.p.) injection of 65 mg/kg STZ was given and for how long?For type 2 diabetes, why the investigators fed the rats high fat diet (HFD: 58% Kcal fat) for a period of 2 weeks before STZ injection? How often the blood glucose levels and body weights of all rats were assessed? Did the investigators measure the blood glucose of all the rats before STZ injection of the diabetes type 2 DM groups? If so, how glucose levels of injected type 1DM, type 2 DM (before injection) compares to the controls, and how type 1 DM compares to type 2 DM (before injection)? Do the authors think that the fat diet may be a confounding parameter in the type 2 DM?Insulin treatmentPlease add when exactly the insulin treatment was started for each group. Explain why the dose of insulin given to the treated rats was the same in type and type 2 diabetic rats. Why the subcutaneous route was used for injection?Histological stainingThe authors wrote that the brains were removed and placed in a new formaldehyde solution for 24 hrs. Please add the concentration of this paraformaldehyde solution. Were the brains left at room temperature or stored at 4°C during those extra 24 hrs prior to paraffin embedding?GFAP immunoreactivity scoring using QWin 500 software requires a more accurate description. For example, describe how immunoreactive areas were chosen? How areas were classified as positive or negative? How thresholds evaluating color and intensity of staining were selected?RESULTSMetabolic parameters in the different studied groupsHow type 2 DM group treated with both insulin and memantine compares with type 2 treated with insulin alone? Hematoxylin and eosin stained sectionsIn figure 8, please give the percentage decrease in thickness of small and large pyramidal cell layers from non-diabetic groups receiving memantine alone. Also add the percentage of enlargement of pyramidal cells.In figure 9, please add the percentage of size decrease in the large pyramidal cells from the type 1 DM group?In figure 10, by how much the level of apoptosis in large cells was decreased? Describe how the number of apoptotic cells was counted?In figure, 11, by how much the level of apoptosis in large cells was decreased in the group treated with memantine? And how dilation of vessels was evaluated?Effect of diabetes and memantine on hippocampal synaptic markersGive % ARC distribution for all experimental conditions vs. respective matched controls.Give the % of GFAP staining for all experimental conditions vs. respective matched controls.IMMUNOHISTOCHEMICAL STAINS GFAP immunostainingThe title of this section is confusing. What is the difference between area of Arc and GFAP distribution and immunostaining of these two proteins? Please clarify.Please add % level or fold change of GFAP and ARC immunostaining for type 1 DM and type 2 DM, as compared to their respective controls.All the figures should have scale bars, not just magnification at which the pictures were taken.DISCUSSIONThe first sentence of this section “Diabetes is associated with several adverse effects on the brain, some of which may result primarily from direct consequences of chronic hyperglycemia” needs reference.The authors should discuss findings that contrast with theirs.", "responses": [] } ]	1	https://f1000research.com/articles/2-151
https://f1000research.com/articles/2-150/v1	09 Jul 13	{ "type": "Research Article", "title": "A fixed-dose randomized controlled trial of olanzapine for psychosis in Parkinson disease", "authors": [ "Michelle J Nichols", "Johanna M Hartlein", "Meredith GA Eicken", "Brad A Racette", "Kevin J Black", "Michelle J Nichols", "Johanna M Hartlein", "Meredith GA Eicken", "Brad A Racette" ], "abstract": "Background: Psychosis is a common and debilitating side effect of long-term dopaminergic treatment of Parkinson disease (PD). While clozapine is an effective treatment, the need for blood monitoring has limited its first-line use. Objective: Since olanzapine shows similar receptor affinity to clozapine, we hypothesized that it might be an effective alternative to clozapine for treatment of drug-induced psychosis (DIP) in PD, and that lower doses than usual might make it tolerable.Methods: In 1998-2003 we conducted a four-week, double-blind, placebo-controlled, parallel group, fixed-dose trial of olanzapine (0, 2.5mg, or 5mg) in 23 PD patients with DIP while allowing for clinically realistic dose adjustments of dopaminomimetic mid-study. The primary outcome measures were Brief Psychiatric Rating Scale (BPRS) ratings scored from videotaped interviews after study termination by an observer blinded to dose assignment and to interview timing, and CGI (Clinical Global Impression). The Unified Parkinson’s Disease Rating Scale motor subscale (UPDRS) was the primary measure of tolerability.Results: Intention-to-treat analysis found no significant differences among treatment groups in study completion or serious adverse events. However, a disproportionate number of olanzapine vs. placebo subjects reported mild side effects (p<0.04), many citing motor worsening. Fourteen patients completed the study (seven on placebo, two on 2.5mg olanzapine, five on 5mg olanzapine). In study completers, analysis by repeated measures ANOVA revealed no significant difference between olanzapine and placebo groups in BPRS psychosis reduction (p=0.536), parkinsonism (p=0.608), or any other measured parameters (CGI, MMSE, Beck Depression Inventory, Hamilton Depression score, PDQ‑39, Schwab-England ADL assessment, and sleep scores).Conclusion: This study adds to other evidence that olanzapine is ineffective in treating medication-induced psychosis in Parkinson disease.", "keywords": [ "Parkinson disease", "psychosis", "olanzapine", "randomized controlled trial", "placebo", "BPRS", "videotape" ], "content": "Introduction\n\nDrug-induced psychosis (DIP) is a significant and disabling complication of long-term treatment of Parkinson disease (PD), affecting a large minority of PD patients receiving chronic dopaminergic therapy1. Visual hallucinations are the most commonly reported psychotic phenomena in this population, with auditory, tactile, somatic, and olfactory hallucinations being much less common. Delusions, when they occur, often antedate visual hallucinations and commonly are paranoid or persecutory in nature2,3. In addition to the increased caregiver burden caused by psychosis and its sequelae, hallucinations in the context of chronically treated PD tend to be progressive in nature, resulting in increased propensity for nursing home placement and subsequent higher mortality4,5. These sobering associations suggest aggressive management of DIP in this population. However, either dose reduction of antiparkinsonian medications or addition of traditional neuroleptics usually increases parkinsonian motor disabilities. Atypical antipsychotics, with their comparatively lower incidence of parkinsonism in schizophrenia, have potential advantages for treatment of hallucinations in this sensitive population1.\n\nUntil recently, the only treatment proven with randomized, placebo-controlled studies to reduce DIP has been clozapine, an agent that does not worsen motor function6–8. Despite these favorable data, use of clozapine has been limited secondary to its rare but potentially serious risk of agranulocytosis and the consequent necessity for frequent blood draws1. Thus alternative treatments have been eagerly sought.\n\nQuetiapine has become the most commonly prescribed antipsychotic in DIP9. Although double-blind, placebo-controlled trials of quetiapine in PD confirmed it is well tolerated in terms of motor side effects, it has not proven significantly more effective than placebo in treating psychosis10–15, and a head-to-head comparison found clozapine superior to quetiapine16. Ziprasidone showed some benefit in open-label experience17, including in a random-assignment open comparison to clozapine18. However, ziprasidone can cause motor side effects in PD and is not generally considered standard therapy for DIP1,19. Other treatments, such as ondansetron, acetylcholinesterase inhibitors, and electroconvulsive therapy are supported by limited data in idiopathic Parkinson disease but are generally not viewed as first-line therapy1,19. Recently, a phase III clinical trial of a serotonin 5HT2A inverse agonist, pimavanserin, showed benefit over placebo, but the drug will not be available in the U.S. at least until late 201420–22.\n\nClozapine’s antipsychotic efficacy is often attributed to its D4 receptor antagonism. It is also posited that its robust 5HT2A receptor antagonism, especially in relation to its relatively weaker D2 receptor blockade, actually increases dopamine transmission in prefrontal cortical and nigrostriatal projections23. This may account for the cognitive improvement as well as paucity of extrapyramidal adverse events observed in clozapine-treated patients with dopaminomimetic-induced psychosis23,24. Olanzapine, therefore, with its ostensibly similar receptor binding profile to clozapine at D2, D4, and serotonergic receptors (especially 5HT2A and 5HT2C), and muscarinic sites, provides a theoretically encouraging alternative to clozapine in this fragile population25.\n\nAn initial open study of olanzapine in Parkinson disease revealed antipsychotic benefit without motor deterioration when drug dosage was optimized in a slow titration (mean daily dose at end of study was 6.5mg) and dopaminomimetic dose adjustments were allowed26. Aarsland and colleagues replicated these findings in a relatively more challenging population of Parkinson disease patients with and without dementia27. Several other small, open-label studies of olanzapine, however, have demonstrated antipsychotic benefit but at the expense of intolerable worsening of gait and bradykinesia, frequently leading to premature termination of the drug28–30. Another small open-label trial and case report series suggested unacceptable Parkinsonian motor deterioration in the context of dubious antipsychotic efficacy31,32. Later, two double-blind placebo-controlled trials revealed equivocal antipsychotic benefit and problematic motor decline in PD patients with DIP treated with 2.5–15mg/day olanzapine (mean final doses 4.1–4.6mg/day)33,34. As a result, experts have recommended against the use of olanzapine in PD1,19.\n\nNone of these studies, however, were parallel-group fixed-dose trials, and some allowed for neuroleptic dose in the same range as approved for schizophrenia; experience with clozapine suggests that an effective antipsychotic dose in PD is often an order of magnitude less than that typical for schizophrenia treatment. In addition, the two double-blind placebo-controlled trials did not permit adjustments of subjects’ dopaminomimetics, which might have alleviated motoric side effects. Finally, some of the studies cited were terminated prematurely due to side effects. Given that the only marketed drug for which efficacy has been shown is clozapine, demonstrating efficacy for an alternative agent would be important, and a fixed low dose of olanzapine (2.5mg/day) may allow a reasonably low incidence of side effects if dopaminomimetic dose adjustments are allowed. We discuss here the findings of a double-blind, placebo-controlled study of fixed, low-dose olanzapine for treatment of DIP in the context of flexible dopaminomimetic dosing. The hypothesis was that olanzapine given in this fashion would reduce DIP in patients with idiopathic PD significantly more than would a placebo, without causing intolerable motor worsening.\n\n\nMethods and materials\n\nThe completed CONSORT checklist35,36 and the original study protocol are available in the Data Files.\n\nAll patients gave written informed consent to participate in the study, which was approved by the Washington University Human Studies Committee (approval # 97-0366). In most cases an appropriate surrogate decision maker also consented. FDA approval was through IND # 53,556. This trial concluded in 2003, so it is exempt from the current ICMJE requirement of prospectively registering clinical trials.\n\nTwenty-four patients were recruited from the Washington University Movement Disorders Center from February 1998 to October 2003. Patients were examined by a movement disorders specialist and diagnosed with idiopathic PD based on presence of at least two of three cardinal manifestations of the disease (rigidity, bradykinesia, rest tremor), response to levodopa or a dopamine agonist, and absence of historical or examination features suggesting secondary parkinsonism. Subjects were treated with levodopa and were experiencing clinically significant hallucinations or delusions, as judged by their treating neurologist or psychiatrist and by the investigator (KJB). Subjects were required to be over 30 years old and have a caregiver who could provide a reliable report. At study entry, patients were required to be treated with the lowest clinically acceptable dose of dopaminomimetic. Patients treated only with a dopamine agonist were not entered in the study, as it was deemed more clinically appropriate to try a switch to levodopa before adding an antipsychotic. Exclusion criteria included a Folstein Mini-Mental State Examination (MMSE) score < 2237, pregnancy, concurrent diagnosis of delirium (unless clearly explained by dopaminomimetics), catatonia or neuroleptic malignant syndrome (NMS)-like syndrome, other confounding central nervous system (CNS) illness or systemic illness with potential CNS effects, antipsychotic use within the last month predating study enrollment (within the past six months for depot neuroleptics), history of olanzapine sensitivity, or any expectation of significant medical or surgical intervention within six weeks after enrollment. Subjects were also excluded if severity of psychosis warranted hospitalization or if, in the investigator’s judgment, psychosis severity would have made randomization to placebo inappropriate.\n\nPatients were randomized 1:1:1 to treatment with placebo or either of two doses of olanzapine. At study initiation, treatment groups consisted of a placebo arm, a 5mg arm, in which patients received this dosage nightly throughout the four weeks of investigation, and a 10mg arm, in which patients received 5mg for the first week and 10mg thereafter. Subjects received matched tablets or capsules provided by Lilly Research Laboratories (Indianapolis, IN), who provided the investigator with sealed, sequentially numbered envelopes containing the medication identity for each subject. The envelopes were not opened until after all data were collected and reviewed for accuracy, and after all decisions about statistical analysis were final, so that both investigators and patients were blind to intervention assignment. The randomization was done by Lilly. KJB enrolled subjects and patients were assigned to treatment packages sequentially by enrollment date.\n\nAfter the first five patients were enrolled, an interim safety analysis was conducted by a reviewer otherwise not involved in the study, in light of reports published since the study initiation that higher olanzapine doses caused intolerable exacerbation of parkinsonism in PD. Though serious adverse events were no more common in the treatment groups than in the placebo group, it was decided at this time that the two active treatment arms would be changed to fixed doses of 2.5mg and 5mg olanzapine, maintained throughout the four weeks of study. New treatment packages were received and the blind was maintained until after data analysis, as above. No other changes to the protocol were made. See Table 1 for a summary of the final study design. The study was planned for 10 subjects in each of three dose arms. This would produce 90% power (at alpha = 0.05) to detect a change of the magnitude and variability seen in the Wolters et al.26 report.\n\nThis table summarizes the study design and timing of assessments and interventions for the last 19 subjects enrolled in the study. ↑ dopaminometic: dose increase allowed for antiparkinsonian medication, if parkinsonism had worsened since starting the study. See Methods and Figure 1 for further details.\n\nSubjects received a baseline evaluation that involved a full psychiatric, neurologic, and medical history and examination, CGI (Clinical Global Impression) by MD38, PDQ-39, a self-rated quality-of-life measure for PD39, videotaped interview for later BPRS (Brief Psychiatric Rating Scale) rating blind to drug dose and blind to which visit was being rated40, Schwab-England ADL assessment41, UPDRS (Unified Parkinson’s Disease Rating Scale), section III (motor)42, MMSE37, HDRS (Hamilton Depression Rating Scale)43, BDI (Beck Depression Inventory)44, and patient/caretaker reported hours and quality of sleep. Repeated measures at the two-week interim visit and the final four-week evaluation included CGI (by MD, patient, and caretaker), videotaped interview for later blinded BPRS, Schwab-England ADL assessment, UPDRS, MMSE, PDQ-39, BDI, sleep questionnaire, and pill counts. All assessments were done at Washington University Medical Center.\n\nPrimary efficacy measures were CGI scores and BPRS ratings of psychosis. At each visit, the coordinator interviewed the patient during videotaping using a semi-structured interview designed to facilitate later scoring of psychopathology using the BPRS40,45,46. After all subjects had completed participation, the videotaped segments were edited to remove references to date or study visit. Author KJB in consultation with a BPRS expert (John G Csernansky, MD) wrote rules for rating “motor retardation” and other BPRS items potentially influenced by parkinsonism (see Supplementary materials), and trained author MJN in BPRS ratings. Videotaped segments were reviewed in random order by MJN, who was unaware of drug assignment or treatment duration at the time of the visit. BPRS ratings used the anchored BPRS and each item was scored from 1–740. Secondary efficacy measures included the PDQ-39, ADL assessments (Schwab-England and UPDRS), BDI, and sleep log. Primary safety measures were UPDRS motor ratings, sleep logs, and MMSE in addition to clinical review of systems.\n\nPrior to unblinding of drug codes, the decision was made to analyze data from weeks 0–2 and weeks 2–4 separately. This a priori decision was made since adjustment of dopaminomimetics was allowed at the interim (week 2) visit. Change from 0 to 2 weeks was chosen to be the primary test of efficacy. An intention-to-treat (ITT) analysis was performed on all enrolled subjects. However, since some subjects dropped out without completing outcome measures at a follow-up visit, the ITT analysis was limited to between-group comparisons of dropout rate, serious adverse events, and reported worsening of parkinsonism or other side effects judged to be at least mild in severity. Adverse events, side effects, and study withdrawal were compared between groups using the chi-squared test.\n\nFor those subjects with data at both time points of an epoch, primary and secondary efficacy measures were tested separately for the two epochs using repeated-measures ANOVA to compare the groups. The decision was made a priori to include any subject in these analyses if that patient had taken at least one week’s worth of drug during an epoch and returned for a follow-up visit. A secondary post hoc analysis of the data from trial completers was also performed across all three visits using repeated-measures ANOVA. Statistical computations used STATISTICA 7.1 (StatSoft, Inc., Tulsa, OK) or Excel (Microsoft, Redmond, WA).\n\n\nResults\n\nA total of 24 patients were enrolled (see Figure 1). Though the original study design sought enrollment of 30 patients, the study was terminated early, secondary to the growing body of literature questioning the safety of olanzapine in the treatment of DIP as well as the increasing difficulty in enrolling antipsychotic-naive patients.\n\nOnly one subject was treated with 10mg (one other was randomized to the 10mg group, but was treated only for one week, so received only 5mg doses). His hallucinations were rated “very much improved” at the study end; he required no adjustment in dopaminomimetic dose mid-study and no side effects were observed. This 10mg subject was not included in statistical analyses. In the remaining 23 subjects, no significant imbalances were present at baseline between placebo and treatment groups on any demographic characteristic or any psychiatric or neurologic measure (Table 2).\n\nValues are given as mean (SD). MMSE, Folstein mini mental test examination; BPRS-T, Brief Psychiatric Rating Scale total score; BPRS-P, psychosis subscale; UPDRS, Unified Parkinson’s Disease Rating Scale; PDQ-39, Parkinson’s disease quality of life questionnaire; BDI, Beck depression inventory; HAM-D, Hamilton depression rating scale; CGI, Clinical global impression; INS, Insomnia score; HYP, Hypersomnia score; SEADL, Schwab-England ADL assessment.\n\nThe intention-to-treat analyses did not show significant differences between groups except for incidence of mild side effects (p<0.04) (Table 3). While spontaneous report of motor side effects was not statistically significant between groups, a disproportionate number of olanzapine vs. placebo group subjects who withdrew did so secondary to reported motor side effects (0% of placebo withdrawers vs. 21% of olanzapine withdrawers). Nine subjects did not complete the study: two from the placebo group, four from the 2.5mg olanzapine group, and three from the 5mg olanzapine group. In the placebo group, one patient died of myocardial infarction and another withdrew from the study secondary to lack of efficacy. In the 5mg olanzapine group, two reported serious adverse events and a third discontinued her medication following the first dose, declaring herself “cured”. Of the 5mg subjects who withdrew for serious adverse events, one was hospitalized with delirium three weeks into the study; the other withdrew after day six due to hospitalization with hip fracture and pneumonia, and reported worsening PD symptoms prior to dropout. Of the four subjects who dropped out of the 2.5mg olanzapine group, two withdrew due to worsening parkinsonian symptoms, one secondary to unspecified side effects, and one secondary to “feeling confused”. Only two subjects in the 2.5mg group completed the study, both requiring increases of their levodopa dose at their interim visit. One each in the placebo and 5mg olanzapine arms also required levodopa adjustment at their two-week assessment. Retention and attrition of study subjects is summarized in Table 3 and Figure 1.\n\nSide effects (SEs) were any complaint of drug spontaneously reported by the patient, independent of whether SE intensity was severe enough to prompt withdrawal from the study. Serious adverse events always prompted withdrawal. SE, side effects; ↑, increase; 1st epoch, week 0–2 analysis; 2nd epoch, week 2–4 analysis, *, p<0.05.\n\nTo assess adequacy of blinding, both the primary investigator and study subjects were asked on study completion (or drop-out) to guess the identity of administered medication (i.e., olanzapine vs. placebo). Both investigator and patient were much more likely than chance would predict to correctly guess the identity of administered medication (for investigator, χ2=12.29, p=0.0021; for study subjects, χ2=6.94, p=0.0312). However, the videotape rater had no information about side effects.\n\n\n\nAnalysis of the psychosis subscale of BPRS scores (the more sensitive of our primary efficacy measures) did not reveal a statistically significant difference between groups (drug doses) in severity of psychosis in either the week 0–2 epoch (p=0.433) or the week 2–4 epoch (p=0.393). Again, post hoc analysis in study completers revealed no statistical significance in psychosis reduction between olanzapine (combined groups) and placebo (p=0.536), as shown in Figure 2.\n\nCurrent effect: F(2, 24)=0.64064, p=0.53573. Effective hypothesis decomposition. Vertical bars denote 0.95 confidence intervals. Olanzapine-blue; placebo-red.\n\nData from the first and second epochs revealed no statistically significant difference in parkinsonian signs across treatment groups, as measured by the UPDRS III (week 0–2 epoch, placebo vs. 2.5mg olanzapine group p=0.172; week 2–4 epoch p=0.677). Post hoc analysis of UPDRS motor scores comparing olanzapine (combined groups) versus placebo across the duration of study found no significant difference in parkinsonism among study completers (p=0.608) (Figure 3).\n\nCurrent effect: F(2, 24)=0.50826, p=0.60787. Effective hypothesis decomposition. Vertical bars denote 0.95 confidence intervals. Olanzapine-blue; placebo-red.\n\nAnalyses were repeated in like fashion for all other psychiatric and neurological parameters (CGI impression, CGI improvement, BPRS total, BDI, MMSE, insomnia score, hypersomnolence score, PDQ-39, and Schwab-England ADL assessment), none of which revealed statistical significance between olanzapine groups and placebo.\n\n\nDiscussion\n\nThe study failed to reject the null hypothesis. This could be a Type II error, but larger studies of olanzapine also failed to demonstrate antipsychotic efficacy of this drug in the PD population14,33. In study completers, we did not observe the motoric exacerbation documented in several studies in the literature28–34, but perhaps this is a function of our allowance for dopaminomimetic increase mid-study as well as a selection bias in some analyses for those subjects who best tolerated the medication and therefore completed the study. After all, of the nine subjects who withdrew from the study, a third identified a worsening of their motor disability prior to dropout, all of whom were discovered on unblinding to have been randomized to olanzapine. Therefore the good retrospective accuracy of investigator and patient guesses of study drug identity is not surprising.\n\nThe subjects enrolled are relatively typical of PD patients with psychotic symptoms with a few exceptions. Subjects with urgent need for treatment were not enrolled for ethical reasons. Although mild dementia was allowed, this sample had relatively high cognitive functioning, with a mean MMSE score > 26 (Table 2). Finally, at this center, some of the patients are referred for subspecialty movement disorders consultation, though a large fraction of the patients are not referred and are typical of PD patients treated in the community. With these caveats, the results appear to be generally applicable to patients with PD and psychosis.\n\nOne methodological innovation in this study was the use of videotape to record semi-standardized interviews for later analysis by a rater blind not only to drug assignment but also to time (i.e., week 0, week 2, or week 4). The rationale was to minimize rater expectation of improvement over time that might reduce our power to detect significantly greater improvement in the active treatment groups. It also reduced the likelihood of rater unblinding.\n\nThis trial supports other evidence suggesting that olanzapine is ineffective for relieving dopaminomimetic-induced psychotic symptoms in Parkinson disease and that it may cause intolerable worsening of motor disability1,19. This trial also underscores the importance of rigorous study design for the assessment of drug effectiveness in special populations, as we and others have not replicated the early, positive open-label experience reported for olanzapine in this population. If clozapine’s prominence in the clinical management of DIP in PD is to be usurped, antipsychotic agents will have to meet the burden of proof of double-blind, randomized, placebo-controlled trials.", "appendix": "Author contributions\n\n\n\nApproximate contributions to this manuscript are as follows. MJN performed 50% of data analysis and 70% of manuscript preparation. JMH performed 50% of data collection. MGAE performed 10% of data analysis. BAR performed 10% of data collection. KJB designed the study, supervised all aspects of the study, takes responsibility for all aspects of the manuscript, and performed 40% of data analysis, 40% of data collection, and 30% of manuscript preparation. All authors reviewed the manuscript.\n\n\nCompeting interests\n\n\n\nThis study was funded by Lilly Research Laboratories (Investigator-Initiated Trial F1D-MC-I012). When this manuscript was submitted for publication (June 2013), KJB was a site investigator on a study of pimavanserin for psychosis in PD funded by Acadia Pharmaceuticals. Neither company influenced the design of the study, the data analysis, the decision to publish, or the manuscript.\n\n\nGrant information\n\nThis study was funded by Lilly Research Laboratories (Investigator-Initiated Trial F1D-MC-I012).\n\n\nAcknowledgements\n\nJoel S. Perlmutter, M.D., gave valuable advice on study design and was instrumental in patient referral. Special thanks to Colleen Taylor, Jonathan Koller, Maria Chushak, Tamara Hershey, Ph.D., and John G. Csernansky, M.D., for their assistance with this project.\n\n\nSupplementary materials\n\nGuidelines for rating selected BPRS items in a treatment study of psychosis in Parkinson disease.\n\n1. Emotional withdrawal = interpersonal relatedness during interview.\n\n2. Tension:\n\na. Ignore: rest tremors, postural tremors, chorea, athetosis, dystonia.\n\nb. Include: tardive dyskinesia and akathisia.\n\n3. Depressive mood rating does not consider “pure apathy” (i.e., apathy w/o other depressive signs or symptoms), but apathy can contribute to the total judgment of depressive mood if other signs or symptoms are present.\n\n4. Hallucinatory behavior:\n\na. 2 = illusions and “shadow in the corner of the eye”.\n\nb. 3 = e.g., colors on the wall.\n\nc. ≥ 4 = definitively abnormal sensory perceptions.\n\n5. Motor retardation: Speed of movement, not amplitude (also, depressive retardation is not substantially helped by external cues; if slowed movement is substantially helped by external cues, then it may be more parsimoniously attributed to PD).\n\n6. Unusual thought content: Ratings ≥ 5 require action on delusion.\n\n7. Blunted affect: Rate according to scale, considering emotional variance, regardless of amplitude; remember that flat/blunted affect is not equivalent to depressed affect.\n\n8. Disorientation: Off by one day of week = 3.\n\nMotor hyperactivity: Limit rating to pressured speech and voluntary movement; festination does not count.\n\nKevin J Black MD consulted with John G Csernansky MD to write these additional rules for scoring BPRS items potentially influenced by motor signs in Parkinson disease patients.\n\n\nReferences\n\nFriedman JH: Parkinson disease psychosis: Update. Behav Neurol. 2013; 27(4): 469–77. 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PubMed Abstract" }	[ { "id": "1054", "date": "11 Jul 2013", "name": "Irene Richard", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 (completed in 2003) was well designed, albeit with a relatively small sample size, and intended to answer an important and clinically relevant question. After reviewing the manuscript I would agree that it probably does support the notion that olanazapine may cause intolerable worsening of motor disability but I do not think that one can draw any conclusions regarding efficacy or lack thereof based on this study.\n\nOne element of the trial design (i.e. permitting changes in dopaminergic medications) may have created some challenges when interpreting the data. The investigators speculated that olanzapine may be better tolerated if adjustments in dopaminergic medications were allowed (and therefore permitted dopaminergic medication adjustment at the 2 week visit). While this may be true, it could also increase the chances that dopaminergic drug induced psychosis could worsen (if dopaminergic medication were increased in an effort to improve motor worsening). This could potentially be a \"set up\" for decreased efficacy (if dopaminergic medications were changed more frequently in active vs. placebo). It is noted that medications were adjusted in one of the placebos, two of the 2.5 mg active and one of the 5 mg active. The authors note that there was an apriori decision to analyze data from weeks 0-2 and 2-4 separately. Change from 0-2 weeks was chosen to be the primary test of efficacy, apparently in order to limit the confound of changes in dopaminergic medications allowed at week two. However, one could question if 2 weeks is long enough to demonstrate efficacy.\n\nIn addition, a series of unplanned events contributed to challenges with data interpretation. These events included a change in design after study initiation, lower than expected enrollment and high dropout rate. The change in study design was a decrease in study drug dosage after enrollment of 5 subjects (\"in light of reports published since study initiation that higher olanzapine doses cause intolerable exacerbation of parkinsonism in PD\"). This resulted in one subject being excluded from analyses (see below) and perhaps, decreased the chance of demonstrating study drug efficacy (if higher dosages were required).\n\nOnly 24 (of an anticipated 30) subjects were enrolled and 9 withdrew (39%) which is a fairly high dropout rate. One of the 24 subjects was not included in the analyses because he was the only one to receive the initially planned dosage of 10 mg.\n\nWhile spontaneous reports of motor side effects were not statistically significant between groups, a disproportionate number of olanzapine vs. placebo who withdrew did so due to motor side effects. This finding does suggest that olanzapine may be associated with worsening motor function. However this may not be true for every patient, as exemplified by the one subject who was the only to receive the initially planned dosage of 10 mg. He had no worsening of motor function (and an improvement in psychosis).", "responses": [ { "c_id": "500", "date": "11 Jul 2013", "name": "Kevin J Black", "role": "Author Response", "response": "I agree with Dr. Richard's comments about the limitations and possible conclusions from this report." } ] }, { "id": "1567", "date": "22 Aug 2013", "name": "Eric Molho", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 conceived and rigorously carried out clinical trial addressing an important issue in a difficult to study patient population. Although the data was collected a decade ago, the information is still relevant because remarkably little progress has been made since 2003 in the treatment of PD related psychosis. Dr. Richard’s referee response presents the methodological shortcomings and correctly points out resulting limitations in data interpretation. I agree with all of the comments in her review.In addition, I would like to add the following comments/observations: In the abstract and introduction, the authors suggest that because olanzapine has a similar receptor binding profile to clozapine, it is a good candidate drug to study in the PD population and they cite Seeman et al. 1993. More recent work by Seeman shows that what is unique about clozapine is that it binds D2 receptors “loosely” and that its receptor occupancy is short-lived. Quetiapine has a similar D2 binding profile to clozapine but, olanzapine behaves much more like haloperidol and risperidone in this regard. This may explain the motor worsening that seems to be associated with olanzapine use in PD. (Kapur and Seeman. Am J Psychiatry 2001;158:360-369. Seeman and Tallerico. Am J Psychiatry 1999;156:876-884.)I disagree with the statement in the introduction that “Delusions, when they occur, often antedate visual hallucinations....” The natural history of hallucinations in PD has been looked at in only a few longitudinal studies and the interaction between hallucination and delusions has not been adequately studied but my clinical experience and the bulk of what is presented in the literature suggests that minor hallucinatory phenomena (illusions, passage or presence hallucinations, benign hallucinations with insight) are much more common and occur earlier in PD than delusions. Delusions are generally associated with more severe underlying cognitive impairment, by definition are associated with lost insight and are generally considered to indicate a more advanced stage of disease progression than isolated visual hallucinations. (Fenelon et al. Brain 2000;123:733-745).I would also question the premise that allowing for a dopaminergic medication dose adjustment in the study protocol (to reverse any worsening of PD motor symptoms due to the introduction of low dose olanzapine) might produce study results that are a more appropriate way to evaluate how this drug might work in the clinic. My experience is that this strategy is unsustainable even in the short term in this population. One reason is that each dose increase of dopaminergic medication is prone to reignite symptoms of psychosis and thus lead to a need for higher doses of the antipsychotic, setting up a cycle of overall worsening. Secondly, these patients tend to have more advanced motor symptoms including high risk symptoms such as postural instability and dysphagia. It may be very difficult to continue a drug known to worsen PD motor symptoms after the first fall or choking episode even if one cannot be sure it was a direct result of the antipsychotic drug.\n\nI would suggest more clearly pointing out that this study cohort had relatively mild cognitive impairment (MMSE >26) which may be quite different than what is encountered in practice. More demented patients may be more susceptible to side effects of atypical neuroleptics including sedation, encephalopathy and motor worsening. More demented patients may also have more severe psychosis which is likely to be less responsive to the low doses used in this and some other clinical trials. Finally, I suspect that some readers may not have a full grasp of “Type II error”. This should probably be spelled out without jargon in the discussion.", "responses": [ { "c_id": "532", "date": "22 Aug 2013", "name": "Kevin J Black", "role": "Author Response", "response": "Dr. Molho's comments are well thought out and much appreciated. I agree with the discussion about clozapine pharmacology with reference to higher dissociation rate/decreased binding avidity as a better explanation for clozapine's superior tolerability in PD.I agree that delusions usually follow hallucinations in this population; my error. Dr. Molho's comments on mid-protocol antiparkinsonian dose adjustment are reasonable. Nevertheless the rationale described explains the original protocol design, and as he points out, we need more controlled data to definitively answer this and other questions about treatment. I agree that the mild cognitive impairment in this sample limits the application of the results to a total clinical population of psychosis in PD. However, it can also be seen as a strength of the study, in that psychosis with dementia may conceivably differ in etiology and optimal treatment, so in that respect this sample may be considered more \"pure.\"" } ] } ]	1	https://f1000research.com/articles/2-150
https://f1000research.com/articles/2-149/v1	08 Jul 13	{ "type": "Research Article", "title": "Determining the efficacy of full-time occlusion therapy in severe amblyopia at different ages", "authors": [ "Sameera Irfan", "Nausherwan Adil", "Haris Iqbal", "Nausherwan Adil", "Haris Iqbal" ], "abstract": "Objective: To find out how much visual improvement is possible in severe amblyopia using full-time occlusion therapy and if improvement is influenced by the patient’s age.\nMethods: A trial of 115 consecutive cases with unilateral, severe amblyopia was conducted at a tertiary referral center from Jan 2010 to Oct 2012. Patients were divided into three age groups: 3-7 years (n= 38), 8-12 years (n=41), 13-35 years (n=36). After a complete ophthalmological examination by a single ophthalmologist, cases with organic visual loss were excluded; cases with previous part-time occlusion therapy that had failed were included in the study. Patients were given optimal refractive correction for a month, followed by full-time occlusion therapy along with near visual activities for 3-4 hours/day. The therapy was continued until maximum visual recovery was achieved (6/6 Snellen’s). Therapy was gradually reduced and stopped. Patients were followed-up regularly for the next 18 months.\nResults: There was 100% success in the 3-7 year group, 92.68% in the 8-12 year group and 97.22% in the 13-35 year group.\nConclusion: Visual improvement is possible in almost all patients with severe amblyopia irrespective of their age with full-time occlusion therapy.", "keywords": [ "Amblyopia", "occlusion therapy", "age", "lazy eye" ], "content": "Introduction\n\nAmblyopia, (‘blunt vision’ in Ancient Greek), also known as lazy eye1, is a visual deficiency in an eye that is otherwise physically normal, or that is greater than would be expected from any structural abnormalities of the eye. It is thought that amblyopia results from inadequate stimulation of the fovea or peripheral retina and/or abnormal binocular interaction, resulting in different visual input from the foveae2. It has been estimated to affect 2–5% of the population3. The global prevalence of amblyopia has not significantly changed over the years4.\n\nAmblyopia results in the loss of binocular vision, which is manifested as absent stereoscopic depth perception, poor spatial acuity, low contrast sensitivity and reduced sensitivity to motion5. This can be detected clinically by assessing whether the patient has difficulty seeing three-dimensional images on autostereograms6.\n\nAmblyopic patients can have poor spatial acuity, low contrast sensitivity, and reduced sensitivity to motion5. They may also have poor binocular vision and limited depth perception which can be detected on autostereograms6.\n\nThe largest study on patient response to amblyopia therapy to date was conducted by the Pediatric Eye Disease Investigator Group (PEDIG) in the USA7, which reported that amblyopia due to strabismus and/or anisometropia responds similarly to part-time occlusion therapy in children between the ages of three and seven years. PEDIG also reported that about 75% of children with previously untreated amblyopia respond to treatment with only two or more lines of improvement on the Early Treatment Diabetic Retinopathy Study (ETDRS) chart up to 18 years of age8. The upper age limit for optimal therapy was considered to be in the range of nine to ten years9–11. The American Academy of Ophthalmology recommends that all children be considered for treatment of amblyopia, regardless of age12.\n\nRecent findings in neuroplasticity have replaced the formerly-held position that the brain is a physiologically static organ and have shown that it changes throughout life13. Much evidence of neuroplasticity has been found in adults14,15. According to Levi and Polat, significant improvement of Vernier acuity in adult amblyopes occurred following stereoacuity by using 3-D video games14. Similar findings were announced by researchers of the Goldschleger Eye Research Institute, Tel Aviv University16. The mean improvement in distance and near acuity in amblyopic eyes by 12 months was 3.3 and 1.9 lines log MAR (minimum angle of resolution) respectively.\n\nIn 2009, a study conducted by MK Mallah and coworkers at Queen's University17, Royal Victoria Hospital, Belfast, Ireland, showed that older people (60–80 years) with a history of amblyopia who develop visual loss in the previously normal eye can experience recovery of visual function in the amblyopic eye over a 12 month period. This recovery in visual function occurred following visual loss in the other, previously normal, eye; distance improved to 3.3 lines and near to 1.9 lines log MAR. This improvement appeared to be sustained.\n\nThere are unsubstantiated beliefs that it is more difficult to treat amblyopia in older age groups, that children that have already received failed amblyopia therapy do not respond to treatment and that full-time occlusion therapy may result in occlusion (disuse) amblyopia of the good eye. The aim of this study was to assess whether these beliefs are true.\n\n\nMaterials and methods\n\nThis was a prospective trial of 115 consecutive cases with unilateral, severe amblyopia conducted at a tertiary referral center in Lahore, Pakistan, from January 2010 to October 2012 after obtaining ethical committee approval by the University of Health Sciences. Eligibility criteria included an age of over three years, visual acuity in the amblyopic eye from 6/60 Snellen’s or even counting fingers only, visual acuity in the sound eye of 6/6, an inter eye acuity difference of three or more lines, and the presence or history of an amblyogenic factor such as refractive, deprivational and strabismic components that met study-specified criteria for strabismus and/or anisometropia. A complete ophthalmic examination was performed by only one ophthalmologist (SI). This included examining the fixation pattern of both eyes, presence or absence of a phoria or a tropia by a cover-uncover test, fundus examination and color vision using Ishihara color plates. Any case with an organic cause for visual loss was excluded from the study after a thorough ophthalmological examination. Assessment of visual acuity of either eye for both near and distance acuity (Snellen’s in literate patients and Kays picture chart for illiterate children (3–5 year olds)), refraction and Best Corrected Visual Acuity (BCVA) was performed by a trained optician who was masked to the study and patient’s demographics.\n\nThere were 59 female and 56 male patients and the cases were divided into three age groups:\n\nGroup A: age 3–7 years (mean age 5.38±2, median 5 yrs) = 38 cases (37.25% of the total number).\n\nGroup B: 8–12 years (mean age 9.73±1.73, median 10 yrs = 41 cases (40.20% of the total number).\n\nGroup C: 13–35 years (mean age = 19.26±6, median 17 yrs = 36 cases (22.55% of the total number).\n\nAll patients had some degree of anisometropia; two children had stimulus deprivation amblyopia due to traumatic cataract (aged five and seven years) and 82 patients presented with strabismus.\n\nAll cases were prescribed an optimal refractive correction for a month after which full-time occlusion therapy was started. After verbal informed consent was obtained from either the parents or the guardians, patients were provided with stick-on commercial eye patches and were instructed to wear the patch over the good eye as soon as possible after waking up in the morning. They were strictly instructed not to take it off during the day, only when they were about to sleep at night. Patients were instructed regarding the near visual activities they should perform for 3–4 hours/day. All patients and both of their parents or care-takers were thoroughly counseled about regular follow up, compliance towards the patching schedule and engaging the children towards near visual activities. The latter included coloring, drawing, reading large prints initially and then as near vision improved, shifting to smaller prints, playing video games on their personal computers and hand held video games inter alia.\n\nPatients were closely followed-up at regular intervals of one day/year age; for instance four year olds were checked every fifth day and six year olds, every seventh day. Children more than 7 years old were followed up every two weeks. Patients were instructed to come into the clinic wearing the occlusive patch. First the vision of the amblyopic eye was checked and then that of the occluded eye; children were instructed to wear the patch immediately thereafter. Any change in fixation pattern of the two eyes was noted after removing the patch.\n\nThe criteria for a successful therapeutic outcome in this study was regarded as a maximum visual recovery (achieving 6/6 Snellen’s). Occlusion therapy was continued until this was achieved in all age groups. If after two months of full-time patching no visual improvement was noted, further therapy was stopped. If therapy was successful it was gradually reduced over the next seven weeks and then stopped. The weaning protocol adopted was one day off in the first week, two days off the second week, three days off the third week and so on until the seventh week, when occlusion therapy was discontinued.\n\nChildren in the 3–7 year age group were followed-up weekly after therapy discontinuation and any drop in visual acuity during weaning was monitored. Patients over the age of seven were followed-up after every two weeks until occlusion therapy was successfully finished in the seventh week.\n\nPatients were followed-up at two weeks, one month, two months and then every three months for the next 18 months after stopping full-time occlusion.\n\n\nResults\n\nThe clinical demographics of the patients are summarized in Table 1. Success was defined as equalization of visual acuity in both eyes, i.e. 6/6 (Snellen’s).\n\nImprovement in visual acuity noted with full-time occlusion therapy was 6/6 Snellen’s in 38/38 (100% success) in group A. In group B, 38 cases out of 41 achieved 6/6 vision (92.68% success). In group C, 35 cases out of 36 achieved 6/6 (97.22%) success (Table 2). The average time duration for successful amblyopia therapy in group A was 8 ± 1 weeks, in group B was 9 ± 2 weeks and in group C was16 ± 2 weeks (Figure 1).\n\nThe blue line represents group A (3–7 years); red represents group B (8–12 years); green represents group C (13–35 years). Visual acuity axes are a conversion of Snellen’s scores to numerical values: 6/60–0.1; 6/36–0.167; 6/24–0.25; 6/18–0.333; 6/12–0.5; 6/9–0.667; 6/6–1.\n\nVisual acuity improved even in the \"unsuccessful cases\" (not achieving 6/6). The three unsuccessful cases in group B improved from 6/60 to 6/12 and the one unsuccessful case in group C was found to be due to eccentric fixation. Hence an improvement of five lines did occur even with eccentric fixation.\n\nPatient compliance to therapy was found to be an important factor influencing clinical improvement in vision in this study. Groups A and C were noted to be more compliant to therapy than Group B in spite of regular counseling of both the parents and the patients (Figure 2).\n\nThe blue line represents group A (3–7 years); red represents group B (8–12 years); green represents group C (13–35 years). Visual acuity axes are a conversion of Snellen’s scores to numerical values: 6/60–0.1; 6/36–0.167; 6/24–0.25; 6/18–0.333; 6/12–0.5; 6/9–0.667; 6/6–1.\n\nAll cases showed improvement in near vision prior to distance vision. Color vision in all cases was found to be normal as checked by the Ishihara color plates before and after therapy. We noticed an increase in stereopsis, determined at each follow-up visit after successful amblyopia therapy by the TNO test, but this is beyond the scope of our present study.\n\nA more interesting outcome of the study was that out of the 115 amblyopic cases included in our study, 82 (71.30%) presented with strabismus. Out of these 82 cases, 51 cases (62.19%) became orthophoric once their amblyopia was fully corrected while only 31 cases (37.80%) needed surgery for strabismus correction once their vision was restored in the amblyopic eye (Table 3).\n\nFurther analysis of the outcome of amblyopia therapy on strabismus presenting in each group revealed that in group A (38 cases), 33 had strabismus; out of these, 28 cases (84.84%) became orthophoric with amblyopia therapy alone and only 5 (15.15%) needed strabismus surgery. In group B (41 cases), 25 had strabismus; out of these, 16 cases (64%) became orthophoric and 9 (36%) needed surgery for squint correction. In group C (36 cases), 24 had strabismus; out of these, 7 (29.16%) became orthophoric and 17 cases (70.83%) needed strabismus surgery.\n\nReversal of amblyopia was noted in two cases in group A. One stopped wearing glasses after two months of completing the weaning and visual acuity dropped to two lines when she came for follow-up. She was started again on full-time patching and the visual acuity returned to 6/6. The second case stopped patching abruptly during weaning. On follow-up, the visual acuity had dropped by two lines. It was controlled by resuming full-time patching again. One patient in group C behaved similarly and reversal of amblyopia was controlled by resuming full-time patching and continuous spectacle wear.\n\nPatch related mild contact dermatitis was noted in 60% patients, which was managed by steroid skin cream at night on the rash while the patch was off. No other patch related complication was noted.\n\n\nDiscussion\n\nAmblyopia is the most common cause of monocular visual impairment among children, young, and middle-aged adults18. Occlusion of the good eye by a stick-on eye-patch is the main therapy, which may be applied full-time or part-time. The standard teaching has been that children being treated with full-time occlusion therapy need to be observed at intervals of one day per year of age so as to avoid occlusion amblyopia of the good eye. The Amblyopia Treatment Studies (ATS) have helped to provide new information on the effects that result from patching of various durations (2–6 hours per day)19. Studies have shown that patching or occlusion therapy compliance is a major factor that influences the outcome of treatment19,20. Most of the visual improvement in this study was obtained in the first four hundred hours of occlusion therapy.\n\nFull-time patching is quite difficult, particularly when a teenager or an adult is asked to manage with a poorly sighted amblyopic eye; it needs a major life style modification for at least 2–3 weeks after which the near vision is improved to such an extent that the patient can manage on his own. Hence a positive approach by the patient, the family and the treating ophthalmologist is very important for a successful outcome following full-time patching.\n\nIn our study, a 100% positive outcome was seen in children between age three to seven years. This seems to be correlated with excellent compliance due to education and regular counseling of both parents to fully comply with the therapy and to return for regular follow-ups. All of these cases had severe amblyopia with vision of counting fingers at 6/60. They achieved 6/6 Snellen’s visual acuity after six weeks of constant full-time patching in all three literate cases, (100%) in this age group. In the remaining eight children, visual acuity was checked by Kay's picture test and a 100% improvement was noted. Results from a study done by MX Repka and coworkers7 showed that improvement of visual acuity in children between three and seven years of age with moderate amblyopia was a mean of 3.7 lines in the part-time patching group and 3.6 lines in the atropine group, from baseline. A study conducted by Scheiman and coworkers21, in 2004 on 404 patients aged 7–17 years found 49% of treatments were successful in 7–12 year olds and 23% were successful in 13–17 year olds. They observed that near vision activities had a beneficial impact on visual acuity in the 13–17 year age group even when amblyopia was not being treated; this was more pronounced if amblyopia was treated by 2–6 hours patching/day. Their study confirms that near visual activities have a positive visual outcome. However, the difference in their results in terms of success differs from our study because of the different patching technique; 2–6 hour patching in their study had no significant impact and only resulted in 2–4 lines improvement in visual acuity. Our study implemented full time patching, which was shown to give promising results even in severe amblyopia in adults in 97.22% cases achieving 100% improvent in vision.\n\nFull time patching can be accomplished if the child and their parents are strongly motivated at each follow-up visit; parents need to make sure that the child is doing near visual activities for 3–4 hours per day for the amblyopic eye. Holmes and coworkers22, also suggested the involvement of parents and children results in better compliance in their research on amblyopia patching therapy.\n\nThe time taken to achieve improvement in visual acuity in our study is consistent with the studis by Levi and Li23,24, using perceptual learning technique in adults. Perceptual learning involves practicing visual tasks to improve performance using special computer programmes in hospitals during which the good eye is occluded for only a short period of time and is considered a form of neuroplasticity, reflecting an alteration of neural response in the visual pathway. But this group achieved a visual acuity improvement of only 33% while with full time patching and active use of the amblyopic eye by the patient at home in his own comfortable sorroundings, our study achieved marked improvement in visual acuity from 95–100%. Patients were only called in for a brief follow-ups in our study rather than extensive and repeated clinical sessions in the hospital in the perceptual learning technique; hence it was both cost-effective and time-saving for the patient and the treating clinician.\n\nA study by Carl Kupfer (1957)25, showed marked improvement in visual acuity in seven adult strabismica amblyopes, aged 18 to 22 years from hand movements (they could not even count fingers due to dense amblyopia) to 20/25 after four weeks of intensive therapy. The patients were hospitalized for four weeks, during which time they were given full-time occlusion therapy and fixation training. Results of our study in groups B and C were much better, achieving 6/6 Snellen’s vision in 95.65% of the cases in Groups B and C. The remaining 4.35% in our study had eccentric fixation in whom visual improvement stopped at 6/18–6/12 from 6/60. They were asked to see through a pinhole applied on their correcting glasses while the good eye was patched. Their vision improved to 6/6 in further 3–4 weeks of therapy. We did not hospitalize our patients but focused on motivating them at each follow-up visit.\n\nThe fact highlighted by this study is that amblyopia can also be successfully treated in adults and that there is no clear upper age limit for recovery of vision though older patients may require two to three months of full-time patching compared to younger age groups who achieved 100% improvement within two months of therapy.\n\n\nConclusion\n\nThis study shows that any severity of amblyopia can be reversed at any age with full-time occlusion therapy. A 100% improvement in visual acuity is possible within a period of 2–3 months. In comparison only a 3–4 line improvement (30–50%) in visual acuity occurs after a period of six months to one year with part time patching. The only factor that determines a successful outcome following full-time occlusion therapy is patient's total compliance to therapy.\n\nOther recent technological approaches such as the perceptual learning technique, Pac-man technique, racing games and other computer soft-ware tools are not only complicated but a financial burden on the economy of under-developed countries. In comparison, occlusion therapy by a patch is very an economical, affordable and the only feasible option in countries such as Pakistan. The extent of visual improvement achieved by full time occlusion therapy is more effective than other techniques.", "appendix": "Author contributions\n\nSameera Irfan formulated the study, ophthalmological examination of patients, parent's and patient's counselling, wrote the final study. Nowsherwan Adil made the data spread sheet, did the statistical analysis, plotted the graphs. Haris Iqbal: helped with the clinical study, did literature search, kept patient's records throughout their follow-up. All authors actively revised the manuscript.\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\nAmerican Academy of Family Physicians Information from your family doctor. Amblyopia (\"lazy eye\") in your child. Am Fam Physician. (2007); 75(3): 368.\n\nAmerican Academy of Ophthalmology Amblyopia: Basic and Clinical Science Course: Pediatric Ophthalmology and Strabismus.1997; 259–65.\n\nWebber AL, Wood J: Amblyopia: Prevalence, Natural History, Functional Effects and Treatment. Clin Exp Optom. 2005; 88(6): 365–375.\n\nSimons K: Preschool vision screening: rationale, methodology and outcome. Surv Ophthalmol. 1996; 41: 3–30.\n\nAstle AT, McGraw PV, Webb BS: Can human amblyopia be treated in adulthood? Strabismus. 2011; 19(3): 99–109.\n\nHess RF, Mansouri B, Dakin SC, et al:Integration of local motion is normal in amblyopia. J Opt Soc Am A Opt Image Sci Vis. (2006); 23(5): 986–992.\n\nRepka MX, Wallace DK, Beck RW, et al:Two-year follow-up of a 6-month randomized trial of atropine vs patching for treatment of moderate amblyopia in children. Arch Ophthalmol. 2005; 123(2): 149–57.\n\nScheiman MM, Hertle RW, Beck RW, et al:Randomized trial of treatment of amblyopia in children aged 7 to 17 years. Arch Ophthalmol. 2005; 123: 437–447.\n\nFlynn JT, Schiffman J, Feuer W, et al:The therapy of amblyopia: an analysis of the results of amblyopia therapy utilizing the pooled data of published studies. Trans Am Ophthalmol Soc. 1998; 96: 431–50.\n\nAssaf AA: The sensitive period: transfer of fixation after occlusion for strabismic amblyopia. Br J Ophthalmol. 1982; 66: 64–70.\n\nSimons K, Preslan M: Natural history of amblyopia untreated owing to lack of compliance. Br J Ophthalmol. 1999; 83: 582–587.\n\nAmerican Academy of Ophthalmology Pediatric Ophthalmology/Strabismus Panel. Preferred Practice Pattern Guidelines. Amblyopia. American Academy of Ophthalmology, San Francisco 2007.\n\nPascual-Leone A, Amedi A, Fregni F, et al:The plastic human brain cortex. Annu Rev Neurosci. (2005); 28: 377–401.\n\nLevi DM, Polat U: Neural plasticity in adults with amblyopia. Proc Natl Acad Sci U S A. 1996; 93(13): 6830–4.\n\nLevi DM, Li RW: Improving the performance of the amblyopic visual system. Philos Trans R Soc Lond B Biol Sci. 2009; 364(1515): 399–407.\n\nPolat U: Restoration of underdeveloped cortical functions: evidence from treatment of adult amblyopia. Restor Neurol Neurosci. 2008; 26(4–5): 413–24.\n\nEl Mallah MK, Chakravarthy U, Hart PM: Amblyopia: is visual loss permanent? Br J Ophthalmol. 2000; 84(9): 952–6.\n\nMenon V, Chaudhuri Z, Saxena R, et al:Factors influencing visual rehabilitation after occlusion therapy in unilateral amblyopia in children. Indian J Med Res. 2005; 122(6): 497–505.\n\nHolmes JM, Kraker RT, Beck RW, et al:A randomized trial of prescribed patching regimens for treatment of severe amblyopia in children. Ophthalmology. 2003; 110(11): 2075–87.\n\nRepka MX, Beck RW, Holmes JM, et al:A randomized trial of patching regimens for treatment of moderate amblyopia in children. Arch Ophthalmol. 2003; 121: 603–11.\n\nScheiman MM, Hertle RW, Beck RW, et al:Randomized trial of treatment of amblyopia in children aged 7 to 17 years. Arch Ophthalmol. 2005; 123(4): 437–47.\n\nHolmes JM, Repka MX, Kraker RT, et al:The treatment of amblyopia. Strabismus. 2006; 14(1): 37–42.\n\nLevi DM, Li RW: Perceptual Learning as a potential treatment for amblyopia: a mini-review. Vision Res. 2009; 49(21): 2535–49.\n\nLevi DM: Perceptual Learning in Adults with Amblyopia: A Reevaluation of Critical Periods in Human Vision. Dev Psychobiol. 2005; 46: 222–32.\n\nKupfer C: Treatment of amblyopia exanopsia in adults: A preliminary report of seven cases. Am J Ophthalmol. 1957; 43(6): 918–922." }	[ { "id": "1187", "date": "31 Jul 2013", "name": "Robert Taylor", "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 publication is a prospective case series of patients who are reported to have amblyopia, who underwent full time occlusion following one month of refractive adaption. The authors have divided the patients in to three age groups. They claim 100% success in achieving 6/6 Snellen in the younger group, with high percentages claimed in the other two age groups exceeding 90%. The authors also claim that the full time occlusion treated heterotropia in 85% of the younger age group who had a strabismus, 64% of the middle age group (age 8-12) and 29% of the older age group. The latter claim is not discussed further. Very few visually related problems are aired, with two children, and one patient in the older age category relapsing, with vision being restored with further occlusion. The authors have reported a high incidence of contact dermatitis - treated with topical steroid.\nOn the face of it the authors seem to have solved the problem of amblyopia management, declaring results far in excess of other published studies. They have also managed in some way to treat strabismus, even though occlusion is not normally thought to be a treatment for strabismus.\n\nThere are a number of observations that might explain the discrepancy between this publication’s results and those of others.\n\nThe first is the case selection. Paragraph 1 of the methods is confusing. If the better eye has to be 6/6 for inclusion, it would be much more simple to state that all amblyopic eyes were equal to or less than 6/12 (assuming a three line difference of 6/7.5, 6/9 and 6/12). If all amblyopic eyes were 6/60 or less (as is suggested later in the discussion) than this is not a difference of three lines. We are not told how many lines there are on their charts, which can be highly variable. We are also told that there are “study specified criteria for strabismus and/or anisometropia”, but are not told what these are. We are told that those with organic visual loss are excluded, although cases of stimulus deprivation are included. One has to assume therefore that in these cases the cause of the stimulus deprivation has been removed. The cases have been referred to a tertiary centre, so it is not clear how much treatment might have been instituted in the past, and again an assumption that many of these cases have been treated by other methods could be made, possibly with an initial good outcome at some point in the past. Although the cases selected are probably all amblyopic, it is possible that some may not be, if they have not been refracted for many years. Lastly by selecting patients with 6/6 in the better eye, cases of bilateral ammetropic amblyopia have been excluded.\n\nThe second concern is the method of assessing visual acuity. All studies of amblyopia currently use crowded logMAR testing on all patients, even those with the inability to read letters. The use of Snellen does not lend itself to statistical analysis (as the authors have done to plot their graphs) as it is a non linear scale. Furthermore the authors have stated that Kay’s pictures are used for those who are unable to read letters. It is assumed that these are single optotypes and not crowded. This method of assessment is highly correlated to over-estimations of visual acuity, and may go some way to explain the apparent good results. We are not told of the proportion of those who have been tested using this method.\n\nThe third concern is the length of time allowed for refractive adaption. Most authors would agree that a month is not long enough, with much variation in the literature, but up to 18 weeks being common. It is possible that some of the reported improvement in acuity is as a result of the refractive adaption rather than occlusion.\nThe authors might have given more information on how compliance was assessed. Many studies report poor compliance. Having said this the apparent high association of contact dermatitis suggests good compliance.\n\nThere are a number of omissions that are of concern. In such a group of children with strabismus and amblyopia, I would have expected some children to have small angle, constant esotropia with no binocular vision (and suppression). The paragraph on page 4 (last paragraph in right hand column) seems to divide patients into those with orthophoria and those that had surgery. It seems odd that there are no patients in the group that have small angle manifest strabismus, but did not need surgery. Are we to assume the patients with orthophoria have binocular vision? It is unlikely in patients who started the study with dense amblyopia.\n\nAnother omission is any information on binocular vision. In the UK, the practice of occlusion therapy is associated with assessments of the depth of suppression, albeit without much data to support it. The concern is the complication of diplopia, by removing suppression with occlusion. Any publication expounding the virtues of full time occlusion needs to be careful not to create an environment where diplopia is created to the detriment of the patients overall vision.\n\nA further concern is occlusion amblyopia. It would appear from the graphs that occlusion was applied for a mean of 14 weeks. Full time occlusion in patients as young as 4 might be associated with this complication, which it would appear did not occur.\n\nLastly it would be of interest if there were any patients who were lost to follow up. These patients are important as often associated with treatment failure. Again it would appear not to have occurred.\n\nIn the discussion, much of the first paragraph could be edited. On page 5, the first complete paragraph, it is confusing as the authors describe group A, as being 3 patients (“three literate cases”) and “the remaining eight children” - which makes 11 cases, however there were 38 in this group.\nLanguage and Typographical errors: Page 4, first paragraph on the right, talks about reversal of amblyopia. I think the authors are talking about the amblyopic eye visual acuity decreasing. I think “regression” might be a better term as reverse amblyopia is a term used to describe the reduction of vision in the previously better eye, described above as occlusion amblyopia.\n\nPage 5 first paragraph - “can manage on his own” would be better “can manage independently”.\n\nPage 5 right hand column talks about “100% improvement”. I do not understand this. 100% of what?\nPage 5, second to last full paragraph on left, word \"improvent\" should be improvement, and first full paragraph on the right, strabismica - should be strabismic. Third to last line on page 5 - “is very an economical” - delete \"an\".\nPage 6, column on the right, first line, “performed the literature search, kept the patients’ records...” would read better.\nOverall the authors have produced a piece of work that is thought provoking. Although the overall results are challenged, they are still impressive. Consideration of full time occlusion, with care to avoid occlusion amblyopia and diplopia, may be an accepted form of treatment in selected cases.\n\nDeclaration: Sameera Irfan (first author) is a former trainee of the reviewer (1995 - 1996).", "responses": [] }, { "id": "1932", "date": "30 Sep 2013", "name": "James Loughman", "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\nIrfan et al. have reported on the efficacy of occlusion therapy in severe amblyopia among subjects aged 3 to 35. The authors present a series of interesting, but somewhat surprising results. Most notably the authors conclude that (a) any degree of amblyopia can be resolved with full time occlusion therapy, with almost 100% success even in those aged 13 to 35, in reaching and maintaining 6/6 acuity, and (b) that amblyopia correction is an effective treatment for strabismus, creating orthophoria in almost two-thirds of cases. These findings are potentially groundbreaking, but are so contrary to conventional clinical experience (including experience in the days when full time occlusion was routine), and to the extensive body of prior research evidence (some of which, including the PEDIG study are cited by the authors), that a serious and in-depth evaluation of the scientific robustness of the study methods and interpretation of reported findings is needed.\n\nAlthough it is beyond the scope of a short review such as this to explore the flaws in substantial detail, I will outline in brief, a number of significant flaws in the study methods and interpretation of findings as presented in this manuscript.\n\nThe study sample is very poorly described. For example, we are not given any idea as to the degree of anisometropia, the type (e.g. accommodative) and magnitude of strabismus, or participants’ past ocular history. Patient history is a very important potential confounder that is not at all addressed in this study. The reader is left clueless as to key factors such as why the participants have been referred to the clinic, whether they have undergone any previous ocular treatment (possibly including occlusion therapy) or whether they have previously worn spectacles among many other unknown factors. The inclusion criteria allow for participation of subjects having “failed” previous occlusion therapy, yet there is no information at all as to how many of such participants were included, or how the investigators deemed that past therapy had indeed “failed”. Without such information, the reader simply cannot be certain as to whether the study participants have true amblyopia, or simply took some time to adapt to new spectacles.\n\nThe study methodology is certainly unclear, and most likely flawed. For example, the authors state that the optician was masked as to the subjects’ demographics. Quite how an investigator can be masked to patient demographics is beyond me (e.g. a child age 3 versus an adult age 31).\n\nAnother important aspect is the acuity chart used. Firstly, the Snellen and Kay’s picture charts are inappropriate for amblyopia assessment, but that remains a minor point. The major concern is whether the same chart and series of letters was used throughout the study. The methods describe that subjects were assessed at a minimum of every 2 weeks (and much more frequently for those under 7, such as every 5 days for 4 year olds). VA in the amblyopic eye was assessed first, then in the normal eye. Surely, the failure to randomise charts and letters is potentially a major flaw in the study (the reader must assume that letter or chart randomisation did not occur as it is not stated as a prescribed method). The effect of learning could be truly substantial, with subjects becoming increasingly familiar with the charts and letters at every visit. Despite even the best of participant intentions, such a learning effect would be impossible to avoid and could entirely explain the results observed in older observers. Although the authors dismiss it as not within the scope of their paper, I am unclear as to the reasons why the reported improvements in stereopsis are not explored in detail in the manuscript. Surely, a paper concerned with neural plasticity in older age groups should report on stereopsis when the test has been included in the study protocol.\n\nI am further concerned by the authors’ interpretation of the effect of occlusion therapy on strabismus, stating that “Out of these 82 cases, 51 cases (62.19%) became orthophoric once their amblyopia was fully corrected while only 31 cases (37.80%) needed surgery for strabismus correction once their vision was restored in the amblyopic eye”. The effect of refractive correction on the reported strabismus is not explored in the paper. Inspection of the amblyopia data provided reveals, as would be expected, a strong relationship between refractive error type and strabismus type (i.e. esotropia mostly associated with hyperopia, and exotropia with myopia). It would seem entirely more likely that the actual reason that strabismus is rendered orthophoric, simply relates to the provision of appropriate spectacles, and not to occlusion therapy success as described. Accommodative esotropia, for example, remains the most common cause of childhood esotropia, and is readily treated with spectacle provision, not requiring surgery.\n\nThis study simply does not stand up to proper scientific scrutiny. The methods are flawed, the interpretation is inaccurate, and the results are most likely explained away by factors not explored in the paper. I am concerned that this article could potentially create confusion among the general public and possibly even among clinicians inexperienced in the art of evaluating scientific publications.", "responses": [] } ]	1	https://f1000research.com/articles/2-149
https://f1000research.com/articles/2-108/v1	10 Apr 13	{ "type": "Research Article", "title": "Differences in antibiotic use and knowledge between adolescent and adult mothers in Ecuador", "authors": [ "Arturo Quizhpe P", "Martyna Gassowski", "Lorena Encalada T", "Francoise Barten", "Martyna Gassowski", "Lorena Encalada T", "Francoise Barten" ], "abstract": "Objectives: To investigate the differences in antibiotic use and knowledge between adolescent and adult mothers of children under the age of 5 years in Ecuador.Methods: A cross sectional study was performed in four health centers and hospitals. Mothers of children under five years, seeking medical attention their child's upper respiratory tract infection (URI), were included. The data was collected through interviews, using a structured questionnaire. The questionnaire covered the topics knowledge of antibiotic treatment, risk and resistance. Results: 777 mothers were included in the study, of which 15.8% were adolescent and 84.1% adult mothers. There were significant differences in the social and economic characteristics of the mothers (p ≤ 0.05), with adolescent mothers being more likely to have an incomplete high school education and lack of basic services in their home. Significant differences between these groups were found in adherence to treatment, knowledge about risks associated with antibiotic use, and having heard of antibiotic resistance. Among the adult mothers, 83.5% reported correct adherence, 28.5% were knowledgeable about risks associated with antibiotic use, and 29.3% had heard of antibiotic resistance. Among the adolescent mothers, these numbers were 75.4%, 15.0%, and 19.8%, respectively.Conclusions: To develop successful interventions, it is crucial to understand the factors causing differences in antibiotic use and knowledge between mothers.", "keywords": [ "Child care", "child health", "maternal educational status", "patient education", "antibiotics", "Latin America" ], "content": "Introduction\n\nAntibiotics are potent drugs, which since their discovery have saved millions of lives. However, when used incorrectly, they can pose a health risk to the patient, as well as contribute to the development of bacterial resistance to these drugs. Considering the already alarming levels of resistance reported worldwide1,2 an improvement in use, in terms of indications for treatment, choice of antibiotic, dosage, duration and enhanced knowledge, are crucial steps towards containment of the problem of resistance.\n\nThe majority of antibiotics are consumed in the community3, and in this setting there is plenty of opportunity for incorrect use. Particularly in countries with lack of access to and availability of health care, poor drug control, and lack of education of the population, self-medication is often common practice4,5. The high cost of medicines, incomplete treatment courses, and patient pressure on the physician for a prescription, contribute to the misuse of antibiotics. Studies from both developed and developing countries show that misconceptions about antibiotics and their use are prevalent in the lay populations6–9. It is for example frequently thought that antibiotic treatment is indicated in conditions such as the common cold, which in most cases is a condition of viral etiology4–6. The financial aspect also influences the use of antibiotics, and poor patients will often not be able to afford a doctor visit or full treatment10. On the other hand, many health care systems do not make an effort to educate either the lay population about the importance of correct use of antibiotic, or the physicians about how to best deal with these problems.\n\nIn Ecuador, little is known about the use of antibiotics and the relevance of educational efforts made. In a review of the use and misuse of antibiotics in Latin America, M. J. Wolff described an \"antibiotic culture\" where treatment often is thought to be indicated at any sign of a suspected infection, which results in frequent self-medication4. In order to be able to address this and other incorrect antibiotic use through interventions, it is important to first identify the causes of these behaviors in the local context, as well as the differences between different groups in the society. Preschool children are a group that are highly exposed to antibiotics in the community11,12. However, in the case of such young children, the final decision about antibiotic treatment is taken by the child’s care giver, usually the mother. In Ecuador, despite an over-all decreasing fertility rate, the group of adolescent mothers is steadily increasing13. Thus, the aim of this study was to investigate whether there are differences in antibiotic use and knowledge between adolescent and adult mothers, seeking care for an upper respiratory tract infection (URI) in their child, aged less than five years. URIs are the leading cause of morbidity and doctor consultations in children under the age of five in Ecuador14, and it was therefore chosen as a reference condition for the study.\n\n\nMethods\n\nPatients were informed about the study and its goals and oral informed consent was obtained.\n\nThis cross sectional study was performed between February and April 2011, in four health centers and basic hospitals in the provinces of Azuay and Guayas in southern Ecuador. The sites were selected to represent a variety of ethnicities, local cultures, and climatic regions. The selected study sites are located in the highlands and in the coast and cover both urban and rural populations. Table 1 gives an overview of the characteristics of each of the health facilities and the populations they cover.\n\nThe data analyzed in this article came from a larger cross-sectional survey aimed at investigating perceptions of disease severity, treatment and antibiotic use in caregivers of children under the age of five. The data was collected through interviews, using a structured questionnaire. All mothers of children under five years of age, seeking medical attention in one of the study sites for a respiratory tract infection in the child, were eligible for the study. Due to low numbers of attendance in the health center in Nabon, the approach was changed and the participants from this study site were recruited during medical visits in the home. The aim was to include 200 participants from each of the study sites.\n\nThe questionnaire included questions about demographic and socioeconomic characteristics of the child, mother and family, and questions about antibiotic knowledge and use by the mother. The main outcome measures were if the mother had ever used antibiotics without previously consulting a physician; always gave the complete treatment course to her child; knew of any risk associated with antibiotic use; and had ever heard of antibiotic resistance. The secondary outcome measures were the source of antibiotics if not consulting a physician before use; reason for giving incomplete treatment course if ever doing so; source of information about risks associated with antibiotic use if having this knowledge; and source of information about antibiotic resistance if they had this knowledge.\n\nThe statistical analysis preformed consisted of a descriptive analysis of the study population. To assess the significance of the differences between the adolescent and adult mothers, a Chi-square test was used, with level of significance set to p≤0.05. The data were analyzed using SPSS 17 for Windows.\n\n\nResults\n\nIn total, 777 participants were included in this study. Of these, 123 (15.8%) were adolescent mothers (aged 19 years or younger at the birth of their child), while 654 (84.2%) were adult mothers. The mean age of the adolescent mothers was 17.5 years (s = 1.3), and 27.1 years (s = 5.3) for the adult mothers at the time of the study. Table 2 shows the basic characteristics of the mothers and of their living conditions.\n\na Basic services is defined as having electricity, running water and connection to a sewage system in the home.\n\nb Gross minimum wage in Ecuador.\n\nThe groups differed significantly in social and economic characteristics. Adolescent mothers were more likely to have an incomplete high school or primary school education and to live in a rural area. They also were more likely than adult mothers to live in a home lacking access to at least one of the basic services (electricity, running water or a connection to a sewage system). In addition, the number of children was lower in the group of adolescent mothers.\n\nAdult mothers were significantly more likely to have at least two children under the age of five in the family and they were also more likely to have a family income exceeding one minimum wage per month, than were the adolescent mothers.\n\nTable 3 shows the responses to the questions concerning antibiotic use and knowledge of the mothers. For all questions, less than 1% of the data was missing. Having used antibiotics without previously consulting a physician was common in both groups, with 30 (24.6%) of the adolescent mothers and 191 (29.2%) of the adult mothers having ever done so, but no significant difference between the groups was found. However, a significant difference was found in adherence to treatment, where 543 (83.5%) of the adult mothers stated that they always gave the complete antibiotic course to their child, compared to 92 (75.4%) of the adolescent mothers. The reasons behind not completing a treatment were also investigated, and the most frequently given reason in both groups was that the child was feeling better, stated by 19 (61.3%) in adolescent mothers and 60 (55.0%) in adult mothers. However, no significant differences were found between the groups in this aspect (p< 0.061).\n\nOf the adult mothers, 185 (28.5%) stated that they knew of any of the risks associated with antibiotic use, such as antibiotic resistance, allergy and secondary effects compared to 18 (15.0%) of the adolescent mothers, a difference that was significant.\n\n24 (19.8%) of the adolescent mothers and 191 (29.3%) of the adult mothers had heard of antibiotic resistance, and this difference was significant. The majority in both groups (56% of the adolescent mothers and 68.1% of the adult mothers) stated the physician as the source of this knowledge. There was no significant difference found between the groups either for this source, or for others.\n\n\nDiscussion\n\nThis study found that adolescent mothers more frequently used antibiotics incorrectly and had less antibiotic knowledge compared to adult mothers. A number of differences in the characteristics of the mothers and their living conditions were identified, showing a tendency of the adolescent mothers to have a lower level of education and income, and to be more likely to live in rural areas.\n\nIn regard to the level of education of the mothers, as one of the determinants of this study demonstrates, only 23.6% of adolescent mothers completed high school compared with 41.9% of adult mothers. A study in Cuba on educational level of mothers and their knowledge, attitudes and practices concerning acute respiratory infections in their children concluded that the most important variable for the adequacy of knowledge was the educational level, which highlights the fundamental and positive influence of this factor on the preventive and curative care that mothers provide for their children, results by regression of age of the mother and level of education were RP 0.91 95% (0.85–0.98)15.\n\nAnother study in Spain on appropriate use of antibiotics in primary health care points out that an adequate level of beliefs in parents about the indications of antibiotic use in several illnesses are associated with a high level of parental education (Odds Ratio, 2.04; 95% CI, 1.16 -3,06). In relation to the level of education, 45.8% of mothers had only a primary school education. 73 children (21%) received antibiotics in the 3 months before the study. 27 parents (7.8%) bought antibiotics without prescription. 82 respondents (23.6%) stated that their pediatrician did not prescribe antibiotics in situations where they expected them to do so16.\n\nAs found in our study, the education of mothers is an influential factor in the knowledge and use of antibiotics in case of URIs, because it allows greater access to the basic information on health education in order to recognize the symptoms and signs that require urgent attention at a health care centre and adopt healthier lifestyles to decrease the level of child morbidity. Another study in 2008–2009 in the Ahmedabad district in India, found that the prevalence of acute respiratory tract infections (ARI) was higher in low social classes (III, IV and V) (26.56%), illiterate mothers (24.4%) and first-time mothers (23.9%) and those living in overcrowded houses (28.5%). At the same time, the prevalence of ARI was lower in urban areas (17.2%) as compared to rural areas (26.8%). This difference is more likely due to a lack of availability of basic health services and other associated factors like overcrowding, low socio-economic status, absence of cross ventilation and indoor air pollution.\n\nAbout one third (30%) of children belonged to upper social classes (I, II) and the remaining were in low social classes (III, IV, V). According to social class, prevalence of ARI was higher in lower social classes (III-31.4%, IV-22.1%, and V-26.2% respectively) and was statistically significant (p<0.001). Prevalence of ARI was highest in children of illiterate (24.4%) and primary school educated (23.9%) mothers. This difference was not statistically significant.\n\nThe above study concluded that strengthening reproductive and child health phase II (RCH-2) or integrated management of childhood illness (IMCI) programs and raising female literacy levels will go a long way in preventing ARI and morbidity amongst children in general. Reorientation of IMCI strategy and training of health workers in peripheral areas regarding identification, management and timely referral of cases of ARI and strong supervision, monitoring and evaluation of rural care health services specifically for ARI are transcendental to improving the quality of the health care centers and decreasing child morbidity and mortality17. A study conducted in eight countries in Latin American (Bolivia, Colombia, Ecuador, Guatemala, Nicaragua, Panama, Peru and Dominican Republic) in 2006 assessed the possible independent and combined action of the parent’s educational level and the economic situation of the family with the risk of diarrheal and respiratory diseases among children under 5 years. For ARIs, the risk of illness decreased as the economic situation of the family improved. However, the mother’s educational level played a contradictory role: the children of mothers with some education had a higher risk of respiratory illnesses than children of mothers without education. This may be because the women with lower educational levels have breastfeed their children for longer or because women with some education could recognize milder respiratory conditions better.\n\nWe did not find synergistic effects between the father’s educational level and the family's financial situation.\n\nThe Latin American study meantioned above found that better family economic situations reduced the risk of disease in the children of mothers with higher education than in mothers without formal education. Similarly, better education of mothers had a greater protective effect in the children of mothers with a better educational level than in poor families. A better economic situation can allow women to make better choices in regards to child health and helps them to provide their children with better hygiene, a healthy lifestyle and greater access to health care, while educated women living in more severe economic conditions do not always have access to these options for their child’s health. The father’s higher level of education had a protective effect, independent of the economic situation of the family. These results demonstrate that efforts to decrease poverty will have a greater protective effect on child health if they include efforts to improve the educational level of women and girls than if any of these efforts were to be made separately18.\n\nIt is believed that the age of the mother may be associated with the level of knowledge about URIs and the use of antibiotics. An analysis of a bivariate study in Spain showed that the level of knowledge was directly related to the age of the person who answered the survey: the older the mother, the higher the level of knowledge (34.5 years compared to 32.8 years) p = 0.00319.\n\nThe results of our study show that the knowledge about resistance and risks of antibiotic use is also not very high for adult mothers, where less than a third had heard of either. This points towards a generally low level of knowledge in the population and the lack of education programs at the community and hospital level.\n\nAs described in the results, the physician was stated as the most common source of information about antibiotic resistance by both the adult and adolescent mothers. This shows the potential of physicians as educators, and it should be taken into account when developing interventions targeting the lay population. We believe that the leadership of the medical professional is a key point for the knowledge of the appropriate use of antibiotics.\n\nIn a study on the general public’s perceptions and use of antimicrobials in Trinidad and Tobago, it was shown that it was not uncommon for the study participants to incorrectly classify different painkillers and cough and cold formulations as antibiotics20. It is possible that this occurred to a certain extent in this study as well, as the mothers’ understanding of the term \"antibiotics\" was not investigated. Also, it is possible that some of the participants might have been giving desired answers, rather than truthful ones, when asked about self-medication and early termination of treatment.\n\nDifferent studies show the importance of knowledge on the appropriate prescribing of antibiotics in children under 5 years, on the economic impact of respiratory infections on the family budget, and on the beliefs of mothers in regards to using antibiotics for acute respiratory infections without a prescription; however, the one factor that contributed to treatment failure was maternal education (p = 0.05)21–23. This highlights the importance of ensuring a higher level of education in this population group.\n\nThe mis- and overuse of antibiotics in the community are behaviors of a complex nature. They are the result of different socioeconomic, cultural, demographic and other factors interacting in ways specific to the local context. In order to identify these factors, multivariate analysis is needed, but even with the significant factors identified, only half the battle is won. In order to be able to develop successful interventions, it is crucial to first understand the mechanisms behind how these factors influence each other and the behaviors and attitudes towards antibiotics.\n\nLittle research has been done on the determinants of antibiotic use in the community, and the results are often contradictory6,15,16,19–21. More attention urgently needs to be given to this part of the combat against antibiotic resistance, if the battle shall be won.", "appendix": "Author contributions\n\n\n\nAQ and FB conceived the study. AQ, MG and LE carried out the research. AQ, MG, FB and LE prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research resulted from a partnership between the Faculty of Medical Sciences, University of Cuenca, Ecuador and Radboud University Nijmegen Medical Centre in the Netherlands. The Radboud University Nijmegen Medical Centre funded the travel and hosting of the student in Ecuador. The University of Cuenca helped with logistic support of the student in the health care institutions in Ecuador.\n\n\nReferences\n\nWorld Health Organization: The World Health Report 1996--fighting disease, fostering development. World Health Forum. 1997; 18(1): 1–8. PubMed Abstract\n\nFrench GL: The continuing crisis in antibiotic resistance. Int J Antimicrob Agents. 2010; 36(Suppl 3): S3–S7. PubMed Abstract \| Publisher Full Text\n\nFuruya EY, Lowy FD: Antimicrobial-resistant bacteria in the community setting. Nat Rev Microbiol. 2006; 4(1): 36–45. PubMed Abstract \| Publisher Full Text\n\nWolff MJ: Use and misuse of antibiotics in Latin America. Clin Infect Dis. 1993; 17(Suppl 2): S346–S351. PubMed Abstract \| Publisher Full Text\n\nMulticenter study on self-medication and self-prescription in six Latin American countries. Drug Utilization Research Group, Latin America. Clin Pharmacol Ther. 1997; 61(4): 488–93. PubMed Abstract \| Publisher Full Text\n\nCalva J, Bojalil R: Antibiotic use in a periurban community in Mexico: A household and drugstore survey. Soc Sci Med. 1996; 42(8): 1121–8. PubMed Abstract\n\nTan YS, Hong CY, Chong PN, et al.: Knowledge that upper respiratory tract infection resolves on its own is associated with more appropriate health-seeking behaviour and antibiotic cognition. Singapore Med J. 2006; 47(6): 518–24. PubMed Abstract\n\nChan GC, Tang SF: Parental knowledge, attitudes and antibiotic use for acute upper respiratory tract infection in children attending a primary healthcare clinic in Malaysia. Singapore Med J. 2006; 47(4): 266–70. PubMed Abstract\n\nLee GM, Friedman JF, Ross-Degnan D, et al.: Misconceptions about colds and predictors of health service utilization. Pediatrics. 2003; 111(2): 231–6. PubMed Abstract \| Publisher Full Text\n\nHart CA, Kariuki S: Antimicrobial resistance in developing countries. BMJ. 1998; 317(7159): 647–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClavenna A, Bonati M: Drug prescriptions to outpatient children: a review of the literature. Eur J Clin Pharmacol. 2009; 65(8): 749–55. PubMed Abstract \| Publisher Full Text\n\nMajeed A, Moser K: Age- and sex-specific antibiotic prescribing patterns in general practice in England and Wales in 1996. Br J Gen Pract. 1999; 49(446): 735–6. PubMed Abstract \| Free Full Text\n\nCEPAR: ENDEMAIN, National Demographic and Maternal and Child Health [National Demographic and Maternal Health Survey, 2004]. Quito: Center for Population Studies and Social Development 2004.\n\nOPS/OMS Ecuador's representation. Health situation in Ecuador 2006.\n\nValdez R, Ana I, Martínez CH, et al.: [Educational level of mothers and their knowledge, attitude and practices concerning acute respiratory infections of their children]. Rev Panam Salud Publica. 1999; 6(6), pp. 400–407 [Article in Spanish]. PubMed Abstract\n\nBuñuel ÁJC, Fortea GE, Cortés MRB, et al.: [Antibiotic use in primary care. Do we know what parents think?] An Pediatr (Barc). 2004; 61(4): 298–304. PubMed Abstract\n\nBipin P, Nitiben T, Sonaliya KN, et al.: A study on prevalence of acute respiratory tract infections(ari) in under five children in urban and rural communities of Ahmedabad district, Gujarat, India. National Journal of Community Medicine. 2011; 2(2): 255. Reference Source\n\nRegional de la Organización Mundial de la Salud para las Américas: Efecto sinérgico del nivel educacional de los padres y la situación económica de la familia en la salud infantil en América Latina. Rev Panam Salud Publica. 2006; 19(2): pp. 124–125. Reference Source\n\nParimi N, Pinto PLM, Prabhakar P, et al.: The general public's perceptions and use of antimicrobials in Trinidad and Tobago. Rev Panam Salud Publica. 2002; 12(1): 11–8. PubMed Abstract\n\nDelpiano ML, Kabalan BP, Diaz VC, et al.: [Acute respiratory infections in children of day care center: characteristics and costs]. Rev Chilena Infectol. 2006; 23(2), pp. 128–133 [Article in Spanish]. PubMed Abstract \| Publisher Full Text\n\nGutierrez L: Beliefs of mothers of children between 2 and 5 years on the treatment of acute respiratory infections in San Antonio-Ate Health center. Lima-Perú. 2009; pp. 10–120.\n\nSantos M: Compliance the antibiotic therapy in children with pneumonia. Federal University of Rio de Janeiro. 1999; pp.145–163. Reference Source\n\nLópez Y: Incidence of acute respiratory infections in children under five years old. Electronic Journal of PortalesMedicos.com 2010. Reference Source" }	[ { "id": "891", "date": "15 Apr 2013", "name": "Andrew Gilbert", "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 and important topic area to study. The paper is well written and the conclusions match the results of the data analysis. Basically it is stating the obvious: Older, more experienced mothers, who are generally better educated, have more money and have more children are better at using antibiotics and know about antibiotic resistance. The paper would benefit from having a statistician provide advice on data analysis; a better story could be told about the influence of age etc if logistic regression models were used.One of main concerns in the interpretation provided in that it has missed the obvious question about, “were the older mothers the same as the young mums in terms of actions, knowledge and use of antibiotics. How would the older mums compare if those with only one child were compared with the younger mums; ie is knowledge about antibiotics and obtaining antibiotics for medical practitioners learnt “on the job”. Planned regression modelling can answer questions such as this.If no statistical support is available, the authors should at least acknowledge this and include in the discussion a statement, such in the paragraph above, on weaknesses of this study. Any future study should include a statistician at the very first study planning session to ensure adequate data are collected via the questionnaire to answer the research question(s), and the appropriate statistical procedures. There is no mention in the study of basic statistical requirements such as a power calculation, for example.", "responses": [ { "c_id": "435", "date": "23 Apr 2013", "name": "Diana Andrade", "role": "Reader Comment", "response": "Dr. Andrew Gilbert,Your perspective is very interesting and your report has opened us up to a new way to show the results in the article, we hope to send it as soon as possible addressing all the suggestions contained in your review.Thanks a lot Dr. Gilbert for the comments and support.Kind regards,Dr. Arturo Quizhpe, Dean at the Faculty of Medical Sciences, University of Cuenca." }, { "c_id": "463", "date": "20 May 2013", "name": "Diana Andrade", "role": "Reader Comment", "response": "Dear Dr. Gilbert:We have proceeded to perform the review and corresponding analysis according to your comments and suggestions. In a revised version, we will incorporate this and will be adding the following paragraph at the end of the text of the study: “This is a descriptive study. It is a pioneer in addressing this subject in Ecuador. The results show interesting differences in the knowledge and use of antibiotics between teenage and adult mothers. However, there were limitations to what could be statistically analyzed. Therefore a new study design is needed to confirm and quantify the association. Given the current importance of the subject matter and the high prevalence of teenage mothers in our environment, this line of research must be continued and requires the validation of a more analytical research instrument and integration of an expert in statistics into the team for the duration of the study”. We thank you for your support. Kind regards, Dr. Arturo Quizhpe" } ] }, { "id": "994", "date": "10 Jun 2013", "name": "Liliana Clara", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 that education efforts should be targeted to concentrate more on these specific age groups.", "responses": [] } ]	1	https://f1000research.com/articles/2-108
https://f1000research.com/articles/2-148/v1	08 Jul 13	{ "type": "Research Article", "title": "Extracellular histone H1 is neurotoxic and drives a pro-inflammatory response in microglia", "authors": [ "Jonathan D Gilthorpe", "Fazal Oozeer", "Julia Nash", "Margarita Calvo", "David LH Bennett", "Andrew Lumsden", "Adrian Pini", "Jonathan D Gilthorpe", "Fazal Oozeer", "Julia Nash", "Margarita Calvo", "David LH Bennett", "Andrew Lumsden" ], "abstract": "In neurodegenerative conditions and following brain trauma it is not understood why neurons die while astrocytes and microglia survive and adopt pro-inflammatory phenotypes. We show here that the damaged adult brain releases diffusible factors that can kill cortical neurons and we have identified histone H1 as a major extracellular candidate that causes neurotoxicity and activation of the innate immune system. Extracellular core histones H2A, H2B H3 and H4 were not neurotoxic. Innate immunity in the central nervous system is mediated through microglial cells and we show here for the first time that histone H1 promotes their survival, up-regulates MHC class II antigen expression and is a powerful microglial chemoattractant. We propose that when the central nervous system is degenerating, histone H1 drives a positive feedback loop that drives further degeneration and activation of immune defences which can themselves be damaging. We suggest that histone H1 acts as an antimicrobial peptide and kills neurons through mitochondrial damage and apoptosis.", "keywords": [ "H1", "histone", "neurodegeneration", "inflammation", "microglia", "neurons", "neurotoxicity" ], "content": "Introduction\n\nAll of the major neurological diseases and trauma to the central and peripheral nervous systems carry the same cytopathological features, namely neuronal death and reactive gliosis. These differential responses occur simultaneously within the same milieu but why this happens is poorly understood. The degenerating nervous system is not a non-specific killing field.\n\nThe observations described here derive from experiments designed to identify axonal chemorepellents in which explants of degenerating adult rat brain were co-cultured with embryonic brain explants in collagen gel matrices. Unexpectedly, we found that adult brain causes diffusion-mediated degeneration of embryonic axons and have shown that the degenerating adult brain releases histone proteins. Histone H1, but not core histones, selectively kills embryonic cortical neurons and causes microglial cells to become reactive.\n\nHistones are highly basic proteins that are present in the nucleus where they form major components of eukaryotic chromatin and function to regulate transcription. The core histones, H2A, H2B, H3 and H4 have relatively similar structures containing a histone fold domain and together form the histone octamer. The winding of genomic DNA around this octamer forms the basic unit of DNA packaging termed the nucleosome. Members of the more divergent histone H1/H5 family are structurally unrelated to core histones and function to link nucleosomes. It has usually been assumed that histones are restricted to the nucleus but a growing body of evidence indicates that histones, histone-DNA complexes and histone-derived peptides have cytoplasmic and extracellular locations and actions1.\n\nCore and linker histones and nucleosomes have been detected both at the cell surface2–6 and in the cytoplasm7,8 and have also been linked to apoptosis7–11. Several lines of evidence support the idea that extracellular histones may contribute to human disease mechanisms. Anti-histone antibodies are present in vascular dementia and pre-senile Alzheimer’s Disease (AD)12. In systemic lupus erythematosus (SLE), many different nuclear antigens are released and provoke high titres of destructive anti-histone autoantibodies8. Indeed, a significant proportion of patients with SLE have associated neurodegeneration. More recent evidence indicates that extracellular histones can mediate endothelial cell death and sepsis13, and cause thrombocytopenia through platelet aggregation14.\n\n\nMaterials and methods\n\nSprague Dawley and Wistar rats were obtained from Charles River and kept on a 12-h light-dark cycle with food and drinking water provided ad libitum. Animals were killed under Schedule 1 by cervical dislocation conforming to British Home Office Regulations. For neuronal cultures cerebral cortices were removed from embryonic day (E) 15–17 Sprague Dawley rat embryos and adult brain explants were obtained from the mother.\n\nFor microglial cultures the mother and her litter were housed in one cage and post-natal day 3 (P3) pups were killed under British Home Office Schedule 1 regulations by rapid decapitation.\n\nFor substratum experiments mouse cortical neurons were prepared from E16.5 CBA x C57BL/6 mice (bred in house Umeå University) in accordance with approval granted by Umeå University Ethical Committee for Animal Experimentation (Permit Number: A113-11). Animals were maintained as part of a breeding colony in a designated facility on a 12-h light-dark cycle with food and drinking water provided ad libitum. Neuronal isolation was performed after the mother had been sacrificed by cervical dislocation (the morning a vaginal plug was discovered was determined to be E0.5).\n\nAll animal experiments were designed and performed according the mandated principles of reduction, refinement and replacement.\n\nCortical explants were dissected into pieces of about 200–400 μm2 (embryonic) and 500–1200 μm2 (adult) using fine tungsten needles and kept on ice-cold minimum essential medium (MEM, Gibco UK). For dissociated cultures embryonic cortices were also cut into small pieces and dissociated with the Papain Dissociation System (Worthington Biochemicals) according to the manufacturer’s instructions. Neurons were plated on 13 mm diameter glass coverslips coated first with poly-D-lysine (10 μg/ml in PBS) followed by laminin (10 μg/ml in PBS) (Gibco) and cultured at 37°C in a humidified 8% CO2 (v/v) atmosphere for 24–48 hrs in Neurobasal medium plus 1% (v/v) Antibiotic-Antimycotic (Gibco). To make conditioned media, the meninges were removed from adult and embryonic brains and were then cut into small pieces (~2 mm3) and kept on ice-cold MEM.\n\nCollagen was prepared by mixing 90 µl of filtered rat tail collagen as described in15 with 10 µl of 10× concentrated Dulbecco's Modified Eagle Medium (DMEM, Gibco), which was kept on ice until required15,16, and set by mixing with 2–3 μl of 7.5% (w/v) sodium bicarbonate (Gibco). Explants were placed on 35 mm Falcon dishes, excess liquid aspirated with a fine glass capillary tube and covered with 30 μl of the setting collagen solution. Co-cultures of adult and embryonic cortex were now positioned ~0.5–1 mm apart and lectin-coated agarose beads (Vector Laboratories) (1–2 μl) injected between them when required. Co-cultures and dissociated cells were incubated for 24–48 hrs in DMEM plus 1% (w/v) Antibiotic-Antimycotic (Gibco) at 37°C in a humidified 8% (v/v) CO2 atmosphere and examined by phase contrast microscopy (Nikon T800 and Lucia software).\n\nAdult conditioned medium was made by incubating two chopped cortices per 20 ml culture medium while for embryonic conditioned medium four E15 whole brains per ml were used. Incubations were performed for 48 hrs as above but the medium was replenished after 24 hrs. Conditioned media were centrifuged for 3 mins at 900g and stored at -20°C. Careful checks were made to ensure that pH remained physiological.\n\nHistones and other proteins were isolated and identified from adult and embryonic conditioned media. Affinity chromatography columns were constructed using agarose beads as the solid phase to which Sambucus nigra lectin (Vector Laboratories, 3 mg lectin/ml gel) had been covalently bound. Columns were loaded with 5 ml of conditioned media and incubated for 1 hr at room temperature before being washed with 5 mls of PBS and eluted with 5 mls of PBS and alpha-lactose (500 mM) followed by 5 mls PBS and 2% (w/v) acetic acid according to the manufacturer’s instructions. Elution was by gravity (1 ml column) or by pump (10 mls column 0.4 ml/min). Eluates were concentrated in two steps, first using 5,000 NMWL (nominal molecular weight limit) 15 ml filters (Microcon, Millipore) centrifuged at 900g at room temperature until 2 mls remained. These aliquots were further concentrated using 10,000 nominal molecular weight limit NMWL Microcon 1.5 ml filters centrifuged at 13,000g at room temperature until dry. Material was reconstituted from the membrane filters in 10–40 mls of sample buffer made according to Laemmli17 and stored at -20°C.\n\nDenaturing gel (Laemmli) electrophoresis was performed using 10% (w/v) SDS-polyacrylamide gels (SDS-PAGE). Samples were denatured prior to loading and electrophoresis was performed at a constant current of 25 mA, using a Tris-glycine buffer system with SDS (192 mM glycine (Sigma), 25 mM Trizma base (Sigma) and 0.1% SDS (Fisher Scientific)). Gels were stained with R250 Coomassie Brilliant Blue (1g Coomassie in 50% (v/v) methanol/10% (w/v) acetic acid) and destained with 12% (v/v) methanol in 7% (w/v) acetic acid for 1 minute. Molecular weight markers were run with all gels. Bands of interest were excised from the gels and stored at -20°C until sequenced.\n\nCoomassie-stained bands resolved by SDS-PAGE were excised and digested with 200 ng trypsin (Promega) for 4 hrs at 37°C. The resultant peptides were separated by reverse-phase chromatography (Vydac C18 0.8 × 150 mm) and were analysed by Matrix-assisted laser desorption/ionization-time of flight (MALDI TOF) mass spectrometry using an Applied Biosystems 4700 Proteomics Analyser. Peptide masses were searched against the NCBI (National Center for Biotechnology Information) non-redundant database using Protein Prospector MS-FIT software (University of California San Francisco).\n\nHuman recombinant histones H1, H2A, H2B, H3 and H4 were obtained from New England Biolabs. Additionally, histone H1 purified from calf thymus, and Xenopus laevis recombinant histones H2A, H2B, H3 and H4 were obtained from Millipore. Histones were diluted in culture media as indicated.\n\nPrimary cortical neurons were seeded onto poly-D-lysine and laminin (both 10 μg/ml) coated black 96-well plates (Greiner) at 3 x 104 cells per well in 100 μl. Cells were incubated for 48 hrs in Neurobasal medium supplemented with Glutamax, B27 and 1% (v/v) Antibiotic-Antimycotic (all Gibco) and then treated with histones at 0, 25, 50, 100 and 200 nM for a further 24 hrs. Histone-induced cell death was determined by the CytoTox-Glo™ Cytotoxicity Assay (Promega). Briefly, degenerating cells release intracellular proteases that cleave acetylaminofluorene (AAF) to liberate aminoluciferin, which is a substrate for luciferase. This reaction generates luminescence proportional to the number of dead cells18 and was measured using a Promega GloMax 96 luminometer. Readings were made within 24 hrs to prevent false negatives due to protease degradation.\n\nMouse cortical neurons were prepared from E16.5 CBA x C57BL/6 mice using a similar protocol to rat except 0.05% (w/v) trypsin digestion for 10 min at 37°C in Hank’s buffered saline (HBS, Gibco) was used instead of papain. Cells were plated at 3 x 103 cells per well in 96-well plates in Neurobasal media supplemented with 2% (v/v) NS21 supplement 0.25 mM L-glutamine and 10 µg/ml gentamicin19. Wells had been previously coated with poly-D-lysine, histone H1, or histone Type VIII-S (arginine rich fraction of calf thymus histone, predominantly histone H3, Sigma). Cells were grown for 72 hrs and the number of living neurons was estimated using calcein acetoxymethyl ester (AM) staining. Cells were incubated with 1.1 nM calcein AM for 30 min at 37°C in a humidified incubator. Hoechst 33342 (16 µM) (Molecular Probes, Invitrogen) was added to counterstain the nucleus of both live and dead cells. Haemoglobin (15 mg/ml) was then added to quench fluorescence generated by the culture medium and the plate was imaged using a TROPHOS Plate RUNNER HD cell fluorescence imaging instrument. Total cell fluorescence from calcein AM, a measure of metabolic activity from live cells, was determined using the Tina analysis package (Trophos).\n\nCortical astrocytes were cultured from 2–3 days post-natal rat pups. Two cortices were removed and cut into small pieces (~2mm2) before being digested with 5 ml of 0.2% (w/v) trypsin, BSA, DNase I (Sigma) in Earle’s buffer (Worthington) at 37°C for 20 mins. Digestion was terminated by addition of an equal volume of DMEM followed by centrifugation for 5 mins at 1,500 rpm. The pellet was re-suspended in 10 ml of DMEM with 10% (v/v) FCS and 1% (v/v) penicillin and streptomycin (Gibco) which was divided between two 5 ml flasks coated with poly-L-lysine (0.5 mg/ml Sigma). Flasks were incubated as described above for 10 days with media changed at 48 and 96 hrs. Cells were passaged and plated on poly-D-lysine coated coverslips for a further 6 days in 4-well plates prior to treatment with histones and staining with antibodies.\n\nCells were fixed in 4% (w/v) paraformaldehyde (PFA) and incubated for 1 hr with anti-glial acidic fibrillary protein GFAP rabbit polyclonal antibody (Z0334 Dako) diluted at 1:2500 in PBS or cleaved caspase 3 antibody (Abcam) diluted 1:500 in PBS. Cells were washed and incubated for 1 hr in polyclonal anti-rabbit TRITC (Dako) diluted 1:2500 (for GFAP) or goat anti rabbit polyclonal Alexa Fluor 488 (Invitrogen) for caspase 3 in PBS prior to washing and mounting. Some neuronal cultures were incubated with 15 μM propidium iodide (Invitrogen) for 10 mins before fixing and with 0.5 μM DAPI (4′, 6-diamidino-2-phenylindole) (Sigma) for 5 mins after fixing during washing with PBS.\n\nMixed glial cultures were made from explants of cerebral cortices of P3 (3 day post-natal) Wistar rats as described previously20,21. Briefly, after mechanical and chemical dissociation cells were plated in DMEM 10% (v/v) fetal bovine serum at a density of 500,000 cells/ml and cultured at 37°C in humidified 5% (v/v) CO2/95% (v/v) air. Reagents were obtained from Invitrogen. Confluence of the mixed glial culture was achieved after 5 days in vitro. Cultures were gently shaken and the floating cells were pelleted and sub-cultured. This method resulted in 96–99% purity as assessed by staining with the rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1) polyclonal antibody at 1:1000 (019-19741, Wako Chemicals, Japan) and 4′, 6-diamidino-2-phenylindole (DAPI).\n\nChemotaxis was assessed using a Boyden22 chamber (Neuroprobe, Bethesda, MD). Polycarbonate filters (5 μm pore diameter) were installed in the chamber, whose bottom wells were filled with serum-free DMEM with or without histone H1 at various concentrations. Freshly prepared microglia were suspended in serum-free DMEM and were placed into the top wells at 50000 cells/well. The chamber was maintained in a CO2 incubator at 37°C for 3 hrs. The filter was removed and stained with RapiDiffII (Biostain RRL, UK), cells on the top side of the filter were wiped off, and the numbers of cells that had migrated to the bottom side were counted as described in20. Four independent experiments were performed (4–8 filters were counted for each condition in each experiment) and the data expressed as cell counts.\n\nMicroglial cells were plated at around 50,000 cells/well on glass coverslips. The following day cells were left in serum-free DMEM for 4 hrs and then histone at different concentrations was added to the medium. Twenty-four hours later cells were fixed in 4% (w/v) PFA for 30 min and with ice cold methanol for 5 min. Microglia were labeled using a polyclonal antibody to ionized calcium binding adapter molecule 1 (Iba1) (Wako). For detecting MHC class II antigens we used a monoclonal mouse anti-rat RT1B antibody (OX-6) (Serotec clone 554926), BD Bioscience diluted 1:100. The percentage of cells that were OX-6 positive over the total number of Iba1 positive cells was determined. Three independent experiments, each of them run in triplicate were analysed. Cultures were then treated with lipopolysaccharide (LPS) (Sigma UK) at 1 μg/ml or histone H1 at 25–200 nM for 24 hrs. Primary microglial cultures were incubated in serum-free medium for 24 hrs and identified with Iba1 (red) while reactive microglia were identified with OX-6, which labels MHC-class II expressing cells in green so that double-stained cells appear yellow.\n\nWhen microglial cells are kept in serum-free conditions and in relatively low concentration (approximately 2000 cells/mm2), apoptotic cell death is observed within 24 hrs21. Therefore, to assess survival, microglia were plated at low density and incubated for 24 hrs in serum-free medium supplemented with increasing doses of histone H1 (50, 100 and 200 nM). Cells were then fixed and immunostained with Iba1 and DAPI. The number of Iba1-stained cells was counted per coverslip in four independent experiments.\n\nThe possibility that histone H1 induces apoptosis was tested by staining for activated caspase 3 at 6 and 24 hrs following application of histone H1.\n\nAll statistical tests were performed in Sigmaplot v. 12.3 (Systat Software). Either one-way analysis of variance or ANOVA on ranks was used to test differences between groups. All post hoc tests were made with the appropriate Bonferroni/Holm-Sidak correction. Dose-dependent relationships were tested using linear regression where appropriate.\n\n\nResults\n\nDuring the course of experiments using collagen gel co-cultures to identify axonal chemorepellents from different brain regions we observed that adult cortical explants caused embryonic neural explants to degenerate. Having shown that medium conditioned with adult brain explants also caused neurodegeneration, we set about using this system to identify neurotoxic activities and demonstrated that the linker histone H1, but not core histones, caused degeneration of cortical neurons. As neurodegeneration is accompanied by glial activation and subsequently inflammation and activation of the immune system, we investigated whether histone H1 modulates the phenotype of astrocytes and microglia and have also shown that indeed it does.\n\nCultured alone, embryonic explants extend axons vigorously over 24–48 hrs but in the presence of nearby adult brain start to degenerate within 24 hours. We reasoned that this might be caused by diffusible glycosylated proteins or peptides which would bind differentially to lectins. Thus we devised a co-culture assay in which lectin-coated agarose beads were placed between the adult and embryonic explants to see whether degeneration could be inhibited.\n\nBy trial and error we found that Sambucus nigra lectin (SNA)-coated beads greatly reduced or blocked degeneration in collagen gel co-cultures (6/6 experiments, Figure 1). We therefore used SNA-coated beads to construct affinity chromatography columns to adsorb glycosylated moieties which could then be isolated.\n\n(a) Shows an adult cortical explant to the left (AD) and a degenerating E 15 embryonic cortical explant (E) to the right; arrows indicate blebbing of degenerating axons. (b) Degeneration is inhibited by placing agarose beads coated with Sambucus nigra lectin between the explants.\n\nFollowing SDS-PAGE of the column eluates, a variety of proteins was identified by MALDI TOF/TOF/QTOF analysis and included malate dehydrogenase 1, lactate dehydrogenase A, complement component factor h, Ig kappa chain C, ubiquitin-conjugating enzyme E2N, prostaglandin D synthase, prostaglandin D2, serotransferrin precursor protein, glycolipid transfer protein, transferrin, apolipoprotein A, apolipoprotein E and histones, H2A, H2B and H3.\n\nHaving demonstrated the presence of histones in column equates, we found by western blotting that Histones H1 and H2B were present in medium conditioned by adult brain (Figure 2).\n\nAdult conditioned medium (ACM) was made by incubating ischaemic brain slices in defined culture medium that was subjected to affinity chromatography. Western blots using antibodies specific to histones H1 and H2B show the presence of histone H1 at 32 kDa (lane 1) and histone H2B at 16 kDa in ACM (lane 3). Protein standards are shown at 32 kDa (H1 lane 2) and at 16 kDa for the monomer and 32 kDa for the dimer (H2B lane 4).\n\nDissociated embryonic cortical neurons were cultured for 48 hrs and then for a further 24 hrs in the presence of histones H1, H2A, H2B, H3 and H4. Only histone H1 demonstrated neurotoxicity at concentrations below 200 nM (Figure 3–Figure 5). Histone H1 caused significant degeneration from 100 nM (ANOVA on ranks p<0.001) and a dose-dependent relationship is evident between 0 and 200 nM (R2 = 0.966, n = 6, p<0.001).\n\nDissociated cortical neurons were cultured for 48 hrs and histones were applied for a further 24 hrs. Cell death was determined by luminescence to measure protease release and was expressed as relative luminescence units (RLU). For clarity RLU means are expressed as +/- 2 x standard deviation (SD). Histone H1 caused dose-dependent death of cortical neurons (a) while core histones H2A, H2B, H3 and H4 were without effect up to 200 nM (b–e). Cell death as measured by luminescence was confirmed directly via phase contrast microscopy of dissociated cortical neurons in culture (Figure 4).\n\nCortical neurons were plated on laminin-coated coverslips for 48 hrs. Cultures were treated with histone H1 at 50, 100 or 200 nM or left as controls for a further 24 hrs when they were incubated with propidium iodide (PI) for 10 mins prior to fixing and incubation with DAPI. As propidium iodide enters dying cells and binds to nuclear DNA its red fluorescence is greatly enhanced. Top left (control) and lower left (100 nM histone H1) show phase bright examples with their corresponding DAPI (blue) and PI (red) merged images which indicate the numbers of dying cells (magenta) and total nuclei (magenta and blue).\n\nCortical neurons were plated on laminin-coated coverslips and treated with histone H1 at 50, 100 and 200 nM for 6 hrs and 24 hrs. Cells were fixed and stained with DAPI (total nuclei) and for activated caspase 3 to detect apoptosis. Counts for both DAPI and caspase 3 were made for between 11–14 random fields per coverslip for each condition using Image J (University of California San Francisco UCSF) and expressed as % caspase 3/DAPI. Histone H1 induced significant upregulation of activated caspase 3 at 6 and 24 hrs for neurons treated at 100 and 200 nM respectively (ANOVA).\n\nCell death was determined from protease release and expressed as relative luminescence units (RLU). This was confirmed at a microscopic level and by using the vital dye propidium iodide (which is only taken up by dying cells as their membranes break down) and DAPI which stains all nuclei both in living and dying cells.\n\nWe considered the possibility that the presentation of histones might be a crucial determinant of their actions and investigated the effects of substratum-bound histones on neuronal survival. When bound to the dish however, histone H1 and poly-D-lysine significantly increased neuronal survival compared with controls whereas histone H3 was without effect (Figure 6).\n\nDissociated neurons were plated on substrate formed by binding histone H1, histone H3, and poly-D lysine to culture dishes at 1.25, 2.5, 5 and 10 μg/ml. Growth on immobilized histone H1 and poly-D-lysine was significantly increased at 5 and 10 μg/ml (one way ANOVA with Bonferroni’s post hoc correction) but was independent of substratum levels of histone H3.\n\nIt is well known that glial cells can proliferate and become activated in regions where neurons are degenerating so we made preliminary qualitative observations of the effects of histone H1 on cortical astrocytes. Control cultures mainly exhibit a flat ‘type 1’ rather than a stellate ‘type 2’ morphology. Histone H1 greatly increased the numbers of astrocytes with a stellate morphology when applied at concentrations as low as 15 nM (Figure 7). These observations are consistent with a previous report23 and we went on to look at the effects of histone H1 on microglia that mediate the innate immune response of the central nervous system (CNS).\n\nHistone H1 applied for 24 hrs at 50 nM on 6-day cultured cortical astrocytes (visualized using antibodies to GFAP) promoted differentiation of stellate (type 2) over flat (type 1) morphologies.\n\nMicroglial cells are resident monocytes in the CNS and local damage induces them to adopt an effector, or ‘activated’ state, which is associated with the development of an amoeboid morphology, pro-inflammatory cytokine expression and expression of MHC class II antigens20. We looked first at MHC class II expression using OX-6 as a marker following application of histone H1 (Figure 8a, b) or LPS (Figure 8b). Around 10% of untreated microglia were OX-6 positive but this increased to 58% when cells were treated with histone H1 at 25 nM and rose to almost 80% with increasing doses such that the effect of 50 nM histone H1 was not different from dosage of LPS which causes a maximal response (8a and b).\n\n(a) Primary microglial cultures were incubated in serum-free medium for 24 hrs and identified with Iba1 (red). (b) Cultures were then treated with histone H1 (100 nM) for 24 hrs. Activated microglia were identified with OX-6 (which labels MHC-class II, green) so that double-stained cells appear yellow. Scale bars: 100 μm. (c) Histone H1 dose-dependently increased the expression of MHC class II to a level similar to that elicited by LPS (1 μg/ml). 25 nM H1 produced a 6-fold increase in expression of OX-6 (p<0.001) while the effects of 50 nM histone H1 were not different from the maximal response to LPS at 1 μg/ml. Error bars represent ± SEM for all conditions, one-way ANOVA, Bonferroni post-hoc correction.\n\nMicroglia migrate towards sites of CNS damage that release chemoattractants. Using a Boyden chamber in which microglia migrate up a concentration gradient of attractant through pores in a polycarbonate filter, histone H1 significantly increased chemotaxis in a dose dependent manner (Figure 9a and b).\n\n(a and b) Chemotaxis towards histone H1 was measured using a Boyden chamber assay and cells that migrated through the filter are labelled in blue. The addition of histone H1 to the lower well of the chamber (b) increased microglial migration to the inner membrane surface compared to controls (a) demonstrating that histone H1 is a potent chemoattractant for microglia. The increases in microglial migration caused by histone H1 were significant (p<0.007 at 50 nM, p<0.001 at 100 and 200 nM one-way ANOVA, Holm-Sidak posthoc correction). Error bars represent ± SEM.\n\nMicroglial cells were incubated at low concentration in serum-free medium for 24 hrs to assess survival. Previous work indicates that serum deprivation results in a loss of around 50% of microglial cells by 24 hrs21. We quantified the number of cells after 24 hrs of incubation in serum free medium by fixing them and immunostaining with Iba1. For microglia treated with histone H1 at 50 and 200 nM survival was increased significantly but there was no increased effect of histone H1 with increasing dose confirming that in the range over which it is highly toxic to neurons it has the opposite effect on microglia (Figure 10). We were unable to distinguish between increased longevity and proliferation but experiments were conducted under conditions that lead to cell loss and it seems most plausible that the increased numbers of cells remaining after 24 hrs in culture are due to improved survival rather than increased proliferation.\n\nTreatment with histone H1 increased microglial survival in all the treatment groups considered together (ANOVA p=0.011 Holm-Sidak post hoc correction) and in multiple comparisons survival was increased significantly at both 50 nM and 200 nM H1 compared to control but just failed to reach significance at 10 nM (p=0.06).\n\nHistone H1 released from dying neurons causes cell death of neighbouring neurons and simultaneously promotes pro-inflammatory responses in microglia and astrocytes, which may then exacerbate neurodegeneration.\n\n\nDiscussion\n\nWe show that histone H1 is released from damaged brain explants and that it causes neuronal death and activation of the innate immune response via microglia. Linker histone H1 caused dose-dependent death of cortical neurons and but core histones H2A, H2B, H3 and H4 were without effect up to 200 nM. The effect of histone H1 is therefore selective and argues against histone-induced neurodegeneration being caused by membrane disruption due their highly basic nature. Indeed, histone H1 does not kill astrocytes and microglia but causes them to become reactive. This differential toxicity differs from that observed in sepsis where histones H3 and H4 rather than histone H1 are mediators of endothelial cell death13 and from platelet aggregation where histone H4 is an activator14.\n\nWe have shown the release of histones H2A, H2B and H3 from adult brain by sequencing and the release of histones H1 and H2B by western blotting. These observations are thus consistent with previous reports of the extracellular release of histones1,7 and strengthen the notion that the appearance of histones in the extracellular space can be linked with pathophysiological effects in neighbouring cells.\n\nIt has been shown that histone H1 binds directly to polysialic acid and promotes the survival of primary sensory neurons23, an effect opposite to that reported here for soluble histone H1. However, when immobilized on tissue culture plastic histone H1, but not H3, does significantly increase neuronal survival in a dose-dependent manner and is consistent with the effects reported on sensory afferents implying that binding to polysialic acid or plastic masks neurotoxic cell-surface binding or prevents internalization.\n\nWe isolated histones using Sambucus nigra lectin, which binds sialylated glycoproteins through recognition of Neu5Ac(α2–6)Gal/GalNAc sequences and has particularly high affinity for N-acetylneuraminic acid linked by α2,6-linkages to galactose or N-acetylgalactosamine. Histones have not been reported to be sialylated and it is possible that we co-purified histones bound to sialylated moieties from which they would dissociate under the reducing conditions of SDS PAGE.\n\nWe have shown that low doses of histone H1 dramatically up-regulate the expression of MHC class II antigens, which present pathogen-derived fragments to CD4+ T ‘helper’ cells. Thus, when histone H1 is released from the dying or injured nervous system it drives an inflammatory response via the innate immune system. We also show for the first time that histone H1 is a potent chemoattractant for microglia which are recruited to sites of inflammation and infection by chemokines. Histone H1 also promoted the survival of microglia under stress conditions and taken together these findings are consistent with a novel contribution of extracellular histone H1 to innate immunity in defence of the nervous system. The transition of microglia from a ‘surveying’ (also termed resting) to a pro-inflammatory or ‘effector’ state is widely regarded as a double-edged sword24–26 within the damaged nervous system and it is now plausible to think that the effects of histone H1 may contribute to this.\n\nLittle is known of the mechanisms or circumstances by which histone H1 appears in the extracellular space although core histones have been observed in exosomes27. It is possible that there are clearance mechanisms for histones following cell death or apoptosis but that under certain conditions these become overloaded. Histone up-regulation is known to occur in AD, where ectopic non-nuclear H1 is found at the neuronal cell-surface and in activated astrocytes28. Phosphorylated histone H3 is a nuclear marker for the M-phase of normal cell division but becomes activated and re-localised to the cytoplasm of neurons in AD29. There is compelling evidence that aberrant re-entry into the cell cycle may be a significant cause of neuronal cell death in AD30. Moreover, following UV-induced damage, the histone isoform, H1.2, translocates from the nucleus to the cytoplasm and triggers apoptosis10. Thus, one source of extracellular histones including histone H110,31 might be those re-localised to the cytoplasm while another could be those released from disintegrating nuclei. Potentially, extracellular histones could act both at the cell surface and within the cytoplasm following uptake. Hence an accumulation of extracellular histones could exacerbate neurotoxicity and as cell death increases produce an auto-catalytic cascade of neuronal cell death. Histones are also implicated in the formation of insoluble protein deposits. They bind to the Parkinson’s disease-associated protein α-synuclein and increase its fibrillation rate32–34. Histones also bind Alzheimer β-amyloid precursor protein (APP) and to β-amyloid with high affinity32,35. Since histones are found within amyloid plaques in AD brains36 an increase in their expression could be a catalyst for neuronal death and would be consistent with the findings of raised anti-histone antibody titres12.\n\nUnder cold stress, cortical slices lose their ability to increase respiration in response to electrical stimulation37,38. This respiratory depression is accompanied by migration of histones from the nucleus into the cytoplasm where they associate with microsomes and mitochondria, which swell. Respiratory depression is most likely to result from inhibition of oxidative metabolism of ATP and is reproduced by addition of soluble histones to the culture medium. Thus, under certain conditions histones can be liberated from the nucleus and taken up with both events leading to neuronal toxicity. These early observations exactly mirror those following UV-induced damage where histone H1.2 translocates from the nucleus to mitochondria via the cytoplasm and triggers apoptosis10. Significantly it has been shown that while linker and core histones bind to mitochondria and release pro-apoptotic proteins only linker histone H1.2 disrupts the inner membrane potential and causes release of the mitochondrial NAD+ pool39. Our observation that histone H1 causes apoptosis via activated caspase 3 is entirely consistent with observations of mitochondrial damage and dysfunction.\n\nFree histones have been reported in blood13,40 and it is reasonable to expect that their levels would increase, as do those of nucleosomes, following neuronal cell death from whatever cause. It has been known for a considerable time that histones can be cytotoxic to a variety of bacteria and mammalian cells13,41–43. Histones may contribute to defence against bacterial and viral infection by acting as cell surface receptors for bacterial and viral proteins and as antimicrobial agents in the gut. Extracellular histones and histone peptides (e.g. ncamp-1, Hipposin, Onchorynciin II, Parasin I, Buforin I/II, MUMP1-3) are non-specific antimicrobial agents of the immune response in fish, amphibians and mammals42,44–55. Histones in blood entrap pathogens when neutrophils release parcels of granule proteins and chromatin known as ’neutrophil extracellular traps’ that bind bacteria, fungi and yeast as a part of the innate immune response56–58. However, as discussed, it may be that the contribution of histone H1 to immune defence in the nervous system is in fact damaging.\n\nWe propose that in neurodegenerative diseases and following trauma, histone H1 acts in the brain as an antimicrobial peptide that drives the innate immune system and targets neurons as foreign bodies. We suggest that histones recognise binding sites in the nervous system that are shared with those of bacteria and viruses and that mitochondria, which were evolutionarily derived from bacteria, are the primary intracellular targets of histone H1 within the nervous system.", "appendix": "Author contributions\n\n\n\nAP conceived the study, performed collagen gel experiments, cell counting; interpretation and development of results and wrote the paper. AL was involved in the interpretation and development of results and revised the paper. JG performed neuron experiments, interpreted and developed the results and revised the paper. FO performed neuron, astrocyte culture and luciferase assays and revised the paper. JN performed protein isolation and purification. DB and MC performed microglial assays and revised the paper. All authors approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by MRC and BBSRC project grants to AP and by an MRC Programme Grant to AL (G0601064).\n\n\nAcknowledgements\n\nIt is a pleasure to thank Grant Wray for IT support, Sam Barnes for statistical analysis, Anthony Graham for the caspase 3 antibody as well as Geoff Cook and Caroline Formstone for helpful discussions. Sequence analysis was performed by Nick Totty and Sarah Hanrahan at the Protein Analysis Laboratory, London Research Institute, Cancer Research UK London WC2A 3PX.\n\n\nReferences\n\nParseghian MH, Luhrs KA: Beyond the walls of the nucleus: the role of histones in cellular signaling and innate immunity. Biochem Cell Biol. 2006; 84(4): 589–604. PubMed Abstract \| Publisher Full Text\n\nBolton SJ, Perry VH: Histone H1; a neuronal protein that binds bacterial lipopolysaccharide. 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PubMed Abstract \| Publisher Full Text\n\nKontopoulos E, Parvin JD, Feany MB: Alpha-synuclein acts in the nucleus to inhibit histone acetylation and promote neurotoxicity. Hum Mol Genet. 2006; 15(20): 3012–23. PubMed Abstract \| Publisher Full Text\n\nPotempska A, Ramakrishna N, Wisniewski HM, et al.: Interaction between the beta-amyloid peptide precursor and histones. Arch Biochem Biophys. 1993; 304(2): 448–53. PubMed Abstract \| Publisher Full Text\n\nIssidorides MR, Chrysanthou-Piterou M, Kriho V, et al.: Histones are components of senile plaques in Alzheimer's disease. Biological Psychiatry. 1995; 37(9): 643–643. Publisher Full Text\n\nMcIlwain H: Protein interactions and metabolic response to stimulating agents in isolated cerebral tissues: histones as inhibitors. Biochem J. 1959; 73(3): 514–21. PubMed Abstract \| Free Full Text\n\nWolfe LS, McIlwain IH: Migration of histones from the nuclei of isolated cerebral tissues kept in cold media. Biochem J. 1961; 78: 33–40. PubMed Abstract \| Free Full Text\n\nCascone A, Bruelle C, Lindholm D, et al.: Destabilization of the outer and inner mitochondrial membranes by core and linker histones. PLoS One. 2012; 7(4): e35357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaga S, Tan EM, Rubin RL: Identification and isolation of soluble histones from bovine milk and serum. Biochem J. 1987; 244(3): 675–82. PubMed Abstract \| Free Full Text\n\nAlström L, von Euler H: Toxic action of histones and protamines from thymus. Ark Kemi Mineral och Geol. 1946; 23A(3).\n\nHirsch JG: Bactericidal action of histone. J Exp Med. 1958; 108(6): 925–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller BF, Abrams R, Dorfman A, et al.: Antibacterial Properties of Protamine and Histone. Science. 1942; 96(2497): 428–430. PubMed Abstract \| Publisher Full Text\n\nBirkemo GA, Lüders T, Andersen Ø, et al.: Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.). Biochim Biophys Acta. 2003; 1646(1–2): 207–15. PubMed Abstract \| Publisher Full Text\n\nEvans DL, Kaur H, Leary J 3rd, et al.: Molecular characterization of a novel pattern recognition protein from nonspecific cytotoxic cells: sequence analysis, phylogenetic comparisons and anti-microbial activity of a recombinant homologue. Dev Comp Immunol. 2005; 29(12): 1049–64. PubMed Abstract \| Publisher Full Text\n\nFernandes JM, Molle G, Kemp GD, et al.: Isolation and characterisation of oncorhyncin II a histone H1–derived antimicrobial peptide from skin secretions of rainbow trout, Oncorhynchus mykiss. Dev Comp Immunol. 2004; 28(2): 127–38. PubMed Abstract \| Publisher Full Text\n\nHiemstra PS, Eisenhauer PB, Harwig SS, et al.: Antimicrobial proteins of murine macrophages. Infect Immun. 1993; 61(7): 3038–46. 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PubMed Abstract \| Publisher Full Text\n\nPark IY, Park CB, Kim MS, et al.: Parasin I an antimicrobial peptide derived from histone H2A in the catfish, Parasilurus asotus. FEBS Lett. 1998; 437(3): 258–62. PubMed Abstract\n\nRichards RC, O'Neil DB, Thibault P, et al.: Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar). Biochem Biophys Res Commun. 2001; 284(3): 549–55. PubMed Abstract \| Publisher Full Text\n\nRose FR, Bailey K, Keyte JW, et al.: Potential role of epithelial cell-derived histone H1 proteins in innate antimicrobial defense in the human gastrointestinal tract. Infect Immun. 1998; 66(7): 3255–63. PubMed Abstract \| Free Full Text\n\nTamura M, Natori K, Kobayashi M, et al.: Inhibition of attachment of virions of Norwalk virus to mammalian cells by soluble histone molecules. Arch Virol. 2003; 148(9): 1659–70. PubMed Abstract \| Publisher Full Text\n\nBrinkmann V, Reichard U, Goosmann C, et al.: Neutrophil extracellular traps kill bacteria. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "1045", "date": "08 Jul 2013", "name": "Alain Prochiantz", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 convincing. It would be nice to know if neutralizing H1 in the culture medium modifies axon degeneration and/or cell death.", "responses": [] }, { "id": "1056", "date": "12 Jul 2013", "name": "Roger Keynes", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a very nice paper. This interesting study identifies a novel mechanism to explain the differential susceptibilities of neurons and glia in neurodegenerative disease and injury, and so makes a significant advance. In vitro experiments implicate Histone H1, but not core histones, as selectively toxic for neurons while simultaneously activating astrocytes and microglia. The data are convincing and the proposed H1 positive feedback model (Figure 11) warrants future investigation in vivo.Suggested revisions:Figure 1 The caption should read 'Diffusion-mediated degeneration of embryonic neocortex is...' The blebbing of degenerating axons is indistinct as shown and would be more convincing at a higher magnification.It would also be interesting to know whether neuronal cell bodies, and not just their axons, are susceptible to H1 toxicity in the collagen gel co-culture; and to confirm that H1-antibody-coated beads pull off the neurotoxic activity. Figure 7The type 1 morphology is indistinct - it would be helpful to show or state the typical appearance of astrocytes without added H1.", "responses": [] }, { "id": "1059", "date": "15 Jul 2013", "name": "Jan-Marino Ramirez", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 fantastic paper and a very important contribution to our understanding of neurodegenerative disease and the response of the brain to injury. The authors have done a marvellous job in demonstrating that the adult brain releases histone H1 that were neurotoxic. The toxicity was specific for the H1 histone, while no toxicity was seen for H2, H2B, H3 and H4. An exciting finding is that H1, while neurotoxic to cortical cells promotes the survival of microglia, and acts as a potent chemoattractant; conceptually this is an important finding. The study is well done, the conclusions are fair and balanced and the techniques used in the study are very rigorous. The final figure nicely summarizes the proposed role of histones in inducing neuronal cell death whilst activating microglia. I am confident that this study will be a much cited contribution to the field of traumatic brain injury. The work is well written and the figures are outstanding.", "responses": [] }, { "id": "1259", "date": "30 Jul 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\nI think this paper excellent and is an important addition to the literature. I really like the conceptualization of a self-replicating cycle as it illustrates the concept that the “problem” starts with the neuron, i.e., due to one or more of a variety of insults, the neuron is negatively impacted and releases H1, which in turn activates microglia with over expression of cytokines that may, when limited, foster repair but when activated becomes chronic (as is demonstrated here with the potential of cyclic H1 release) and thus facilitates neurotoxicity. I hope the authors intend to measure cytokine expression soon, especially IL-1 and TNF in both astrocytes and microglia, and S100B in astrocytes.02/08/2013 - additional commentsIn more detail, Gilthorpe and colleagues provide novel experimental data that demonstrate a new role for a specific histone protein—the linker histone, H1—in neurodegeneration. This study, which was originally designed to identify axonal chemorepellents, actually provided a previously unknown role for H1, as well as other novel and thought provoking results. Fortuitously, as sometimes happens, the authors had a pleasant surprise: their results set some old dogmas on their respective ears and opened up new avenues of approach for studying the role of histones in self-amplification of neurodegenerative cycles. In point, they show that H1 is not just a nice little partner of nuclear DNA as previously thought. H1 is released from ‘damaged’ (or leaky) neurons, kills adjacent healthy neurons, and promotes a proinflammatory profile in both microglia and astrocytes. Interestingly, the authors’ conceptualization of a damaged neuron → H1 release → healthy neuron killing cycle does not take into account the H1-mediated proinflammatory glial response. This facet of the study opens for these investigators a new avenue they may wish to follow: the role of H1 in stimulation of neuroinflammation with overexpression of cytokines. This is interesting, as neuronal injury has been shown to set in motion an acute phase response that activates glia, increases their expression of cytokines (interleukin-1 and S100B), which, in turn, induce neurons to produce excess Alzheimer-related proteins such as βAPP and ApoE (favoring formation of mature Aβ/ApoE plaques), activated MAPK-p38 and hyperphosphorylated tau (favoring formation of neurofibrillary tangles), and α synuclein (favoring formation of Lewy bodies). To date, the neuronal response shown responsible for stimulating glia is neuronal stress related release of sAPP, but these H1 results from Gilthorpe and colleagues may contribute to or exacerbate the role of sAPP.", "responses": [] } ]	1	https://f1000research.com/articles/2-148
https://f1000research.com/articles/2-64/v1	28 Feb 13	{ "type": "Research Article", "title": "Stability, orientation and position preference of the stem region (residues 689-703) in Hepatitis C Virus (HCV) envelope glycoprotein E2: a molecular dynamics study", "authors": [ "Rahmad Akbar", "Siti Azma Jusoh", "Rahmad Akbar" ], "abstract": "Envelope glycoproteins of Hepatitis C Virus (HCV) play an important role in the virus assembly and initial entry into host cells. Conserved charged residues of the E2 transmembrane (TM) domain were shown to be responsible for the heterodimerization with envelope glycoprotein E1. Despite intensive research on both envelope glycoproteins, the structural information is still not fully understood. Recent findings have revealed that the stem (ST) region of E2 also functions in the initial stage of the viral life cycle. We have previously shown the effect of the conserved charged residues on the TM helix monomer of E2. Here, we extended the model of the TM domain by adding the adjacent ST segment. Explicit molecular dynamics simulations were performed for the E2 amphiphilic segment of the ST region connected to the putative TM domain (residues 683-746). Structural conformation and behavior are studied and compared with the nuclear magnetic resonance (NMR)-derived segment of E2 (2KQZ.pdb). We observed that the central helix of the ST region (residues 689 - 703) remained stable as a helix in-plane to the lipid bilayer. Furthermore, the TM domain appeared to provide minimal contribution to the structural stability of the amphipathic region. This study also provides insight into the orientation and positional preferences of the ST segment with respect to the membrane lipid bilayer interface.", "keywords": [ "Envelope glycoproteins E1 and E2 are essential for the initial binding and internalization of Hepatitis C Virus (HCV) into the host cells. Both glycoproteins have been shown to interact as a non-covalent heterodimer during biosynthesis1. Several conserved charged residues located in the TM domains of E1 and E2 were shown to function not only as membrane anchors", "but were also essential for dimerization", "endoplasmic reticulum retention and viral envelope formation2", "3." ], "content": "Introduction\n\nEnvelope glycoproteins E1 and E2 are essential for the initial binding and internalization of Hepatitis C Virus (HCV) into the host cells. Both glycoproteins have been shown to interact as a non-covalent heterodimer during biosynthesis1. Several conserved charged residues located in the TM domains of E1 and E2 were shown to function not only as membrane anchors, but were also essential for dimerization, endoplasmic reticulum retention and viral envelope formation2,3.\n\nIn our previous studies, we demonstrated the unfolding behavior of the E2 TM helix monomer was attributed to the charged Asp728 which was located in the hydrophobic core. The main contribution of Asp was postulated to be located at the helix-helix interface and involved the formation of a salt bridge with the Lys of the E1 envelope glycoprotein2. The ion-pair interaction of the E1–E2 heterodimer was captured in the molecular dynamics (MD) simulation studies based on the model that placed the charged Asp and Lys at the helix-helix interface4,5.\n\nE2 envelope glycoprotein is known to be required for interactions with cellular receptors involved in endocytosis and membrane fusion6–8. E2 is composed of domain I-III, followed by the ST region and the TM domain7. Recent studies based on circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy revealed that the soluble region located adjacent to the TM domain of E2 was involved in the initial virus entry. This highly conserved ST region was showed to fold as a helix upon membrane binding9.\n\nIn this work, we carried out MD simulations for three E2 structures: (1) a model generated by the I-Tasser server, (2) an ideal helix model and (3) an NMR derived structure of the E2 segment (2KZQ.pdb). The first two models include the TM domain of E2 but the TM domain is not present in the NMR structure. We observed consistent structural stability in the ST region amphiphilic segment (residue 689–703) across all simulations suggesting that the contribution of the TM domain to the segment structure stability is minimal. In addition, we demonstrated the orientation and positional preferences of this amphiphilic segment.\n\n\nMethods\n\nThe protein sequence of HCV E2 genotype 1a (H77 strain) (Uniprot ID P27958) used to prepare the models for MD simulations was obtained from UniProtKB/Swiss-Prot database (www.uniprot.org)10. The following is the sequence segment of HCV E2 used to prepare the first model for this work, referred to as an ideal helix model: 683PALSTGL 690IHLHQNIVDV 700QYLYGVGSSI 710ASWAIKWEYV 720VLLFLLLADA 730RVCSCLWMML 740LISQAEA. The ideal helix model was generated using Pymol (http://pymol.sourceforge.net) by orienting the ST segment (residue 683–714) perpendicular to the TM domain (residues 715–746). The protein structure images in this work were prepared with the Pymol program. The I-Tasser webserver11 was used to generate the second model. The complete sequence of E2 was submitted to the I-Tasser server. Five models were generated and the model with a low root mean square deviation (RMSD) with the available NMR structure (2KZQ.pdb) and consisting of a helical TM domain was selected. Only the same segment as examined with the ideal-helix model was used for further MD simulations. The third model studied was the NMR-derived structure of E2 (PDBID: 2KZQ) that was obtained from the Protein Databank (PDB)12. This E2 protein segment was based on the HCV genotype 2a (JFH-1) (Uniprot ID Q99IB8)9.\n\nPre-equilibrated dipalmitoylphosphatidylcholine (DPPC) lipid bilayer was retrieved from the web of Prof. Tieleman (http://moose.bio.ucalgary.ca/). Peptide orientation in DPPC lipid bilayer was done by aligning hydrophobic belt of the peptide, parallel to the membrane plane using LAMBADA13. The optimal number of overlapping lipid molecules was subsequently calculated and removed followed by lipid expansion (inflation) and alternating twenty steps of deflation and energy minimization to allow the peptide to be embedded within the bilayer using inflateGRO213. A short 100 ps energy minimization was employed to relax possible steric conflicts. Ions and counter ions were added to neutralize the system followed by 20 ns position-restrained simulation allowing the bilayer to re-equilibrate around the protein. Production MD simulations were carried out for 100 ns in the I-Tasser model, and 20 ns for both the ideal helix model and NMR structure (2KZQ.pdb).\n\nThe DPPC lipid bilayer interactions were described using the Berger force-field parameters14. The TM helices were modeled with the united atom force-field GROMOS96 53a615. Simulations were performed with the Gromacs 4.5.5 package16 using 2-fs time steps. Periodic boundary conditions were used in all directions. Bonds to hydrogen atoms were constrained using the LINCS algorithms17. For the short-range van der Waals interactions, a cut-off distance of 1.0 nm was used. The long-range electrostatic interactions were treated using the particle mesh Ewald (PME) method with a grid spacing of 0.12 nm and cubic interpolation. The non-bonded pair list was generated every 10 steps with a cut-off of 1.0 nm. Water, lipid and peptide systems were coupled separately to temperature baths with 323 K for the DPPC using the Berendsen algorithm with a time constant of τT = 0.1 ps18. To maintain constant pressure, semi-isotropic coupling was employed separately for the lateral and for the normal directions with Berendsen weak coupling and a τp = 1 ps time constant. The compressibility was set to 4.5 × 10-5 bar-1 18.\n\nAnalyses of the trajectories were primarily performed with tools included in the Gromacs 4.5.5 suite16. RMSD analyses were based on the coordinates of all atoms of the peptides. The bilayer thickness was measured by averaging the distances between lipid headgroups in the upper and lower leaflets of the lipid membrane with the tool GridMAT-MD19.\n\n\nResults and discussion\n\nA stable helical conformation of the E2 ST region (residue 689–703) was consistently observed with some uncharacteristic spikes in the early stages and towards the end of the MD simulation of I-Tasser model and the NMR derived structure (2KZQ.pdb), respectively. RMSD of this amphipathic segment was also consistently observed to be progressing within the commonly accepted 2 Å range for the ideal helix and NMR structures throughout the simulations. On the other hand, the I-Tasser model showed subtly higher RMSD progression over the simulation time (Figure 1, Figure 2 and Figure 3a, 3b).\n\nThe most stable helical region of the E2 amphiphilic segment during molecular dynamics simulations. 2KZQ.pdb in red, I-Tasser model in black and ideal helix model in gray.\n\nBoth amphiphilic region (segment 689–703) and TM domain located in the hydrophobic core of the bilayer. Length of residues 689–703 is plotted in red, root mean square deviation of the same residues with respect to the starting structure is plotted in black.\n\n(a) Molecular dynamcs simulation of the ideal helix model. The amphiphilic segment was positioned in water-lipid interface and the TM domain was oriented in hydrophobic core of the bilayer. The length of the helix of residues 689–703 is plotted in red, root mean square deviation of the same residues with respect to the starting structure is plotted in black. (b) Molecular dynamics simulation of nuclear magnetic resonance derived structure (2KZQ.pdb). 2KZQ was positioned in the water-lipid interface. The length of the helix segment is plotted in red, root mean square deviation of the same residue with respect to starting structure is plotted in black.\n\nSecondary structure stability observed in these three contrasting simulation systems can be attributed to the amphiphilic nature of the residues allowing the helix to retain its structure on both the hydrophobic core of the lipid bilayer and the hydrophilic environment of the solvent. This amphiphilic characteristic of the residues has been discussed and described to great extent by Albecka et al. in a previous study9. Interestingly, the presence of the TM domain does not appear to contribute significantly to the helix stability of the amphiphilic region.\n\nThe helical length of this region does not vary much between the I-Tasser and the ideal-helix models (Figure 2 and Figure 3a), which include both the ST region and TM domain, compared with the 2KZQ.pdb (Figure 3b) which does not include the TM domain. This observation suggests that lipid-peptide interactions play a larger role in stabilizing the secondary structure of this amphipathic segment compared with the TM domain. These data provide evidence of the contribution of lipid to structural stability modulation and are in good agreement with the hypothesized lipid and/or protein contribution to structural stability9. The higher RMSD value observed in the I-Tasser model simulation was well anticipated and was mainly attributed to the relative positioning of the ST region, which was sandwiched in between lipid leaflets, forcing the segment to reorganize its structure conformation and having only a minimal effect on the helical integrity of the secondary structure. Examining this reorganization further by monitoring the distance of the amphiphilic segment to lipid leaflets led to another interesting observation described in the next section of this article.\n\nMonitoring the movement of the amphiphilic segment during the simulations led to another interesting observation. Segment of residues 689–703 in the I-Tasser model appeared to move towards the hydrophobic core of the lipid bilayer as depicted by steady progression in the distance to both the upper and lower lipid leaflets depicted in Figure 4. This reorganization is surprisingly interesting because we would have previously assumed that the amphiphilic region exposed to the solvent would hold the structure steady, despite some part of the amphiphilic region being initially vertically positioned in the hydrophobic core of the lipid (Supplementary Figure 1). In addition, given the amphiphilic nature of the residues in this segment, one could postulate that the residues would remain at this position. However, over the period of the simulation the segment reoriented by moving away from the lipid leaflets, while at the same time retaining structural integrity. The structural stability of the segment, as discussed in the previous section of this article, is attributed to the amphiphilic nature of the residues but this does not explain the movement towards the hydrophobic core of the lipid. The systematically orchestrated movement towards the hydrophobic core of the lipid leaflets indicates a strong orientation preference of the amphipathic segment, which in this specific case was parallel to the lipid leaflets. We then monitored the segment movement relative to the lipid leaflets with the other two simulations (the ideal helix model and the 2KZQ structure). The segment was initially positioned horizontally to the lipid leaflets. The results showed that the distance of the amphiphilic segment in both simulations was consistently within 4 Å to the lipid phosphate head group throughout the simulation (Figure 5). These data further clarify the orientation preference of the amphipathic segment with respect to the lipid leaflets and suggest that the residues are positioned in the membrane interface in a very stable manner. Interestingly, Albecka et al.9 speculated that these residues could have an in-plane topology or orientation and suggested that the ST region would ideally be positioned in the membrane interface, which is again in agreement with our data9. We have described the behavior and provided a detailed insight into the dynamics of this amphipathic segment in a lipid bilayer environment.\n\nThe distance to the upper lipid leaflet is plotted in red and lower leaflet is plotted in black.\n\nThe 2KZQ structure is colored in black while the ideal helix model is colored in red. Amphiphilic residues remain within 4 Å from phosphate head group throughout the simulations.\n\n\nConclusion\n\nIn this study, the atomistic MD simulations provide insightful structural data for the E2 segment. The amphiphilic segment of E2 was able to remain as a stable helix in a lipid bilayer environment even without the respective TM domain. The results also revealed the orientation and positional preferences of the amphiphilic segment in relation to the water-bilayer interface that further clarify speculations from experimental studies.", "appendix": "Author contributions\n\n\n\nSAJ analyzed the sequences. RA performed the molecular dynamics simulations. SAJ and RA carried out the research, were involved in the drafting of manuscript and have approved the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Universiti Teknologi MARA (UiTM) Dana Cluster 600-RMI/DANA 5/3/CG (2/2012).\n\n\nAcknowledgments\n\nWe are grateful to Faculty of Pharmacy, Universiti Teknologi MARA (UiTM) for providing the computational facilities in the Bioinformatics Lab. We acknowledge financial and administrative support from the Research Management Institute (RMI), UiTM and Ministry of Science and Technology Malaysia (MOSTI).\n\n\nSupplementary figure\n\n(a) Initial structure; (b) 100 ns snapshot. (Red – residues 689–703).\n\n\nReferences\n\nDubuisson J, Duvet S, Meunier JC, et al.: Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein. J Biol Chem. 2000; 275(39): 30605–30609. PubMed Abstract \| Publisher Full Text\n\nCocquerel L, Wychowski C, Minner F, et al.: Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins. J Virol. 2000; 74(8): 3623–3633. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOp De Beeck A, Montserret R, Duvet S, et al.: The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization. J Biol Chem. 2000; 275(40): 31428–31437. PubMed Abstract \| Publisher Full Text\n\nJusoh SA, Welsch C, Siu SW, et al.: Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C virus. J Mol Model. 2010; 16(10): 1625–1637. PubMed Abstract \| Publisher Full Text\n\nJusoh SA, Helms V: Helical integrity and microsolvation of transmembrane domains from Flaviviridae envelope glycoproteins. Biochim Biophys Acta. 2011; 1808(4): 1040–1049. PubMed Abstract \| Publisher Full Text\n\nOp De Beeck A, Voisset C, Bartosch B, et al.: Characterization of functional hepatitis C virus envelope glycoproteins. J Virol. 2004; 78(6): 2994–3002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrey T, D’Alayer J, Kikuti CM, et al.: The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule. PLoS Pathog. 2010; 6(2): e1000762. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPenin F, Dubuisson J, Rey FA, et al.: Structural biology of hepatitis C virus. Hepatology. 2004; 39(1): 5–19. PubMed Abstract \| Publisher Full Text\n\nAlbecka A, Montserret R, Krey T, et al.: Identification of new functional regions in hepatitis C virus envelope elycoprotein E2. J. Virol. 2011; 85(4): 1777–1792. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMagrane M, Consortium U: UniProt Knowledgebase: a hub of integrated protein data. Database (Oxford). 2011; 2011: bar009. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Y: I-TASSER server for protein 3D structure prediction. BMC Bioinformatics. 2008; 9: 40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerman HM, Westbrook J, Feng Z, et al.: The Protein Data Bank. Nucl Acids Res. 2000; 28(1): 235–242. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmidt TH, Kandt C: LAMBADA and lnflateGRO2: efficient membrane alignment and insertion of membrane proteins for molecular dynamics simulations. J Chem Inf Model. 2012; 52(10): 2657–2669. PubMed Abstract \| Publisher Full Text\n\nBerger O, Edholm O, Jähnig F: Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature. Biophys J. 1997; 72(5): 2002–2013. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOostenbrink C, Soares TA, van der Vegt NF, et al.: Validation of the 53A6 GROMOS force field. Eur Biophys J. 2005; 34(4): 273–284. PubMed Abstract \| Publisher Full Text\n\nHess B, Kutzner C, Van der Spoel D, et al.: GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J Chem Theory Comput. 2008; 4(3): 435–447. Publisher Full Text\n\nHess B, Bekker H, Berendsen H, et al.: LINCS: A linear constraint solver for molecular simulations. J Comput Chem. 1997; 18(12): 1463–1472. Publisher Full Text\n\nBerendsen HJC, Postma JPM, Van Gunsteren WF, et al.: Molecular dynamics with coupling to an external bath. J Chem Phys. 1984; 81(8): 3684–3690. Publisher Full Text\n\nAllen WJ, Lemkul JA, Bevan DR: GridMAT-MD: A grid-based membrane analysis tool for use with molecular dynamics. J Comput Chem. 2009; 30(12): 1952–1958. PubMed Abstract \| Publisher Full Text" }	[ { "id": "866", "date": "26 Mar 2013", "name": "Martin Zacharias", "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 paper describes comparative MD simulations on the E2 TM region embedded in a lipid membrane. Different start conformations have been considered based on different molecular modelling methods + a structure from the pdb. I believe the simulations have been performed properly. Some interesting results on orientational preference of the helices have been reported. However, it should be emphasized that the simulations are too short to achieve convergence of the simulation results. This should be discussed in the Results & Discussion section or in the Conclusion section. Also, comparison to other simulation studies on peptide helices in membranes are missing (e.g. of the Thielemann group). At the end of the conclusion section the authors state “The results also revealed the orientation and positional preferences of the amphiphilic segment in relation to the water-bilayer interface that further clarify speculations from experimental studies.” The authors mention “.. speculations from experimental studies.”, however, no reference to such speculations have been given and the details of the speculations are not discussed. The authors should give proper reference and should in detail explain what is meant by their statement.", "responses": [] }, { "id": "900", "date": "18 Apr 2013", "name": "Gloria Fuentes", "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 carried out MD simulations in lipid bilayer for three different models for the C-terminal region of the envelope glycoprotein E2. The use of different structures provides a priori a good benchmarking for the observations claimed in the article. However, the trajectories presented in this work seem not to have converged completely. The plots will show the trend more clearly if running average is used rather than the raw data. With the current advances in hardware and software, it seems to me that the authors have explored a limited conformational space of the system, which might be masking some other biological conformations for the region in the study.I am missing a more elaborated discussion in context with the available experimental data that they refer to in the conclusion. So far the correlation done in this study is too vague. The paper with experimental data the authors mentioned also suggests that this amphipathic helix could fold upon lipid binding. A MD simulation in water could help to elucidate this experimental observation. It would be convenient if the authors could specify the exact definition of membrane interface; for those people not working in the field, it could be a bit confusing. This concept could suggest the interface of the phospholipid bilayer (membrane core) or the lipid-water interface.The legend for the Supplementary Figure is missing the colour code used.", "responses": [] }, { "id": "888", "date": "18 Apr 2013", "name": "Victor Munoz", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments and Recommendation: The title is appropriate, the design of the simulations, and the methods and analysis employed are state of the art. The data provided is sufficient. However, the conclusions are overstretched and much longer simulations are needed to substantiate the claims. More detailed review:The article describes simulation studies of a 64-residue transmembrane fragment of the envelope glycoprotein E2 domain consisting of two segments: TM (residues 715-746) and ST (residues 683-714). The objective of the study is mainly to understand the role and structural dynamics of the ST segment. MD simulations have been performed on three different starting points: 2 models and one experimentally derived structure. The experimental structure 2KZQ is only 36 residues long itself, lacking the TM segment, and has large dynamics within the NMR structural ensemble (36 models with mean RMSD of 11.32). It is the homolog that produced the best model in I-Tasser, the authors have used in the study. The authors do not mention which model from the NMR ensemble they used in their MD simulations. Though the authors refer to previous experimental studies that show that the ST segment is helical upon membrane binding, a simple sequence-based helical propensity prediction of the HCV E2 genotype 1a (H77 strain) sequence (Uniprot ID P27958) could be included (to further substantiate their 'ideal helix' model used for the MD simulations). One of the main results of the article is that the ST segment has a stable helical conformation, stabilized more by the lipid-peptide interactions than the TM-ST peptide-peptide interactions. The similarity between the three different simulations, even with one of them lacking the TM segment, forms the basis of their conclusion. The authors observe uncharacteristic spikes towards the end in the simulations of NMR-structure and the I-Tasser model MD simulations (Figures 2, 3a, 3b). Since the NMR-structure simulation is very short (just 20ns) compared to the I-Tasser model simulation, the authors could perform a longer, equivalent 100ns simulation to compare them and ascertain further the importance of lipid-peptide interactions in the ST helical segment stability. Also, given that in the I-Tasser simulation, there is a lot of dynamics observed after the 20ns timepoint until the end of the simulation, a longer simulation of the NMR-structure is recommended.\n\nThe other key result which we found interesting is the orientational and positional preference of the amphipathic ST segment, which seems to be parallel to the lipid leaflets. Reorganization and movement of the ST segment towards the hydrophobic core of the lipids, as shown in Fig S1, is observed clearly in the I-Tasser model simulation, whereas the initial horizontal orientation is maintained in the other simulations. The authors could identify the key residues involved in this re-organization and highlight them", "responses": [] } ]	1	https://f1000research.com/articles/2-64
https://f1000research.com/articles/2-146/v1	02 Jul 13	{ "type": "Correspondence", "title": "Pollinator declines: reconciling scales and implications for ecosystem services", "authors": [ "Ignasi Bartomeus", "Rachael Winfree", "Rachael Winfree" ], "abstract": "Despite the widespread concern about the fate of pollinators and the ecosystem services they deliver, we still have surprisingly scarce scientific data on the magnitude of pollinator declines and its actual contribution to crop pollination and food security. We use recently published data from northeastern North America to show that studies at both the local and regional scales are needed to understand pollinator declines, and that species-specific responses to global change are broadly consistent across scales. Second, we show that bee species that are currently delivering most of the ecosystem services (i.e. crop pollination) are not among the species showing declining trends, but rather appear to thrive in human-dominated landscapes.", "keywords": [ "bee", "pollinator", "decline", "extinction", "ecostystem provider", "crops", "decline" ], "content": "Main text\n\nThere is widespread concern regarding the fate of pollinators and the ecosystem services they deliver1. However, the information we have is still limited and at times appears contradictory. Four recent articles, three from Science and one from PNAS, highlight this point2–5. Burkle et al.2 show that 50% of the bee species in one locality in the Midwestern USA became locally extinct during the last century, which in combination with recent evidence that wild pollinators are critical to global crop pollination3, has led some to conclude that we might face an imminent collapse of crop pollination4. In contrast, Bartomeus et al.5 explored bee declines over a similar time scale but at a regional scale (the northeastern USA) and reported only a 15%, non-significant decline in bee species richness. Here we present new analyses that help to reconcile this apparent contradiction in the magnitude of bee declines, while also suggesting that any effects on crop pollination might be less than previously thought.\n\nFirst, we used the 67 bee species included in both the regional-scale5 and the local-scale2 analyses (see data file below) to show that the two studies in fact found broadly consistent results: the locally extinct species of Burkle2 tend to be declining regionally, whereas the locally persistent species tend to be increasing regionally (Figure 1A, ANOVA: F = 5.89, df = 1,65, P = 0.01). Second, we used data from Garibaldi et al.3 on the bee species that provide ecosystem services to four crops in the region covered by Bartomeus et al.5 to show that these ecosystem service providers tend to have increasing population trends compared to non-ecosystem service providers (Figure 1B, F = 7.12, df = 2,184, P = 0.001). All analyses were conducted in R6.\n\nA) For species that either became locally extinct or persisted in Carleville, Illinois. B) For species that either are not ecosystem-service providers to crops (non-ESP), are at least occasionally ecosystem-service providers to crops (ESP), or are among the species cumulatively responsible for 90% of the pollinator visitation to at least one crop (main ESP). Regional data from Bartomeus et al.5, local data from Burkle et al.2 and crop pollinator data from Garibaldi et al.3.\n\n\n\nThus, our analyses demonstrate that, as one would expect, local-scale extinctions do not imply regional-scale extinctions; and that bee species that are important crop pollinators are less likely to be declining at the regional scale. It is important to remember that all bee species may well be crucial to providing ecosystem functions in natural systems and therefore merit conservation attention.", "appendix": "Author contributions\n\n\n\nIB analyzed the data and IB and RW wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests disclosed.\n\n\nGrant information\n\nThis work was supported by United States Department of Agriculture NIFA-AFRI 2009-65104-05782 to R Winfree and N M Williams, Multi-state project 08204 to R Winfree and NSF BIO DEB collaborative grant #0554790/0516205 to C Kremen, N M Williams, and R. Winfree.\n\n\nReferences\n\nPotts SG, Biesmeijer JC, Kremen C, et al.: Global pollinator declines: Trends, impacts and drivers. Trends Ecol Evol. 2010; 25(6): 345–353. PubMed Abstract \| Publisher Full Text\n\nBurkle LA, Marlin JC, Knight TM: Plant-pollinator interactions over 120 years: loss of species, co-occurrence, and function. Science. 2013; 339(6127): 1611–1615. PubMed Abstract \| Publisher Full Text\n\nGaribaldi LA, Steffan-Dewenter I, Winfree R, et al.: Wild pollinators enhance fruit set of crops regardless of honey bee abundance. Science. 2013; 339(6127): 1608–1611. PubMed Abstract \| Publisher Full Text\n\nTylianakis JM: Ecology. The global plight of pollinators. Science. 2013; 339(6127): 1532–1533. PubMed Abstract \| Publisher Full Text\n\nBartomeus I, Ascher JS, Gibbs J, et al.: Historical changes in northeastern US bee pollinators related to shared ecological traits. Proc Natl Acad Sci U S A. 2013; 110(12): 4656–4660. 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, Vienna, Austria. ISBN 3-900051-07-0, URL 2008. Reference Source" }	[ { "id": "1039", "date": "03 Jul 2013", "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 short article comes to the important conclusion that while declines in pollinator [bee] abundance at local and regional scales are generally consistent, these declines are not occurring among those species responsible for delivering the majority of pollination services to particular crops. This insight makes a valuable contribution to the recent debate on the implications of pollinator declines for food production and will hopefully spur more studies that look closely at the relative contribution of different species to delivering pollination services across different crop types, and how the abundance of these species has changed over time.", "responses": [] }, { "id": "1040", "date": "03 Jul 2013", "name": "Ryan Chisholm", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 straightforward analysis and the interpretations are justified. I have two minor comments:Figure 1B shows that ecosystem-service-providing (ESP) bee species tend to be increasing whereas non-ESP species tend to be decreasing. But how is ESP measured? Is there any possibility that this result could be artefact, in that declining species are now rare and that therefore their ESP behaviour is less likely to be recorded? I would like to see this issue addressed briefly.The final sentence “It is important to remember that all bee species may well be crucial to providing ecosystem functions in natural systems and therefore merit conservation attention.” seems like a non-sequitur, because the authors have just finished talking about how important pollinators are less likely to be declining. Presumably the authors mean that we have incomplete information and that it would be risky to assume that apparent non-pollinators are genuinely playing no useful ecosystem-service role. This could be expressed better.", "responses": [ { "c_id": "499", "date": "10 Jul 2013", "name": "Ignasi Bartomeus", "role": "Author Response", "response": "Regarding your first comment, ESP is measured as any bee species visiting flowers of at least one of four main crops the study area, based on data we collected in the recent period (2004-2011) and published in Garibaldi et al., 2013. Thus the reviewer is correct that there could have been species that were ESP in the past but, due to population decline, were not recorded as ESP in our recent surveys, and our results should be interpreted in this light. However, we believe that our finding, that ESP are declining less than other species, has generality for two reasons. 1) We report in Bartomeus et al. 2013 that most of the bee species that have been declining over the past 140 years in our study region are still frequently recorded, hence our measure of ESP as \"any species observed visiting crops at least once\" should capture declining species as well if they commonly visit crops 2) Even the current ESP are a subset of the historical ESP, Garibaldi et al., 2013 shows that the under this current situation, wild pollinators enhance fruit set, suggesting that the resilience of the current ESP to global change may buffer the effects of a pollinator decline. Lastly, on a priori grounds it would not be surprising if ESP were more robust to land use change (a primary form of global change in our study region) than non-ESP because by definition species found pollinating crops are able to persist in agricultural areas.Regarding your second question, it is important to note that the last sentence refers to \"natural systems.\" While our data and analyses refer to simplified crop systems, where a few dominant pollinators are responsible for most of the function delivered to a single plant species (the crop), natural systems are far more complex and high levels of bee diversity are likely to be necessary to provide function to the full community. Hence our results should not be extrapolated to other, non-agricultural systems. Ignasi Bartomeus & Rachael Winfree" } ] } ]	1	https://f1000research.com/articles/2-146
https://f1000research.com/articles/2-145/v1	27 Jun 13	{ "type": "Research Article", "title": "Thiazolidinedione use and risk of hospitalization for pneumonia in type 2 diabetes: population based matched case-control study ", "authors": [ "Sonal Singh", "Hsien Yen Chang", "Thomas Richards", "Jonathan P Weiner", "Jeanne M Clark", "Jodi B Segal", "Hsien Yen Chang", "Thomas Richards", "Jonathan P Weiner", "Jeanne M Clark", "Jodi B Segal" ], "abstract": "Objective: Previous randomized clinical trials and their meta-analyses have raised the possibility that thiazolidinediones (rosiglitazone and pioglitazone) may increase the risk of pneumonia. We aimed to test the hypothesis that thiazolidinediones may increase the risk of pneumonia.Design: Population based case-control study using a new user design.Setting: A large administrative database in the United States from 2002 to 2008.Population: Adults with type 2 diabetes aged 18-64; restricted to 6129 hospitalized pneumonia cases and 6129 controls without congestive heart failure matched on age, sex, enrollment pattern and diabetes complication severity index matched controls. Conditional logistic regression was used to analyse the data.Results: Compared with controls, cases were more likely to have chronic obstructive pulmonary disease (COPD), tobacco use, cancer and have received influenza and pneumococcal vaccination. After adjusting for COPD, cancer, tobacco use, and receipt of influenza and pneumococcal vaccination, and exposure in other periods, neither recent exposure to pioglitazone (adjusted Odds Ratio [aOR], 1.15, 95% Confidence intervals 1.00 – 1.32) or rosiglitazone (aOR 1.09, 95% CI, 0.83 – 1.44) nor current exposure to pioglitazone within 60 days (aOR, 1.04, 95% CI, 0.60 – 1.79) was associated with a statistically significant odds of pneumonia. Current exposure to rosiglitazone was associated with a statistically significant reduction in the odds of pneumonia (aOR, 0.33, 95% CI 0.11-0.95).Conclusion: In this study of US adults with type 2 diabetes we did not detect a significant increased risk of pneumonia with the thiazolidinediones. The unusually large protective effect of current exposure to rosiglitazone reflects the healthy user effect or unmeasured confounding.", "keywords": [ "Thiazolidinediones", "pneumonia", "type 2 diabetes", "case-control study" ], "content": "Introduction\n\nThe thiazolidinediones, rosiglitazone and pioglitazone, are peroxisome proliferator-activated receptor γ (PPARγ) agonists. These drugs effectively lower glycated hemoglobin levels among patients with type 2 diabetes1. Both these agents have relatively similar efficacy, but may have different adverse effect profiles1–8. Since regulatory approval, the thiazolidinediones have been associated with an increase in the risk of fractures in women, heart failure and, in the case of rosiglitazone, myocardial infarction1–6. Certain immunological adverse effects such as increased risk of bladder cancer7, or acute cholecystitis9, are specific to pioglitazone.\n\nRosiglitazone is a PPARγ agonist, whereas pioglitazone has both α and γ effects. PPARγ activation may result in glucorticoid like effects in the airways10–12. These effects could induce susceptibility to pneumonia similar to the effect of glucorticoids in chronic obstructive pulmonary disease13. In human macrophages the thiazolidinediones have off-target effects on PPARδ at clinically relevant doses14–17. This augmentation of PPARδ signaling by the thiazolidinediones may potentially exhibit proinflammatory activities such as pneumonia14,15.\n\nPatients with type 2 diabetes are known to be at a higher risk of pneumonia18,19. A large, long term clinical trial7, and a meta-analysis of long term clinical trials raised the possibility that long term use (> one year) of the thiazolidinediones may increase the risk of pneumonia6. We aimed to test the hypothesis that the use of rosiglitazone or pioglitazone was associated with an increased risk of pneumonia in patients with type 2 diabetes.\n\n\nMethods\n\nWe used administrative claims data from seven US Blue Cross and Blue Shield (BCBS) plans. These were the BCBS of Tennessee, Hawaii, Michigan, North Carolina; Highmark, Inc. of Pennsylvania, Independence Blue Cross of Pennsylvania and Wellmark, Inc. of Iowa and of South Dakota.\n\nWe assembled an incipient cohort of all patients with type 2 diabetes who filled at least one prescription for any hypoglycemic agent between 2002 and 2008. To be eligible for inclusion, individuals had to be: a) between 18 and 64 years on their first date of diagnosis of diabetes, b) contribute at least 6 months of medical or pharmacy coverage in the calendar year of diabetes diagnosis; c) of known sex; d) without any claim for congestive heart failure. We restricted the analytical sample to cases of pneumonia without a history of diagnosis of congestive heart failure to reduce outcome misclassification because congestive heart failure is a known adverse effect of thiazolidinedione use and shares many clinical and radiographic similarities to pneumonia4. We determined eligibility for the study from computerized encounter data including enrollment files for administrative data; benefits information to determine medical and pharmacy coverage; and inpatient, outpatient, and pharmacy claims records containing Common Procedural Terminology (CPT) codes, International Classification of Disease-9 Clinical Modification (ICD-9-CM) codes, and National Drug Codes (NDC) or Diagnosis Related Group (DRG) codes and costs and charges (submitted, allowed, and paid).\n\nBriefly, we identified presumptive cases by using an algorithm of ICD-9 codes for inpatient pneumonia (Supplementary Table 1). These were defined as individuals with an inpatient code for pneumonia. For people with multiple episodes of pneumonia, we included only the first episode.\n\nWe selected one control for each case from the eligible source population matched on age (10 year intervals), sex, insurance plan site, and Diabetes Complication Severity Index (0, 1, and 2)20,21, and enrollment pattern or duration of pneumonia-free follow-up (incidence density sampling). Pneumonia cases were not eligible to be resampled as controls.\n\nPrescription data were used as an indicator of drug exposure using NDC codes. We obtained information about thiazolidinedione use from a computerized pharmacy database containing the date the prescription was filled and number of days supplied. We used these data elements to determine the dates a patient was exposed to the drug prior to the first observed diagnosis of pneumonia.\n\nDrug exposure was defined as having filled a prescription on any day for rosiglitazone or pioglitazone prior to the first observed diagnosis of pneumonia. A first exposure after the index diagnosis of pneumonia with thiazolidinediones was counted as unexposed. We defined four categories of exposure. Any users were those with exposure to the thiazolidinediones after the diagnosis of diabetes before the index date of pneumonia. Current users were those who were exposed to the thiazolidinediones within 60 days prior to the index date of onset of pneumonia. Recent users were those with a claim for the thiazolidinediones from 61 days to 2 years prior to the index date of pneumonia. Non-users are defined as those for whom there was no thiazolidinedione prescription or a prescription more than 2 years prior to the index date of pneumonia. Current users, recent users and non-users were mutually exclusive categories of exposure. However, any users included current and recent users.\n\nWe calculated proportions for categorical variables and means or medians for continous variables according to the case status. We used conditional logistic regression to estimate the McNemars Odds Ratio for exposure to rosiglitazone or pioglitazone to account for the matching design of the study. We evaluated rosiglitazone and pioglitazone separately. However, we did not distinguish between single agent vs. combinations of the thiazolidinediones with other oral hypoglycemic agents. We conducted analyses for recent and current rosiglitazone and pioglitazone users and for any users in a series of statistical models. The most parsimonious model only took into account the matched design of the study (age, sex, enrollment pattern, and Diabetes Complication Severity Index). Subsequent statistical models adjusted for potential confounders specified a priori using a review of the literature including chronic obstructive pulmonary disease, tobacco use, receipt of influenza or pneumococcal vaccination, and an indicator of general morbidity level (the resource utilization band from the Johns Hopkins Adjusted Clinical Group case-mix system; Supplementary Table 2)23. The fully adjusted models adjusted for matching variable, confounders and recent and current exposure of both rosiglitazone and pioglitazone. We used SAS version 9.2 for analysis. Statistical significance was set at two sided P=0.05.\n\n\nResults\n\nWe found 1,100,899 patients with type 2 diabetes who were potentially eligible during the study period. Within this group 5.8% of participants (n=64,157) experienced a first episode of inpatient pneumonia during the study period. After excluding participants above 64 or below the age of 18 years (n=31,347), participants with incomplete coverage (n=24,402), missing information on sex (1,761) a history of congestive heart failure (34,589) or a date of diabetes diagnosis later than or equal to date of pneumonia (14,150), 8883 cases of pneumonia remained eligible for inclusion. We removed two pairs of cases and controls for using both rosiglitazone and pioglitazone in the same study period. We restricted the analysis to 6129 cases of pneumonia without a history of congestive heart failure. 6129 controls matched for age, sex, and enrollment pattern and diabetes complication severity index were randomly selected from a pool of 428,826 potentially eligible controls. The flow of participants through the study is shown in Figure 1.\n\nBCBS – Blue Cross and Blue Shield; CHF – coronary heart failure; DCSI – Diabetes Complication Severity Index.\n\nThe characteristics of the cases and controls are shown in Table 1. The mean age of participants in the study was 52 years. Just over 50% of participants were male. Cases with pneumonia were more likely than controls to have chronic obstructive pulmonary disease, cancer, or be tobacco users, or to have received influenza and pneumococcal vaccination. More cases than controls were exposed to the thiazolidinediones during the study period (any exposure). The exposure of cases and controls to rosiglitazone and pioglitazone in various exposure windows is shown in Table 2.\n\nACG = adjusted clinical group; SD = Standard deviation.\n\nValues are percentages unless otherwise noted.\n\nOur results for Model 1, Model 2, Model 3 and Model 4 are shown in Table 3. After adjusting for COPD cancer, tobacco use, and receipt of influenza and pneumococcal vaccination, and exposure in other periods in the fully adjusted model, neither recent exposure to pioglitazone (adjusted Odds Ratio (aOR), 1.15, 1.27, 95% confidence intervals 1.00–1.32), rosiglitazone (aOR 1.09, 95% CI, 0.83 1.44) nor current exposure to pioglitazone within 60 days (aOR, 1.04, 95% CI, 0.60–1.79) was associated with statistically significant odds of pneumonia. Current exposure to rosiglitazone was associated with statistically significant reduction in the odds of pneumonia (aOR, 0.33, 95% CI 0.11–0.95).\n\n† Adjusted for the matching design of the study [age, sex, enrollment pattern and diabetes complication severity index].\n\n* Model 1 plus chronic obstructive pulmonary disease, tobacco use, cancer, pneumococcal and influenza vaccination and a general index of comorbidity Johns Hopkins adjusted clinical group case-mix index.\n\n‡ Model 2 plus recent exposure and current exposure of rosiglitazone or pioglitazone.\n\n# Model 3 plus recent exposure and current exposure of both rosiglitazone and pioglitazone.\n\nBold signifies statistical significance.\n\n\nDiscussion\n\nWe did not find any evidence to support our initial hypothesis that either rosiglitazone or pioglitazone was associated with a statistically significant increased risk of hospitalization for pneumonia in a restricted sample of adult patients with type 2 diabetes without a history of congestive heart failure. We also noted some unexpected findings such as a statistically significant reduction in the risk of pneumonia with rosiglitazone of ≈ 70%. There is no biological evidence to support such a beneficial effect of rosiglitazone on infections such as pneumonia. Such post-hoc findings from observational studies should be noted with caution. It is also possible that some unmeasured confounders or potentially a healthy user effect could explain these results. We did not have access to clinical records to fully evaluate the healthy user effect.\n\nOur findings need to be interpreted in the context of other studies. The largest 4 year clinical trial of pioglitazone, the PROspective pioglitAzone Clinical Trial In macroVascular Events (PROactive) study NCT00174993) reported a statistically significant increased risk of pneumonia reported as serious adverse events ( Relative Risk (RR) 1.53, 95% CI 1.00–2.34: P=0.047) compared to placebo7. However a similar, large, long term clinical trial of rosiglitazone, the Rosiglitazone Evaluated for Cardiovascular outcomes in ORal agent combination therapy for type 2 Diabetes (RECORD) study did not detect a significantly increased risk of pneumonia with rosiglitazone as compared to metformin or sulfonylureas (RR 1.18, 95% CI 0.75–1.84; P=0.56)8. A previous meta-analysis of thirteen long-term (>1 year) randomized trials of the thiazolidinediones, which included the two long-term studies just mentioned, reported a ≈ 40% increased risk of pneumonia or lower respiratory tract infection adverse events or serious adverse events with statistically significant RRs for pioglitazone (RR 1.63; 95% CI 1.09–2.46) but not for rosiglitazone (RR 1.26; 95% CI 0.90–1.76)6.\n\nThere are several possible reasons for the divergent findings between this observational study and the previous meta-analysis. It is possible that participants in the trials were different from this observational study. Some trials did not report on pneumonia events, and missing data are possible in both the trial and the observational study. Although chest X-rays were adjudicated in the largest trial for pioglitazone7, it is possible that the clinical trials reported more events of pneumonia associated with the thiazolidinediones because patients on the thiazolidinediones in the trials probably received more radiographic studies to evaluate congestive heart failure associated with the thiazolidinedione (i.e. detection bias)22. In this study, we restricted our analysis to patients without congestive heart failure, which could have accounted for misclassification of outcomes.\n\n\nStrengths of the study\n\nThe present study minimized the impact of the potential misclassification of congestive heart failure, a known adverse effect of thiazolidinedione use25, by restricting the analysis to hospitalized patients with pneumonia and without congestive heart failure. We also used a new user design, which is important in studying the effects of drugs to remove prevalent user biases. In addition we were able to control for several available confounders.\n\n\nLimitations of the study\n\nOur study has some limitations. Misclassification of exposure is possible because information on exposure was derived from pharmacy claims, and we cannot guarantee that the drugs were ingested. Differential misclassification of exposure ascertainment among cases and controls is unlikely but cannot be definitively ruled out. We did not impute for missing data. Although we restricted our analysis to participants without congestive heart failure to minimize potential differential misclassification of heart failure, we did not have access to the clinical records to confirm the diagnosis of pneumonia so potential outcome misclassification of congestive heart failure as pneumonia is still possible. Other database studies suggest that the positive predictive value of using a claims database to detect individuals with pneumonia is around 93%24. Our findings are not generalizable to patients above the age of 65 who were excluded from the analysis. We were not able to evaluate dose-responsiveness of the association or the specific subtypes of pneumonia (viral or bacterial) and the subsequent outcomes from the pneumonia. Residual confounding is always possible in a database study as administrative databases are limited in their ability to identify occupational exposures and other risk-factors for pneumonia such as smoking. We did not adjust for inhaled corticosteroid use, which has been shown to increase the risk of pneumonia in patients with COPD to avoid over adjustment of proximal confounders13.\n\nWhat is already known on this topic and what this study adds\n\nRosiglitazone and pioglitazone are used to lower glycated haemoglobin in patients with type 2 diabetes. Previous clinical trials and meta-analysis have raised the possibility that thiazolidinedione use may increase the risk of infections such as pneumonia. In this study of US adults with type 2 diabetes thiazolidinedione use was not associated with a statistically significant increased risk of pneumonia.\n\n\nUnanswered questions and future research\n\nFurther studies with validation of pneumonia diagnosis with clinical and radiographic information are needed. Other methods, such as the use of instrumental variables, to control for confounding by indication may offer additional insight. Further studies are required to elucidate the role of PPAR agonists off-target and glucocorticoid effects in the development of serious infections such as pneumonia.\n\n\nConclusions\n\nDespite these limitations, our findings have implications. In this administrative database study of US adults with type 2 diabetes, we did not detect a significant increased risk of pneumonia with the thiazolidinediones. The unusually large protective effect of current exposure to rosiglitazone may reflect the healthy user effect or unmeasured confounding. Clinicians should carefully balance the benefits of thiazolidinediones on glycemic control against their known risks, after eliciting patient preferences for various outcomes in a shared decision making context.", "appendix": "Author contributions\n\nSingh and Segal conceived and designed the study. Clark, Richardson and Segal acquired the data. Hsien-Yen, Singh and Richards analyzed the data and all authors interpreted the data. Singh drafted the initial manuscript and all authors critically revised the manuscript.\n\n\nCompeting interests\n\nThe authors have no competing interests. The dataset used in this study was created for a research project on the patterns of obesity care within selected BlueCross BlueShield health insurance plans. The original development of the dataset was funded by unrestricted research grants from Ethicon Endo-Surgery, Pfizer, and GlaxoSmithKline. Data and database development support were provided by the BlueCross BlueShield Association (Tennessee, Hawaii, Michigan, and North Carolina), Highmark (Pennsylvania), Independence BlueCross (Pennsylvania), and Wellmark BlueCross BlueShield (Iowa and South Dakota). The BlueCross BlueShield plans were invited to review the manuscript but they did not have any direct role in the design and conduct of the study, data management or analysis, interpretation of the data, or preparation of the manuscript.\n\n\nGrant information\n\nSS was supported by the Johns Hopkins Clinical Research Scholars Program. This publication was made possible by Grant Number 1KL2RR025006-03 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Information on NCRR is available at http://www.ncrr.nih.gov/. Information on Re-engineering the Clinical Research Enterprise can be obtained from http://nihroadmap.nih.gov/clinicalresearch/overview-translational.asp.\n\nThe design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript were independent of any other sources of funding. Dr Singh had full access to all of thedata in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.\n\n\nSupplementary tables\n\n* Denotes a digit that can be anything, even missing i.e. 482** could be 482 or 482.2 or 482.49.\n\nICD-9 = International Classification of Disease=9th revision.\n\n* Denotes a digit that can be anything, even missing.\n\n\nReference\n\nBennett WL, Maruthur NM, Singh S, et al:Comparative effectiveness and safety of medications for type 2 diabetes: an update including new drugs and 2 drug combinations. Ann Intern Med. 2011; 154: 602–613.\n\nNissen SE, Wolski K: Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. N Engl J Med. 2007; 356: 2457–2471.\n\nSingh S, Loke YK, Furberg CD: Long-term risk of cardiovascular events with rosiglitazone: a meta-analysis. JAMA. 2007; 298: 1189–1195.\n\nSingh S, Loke YK, Furberg CD: Thiazolidinediones and heart failure: a teleo-analysis. Diabetes Care. 2007; 30: 2148–2153.\n\nLoke YK, Singh S, Furberg CD: Long-term use of thiazolidinediones and fractures in type 2 diabetes: a meta-analysis. CMAJ. 2009; 180: 32–39.\n\nSingh S, Loke YK, Furberg CD: Long-term use of thiazolidinediones and the associated risk of pneumonia or lower respiratory tract infection: systematic review and meta-analysis. Thorax. 2011; 66: 383–388.\n\nDormandy JA, Charbonnel B, Eckland DJ, et al:PROactive Investigators. Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial in macroVascular Events): a randomized controlled trial. Lancet. 2005; 366: 1279–1289.\n\nHome PD, Pocock SJ, Beck-Nielsen H, et al:RECORD Study Team. Rosiglitazone evaluated for cardiovascular outcomes in oral agent combination therapy for type 2 diabetes (RECORD): a multicentre, randomised, open-label trial. Lancet. 2009; 373: 2125–2135.\n\nBolen S, Feldman L, Vassy J, et al:Systematic review: comparative effectiveness and safety of oral medications for type 2 diabetes mellitus. Ann Intern Med. 2007; 147: 386–399.\n\nMatthews L, Berry A, Tersigni M, et al:Thiazolidinediones are partial agonists for the glucocorticoid receptor. Endocrinology. 2009; 150: 75–86.\n\nSpears M, Donnelly I, Jolly L, et al:Bronchodilatory effect of the PPAR-gamma agonist rosiglitazone in smokers with asthma. Clin Pharmacol Ther. 2009; 86: 49–53.\n\nNarala VR, Ranga R, Smith MR, et al:Pioglitazone is as effective as dexamethasone in a cockroach allergen-induced murine model of asthma. Respir Res. 2007; 8: 90.\n\nSingh S, Amin AV, Loke YK: Long-term Use of Inhaled Corticosteroids and the Risk of Pneumonia in Chronic Obstructive Pulmonary Disease - A Meta-analysis. Arch Intern Med. 2009; 169: 219–229.\n\nHall JM, McDonnell DP: The molecular mechanisms underlying the proinflammatory actions of thiazolidinediones in human macrophages. Mol Endocrinol. 2007; 21: 1756–1768.\n\nDesmet C, Warzee B, Gosset P, et al:Pro-inflammatory properties for thiazolidinediones. Biochem Pharmacol. 2005; 69: 255–265.\n\nWelch JS, Ricote M, Akiyama TE, et al:PPAR gamma and PPAR delta_ negatively regulate specific subsets of lipopolysaccharide and IFN-gamma target genes in macrophages. Proc Natl Acad Sci USA. 2003; 100: 6712–6717.\n\nvon Knethen A, Soller M, Brüne B:Peroxisome proliferator-activated receptor (PPAR.) and sepsis. Arch Immunol Ther Exp. 2007; 55: 19–25.\n\nMuller LM, Gorter KJ, Hak E, et al:Increased risk of common infections in patients 475 with type 1 and type 2 diabetes mellitus. Clin Infect Dis. 2005; 41: 281–288.\n\nShah BR, Hux JE: Quantifying the risk of infectious diseases for people with diabetes. Diabetes Care. 2003; 26: 510–513.\n\nYoung BA, Lin E, Von Korff M, et al:Diabetes complications severity index and risk of mortality, hospitalization, and healthcare utilization. Am J Manag Care. 2008; 14(1): 15–23.\n\nChang HY, Weiner JP, Richards TM, et al:Validating the adapted Diabetes Complications Severity Index in claims data. Am J Manag Care. 2012; 18(11): 721–6.\n\nHealth Services Research & Development Center at The Johns Hopkins University Bloomberg School of Public Health. The Johns Hopkins ACG Case-Mix System Reference Manual Version 7.0. Baltimore, MDThe Johns Hopkins University Bloomberg School of Public Health. 2005.\n\nSingh S, Loke YK: Drug safety assessment in clinical trials: methodological challenges and opportunities. Trials. 2012; 13: 138.\n\nWhittle J, Fine MJ, Joyce DZ, et al:Community-acquired pneumonia: can it be defined with claims data? Am J Med Qual. (1997); 12: 187–93.\n\nLoke YK, Kwok CS, Singh S: Comparative Cardiovascular Effects of Thiazolidinediones: A systematic review and meta-analysis of observational studies. BMJ. 2011; 342: d1309." }	[ { "id": "1375", "date": "09 Aug 2013", "name": "Stephan F. Lanes", "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\nThiazolidinedione use and risk of hospitalization for pneumonia in type 2 diabetes: population based matched case-control study. ReviewBackground Results from a recent meta-analysis of randomized controlled trials suggest both drugs increase the risk of pneumonia (Thorax 2011; 66:383-8): Pioglitazone: RR=1.63; 95% CI 1.09, 2.46. Rosiglitazone: RR=1.26; 95% CI 0.90 1.76. An undue emphasis on statistical significance leads the authors to suggest these results are inconsistent, but both results show a large overlap in the confidence intervals in a range indicating an increased risk that is perhaps greater for pioglitazone than rosiglitazone. Package inserts on both pioglitazone and rosiglitazone carry warnings for heart failure and edema, along with increased risk of upper respiratory tract infection. Further, heart failure increases the risk of hospitalization for pneumonia (Eur J Intern Med 2013 Jun; 24 (4): 349-53) as does diabetes, especially with poor glycemic control (Diabetes Care 2008 August; 31 (8): 1541–1545). ResultsThis study reports small increases in risk of hospitalization for pneumonia with recent exposure to pioglitazone (OR=1.15; 95% CI 1.00, 1.32) and pioglitazone (OR=1.09; 95% CI 0.83, 1.44), but not for current exposure to pioglitazone (OR=1.04; 95% CI 0.60, 1.79). Current exposure to rosiglitazone was associated with a reduced risk of pneumonia (OR=0.33; 95% CI 0.11, 0.95). The relatively higher rate of pneumonia with pioglitazone compared with rosiglitazone is consistent with the results from clinical trials, but the magnitudes of the effect estimates reported here are markedly reduced, and recent exposure is associated with greater risk than current exposure. This pattern of results is puzzling and the authors offer no compelling explanation.\n\nMethodology The study description raises no obvious bias to account for a systematic reduction in risk estimates nor the greater risk with recent versus current exposure. There are, however, several uncertainties in the study methods that may have a bearing on validity:The study is described as a new user design. The new user design was described by Ray (Am J Epidemiol 2003; 158: 915–920): ‘A new-user design begins by identifying all of the patients in a defined population (both in terms of people and time) who start a course of treatment with the study medication. Study follow-up for endpoints begins at precisely the same time as initiation of therapy, or t0. The study is further restricted to patients with a minimum period of nonuse (washout) prior to t0. The study should include all patients in the study population meeting these criteria. Data for all patient characteristics are obtained at a time just before t0’. The new user design is described for cohort studies, but can be adapted easily to a case-control study nested within a cohort of new users. Nevertheless, it is not clear that the current study follows the new user design described above by Ray. First, key to a new user design is that “new use” is defined by a drug dispensing following a period of minimum duration (e.g., 6 months or 1 year) with no dispensing of the drug. In the current study, the inclusion criteria as described do not include any minimal drug-free period. This raises a question of whether the study population truly comprises new users.The current study indicates that the start of follow-up or index date corresponds to a diagnosis of type 2 diabetes (Table 1). This is also consistent with Figure 1, in which the cohort is defined by a diagnosis and without regard to therapy. However, the study methods indicate that therapy was required: “We assembled an incipient cohort of all patients with type 2 diabetes who filled at least one prescription for any hypoglycemic agent between 2002 and 2008” (emphasis added). The methods go on to list as an inclusion criterion that individuals had to “contribute at least 6 months of medical or pharmacy coverage in the calendar year of diabetes diagnosis.” This strategy is problematic in several respects. First, the required therapy for cohort entry is any hypoglycemic agent and does not specify those medications which may be suitable therapeutic alternatives to glitazones. According to the new user design, the index date should be the first prescription of one of the glitazones or a therapeutic alternative (with analyses conditional on use of other medications). Secondly, the six months of required observation time is not specified as occurring before the start of therapy or being drug-free. Instead, the required period is defined without regard to the date of initiation of therapy and only as occurring during the same calendar year as a diagnosis of type 2 diabetes (the apparent index date). This means that the six months of observation could occur before, after, or spanning the diagnosis date and putative start of follow-up. To the extent that the required observation time before the diagnosis was less than six months, even if it was a diagnosis-free and drug-free period, the probability is diminished that the patient was free of the diagnosis prior to inclusion and, therefore, that this is not a cohort of newly diagnosed patients. Further, to the extent that the inclusion criteria require observation time after a diagnosis and start of follow-up, the corresponding follow-up time is “immortal” and represents a source of bias (J Clin Oncol 2009; 27: e55-6). Finally, to detect diagnoses, patients would have to have medical coverage, and to detect prior therapy, patients would have to have pharmacy coverage. But the requirement of medical or pharmacy coverage does not ensure that either diagnosis or therapy would be detectable. If the patients had only medical coverage during this period, for instance, prescriptions of antidiabetic therapy would not be reimbursable and would not appear on the claims database. For this reason, claims database studies such as this one are typically restricted to patients having both medical and pharmacy benefits. To summarize, the inclusion criteria do not define a cohort that is either newly diagnosed or newly treated. There are at least two potential concerns with the study as described. First, if it is not a new user design, then the cohort would include prevalent users of glitazone therapy. Prevalent users represent a “survivor” cohort of patients who have a good experience with therapy, by obtaining effectiveness and also by tolerating the therapy well; patients who did not do well are more likely to discontinue therapy. The inclusion criteria provide no assurance that the study cohort failed to exclude prevalent users. The new user design was developed in part to eliminate the “healthy user” effect, but this type of selection remains a concern with inclusion criteria described for this study. Another important feature of the new user design is that of defining a minimum drug-free period before the start of therapy (which should correspond to the start of follow-up), also defines the minimal baseline period during which risk factors (i.e., covariates) are identified as potential confounding variables. The current study does not describe the time period during which risk factors are identified. If the authors defined risk factors as occurring before the outcome (i.e., pneumonia) instead of before the start of glitazone therapy, then covariates such as the “Diabetes Complication Severity Index” and “enrolment pattern” occurred (at least in part) after initiation of glitazone therapy. It is not advisable to control for factors that occur after the study exposure because they could be a result of exposure and, therefore, in the causal pathway between exposure and outcome. Analytic control of such factors would inappropriately attenuate the true relation between the exposure and the endpoint.The authors included patients “without any claim for congestive heart failure,” noting that heart failure shares similar clinical and radiographic findings as pneumonia. Their goal was to reduce outcome misclassification, presumably concerned that cases of heart failure could be confused with cases of pneumonia (and vice versa). I have two comments on this strategy. First, if the authors are concerned that claims data cannot accurately distinguish heart failure from pneumonia, then how did they exclude patients with heart failure? How do they know that patients with heart failure who were excluded did not have pneumonia? Secondly, including patients “without any claims for congestive heart failure” would exclude all patients with a claim of heart failure without regard to timing of the heart failure diagnosis. Specifically, cases of heart failure that occurred after initiation of glitazone therapy could be the result of glitazone therapy. Moreover, even if there was no misclassification, because heart failure increases the risk of hospitalization due to pneumonia (Eur J Intern Med 2013 Jun; 24 (4): 349-53), excluding cases of heart failure that occurred after the start of therapy would also exclude cases of pneumonia etiologically related to glitazone therapy (because heart failure lies in a causal pathway). The impact would be to attenuate the relation between glitazone therapy and pneumonia. The authors state that they matched controls to cases on duration of follow-up, according to the method of incidence density sampling. Actually, incidence density sampling stipulates that controls be in the cohort (and contributing person-time to the denominator of a theoretical incidence rate) at the time a case occurred, but does not require the control have the same duration of follow-up as the case (Am J Epidemiol 1982; 116; 547-53). Controls should be sampled to represent the exposure frequency in the entirety of person-time at risk, which includes people with any duration of follow-up (both longer than and shorter than the case’s duration of follow-up). To the extent that glitazone therapy may be related to duration of follow-up, matching by duration of follow-up would have provided an exposure distribution that was not representative of the base population, creating a control selection bias. A causal effect of an exposure is always measured relative to some alternative. A drug may cause an effect (RR>1) compared with one drug, and prevent the same effect (RR<1) when compared with another drug (that has an even stronger causal effect). These findings, especially that of a reduced risk for rosiglitazone, should be understood in terms of the reference category. The current study measures current and recent use of each glitazone relative to non-use of glitazones within two years. Non-use of glitazones is consistent with the absence of therapy, as well as many possible combinations of concomitant antidiabetic and other medications (some of which may be related to risk of pneumonia). It might be preferable to specify the comparator as a therapeutic alternative(s) to the glitazones. A related issue is that effects of other medications evidently were not controlled. Glycemic control has been related to risk of pneumonia. Although no direct measure of glycemic control is likely available in the claims database, data on the dispensing of and adherence to antidiabetic agents is available. The authors call for additional studies using other methods to better control for confounding, but it appears that better control of confounding is possible with the data used in this study. The higher risk for recent exposure than current exposure is peculiar and seems to indicate an increased risk of recent discontinuation. Several explanations are possible, including an increased risk due to loss of glycemic control after discontinuation, or discontinuation due to early prodromal symptoms of adverse events that were diagnosed after discontinuation. If drug exposure caused the outcome, one would expect higher risks during exposure than after exposure. Further analyses aimed at identifying reasons for discontinuation would be of interest. In addition, duration of therapy might be important, and analyses of risk by duration of therapy might be illuminating.", "responses": [] }, { "id": "4687", "date": "09 May 2014", "name": "Hans-Peter Hammes", "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 manuscript addresses the question whether PPAR-γ agonists can induce pneumonia as indicated by prior RCTs. The authors used administrative data sets and a new user design recruiting more than one million patients. Of these patients, a subgroup of > 6000 “cases” (i.e. age between 18 and 64, diabetes prior to pneumonia, one episode of pneumonia, no CHF) was matched with an identical number of diabetic patients without pneumonia “controls”.Statistical analysis revealed a higher number of COPD patients in the pneumonia group (which is expected). After adjusting for important confounders of pneumonia (such as COPD, cancer, tobacco use and vaccination) the use of PPAR-γ agonists was not associated with higher incidence of pneumonia. In fact, a decreased odds ratio was found when current exposure of rosiglitazone was plotted against pneumonia. Overall, the authors conclude that there is no increased risk of pneumonia, when using PPAR-γ agonists.In general, the paper is of high clinical relevance, only presently limited by the fact the TZDs are almost off market because the spectrum of side effects. There are a few points of possible amendments:Introduction: the difference between the two TZDs is likely not relevant with regard to pneumonia development. This should be mentioned. Introduction: reference 19 involves Type 1 diabetic patients – the pathogenesis of increased susceptibility is different, therefore a ref. restricted to type 2 diabetes is preferable. M&M and result section: one of the most important determinants of infections in diabetic patients is metabolic control. Are there data on blood glucose, or HbA1c of the two groups to identify a possible additional confounder? Moreover, data on the current use of steroids would be helpful as the proportion of COPD is different between the two groups. M&M and result section: important anthropometric data from both patient groups are lacking, such as age, known diabetes duration, and comorbidities. Are there any available data that could be incorporated? Discussion section: specify the confounders that were explicitly addressed. Future research: the current status of TZD approval with the FDA and EMA should be incorporated here.", "responses": [] } ]	1	https://f1000research.com/articles/2-145
https://f1000research.com/articles/1-67/v1	17 Dec 12	{ "type": "Research Article", "title": "Respiratory functions of conservancy workers working in solid waste management sector of Chennai, India", "authors": [ "Srinivasan Roopa", "Ramaswamy Padmavathi", "Avinash Akolkar", "Sambandam Sankar", "Pitani Ravishankar", "Thanasekharaan Vijayalakshmi", "AS Subhashini", "Balakrishnan Kalpana", "Ramaswamy Padmavathi", "Avinash Akolkar", "Sambandam Sankar", "Pitani Ravishankar", "Thanasekharaan Vijayalakshmi", "AS Subhashini", "Balakrishnan Kalpana" ], "abstract": "Chennai is the fourth largest metropolitan city in India. Around 10,000 conservancy workers of the Chennai Corporation handle about 4500 to 5200 tons of solid wastes per day. These workers are exposed to a lot of environmental and occupational hazards affecting the respiratory system. This paper presents the results of pulmonary function assessment in 178 conservancy workers (100 sweepers & 78 loaders) of the Chennai Corporation. Detailed medical and occupational history was obtained and clinical examination was performed after obtaining informed consent. Pulmonary functions (forced vital capacity [FVC], forced expiratory volume in 1 second [FEV1] and peak expiratory flow rate [PEFR]) were measured using a portable spirometer. Since normal pulmonary function values for healthy non-smoking South Indian adults are available through previously published studies, the pulmonary function test (PFT) values from the study group were compared with the predicted values after corrections for age and anthropometry. The pulmonary functions of the conservancy workers were significantly lower than their predicted values. Moreover, the pulmonary functions declined with increasing years of working. Among both the groups of conservancy workers, the pulmonary functions were significantly lower in sweepers than loaders (P<0.01). This study has generated lung function data of the Chennai Corporation conservancy workers that can aid the concerned authorities to implement specific interventions to reduce the exposure and improve the health status of the workers.", "keywords": [ "The Chennai metropolitan area is the fourth largest metropolis in India. Urban development has been rapid over the last two decades. The development process", "however", "has had an adverse impact on the environment in the metropolis1. Municipal solid waste management (MSWM) is one of the major environmental problems of Indian cities. Increasing volumes", "entry of select hazardous waste streams", "manual handling of wastes", "inadequate personal protective equipment", "lack of awareness about health and sanitation and inadequate environmental management at the landfill sites expose conservancy workers to a multitude of environmental and occupational hazards." ], "content": "Introduction\n\nThe Chennai metropolitan area is the fourth largest metropolis in India. Urban development has been rapid over the last two decades. The development process, however, has had an adverse impact on the environment in the metropolis1. Municipal solid waste management (MSWM) is one of the major environmental problems of Indian cities. Increasing volumes, entry of select hazardous waste streams, manual handling of wastes, inadequate personal protective equipment, lack of awareness about health and sanitation and inadequate environmental management at the landfill sites expose conservancy workers to a multitude of environmental and occupational hazards.\n\nChennai city is divided into 10 zones2. More than 10,000 conservancy workers of the Chennai Corporation handle 4500 to 5200 tons of municipal solid wastes per day. Per-capita generation of solid waste is 0.7 kg per day3. The waste disposal sites of Chennai are the Kodungaiyur and the Perungudi dumping grounds. Although the incidence and prevalence of various hazards in formal solid waste sector workers are high, very few studies have been conducted in developing countries4. Studies have been published from the developed high-income countries5. The data from these developed countries cannot be directly extrapolated to developing countries, as the entire scenario is different between the two. The health profile in the Solid Waste Management sector needs to be generated region wise in order to implement the appropriate preventive and corrective measures. This study was not designed to establish the causative role of particular workplace exposures for the observed health impairments, but rather to evaluate the pulmonary function of workers in this environment with the intention of aiding subsequent environmental health management initiatives aimed at preventing such job-related exposures.\n\n\nMethods\n\nThe present cross sectional study was carried out among 178 conservancy workers of the Chennai Corporation. This project was executed in collaboration with the Chennai Metropolitan Development Authority and the Corporation of Chennai. Permissions were obtained from the Commissioner of Chennai Municipal Corporation, Superintending Engineer of Solid Waste Management and the Chennai Corporation Health Officer. The Solid Waste Management sector in the Corporation of Chennai is directly under supervision of the Superintending Engineer.\n\nThe 10,896 conservancy workers of the Chennai Corporation are spread all over the city across 10 zones and 155 wards. 178 conservancy workers were recruited for this study. Using Probability Proportionate to Sample (PPS), six zones (zones 2, 3, 4, 5, 7 and 9) were identified for the study purpose. Workers were then selected randomly from the roster.\n\nThe study proposal was approved by the corresponding author’s Institutional Ethics Committee. Written informed consent was obtained from all the subjects involved in the study for publications of their clinical details, prior to administration of medical examination. A validated questionnaire was used for obtaining medical history and occupational history of patients. A detailed clinical examination was performed on all the subjects, which included a general examination and respiratory system examination.\n\nPulmonary function tests (PFT) were performed using portable data-logging Spirometer (MIR SPIROBANK – Model A23). This spirometer works on the infrared interruption principle, where an infrared miniflow sensor is used for measurement of both flow and volume. All the workers were properly trained to perform the pulmonary function test. All the subjects underwent an anthropometric assessment, which included height and weight. A nose clip was fixed and the test was performed in a sitting position. The best value of three attempts was taken. A complete flow – volume loop was obtained from the spirometer. The spirograms (flow – volume loop) were directly downloaded from the instrument and printed, and the values were also manually recorded. The Spirometer that was used performs as per the equipment specifications of the American Thoracic Society. All volumes were corrected to conditions of body temperature and pressure saturable with water vapour (BTPS). The best values of Forced Vital Capacity (FVC), Forced Expiratory Volume at the end of one second (FEV1) and Peak Expiratory Flow Rate (PEFR) were used for analysis.\n\nData are represented as Mean ± Standard Deviation. The comparison test of significance used was the independent ‘t’ test. Pearson’s correlation analysis and linear regression were used for assessing the association of pulmonary function parameters with total duration of employment. The level of significance was taken at the 5% level. Data were analysed using SPSS version 16.\n\n\nResults\n\nThe age, height, weight and BMI (Body mass index) of the 178 conservancy workers (study subjects) are provided in Table I.\n\nAll values are represented as Mean ± Standard deviation; BMI = Body mass index.\n\nResults of Pulmonary function assessment: The key pulmonary function parameters chosen for analysis were FVC, FEV1 and PEFR. Females had lower pulmonary function than males. The pulmonary function parameters of the study subjects are provided in Table II.\n\n* P<0.01; Sweepers had significantly lower values compared to loaders.\n\nAll values are represented as Mean ± Standard deviation.\n\nFVC = Forced Vital Capacity; FEV1 = Forced Expiratory volume at the end of one second; PEFR = Peak expiratory flow rate.\n\nSince normal pulmonary function values for healthy non-smoking South Indian adults are available through previously published studies6,7, the PFT values of non smoking men and women from the study group were compared with the predicted values after corrections for age and anthropometry. The study population, both males and females had lower observed values than their own predicted values, which were statistically significant.\n\nIn order to discern differences among both the categories of workers, who have varying degrees of occupational exposures, PFT values of both the job categories were compared. Sweepers had the lower PFT (FVC, FEV1 and PEFR) values compared to loaders, which were statistically significant, as shown in Table II.\n\nPFT values were also compared across both the groups of workers with varying duration of work experience (resulting in differences in duration of exposure) as shown in Figure 1. The workers were classified into three groups based on the duration of working (Group I: work duration <10yrs; Group II: work duration 10–20yrs; Group III: work duration >20yrs). Pulmonary functions were significantly lower in the group III, compared to group I and group II. FVC and FEV1 were significantly different among the three groups. PEF of Group I was significantly different when compared with Group II and Group III, whereas there was no difference between Group II and Group III. Pulmonary function significantly declined with increasing years of working as assessed by linear regression.\n\n(Group I: <10yrs; Group II: 10–20yrs; Group III: >20yrs) FVC = Forced Vital Capacity; FEV1 = Forced Expiratory volume at the end of one second; PEFR = Peak expiratory flow rate.\n\n\nDiscussion\n\nThis study has generated the pulmonary function profile of the conservancy workers of the Solid Waste Management sector of Chennai. There is higher prevalence of both respiratory symptoms and respiratory impairments (as established through pulmonary function tests) in the study population. The decrement in pulmonary function with increasing duration of working strongly suggests that workplace exposures may be contributing significantly to such impairments. A very high prevalence of respiratory symptoms, much lower lung function values than predicted for a normal healthy population, increasing impairments with increasing years of working all point to a pattern of increased potential for respiratory morbidity among these workers due to occupational exposures. Exposures to air pollutants including dusts, air toxins and bio-aerosols from fugitive and occupational sources are a major health concern for conservancy workers. It is likely that operations such as sweeping, loading and unloading solid wastes could potentially expose workers to greater than safe levels, as all such operations were being performed in the absence of any engineering controls or use of personal protective equipment (PPE). In the present study, the conservancy workers did not use any PPE.\n\nStudies have reported higher respiratory morbidity among conservancy workers. In a study conducted by Athanasiou et al., municipal solid waste workers had increased symptoms pertaining to respiratory system and a significant reduction in FVC compared with controls8. Ray et al. also reported that landfill workers had a significantly higher prevalence of respiratory symptoms and increased impairment of lung function compared with controls. The landfill workers also had airway inflammation and a lot of complaints pertaining to general health9. Rajnarayan R. Tiwari reported that the abnormal respiratory functions found in sanitary workers may be due to exposure to endotoxins and airborne bacteria by way of bioaerosols10. Zuskin et al. reported that forced end expiratory flow FEF50 and FEF25 were reduced, probably due to small airway obstruction11. In another study, Zuskin et al. have demonstrated reduced FVC and FEV1, which was significant in sanitation workers compared with controls12. Similar significant reductions in lung function parameters have been reported in solid waste collectors in other studies13.\n\nThe present study demonstrates the degree of pulmonary impairments prevalent in workers of this sector. Given the similarity in process operations across other districts in the state, as well as other states in South India, this may be a fair representation of the sector in South India.\n\nBaseline health assessments in these work environments further provide the necessary inputs for designing surveillance programs. Pulmonary functions assessment is also a pre-requisite for assessing fitness to wear respirators and hence must be periodically conducted. It is important to implement control strategies at an early stage to prevent the disease process from setting in and also it would be crucial for these workers to be covered under a regular occupational health-monitoring program that would keep them under routine surveillance. Sector-specific exposure and health profiling is an important element for creation of local occupational health databases and the results of the study have provided baseline information to serve as an input to the development of such databases.\n\nLongitudinal studies can be planned with the input available from this study to assess the chronic or permanent functional loss resulting from exposure. Further work is also needed to clarify potential reversibility after cessation of exposure. Medical institutions and the Occupational Health Institute need to be encouraged to study the health of conservancy workers with an appropriate baseline control population, since epidemiological data from this sector is lacking. Apart from all these, an environmentally sound garbage management system is required for urban waste management14. A proper method should be used to dispose of solid waste15.\n\n\nConclusion\n\nThis profile can be used to address the hazards identified in solid waste management, so that appropriate preventive and corrective measures can be undertaken. The issue of occupational health in India is deeply embedded in a matrix of environmental, health, and economic/developmental considerations. Understanding the potential for health risks is necessary to ensure that the most vulnerable communities need not suffer. Indeed if human development is the goal, addressing health risks is an important mechanism to ensure equity in quality of life for all and it is hoped that the information presented here represents a small incremental step towards achieving the same.", "appendix": "Author contributions\n\nRoopa S, R. Padmavathi, B. Kalpana and A. Akolkar conceived and designed this paper. Roopa S and S. Sankar acquired the data and drafted the article. R. Padmavathi revised it for intellectual content. P. Ravishankar analysed and interpreted the data. T. Vijayalakshmi and Subhashini A.S. helped in final approval of the version to be published.\n\n\nGrant information\n\nThe present study was funded by the Central Pollution Control Board, New Delhi, India.\n\n\nCompeting interests\n\nThere are no competing interests.\n\n\nConsent\n\nWritten informed consent for publications of their clinical details was obtained from all the subjects involved in the study.\n\n\nReferences\n\nSujatha P, Janardhanam PVS: Solid waste management in Chennai city. Indian J Edu Inf Manage, 2012; 1(3): pp. 115–125.\n\nSudhir V, Muraleedharan VR, Srinivasan G, et al:Integrated solid waste management in urban India: A critical operational research framework. Socio-Econ. Plann. Sci, 1996; 30(3): pp. 163–181.\n\nhttp://www.chennaicorporation.gov.in/departments/solid-waste-management/index.htm.\n\nMisra V, Pandey SD: Hazardous waste, impact on health and environment for development of better waste management strategies in future in India. Environ Int, Apr 2005; 31(3): pp. 417–31.\n\nLennart Friis, Dan Norback, Christer Edling, et al:Self-Reported Asthma and Respiratory Symptoms in Sewage Workers. J Occup Health, 1999; 41: pp. 87–90.\n\nVijayan VK, Kuppurao KV, Venkatesan P, et al:Reference values and prediction equations for maximal expiratory low rates in non-smoking normal subjects in Madras. Indian J Physiol Pharmacol, 1993; 37(4): pp. 291–297.\n\nVijayan VK, Kuppurao KV, Venkatesan P, et al:Pulmonary functions in healthy young adult Indians in Madras. Thorax 1990; 45: pp. 611–615.\n\nAthanasiou M, Makrynos G, Dounias G, et al:Respiratory health of municipal solid waste workers. Occup Med (Lond), Sep 2010; 60: pp. 618–623.\n\nRay MR, Roychoudhury S, Mukherjee G, et al:Respiratory and general health impairments of workers employed in a municipal solid waste disposal at an open landfill site in Delhi. Int J Hyg Environ Health, 2005; 208(4): pp. 255–62.\n\nRajnarayan R Tiwari: Occupational health hazards in sewage and sanitary workers. Indian J Occup Environ Med, 2008 Dec; 12(3): pp. 112–115.\n\nZuskin E, Mustajbegovic J, Schachter EN, et al:Resiratory function in sewage workers. Am J Ind Med, 1993; 23: pp. 751–61.\n\nZuskin E, Mustajbegovic J, Schachter EN, et al:Airway function and respiratory symptoms in sanitation workers. J Occup Environ Med, May 1996; 38(5): pp. 522–7.\n\nHeldal KK, Halstensen AS, Thorn J, et al:Airway inflammation in waste handlers exposed to bioaerosols assessed by induced sputum. Eur Respir J, 2003; 21: pp. 641–645.\n\nDhande AD, Ingle ST, Attarde SB, et al:Ecofriendly approach of urban solid waste management: a case study of Jalgaon city, Maharashtra. J Environ Biol, Oct 2005; 26(4): pp. 747–52.\n\nAvinash Puri, Manoj Kumar, Eonkar Johal, et al:Solid-waste management in Jalandhar city and its impact on community health. Indian J Occup Environ Med, Aug 2008; 12(2): pp. 76–81." }	[ { "id": "403", "date": "19 Dec 2012", "name": "Youcheng Liu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "727", "date": "24 Jan 2013", "name": "Benoit Nemery de Bellevaux", "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 methodology of the field survey appears to have been adequate. However, the analysis is not adequate.The authors should not base their analysis on absolute levels of pulmonary function parameters. Indeed the differences observed between sweepers and loaders (table 2) and between age groups (figure 1) could simply be due to differences in sex (how many men and women were there in each group?), height (table 1 shows a 7 cm difference in mean height between sweepers and loaders!) or even age distribution. In other words, the authors should have analyzed their data taking into account these 3 factors, either by doing a multiple correlation analysis or by taking values in percent predicted (which they probably did, but they don’t present the data).", "responses": [ { "c_id": "474", "date": "05 Jun 2013", "name": "Srinivasan Roopa", "role": "Author Response", "response": "As Dr Nemery de Bellavaux has mentioned, analyses of data was actually done by comparing pulmonary function values of the study subjects with their predicted values. Normal pulmonary function values for healthy non-smoking South Indian adults are available through previously published studies (References 6,7 and the equations of Vijayan et al). The data was not presented earlier, but now the data is presented as Table II. The PFT of the study group were compared with the predicted values (for men and women respectively) after corrections for age and anthropometry. The study population, both males and females had lower observed values than their own predicted values, which were statistically significant as shown in the Table II. Table II: Observed and predicted pulmonary function values of the study population Parameter Observed values Predicted values Males FVC 2.8 ± 0.7 3.2 ± 0.5* FEV1 2.4 ± 0.5 3.0 ± 0.4* Females FVC 1.9 ± 0.5 2.3 ± 0.2* FEV1 1.6 ± 0.5 1.9 ± 0.2* Data expressed as Mean ± SD * P < 0.05. Figure 1 shows the Pulmonary functions in both the groups of conservancy workers (i.e. sweepers and loaders) based on duration of working and not age groups. The authors thank the referees for their responses and their comments are well taken." } ] } ]	1	https://f1000research.com/articles/1-67
https://f1000research.com/articles/2-141/v1	19 Jun 13	{ "type": "Commentary", "title": "Tissue-resident Sca1+ PDGFRα+ mesenchymal progenitors are the cellular source of fibrofatty infiltration in arrhythmogenic cardiomyopathy", "authors": [ "Ben Paylor", "Justin Fernandes", "Bruce McManus", "Fabio Rossi", "Justin Fernandes", "Bruce McManus", "Fabio Rossi" ], "abstract": "Arrhythmogenic cardiomyopathy (AC) is a disease of the heart involving myocardial dystrophy leading to fibrofatty scarring of the myocardium and is associated with an increased risk of both ventricular arrhythmias and sudden cardiac death. It often affects the right ventricle but may also involve the left. Although there has been significant progress in understanding the role of underlying desmosomal genetic defects in AC, there is still a lack of data regarding the cellular processes involved in its progression. The development of cardiac fibrofatty scarring is known to be a principal pathological process associated with ventricular arrhythmias, and it is vital that we elucidate the role of various cell populations involved in the disease if targeted therapeutics are to be developed. The known role of mesenchymal progenitor cells in the reparative process of both the heart and skeletal muscle has provided inspiration for the identification of the cellular basis of fibrofatty infiltration in AC. Here we hypothesize that reparative processes triggered by myocardial degeneration lead to the differentiation of tissue-resident Sca1+ PDGFRα+ mesenchymal progenitors into adipocytes and fibroblasts, which compose the fibrofatty lesions characteristic of AC.", "keywords": [ "hypothesis", "arrhythmogenic cardiomyopathy", "desmosomal mutations", "myocardial dystrophy", "fibrofatty infiltrate", "tissue-resident mesenchymal progenitors", "PDGFRα", "Sca1" ], "content": "Introduction\n\nArrhythmogenic cardiomyopathy (AC) is a heterogeneous disease of the heart associated with an increased risk of both ventricular arrhythmias and sudden cardiac death. Although the exact pathogenesis of this disease is unknown, mutations in genes coding for the five major proteins of the desmosome, namely plakoglobin, desmoplakin, plakophilin-2, desmoglein1 and desmocollin-22, have been strongly implicated. The penetrance of these desmosomal mutations in AC patients has been estimated to be between 40 and 90% by different studies3–5, making identification of at-risk individuals by clinical genotyping difficult. As such, there is currently no single test to diagnose AC, although improved quantitative functional parameters associated with the disease as well as identification of pathogenic mutations in first-degree relatives have improved both the sensitivity and specificity of current diagnostic criteria6. Current therapeutic and management paradigms rely on symptomatic impact including anti-arrhythmic drugs and lifestyle modifications, and it is hoped that probing the link between desmosomal gene defects and the progression of AC will lead to more effective disease-targeted therapies7.\n\nThe name, arrhythmogenic cardiomyopathy, has gradually evolved since the initial classification of this disease in 1982 when it was called \"right ventricular dysplasia\"8 as it was thought to be caused by a developmental defect of the heart before birth. It was soon determined that the symptoms and signs were in fact a progressive cardiac disease, and thus dysplasia was replaced with \"cardiomyopathy\"9. Clinically, since the main symptoms that appear during the progression of the disease are right ventricular arrhythmias, the term arrhythmogenic right ventricular cardiomyopathy (ARVC) became the most commonly used name. With a growing body of evidence demonstrating left ventricular involvement in this disease10,11, the current terminology of arrhythmogenic cardiomyopathy (AC) was adopted in 20107. Several disease patterns are encompassed by this definition; including right dominant, left dominant and biventricular AC6,7.\n\n\nPathophysiological mechanisms of arrhythmogenic cardiomyopathy\n\nWhile several etiopathogenic theories for the development of AC have been proposed12,13, including dysontogenic (dysplasia)14, apoptotic15,16 and transdifferentiative17 processes, it is most widely accepted today that degenerative and dystrophic mechanisms underlie its progression7. This latter model, proposed well before the discovery of associated desmosomal-mutations, draws from histopathological similarities between AC and skeletal-muscular dystrophies, which are both characterized by progressive muscle damage with associated replacement with fibrofatty connective tissue. Experimental data have demonstrated that cardiomyocyte death, either by apoptosis or necrosis, is the primary initiating trigger that eventually is followed by fibrofatty replacement of functional myocardium18,19. The molecular pathways that underlie the progressive loss of cardiomyocytes in AC continue to be investigated (reviewed in Shirokova and Niggli (2012)20), with the ultimate goal of developing targeted preventives or therapies. It should be noted that while the presence of fibrofatty tissue is pathognomonic for this disease, Burke et al.21 suggest that the fibrofatty infiltration is most likely secondary to associated desmosomal gene mutations. Progressive loss of cardiomyocytes due to mutated desmosomal gene products may activate reparative processes and lead to progressive accumulation of diffuse fibrofatty tissue, with concomitant alterations in cardiac electrophysiological and contractile function. Although it is widely accepted that a key pathological link between desmosomal mutations and the often fatal ventricular arrhythmias of this disease is the development of the characteristic fibrofatty infiltrate, the cellular source of the fibroblasts and adipocytes that compose this connective tissue currently remains unclear. Two recent studies in murine models of the disease have implicated cardiac progenitor cell (CPC) populations as the most likely candidate, but noted that further work was necessary to clearly identify the cells involved22,23.\n\n\nThe hypothesis\n\nWe propose that reparative processes in the heart triggered by myocardial dystrophy lead to the differentiation of tissue-resident cardiac Sca-1+ PDGFRα+ mesenchymal progenitors into both fibroblasts and adipocytes, resulting in the characteristic fibrofatty lesion observed in AC.\n\n\nCellular source of fibrofatty infiltration in arrhythmogenic cardiomyopathy\n\nRecent human genetic analyses of AC patients24 have identified the causative role of desmosomal mutations in its progression and have aided in the clinical diagnosis of the disease, but it has been the development of numerous in vivo transgenic murine models of AC25 that have greatly furthered our fundamental understanding of the disease.\n\nUtilizing lineage tracing and genetic fate mapping, Lombardi et al. showed recently that second heart field CPCs are a source of adipocytes23 in a murine model of AC. Through use of a series of conditionally expressed reporter strains, which concomitantly delete the desmosomal protein desmoplakin in cardiac myocyte lineages and permanently activate yellow fluorescent protein expression in the deleted cells, this group elegantly demonstrated a contribution of second heart field CPCs to adipogenesis. Further, they provided strong evidence implicating perturbations in Wnt/Tcf712 signaling as a molecular mechanism underlying this progression. Although these experiments have provided compelling data to support the molecular mechanisms governing the development of fibrofatty scar tissue in AC, other than demonstrating the involvement of Isl-1+ second heart field progenitors, the lineage tracing strategies were unable to provide substantive evidence as to the identity of cells involved in fibrofatty scar development. Further, they were unable to conclude that the second heart field CPCs are the sole cellular source since the Cre-drivers used (Nkx2.5, Mef2C, α-MyHC) are unable to distinguish the involvement of pericytes, fibroblasts or circulating cells.\n\nIt was demonstrated long ago that CPCs identified using the marker Sca-1 can give rise to adipocytes in vitro26, yet evidence implicating Sca-1+ CPCs as the cellular source of adipocytes in models of AC in vivo is still lacking. Lombardi et al.22 have identified a role for Sca-1+ CPCs in the enhanced adipogenesis of AC mice harbouring mutations in the desmosomal protein plakoglobin. They did not, however, perform the strict lineage tracing experiments required to quantitatively assess the relative contribution of CPCs to the adipogenesis in their models. The long standing dogma of AC being primarily a right ventricular disease was supported by demonstration of the involvement of second-heart field progenitors, but this proposed model of AC fails to accommodate recent reports of similar pathological processes in the left ventricle17,27. Indeed, data demonstrating that fibrofatty scarring and functional deterioration occur in both ventricles in AC argues that the progenitor cell subset responsible for generation of fibroblasts and adipocytes in this disease is distributed throughout the heart. If such is true, a population of subepicardial progenitors may be a good candidate28. Such a notion is strongly supported by recent studies demonstrating that both the murine29 and human30 heart harbour a population of pro-epicardially-derived tissue-resident mesenchymal progenitors, which express Sca1 and PDGFRα. These studies demonstrated the broad trans-germ layer differentiative capacity of this cardiac-resident progenitor population29 and characterized their presence in both fetal and diseased human myocardium30, but did not thoroughly investigate their role in regeneration and repair of the heart. The notion that the heart harbours a population of Sca1+ PDGFRα+ mesenchymal progenitors able to generate both fibroblasts and adipocytes aligns with the hypothesis that this population is the most significant cellular source of fibrofatty infiltration in AC.\n\nAlthough fibrofatty replacement of the myocardium is the hallmark feature of AC, the consistent observation of small lymphocytic foci in the disease31–33 further supports the involvement of cardiac-resident Sca1+ PDGFRα+ mesenchymal progenitors in its progression, as these progenitors have been recently shown to contribute in vivo to lymph node stroma34 and follicular dendritic cells35. Thus, the milieu of chronic myocardial inflammation may trigger their differentiation not just into adipocytes and myofibroblasts, but also possibly in cells capable of attracting and supporting lymphocytes.\n\nAlthough studies in murine models of AC employing the strict lineage tracing methods required to determine the cellular source of adipocytes have yet to be performed, studies in skeletal muscle (SM) further support our hypothesis in implicating Sca1+ PDGFRα+ tissue-resident mesenchymal progenitors. Recent studies36,37 accomplish the prospective isolation and purification of a population of SM resident Sca1+ PDGFRα+ mesenchymal progenitors, which were further shown to be the sole SM-derived population with fibro-adipogenic potential. Additional evidence from mdx mice, a murine model of Duchenne muscular dystrophy38, strongly supports the notion that tissue-resident Sca1+ PDGFRα+ cells are the principal cell population involved in the generation of fibrofatty scars in situations of chronic muscle damage. Interestingly, a robust regenerative capacity of SM was demonstrated by these studies, and others39,40, to be at least partly due to paracrine roles of these tissue-resident mesenchymal progenitors. In light of this evidence, the therapeutic potential of modulating proliferation and differentiation of cardiac Sca1+ PDGFRα+ progenitor cells in situations of acute or chronic damage is certainly intriguing. With a large body of clinical evidence demonstrating the beneficial effects of transplanting bone-marrow derived mesenchymal stromal cells (MSCs) into patients afflicted with a variety of cardiovascular disorders41, and numerous data highlighting the similarities between MSCs derived from different tissues42, the identification of a tissue-resident counterpart presents an attractive candidate for pharmacological modulation. Such manipulation could lead to therapeutic benefits, similar to those observed with exogenous delivery of similar cells but devoid of the adversities stemming from ex vivo manipulations, including (but not limited to) transplantation, immunogenicity issues and challenges involved in the use of good manufacturing practice facilities for clinical-grade cell preparations.\n\n\nResearch required to evaluate the hypothesis\n\nIn order to unequivocally demonstrate the contribution of cardiac-resident Sca1+ PDGFRα+ mesenchymal progenitors to fibrofatty scarring in AC, a number of questions must be addressed.\n\nFirst, a more thorough characterization of these progenitor cells must be performed to determine both their functional role in health and disease, as well as determine the homogeneity of this population. It is very possible that within this phenotypic identity, several functionally different cellular subsets are present, and the unravelling of these hierarchies could provide significant insight into cellular processes in the regenerating or degenerating myocardium. Dularoy et al.43 identified a fibrogenic subpopulation within Sca1+ PDGFRα+ progenitor in the SM using ADAM12+, highlighting heterogeneity, and supporting the need to further distinguish between functional subsets of this mesenchymal population. It will also be highly important to determine whether the desmosome plays a direct role in Sca1+ PDGFRα+ progenitor cell function, or whether the arrhythmogenic reparative disorder observed in AC is to be ascribed solely to continued loss of cardiomyocytes due to desmosomal gene defects.\n\nSecond, further lineage tracing experiments using several inducible Cre-drivers such as PDGFRα-CreER in combination with either previously described25 or novel models of AC will allow identification of the role of this population in AC.\n\nFinally, the limitations of the murine Sca-1 (Ly6A/E) as a marker of tissue-resident progenitors should be addressed since there is currently no known human homolog for this gene. Such is the case despite the hypothesis that a broad range of functions mediated by this marker are probably assumed by other Ly6 proteins in humans44.\n\n\nConsequences of the hypothesis\n\nWith no current treatment available for AC, it is highly attractive to consider that manipulation of molecular mechanisms underlying the development of the cardiac fibrofatty infiltration could mitigate both functional deterioration as well as survival of AC patients. Clinical trials in patients suffering from Duchenne muscular dystrophy utilizing anti-fibrotic strategies (NCT01521546) may provide valuable insight into the beneficial effects of preventing development of fibrofatty scars in the functional myocardium45. Additionally, with several recent reports describing in vivo reprogramming of cardiac fibroblasts into functional cardiomyocytes46–48, it could be postulated that targeting of existing fibrofatty scars could be therapeutically beneficial. With a growing recognition of functional and theoretical overlap between fibroblasts and mesenchymal stem cells49,50 there is a strong possibility that the beneficial effects seen in recent cardiac studies are due to reprogramming of these tissue-resident progenitor cells. Recent technical advances involving patient-specific induced pluripotent stem cells (iPSCs)51 as well as further development of aforementioned reprogramming strategies may enable novel targeted approaches to specific cell populations with enhanced transdifferentiative capacities, and could provide a basis for next-generation therapy of AC.\n\n\nConclusion\n\nIn summary, recent evidence demonstrating that the heart harbours a population of Sca-1+ PDGFRα+ mesenchymal progenitors provides new directions for defining the cellular source of the fibrofatty infiltrate that is characteristic of AC. A growing understanding of the role of tissue-resident mesenchymal progenitors in numerous other tissues, most notably SM, offers strong support for our hypothesis. Potential avenues for novel targeted therapeutics may emerge to benefit patients with AC.", "appendix": "Author contributions\n\n\n\nBP conceived the hypothesis, co-wrote the initial manuscript, implemented all revisions from co-authors and finalized the manuscript. JF co-wrote the initial manuscript. BM provided expertise in cardiovascular pathology and edited the manuscript. FR aided in the development of the hypothesis and helped critically revise the manuscript by providing editorial support.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nYang Z, Bowles NE, Scherer SE, et al.: Desmosomal dysfunction due to mutations in desmoplakin causes arrhythmogenic right ventricular dysplasia/cardiomyopathy. Circ Res. 2006; 99(6): 646–55. 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Nature. 2013; 494(7435): 105–10. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "1013", "date": "20 Jun 2013", "name": "James Chong", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 considered hypothesis for cellular origins of the fibro-fatty myocardial scarring underlying the poorly understood clinical disease - Arrhythmogenic Cardiomyopathy. The authors are a group with considerable experience investigating fibroadipogenic and skeletal muscle progenitor cells. The points below address the questions posed to reviewers:The title is perhaps too strongly worded. Whilst the hypothesis is sound and intriguing, there is currently little or no data proving that the title/hypothesis is true. This is appropriately discussed in the manuscript.The abstract does represent a suitable summary of the work.The article content provides a thorough review of data supporting the proposed hypothesis. If the hypothesis were proven true this would be a substantial advance for this field and for clinical cardiologyThe conclusions are sensible and justified.", "responses": [] }, { "id": "1015", "date": "20 Jun 2013", "name": "Richard Harvey", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 synthesis of the literature surrounding the history, clinical manifestations, genetic basis and aetiology of arrhythmogenic cardiomyopathy (AC), and in particular, proposes a role for the resident Sca1+ PDGFRα+ interstitial population that includes mesenchymal (MSC-like) progenitor cells in the fibrofatty infiltrates that develop during the disease. This hypothesis piece notes carefully the limitations of conclusions arising from published studies using animal models that hint at a role for second heart field cardiomyocyte progenitors in the development of disease. Of importance, is whether the fibrofatty infiltrates are the primary or secondary target of the mutations in desmosomal component genes that underpin the disease. The piece is scholarly, and well written. A dimension not explored extensively is the possibility that the fibrofatty cells arise from the epicardium of the adult heart, which has been shown by a number of groups to become activated after injury. Activated epicardium re-expressed genes involved in the fetal epicardial program and reveals a latent lineage reserve that can contribute fibroblasts and smooth muscle cells to the infarct zone of a chronically injured ischaemic heart. While the Sca1+ PDGFRα+ interstitial mesenchymal cells and epicardial cells share a lineage relationship in development, an additional challenge in the lineage mapping studies proposed is to distinguish whether fibrofatty infiltrates in AC arise from one or the other, or both, of these cellular compartments. Nonetheless, the ideas explored in this article overlap with interesting and important issues in cardiac developmental and stem cell biology, and with the potential for targeting fibrofatty progenitor cells as a therapy in arresting the progression of AC in the clinical setting. An excellent work that should be a reference point for future studies.", "responses": [] } ]	1	https://f1000research.com/articles/2-141
https://f1000research.com/articles/2-60/v1	25 Feb 13	{ "type": "Research Article", "title": "Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site", "authors": [ "Jasmina Kurepa", "Jan A. Smalle", "Jasmina Kurepa", "Jan A. Smalle" ], "abstract": "Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin.Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity.Results: We describe the isolation and analyses of suppressor of rpn12a-1 (sor1). The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation.Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin regulation of plant growth.", "keywords": [ "Arabidopsis", "exon trap mutagenesis", "chimeric introns", "cytokinin responses" ], "content": "Introduction\n\nThe 26S proteasome (26SP) is a multisubunit protease responsible for the degradation of proteins that are covalently labeled with a polyubiquitin (Ub) chain via the combined action of Ub activating enzymes, Ub conjugating enzymes and Ub ligases1. The 26SP is localized in the cytosol and the nucleus, and it degrades proteins involved in many signaling and metabolic pathways1,2. The 26SP is also essential for the destruction of misfolded proteins that are generated by mistranslations and during stress2–4.\n\nStudies with proteasome mutants in Arabidopsis have revealed that the 26SP is required for both male and female gametogenesis, confirming its essential role in cellular viability2,5,6. Partial loss-of-function mutants, on the other hand, have been indispensable for uncovering pathways in which key components are regulated by proteasome-dependent degradation7–13.\n\nThe rpn12a-1 mutant, which carries an insertion in the RPN12a gene (At1g64520) encoding regulatory particle non-ATPase subunit (RPN) 12a, was isolated from a collection of exon-trap lines14,15. These lines were generated by transforming Arabidopsis plants (C24 accession) with a T-DNA construct that contains a promoterless neomycin phosphotransferase (NPTII) without a starting methionine which is preceded by a 3´ splice site of the first intron of the apurinic endonuclease (APR)14. Kanamycin-resistant exon-trap lines are therefore predicted to have the APR-NPTII construct inserted downstream of an active promoter either in frame with the coding region or in a position that allows the formation of a novel, chimeric intron. The rpn12a-1 mutation is unusual because the T-DNA is inserted downstream of the RPN12a gene, and both the full-length RPN12a cDNA and a chimeric RPN12a-NPTII cDNA are produced15. This suggested that two types of cis signals involved in the pre-mRNA processing of RPN12a are competing. Because the wild-type transcript is produced in the mutant and is stable enough to be detected using routine RNA analytic procedures, the poly(A) signal of the RPN12a gene must be intact and active. On the other hand, since a chimeric RPN12a-NPTII transcript is produced, the 3´ splice site of the inserted T-DNA must have recruited a latent 5´ splice site in the RPN12a gene. We have previously shown that this predicted latent 5´ splice site is STOP codon-associated, and that the pre-mRNA splicing of the chimeric intron leads to the production of the fusion mRNA15. As a result of the action of these two opposing pre-mRNA processing mechanisms, a fraction of the mRNA species transcribed from the mutant RPN12a gene are translated into a functional RPN12a protein, and the rest encodes a chimeric RPN12a-NPTII fusion protein. Because both RPN12a forms are incorporated into the 26SP, the total proteasome activity in these mutant seedlings is reduced, but not abolished15.\n\nThe reduction of 26SP activity in rpn12a-1 caused a pleiotropic phenotype, which included altered responses to cytokinins15. Cytokinins are plant hormones that are essential for every aspect of growth and development16–19. For example, cytokinins control the development of meristems and vasculature, and play an important role in senescence and nutrient allocation19,20. To gain better insight into the cytokinin insensitivity of rpn12a-1 seedlings, we screened for suppressor mutants that have a wild-type cytokinin growth response. Here we describe the intragenic suppressor of rpn12a-1 (sor1) that disrupts the latent 5´ STOP-associated splice site. Sor1 reduced the expression of the RPN12a-NPTII fusion mRNA with a concomitant increase in RPN12a transcript level. As a result, RPN12a accumulation in sor1 seedlings was identical to the wild-type and was accompanied by wild-type cytokinin sensitivity. These results validate our transcript processing interpretation of the rpn12a-1 exon-trap effect and accentuate the importance of optimal RPN12a expression for cytokinin signaling.\n\n\nMaterials and methods\n\nThe Arabidopsis thaliana rpn12a-1 mutant in the C24 background was described by us previously15. To grow plants on soil and in axenic cultures, seeds were surface-sterilized in 70% ethanol followed by 50% bleach and plated on MS/2 medium that contained half-strength MS salts (pH 5.7, Sigma, St. Louis, MO) and 1% (w/v) sucrose. The seeds were kept for 4 days in darkness at 4°C, and either plated on MS/2 or on soil (Miracle-Gro potting mix:Perlite at 1:1 ratio). Plants were grown in continuous light at 22°C.\n\nThe rpn12a-1 seeds were pre-incubated in 1.0% KCl for 12 hours, and then mutagenized for 5 hours in 100 mM sodium phosphate buffer (pH 5) containing 5% DMSO and 80 mM ethyl methanesulfonate (EMS; Sigma-Aldrich, St. Louis, MO). Seeds were washed twice in 100 mM sodium thiosulphate and then twice in distilled water. Seeds were incubated and chilled in 0.1% agar and sown directly to soil. All the seeds in the M2 generation were pooled upon harvest, surface-sterilized and plated on the MS/2 medium containing 0.1 µM kinetin (6-furfurylaminopurine; obtained from Duchefa Biochemie (Gold Biotechnology, St. Louis, MO, USA). The putative suppressor mutants were transferred from the selection medium onto MS/2 medium to allow recovery, and were then transferred to soil.\n\nCytokinin treatments were as previously described15. For fresh weight analyses, seedlings were germinated and grown on kanamycin-containing media, and their weight was measured in pools of 5 seedlings after 24 days of growth. Kanamycin monosulfate was obtained from Gold Biotechnology.\n\nFor the RT-PCR experiments, total RNA was extracted using TRIzol reagent (Invitrogen. Carlsbad, CA, USA). The reverse transcription was performed using 1 µg of TURBO DNAse (Ambion, Austin, TX, USA), pre-treated total RNA and iScript kit (BioRad, Hercules, CA, USA). The primers used for the amplification of wild-type cDNA fragment (306 bp in length) were F1: 5´-GGGTGCCTATAACCGTGTGTTGAGTGCTAG-3´ and R1: 5´-ATACGCTCCAGCTCTCTGGCGTAGCTTAGA-3´. The RPN12A-NPTII fusion transcript fragment was amplified with F1 and NPTII primer R2: 5´-CCCCTGCGCTGACAGCCCGGAACA-3´. PBA1 (At4g31300) was amplified using forward and reverse primers that contained the first and last 25 bp of the cDNA. The primer set used to amplify the Arabidopsis elongation factor 1-α (EF-1-α; At5g60390) was previously described9.\n\nFor immunoblotting analyses, total proteins were isolated, separated and transferred to nitrocellulose membranes as described15. Rabbit polyclonal anti-RPN12a and anti-PBA1 antibodies (used at 1:1000 dilution) were purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA). The anti-NPTII antibodies (rabbit, polyclonal) were obtained from Abcam (Cambridge, MA, USA).\n\nGenomic DNA fragments from rpn12a-1 and sor1 were amplified using F1 and R2 primers and sequenced using dye-termination chemistry (Perkin-Elmer, Foster City, CA, USA) at Advanced Genetic Technologies Center (AGTC, KY, USA). Sequences were analyzed using Vector NTI Suite (Invitrogen, Carlsbad, CA).\n\n\nResults and discussion\n\nTo obtain rpn12a-1 suppressors, we mutagenized seeds with EMS and plated ~50,000 M2 seeds on a medium with 0.1 µM of the cytokinin kinetin. Wild-type plants grown on 0.1 µM kinetin are chlorotic and smaller compared to rpn12a-115, and thus we selected chlorotic and smaller M2 seedlings after 14 days of growth. The putative suppressors were first transferred to cytokinin-free media to recover, and subsequently to soil for self-pollination.\n\nAnalyses of the M3 generation showed that in suppressor of rpn12a-1 1 (sor1), all visible phenotypes of rpn12a-1 were reverted back to the wild-type (Figure 1). For example, the rpn12a-1 mutant has a smaller rosette than the wild-type and a reduced leaf initiation rate15. The sor1 plants had a leaf number and rosette size similar to the C24 wild-type plants (Figure 1). The sor1 mutant plants also displayed wild-type sensitivity to cytokinin. After three weeks of growth on a medium with 0.2 µM kinetin, both wild-type and sor1 seedlings were chlorotic and their growth was severely inhibited, while the rpn12a-1 seedlings were green and larger (Figure 1).\n\nPlants were grown for three weeks on MS/2 media (control) or MS/2 media containing 0.2 µM kinetin in continuous light. Representative seedlings are shown.\n\nNext, we analyzed the kanamycin (Km) resistance of the sor1 mutant line. The Km resistance of the rpn12a-1 mutant is completely linked to the proteasome-related phenotypes and thus, all the progeny of a plant homozygous for the rpn12a-1 mutation should be Km resistant. All sor1 seedlings were indeed resistant to Km, but the levels of resistance were significantly lower compared to rpn12a-1 (Figure 2). While Km did not affect the growth of rpn12a-1 seedlings, both root and shoot growth of sor1 were partially inhibited (Figure 2). We did not observe any attenuation of Km resistance over several generations, a phenomenon that has been documented for a number of T-DNA insertion mutant collections21 (see also the Salk Institute Genomic Analysis Laboratory Arabidopsis sequence indexed T-DNA insertion Project FAQ). An explanation for the change in Km tolerance in sor1 is that the mutation affects the expression of the NPTII gene which is an integral part of the exon-trap (Figure 3a and Babiychuk et al. 199714). When the sor1 mutant was outcrossed to the C24 wild type, none of the plants of the F2 population displayed an rpn12a-1 phenotype, indicating that sor1 is intragenic and tightly linked with the rpn12a-1 mutation.\n\n(a) Wild-type (C24), rpn12a-1 and sor1 seeds were sown and grown on MS/2 media containing 35 µg/ml kanamycin (Km). Representative plants were photographed after two weeks of growth. (b) Fresh weight (FW) of seedlings grown on Km media was measured after two weeks of growth. FW of the wild-type plants grown on control MS/2 media was calculated as 100%. Seedlings were measured in pools of five, and mean ± SD is presented (n≥7).\n\n(a) Simplified schematic representation of the RPN12a gene and the inserted T-DNA in the rpn12a-1 mutant15. The T-DNA contains the first intron and second exon of the apurinic endonuclease gene (ARP) fused in frame to the neomycin phosphotransferase II (NPTII) coding region. Exons are represented by gray boxes and introns as lines. Positions of the forward (F1) and reverse (R1 and R2) primers used for the RT-PCR are indicated. (b) Total RNA was extracted, reverse transcribed and used to amplify the RPN12a-NPTII (42 cycles) and wild-type RPN12a transcripts (35 cycles). The primers used for the reaction are indicated. Ampliﬁcation of proteasome β subunit 1 (PBA1) and ACTIN2 serve as controls.\n\nTo obtain further insight into the nature of the sor1 mutation, we analyzed the expression of the RPN12a gene and the accumulation of the RPN12a protein. RT-PCR analyses showed that the RPN12a-NPTII fusion transcript was not detectable in sor1 seedlings (Figure 3b). The abundance of the RPN12a cDNA, which was lower in the rpn12a-1 mutants, was restored back to the wild-type level.\n\nReductions in proteasome activity typically lead to the activation of a feedback mechanism that induces the transcription of proteasome subunit genes. This mechanism is operational in all eukaryotes, including yeasts, Drosophila, mammals and plants7,22–26. Due to this global feedback up-regulation of 26SP subunit genes, the 20S proteasome subunit β1 transcript (PBA1) is more abundant in rpn12a-1 compared to the wild-type (Figure 3b). As expected, the PBA1 level in the sor1 mutant was similar to that in the wild type.\n\nImmunoblotting analyses using anti-RPN12a antibodies showed that the sor1 mutant does not accumulate the RPN12a-NPTII fusion protein (Figure 4). The RPN12a abundance in sor1 was increased compared to rpn12a-1 and similar to the wild-type. We were also unable to detect the RPN12a-NPTII fusion in sor1 by using anti-NPTII antisera (Figure 4). In the rpn12a-1 mutant, a fraction of the assembled 26SP contains the fusion protein leading to a decrease in total cellular 26SP activity and a compensatory increase in the expression of proteasome subunit genes15,26. In the sor1 mutant, with no or little fusion protein, 26SP function is expected to be restored back to the wild-type level. Indeed, immunoblotting analyses with the anti-PBA1 antibodies showed that the abundance of the 20S proteasome subunit PBA1 in sor1 seedlings was comparable to that of the wild-type, indicating that proteasome activity was restored to optimal levels and that feedback up-regulation of proteasome subunit genes was halted (Figure 4).\n\nTotal protein was isolated from two-week-old wild-type (C24), rpn12a-1 and sor1 seedlings and used for immunoblotting analyses with anti-RPN12, anti-NPT and PBA1 antisera. In addition to the RPN12a and RPN12a-NPTII fusion proteins, the anti-RPN12 antisera also recognized two proteins (cross) that are not related to RPN12a. Ponceau S-stained membrane showing the large RuBisCO subunit (LSU) is presented as a loading control. The size of the proteins used as molecular mass standards is shown on the right-hand side.\n\nTaking into account both the result of the Km resistance tests (Figure 2) and the expression data (Figure 3 and Figure 4), we concluded that the sor1 mutation suppresses the formation of the RPN12a-NPTII fusion transcript which was sufficient to restore 26SP function back to the wild-type level.\n\nTo find the mutation that causes the sor1 phenotype, we amplified and compared the sequences of the RPN12a-NPTII chimeric gene from sor1 and rpn12a-1. No mutations were found in NPTII, indicating that the loss of Km resistance and NPTII abundance was not caused by any disruption of the NPTII coding region. We also did not detect any changes in the RPN12a coding region, but did find a single nucleotide change immediately downstream of the RPN12a STOP codon (Figure 5). Sequencing of the entire region between RPN12a and NPTII did not reveal any additional mutations, confirming that the RPN12a STOP codon-associated G to A mutation was indeed sor1.\n\nGenomic DNA fragment was amplified using F1 and R2 primers (presented in Figure 3), sequenced and the sequence was aligned using Vector NTI suite. Alignment of the region starting with base pair 1615 and ending with base pare 1804 of the annotated RPN12a gene is presented using BoxShade 3.2. The red arrowhead points to the sor1 mutation and the RPN12a STOP codon is boxed in red.\n\nTo analyze how this G-to-A substitution leads to reversion of the rpn12a-1 phenotype, we manually compared the consensus sequence for 5´ splice sites in Arabidopsis27 with the sequence of the exon/intron junction that precedes the RPN12a STOP codon in rpn12a-1 and sor1 (Figure 6a). The alignment revealed that both the intron and exon residues adjoining the splice junction of the mutants match the consensus well. Interestingly, the sor1 mutation changes a consensus G at the fifth position of the intron into an A, thus weakening the 5´ splice site of the chimeric intron. The G at the position +5 is thought to be required for efficient binding of U1snRPN27. Reduced splicing of the chimeric intron between the RPN12a and NPTII coding regions is predicted to lead to a reduced accumulation of the RPN12a-NPTII transcript and protein (Figure 6b and 6c). The combination of reduced intron splicing and unaffected 3´ end processing is therefore predicted to lead to a dramatic shift in favor of the formation of the wild-type RPN12a transcript, and thus to the accumulation of the RPN12a protein back to the wild-type level, which is what we observed in sor1 seedlings.\n\n(a) Sequence alignment of the terminal exonic tetranucleotides and proximal intronic hexanucleotides of the Arabidopsis consensus sequence27, and rpn12a-1 and sor1 sequences surrounding the STOP codon. Numbers next to the nucleotides of the consensus sequence refer to the frequency (%) for the noted nucleotide to be found at a given position. (b), (c) Schematic representations of splicing types in rpn12a-1 (c) and sor1 (d). aa, amino acids.\n\n\nConclusions\n\nCollectively, the results shown here validate our earlier interpretation of the effects of the rpn12a-1 mutation on RPN12a expression and 26SP function14. In the original study, we proposed that the partial loss of 26SP function in rpn12a-1 seedlings is caused by the competition between RPN12a and RPN12a-NPTII transcript processing that leads to a decrease of RPN12a protein levels and thus, to a decrease in the abundance of wild-type 26SP particles14. Our finding that suppression of RPN12a-NPTII accumulation was sufficient to restore RPN12a accumulation and reverse the plant development and cytokinin sensitivity back to the wild-type level validates the proposed interpretation and accentuates the importance of optimal 26SP abundance for Arabidopsis growth and cytokinin regulation1,2,15,28,29.", "appendix": "Author contributions\n\n\n\nJK and JAS designed the experiments, performed the experiments, analyzed the data and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by grants from NIFA/NRI (2005-35304-16043), NSF (IOS-0919991) and the Kentucky Tobacco Research and Development Center.\n\n\nReferences\n\nSmalle J, Vierstra RD: The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol. 2004; 55: 555–90. PubMed Abstract \| Publisher Full Text\n\nKurepa J, Smalle JA: Structure, function and regulation of plant proteasomes. 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PubMed Abstract \| Publisher Full Text\n\nSmalle J, Kurepa J, Yang P, et al.: The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling. Plant Cell. 2003; 15(4): 965–80. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "801", "date": "27 Feb 2013", "name": "Vitaly Citovsky", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nicely executed and clearly written work. The results are clear, and they support the authors’ conclusions and previously published hypotheses of proteasome involvement in cytokinin response. One potential enhancement would be to use qPCR to quantify the amount of transcripts, especially since these data represent one of the major findings of the paper.", "responses": [] }, { "id": "812", "date": "06 Mar 2013", "name": "Patrick Masson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nicely documents the molecular basis for an intragenic suppressor of the rpn12a-1 exon-trap mutation of Arabidopsis, which weakens a chimeric 5’ splice site that fuses the RPN12 open reading frame to the NPTII coding region in the original mutation. These authors had previously shown that the T-DNA insertion of rpn12a-1 results in a competition between 3’ splicing of RPN12A RNA (using a donor splice site that overlaps with the stop codon and the acceptor splice site upstream of its NPTII coding region of the T-DNA) and its normal polyadenylation. They had suggested that a fraction of the mutant transcripts encoded a non-functional RPN12A-NPTII fusion protein that, upon insertion into the proteasome, altered its activity. Hence, in the original mutant, overall altered proteasome activity resulted in pleiotropic phenotypes associated with cytokinin resistance compared to wild type. In this suppressor line, a point mutation 5 nucleotides within the cryptic intron altered this competing splicing, thereby restoring more efficient polyadenylation and production of enough functional RPN12a protein to restore fully functional proteasome activity. Hence, this analysis confirms the initial interpretation of the source of phenotypes associated with rpn12a-1, and documents an interesting example of alteration through mutation of a balance between 3’ splicing and polyadenylation of a precursor RNA.The design of this work, protocols and results are well presented and justify the conclusions. However, I had a few minor questions on this work:How many suppressors were identified in this analysis? Were other intragenic suppressors identified?Considering the information provided here, one would suspect that sor1 is a dominant mutation. Is it? If it is, has an experiment been carried out to show that a transgenic copy of the suppressed rpn12a-1 sor rescues the cytokinin-resistance phenotype of rpn12a-1?Analysis of kanamycin resistance in wild type C24, rpn2a-1 and sor1 seedlings showed that sor1 retains a reasonably high level of resistance compared to wild type (Fig 2). Yet, the molecular characterization described in Figures 3 and 4 shows no evidence of NPTII transcript or protein being produced in this suppressor. Is this a problem of experimental sensitivity? A brief discussion of this observation should be included in the text.", "responses": [] } ]	1	https://f1000research.com/articles/2-60
https://f1000research.com/articles/2-126/v1	10 May 13	{ "type": "Research Article", "title": "Demonstration of natriuretic activity in urine of neurosurgical patients with renal salt wasting", "authors": [ "Steven J Youmans", "Miriam R Fein", "Elizabeth Wirkowski", "John K Maesaka", "Miriam R Fein", "Elizabeth Wirkowski" ], "abstract": "We have utilized the persistent elevation of fractional excretion (FE) of urate, > 10%, to differentiate cerebral/renal salt wasting (RSW) from the syndrome of inappropriate antidiuretic hormone secretion (SIADH), in which a normalization of FEurate occurs after correction of hyponatremia. Previous studies suggest as well that an elevated FEurate with normonatremia, without pre-existing hyponatremia, is also consistent with RSW, including studies demonstrating induction of RSW in rats infused with plasma from normonatremic neurosurgical and Alzheimer’s disease patients. The present studies were designed to test whether precipitates from the urine of normonatremic neurosurgical patients, with either normal or elevated FEurate, and patients with SIADH, display natriuretic activity. Methods: Ammonium sulfate precipitates from the urine of 6 RSW and 5 non-RSW Control patients were dialyzed (10 kDa cutoff) to remove the ammonium sulfate, lyophilized, and the reconstituted precipitate was tested for its effect on transcellular transport of 22Na across LLC-PK1 cells grown to confluency in transwells.Results: Precipitates from 5 of the 6 patients with elevated FEurate and normonatremia significantly inhibited the in vitro transcellular transport of 22Na above a concentration of 3 μg protein/ml, by 10-25%, versus to vehicle alone, and by 15-40% at concentrations of 5-20 μg/ml as compared to precipitates from 4 of the 5 non-RSW patients with either normal FEurate and normonatremia (2 patients) or with SIADH (2 patients).Conclusion: These studies provide further evidence that an elevated FEurate with normonatremia is highly consistent with RSW. Evidence in the urine of natriuretic activity suggests significant renal excretion of the natriuretic factor. The potentially large source of the natriuretic factor that this could afford, coupled with small analytical sample sizes required by the in-vitro bioassay used here, should facilitate future experimental analysis and allow the natriuretic factor to be investigated as a potential biomarker for RSW.", "keywords": [ "The unresolved controversy over the prevalence of the cerebral salt-wasting", "or the preferred term", "renal saltwasting (RSW) syndrome", "can be ascribed", "in large part", "to difficulties in differentiating RSW", "where extracellular volume is depleted", "from the syndrome of inappropriate secretion of antidiuretic hormone (SIADH)", "where volume is normal or expanded. The overlapping laboratory characteristics", "clinical associations and our inability to assess accurately", "but non-invasively and rapidly", "the volume status of these patients are at the root of this diagnostic and consequential therapeutic dilemma1–4. In general", "neurosurgeons consider RSW to be more common than SIADH in neurosurgical patients", "whereas internists consistently regard RSW as a rare clinical entity. As reviewed elsewhere", "RSW has in fact been found to be much more common than SIADH in neurosurgical patients and our reports of RSW occurring in patients without clinical cerebral disease introduce new challenges to determine the true prevalence of RSW5", "6. This diagnostic dilemma poses a therapeutic dilemma that has significant clinical outcomes", "such as the risk inherent in fluid restricting a volume-depleted patient with RSW. In addition", "the recent awareness that even mild hyponatremia induces symptoms with potentially serious consequences including attention deficits", "mental confusion", "unsteadiness", "and heightened risk for falls and fractures7–9", "underscores the urgency needed to resolve this diagnostic dilemma so that the proper therapy can be instituted on first encounter with the patient7–9. Because of the erroneous perception that RSW is rare in neurosurgical patients and the unknown prevalence of RSW in patients without cerebral disease", "the inappropriate fluid restriction or use of vasopressin receptor blockers in patients with RSW for an erroneous diagnosis of SIADH is probably much more common than what has been documented in the literature. Indeed", "inappropriate fluid restriction of volume-depleted patients with RSW has been reported to have higher morbidity and mortality rates10–12. Our recent proposal to replace the inappropriate and outdated term", "cerebral salt wasting", "with RSW", "therefore", "has great clinical relevance because RSW would not in the past be considered unless the patient had evidence of cerebral disease1." ], "content": "Introduction\n\nThe unresolved controversy over the prevalence of the cerebral salt-wasting, or the preferred term, renal saltwasting (RSW) syndrome, can be ascribed, in large part, to difficulties in differentiating RSW, where extracellular volume is depleted, from the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), where volume is normal or expanded. The overlapping laboratory characteristics, clinical associations and our inability to assess accurately, but non-invasively and rapidly, the volume status of these patients are at the root of this diagnostic and consequential therapeutic dilemma1–4. In general, neurosurgeons consider RSW to be more common than SIADH in neurosurgical patients, whereas internists consistently regard RSW as a rare clinical entity. As reviewed elsewhere, RSW has in fact been found to be much more common than SIADH in neurosurgical patients and our reports of RSW occurring in patients without clinical cerebral disease introduce new challenges to determine the true prevalence of RSW5,6. This diagnostic dilemma poses a therapeutic dilemma that has significant clinical outcomes, such as the risk inherent in fluid restricting a volume-depleted patient with RSW. In addition, the recent awareness that even mild hyponatremia induces symptoms with potentially serious consequences including attention deficits, mental confusion, unsteadiness, and heightened risk for falls and fractures7–9, underscores the urgency needed to resolve this diagnostic dilemma so that the proper therapy can be instituted on first encounter with the patient7–9. Because of the erroneous perception that RSW is rare in neurosurgical patients and the unknown prevalence of RSW in patients without cerebral disease, the inappropriate fluid restriction or use of vasopressin receptor blockers in patients with RSW for an erroneous diagnosis of SIADH is probably much more common than what has been documented in the literature. Indeed, inappropriate fluid restriction of volume-depleted patients with RSW has been reported to have higher morbidity and mortality rates10–12. Our recent proposal to replace the inappropriate and outdated term, cerebral salt wasting, with RSW, therefore, has great clinical relevance because RSW would not in the past be considered unless the patient had evidence of cerebral disease1.\n\nWe have utilized the persistence of an increased fractional excretion (FE) of urate after spontaneous or therapeutic correction of hyponatremia in RSW to differentiate it from SIADH where FEurate normalizes after correction of hyponatremia2,12–14. This relationship between FEurate and serum sodium has been corroborated in several publications2,10–18. The coexistence of an elevated FEurate with normonatremia appears to also identify patients with RSW without the need to correct a pre-existing hyponatremia. This notion was supported by earlier studies, which demonstrated natriuretic activity when the plasma of largely normonatremic neurosurgical and Alzheimer's disease patients with elevated FEurate were administered to rats19,20. In those rat studies, natriuretic activity was noted when the plasma was administered simultaneously by IV infusion and injection into the peritoneum, suggesting that some of the natriuretic factor may have entered the rats’ circulation by the latter route. We entertained the possibility then that the factor could be a protein small enough to be absorbed from the peritoneum, and if so, small enough to be filtered by the glomerulus as well. We postulated that the circulating protein would be filtered at the glomerulus, possibly saturating receptor sites in the renal tubule and thereby making its way into the urine. The present studies were therefore designed to investigate the presence of natriuretic activity in ammonium sulfate precipitates of urine from patients admitted predominantly to our neuroscience intensive care unit with normal or increased FEurate with or without hyponatremia in addition to SIADH.\n\n\nMaterials and methods\n\nPatients included in this report were recruited mainly from the neuroscience intensive care unit of Winthrop-University Hospital. The protocol was approved by the Institutional Review Boards for human research at Winthrop-University Hospital and the New York College of Osteopathic Medicine (recently renamed the New York Institute of Technology College of Osteopathic Medicine) where the in vitro sodium transport studies were performed. Signed consent was obtained for each patient before initiating the study.\n\nThe clinical portion of the study consisted of the inclusion of tests for serum uric acid and urine osmolality, and determination of sodium, potassium, uric acid, and phosphate levels in addition to the routine tests for each patient. Exluded from the study were patients with serum creatinine greater than 1.1 mg/dl, those with edematous states, pregnant females, and those with abnormal thyroid or adrenal function. All urine passed was kept on ice at the bedside, with the addition of 80% by volume ammonium sulfate, before being transferred to the refrigerator and laboratory.\n\nLLC-PK1 cells, an epithelial line derived from porcine proximal tubules, were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen), 100 U/ml penicillin-100 µg/ml streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen) and 7.5% (w/v) sodium bicarbonate (Invitrogen). Cultures were grown at 37°C in a 5% CO2 atmosphere. Subculturing was performed every 3–4 days by trypsinization and reseeding at 1:20 dilution, and complete medium was replaced every 2 days. For experiments, cells were seeded on permeable supports that exposed both mucosal and serosal surfaces to the bathing media (12-well transwell filter inserts; Becton Dickenson Biocoat Control Inserts, 3 micron pore size, #354573) at a density of ≤ 2.5 × 105 cells/well and grown to confluence. All cells were serum-starved at least 24 hours prior to experiment.\n\nUrine samples were collected from normal, SIADH, and salt-wasting patients. The natriuretic factor(s) was precipitated out with ammonium sulfate (80% w/v). The precipitate was dialyzed for 48 hours, 20x the volume with > 3 changes, using a 10kDa membrane against deionized water (dH2O) to remove the ammonium sulfate, and the sample was then lyophilized and reconstituted in dH2O and stored at -80°C until use. The precipitate was quantitated on the basis of its protein content by the Bradford method31 and concentrations in the in vitro experiments were based on the protein concentration.\n\nAt the start of each experiment, the culture medium of the LLC-PK1 cells was replaced by a transport medium composed of NaCl 140 mM, KCl 5 mM, CaCl2 2 mM, MgCl2 1 mM, HEPES buffer 15 mM, and L-glutamine 2 mM, pH 7.4. In some experiments, 5 mM glucose was also included. Calcein, a fluorescent derivative of fluorescein, was used to evaluate transepithelial passive leak rates across cultures. Calcein at a concentration of 16 micromolar was added to the serosal (lower) compartment of each well (Becton Dickenson, Falcon 12-well plates, #353503; 1 cm wells) and timed samples were taken from the mucosal side. Fluorescence of the samples was read at 495/515 nm excitation/emission wavelengths in a Turner Model 430 Spectrofluorometer, calibrated against graded concentration standards over the range 0–0.4 micromolar, and leak-down rates per hour calculated. To measure sodium transport, 22Na (Perkin Elmer) was diluted in serum-free DMEM/F12 (Invitrogen/GIBCO, cat. #11330) and added to the mucosal side of the transwells (final specific activity 1 to 6 microCi/mmol Na; 0.1–0.6 µCi/ml). Urinary precipitates (typically 5–20 µl/ml in dH2O) when applied were resuspended and introduced to the media bathing both the mucosal and serosal surfaces of the epithelial cultures. Sodium transport was measured based on liquid scintillation counting (Packard PE Ultima Gold Liquid Scintillation Cocktail, Cat. #6013329, in a Packard Tri-Carb 2900 LS counter) of aliquots taken from the serosal side at specified time points. Aliquots of 10 microliters were taken from each 2 ml serosal compartment every 30–60 minutes for 1–2 hours prior to sample addition (baseline) and for 3–4 hours after (experimental). Preliminary experiments indicated that a 30 to 60 minute period was sufficient to achieve an isotopic steady-state. The steady-state rate of transport under any condition was determined as the slope (change in cpm/time) of the linear regression line of the individual cold-side activity determinations (cpm/10 µl) versus time. Each individual patient was considered n = 1. Statistical significance was determined based on Students’ t-test with p = 0.05 or less being considered significant.\n\nThe effect of any treatment on sodium transport was determined as follows. After the initial baseline-establishing period, resuspended urinary precipitate was added to the desired concentration of protein in the mucosal and serosal bathing solutions of the appropriate culture wells. (In some experiments bovine serum albumin (BSA) as a control rather than urinary precipitates was added. The BSA (Sigma Chemical #A7030) was made as a stock solution of 1 mg/ml in deionized water and added to final experimental concentrations of 2.5, 5, or 10 micrograms/ml in the mucosal and serosal bathing solutions.) Equal volumes of Vehicle only (dH2O) were added at the same time to Vehicle wells run side-by-side in each culture plate. Each experimental condition (RSW patient urine precipitate, non-RSW Control patient precipitate, BSA, or Vehicle) was run in quadruplicate wells in a given experiment.\n\nThe effect of any experimental addition on sodium transport was determined by comparison of the baseline slope to the post-addition slope (delta-m, change in slope, = (slope after treatment) - (slope before)). The specific effect of urine precipitate or BSA on sodium transport in a given experiment was calculated versus any effect seen with vehicle in the same experiment, i.e. as the deviation of the slope change for Experimental wells (precipitate or BSA) from that for Vehicle-only wells, the deviation being given by the expression [(ΔmE/ΔmV) – 1] × 100%, where ΔmE is the slope change for Experimental wells caused by precipitate (or BSA) addition and ΔmV is that for wells to which only vehicle was added.\n\nThe effects of urine precipitates from RSW patients and from unaffected (non-RSW) Control patients was determined in separate experimental runs, each versus Vehicle-only wells run side-by-side in each experiment.\n\nFinally, the specific effect of urine from the affected group of RSW patients was calculated versus any effect seen with the group of Control patient urines, i.e. as the mean effect for RSW/Experimental urine precipitates versus the mean effect for Control-patient urine precipitates.\n\nThe effect of RSW or Control urine precipitates on sodium movement was alternatively calculated as the slope after addition of precipitates versus the slope before addition in the same wells (without reference to Vehicle wells). However, that calculation proved to yield a higher degree of statistical scatter and was not ultimately used.\n\nData are reported as the means ± S.E.M for both the number of patients and the number of experimental runs. Within a given run, data points are the mean values from four individual culture wells. Statistical differences were calculated based on the number of patients and were determined by unpaired Student's t test or analysis of variance where appropriate using Sigma Plot 9. Values of p < 0.05 were considered statistically significant.\n\n\nResults\n\nThe study population was composed of patients with neurosurgical diseases of various etiologies who were admitted to the Winthrop-University Hospital neurosurgical intensive care unit, Table 1. After obtaining a signed informed consent, patients recruited into the study were divided into three groups, based on whether or not the patient had a normal or increased FEurate (normal 5–10%) or SIADH. Group I, controls, consisted of those with normal FEurate. Group II, SIADH, had increased FEurate, hyponatremia, and decreased baseline plasma renin and aldosterone levels. Group III, RSW, had increased FEurate with normonatremia, a combination that is consistent with RSW but not SIADH. Table 1 summarizes the pertinent clinical information, including diagnoses, age, gender, serum electrolytes, creatinine, phosphate and urate, urine sodium, and fractional excretion (FE) of sodium, urate and phosphate.\n\nInitial experiments revealed that certain culture conditions had to be met to achieve consistency of transport pattern from day to day. In order to achieve this consistency, the cell cultures had to go through a minimum of 10–12 passages after initial growth from frozen stocks, be plated at initial concentrations of 2.5 × 105 per 2.3 cm diameter well [6 × 104/cm2] or less, and be utilized for transport studies 12–16 days after plating. The cultures were tested for confluence and transepithelial leakage using the fluorescent marker calcein, as described in the Methods. Using this method, cultures were shown to have passive leakage rates of < 1% per hour (0.7 ± 0.7%/hr, mean ± SD, range 0–2.2%/hr; n = 17 plates of 12 wells each). The cell monolayers also showed a high degree of restriction to movement of sodium isotope over a period of hours, indicating complete confluence and consistent formation of intercellular tight junctions throughout the study, as evidenced by the results of six consecutive experiments in which vehicle was the only addition to the bathing medium, shown in Figure 1. The results indicate that there was isotopic equilibrium within 30 minutes and consistent linear transport over 5 hours that was reproducible from day to day (Figure 1). Transcellular transport became erratic and nonlinear after 5 hours, thereby limiting all subsequent experiments to < 5 hrs.\n\nValues at each time point are means ± SEM for 6 consecutive experiments with 3 or 4 wells per experiment at each time point. Number of data points at each time point are: 0.5 h (13); 1 h (19); 2 h (19); 3 h (20); 4 h (20); 5 h (20); 6 h (6).\n\nTransepithelial movement of 22Na in cells exposed to precipitates from patient urine. Because data from Groups I and II were indistinguishable from each other, the data from both groups were pooled as a non-RSW control group. After attaining suitable culture conditions, resuspended precipitates from urine of Group III patients, applied to both mucosal and serosal bathing media, significantly inhibited sodium fluxes as compared to vehicle alone, outcomes that were distinctly different from those for Groups I and II. Of 6 patients initially in Group III, precipitates from 5 patients (Table 1) showed a decrease in sodium transport of 10–25% at concentrations greater than 3 µg protein/ml, as compared to vehicle alone in wells run side-by-side in each experiment, Figure 2 (P < 0.05 vs. vehicle).\n\nData are from 5 of 6 RSW patients with 6–16 experiments at each of the protein concentrations 5, 10, and 20 µg/ml. Values are versus vehicle set = 0 at each concentration. Significance levels vs. vehicle: At 5 µg protein/ml, P = 0.03 (); 10 µg, P = 0.03 (); 20 µg, P = 0.05 (#).\n\nIn contrast, urinary precipitates from 4 out of the 5 patients in Groups I/II (Table 1) did not inhibit sodium transport versus vehicle. In fact, mean sodium transport for Group I/II were mostly positive or increased, although statistical significance versus vehicle was not reached at any concentration in the ranges tested, Figure 3 (P = 0.09 to 0.82 @ 1–20 µg).\n\nData from 4 non-RSW control patients (2 neurosurgical patients with normal FEurate and normonatremia and 2 SIADH) with 4–10 experiments at each protein concentration. Values are versus vehicle set = 0 at each concentration. Significance levels vs. vehicle: At 1 µg, protein/ml, P = 0.42; 5 µg, P = 0.09; 10 µg, P = 0.21; 20 µg, P = 0.82.\n\nWhen the results for Group III patients were compared directly with Group I/II control patients, the differences were striking, as shown in Figure 4 and Table 2. Sodium transport by the Group III urine precipitates decreased significantly by 15–40% compared to Group I/II (P < 0.02 at 5 and 10 µg/ml).\n\nValues are versus Controls set = 0 at each concentration. Significance levels, Group III/RSW vs. Group I-II/Control: At 5 µg protein/ml, P = 0.012 (); 10 µg, P = 0.013 (); 20 µg, P = 0.24.\n\nEffect on absorptive sodium flux of urine precipitates from Group I-II vs. vehicle; Group III/RSW vs. vehicle; and Group III vs. Group I-II.\n\n(a), Group I-II, 4 patients except for 1 µg/ml point (2 patients, 5 experimental runs).\n\n(b), Group III, 5 patients except for 1 µg/ml point (1 patient, 3 experimental runs).\n\nTransepithelial transport of 22Na determined at different concentrations of precipitate on the same day for each patient in Group III. In these experiments, Group III precipitates consistently inhibited 22Na transport in a dose-dependent manner at concentrations of 5 to 10 µg protein and 10 to 20 µg (Figure 5). We also tested bovine serum albumin (BSA) at similar concentrations to assess whether a non-specific protein effect might be occurring. BSA did not significantly alter sodium transport from a baseline of zero (P = 0.2 to > 0.95 at concentrations of 2 to 10 µg/ml, data not shown). Therefore, the data indicate that a natriuretic factor(s) exists in the urine of Group III patients that inhibits transcellular sodium transport in cultured LLC-PK1 cells in a dose-dependent manner. These data are also consistent with previous demonstration of a dominant effect of plasma from neurosurgical and Alzheimer disease patients on rat proximal tubule transport of sodium and lithium19,20.\n\nWithin a given experimental run, the effect of 10 µg protein was compared to that of 5 µg (each measured vs. vehicle) or the effect of 20 µg was compared to that of 10 µg (each versus vehicle). Significance levels: 10 µg vs. 5 µg, P = 0.046 (3 patients/6 experimental runs); 20 vs. 10 µg protein, P = 0.0038 (3 patients/5 experimental runs); 10 to 20 µg step versus 5 to 10 µg step, P = 0.045 (group comparison). Control patients showed no significant effect for either 5 to 10 or 10 to 20 µg concentration step (P > 0.3). P values calculated based on number of patients.\n\n\nDiscussion\n\nThis study demonstrates the presence of a factor, in the urine of 5 of 6 neurosurgical patients with increased FEurate and normonatremia, that inhibits transcellular transport of 22Na in LLC-PK1 cells grown to confluency in transwells (Group III). By contrast, ammonium sulfate precipitates of urine from neurosurgical patients with normal FEurate and normonatremia and those with SIADH did not inhibit transcellular transport of 22Na, suggesting that the natriuretic factor was present only in the urine of patients with increased FEurate and normonatremia. These data are consistent with previous rat clearance studies, which demonstrated natriuretic activity in the plasma of neurosurgical and Alzheimer’s disease patients who had increased FEurate and normonatremia as compared to two control groups, composed of (1) age- and gender-matched controls and (2) patients with multi-infarct dementia, both groups with normal FEurate and normonatremia19,20. These studies collectively support our proposal that an increased FEurate with normonatremia is consistent with RSW and represent an initial step to utilize the natriuretic factor as a biomarker for RSW1,2.\n\nThe plasma natriuretic factor in those previous rat clearance studies significantly increased FElithium and FENa without change in glomerular filtration rate, blood pressure, or urine flow rate. FElithium increased from control means of 24.0% and 27.2% to 36.6% and 41.7%, respectively, when plasma from neurosurgical and Alzheimer disease patients were injected into rats, and FENa changed from controls of 0.29% and 0.33% to 0.59% and 0.63% respectively19,20. Since lithium, in the absence of nonreabsorbable solutes such as mannitol, is transported over sodium pathways in the proximal tubule, the increases in FElithium suggested that the natriuretic factor had its major effect in the proximal tubule21,22. These data support our clinical impression that the increase in FEurate, a solute transported exclusively in the proximal tubule, indicated both a defect in solute transport in the proximal tubule of these patients and a defect involving more than one solute14.\n\nIn those rat clearance studies, we administered 0.5 ml of plasma intraperitoneally 90 minutes prior to anesthetizing the animal and, under anesthesia, we rapidly infused 0.2 ml plasma as a primer and infused 1.8 ml of plasma at a rate of 0.01 ml/min over the next 3 hours. This maneuver was intended to increase the time of exposure of the natriuretic factor while minimizing duration of anesthesia to assure physiological integrity of the animal. FElithium and FENa increased progressively, attaining significance approximately 4 hours after intraperitoneal injection of plasma19,20. The fact that some of the human plasma was administered peritoneally raised the possibility that some of the natriuretic factor may have entered the rats’ circulation via that route. We reasoned that the natriuretic factor could be a protein of small enough size to pass through the peritoneal membrane, and hence small enough to be filtered at the glomerulus, possibly saturate downstream tubular receptor sites, and be excreted in the urine. In the present study then, we therefore collected urine from prospective study patients and tested ammonium sulfate precipitates of urine for natriuretic activity in a transwell culture system. The inhibition of transcellular sodium transport by urinary ammonium sulfate precipitates of Group III patients suggests that the kidneys had indeed excreted the natriuretic factor and perhaps concentrated it as well. Clearly, the combination of small sample-sizes required by our in-vitro bioassay, together with a ready experimental source of the natriuretic factor in urine, should simplify purification and identification of the natriuretic factor going forward.\n\nAtrial/brain natriuretic peptides (A/BNP) have been suggested as a possible cause of RSW. A/BNP has in fact been shown to be increased in conditions often associated with RSW such as subarachnoid hemorrhage, as well as in non-salt-wasting conditions such as SIADH and in salt-retaining conditions such as congestive heart failure27–29. However, the precipitates employed in the in vitro sodium transport experiments reported here were exhaustively dialyzed against 10 kDa-cutoff membranes. Hence, it’s unlikely that ANP (human MW_3080) or BNP (human MW_3464) were retained and present to any appreciable extent in the retentate to which the cultured cells were exposed. Furthermore, the physiological effects of ANP and BNP are distinctly different from those of the natriuretic factor observed in the earlier rat clearance studies23–26. A/BNP increase GFR, decrease renal blood flow and blood pressure, and increase urine flow rate and sodium excretion but have a greater decreasing effect on distal as compared to proximal tubule sodium transport. The natriuretic factor in our previous rat clearance studies did not increase GFR, decrease blood pressure or increase urine flow rate, and the increase in FElithium far exceeded the increase in FENa, suggesting that the natriuretic factor had a greater effect on proximal than distal tubule sodium transport19,20, concordant with the other considerations discussed above which indicate defects in proximal tubule solute transport in RSW. The properties then of the natriuretic effect noted in the plasma of patients in the earlier rat study and urine of patients in the present study with a very high probability for RSW strengthens our proposal that A/BNP is an unlikely contributor to RSW.\n\nIt should also be noted that the predominant stimuli for increased A/BNP release are related to increases in extracellular volume and blood pressure, the opposite of what is seen in RSW23,24. This was seen strikingly in the low normal value of 35 pg/ml of ANP we reported in a volume-depleted patient with unequivocal RSW6, and does not support the common perception that A/BNP is the major natriuretic factor in RSW. This instructive case of RSW without cerebral disease had a decreased blood volume of 7.1% as determined by the gold standard radioisotope dilution method, using 51Cr-tagged red blood cells and 131I-tagged albumin, and increased baseline plasma renin and aldosterone levels, features all consistent with RSW. The subsequent infusion of saline removed the stimulus for ADH secretion and permitted the coexisting hypo-osmolality to inhibit ADH secretion, increase free water excretion and result in prompt correction of hyponatremia within 48 hours after initiation of saline therapy6, responses again consistent with RSW.\n\nBecause of divergent therapeutic goals for SIADH and RSW (fluid restriction for SIADH and fluid supplementation for RSW), it is important to differentiate SIADH from RSW and thus avoid selecting the incorrect mode of therapy that can lead to increased morbidity and mortality. The patient with an increased FEurate with normonatremia without going through a phase of hyponatremia probably has RSW and should be treated with saline. In the case of the patient who presents with hyponatremia, there are ample clinical data to support the notion that the persistence of an elevated FEurate in RSW can be contrasted to normalization in SIADH after correction of the hyponatremia. However, this unique association between FEurate and hyponatremia is limited by the need to correct a preexisting hyponatremia, which is not always present, as just noted, and in any case may be difficult to achieve on a timely basis, and is hence not ascertainable on first encounter with the patient when therapeutic decisions are being made. On the other hand, a normal FEurate in a nonedematous hyponatremic patient consistently identifies patients with a reset osmostat, who fall into a different physiological and treatment group than the patients being addressed in this communication30. The unique relationship between serum sodium and FEurate can, therefore, be utilized to establish or rule out the diagnosis of a reset osmostat and to differentiate RSW from SIADH, but for practical reasons can not be applied in every case2. Diagnostic dilemmas that remain could be resolved simply if the natriuretic factor in the current study can be identified and validated as a biomarker of RSW. We believe the data from the current study are a major advancement to that ultimate identification of the natriuretic factor and developing the factor as a potential biomarker of RSW.", "appendix": "Author contributions\n\n\n\nJKM conceived the study, recruited patients, and drafted the manuscript; SJY contributed to writing and editing the manuscript, prepared the figures, and executed many of the experiments; MFC executed many of the experiments and contributed to writing and editing the manuscript; EW contributed to the design of the study and recruited patients.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThe study was supported in part by research grant GA2580F8 from Pfizer.\n\n\nAcknowledgments\n\nThe authors wish to thank John H. Durham, M.D./Ph.D. (Palmetto Nephrology PA, Orangeburg, SC 29118), for valuable discussions regarding the design of the study and interpretation of results.\n\n\nReferences\n\nMaesaka JK, Imbriano LJ, Ali NM, et al.: Is it cerebral or renal salt wasting? Kidney Int. 2009; 76: 934–938. PubMed Abstract \| Publisher Full Text\n\nMaesaka JK, Imbriano L, Shirazian S, et al.: Complexity of differentiating cerebral-renal salt wasting from SIADH, emerging importance of determining fractional urate excretion. In: Vijayakumar S, editor. Novel Insights on chronic kidney disease, acute kidney injury and polycystic kidney disease. Croatia: InTech. Reference Source\n\nOh MS, Carroll HJ: Cerebral salt-wasting syndrome, we need better proof of its existence. Nephron. 1999; 82: 110–114. PubMed Abstract \| Publisher Full Text\n\nChung HM, Kluge R, Schrier RW, et al.: Clinical assessment of extracellular fluid volume in hyponatremia. Am J Med. 1987; 83: 905–908. PubMed Abstract\n\nBitew S, Imbriano L, Miyawaki N, et al.: More on renal salt wasting without cerebral disease: response to saline infusion. Clin J Amer Soc Nephol. 2009; 4: 309–315. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaesaka JK, Miyawaki N, Palaia T, et al.: Renal salt wasting without cerebral disease: diagnostic value of urate determining in hyponatremia. Kidney Int. 2007; 71: 822–826. PubMed Abstract \| Publisher Full Text\n\nGankam Kengne F, Andres C, Sattar L, et al.: Mild hyponatremia and risk of fracture in the ambulatory elderly. QJM. 2008; 101(7): 583–588. PubMed Abstract \| Publisher Full Text\n\nRenneboog B, Musch W, Vandemergel X, et al.: Mild chronic hyponatremia is associated with falls, unsteadiness, and attention deficits. Am J Med. 2006; 119: 71.e1–8. PubMed Abstract \| Publisher Full Text\n\nSchrier RW: Does 'asymptomatic hyponatremia' exist? Nat Rev Nephrol. 2010; 6: 185. 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PubMed Abstract\n\nDorhout Mees EJ, Blom van Assendelft P, Nieuwenhuis MG: Elevation of uric aicd clearance caused by inappropriate antidiuretic hormone secretion. Acta Med Scand. 1971; 189: 69–72. PubMed Abstract\n\nBeck LH: Hypouricemia in the syndrome of inappropriate secretion of antidiuretic hormone. N Engl J Med. 1979; 301: 528–530. PubMed Abstract \| Publisher Full Text\n\nSonnenblick M, Rosin A: Increased uric acid clearance in the syndrome of inappropriate secretion of antidiuretic hormone. Isr J Med Sci. 1988; 24: 20–23. PubMed Abstract\n\nDecaux G, Prospert F, Cauchie P, et al.: Dissociation between uric acid and urea clearances in the syndrome of inappropriate secretion of antidiuretic hormone related to salt excretion. Clin Sci (Lond). 1990; 78(5): 451–5. PubMed Abstract\n\nMaesaka JK, Venkatesan J, Piccione JM, et al.: Plasma natriuretic factor(s) in patients with intracranial disease, renal salt wasting and hyperuricosuria. Life Sci. 1993; 52(23): 1875–1882. PubMed Abstract\n\nMaesaka JK, Wolf-Klein G, Piccione JM, et al.: Hypouricemia, abnormal renal tubular urate transport, and plasma natriuretic factor(s) in patients with Alzheimer's disease. J Am Geriatr Soc. 1993; 41: 501–506. PubMed Abstract\n\nLeyssac PP, Holstein-Rathlou NH, Skøtt P, et al.: A micropuncture study of proximal tubular transport of lithium during osmotic diuresis. Am J Physiol. 1990; 258(4 Pt 2): F1090–F1095. PubMed Abstract\n\nDorhout Mees EJ: History of the \"lithium concept\". Kidney Int Suppl. 1990; 28: S2–S3. PubMed Abstract\n\nde Zeeuw D, Janssen WM, de Jong PE: Atrial natriuretic factor: its (patho)physiological significance in humans. Kidney Int. 1992; 41: 1115–1133. PubMed Abstract \| Publisher Full Text\n\nJensen KT, Carstens J, Pedersen EB: Effect of BNP on renal hemodynamics, tubular function and vasoactive hormones in humans. Am J Physiol. 1998; 274: F63–F72. PubMed Abstract\n\nEllison DH, Berl T: Clinical practice. The syndrome of inappropriate antidiuresis. N Engl J Med. 2007; 356: 2064–72. PubMed Abstract \| Publisher Full Text\n\nPalmer BF: Hyponatremia in patients with central nervouos system disease: SIADH versus CSW. Trends endocrinol metab. 2003; 14: 182–187. PubMed Abstract \| Publisher Full Text\n\nFukui S, Nawashiro H, Otani N, et al.: Focal brain edema and natriuretic peptides in patients with subarachnoid hemorrhage. Acta Neurochir Suppl. 2003; 86: 489–91. PubMed Abstract\n\nManoogian C, Pandian M, Ehrlich L, et al.: Plasma atrial natriuretic hormone levels in patients with the syndrome of inappropriate antidiuretic hormone secretion. J Clin Endocrinol Metab. 1988; 67(3): 571–5. PubMed Abstract \| Publisher Full Text\n\nNakaoka H, Imataka K, Amano M, et al.: Plasma levels of atrial natriuretic factor in patients with congestive heart failure. N Engl J Med. 1985; 313(14): 892–3. PubMed Abstract \| Publisher Full Text\n\nImbriano LJ, Ilamathi E, Ali NM, et al.: Normal fractional urate excretion identifies hyponatremic patients with reset osmostat. J Nephrol. 2012; 25(5): 833–8. PubMed Abstract\n\nBradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248–54. PubMed Abstract \| Publisher Full Text" }	[ { "id": "942", "date": "13 May 2013", "name": "Pravin C Singhal", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 investigated salt wasting properties in the urine of neurological patients with salt wasting. They have used in vitro techniques to achieve their outcome. These studies are novel though preliminary and worth publishing.", "responses": [] }, { "id": "956", "date": "20 May 2013", "name": "Albert Dreisbach", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a well designed study of a potentially significant and novel natriuretic factor in the urine of patients with elevated fractional urate excretion with renal salt wasting. This factor appears to have properties that distinguish it from ANP and BNP and may be entirely unique.Minor criticisms:Methodological descriptions seem to run into the results and discussion which maybe more appropriate in the methods section. For example the first paragraph under the “Transcellular Transport of 22Na” in the results section describes the calibration of system might be more appropriate under methods.If any objective blood volume data available it would support the differentiation of the RSW and SIADH patients.The use Uosm instead of meq/L or mmole/ L for Na and K is not the usual convention.", "responses": [] } ]	1	https://f1000research.com/articles/2-126
https://f1000research.com/articles/2-77/v1	05 Mar 13	{ "type": "Research Article", "title": "Continuous and intermittent exposure of neonatal rat calvarial cells to PTHrP (1-36) inhibits bone nodule mineralization in vitro by downregulating bone sialoprotein expression via the cAMP signaling pathway", "authors": [ "Suzan A Kamel", "John A Yee", "Suzan A Kamel" ], "abstract": "The development and growth of the skeleton in the absence of parathyroid-hormone-related protein (PTHrP) is abnormal. The shortening of appendicular bones in PTHrP gene null mice is explained by an effect of PTHrP on endochondral bone growth. Whether or not PTHrP influences intramembranous ossification is less clear. The purpose of this study was to determine the effect of exogenous PTHrP on intramembranous ossification in vitro. Neonatal rat calvarial cells maintained in primary cell culture conditions that permit spontaneous formation of woven bone nodules by intramembranous ossification were studied. The expression of PTHrP, parathyroid hormone 1 receptor (PTH1R), and alkaline phosphatase (AP) by osteogenic cells in developing nodules and the effects of PTHrP (1-36) on nodule development was determined over 3-18 days. PTHrP and PTH1R were detected colonies of osteogenic cells on culture day three, and AP was detected on day six. PTHrP and its receptor were localized in pre-osteoblasts, osteoblasts, and osteocytes, and AP activity was detected in pre-osteoblasts and osteoblasts but not osteocytes. Continuous and intermittent exposure to PTHrP (1-36) decreased the number of mineralized bone nodules and bone sialoprotein (BSP) mRNA and protein, but had no effect on the number of AP-positive osteogenic cell colonies, cell proliferation, apoptosis, or osteopontin (OPN) mRNA. These results demonstrate that osteogenic cells that participate in the formation of woven bone nodules in vitro exhibit PTHrP and PTH1R before they demonstrate AP activity. Exogenous PTHrP (1-36) inhibits the mineralization of woven bone deposited during bone nodule formation in vitro, possibly by reducing the expression of BSP.", "keywords": [ "Parathyroid-hormone-related protein (PTHrP) is the causative factor of humoral hypercalcemia of malignancy1", "2. Although originally associated with certain types of cancer", "it is now known that PTHrP is expressed by a variety of normal cell types in many fetal and adult tissues", "including bone3", "4. Stunted growth of the limbs and abnormalities in the craniofacial skeleton of newborn PTHrP-gene null mice served as the first evidence that PTHrP was necessary for normal skeletal development and growth5. Subsequent studies using knockout and transgenic mice revealed that PTHrP influences longitudinal bone growth by controlling chondrocyte differentiation in the epiphyseal growth cartilage6", "7. While development of membrane bones is also altered in PTHrP knockout mice5", "how the peptide contributes to intramembranous ossification has not been clarified." ], "content": "Introduction\n\nParathyroid-hormone-related protein (PTHrP) is the causative factor of humoral hypercalcemia of malignancy1,2. Although originally associated with certain types of cancer, it is now known that PTHrP is expressed by a variety of normal cell types in many fetal and adult tissues, including bone3,4. Stunted growth of the limbs and abnormalities in the craniofacial skeleton of newborn PTHrP-gene null mice served as the first evidence that PTHrP was necessary for normal skeletal development and growth5. Subsequent studies using knockout and transgenic mice revealed that PTHrP influences longitudinal bone growth by controlling chondrocyte differentiation in the epiphyseal growth cartilage6,7. While development of membrane bones is also altered in PTHrP knockout mice5, how the peptide contributes to intramembranous ossification has not been clarified.\n\nThe amino terminus of PTHrP has strong homology with the comparable region of parathyroid hormone (PTH)8 and both peptides are physiologic ligands for the parathyroid hormone 1 receptor (PTH1R)9, a member of the B family of heterotrimeric G-protein-coupled receptors. The binding of either ligand to PTH1R in bone or kidney induces a conformational change in the receptor that results in the formation of a high affinity receptor-G-protein complex that catalyzes guanine nucleotide exchange on the α-subunit of the G-protein10–12. This causes the G-protein to dissociate from the receptor-ligand complex and transduce the extracellular signal from the receptor to at least two signaling pathways, the adenylate cyclase/cAMP/protein kinase A (PKA) pathway that is mediated through the Gαs subunit, and the phospholipase C/diacylglycerol/protein kinase C (PKC) pathway that is mediated through the Gαq subunit13. It is now recognized that exogenously administered amino terminal PTH has anabolic and catabolic effects in the skeleton. Whether bone formation or bone resorption occurs depends to some extent on the pattern or duration of hormone exposure. Intermittent exposure to PTH (1-34) in one or two daily subcutaneous injections increases bone formation and results in a net gain in bone mass in rats and humans14,15. By contrast, continuous infusion of PTH (1-34) preferentially stimulates bone resorption and increases serum calcium in humans16 and rats15,17, with smaller effects on bone formation. The net response to continuous infusion is a decrease in bone mass15,18. Although amino terminal PTH and PTHrP bind with equal affinity to the PTH1R, intermittent PTHrP has been reported to be less effective in stimulating bone formation than equal concentrations of PTH15. This finding is interesting in light of the observation that locally produced PTHrP is required for bone formation in PTH-null mice19. How the contrasting effects of exogenous PTHrP are related to its physiologic function remains to be clarified.\n\nIn several tissues, including bone, it has been reported that PTHrP and PTH1R mRNAs and/or proteins are expressed by adjacent, but different, cell populations20–22. This has led to the concept that PTHrP is a paracrine factor that regulates the proliferation, differentiation, lifespan, or function of its target cells in these tissues23. We previously found that PTHrP and PTH1R are co-expressed by skeletal and extraskeletal cells before and during intramembranous ossification of the chick mandible24. On the basis of their temporal and spatial expression we proposed that PTHrP influences the histogenesis and/or growth of skeletal tissues in the chicken mandible via an autocrine pathway mediated by the PTH1R. The present study was designed to investigate the role of PTHrP during intramembranous ossification using neonatal rat calvarial cell cultures as an in vitro model. First we compared the temporal and spatial expression of PTHrP and PTH1R with the osteoblast marker protein alkaline phosphatase (AP) during the differentiation of osteoblast cell colonies and formation of woven bone nodules. Next, we examined the effect of continuous and intermittent treatment with amino terminal PTHrP on osteoblast differentiation and bone nodule formation. Finally, possible mechanisms underlying PTHrP effects on bone nodule formation were examined by assessing cell proliferation, apoptosis, and gene expression.\n\n\nMaterials and methods\n\nPTHrP (1-36) and PTHrP (7-34) were purchased from Bachem (Torrance, CA, USA). Rabbit anti-PTHrP antibody was from Oncogene Research Products (Boston, MA, USA), rabbit anti-PTH1R antibody was from BAbCo (Richmond, CA, USA), and rabbit anti-BSP antibody was from Chemicon International (Temecula, CA, USA). Alexa fluor 488 conjugated goat anti-rabbit immunoglobulin-G (IgG) antibody was purchased from Molecular Probes, Inc. (Eugene, OR, USA). The Taqman reverse transcription kit, SYBR green polymerase chain reaction (PCR) master mix and PCR primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), osteopontin (OP), and bone sialoprotein (BSP) were from Invitrogen (Carlsbad, CA, USA). Supersignal west pico chemiluminescent peroxidase substrate kit was from Thermo Scientific (Rockford, IL, USA). The vectastain ABC kit and diaminobenzidine (DAB) peroxidase substrate kit were from Vector Laboratories (Burlingame, CA, USA). All other chemicals were from Sigma Chemical Co. (St. Louis, MO, USA).\n\nThe Creighton University Institutional Animal Care and Use Committee (Vertebrate Animal Assurance Number A3348-01) approved all animal procedures employed in this study. Pregnant Sprague-Dawley rats were purchased from Charles River, housed in the Association for Assessment and Accreditation of Laboratory Animal Care International- (AAALAC) accredited Creighton University animal resource facility, and allowed to deliver their offspring. Rat calvarial (RC) cells were isolated from calvariae of 2- to 3-day-old rat pups by digestion with crude collagenase and cultured in Dulbecco’s minimum essential medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), ascorbic acid (10 µg/ml), and β-glycerophosphate (5 mM). These cell culture reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA. In order to determine the effects of PTHrP on colony producing cells, cultures were initially seeded at a plating density of 75 cells/cm2. Two treatment protocols were used to examine the effects of PTHrP on colony-forming RC cells. For continuous exposure, PTHrP (1-36) was present in the culture medium for the entire treatment period and, for intermittent exposure, PTHrP (1-36) was present for the first 1 or 6 hr of each 48-hr treatment period. After these brief exposures, the cells were maintained in control medium for the remainder of the 48-hr treatment cycle.\n\nRC cell cultures were fixed in cold 4% paraformaldehyde, washed with distilled water, and incubated in 1% aqueous silver nitrate for 30 min in the dark. After removing the staining solution the cells were exposed to bright light for 1 hr. Mineralized bone nodules were counted at a 100X magnification using a Nikon inverted phase-contrast microscope.\n\nFor confocal microscopy, RC cells were grown on glass cover slips. Specimens were collected on culture days 3, 6, 9, 12, 16, 18 and 20. Cells were fixed in cold 4% paraformaldehyde and stained for AP (see below). The cover slips were then placed in a 24-well culture plate and washed with phosphate buffered saline (PBS) containing 1% BSA and 0.1% Tween 20. Samples collected on days 16, 18, and 20 were permeabilized with 0.1% Triton X-100 in 0.1% (w/v) sodium citrate buffer. The cells were incubated with rabbit anti-PTHrP antibody (2.5 μg/ml) or rabbit anti-PTH1R antibody (1:100) overnight at 4°C, washed with the blocking buffer, and incubated with Alexa fluor 488 conjugated goat anti-rabbit antibody (1:500) for one hr at room temperature. Washed cover slips were mounted on glass slides and examined by confocal microscopy (Radiance 2000, Bio-Rad). Immunostaining was visualized using 488 nm excitation and 515/30 nm emission filters. The AP reaction product, which is autofluorescent, was visualized using 568 nm excitation and 600/40 nm emission filters. Individual images were merged using BioRad software.\n\nIntracellular cAMP was measured using an enzyme immunoassay (EIA) kit purchased from Sigma Chemical Co. (St. Louis, MO, USA). For this experiment RC cells were plated into a 96-well plate at 10,000 cells/cm2 and incubated overnight at 37°C. The experiment was initiated by adding fresh culture medium containing 0.5 mM 3-isobutyl-1-methylxanthine and 0, 0.1, 1, 10, or 100 nM PTHrP (1-36). After 10 min the medium was replaced with cell lysis reagent. cAMP was measured according to the instructions provided with the EIA kit.\n\nCells that expressed AP were identified by enzyme histochemistry. Paraformaldehyde-fixed cells were incubated in 0.1 M Tris-HCl (pH 8.5) buffer containing 0.01% (w/v) naphthol AS-MX phosphate and 0.07% (w/v) fast red violet LB salt for 30 min in the dark.\n\nCell proliferation was determined by measuring the incorporation of BrdU. RC cells plated in a 96-well plate at 200 cells/cm2 were treated with 100 nM PTHrP (1-36) intermittently or continuously for four days. On day four, BrdU (10 µM) was added to each well. After 5 hr the cells were fixed with pre-cooled acidic 70% ethanol, treated with nuclease (1:100) for 30 min at 37°C, and BrdU incorporation was detected as recommended by the manufacturer of the BrdU ELISA kit.\n\nApoptotic cells were identified using an in situ cell death detection staining kit. Cells fixed with 4% paraformaldehyde were washed with 3% H2O2 in methanol, rinsed with PBS, permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice, and incubated in TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark. After washing, the cells were incubated with peroxidase-conjugated anti-fluorescein antibody for 30 min at 37°C. Peroxidase histochemistry using DAB was used to visualize apoptotic cells. Apoptosis is expressed as the percentage of TUNEL-positive cells in RC cell colonies.\n\nTotal RNA was extracted from RC cells using a high purity RNA isolation kit and cDNA was synthesized using a Taqman reverse transcription kit. Quantitative PCR was performed using an ABI prism 7700 sequence detector. Amplified products were detected using SYBR Green PCR Master Mix. The forward and reverse primer sequences for OP were 5 AAAGTCGCTGACTTTGGCAG 3 and 5 AAGTGGCTACAGCATCTGAGTGT 3, and for BSP were 5 CCGGCCACGCTACTTTCTT 3 and 5 CCTGGACTGGAAACCGTTTC 3.\n\nRC cells were homogenized in lysis buffer (10 mM Tris pH 7.4, 1% SDS, 1 mM sodium orthovanadate), boiled for 5 min, and centrifuged at 15,000Xg. The protein concentration in the supernatant was measured using the bicinchoninic acid (BCA) assay. Samples containing 30 μg protein were loaded on 10% polyacrylamide minigels and electrophoresed at a constant current of 25 ma. Proteins were then transferred to nitrocellulose membrane. The blots were blocked in 5% nonfat dry milk and 0.1% Tween 20 in PBS for one hr and then incubated with a rabbit anti-BSP antibody (1:2500) overnight at 4°C. After washing, the membrane was incubated in horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:40,000) [Amersham Biosciences, Amersham, UK] for one hr at room temperature. Peroxidase was detected using the supersignal west pico chemiluminescent kit and recorded on BioMax X-ray film.\n\nDifferences between the control and treated groups were determined by one-way ANOVA and the Bonferroni post-hoc test using Prism software (v4.00 for windows, GraphPad Software, San Diego, CA). A P<0.05 was considered significant.\n\n\nResults\n\nThe temporal expression of PTHrP, PTH1R, and AP in cell colonies that developed in cultures plated at low density is shown in Figure 1A. Cell colonies immunopositive for PTHrP and PTH1R were observed on culture day three (Figure 1A, Images A and E); however, AP-positive cell colonies were not present at this time. By day six and later, some, but not all cells in the PTHrP- and PTH1R-positive colonies exhibited AP activity (Figure 1A, Images C-D and Images G-H). A single bone nodule from a 20-day culture that was double-stained for PTHrP (Images A-E) and AP (Images F-J) and optically sectioned by confocal microscopy is shown in Figure 1B. All of the cells near the top of the nodule were positively stained for PTHrP and AP (Images A, B, F, G, K and L). Cells located deeper within the nodule in the area that contained mineralized matrix (Images C-E, H-J, and M-O) were positive for PTHrP, but AP negative. A similar pattern of staining was observed in nodules double-stained for PTH1R and AP (Figure 1C).\n\nA. Cells were double-stained for PTHrP (A–D) or PTH1R (E–H) immunoactivity and AP after 3 and 6 days in culture. At three days cells stained positively for PTHrP (A) and PTH1R (E), but were negative for AP (not shown). Cells exhibiting AP activity were detected on day 6 (C and G). The merged images of B-C and F-G are shown in D and H respectively. B. A single bone nodule from an 18 d culture double stained for PTHrP (A–E) and AP (F–J) and optically sectioned from top to bottom. The darkened area in the center of the nodule (panels C-E/H-J/M-O) indicates the mineralized region of the nodule. Cells peripheral to the mineralized region were PTHrP and AP positive. PTHrP immunostaining was less intense but persisted in cells within the mineralized area while AP staining was absent. C. A single bone nodule from an 18 d culture double stained for PTH1R (A–D) and AP (E–H) and optically sectioned from top to bottom. The darkened area in the center of the nodule (panels C,D/G,H/K,L) indicates the mineralized region of the nodule. Cells peripheral to the mineralized region were PTH1R and AP positive. PTH1R immunostaining was less intense but persisted in cells within the mineralized area while AP staining was absent.\n\nAfter 18 days, there were 131±5 bone nodules in high initial plating density control cultures. Adding PTHrP (1-36) at concentrations of 0.1 nM to 100 nM caused a dose-related decrease in the number of bone nodules (Figure 2A). The inhibitory effect of PTHrP (1-36) on bone nodules was reversible. The number of bone nodules in cultures exposed to 1 nM PTHrP (1-36) for ten days was 80% fewer than in controls. When PTHrP-treated cultures were transferred to control culture medium the number of bone nodules rebounded and was not different from control 8 days after transfer (Figure 2B)25.\n\nA. The effect of exposure to 0 to 100 nM PTHrP (1-36) for 18 d on the number of bone nodules in RC cell cultures. B. RC cells were grown in the absence (control) or presence of 1 nM PTHrP (1-36). After 10 d all cultures were maintained in control medium and the nodule number was determined on day 10, 12, 14, 16, 18 and 20. Results are the mean±SEM (n=4), P≤0.05, P≤0.01, and P≤0.001.\n\nThe concentrations of PTHrP (1-36) that decreased bone nodule formation increased cAMP (Figure 3A). Treatment with dBcAMP or forskolin, agents that increase intracellular cAMP by diffusion through the plasma membrane or by activation of adenylate cyclase respectively, reduced bone nodule number (Figure 3B). Treatment with 50 nM PTHrP (7-34), a PTH1R ligand that activates the phospholipase C/diacylglycerol/protein kinase C pathway but doesn’t increase cAMP, had no effect on bone nodule number (Figure 3C).\n\nA. RC cells were plated into a 96-well plate, maintained overnight, and exposed to 0 to 100 nM PTHrP (1-36) for 10 min. The amount of cAMP in each well was measured by EIA. B. The effect of treatment with 0 to 1 mM DBcAMP and 0 to 10 mM forskolin (Fsk) on the number of bone nodules in 18 d RC cell cultures. C. The effect of treatment with 50 nM PTHrP (7-34) for 15 to 19 days on the number of bone nodules in RC cell cultures. Results are the mean±SEM (n=4), P≤0.01, and *P≤0.001.\n\nPTHrP (1-36) caused a dose-related decrease in the number of bone nodules in low plating density cultures (Figure 4A), but did not affect the number of AP-positive cell colonies (Figure 4B). To determine if the effect of PTHrP (1-36) on bone nodule development was affected by the duration of exposure to the peptide, low plating density cultures were exposed to 100 nM PTHrP (1-36) continuously or intermittently for 14, 16 or 18 days. While bone nodule formation was prevented by the three exposure regimens, none affected the number of AP-positive colonies (Figure 5A and 5B). Continuous or intermittent exposure to 100 nM PTHrP (1-36) for four days had no effect on the proliferation of RC cells (Figure 6A), and no effect on apoptosis was observed after exposure to PTHrP (1-36) for nine days (Figure 6B).\n\nRC cells were exposed to 0 to 100 nM PTHrP (1-36) for 18 days and then fixed and stained by the von Kossa method to identify mineralized bone nodules (A). or histochemically stained to identify AP-positive cell colonies (B). Results are the mean±SEM (n=3), P≤0.01, and *P≤0.001.\n\nThe number of bone nodules (A) and AP-positive cell colonies (B) in cultures exposed to 100 nM PTHrP (1-36) for 1, 6, or 48 hr during each 48 hr treatment period. Cells were exposed to a total of seven to nine 48 hr treatment cycles over 14 to18 d. Results are the mean±SEM (n=6), P≤0.001.\n\nCell proliferation was determined in RC cells treated with 100 nM PTHrP (1-36) for 1 hr, 6 hrs, or 48 hrs for two 48 hr treatment cycles and pulse-labeled with BrdU for the last 2 hrs of the experiment (A). Apoptosis was measured by TUNEL staining in low plating density cultures exposed to 100 nM PTHrP (1-36) for four 48 hr intermittent treatment cycles (B). Results are the mean±SEM (n=12 for cell proliferation assay and n=3 for apoptosis assay).\n\nThe number of bone nodules that developed in high plating density cultures was reduced by approximately 50% when transferred to medium containing 50 nM PTHrP (1-36) following ten days in control conditions (Figure 7A). By contrast, bone nodule number was increased by approximately 9-fold when cultures were transferred to control medium after an exposure to 50 nM PTHrP (1-36) for the first ten days of culture (Figure 7A). Neither of these exposure protocols affected the AP activity (Figure 7B).\n\nRC cell cultures were maintained in control medium for 21 days (C/C), maintained in control medium and changed to medium containing PTHrP (1-36) on day 11 (C/P), treated with 100 nM PTHrP (1-36) for 21 days (P/P), or exposed to 100 nM PTHrP (1-36) for 10 days and then transferred to control medium on day 11 (P/C). The number of bone nodules or AP activity was determined after 21 d. Results are the mean±SEM (n=4), P≤0.001.\n\nBoth continuous and intermittent treatment with 100 nM PTHrP (1-36) for 15 days caused a significant decrease in BSP mRNA (Figure 8A), but did not affect OPN mRNA (Figure 8B). Continuous exposure to 10 nM PTHrP (1-36) for 15 days also reduced the amount of BSP protein detected by immunoblotting (Figure 8C).\n\nThe BSP (A) and OPN (B) mRNA in RC cells exposed to 100 nM PTHrP (1-36) for 1 hr, 6 hrs, or 48 hrs for seven 48 hr treatment cycles (15 days) was measured by real-time RTPCR. The effect of 0.01 and 100 nM PTHrP (1-36) on BSP in lysates of RC cells was assessed by immunoblotting (C). Continuous exposure to 100 nM PTHrP (1-36) for 14 d decreased immunodetectable 75 kD BSP compared to the control and 0.01 nM PTHrP (1-36) treated RC cells. The Ponceau S-stained gel shows relative amounts of loaded protein. Real-time RTPCR results are the mean±SEM (n=3), *P≤0.001.\n\n\nDiscussion\n\nTemporal expression of PTHrP, PTH1R, and AP associated with the maturation of cells in the osteoblast lineage during intramembranous bone formation in vitro was studied using primary cultures of cells isolated from calvariae of neonatal rat pups. Limiting dilution analysis has revealed that approximately 0.25–0.5% of the cells isolated from neonatal rat calvaria are osteoblast colony forming units (CFU-Os)26,27. When cultured under appropriate conditions, each CFU-O gives rise to a colony of functionally mature osteoblasts that produce a nodule of mineralized woven bone. Our rational for culturing the primary rat calvarial cells at a low plating density was to study the temporal events associated with osteoblast differentiation and woven bone formation in individual colonies in the absence of intervening cells that do not belong to a colony28. Using this approach, we found small colonies of PTHrP and PTH1R immunopositive cells on the third day of culture while AP-positive cells were not detected until the sixth culture day. Although co-immunolocalization of PTHrP and PTH1R was not performed, the coincident appearance of AP in 100% of the PTHrP- and PTH1R-positive cell colonies on day six is consistent with the interpretation that the ligand and receptor are co-expressed by early progeny of neonatal rat calvarial CFU-Os. This observation confirms results reported by Kondo et al.29 that PTHrP and PTH1R are expressed earlier than AP during osteoblast differentiation. Our observations demonstrate that osteoblasts that produce mineralized nodules of woven bone are both a source and target for PTHrP for the duration of this process. Combining the temporal/spatial expression of PTHrP and PTH1R during bone nodule formation with observations made when individual nodules were optically sectioned by confocal microscopy, our results indicate that preosteoblasts, osteoblasts, and osteocytes express PTHrP and PTH1R. This confirms our previous finding in the developing chick mandible24. The co-expression of PTHrP and its receptor by osteogenic cells supports the conclusion that the peptide may serve as an autocrine/paracrine regulator of osteoblast proliferation, differentiation, survival, and/or function.\n\nAs previously reported for amino terminal PTH (1-34)30, continuous exposure to PTHrP (1-36) decreases the number of bone nodules that develop in primary cultures of neonatal rat calvarial cells. Combined with the dose-related increase in cAMP stimulated by PTHrP (1-36), this establishes the functional activity of PTH1R expressed by the osteogenic cells that give rise to the woven bone nodules. As expected, several features of how PTHrP and PTH affect bone nodule formation are similar. The inhibitory effect of both peptides is dose dependent over a similar range of concentrations, the effect is reversible, and its magnitude is related to when the peptide is added to the culture. Ligand binding to PTH1R activates two intracellular signaling pathways, cAMP/PKA and PLC/DAG/PKC. That dBcAMP and forskolin, but not PTHrP 7-34, decreased bone nodule number indicates that the inhibition of nodule mineralization is mediated by cAMP. This is consistent with the finding that forskolin inhibited bone nodule formation in RC cell cultures31.\n\nThe skeletal effect of exogenous amino terminal PTH and PTHrP in humans and experimental animals is affected by how the peptide is delivered. Bone resorption (catabolic effect) is the predominant effect when PTH is chronically elevated by continuous infusion15–18. By contrast, bone formation (anabolic effect) is stimulated when PTH or PTHrP are delivered in a single daily injection14,15,18. While the anabolic effect is a consistent outcome of intermittent exposure to amino terminal PTH/PTHrP in vivo, this effect has been more difficult to produce in vitro. Ishizuya et al.32 have reported that exposure to PTH (1-34) for the first six hours of eight consecutive 48-hr treatment cycles (17 days) increased bone nodule number and AP activity in cultures of RC cells. Bone nodule number and AP activity were decreased in cultures exposed to PTH (1-34) continuously or for one hour of each 48-hr treatment period. Using a similar design, we found that continuous as well as one- or six-hr periods of intermittent exposure to PTHrP (1-36) decreased the number of mineralized bone nodules. Interestingly, in the present study neither continuous nor intermittent treatment with PTHrP (1-36) for 14 to 18 days had an effect on the number of AP-positive cell colonies, which are derived from the same CFU-O’s that produce bone nodules. This indicates that the decline in bone nodules is not due to a decrease in the number of CFU-O’s or their ability to produce osteoblast cell colonies. Thus, the inhibitory effect of PTHrP on bone nodule formation appears to occur downstream of both cell colony formation and when AP is expressed by differentiating osteoblasts.\n\nIn this regard it is relevant that both continuous and intermittent treatment with PTHrP (1-36) decreased BSP mRNA and protein levels in RC cell cultures. BSP is a phosphoprotein that initiates nucleation of hydroxyapatite during bone matrix mineralization33. The inability to mineralize woven bone matrix because of a decrease in BSP would account for the decrease in mineralized nodules identified by von Kossa staining. The finding that PTHrP (1-34) down-regulates BSP expression and inhibits biomineralization in cultures of a cementoblast cell line34 is consistent with this conclusion. A functional relationship between PTH1R ligands and bone matrix mineralization is further supported by the observation that PTH and PTHrP reduce expression of PHEX/Phex, a gene associated with phosphate metabolism, by clonal osteoblast-like UMR 106 and MC3T3-E1 cells35,36. The decrease in Phex expression by MC3T3-E1 cells was associated with an inhibition of the initiation and progression of matrix mineralization35. While the physiologic significance of this effect is currently unknown, it is interesting that misexpression of PTHrP by osteoblasts leads to reduced mineralization of membrane bone in developing chick embryos at Hamilton Hamburger stage 4437 and that mineralization of endochondral bones is increased in PTHrP gene null mice5. Taken together, these findings suggest that PTHrP may regulate the mineralization of bone matrix during skeletal morphogenesis. Moreover, it is reasonable to speculate that a transient reduction in bone mineralization might be a way to increase the local concentration of calcium and phosphate needed for other metabolic or homeostatic functions.\n\nIt is unclear why the 6-hr intermittent exposure to PTHrP (1-36) and PTH (1-34), reported to be equipotent ligands for PTH1R, had different effects in this study and in the study reported by Ishizuya et al.32. However, it has been reported that there are differences in the ability of these ligands to interact with G-protein coupled (RG) and uncoupled (R0) states of the receptor38. While both ligands bind to RG with equal affinity, PTH (1-34) binds to the G-protein uncoupled receptor with a greater affinity than PTHrP. Ligands that bind stably to G-protein uncoupled PTH1R [e.g. PTH (1-34)] produce a greater cAMP response for extended times after initial binding than ligands that bind with lower affinity [e.g., PTHrP (1-36)]. Thus, a ligand that binds stably to R0 has the potential to produce a downstream signal for longer times after initial binding. In addition, ligands that are bound more stably to the R0 may also have a greater probability for coupling to secondary G proteins and activate alternative signaling pathways. Differential responses under seemingly identical experimental conditions might result since cAMP/PKA and PLC/DAG/PKC signaling is activated by different G-coupled proteins (Gαs and Gαq respectively). The recognition that PTHrP has a role in skeletal development was the result of observations in homozygous PTHrP gene null mice and PTHrP overexpressing transgenic mice5–7. The overt skeletal phenotypes exhibited by these animals indicated that PTHrP is an important regulator of embryonic endochondral ossification. It was subsequently demonstrated that longitudinal bone growth driven by interstitial expansion of the epiphyseal growth plate is controlled, at least in part, by a negative feedback loop involving Indian hedgehog protein (IHH) and PTHrP6,7. In regulating the morphogenesis and growth of endochondral bones, the source and target for PTHrP are perichondrial cells and prehypertrophic chondrocytes respectively39. Although somewhat more subtle, PTHrP also influences intramembranous ossification as well as bone formation associated with skeletal modeling in postnatal mice. For example, PTHrP gene null mice exhibit craniofacial anomalies that may occur as a consequence of altered intramembranous ossification5. Moreover, the expression of PTHrP and PTH1R mRNA and/or protein by osteoblasts has been associated with the morphogenesis of membrane bone formation in chicks24, rats21,40, and rabbits20. PTHrP mRNA and protein have also been detected in cultures of osteoblast-like cells isolated from fetal rat calvaria22. It has been reported that PTHrP and PTH1R are expressed by morphologically and presumably functionally distinct populations of cells in the osteoblast lineage21,22. While we did not quantitate the expression of PTHrP and PTH1R in bone nodule forming colonies of osteoblasts, immunohistochemical staining results suggest that both genes are expressed by preosteoblasts, mature osteoblasts, and osteocytes. Kartsogiannis et al.20 have reported similar findings in an experimental model of intramembranous bone formation in adult New Zealand white rabbits. This result is consistent with the conclusion that PTHrP may act as an autocrine/paracrine regulator of osteoblast differentiation and/or function during intramembranous ossification.\n\nWhile there is substantial evidence to conclude that PTHrP decreases extracellular matrix mineralization during morphogenesis of intramembranous and endochondral skeletal tissues in embryos/fetuses5,37, it appears to increase bone formation by stimulating the proliferation, recruitment, and survival of osteoblasts in adult animals19,41,42. The latter is known as the anabolic action of intermittent exposure to exogenous amino terminal PTHrP and PTH in the adult skeleton. This apparent age-associated relationship between PTHrP and its skeletal effects should be considered when evaluating the role of this protein in local regulation of skeletal cells.", "appendix": "Author contributions\n\n\n\nDr. Kamel performed this work as part of the requirements for earning a doctorate from Creighton University. Dr. Yee was Dr. Kamel’s primary advisor and the work was performed in his laboratory.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work was funded in part by an educational scholarship to Suzan kamel from the Arab Republic of Egypt who had no involvement in the design of the studies, the collection, analysis or interpretation of data, preparation of the manuscript, or decision to submit the work for publication.\n\n\nAcknowledgements\n\nThe authors acknowledge the contributions of Drs. Philip Brauer, Diane Cullen, Mark Johnson and Rita Meyer who served as Dr. Kamel’s doctoral advisory committee.\n\n\nReferences\n\nBurtis WJ, Brady TG, Orloff JJ, et al.: Immunochemical characterization of circulating parathyroid hormone-related protein in patients with humoral hypercalcemia of cancer. N Engl J Med. 1990; 322(16): 1106–1112. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao Q, Brauer PR, Xiao L, et al.: Expression of parathyroid hormone-related peptide (PTHrP) and its receptor (PTH1R) during the histogenesis of cartilage and bone in the chicken mandibular process. J Anat. 2002; 201(2): 137–153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShearon CC: Parathyroid Hormone-Related Peptide Influences Intramembranous Bone Formation In Vitro, in Biomedical Sciences Department 1999, Creighton University, Omaha NE USA. p. Figure 9, 44. Reference Source\n\nBellows CG, Aubin JE: Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro. Dev Biol. 1989; 133(1): 8–13. PubMed Abstract \| Publisher Full Text\n\nTang LY, Kimmel DB, Jee WS, et al.: Functional characterization of prostaglandin E2 inducible osteogenic colony forming units in cultures of cells isolated from the neonatal rat calvarium. J Cell Physiol. 1996; 166(1): 76–83. PubMed Abstract \| Publisher Full Text\n\nMalaval L, Liu F, Roche P, et al.: Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro. J Cell Biochem. 1999; 74(4): 616–627. PubMed Abstract \| Publisher Full Text\n\nKondo H, Ohyama T, Ohya K, et al.: Temporal changes of mRNA expression of matrix proteins and parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor in bone development. J Bone Miner Res. 1997; 12(12): 2089–2097. PubMed Abstract \| Publisher Full Text\n\nBellows CG, Ishida H, Aubin JE, et al.: Parathyroid hormone reversibly suppresses the differentiation of osteoprogenitor cells into functional osteoblasts. Endocrinology. 1990; 127(6): 3111–3116. PubMed Abstract \| Publisher Full Text\n\nTurksen K, Grigoriadis AE, Heersche JN, et al.: Forskolin has biphasic effects on osteoprogenitor cell differentiation in vitro. J Cel Physiol. 1990; 142(1): 61–69. PubMed Abstract \| Publisher Full Text\n\nIshizuya T, Yokose S, Hori M, et al.: Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells. J Clin Invest. 1997; 99(12): 2961–2970. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunter GK, Goldberg HA: Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci U S A. 1993; 90(18): 8562–8565. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOuyang H, Franceschi RT, McCauley LK, et al.: Parathyroid hormone-related protein down-regulates bone sialoprotein gene expression in cementoblasts: role of the protein kinase A pathway. Endocrinology. 2000; 141(12): 4671–4680. PubMed Abstract \| Publisher Full Text\n\nAlos N, Ecarot B: Downregulation of osteoblast Phex expression by PTH. Bone. 2005; 37(4): 589–598. PubMed Abstract \| Publisher Full Text\n\nVargas MA, St-Louis M, Desgroseillers L, et al.: Parathyroid hormone-related protein (1-34) regulates Phex expression in osteoblasts through the protein kinase A pathway. Endocrinology. 2003; 144(11): 4876–4885. PubMed Abstract \| Publisher Full Text\n\nAbzhanov A, Rodda SJ, McMahon AP, et al.: Regulation of skeletogenic differentiation in cranial dermal bone. Development. 2007; 134(17): 3133–3144. PubMed Abstract \| Publisher Full Text\n\nDean T, Vilardaga JP, Potts JT Jr, et al.: Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor. Mol Endocrinol. 2008; 22(1): 156–166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNissenson RA: Parathyroid hormone-related protein. Rev Endocr Metab Disord. 2000; 1(4): 343–352. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "827", "date": "11 Mar 2013", "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\n\nThis is an interesting and well written manuscript demonstrating that PTHrP blocks the mineralization of bone nodules developing in cultures of rat neonatal calvarial osteoblasts. Although similar data have been observed with PTH, and indeed both PTH and PTHrP act via the same receptor, this study demonstrates that the calvarial osteoblasts produce PTHrP as well as expressing the receptor so that they are examining a paracrine situation rather than an endocrine one. Their results were the same whether intermittent or continuous exposure to PTHrP was utilized. Surprisingly, PTHrP did not inhibit alkaline phosphatase activity, proliferation, or apoptosis indicating that the final stages of mineralization was the point at which PTHrP was working. The authors found that BSP expression, but not OPN expression, was reduced by PTHrP, and concluded somewhat prematurely that decreased BSP expression was the mechanism for the inhibition of mineralization. There are other candidates, and the authors did not demonstrate that blocking BSP production as by siRNA would reproduce their findings with PTHrP. Thus the title of the abstract and the conclusion that BSP is the mechanism represent over interpretations. The reduction in BSP is associated with but not necessarily the reason for inhibition of mineralization following PTHrP. One would have liked to see other proteins involved with mineralization examined, and each tested for its role.", "responses": [] }, { "id": "853", "date": "19 Mar 2013", "name": "Joseph Bidwell", "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 experiments presented in this manuscript appear to have been carefully executed. The manuscript is well written. However, the findings in this study appear to contradict an earlier report in which PTHrp induces proliferation in calvarial-derived osteoblasts (see Datta et al., 2007). The present authors do not discuss this previous result in their manuscript. These conflicting observations demonstrate the problems inherent in using in vitro culture systems for characterizing growth factor and hormone action on osteoprogenitors.", "responses": [ { "c_id": "420", "date": "22 Mar 2013", "name": "Suzan Kamel", "role": "Author Response", "response": "Thank you Dr. Bidwell for reviewing our manuscript and we appreciate your thoughtful comments. The difference in the effect of PTHrP (1-36) on cell proliferation in our study and the outcome reported by Datta et al. (2007) may be related to several differences between the two studies. Datta et al. (2007). reported that treatment with 1-100 nM PTHrP (1-34) stimulated cell proliferation in low-density cultures of a MC3T3-E1 subclone cell line (Figure 1A,C). Interestingly, PTHrP (1-34) decreased proliferation in high-density cultures (Figure 1B). This illustrates that culture conditions impact the nature of the proliferative effect of PTHrP on the cells used in their study. By comparison, we found that treatment with 100 nM PTHrP (1-36) for 5 days had no effect on cell proliferation (assessed by BrdU incorporation) in primary cultures of neonatal rat calvaria cells. Differences between the two studies that might have contributed to the varied outcomes include: the cells studied were from different species (rat versus mouse), and our experiments were done on primary cultures of rat calvaria cells while they used a subclone of a mouse calvaria osteoblast-like cell line. A potentially important difference is that the cells used in our experiments were plated, grown, and maintained in the presence of 10% FBS, whereas Datta et al. employed serum-starvation to synchronize cells in the cell cycle and conducted their experiments in presence of 0.5-2% FBS during the period of peptide exposure. These differences in culture conditions might be a significant factor since they noted that the stimulatory effect of PTHrP on cell number and doubling time in the presence of 0.5-2% serum was not observed in cultures supplemented with 10% serum (see results section of Datta et al., 2007). Differences in the observed effect of PTHrP and/or PTH peptides on cell proliferation (see Sabatini et al., 1996; Lomri et al., 1997; Pasquini et al., 2002, and others) and other endpoints of osteoblast differentiation and/or function (see Jongen et al., 1993 and others) in different osteoblast-like cell models and under somewhat different experimental conditions is not uncommon. The fact that PTHrP prevented mineralization of bone nodules under conditions that did not affect cell proliferation is most significant in that it suggests that the effect on mineralization was not related to effects of the peptide on cell proliferation." } ] } ]	1	https://f1000research.com/articles/2-77
https://f1000research.com/articles/2-140/v1	12 Jun 13	{ "type": "Research Article", "title": "Environmental enrichment does not impact on tumor growth in mice", "authors": [ "Jennifer A Westwood", "Phillip K Darcy", "Michael H Kershaw", "Jennifer A Westwood", "Phillip K Darcy" ], "abstract": "The effect of environmental enrichment (EE) on a variety of physiologic and disease processes has been studied in laboratory mice. During EE, a large group of mice are housed in larger cages than the standard cage and are given toys and equipment, enabling more social contact, and providing a greater surface area per mouse, and a more stimulating environment. Studies have been performed into the effect of EE on neurogenesis, brain injury, cognitive capacity, memory, learning, neuronal pathways, diseases such as Alzheimer’s, anxiety, social defeat, emotionality, depression, drug addiction, alopecia, and stereotypies. In the cancer field, three papers have reported effects on mice injected with tumors and housed in enriched environments compared with those housed in standard conditions. One paper reported a significant decrease in tumor growth in mice in EE housing. We attempted to replicate this finding in our animal facility, because the implications of repeating this finding would have profound implications for how we house all our mice in our studies on cancer. We were unable to reproduce the results in the paper in which B16F10 subcutaneous tumors of mice housed in EE conditions were smaller than those of mice housed in standard conditions. The differences in results could have been due to the different growth rate of the B16F10 cultures from the different laboratories, the microbiota of the mice housed in the two animal facilities, variations in noise and handling between the two facilities, food composition, the chemical composition of the cages or the detergents used for cleaning, or a variety of other reasons. EE alone does not appear to consistently result in decreased tumor growth, but other factors would appear to be able to counteract or inhibit the effects of EE on cancer progression.", "keywords": [ "Environmental enrichment", "cage", "cancer", "tumor", "mice" ], "content": "Introduction\n\nEnvironmental enrichment (EE) for mice in laboratory conditions provides enlarged cages for large groups of mice and provides objects which stimulate enhanced sensory, cognitive, social and physical activity compared with mice housed in standard conditions. The positive effects of EE on mice (reviewed in Nithianantharajah and Hannan1) have been reported from numerous studies (from a PubMed search on 9 May 2013 using the phrases “environmental enrichment” and “enriched environment”): at least 150 papers have been published showing enhanced neurogenesis, cognitive capacity, memory, learning, neuronal pathways, and improvements in diseases such as Alzheimer’s, Huntington’s, amyotrophic lateral sclerosis and brain injury. Approximately 100 papers have been published showing reduction in anxiety levels, social defeat, emotionality, depression, drug addiction, alopecia, and stereotypies. In addition, approximately 12 studies have investigated the effect of EE in infectious disease, immunity, atherosclerosis, lifespan, inflammation, asthma and obesity; and about 10 studies have dealt with its effects on olfaction, hearing, photoreceptors, and sight. Only three studies in mice have been performed on the effect of EE on cancer development or treatment.\n\nThree papers have reported on tumor growth in mice housed in enriched environments compared with those housed in standard conditions2–4. Cao et al.2 reported significantly decreased growth in subcutaneous (s.c.) B16 (by 43%), B16F10 (by 77%) and MC38 (by 55%) tumors in mice housed in EE conditions. Nachat-Kappes et al.3 reported significantly smaller s.c. E0771 mammary tumors up to 10 days after tumors were inoculated orthotopically in EE housed mice, but thereafter there was no statistically significant difference in tumor size. Benaroya-Milshtein et al.4 reported no significant difference in size of untreated s.c. 38C-13 B-cell lymphoma tumors. However, all three studies have reported statistically significant differences in other parameters between EE housed mice and standard housed mice: Cao et al.2 reported markedly lower leptin levels and upregulation of brain-derived neurotrophic factor in EE mice, Nachat-Kappes et al.3 reported a statistically significant increase in caspase-3 levels in the tumors of EE housed mice, and Benaroya-Milshtein et al.4 reported reduced tumor growth and significantly greater survival of EE housed mice after immunization with an idiotype-vaccine prior to tumor injection, with 44% disease-free compared with 0% in standard cages. Thus all three studies report an influence of EE on tumors.\n\nOur primary goal was to see if we could replicate the significant difference in tumor size found in the EE group by Cao et al.2, who injected B16F10 s.c. and found a 77% reduction in tumor mass in EE mice and that 17% of mice had no visible tumors, compared with 0% of mice in standard caging. If we could replicate these results, this finding would have important consequences for the way in which we would need to house mice to perform future experiments when testing therapies to combat cancer.\n\n\nMaterials and methods\n\nThe mouse (C57BL/6) B16F10 melanoma tumor cell line5 (from NP Restifo, National Cancer Institute, Bethesda, USA) was maintained in complete medium consisting of DMEM (Gibco, Life Technologies, Grand Island, NY) with 10% heat-inactivated fetal calf serum (FCS; MultiSer, Thermo Trace, Melbourne) and additives (2 mM glutamine (Gibco), 100 μg/ml streptomycin (Sigma-Aldrich, St Louis, MO) and 100 U/ml penicillin (Sigma-Aldrich) in a humidified incubator at 37°C with 5% CO2.\n\nThe black low density polyethylene plastic EE cage (Plastime, Castegnero, Italy) measured 81 cm (length) × 57 cm (width) × 34 cm (height) internally, and had a wire cage lid (Figure 1). It was stocked with the following stimulatory equipment: 2 exercise wheels, 3 PVC plumbing elbow pipes (2.54 cm diameter) bent at 90°, a 2.54 cm plumbing T-piece, 2 standard cages with holes drilled in their sides to allow mouse access and with tissues inside and pellet food provided on their wire lids, 2 cardboard boxes of tissues cut in half and inverted (refuges) and about 20 tissues scattered around. An extra EE cage was also purchased and when mice cages were cleaned fortnightly, all equipment and mice were transferred to the new cage which had clean bedding. Mice were allowed to acclimatize to their conditions for six weeks, prior to injection with tumors. The mice were not handled except for transferring during cage cleaning, and tumor measurement (on day 13 after tumor injection).\n\nSetup shows the refuges, exercise wheels, and tunnels in the environmental enrichment cage.\n\nThe four standard cages used measured 28 cm (length) × 14 cm (width) × 12 cm (height) internally and were made of polycarbonate plastic (Wiretainers, Melbourne, Australia).\n\nFortnightly cleaning of cages was as follows: The EE cage was scraped out manually, then cage and toys were soaked for 10 mins in hot water with 2–5% Decon 90 (Decon Laboratories Ltd, East Sussex, UK) solution, scrubbed with a brush, and rinsed in hot water. The cage was left for 2 weeks before being used again, as two cages were alternated. The standard cages were scraped out manually, washed in a tunnel washer using washing machine powder, before autoclaving.\n\nBoth EE and standard cages had a layer of FibreCycle (recycled paper pellets; FibreCycle P/L, Yatala, Qld, Australia) animal bedding pellets to a depth of approximately 2 cm on the bottom of the cages, and all mice were fed with irradiated Barastoc mouse food cubes (Ridley AgriProducts, Melbourne, Australia) based on wheat, wheat byproducts, oats, meat meal, canola oil, soyabean meal, skim milk powder, molasses, salt, vitamins, and minerals. Drinking water was filtered tap water adjusted to pH 2.5–3 with hydrochloric acid.\n\nBoth EE and standard cages were housed in the same room of the animal facility. Throughout the experiment mice were maintained on a 13-hour-on : 11-hour-off lighting schedule (lights on at 6.00 am and off at 7.00 pm) in a room thermostatically maintained at 20°C. Food and water were available ad libitum. The air in the facility was not HEPA filtered or humidity controlled, and there were 15 air changes per hour.\n\nThe Specific Pathogen Free (SPF) facility houses sentinel mice in each rack and these are monitored regularly for infectious agents with the aim of detecting any pathogenic agents. The facility was monitored for the microorganisms listed in Table 1, and none of these species were detected during the period of this study. The following microbiota are detected in the mice in this facility and are considered endemic in the SPF-3 rated animal facility: Mouse Norovirus, Rotavirus, Protozoa (Chilomastix bettencourti or Entamoeba muris, which are frequently found in intestinal tracts of normal rodents), Proteus spp. (probably P. mirabilis as this is a common inhabitant of the upper respiratory tract and faeces of normal mice), and Helicobacter spp.\n\nEthics statement: This study was carried out in strict accordance with the recommendations of the Victorian Bureau of Animal Welfare, Department of Primary Industries, and the National Health and Medical Research Council’s Australian code of practice for the care and use of animals for scientific purposes. The protocol was approved by the Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee under Permit number E396. All efforts were made to minimize suffering.\n\nWild type male C57BL/6 mice were purchased from the Walter and Eliza Hall Institute of Medical Research (Bundoora, Australia), at age three weeks, and randomly assigned in either the EE cage (20 mice) or in four standard cages with five mice each (20 mice in this group). Mice were habituated to their cages for 6 weeks prior to tumor injection. During the habituation period two mice died (one found dead and one was culled for hydrocephalus) in the standard housed group, so that this group consisted of 18 mice for tumor injection. After the six weeks habituation, mice were shaved on the flank and inoculated s.c. with 100 μl of a single-cell suspension of 1×105 B16F10 melanoma cells in Ca2+- and Mg2+-free phosphate-buffered saline (Merck, Darmstadt, Germany) (day 0). The same person injected all mice for consistency. Tumor growth was monitored using calipers, and tumor area was calculated as the product of two perpendicular diameters. Mice were culled when tumors reached 200 mm2 in size or at the first signs of stress.\n\nStatistical significance in the experiment compared in vivo tumor growth and was determined by two-tailed Mann-Whitney test in Graphpad Prism (Graphpad Software, version 6.02) San Diego, California).\n\n\nResults\n\nC57BL/6 male mice at three weeks of age were divided into two groups. One group of 20 mice was placed in an enriched environment (Figure 1), which consisted of a large cage (surface area of 231 cm2 per mouse) with numerous pieces of stimulatory equipment (exercise wheels, tunnels, refuges, tissues), and the 18 mice in the other group were placed in four standard cages (4–5 mice/cage with surface area of 78 cm2 per mouse) with tissues only.\n\nAfter six weeks of habituation in their respective cages, all mice were injected subcutaneously with 1 × 105 cells of B16F10 on their flank. Mice were not handled except for transfer to clean cages during routine fortnightly cleaning, until day 13 when all tumors were measured.\n\nFigure 2 shows the tumor measurements on days 13 and 16 after tumor injection. Tumors on 33% (six of the standard group of 18 mice) and 30% (six of the EE group of 20 mice) of mice were ≥ 200 mm2 on day 13 and these mice were culled on this day. The average size of tumors was 164.3 ± 25.4 (SEM) mm2 (standard conditions) and 155.7 ± 29.8 (SEM) mm2 (EE conditions) on day 13. Average tumor size between the two groups was not statistically significant (p=0.69). On day 16, 72% of standard housed mice (13 of the 18 mice) and 65% (13 of the 20 mice) of EE housed mice had been culled as tumors of these mice had reached the 200 mm2 size threshold. All mice except one in each group had developed tumors. The experiment was terminated on day 16 as there was no significant difference between tumor sizes in the two groups.\n\nTumor measurements shown on days 13 (first day of measurement) and 16 after tumor injection. Bar represents average measurement for the group. Error bar is ± SEM.\n\nTable 2 summarizes the conditions for previously published studies on cancer in mice housed in EE cages, compared with the current study, in an attempt to ascertain why the results between the studies were so different. Floor space area per mouse varied from 180 to 1250 cm2, with the floor space largest in the study by Cao et al.2, which was about five times that of our study. The number of mice per EE cage varied between five and 20, but was similar between the Cao et al. study and our study. Enrichment toys and equipment were similar between all studies, as was the age that the mice were introduced into the cages (3–4 weeks) and the time for habituation (6–9 weeks). Tumor lines were different between studies, as were the sites of s.c. injection and the frequency of handling the mice. Mice were male in all studies except one3 and of the C57BL/6 strain in all studies except one4. Other parameters that could have caused variability were not mentioned in all studies, such as food composition, cage material (chemical composition and emission of fumes may vary), cage cleaning (chemical residue may vary), lighting, temperature, whether standard and EE cages were housed in the same room, bedding composition, and microbiota detected in each animal facility. With so many unspecified variables it is difficult to determine what is causing three of the studies to find no durable statistically significant difference in tumor size between EE and standard housed mice, whilst one study found a significant difference.\n\n* for tumor measurement; s.c., sub-cutaneous; N.S., not specified.\n\n\nDiscussion\n\nWe attempted to replicate the interesting findings by Cao et al.2, that tumors in mice housed in EE conditions grew at a significantly reduced rate, compared with mice housed in standard cages and that more EE housed mice were resistant to tumors, with 15% showing no visible tumor at day 19 (all control mice showed visible tumors).\n\nWe were not able to replicate these results, and found no statistical difference in tumor size between the two groups, even though we set up an enlarged cage with much greater floor space per mouse than in the standard cages. We also provided toys and equipment similar to Cao et al. to give an enriching and stimulating environment, housed a similarly large number of mice together so that there was more social interaction, introduced the mice at the same age into the cages (at 3 weeks) and habituated them for the same time (6 weeks) before tumor injection. The same tumor line (B16F10) was used, and we injected the same number of cells s.c., injected the same sex mice (male), and limited handling of the mice to the same day (day 13 post tumor injection except for cleaning).\n\nThere are several differences which may explain why we could not replicate these results. Firstly, there was a noticeable difference in growth kinetics between the B16F10 tumor line cells that we used and those used by Cao et al. The B16F10 tumors in our study grew faster and 30% of EE housed mice had to be culled on day 13, whereas those in the Cao et al. study were all still alive on day 17. In addition, the floor space per mouse was about five times greater in the Cao study than ours. Also, the toys and other objects were not identical in both studies. Problems of not standardizing EE design and lack of reproducibility of results between and within studies is reviewed by Fares et al.6, who have attempted to remedy this by producing a standardized EE cage (for rats).\n\nWe are not claiming that EE housing cannot impact on tumor growth, but our results show that EE housing will not consistently reduce tumor growth in all animal facilities and that there may be factors which override the benefits of EE housing. These factors appear to vary between animal facilities, as other studies3,4 have also found no durable statistical difference in tumor size between the two groups.\n\nEE housing would thus appear to offer some benefits in certain animal facilities, but these benefits may be negated or hindered in other animal facilities by other factors. These factors could consist of, for example, differences in the microbiota of the mice. Tavakkol et al.7 examined the skin flora of mice and found 20 different species of microorganisms on the skin alone of mice in an SPF facility. There is likely to be variability in microbiota of mice in different animal facilities, and this could impact on the immune systems and limit the beneficial effect of EE housing. The impact of microbiota on the immune system, inflammation and cancer has been reviewed extensively8–11. Similarly, the food given to the mice probably varied between facilities. Diet also has an influence on microbiota12. In addition, variables such as noise and number of people accessing the facility may have a negative impact on EE mice despite their enriched conditions, which may vary between animal facilities. There were many variables with no information specified in the three published studies summarized in Table 2, which could have been different in our animal facility and counteracted any benefits of EE conditions in our study. Difficulties with designing EE studies and comparison between studies to draw definitive conclusions are reviewed by Toth et al.13, and the great variability of parameters between EE studies is reviewed in Benefiel et al.14 and Bayne15.\n\nOur study and review of the literature has demonstrated that EE housing 20 mice in a large cage and providing toys and a stimulating environment, does not universally lead to reduced tumor growth, and that other factors appear to be acting either in concert with EE or against EE conditions to provide the variable results found.", "appendix": "Author contributions\n\n\n\nMK conceived the study. MK and JW designed the experiments. JW, PD and MK carried out the research. JW and MK prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMK, PD and JW funded by National Health & Medical Research Council of Australia, Grant #1006188.\n\n\nAcknowledgments\n\nWe wish to thank the Peter MacCallum Cancer Centre Animal Facility staff, especially Shellee Brown, for their care and maintenance of mice used in this study.\n\n\nReferences\n\nNithianantharajah J, Hannan AJ: Enriched environments, experience-dependent plasticity and disorders of the nervous system. Nat Rev Neurosc. 2006; 7(9): 697–709. PubMed Abstract \| Publisher Full Text\n\nCao L, Liu X, Lin EJ, et al.: Environmental and genetic activation of a brain-adipocyte BDNF/leptin axis causes cancer remission and inhibition. Cell. 2010; 142(1): 52–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNachat-Kappes R, Pinel A, Combe K, et al.: Effects of enriched environment on COX-2, leptin and eicosanoids in a mouse model of breast cancer. PLoS One. 2012; 7(12): e51525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenaroya-Milshtein N, Apter A, Yaniv I, et al.: Environmental enrichment augments the efficacy of idiotype vaccination for B-cell lymphoma. J Immunother. 2007; 30(5): 517–22. PubMed Abstract \| Publisher Full Text\n\nFidler IJ: Selection of successive tumour lines for metastasis. Nat New Biol. 1973; 242(118): 148–9. PubMed Abstract \| Publisher Full Text\n\nFares RP, Belmeguenai A, Sanchez PE, et al.: Standardized environmental enrichment supports enhanced brain plasticity in healthy rats and prevents cognitive impairment in epileptic rats. PLoS One. 2013; 8(1): e53888. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTavakkol Z, Samuelson D, deLancey Pulcini E, et al.: Resident bacterial flora in the skin of C57BL/6 mice housed under SPF conditions. J Am Assoc Lab Anim Sci. 2010; 49(5): 588–91. PubMed Abstract \| Free Full Text\n\nCarvalho FA, Aitken JD, Vijay-Kumar M, et al.: Toll-like receptor-gut microbiota interactions: perturb at your own risk! Annu Rev Physiol. 2012; 74: 177–98. PubMed Abstract \| Publisher Full Text\n\nChinen T, Rudensky AY: The effects of commensal microbiota on immune cell subsets and inflammatory responses. Immunol Rev. 2012; 245(1): 45–55. PubMed Abstract \| Publisher Full Text\n\nNicholson JK, Holmes E, Kinross J, et al.: Host-gut microbiota metabolic interactions. Science. 2012; 336(6086): 1262–1267. PubMed Abstract \| Publisher Full Text\n\nKamada N, Seo SU, Chen GY, et al.: Role of the gut microbiota in immunity and inflammatory disease. Nat Rev Immunol. 2013; 13(5): 321–35. PubMed Abstract \| Publisher Full Text\n\nFlint HJ, Scott KP, Louis P, et al.: The role of the gut microbiota in nutrition and health. Nat Rev Gastroenterol Hepatol. 2012; 9(10): 577–89. PubMed Abstract \| Publisher Full Text\n\nToth LA, Kregel K, Leon L, et al.: Environmental enrichment of laboratory rodents: the answer depends on the question. Comp Med. 2011; 61(4): 314–21. PubMed Abstract \| Free Full Text\n\nBenefiel AC, Dong WK, Greenough WT: Mandatory \"enriched\" housing of laboratory animals: the need for evidence-based evaluation. ILAR J. 2005; 46(2): 95–105. PubMed Abstract \| Publisher Full Text\n\nBayne K: Potential for unintended consequences of environmental enrichment for laboratory animals and research results. ILAR J. 2005; 46(2): 129–39. PubMed Abstract \| Publisher Full Text" }	[ { "id": "1003", "date": "14 Jun 2013", "name": "Marc Pellegrini", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a nice study that adds to a growing list in the literature exploring the role of environmental enrichment on tumor pathophysiology in mice. Most of these studies, including the present, are unable to reproduce the findings of Cao et al in Cell 2010. The authors explore the possible explanations for these differences.Title and Abstract: The abstract is a good summary.Article content: Design, methods and analysis of the results are explained well and the science is robust.Conclusions: Conclusions are sensible, balanced and justified.Data: The data Is strong and presented well.In summary, this is a very well written manuscript that attempts to dissect possible confounders in previous studies. It would have been nice if the authors could have measured serum leptin levels in their mice (to compare them with those published by Cao). If levels were similar then this would substantially undermine the conclusions of Cao. However, such measurements may be beyond the scope of the present study.", "responses": [] }, { "id": "1018", "date": "21 Jun 2013", "name": "Stephen Nutt", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 clear study that attempts to investigate the role for environmental enrichment in the control of experimental tumour growth. Unlike the study of Cao et al. (2010), the authors here find that environmental enrichment does not impact on the growth of one well-characterized experimental tumour model, B16F10. The manuscript is well written and the conclusions sound. The authors thoroughly describe the similarities and differences in the environmental enrichment strategies that are employed in this and the previous studies, and provide a variety of plausible possible causes for the different findings.Overall, this study provides a warning about the generality of any impacts of environmental enrichment on cancer cell growth that have been claimed and highlights a number of potential confounding factors. While the abstract is an appropriate summary of the study, the title is very broad and could perhaps be more restricted to encompass only the data in the manuscript, which is solely derived from one cancer cell line, B16F10 cells.", "responses": [] } ]	1	https://f1000research.com/articles/2-140
https://f1000research.com/articles/2-137/v1	10 Jun 13	{ "type": "Research Article", "title": "Benefit packages for chronic disease outpatients in the New Rural Cooperative Medical Scheme in 32 Chinese counties", "authors": [ "Chuangzhou Xu", "Christian A Gericke", "Chuangzhou Xu" ], "abstract": "Introduction: Chronic disease has become a major problem affecting the health of the Chinese population. In response to this situation, the New Rural Cooperative Medical Scheme (NRCMS) has begun to provide health cover for outpatients with chronic disease expenses, made possible by the increased risk pool of previous years. We compare the differences between Benefit Packages for Chronic Diseases Outpatients (BPCDO) in order to produce a reference for policy makers.Methods: Information on the various BPCDO was located by searching the official NRCMS website in Chinese, using certain criteria to select the ideal BPCDO. Population coverage, service coverage and cost of coverage were chosen to form the analytical framework for this paper. The diseases were classified according to the World Health Organisation's (WHO) International Classification of Diseases (ICD-10).Results: To avoid “moral hazard”, complex processes have been created. This has resulted in chronic disease patients finding it very difficult to become beneficiaries. Forty-one types of chronic diseases were listed in 32 different BPCDO. We found that different counties have different co-payment rates, deductible lines, ceilings, coverage of drugs and tests, appointed hospitals and reimbursement frequencies.Conclusion: High mortality diseases and diseases with a heavier cost burden should be the priority on the list of reimbursement. The BPCDO scheme should be introduced urgently at the national level. It should include twenty-one types of disease and eight essential factors.", "keywords": [ "Benefit packages", "outpatients", "chronic diseases", "NRCMS", "rural", "China" ], "content": "Introduction\n\nChina is becoming an ageing society and, along with the changing social economy, high-risk unhealthy behaviors have increased. Among the high risk behaviors, an increase in dietary fat and salt intake, reduced physical activity, and high rates of smoking in the male population are all long standing issues1. All of these factors have contributed to the increasing number and proportion of patients suffering from chronic disease1,2. The fourth report of the National Health Services Survey in 20083, reported that 20% of the Chinese population were living with a chronic disease, an increase of 4.9% since 2003. The report also highlighted the increasingly higher proportion of rural residents living with a chronic disease. The survey estimated that nationally the total number of chronic disease cases reached 260 million in 2008, with rural residents accounting for 163 million3.\n\nChronic disease accounted for almost 80% of all deaths, and 70% of the total disability adjusted life years (DALY) lost in 20052. The challenges caused by chronic diseases are predicted to increase in severity in the foreseeable future4,5. Unfortunately, China has performed poorly in addressing non-communicable diseases6, and rural areas have experienced the heaviest burden of chronic disease7.\n\nSince the implementation of the New Rural Cooperative Medical Scheme (NRCMS) in 2003, the main goal has been to reduce the burden of medical expenses, in particular, for farmers who are inpatients with catastrophic illnesses, for which expenses are very high. Although the NRCMS was initially incapable of covering outpatients with chronic diseases due to limited funds, in recent years the minimum funds for each farmer have increased from 30 Yuan in 20038 to 50 Yuan in 20079, and to 100 Yuan in 200910 (US$1≈6.3 Yuan, Sept 2012). This increased financing has meant that the scheme now has the ability to cover some chronic disease expenses for outpatients, as these expensive outpatient services for are one of the main causes of medical impoverishment11. Reimbursement for chronic disease treatment is a beneficial supplement to the NRCMS’s main achievements.\n\nThe NRCMS is currently organized at county level, resulting in relatively independent policy-making, with each county being responsible for deciding what is contained within its own benefits package. Research from other transition countries strongly suggests that diseases that are the major causes of ill-health should be included in community-based health insurance12. From 2006, a few counties started to provide reimbursement to outpatients with chronic diseases, with an increasing number of counties offering reimbursement from 2008 on. Certain provinces, including Anhui, Yunan and Guangxi, have introduced a new policy in order to provide guidelines for the reimbursement of chronic disease expenses; however, not enough detail is included to assist with its uptake. There is still no national policy at present, and, as for research on NRCMS benefit packages, much of the focus is on inpatients13–15, and there are only few papers related to designing Benefit Packages for Chronic Diseases Outpatients (BPCDO)16,17. Our research compares the differences between the BPCDO offered in different counties in China, in order to measure existing inequalities and to produce a point of reference for national and county government policy decision makers.\n\nBenefit packages aim to focus scarce resources on interventions where they are most needed18. Benefit package design needs to encompass the scope of coverage and include the selection of services and providers, and the beneficiary obligations19. Finiteness is a basic characteristic of any benefit package; it cannot include everything, so identifying the priority medical services is the most important process step20. Some benefit packages are designed for particular populations or disease groups, such as HIV/AIDS prevention or maternal health interventions18.\n\n\nMethods\n\nThe BPCDO we used were found on official websites. Two steps were used when selecting the BPCDO.\n\nStep one: in order to select a representative sample, the geography, number of counties in each area and their economic development were taken into consideration. According to the administrative divisions of China, the whole country has 2,860 counties distributed into Eastern (697), Central (1086) and Western regions (1077)21. According to Chinese National Bureau of Statistics criteria, there are also 592 poverty-stricken counties located in the Eastern (72), Central (204) and Western regions (316). Each part of China also has its own Top One Hundred Rich Counties. According to this information, we selected 32 counties as sample counties which were distributed in the Eastern (eight counties including one rich county and one poor county), Central (12 counties including one rich county and two poor counties) and Western regions (12 counties including one rich county and three poor counties) to model a representative sample of China’s rural areas.\n\nStep two: in order to find the BPCDO statements in step one, we used the Google search input terms “cooperative medical schemes” and “chronic diseases” in Chinese. The selection of 32 plans was based on the following criteria: (1) The plan was on an official website, that is, a website of the NRCMS Office, Health Bureau or local government; (2) The final version of the plan was selected if more than one version was located for the same county; (3) Plans that did not mention the types of chronic disease and the amount of reimbursement were omitted; (4) No more than two counties from the same province were selected in order to avoid the inclusion of more than two counties with the same guidelines; (5) The plan was chosen if it met the demands of step one, until all plans required were obtained.\n\nAccording to the above process, 32 plans (counties) were selected across 20 provinces between the 11 November and the 4 December 2009. The names, province, and economic development statuses of the selected counties are shown in Table 1. The website of each plan is included in the appendix.\n\n(1) Denotes poverty-stricken county; # Denotes rich county.\n\n(2) “In” refers to inpatient reimbursement rates.\n\n(3) The counties in the middle of the table are in the central region, those at the top are in eastern China, those at the bottom are in western China.\n\n(4) ? Denotes that the NRCMS County Office did not provide the relative information.\n\nThe research used the Busse et al. conceptual framework to study coverage decisions as published by the World Bank22. This is based on the idea that a benefit package is decided by coverage and benefit entitlements, including breadth (population coverage), depth (service coverage), and height (cost of coverage). This can be represented by a three-dimensional coverage model (Figure 1).\n\nThis paper compares the different plans according to the three dimensions of coverage. We recorded the main points of every plan including reimbursement scope, chronic diseases selected, the deductible line, co-payment ratio and ceiling, methods of cost control, whether an expert panel had been created, and how many times enrolment of beneficiaries is carried out per year. We have attempted to determine the reasoning behind the different packages and produce recommendations for how it should operate. We classified the types of diseases covered according to the ICD-10.\n\n\nResults\n\nConcerned that the number of beneficiaries could get out of control, the NRCMS offices have put a very strict procedure in place to avoid “moral hazard”. As with all BPCDO, one cannot be a beneficiary unless two conditions are met: the first is that they are a member of the NRCMS; the second is that they have a chronic disease. The first stipulation is easy to authenticate, but for the second, the counties differ in the methods used to judge if a person has a chronic disease or not.\n\nTypically, there are two methods of judgment. One is where the NRCMS office empowers a county-level hospital to make the decision, usually the People’s Hospital in the applicant’s county, although some counties use different county-level hospitals according to the different townships. Another method, more often used, is where the NRCMS office uses a specifically created expert panel made up of delegates from different county-level hospitals (usually the People’s Hospital, Hospital of Traditional Chinese Medicine, Maternity & Child Care Centre etc.) to assess whether patients have a chronic disease. The NRCMS office convenes the experts to decide who should be eligible beneficiaries; generally, different diseases have different experts assigned to make the decision. Once a patient becomes a beneficiary, he/she can receive subsidies in the following years, although they must undergo a regular audit to check that they have continued membership of the NRCMS. Out of our 32 research counties, 14 counties had set up expert panels for this purpose. Of the remaining counties, eight counties had not set up a panel and the other counties did not mention whether they had an expert panel.\n\nThe current procedure to be designated as a beneficiary is very complicated. The applicant has to get certification from his/her village community first, and then go to a township hospital to confirm the basic health information in person, and submit a form with certification that he/she is suffering from at least one type of chronic disease. Certification must be provided by a county-level hospital or higher (Municipal hospital or Provincial hospital) if the applicant has been an inpatient. If the applicant was not an inpatient, they must provide evidence that they are suffering from at least one chronic disease from a level higher than a county hospital.\n\nSometimes, evidence of the severity of the disease must be provided by the applicant. For example, hypertension in phase III (ICD I10.XO5) will receive a subsidy, but not in phase II (ICD I10.X04), so the applicant has to show evidence that his/her condition is serious and should be in phase III. The township hospital will collect the information provided by the applicant and transfer it to the county NRCMS Office. The latter will then decide whether the applicant becomes a beneficiary. It takes a long time for a patient to become a beneficiary as the roll is updated slowly; of our 32 research counties, one county updated their beneficiary roll every month, three counties did this four times per year, three counties did this bi-annually, and ten counties did this annually. The other 15 counties did not mention how often they updated their beneficiary roll.\n\nSelection of diseases. The chronic diseases listed in each BPCDO vary from between 4 and 28 diseases. In total, 41 types of chronic diseases are included in the 32 BPCDO, the frequency of inclusion of each type of chronic disease is listed in Table 2. No single chronic disease has been selected by all benefit packages, and some types of common chronic diseases, like coronary heart disease and chronic obstructive pulmonary disease (COPD), were only selected by under half the counties. Some counties have also selected endemic conditions such as sequelae of earthquake injuries and thalassemia.\n\nThirteen counties reimbursement for hypertension in phase ll, 6 counties in phase lll, others did not specify.\n\nDrugs and tests. Most counties listed some drugs and tests in their BPCDO. Some counties specified the scope of prescription according to disease, some even specified a detailed drug list for each disease, and some counties only prescribed within the Catalogue of Basic Medicines of the State, while others did not mention the scope of prescription. Of the 32 counties, 15 stipulated a maximum number of days for each prescription which varied from between 7 and 60 days. In addition, doctors are asked by the NRCMS office to prescribe in duplicate, so one copy is to be left at the hospital and the other copy is to be forwarded to the NRCMS office as evidence of reimbursement.\n\nHospital choice. Some counties demand that beneficiaries must be treated within their county’s hospital, whereas others demand that special diseases must be treated at approved hospitals only. For example, mental disorders must be treated in the county mental hospital otherwise there can be no reimbursement. No county reimburses patients treated at a village clinic.\n\nTo control the cost of reimbursement, a co-payment rate, deductible line and ceiling have been set up by many counties.\n\nDeductible line and ceiling. Four counties have set a deductible line between 100 and 1000 Yuan, five counties have no deductible line, and others have not mentioned it. As for the ceiling, 15 counties have set a ceiling for reimbursement at 500 to 45000 Yuan depending on the disease. Two of the counties have set the ceiling in conjunction with inpatient expenses.\n\nCo-payment rate. The co-payment rate for most counties is between 60% and 80%, and is applicable for all types of chronic diseases. Some counties stipulate rates according to the disease and the level of the hospital, as different diseases have different reimbursement rates.\n\nReimbursement frequency. Although inpatients can receive reimbursement as soon as they leave hospital, outpatients with a chronic disease are treated differently. Of the 32 counties, only four counties provide immediate reimbursement. Eight counties stated that they provide reimbursements once per quarter; five indicated that reimbursement is issued biannually, and twelve counties indicated that reimbursements are only issued annually. The other three counties did not mention how often they provide reimbursements.\n\nTable 3 lists the generosity of cost-sharing within 32 BPCDO according to the number of selected chronic diseases, deductible line, reimbursement percentage, ceiling, the maximum days per prescription and the reimbursement frequency.\n\n\nDiscussion\n\nAs mentioned in the analysis framework, the population coverage, service coverage, and cost coverage, can be represented as a cube. If any of the sides change, the shape of the cube changes too. Since it is impossible to allow infinity for any of the three dimensions, the BPCDO needs to balance the three factors. Among the three factors, disease selection is the core component as it directly influences the coverage of the population and the service coverage.\n\nOur study shows that chronic diseases included in BPCDO vary, not only in number, but also in type. It is unreasonable to assume that the NRCMS fund could include all chronic diseases, so there needs to be an objective criterion for choosing the types of disease for inclusion. We think that high-mortality diseases, and the heavier-burden diseases (based on DALYs) should be taken into account to determine priorities. According to the China Statistical Yearbook 2007, the top 10 causes-of-death in rural China are malignant tumors (24.8%), cerebral vascular disease (20.6%), diseases of the respiratory system (17.2%), heart disease (14.8%), injury and intoxication (9.0%), digestive system diseases (2.7%), endocrine nutritional/metabolic diseases (1.5%), diseases of the genitourinary system (1.2%), nervous system diseases (0.8%) and mental disorders (0.6%).\n\nAccording to the disease surveillance points system in rural China in 2002 (which can be seen as the initial stage of the death registration system), the highest proportion of deaths due to chronic diseases are caused by cancers, cerebrovascular disease, COPD, ischemic heart disease, hypertensive disease, liver disease and tuberculosis23,24. We were not able to obtain detailed information regarding burden of diseases for rural China, but the World Health Organization listed the leading causes of chronic disease in 2004 as: (according to the percentage of total DALYs in sequence), lower respiratory infections, unipolar depressive disorders, ischemic heart disease, cerebrovascular disease, COPD, refractive errors, hearing loss, adult onset diabetes and diabetes mellitus25. Our study found that most of the previously listed common diseases appeared in some BPCDO. However, there are still counties which have omitted very common chronic diseases, such as ischemic heart disease and COPD. From the national perspective, our opinion is that the cause of death diseases and the heavier burden diseases (DALYs) should be used as primary-priority-setting criteria.\n\nFrom synthesis of the information of leading cause-of-death diseases in rural areas in 2005, and morbidity of chronic diseases in rural areas in 2008 (China Health Statistical Yearbook 2009), we think the following 21 types of chronic diseases should be included at a national level: hypertensive diseases, diabetes mellitus, COPD, malignant tumours, sequelae of cerebrovascular disease, pulmonary heart disease, schizophrenia, chronic rheumatic heart diseases, inflammatory diseases of female pelvic organs, chronic viral hepatitis, fibrosis and cirrhosis of liver, atherosclerotic heart disease, rheumatoid arthritis, chronic nephritic syndrome, leukemia, heart failure, chronic renal failure, cardiomyopathy, Parkinson’s disease, chronic active hepatitis, chronic bronchitis and epilepsy. Patients suffering from other types of chronic diseases should get aid from the Medical Financial Assistance Scheme, not from the NRCMS. From the province perspective, the list can be adjusted according to the local spectrum of disease, which county managers can implement. For each province, the causes of death and burden of diseases in rural areas requires further research.\n\nThe most obvious difference between inpatient and outpatient benefit packages is their reimbursement rate. The overall real reimbursement rate for inpatients was 15% in 2007, but only 4% for outpatients26. Therefore, the introduction of BPCDO intends to increase the reimbursement for chronic disease outpatients.\n\nInpatient benefit packages were introduced at the same time as the NRCMS in 2003 and, with development over the past few years, have developed some common principles which must be adhered to. For example, all inpatient benefit packages have a deductible line and ceiling; the higher the hospital level, the higher the deductible line and the lower the co-payment rate. To some extent, these policies have achieved their goals to induce inpatients to the low-level hospitals, and encourage doctors to use less expensive and higher-quality drugs. However, the BPCDO was introduced later and is less specific. Our study found that some of the BPCDO do not mention their maximum days per prescription or how many times they update their beneficiary roll per year. Forty percent of BPCDO lacked a deductible line, ceiling or co-payment rate. Some BPCDO (which we searched but did not use) did not even list the types of diseases they would reimburse. These obscure BPCDO must create difficulties for the practitioners and patients.\n\nAs well as the BPCDO, there are also benefit packages for general outpatients with reimbursement for minor illnesses. More than half of the counties have general outpatients benefit packages since the NRCMS was implemented. There are two types of general outpatients benefit packages. The Household Medical Saving Account (HMSA) is one of these packages and, as its name suggests, some of the insured contribution (for example, 7–15 Yuan per capita per year, accounts for 40–70% of the premium) is a compulsory deposit in the HMSA. This can then be used for both outpatient and inpatient medical expenses for anyone within the family. This type of benefits package is used more often in Western China which is economically underdeveloped (91% of western counties in 2004)27 as the HMSA means a lower premium, attracting more people to join the NRCMS. However, in the east of China, only 33% of counties chose the HMSA in 200427, the remaining counties which have a general outpatient reimbursement, chose another type of benefit package termed “risk pool”. Risk pools work in the following way: the total fund of the NRCMS is divided into an inpatients and outpatients fund, the outpatients account for about 10–20% of the total fund, and when an insured member sees a doctor as an outpatient they receive 20–30% reimbursement; however, the ceiling is only 50–200 Yuan per capita per year. There were 1% of counties in the west of China and 13% in the east that implemented a risk pool in 200427. Some researchers revealed that the function of the HMSA is similar to private savings, which violates the underlying political principle of “cooperation” and cannot achieve the stated aim of “solidarity” in the NRCMS28. The effect of reduced impoverishment by the HMSA is not as obvious as that of the risk pool29, so there is a tendency for more and more counties to choose risk pools at the present30.\n\nBesides the differences mentioned above, there are three points of difference between the general outpatient benefits package and the BPCDO. Firstly, the general outpatient benefits package allows outpatients get reimbursement from a village clinic (only licensed clinics, normally one village has one licensed clinic), but the BPCDO does not. Secondly, general outpatients benefit packages reimburse patients immediately (the hospital cashier), but the beneficiaries of the BPCDO need to wait for several months and possibly up to a year. Thirdly, the ceiling and reimbursement rates for the general outpatients benefits package are far less than the BPCDO31, which is the main reason why the BPCDO is separate from general outpatients benefits package. However, the percentage of funds that should be allocated to outpatients requires further research.\n\nSaltman and Figueras argue that decentralized management in the health system is possibly beneficial for efficiency and immediate responses to new problems, but the experiences of many countries have demonstrated that major policy framework decisions in health systems should be made at the central level32. Our research confirmed this theory and we found that the BPCDO display great differences between different counties. Another study also found substantial differences in NRCMS policy design between regions33. Only 6 of the 32 BPCDO include all of the essential factors. Some counties’ policies appear reasonable, others do not, and some even include obvious errors. For example, patients who are suffering infectious pulmonary tuberculosis are eligible for free medical treatment at an infectious diseases hospital34, but some of the BPCDO still include this condition in their lists. We think that uniform principles of BPCDOa at the national level might avoid some unanticipated mistakes. A national policy on BPCDO should at a minimum include eight key components: (1) a specific list of types of disease using the ICD classification; (2) a specified deductible line; (3) co-payment rate; (4) a reimbursement ceiling; (5) maximum days per prescription; (6) the process of becoming a beneficiary, such as what evidence patients should provide, and whether it is decided by an expert panel; (7) how many times the beneficiary roll is updated per year; (8) whether beneficiaries receive reimbursement immediately, and if not, when they will receive it. In addition, the percentage of the chronic disease outpatient’s fund in relation to the total NRCMS fund should be specified.\n\nMethodologically, our study had two potential limitations. All of the plans came from websites which may lead to “online bias” as not all counties announce their plans on the internet. We had difficulties in testing whether they carried out their plans according to their written documents. Secondly, the omission of counties with incomplete information on their websites might have led us to depict a more positive picture than exists in reality.\n\n\nConclusions\n\nA comparison of the BPCDO in 32 counties in the NRCMS shows substantial differences in the quality of the BPCDO and the level of reimbursement between counties. There is no gold standard for choosing the types of diseases for inclusion in the BPCDO, so we think it is imperative that a principle for the reimbursement of chronic disease should be set up at a national level. Any national BPCDO should include at least 8 essential factors, and 21 types of chronic diseases for the whole country to be covered.", "appendix": "Author contributions\n\n\n\nCX and CAG designed the study, CX conducted the research and analysis with guidance from CAG. CX wrote the first draft of the paper, CAG edited it and both authors approve the final version of this article.\n\n\nCompeting interests\n\n\n\nNo competing interests to disclose.\n\n\nGrant information\n\nFinancial support for Chuangzhou Xu from the Fundamental Research Funds for the Central Universities in China (Shaanxi Normal University,11SZYB43) is gratefully acknowledged.\n\n\nNotes\n\nSadly, on 24 April 2013 Chuangzhou Xu, PhD passed away aged 38 following an acute myocardial infarction. He had dedicated his professional career as a health economist to improving healthcare in rural China through his research and teaching at the Northwest A&F University in Shaanxi, the University of Adelaide, and Shaanxi Normal University. Chuangzhou would have hoped that this publication contributed to this cause by highlighting persisting large inequities in rural healthcare in China, not only between the urban and rural health insurance schemes but also between rural regions.\n\n\nReferences\n\nYang G, Kong L, Zhao W, et al.: Emergence of chronic non-communicable diseases in China. Lancet. 2008; 372(9650): 1697–705. PubMed Abstract \| Publisher Full Text\n\nWang L, Kong L, Wu F, et al.: Preventing chronic diseases in China. Lancet. 2005; 366(9499): 1821–4. PubMed Abstract \| Publisher Full Text\n\nChinese Ministry of Health. The main results of the 4th National Health Services Survey in China.\n\nZhang YT, Yan YS, Poon CC: Some perspectives on affordable healthcare systems in China. Conf Proc IEEE Eng Med Biol Soc. 2007; 2007: 6155. PubMed Abstract \| Publisher Full Text\n\nYip W, Mahal A: The health care systems of China and India: performance and future challenges. Health Aff (Millwood). 2008; 27(4): 921–32. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Rao K, Wu J, et al.: China's health system performance. Lancet. 2008; 372(9653): 1914–23. PubMed Abstract \| Publisher Full Text\n\nZhang XH, Guan T, Mao J, et al.: Disparity and its time trends in stroke mortality between urban and rural populations in China 1987 to 2001: changing patterns and their implications for public health policy. Stroke. 2007; 38(12): 3139–44. PubMed Abstract \| Publisher Full Text\n\nChinese Ministry of Health, Ministry of Finance, Ministry of Agriculture. Guidance for the Establishment of the New Cooperative Medical Scheme. Beijing, 2003.\n\nChinese Ministry of Health, Ministry of Agriculture, National Development and Reform Commission. Announcement for Accelerating the Establishment of Pilots for the New Cooperative Medical Scheme. Beijing: Chinese Ministry of Health, 2006.\n\nChinese Ministry of Health, Ministry of Finance, Ministry of Agriculture. Guidance for the consolidation and development of the New Cooperative Medical Scheme. Beijing: Chinese Ministry of Health, 2008.\n\nYip W, Hsiao WC: Non-evidence-based policy: How effective is China's new cooperative medical scheme in reducing medical impoverishment? Soc Sci Med. 2009; 68(2): 201–9. PubMed Abstract \| Publisher Full Text\n\nOnwujekwe O, Onoka C, Uguru N, et al.: Preferences for benefit packages for community-based health insurance: an exploratory study in Nigeria. BMC Health Serv Res. 2010; 10: 162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang X, Liu L: A positive analysis of deductible and cost sharing ration in the New Rural Cooperative Medical System. Chinese Health Economics. 2005; (3): 12–14. Reference Source\n\nLuo Jh, Du Kl, Mao Y, et al.: Investigating and study on compensatory scheme for hospitalization expenses under New Rural Cooperative Medical System. Soft Science of Health. 2006; (2). Reference Source\n\nFu Lg, Xue Sf, Ruan Ys, et al.: Flow of inpatient compensation of new rural cooperative medical system. Chinese Rural Health Service Administration. 2009; (3). Reference Source\n\nQiao J, Gao XH, Li XF: Analysis on chronic diseases of people applying for enterprise medical insurance. Chinese Journal of Public Health. 2007; 12. Reference Source\n\nZhang FZ, Li L: Reform of assistance methods for outpatient treatment expenses of chronic diseases in patients enjoying Urban Basic Medical Insurance. Chinese Journal of General Practice. 2010; 6.\n\nWHO. WHO Service Delivery Seminar Series, 2008. Reference Source\n\nAnonymous. Glossary of Health Care Terms and Acronyms. 2010.\n\nWong H, Ricardo B, Bitrány A: DESIGNING A BENEFITS PACKAGE. World Bank Institute-Prepared for the Flagship Course on Health Sector Reform and Sustainable Financing.1999. Reference Source\n\nChen B, Wang Z, Yang Z, et al.: Status quo analysis of regions where the New Rural Co-operative Medical Scheme has not been implemented in China by 2007. Chinese Rural Health Service Administration. 2008; 28(3): 163–5.\n\nBusse R, Schreyogg J, Gericke C: Analysing changes in health financing arrangements in high-income countries – a comprehensive framework approach. Health, Nutrition and Population Discussion Paper. Washington DC World Bank; 2007. Reference Source\n\nRao C, Yang G, Hu J, et al.: Validation of cause-of-death statistics in urban China. Int J Epidemiol. 2007; 36(3): 642–51. PubMed Abstract \| Publisher Full Text\n\nWang L, Yang G, Jiemin M, et al.: Evaluation of the quality of cause of death statistics in rural China using verbal autopsies. J Epidemiol Community Health. 2007; 61(6): 519–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO. The global burden of disease: 2004 update. Geneva: World Health Organization; 2008. Reference Source\n\nYi H, Zhang L, Singer K, et al.: Health insurance and catastrophic illness: a report on the New Cooperative Medical System in rural China. Health Econ. 2009; 18(Suppl 2): S119–27. PubMed Abstract \| Publisher Full Text\n\nZuo Y, Hu S, Fo W, et al.: A study on New Cooperative Medical Scheme in China. Chinese Health Resources. 2006; 9(3): 127–9.\n\nYou X, Kobayashi Y: The new cooperative medical scheme in China. Health Policy. 2009; 91(1): 1–9. Publisher Full Text\n\nYang H: Effect Analysis for New Rural Cooperation Medical Scheme Two Kind of Outpatient Service Compensation in Dali. Chinese Health Economics. 2009; 28(3): 42–45. Reference Source\n\nWu S, Tan Y: Research on the optimization of outpatient reimbursement models in the New Rural Cooperative Medical Scheme in poor regions. Journal of Wuhan University of Technology. 2009; 31(12): 148–51. Reference Source\n\nZuo Y, Hu S, Liu B, et al.: The influence of outpatient compensation to utilization of health service and management approach under new rural cooperative medical scheme. Health Economics Research. 2008; (2).\n\nSaltman RB, Figueras J: European Health Care Reform. Analysis of Current Strategies. Copenhagen: World Health Organization Regional Office for Europe 1997. Reference Source\n\nYu B, Meng Q, Collins C, et al.: How does the New Cooperative Medical Scheme influence health service utilization? A study in two provinces in rural China. BMC Health Serv Res. 2010; 10: 116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang Q, Jia Z: Psychological counseling for free treatment of patients with tuberculosis. Shanghai: Collection of Abstracts of the 10th Chinese Academic Conference of Psychology 2005." }	[ { "id": "1041", "date": "04 Jul 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\n\nThe article is interesting in that it compares chronic conditions benefits package in 32 counties in China. It uses a framework based on three dimensions of coverage: population, service and cost. It shows differences between counties and concludes with the necessity to standardize these across counties.", "responses": [] }, { "id": "1080", "date": "10 Sep 2013", "name": "Qunhong Wu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1) The title of the paper is clear and outlines key information in this article. 2)The abstract has provided a concise and fairly good summary. 3) The description on the study design, method as well as the analysis of the findings are comprehensive. 4) The conclusions are justified based on the results. 5) The limitation of the data has been clearly stated within the article. As many of the descriptions are based on the design instead of the implications of the results, some finding of relevant studies should be cited within the discussion to provide more evidence and to support the conclusion and suggestions. Despite this I would like to 'Approve' this article as it has provided a very comprehensive review on the NCMS's benefit package designs for NCD outpatients in China.", "responses": [] } ]	1	https://f1000research.com/articles/2-137
https://f1000research.com/articles/2-135/v1	04 Jun 13	{ "type": "Commentary", "title": "Pro-oxidant activity of dietary chemopreventive agents: an under-appreciated anti-cancer property", "authors": [ "Asfar S Azmi", "Fazlul H Sarkar", "SM Hadi", "Fazlul H Sarkar", "SM Hadi" ], "abstract": "“Let food be thy medicine and medicine be thy food” was quoted by Hippocrates more than two thousand years ago and since ancient times the health benefits of different natural agents have been exploited. In modern research, the disease preventive benefits of many such natural agents, particularly dietary compounds and their derivatives, has been attributed to their well recognized activity as the regulators of redox state of the cell. Nevertheless, most of these studies have focused on their antioxidant activity. A large body of evidence indicates that a major fraction of these agents can elicit pro-oxidant (radical generating) behavior which has been linked to their anti-cancer effects. This editorial provides an overview of the under-appreciated pro-oxidant activity of natural products, with a special focus on their ability to generate reactive oxygen species in the presence of transition metal ions, and discusses their possible use as cancer chemotherapeutic agents.", "keywords": [ "Plant Derived Dietary Agents", "Chemoprevention", "Antioxidants", "Pro-oxidants", "Oxidative DNA damage", "Anti-cancer Mechanisms", "ROS" ], "content": "Introduction\n\nPlant derived agents (fruit and vegetable derived agents and their derivatives) have been recognized to provide a number of disease preventive properties1. It is now more than 20 years since the anti-cancer properties of some of the major natural agents such as resveratrol, tea catechins (particularly epigallocatechin (EGCG)) and the turmeric compound curcumin were recognized2. Their anti-cancer activity has been primarily attributed to their antioxidant behavior and the popular notion of these agents as antioxidants has driven increased consumption in developed countries3. Further work in independent laboratories has shown that most of these agents can induce cell death through apoptosis, suppress anti-apoptotic pathways, block cell proliferation and modulate a number of important proteins implicated in sustaining the growth of cancer cells4. Nevertheless, there is no single unifying mechanism supporting their anti-cancer effects in vitro and in vivo. Recently, researchers have utilized systems and computational techniques to pinpoint the exact pathways modulated by some of these agents5. The results point to a highly promiscuous or having multiple mechanisms of action that involves modulation of multiple signaling in cancer cells6. Most importantly, researchers are yet to identify the main mechanism underlying the preferential cancer selectivity elicited by these agents, which spare normal cells. These are some of the major reasons for the skepticism surrounding their health benefits, which has become a hurdle in their clinical application. In order to solidify their clinical utility, researchers have to convincingly chalk out the primary mechanism of these promising agents.\n\n\nPro-oxidant activity of natural agents: an underappreciated activity\n\nWhile the antioxidant action of dietary plant derived agents is unquestionable, over the last decade a parallel field has evolved that has convincingly presented evidence in support of their pro-oxidant (oxygen radical generating) behavior7, which may be context dependent. Supporting the significance of pro-oxidant drug action, Jim Watson recently presented a strong case that most of the anti-cancer regimens (ionizing radiation, chemotherapeutics and targeted therapeutics) work either directly or indirectly by generating reactive oxygen species (ROS)8.\n\nThis concept is not new. For example a PubMed search on May 21, 2013 using the keywords ‘natural anti-cancer agents’ and ‘prooxidants’ returns 1159 publications, while the keywords ‘prooxidant’ and ‘anticancer agents’ retrieve more than 11,200 papers. Various plant extract and agents such as resveratrol, catechins, EGCG, quercetin, gossypol, curcumin and caffeic acid have routinely been shown to induce damage to isolated plasmid DNA, albeit under certain conditions (e.g. in the presence of transition metal ions, especially copper9). Copper is an essential metal found in chromatin, and observed to be elevated in a number of malignancies10. In the late 1980s, our group was the first to describe quercetin-induced DNA strand scission in the presence of copper11. Following our line of research, Fukuhara and Miyata were the first to demonstrate that resveratrol can induce oxidative DNA damage in the presence of copper ions12. Such activity is dependent on a Fenton type reaction in which resveratrol oxidation results in the generation of superoxide radicals that re-oxidize to produce hydroxyl radicals in the presence of Cu(II) (elaborated in Zheng’s article13). Our laboratory showed similar behavior for a number of natural agents, which led us to propose the hypothesis that one of the major anti-cancer mechanisms of plant polyphenolic compounds involves their pro-oxidant behavior in the presence of transition metal ions14. Other groups have also shown similar oxidant behavior in the presence of iron15.\n\nUsing an alkaline gel single cell electrophoresis/Comet assay, our group was among the first to demonstrate such pro-oxidant behavior in a cellular/biological system either alone or in the presence of copper16. Since then a number of laboratories have confirmed these results17,18, which resulted in the increasing acceptance of the pro-oxidant behavior of a number of natural agents. Recently, we also showed the pro-oxidant DNA damaging effects of resveratrol in a mice model with supra-copper concentrations19. These and other studies20 unequivocally prove that, in addition to their antioxidant behavior, natural dietary and diet-derived agents can elicit pro-oxidant behavior and this property cannot be ignored if drawing up a design for a clinical trial investigating the anti-cancer activity of natural agents.\n\nIt has been recognized that additional work is needed to further elucidate the mechanism supporting such pro-oxidant behavior of natural agents and the reasons for their cancer cell selectivity. Key questions remain such as:\n\n(a) Is pro-oxidant action the primary mechanism of these agents since ROS generation is a very rapid event that occurs within nano-seconds?\n\n(b) What are the metabolic differences in tumors that switch the plant derived agents’ antioxidant behavior to pro-oxidant?\n\n(c) Apart from DNA damage, what is the effect of such pro-oxidant behavior on other cellular components since it is well known that proteins and lipids are also prone to oxidative damage?\n\n(d) Is there any preferential selectivity of natural agents towards copper to generate reactive oxygen species over other metal ions found in the cellular milieu?\n\n(e) Are supra concentrations of copper or other transitional metals required to achieve such pro-oxidant behavior and does this has any value in actual patients?\n\nSome of the answers came from very recent studies of ours in which we showed that agents such as resveratrol can take advantage of the metabolic differences in solid tumors leading to selective cancer cell killing21. Our findings have demonstrated that in solid tumors, the preferential dependence of cancer cells on glycolysis (Warburg effect) leads to a lowering of tumor cell pH, which loosens the DNA structure thereby leading to exposure of chromatin-bound copper22. Such labile copper is more prone to attack by resveratrol, leading to oxidative DNA damage. In non-cancerous cells, the primary source of ATP generation is through the citric acid cycle, and therefore these cells maintain physiological pH and so have normal DNA integrity. This observation is just one small step in explaining the cancer cell selectivity of natural pro-oxidant agents.\n\n\nThe cell culture artifact conundrum\n\nAs convincing as it sounds, the pro-oxidant activities of natural agents come with caveats. For example, Barry Halliwell and colleagues have suggested that the pro-oxidant behavior is nothing but an artifact of cell culture conditions23. They propose that most natural agents get oxidized in the presence of the cell culture media component phenol red, which can generate oxygen radicals leading to lipid peroxidation. However, these assumptions were proved to be wrong when different dietary agents were tested in phenol-free media and their pro-oxidant behavior was retained24. Such experiment put to rest the controversy involving ROS generation in experimental conditions. In another study, Burkitt and colleagues showed that under normal physiological conditions, i.e. in the presence of either ascorbic acid or glutathione, resveratrol loses its pro-oxidant property and behaves as an antioxidant25. In the ascorbate system, resveratrol had no effect on the rate of hydroxyl radical formation, but protected DNA from damage by acting as a radical-scavenging antioxidant. Through these studies the authors have concluded that the DNA-damaging properties of resveratrol, as identified by Fukuhara and Miyata, will be of no significance under standard physiological conditions. Nevertheless, as described above, we have shown both in cellular and animal models that their pro-oxidant behavior in tumors remains relevant15,16.\n\n\nConclusion and future direction\n\nAs much as the anti-cancer activity of natural agents has intrigued researchers, it has also generated a lot of skepticism and controversy. Even though their promiscuous behavior is gaining some acceptance, the lack of a unifying mechanism supporting their activity has kept them at the periphery of clinical application. These agents are multi-mechanistic in nature and merely focusing on their antioxidant behavior will not do justice to their multifarious health benefits. Similarly, honing in on their effect on only a specific set of cancer hallmarks will not allow a proper understanding of the anti-cancer activity of these agents. Researchers need to pay more attention to the dogma-challenging yet under-appreciated pro-oxidant behavior of dietary agents. This exercise is expected to highlight unanswered questions and may lead to the definition of a single unifying mechanism of action related to their unquestionable anti-cancer effects. This in turn will help in the rapid clinical application of agents or their derivatives from nature’s bounty for the treatment of human malignancies.", "appendix": "Author contributions\n\n\n\nAsfar S Azmi developed the hypothesis, wrote and revised the article. Fazlul H Sarkar wrote and revised the article. SM Hadi conceived the hypothesis and revised the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nWillett WC, Stampfer MJ: Current evidence on healthy eating. Annu Rev Public Health. 2013; 34: 77–95. PubMed Abstract \| Publisher Full Text\n\nKatiyar SK, Agarwal R, Wang ZY, et al.: (-)-Epigallocatechin-3-gallate in Camellia sinensis leaves from Himalayan region of Sikkim: inhibitory effects against biochemical events and tumor initiation in Sencar mouse skin. Nutr Cancer. 1992; 18(1): 73–83. PubMed Abstract \| Publisher Full Text\n\nSkrovankova S, Misurcova L, Machu L: Antioxidant activity and protecting health effects of common medicinal plants. Adv Food Nutr Res. 2012; 67: 75–139. PubMed Abstract \| Publisher Full Text\n\nRaffoul JJ, Kucuk O, Sarkar FH, et al.: Dietary agents in cancer chemoprevention and treatment. J Oncol. 2012; 2012: 749310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan Y, Wu Q, Xia J, et al.: Systems biology approaches in identifying the targets of natural compounds for cancer therapy. Curr Drug Discov Technol. 2013; 10(2): 139–46. PubMed Abstract \| Publisher Full Text\n\nAzmi AS, Mohammad RM, Sarkar FH: Can network pharmacology rescue neutraceutical cancer research? Drug Discov Today. 2012; 17(15–16): 807–809. PubMed Abstract \| Publisher Full Text\n\nUllah MF, Ahmad A, Khan HY, et al.: The prooxidant action of dietary antioxidants leading to cellular DNA breakage and anticancer effects: Implications for chemotherapeutic action against cancer. Cell Biochem Biophys. 2011. PubMed Abstract \| Publisher Full Text\n\nWatson J: Oxidants, antioxidants and the current incurability of metastatic cancers. Open Biol. 2013; 3(1): 120144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHadi SM, Bhat SH, Azmi AS, et al.: Oxidative breakage of cellular DNA by plant polyphenols: a putative mechanism for anticancer properties. Semin Cancer Biol. 2007; 17(5): 370–376. PubMed Abstract \| Publisher Full Text\n\nLinder MC: The relationship of copper to DNA damage and damage prevention in humans. Mutat Res. 2012; 733(1–2): 83–91. PubMed Abstract \| Publisher Full Text\n\nRahman A, Shahabuddin, Hadi SM, et al.: Strand scission in DNA induced by quercetin and Cu(II): role of Cu(I) and oxygen free radicals. Carcinogenesis. 1989; 10(10): 1833–1839. PubMed Abstract \| Publisher Full Text\n\nFukuhara K, Miyata N: Resveratrol as a new type of DNA-cleaving agent. Bioorg Med Chem Lett. 1998; 8(22): 3187–3192. PubMed Abstract \| Publisher Full Text\n\nZheng LF, Wei QY, Cai YJ, et al.: DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: mechanism and structure-activity relationship. Free Radic Biol Med. 2006; 41(12): 1807–1816. PubMed Abstract \| Publisher Full Text\n\nHadi SM, Asad SF, Singh S, et al.: Putative mechanism for anticancer and apoptosis-inducing properties of plant-derived polyphenolic compounds. IUBMB Life. 2000; 50(3): 167–171. PubMed Abstract \| Publisher Full Text\n\nTsai YC, Wang YH, Liou CC, et al.: Induction of oxidative DNA damage by flavonoids of propolis: its mechanism and implication about antioxidant capacity. Chem Res Toxicol. 2012; 25(1): 191–196. PubMed Abstract \| Publisher Full Text\n\nAzmi AS, Bhat SH, Hanif S, et al.: Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties. FEBS Lett. 2006; 580(2): 533–538. PubMed Abstract \| Publisher Full Text\n\nTyagi A, Gu M, Takahata T, et al.: Resveratrol selectively induces DNA Damage, independent of Smad4 expression, in its efficacy against human head and neck squamous cell carcinoma. Clin Cancer Res. 2011; 17(16): 5402–5411. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMichels G, Watjen W, Weber N, et al.: Resveratrol induces apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6 glioma cells. Toxicology. 2006; 225(2–3): 173–182. PubMed Abstract \| Publisher Full Text\n\nKhan HY, Zubair H, Ullah MF, et al.: Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism. Biometals. 2011; 24(6): 1169–1178. PubMed Abstract \| Publisher Full Text\n\nMartin-Cordero C, Leon-Gonzalez AJ, Calderon-Montano JM, et al.: Pro-oxidant natural products as anticancer agents. Curr Drug Targets. 2012; 13(8): 1006–1028. PubMed Abstract \| Publisher Full Text\n\nMuqbil I, Beck FW, Bao B, et al.: Old wine in a new bottle: the Warburg effect and anticancer mechanisms of resveratrol. Curr Pharm Des. 2012; 18(12): 1645–1654. PubMed Abstract \| Publisher Full Text\n\nShamim U, Hanif S, Albanyan A, et al.: Resveratrol-induced apoptosis is enhanced in low pH environments associated with cancer. J Cell Physiol. 2012; 227(4): 1493–1500. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLong LH, Clement MV, Halliwell B: Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of (-)-epigallocatechin, (-)-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochem Biophys Res Commun. 2000; 273(1): 50–53. PubMed Abstract \| Publisher Full Text\n\nSudheer AR, Muthukumaran S, Devipriya N, et al.: Ellagic acid, a natural polyphenol protects rat peripheral blood lymphocytes against nicotine-induced cellular and DNA damage in vitro: with the comparison of N-acetylcysteine. Toxicology. 2007; 230(1): 11–21. PubMed Abstract \| Publisher Full Text\n\nBurkitt MJ, Duncan J: Effects of trans-resveratrol on copper-dependent hydroxyl-radical formation and DNA damage: evidence for hydroxyl-radical scavenging and a novel, glutathione-sparing mechanism of action. Arch Biochem Biophys. 2000; 381(2): 253–263. PubMed Abstract \| Publisher Full Text" }	[ { "id": "984", "date": "05 Jun 2013", "name": "Hasan Mukhtar", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well written. Was a joy to read.", "responses": [] }, { "id": "1406", "date": "04 Sep 2013", "name": "Surinder K Batra", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is well-written manuscript.", "responses": [] } ]	1	https://f1000research.com/articles/2-135
https://f1000research.com/articles/2-133/v1	30 May 13	{ "type": "Clinical Practice Article", "title": "Paradoxical immune reconstitution inflammatory syndrome due to toxoplasmic encephalitis: two cases and review of initiation of antiretroviral timing in toxoplasmic encephalitis IRIS", "authors": [ "Andrew R DiNardo", "Douglas Smoot Lewis", "Hoonmo L Koo", "J Clay Goodman", "Elizabeth Chiao", "Roberto Andrade", "Douglas Smoot Lewis", "Hoonmo L Koo", "J Clay Goodman", "Elizabeth Chiao", "Roberto Andrade" ], "abstract": "Toxoplasma encephalitis immune reconstitution inflammatory syndrome (TE-IRIS) is rare and usually occurs in an unmasking, rather than paradoxical form. To the best of our knowledge, only two cases of paradoxical TE-IRIS and nine cases of unmasking TE-IRIS have been previously described. We present two additional cases of histopathology-consistent paradoxical TE-IRIS, after early initiation of antiretroviral therapy (ART), and review the literature on TE-IRIS. Three of the four reported cases of paradoxical TE-IRIS were associated with early (within one week) initiation of ART, an issue that was not addressed in the 2009 US Department of Health and Human Services guidelines for the treatment of opportunistic infections.", "keywords": [ "Toxoplasmosis", "AIDS", "IRIS" ], "content": "Case 1\n\nA 35 year-old man with AIDS, not on antiretroviral therapy (ART) or prophylaxis, presented with shortness of breath, productive cough and fever for one week, as well as progressive weakness of his left upper extremity over the previous two weeks. His exam was significant for weakness (4/5) in his left-hand grip. His CD4 count was 8 (1%) cells/µl and his viral load was 547,000 copies/ml. Serum Toxoplasma IgG levels were undetectable. His chest X-ray revealed a right middle lobe infiltrate and three sputum samples for acid fast bacilli (AFB) were negative. Respiratory symptoms resolved with ceftriaxone and azithromycin. An MRI of his brain revealed two ring-enhancing lesions in the right precentral and occipital temporal areas (Figure 1A). The patient refused a lumbar puncture and was empirically started on anti-toxoplasmic therapy (pyrimethamine, sulfadiazine and leucovorin) on day two. On day four, the patient was started on ART with abacavir, lamivudine and raltegravir.\n\nBrain MRI of patient 1 at presentation showed an ill-defined 1 cm right medial occipitotemporal gyrus mass and a 1.8×1.7×1.5 cm mass in the subcortical white matter of the right precentral gyrus with surrounding edema (A). After 14 days of anti-toxoplasma treatment and antiretroviral therapy, a follow-up MRI revealed enlargement of the prior right precentral gyrus lesion now to maximum size of 2.2 cm (B).\n\nOver the next two weeks, the patient’s function deteriorated: his left deltoid muscle strength weakened to 2/5 (previously 5/5), he developed a left facial droop and left hip flexor and quadriceps weakness were 3/5 (previously 5/5). He consented to a lumbar puncture and his CSF revealed 6 white blood cells (WBC) per mm3 (96% lymphocytes and 4% monocytes), glucose of 41 g/L and protein of 92 g/L. EBV PCR in the CSF was positive. On hospital day ten, he suffered a tonic-clonic seizure. A repeat MRI on day 14 of empiric anti-Toxoplasma therapy, showed enlargement of the two prior lesions and development of a third lesion (Figure 1B). After twenty days of anti-toxoplasmic therapy, a brain biopsy revealed rare Toxoplasma gondii tachyzoites and numerous bradyzoites (Figure 2A), as well as CD8+ predominant lymphocytic infiltrates (Figure 2B). A repeat CD4 count and viral load measure was 13 cells/µll (1%) and 10,300 copies/ml respectively. Anti-Toxoplasma treatment and ART were continued and the patient was started on corticosteroids (dexamethasone 4 mg every 4 hours tapered to prednisone 10 mg per day over 2 weeks), with gradual improvement but not complete resolution of his symptoms and signs.\n\nSignificant necrosis with perivascular mixed inflammatory cells, with lymphocyte predominance and concentric vascular wall fibroblastic proliferation. Rare individual Toxoplasma gondii bradyzoites and numerous tachyzoites were identified (100X) (A). Immunophenotyping of the inflammatory cells, showing a mixture of mature T- and B-lymphocytes with CD8+ over CD4+ cell predominance. There was no evidence of other infections or CNS lymphoma (100X) (B).\n\n\nCase 2\n\nA 51 year-old man presented with two weeks of fevers, weight loss, frontal headaches, unsteady gait and upper extremity left-sided weakness. Three weeks prior, he had been diagnosed with AIDS and was not on ART or prophylaxis. On exam, he had left deltoid and hand-grip weakness (3/5) and left hip flexor and quadriceps extremity weakness (4/5), as well as decreased light sensation in his left arm.\n\nHis CD4 count was 152 (13%) cells/µl and his viral load was 433,000 copies/ml. Toxoplasma IgG was positive. An MRI revealed multiple ring-enhancing lesions in his frontoparietal region (Figure 3A). CSF showed 6 WBCs (100% lymphocytes), undetectable JC virus DNA and no malignant cells on cytopathology or flow cytometry. On day two, the patient was started on pyrimethamine, sulfadiazine and leucovorin, as well as lopinavir-ritonavir, tenofovir and emtricitabine.\n\nBrain MRI of patient 2 at presentation showed an irregular ring-enhancing lesion in the right frontoparietal region with maximal transverse dimension of 2.2 cm (A). After 10 days of anti-toxoplasma treatment and antiretroviral therapy, follow-up MRI revealed no significant interval change of enhancing frontoparietal lesion (B).\n\nThe patient experienced improvement in his weakness and gait to near-baseline and was discharged to rehabilitation. Ten days after initiation of treatment, the patient’s headache and left-sided weakness returned, in addition to a new left-sided facial droop. A repeat MRI showed no significant change (Figure 3B). A repeat CD4 count was 133 (15%) cells/µl and his viral load was 77,700 copies/ml. A brain biopsy revealed T. gondii by immunohistochemistry (Figure 4A) and a CD8+ lymphocytic infiltrate (Figure 4B). His symptoms progressively improved with continued anti-Toxoplasma therapy, ART and a corticosteroid taper (initially dexamethasone 4 mg every 6 hours for days, tapered to oral prednisone 2.5 mg per day, over a six-week period).\n\nImmunohistochemistry for T. gondii (400X) (A). Immunophenotyping of the inflammatory cells, showing again CD8+ over CD4+ T-cell predominance (100X) (B).\n\n\nDiscussion\n\nDespite Toxoplasma encephalitis being the most common cause of focal brain lesions in AIDS patients in resource-rich countries1, TE-IRIS is rare2,3 due to the elegant mechanisms of immune evasion by T. gondii4. A delicately controlled balance between host immune response and parasite immune evasion characterizes the life cycle of T. gondii. Bradyzoites survive in a dormant intracellular state in a parasitophorous vacuole that, typically, evades intracellular acidification. The tissue cysts are kept in check by a Th1 predominant immune response5 characterized by macrophages and dendritic cells, producing IL-12, triggering CD4 cells to release IL-2, IFN-γ, IL-6 and TNF-α, thereby stimulating a reactive oxygen and nitric oxide-mediated cytotoxic response.\n\nAIDS patients have impaired IFN-γ and IL-2 production that leads to uncontrolled T.gondii growth and abscess formation with necrotic centers and hyperemic edges, which appear as the characteristic multiple ring-enhancing lesions on MRI (Figure 1 and Figure 3)6. Initiation of ART and subsequent immune reconstitution restores the ability to control Toxoplasma7. Pathologic excessive reactivation of the immune system is evident on histopathology by an exuberant lymphocytic infiltration with CD8+ predominance, which is not otherwise seen in TE and is characteristic of TE-IRIS (Figure 2 and Figure 4)4.\n\nFor most opportunistic infections, there is increasing evidence to support early initiation of ART8. However, in the case of severe OIs involving the central nervous system (CNS) there is also evidence supporting delayed initiation of ART. In a retrospective review of 514 patients with HIV and tuberculosis meningitis, there was significant mortality in those started on ART and anti-tuberculosis therapy simultaneously9. In a randomized, double-blinded, placebo-controlled prospective study of tuberculosis meningitis there was significant morbidity associated with immediate initiation of ART10. Recently, the prospective COAT (cryptococcal optimal ART timing) trial, designed to look at the optimal timing of ART initiation in cryptococcal meningitis, was stopped early due to increased mortality with early initiation of ART11. Since TE-IRIS is very rare, there is a paucity of data regarding the timing of ART initiation; therefore this issue is not addressed in the current US Department of Health and Human Services guidelines12.\n\nImmune reconstitution inflammatory response syndrome cases are classified as either unmasking (unmasking of an occult infection not known at the time of ART initiation) or paradoxical (the paradoxical worsening of a known disease despite appropriate therapy). In their case series of TE-IRIS, Martin-Blondel et al. report three cases of unmasking TE-IRIS and 65 cases of TE that did not experience paradoxical TE-IRIS despite virologic and immunologic response2. All three patients had unmasking TE-IRIS (none of them had been diagnosed with TE before the initiation of ART) and they all developed IRIS more than a month after the initiation of ART. None of the 65 patients with TE who did not develop IRIS had received ART before two weeks of anti-toxoplasma treatment (Martin-Blondel personal communication). To our knowledge, we describe only the third and fourth cases of paradoxical TE-IRIS in the literature3,13. In both of our patients, as well as the case of paradoxical TE-IRIS presented by Tremont-Lukats3, ART was started simultaneously or within the first week of anti-toxoplasmic therapy. In the case of paradoxical TE-IRIS described by Cabral et al.13, ART was delayed for twenty days after initiation of anti-toxoplasmic therapy (Ferracini personal communication).\n\nIn conclusion, we present two patients with AIDS and CNS toxoplasmosis, who had disease progression, despite empiric anti-toxoplasmic therapy and a 1-log decrease in their HIV viral load with ART. Since the overwhelming majority of patients with toxoplasmic encephalitis improve after two weeks of anti-toxoplasma therapy14, the worsening symptoms led to a brain-biopsy. In both patients, histopathology showed a vigorous cytotoxic immune response, consistent with TE-IRIS1,4. Both patients improved with continuation of anti-parasitic treatment, ART and high-dose steroids. In these two cases and one of the two previous cases of paradoxical TE-IRIS, initiation of ART took place simultaneous or within the first week of anti-parasitic therapy. While generalizations cannot be made with only four cases, until further data evaluates the issue, we recommend very close monitoring if ART is started before at least two weeks of anti-toxoplasma therapy. Moreover, our cases illustrate that, for CNS toxoplasmosis in AIDS patients who have been recently started on ART, after performing a biopsy, the threshold to administer steroids for possible IRIS should be low, if there is any evidence of clinical deterioration.\n\n\nConsent\n\nInformed consent for publication of the clinical details and clinical images was obtained from the patients.", "appendix": "Author contributions\n\n\n\nARD wrote the manuscript. ARD, DSL and RA contacted previous authors for unpublished data. DSL chose and prepared the radiology slides. JCG chose and prepared the histopathology slides. EC, HK and RA contributed to the design and preparation of the manuscript. All authors were involved in the revisions and 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\nAntinori A, Ammassari A, De Luca A, et al.: Diagnosis of AIDS-related focal brain lesions: a decision-making analysis based on clinical and neuroradiologic characteristics combined with polymerase chain reaction assays in CSF. Neurology. 1997; 48(3): 687–694. PubMed Abstract \| Publisher Full Text\n\nBerkeley JL, Nath A, Pardo CA: Fatal immune reconstitution inflammatory syndrome with human immunodeficiency virus infection and Candida meningitis: case report and review of the literature. J Neurovirol. 2008; 14(3): 267–276. PubMed Abstract \| Publisher Full Text\n\nMartin-Blondel G, Delobel P, Blancher A, et al.: Pathogenesis of the immune reconstitution inflammatory syndrome affecting the central nervous system in patients infected with HIV. Brain. 2011; 134(Pt 4): 928–946. PubMed Abstract \| Publisher Full Text\n\nFarkash AE, Maccabee PJ, Sher JH, et al.: CNS toxoplasmosis in acquired immune deficiency syndrome: a clinical-pathological-radiological review of 12 cases. J Neurol Neurosurg Psychiatry. 1986; 49(7): 744–748. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZolopa A, Andersen J, Powderly W, et al.: Early antiretroviral therapy reduces AIDS progression/death in individuals with acute opportunistic infections: a multicenter randomized strategy trial. PLoS One. 2009; 4(5): e5575. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgarwal U, Kumar A, Behera D, et al.: Tuberculosis associated immune reconstitution inflammatory syndrome in patients infected with HIV: meningitis a potentially life threatening manifestation. AIDS Res Ther. 2012; 9(1): 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMandell GL, Bennett JE, Dolin R: Principles and Practice of Infectious Diseases. 6th ed. London: Churchill Livingstone; 2005. Reference Source\n\nTorok ME, Yen NT, Chau TT, et al.: Timing of initiation of antiretroviral therapy in human immunodeficiency virus (HIV)--associated tuberculous meningitis. Clin Infect Dis. 2011; 52(11): 1374–1383.PubMed Abstract \| Publisher Full Text\n\nHIV Treatment Study in Patients with Cryptococcal Meningitis Ends Enrollment Early. Higher Mortality Rate Found with Early Antiretroviral Therapy 2012. Reference Source\n\nKaplan JE, Benson C, Holmes KH, et al.: Guidelines for prevention and treatment of opportunistic infections in HIV-infected adults and adolescents: recommendations from CDC the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. MMWR Recomm Rep. 2009; 58(RR-4): 1–207; quiz CE1. PubMed Abstract\n\nMartin-Blondel G, Alvarez M, Delobel P, et al.: Toxoplasmic encephalitis IRIS in HIV-infected patients: a case series and review of the literature. J Neurol Neurosurg Psychiatry. 2011; 82(6): 691–693. PubMed Abstract \| Publisher Full Text\n\nTremont-Lukats IW, Garciarena P, Juarbe R, et al.: The immune inflammatory reconstitution syndrome and central nervous system toxoplasmosis. Ann Intern Med. 2009; 150(9): 656–657. PubMed Abstract \| Publisher Full Text\n\nCabral RF, Valle Bahia PR, Gasparetto EL, et al.: Immune reconstitution inflammatory syndrome and cerebral toxoplasmosis. AJNR Am J Neuroradiol. 2010; 31(7): E65–6. PubMed Abstract \| Publisher Full Text\n\nCohen BA: Neurologic manifestations of toxoplasmosis in AIDS. Semin Neurol. 1999; 19(2): 201–211. PubMed Abstract \| Publisher Full Text" }	[ { "id": "977", "date": "31 May 2013", "name": "Bruce Brew", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful addition to the literature and serves as a cautionary note to clinicians. There are some minor issues: the grading scale for weakness is the MRC scale and the viral load referred to in the first patient relates to HIV.", "responses": [ { "c_id": "476", "date": "05 Jun 2013", "name": "Andrew DiNardo", "role": "Author Response", "response": "Thank you for your comments. Yes, the strength scale is MRC & yes the \"viral load\" was meant to imply \"HIV viral load.\"" } ] }, { "id": "978", "date": "31 May 2013", "name": "Matthijs Brouwer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written and interesting observation that is helpful for doctors treating HIV-infected patients. Although the number of patients is low with TE-IRIS, one could argue to delay HAART until the patient has been treated for 2 weeks with anti-toxoplasmosis treatment, which is an important message.", "responses": [] }, { "id": "1260", "date": "30 Jul 2013", "name": "Guillaume Martin-Blondel", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, earlier HAART improves outcome particularly in those with the lowest CD4 counts. However, opportunistic infections of the CNS such as TB and cryptococcosis are an exception, and timing of HAART requires special consideration. The optimal timing of HAART in the setting of toxoplasma encephalitis has not been studied. This well written and informative case report illustrates that initiating HAART (very) early after beginning anti-toxoplasma therapy might be deleterious, deserving further studies.", "responses": [] } ]	1	https://f1000research.com/articles/2-133
https://f1000research.com/articles/2-132/v1	29 May 13	{ "type": "Systematic Review", "title": "Venous thromboembolism prophylaxis in patients with traumatic brain injury: a systematic review", "authors": [ "Yohalakshmi Chelladurai", "Kent A Stevens", "Elliott R Haut", "Daniel J Brotman", "Ritu Sharma", "Kenneth M Shermock", "Sosena Kebede", "Sonal Singh", "Jodi B Segal", "Yohalakshmi Chelladurai", "Elliott R Haut", "Daniel J Brotman", "Ritu Sharma", "Kenneth M Shermock", "Sosena Kebede", "Sonal Singh", "Jodi B Segal" ], "abstract": "Objective: There is considerable practice variation and clinical uncertainty about the choice of prophylaxis for preventing venous thromboembolism in patients with traumatic brain injury. We performed a systematic review to assess both the effectiveness and safety of pharmacologic and mechanical prophylaxis, and the optimal time to initiate pharmacologic prophylaxis in hospitalized patients with traumatic brain injury.Data sources and study selection: MEDLINE®, EMBASE®, SCOPUS, CINAHL, International Pharmaceutical Abstracts, clinicaltrial.gov, and the Cochrane Library were searched in July 2012 to identify randomized controlled trials and observational studies reporting on the effectiveness or safety of venous thromboembolism prevention in traumatic brain injury patients.Data extraction: Paired reviewers extracted detailed information from included articles on standardized forms and assessed the risk of bias in each article.Data synthesis: Twelve studies (2 randomized controlled trials and 10 cohort studies) evaluated the effectiveness and safety of venous thromboembolism prophylaxis in patients with traumatic brain injury. Five of the included studies assessed the optimal timing of initiation of pharmacological prophylaxis. Low grade evidence supports the effectiveness of enoxaparin over control in reducing deep vein thrombosis. Low grade evidence also supports the safety of unfractionated heparin over control in reducing mortality in patients with traumatic brain injury. Evidence was insufficient for remaining comparisons and outcomes including the optimal timing of initiation of pharmacoprophylaxis.Conclusion: There is some evidence that pharmacoprophylaxis improves deep vein thromboses and mortality outcomes in patients hospitalized with traumatic brain injury. Additional studies are required to strengthen this evidence base.", "keywords": [ "There is considerable practice variation and clinical uncertainty about the choice of a prophylaxis modality (pharmacologic and mechanical) and about the optimal pharmacologic agent", "dose", "timing of initiation", "and duration for the prevention of venous thromboembolism (VTE) among patients with traumatic brain injury (TBI)1. This population is at increased risk for VTE due to a combination of factors (i.e.", "the brain injury itself", "other injuries", "intensive care unit admission", "immobilization", "major surgery", "etc.). This increased risk should prompt routine thromboprophylaxis in patients with TBI", "however", "the concern over an associated elevated risk of bleeding in patients with TBI often leads physicians to withhold pharmacological thromboprophylaxis. The American College of Chest Physician guidelines do not specifically address DVT prophylaxis in patients with traumatic brain injury2. To help clarify the practice standards to prevent VTE events in the TBI population", "we performed a comprehensive systemic review of the literature." ], "content": "Introduction\n\nThere is considerable practice variation and clinical uncertainty about the choice of a prophylaxis modality (pharmacologic and mechanical) and about the optimal pharmacologic agent, dose, timing of initiation, and duration for the prevention of venous thromboembolism (VTE) among patients with traumatic brain injury (TBI)1. This population is at increased risk for VTE due to a combination of factors (i.e., the brain injury itself, other injuries, intensive care unit admission, immobilization, major surgery, etc.). This increased risk should prompt routine thromboprophylaxis in patients with TBI; however, the concern over an associated elevated risk of bleeding in patients with TBI often leads physicians to withhold pharmacological thromboprophylaxis. The American College of Chest Physician guidelines do not specifically address DVT prophylaxis in patients with traumatic brain injury2. To help clarify the practice standards to prevent VTE events in the TBI population, we performed a comprehensive systemic review of the literature.\n\n\nMethods\n\nThe protocol for the review was developed and posted online following guidelines for systematic reviews3,4. Additional methodological details are available in our evidence report prepared for the Agency for Healthcare Research and Quality (AHRQ)5.\n\nThe following databases were searched in July 2012 for primary studies: MEDLINE®, EMBASE®, SCOPUS, CINAHL, International Pharmaceutical Abstracts, clinicaltrial.gov, and the Cochrane Library. An analytic framework depicting our population of interest, interventions tested for prevention of VTE, intermediate and patient-oriented outcomes of treatment, as well as the harms of the interventions was developed3.\n\nTitles were reviewed followed by abstracts to identify randomized controlled trials (RCTs) or observational studies with comparison groups reporting on the effectiveness or safety of VTE prevention in TBI patients. Two investigators independently reviewed abstracts meeting our inclusion criteria; abstracts were excluded if both reviewers agreed that the article met one or more of the exclusion criteria (Table 1).\n\nEvidence Partners 2010 web-based database management program, DistillerSR, was used to manage the screening and review process. Standardized forms for data extraction from the articles were created. Paired investigators reviewed all extracted data.\n\nThe risk of bias was assessed independently and in duplicate, using the Downs and Black instrument6. Ten items that were most relevant to this review were prioritized in our assessment of risk of bias. Studies were assessed to have a low risk of bias if all of the following were true: the article completely described the hypothesis, the outcomes (in the introduction or methods section), the characteristics of the included subjects, the distribution of the potential confounders in each group, the interventions and comparisons (if relevant) the main findings, adverse events, and characteristics of the subjects lost to follow up. Additionally, we judged studies to be at low risk of bias if they randomized subjects to the intervention and concealed the assignment until randomization was complete, and if they attempted to blind the study participants and to blind those who measured the main outcomes. By this system, non-randomized studies could only be at moderate or high risk of bias. Studies were rated as having a moderate risk of bias if one of those items was not true, even if all of the others were true, or if the reporting on the distribution of potential confounders in each group was at least partially done. If two of the elements were not true, studies were rated to have a high risk of bias.\n\nA detailed set of evidence tables was created containing all information abstracted from eligible studies. Given the substantial statistical and clinical heterogeneity, we do not report pooled results but display the individual magnitude of effect and statistical significance for the individual studies.\n\nThe effectiveness of pharmacological and mechanical strategies in preventing patient-oriented outcomes such as VTE, deep vein thrombosis (DVT) and pulmonary embolism (PE), mortality and progression of intracranial hemorrhage.\n\nThe quantity, quality, and consistency of the best available evidence was graded by adapting an evidence-grading scheme recommended in the Agency for Healthcare Research and Quality: Methods Guide for Conducting Comparative Effectiveness Reviews7.\n\n\nResults\n\nThe literature search identified 30902 citations. After necessary exclusions and triage to other topics, 12 articles were included for this review (Figure 1).\n\nSeven studies that evaluated the effectiveness of pharmacological and mechanical strategies to prevent VTE in hospitalized patients with TBI were identified8–14, four that evaluated the optimal timing of initiation of pharmacological prophylaxis1,15–17 and one study that evaluated both18. Most of the studies were conducted in North America1,8,9,11–18. Two RCTs were included in this review10,14. The remaining were cohort studies; nine retrospective studies1,8,9,11,13,15–18 and one prospective12. The majority of studies included patients admitted in level 1 trauma centers.\n\nThe number of participants in the included studies ranged from 32 to 812; the mean age of the participants ranged from 36 to 47 years. The Injury Severity Score (ISS) of TBI patients was reported in eight studies; the mean ranged from 15.7 to 33.8 indicating severe multi-system trauma8–12,14,15,18. The ethnicity or race of the participants was not reported in any study (Table 2).\n\nStudy has three arms, we have shown data for all comparisons individually; UFH=Unfractionated heparin; SCD=Sequential Compression Devices; ISS=Injury Severity Score; NR=Not Reported; RCT=Randomized Controlled Trial; PC=Prospective Cohort; RETRO=Retrospective Cohort; ¥Mean reported for overall group.\n\nEight studies were included to assess the effectiveness and safety of pharmacological and mechanical interventions to prevent VTE in patients with traumatic brain injury8–14,18. The interventions compared in these studies were highly heterogeneous; studies varied in drugs compared, the dosages and timing of initiation of therapy. Many studies had a control group in which active therapy was withheld from participants. The dose of pharmacological drugs used was reported in five studies; dalteparin was administered as 5000 U once daily. Unfractionated heparin (UFH) as 5000 U thrice daily, and enoxaparin as 30 mg twice daily or 40 mg daily.\n\nFive studies independently assessed the optimal timing of the initiation of chemoprophylaxis in the same population1,15–18. Although enoxaparin and UFH were the only pharmacological agents employed in these studies, two studies were unclear about the pharmacological agents used and were classified as “any heparin” intervention16,17. Four out of five studies compared the effectiveness and safety of pharmacoprophylaxis in preventing VTE when initiated less than 72 hours (early prophylaxis) of hospital admission versus greater than 72 hours (late prophylaxis).\n\nThere were no studies that assessed the effectiveness of inferior vena cava filters in preventing PE in TBI patients.\n\nMost studies did not routinely screen for VTE1,8–10,13,14,16,18. Weekly surveillance using duplex ultrasound examination was carried out in four studies11,12,15,17, although two of these studies performed it in high risk patients exclusively11,17.\n\nVenous thromboembolism. Five studies assessed the effectiveness of pharmacological agents in preventing VTE in patients with TBI8,11–13,18. One study demonstrated lower rates of VTE in the UFH group compared to the control group (3% vs. 1%, respectively, p=0.019)11; two studies showed increased rates of VTE in the enoxaparin and sequential compression devices group compared to the control group (enoxaparin vs. control, 3.9% vs. 2.2%, p=0.29; Sequential compression devices (SCD) vs. control, 28.6% vs. 22.2%, p=0.7)12,18, while the last study demonstrated no difference in rates of VTE between dalteparin and control groups (0% vs. 0%)13. Head-to-head comparison available in a study demonstrated marginally increased rates of venous thromboses in patients treated with dalteparin compared to those treated with enoxaparin (7.5% vs. 7.0%, p value not significant)8.\n\nA single study demonstrated increased rates of VTE with early enoxaparin prophylaxis when compared to late prophylaxis. (5.56% vs. 2.72% percent, Odds ratio (OR) 2.10, p=0.26)18 (Table 3).\n\nStudy has three arms; UFH=Unfractionated heparin; SCD=Sequential Compression devices; DVT=Deep vein thrombosis; PE= Pulmonary embolism; ICH=intracranial hemorrhage; N=Number; NR=Not Reported; #p value not significant; ¶p value significant; Φ- Of the total PE, 6.6% in the enoxaparin arm and 3.3% in the IPC arm were fatal; Ж- DVT risk per 100 patients.\n\nOverall, the evidence was concluded to be insufficient to comment on the effectiveness and optimal timing of initiation of VTE prophylaxes in TBI patients (Table 4).\n\nUFH=Unfractionated heparin; SCD=Sequential Compression devices; NR=Not Reported; NS=Not significant; *P-values or tests of statistical significance not reported; # Two sided P-estimated using Fishers exact test. Bold-italic text indicates studies with evidence for effectiveness. Non bold-italic text indicates studies with insufficient evidence.\n\nDeep vein thrombosis. Four studies were included to evaluate the efficacy of enoxaparin, UFH and sequential compression devices in preventing the development of DVT in patients with TBI9,10,12,14. A single study demonstrated reduced rates of DVT in enoxaparin and UFH heparin groups compared to control (1% vs. 1% vs. 2% respectively, p value not reported)9. Two more studies demonstrated lower rates of DVT in patients treated with enoxaparin compared to those treated with placebo and sequential compression devices (0% vs. 3.6%, p=0.45 and 5% vs. 6.6%, p=0.07)10,14. In contrast to this, a fourth study demonstrated that patients treated with sequential compression devices experienced fewer events when compared to a control group (0% vs. 11.1%)12.\n\nIn two “any heparin” studies, the rates of DVT were consistently higher in the late prophylaxis group16,17. The same was observed in patients treated with UFH; rates of DVT were higher when UFH was commenced later than 72 hours (4.3% vs. 5.9%, p value not significant)15 (Table 3).\n\nThree individual studies demonstrated that rates of DVT were lower in patients treated with enoxaparin when compared to controls or patients treated with sequential compression devices only9,10,14. Consistent, direct, yet imprecise results, which included one RCT with a low risk of bias, led to the conclusion that low-grade evidence supported the effectiveness of enoxaparin over control/sequential compression devices in reducing DVT in hospitalized patients with TBI. However, the evidence is insufficient to comment on the optimal timing of initiation of chemoprophylaxis in the same population (Table 4).\n\nPulmonary embolism. Five out of the eight included studies assessed the effectiveness of prophylaxis with enoxaparin, dalteparin, UFH and sequential compression devices in preventing development of PE in patients hospitalized with TBI. The results of these studies were equivocal. One study demonstrated that patients treated with enoxaparin failed to develop PE, whilst those in the control and UFH intervention groups did, the rate being lower in the control group9. In contrast, a RCT demonstrated that there was no difference in rates of PE in enoxaparin-treated patients and controls (0% vs. 0%)14. Two studies showed varying outcomes in patients treated with sequential compression devices only; a RCT demonstrated lower rates of PE, all of which were fatal, in this group compared to treatment with enoxaparin (3.3% vs. 6.6%, p=0.04)10. However, in another study, the patients in the sequential compression devices intervention group were reported to have experienced an increase in pulmonary embolic events in comparison to control patients (28.6% vs. 11.1%, p value not reported)12. The last study reported the rate of development of PE in patients treated with dalteparin only, limiting an assessment of comparative effectiveness8.\n\nOptimal timing of initiation of chemoprophylaxis in TBI populations to prevent development of PE was analyzed in three studies. Two studies demonstrated increased incidence of PE with early prophylaxis (3.5% vs. 0% and 4.3% vs. 0%), whereas in the third study, patients treated with enoxaparin within 72 hours of admission experienced fewer pulmonary embolic events (1.5% vs. 2.2%, respectively, p=0.49)1,17,18 (Table 3).\n\nThe evidence was concluded to be insufficient to comment on the effectiveness and optimal timing of initiation of prophylaxes in preventing PE in TBI patients (Table 4).\n\nTotal mortality. Three studies included in this review evaluated the efficacy of prophylaxis with UFH or enoxaparin versus no prophylaxis or treatment with sequential compression devices only. Two studies uniformly demonstrated increased mortality in control groups when compared to patients treated with enoxaparin and UFH9,10. However, the third study demonstrated that rates of mortality were increased in patients treated with enoxaparin when compared to those prescribed sequential compression devices only (13.3% vs. 11.6%, p=0.08)10.\n\nA single cohort study reported increased deaths with early UFH prophylaxis when compared to late prophylaxis (8.5% vs. 5.9%, p=1.0)15 (Table 3).\n\nLow grade evidence supported the effectiveness of UFH over no pharmacoprophylaxis in reducing total mortality in patients hospitalized with traumatic brain injury (Table 4).\n\nProgression of intracranial hemorrhage. The rates of progression of intracranial hemorrhage resulting from prophylaxis with dalteparin, enoxaparin, or UFH were reported in six studies8–11,13,14. Two studies reported that there was no difference in rates of progression of intracranial hemorrhage between the control or sequential compression devices only group and the pharmacoprophylaxis (enoxaparin and dalteparin) group10,13. Another set of two studies that compared prophylaxis with UFH and enoxaparin to control or placebo demonstrated equivocal results11,14; patients treated with UFH had lower rates of progression of intracranial hemorrhage, while those treated with enoxaparin had higher rates. Two other studies demonstrated head-to-head comparisons of two pharmacological agents. According to one study, patients treated with enoxaparin and dalteparin had comparable rates of intracranial bleeding (0.001% vs. 0%)8, while the other demonstrated a statistically significant increase in intracranial bleed in patients treated with UFH compared to those treated with enoxaparin (12% vs. 5%, p<0.05)9.\n\nThree studies evaluating the optimal timing of initiation of pharmacoprophylaxis reported on rates of progression of intracranial hemorrhage in TBI populations1,15,18. Even though all three studies reported increased rates of intracranial hemorrhage when prophylaxis was initiated with enoxaparin or any other heparin after 72 hours of admission, the increase was only minimal in two studies (3.5% vs. 3.8%; 1.46% vs. 1.54%) (Table 3).\n\nOverall, the evidence was insufficient to comment on the effect of pharmacological and mechanical prophylaxis and timing of initiation of pharmacoprophylaxis on progression of intracranial bleeding in TBI patients (Table 4).\n\nOf the twelve studies included in this review, only one RCT was at a low risk of bias14. With the exception of a single cohort study that was at a moderate risk of bias8, ten were estimated to be high risk of bias studies. Most cohort studies had incomplete descriptions of the important confounders and a lack of adjustment for differences between groups. They also had incomplete accounts of losses to follow-up. All of these are important confounders and threaten the internal validity of these studies.\n\nThe participants that these studies recruited were typical of participants admitted to other trauma centers and hence findings are generalizable. The studies were generally representative of patients with TBI in the USA. Gender was inconsistently reported, thus we could not assess the applicability of these findings to females. We did not have details to assess the applicability of this evidence to other racial groups since the studies inconsistently reported on ethnicity or race. Some studies excluded patients with previous VTE1,10 as well as those at higher risk of bleeding, such as those with low platelet counts1,10,14,15, limiting generalizability to these high-risk subgroups.\n\n\nDiscussion\n\nWe found low-grade evidence that enoxaparin reduced rates of DVT and UFH reduced rates of mortality when compared to no pharmacoprophylaxis in TBI patients. The evidence was insufficient to comment on the effectiveness and safety of remaining comparators. Evidence was also insufficient for assessment of optimal timing of initiation of pharmacoprophylaxis for all comparators and outcomes.\n\nWe found only two RCTs that addressed VTE prophylaxis in patients with TBI. The remaining studies were single-center cohort studies, the majority of which were retrospective, having high risk of bias. Although the studies in this review asked similar questions (i.e., enoxaparin vs. heparin, pharmacologic prophylaxis vs. SCDs) and had similar patient populations, the scarcity of good quality studies with low risk of biases prevents definitive conclusions.\n\nWe identified a retrospective cohort study by Kwiatt et al. with a moderate risk of bias, published after our search cutoff date that evaluated the effectiveness of enoxaparin compared to control in reducing venous thrombosis and progression of intracranial hemorrhage in TBI patients19. The results of this study were consistent with other studies included in our review that compared enoxaparin with a control or placebo group. This study demonstrated that the rates of venous thrombosis and progression of intracranial hemorrhage were significantly higher in patients treated with enoxaparin compared to patients in the control group (9.1% vs. 3.1% and 42% vs. 24% respectively, p<0.001 for both outcomes) indicating a potential for more harm than benefit with utilization of enoxaparin in this population. This reiterates the need for good quality studies to establish the effectiveness and safety of VTE prophylaxis in patients with TBI.\n\nOur results should be interpreted in the context of other systematic reviews and existing guidelines. We did not identify any existing systematic reviews about the role of VTE prophylaxis and its optimal timing and initiation in patients with traumatic brain injury. The two organizations, The Eastern Association for the Surgery of Trauma (EAST) and the Brain Trauma Foundation, that provide guidelines for the care of trauma patients and patients with traumatic brain injury, respectively, do not make specific recommendations about DVT prophylaxis in TBI patients. EAST practice guidelines address DVT prophylaxis in the general trauma patient but do not make specific recommendations about patients with brain trauma. In 2007, the Brain Trauma Foundation Guidelines for the Management of Severe Traumatic Brain Injury found no good quality data to support the use of DVT prophylaxis in TBI patients. They found level III evidence for IPC and chemoprophylaxis, while stating that “there is insufficient evidence to support recommendations regarding the preferred agent, dose, or timing of pharmacologic prophylaxis for deep vein thrombosis (DVT)”20.\n\nAdditionally, the American College of Chest Physician guidelines do not specifically address DVT prophylaxis in these patients2.\n\nOur systematic review identified important weaknesses in the literature. We did not identify high quality RCTs for this review. The majority of observational studies included in this review were at a high risk of bias and did not report on several quality items of interest. The studies were heterogeneous in the definition of VTE and bleeding outcomes precluding any meaningful pooling in a meta-analysis. We also did not find data on several pharmacologic comparisons of interest or details about optimal timing of initiation of prophylaxis in this population. We were unable to assess the possibility of publication bias or selective outcomes reporting and its impact on our findings.\n\nStudies among patients with TBI are needed to determine whether pharmacologic DVT prophylaxis should be employed in these patients and the timing of administration. Studies should also determine the role of appropriate classification and severity of TBI when deciding to administer pharmacologic prophylaxis. Our report shows that confounding by indication was a major problem in these studies. Patients at high risk for thrombotic outcomes were more likely to receive prophylaxis and more likely to have events-the treated and untreated patients were not comparable. Future studies should consider the use of appropriate analytic strategies such as instrumental variables that control for unobserved variables if an appropriate instrument can be identified for analysis. High-quality observational studies that control for confounding by indication, such as provider and practice patterns, and confounding by disease severity may be needed as RCTs typically exclude or do not report on these populations.\n\n\nConclusion\n\nLow grade evidence supports the effectiveness of enoxaparin over no pharmacoprophylaxis in reducing the rates of DVT in patients with TBI. Low-grade evidence also supported the safety of UFH over no pharmacoprophylaxis in reducing total mortality in the same population. The evidence was insufficient for the remaining comparators and outcomes assessed such as VTE and PE.", "appendix": "Author contributions\n\n\n\nYC selected articles for inclusion, extracted data, graded the strength of the evidence, drafted the initial manuscript and revised the manuscript. KAS and SS selected articles for inclusion, extracted data, graded the strength of the evidence and reviewed and revised the manuscript. ERH, DJB, KMS and SK selected articles for inclusion, extracted data, graded the strength of the evidence and critically reviewed the manuscript. RS designed the data abstraction forms, coordinated data abstraction and data management, selected articles for inclusion and critically reviewed the manuscript. JBS supervised all steps of the systematic review process and reviewed and revised the manuscript. All authors agreed on the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nDr Haut is the primary investigator of the Mentored Clinician Scientist Development Award K08 1K08HS017952-01 from the Agency for Healthcare Research and Quality entitled “Does Screening Variability Make DVT an Unreliable Quality Measure of Trauma Care?” Dr Haut receives royalties from Lippincott, Williams, & Wilkins for a book he coauthored (Avoiding Common ICU Errors) and has given expert witness testimony in various medical malpractice cases.\n\nDr. Brotman reports having been on an advisory boards for the following companies: Canyon Pharmaceuticals, Cubist Pharmaceuticals, Sanofi-Aventis Pharmaceuticals, Bristol-Myers Squibb/Sanofi Pharmaceuticals Partnership, EMCREG International, Otsuka America Pharmaceutical, Inc., Ortho-McNeil Pharmaceuticals, Johnson & Johnson. He has consulted for Gerson Lehrman Group. The Dunn Group and Quantia Communications, LLC. He has received research support from Siemens Healthcare Diagnostics (formerly Dade Behring), Agency for Healthcare Research and Quality (AHRQ), Center for Medicare & Medicaid Services (CMS), and Amerigroup Corporation. He has been an expert witness in various cases addressing clinical decision making and actions of Hospitalists, nurses, and other healthcare professionals.\n\nAll other authors report no conflicts of interest.\n\nThe authors of this article are responsible for its contents, including any clinical or treatment recommendations. No statement in this article should be construed as an official position of AHRQ or of the U.S. Department of Health and Human Services.\n\n\nGrant information\n\nThis study was funded by a grant from the Agency for Healthcare Research and Quality (AHRQ), Contract number: HHSA-290-2007-10061 I. The AHRQ participated in formulating the key questions and reviewed planned methods and data analyses, as well as interim and final evidence reports. The AHRQ had no role in data collection, data management, data analysis, study selection, quality ratings, or interpretation/synthesis of the evidence.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nKoehler DM, Shipman J, Davidson MA, et al.: Is early venous thromboembolism prophylaxis safe in trauma patients with intracranial hemorrhage. J Trauma. 2011; 70(2): 324–9. PubMed Abstract \| Publisher Full Text\n\nGould MK, Garcia DA, Wren SM, et al.: American College of Chest Physicians. Prevention of VTE in nonorthopedic surgical patients: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest. 2012; 141(2 Suppl): E227S–77S. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nComparative Effectiveness of Pharmacologic and Mechanical Prophylaxis of Venous Thromboembolism Among Special Populations - Research Protocol \| AHRQ Effective Health Care Program 2012. Reference Source\n\nWalther S, Schuetz GM, Hamm B, et al.: [Quality of reporting of systematic reviews and meta-analyses: PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses)]. Rofo. 2011; 183(12): 1106–10. PubMed Abstract \| Publisher Full Text\n\nSingh S, Haut ER, Brotman DJ, et al.: Pharmacological and Mechanical Prophylaxis of Venous Thromboembolism Among Special Populations; Comparative Effectiveness Review. PubMed Abstract\n\nDowns SH, Black N: The feasibility of creating a checklist for the assessment of the methodological quality both of randomised and non-randomised studies of health care interventions. J Epidemiol Community Health. 1998; 52(6): 377–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMethods Guide for Effectiveness and Comparative Effectiveness Reviews. Rockville MD: Agency for Healthcare Research and Quality; August 2011. AHRQ Publication No. 10(11)-EHC063-EF. Chapters Reference Source\n\nDudley RR, Aziz I, Bonnici A, et al.: Early venous thromboembolic event prophylaxis in traumatic brain injury with low-molecular-weight heparin: risks and benefits. J Neurotrauma. 2010; 27(12): 2165–72. PubMed Abstract \| Publisher Full Text\n\nMinshall CT, Eriksson EA, Leon SM, et al.: Safety and efficacy of heparin or enoxaparin prophylaxis in blunt trauma patients with a head abbreviated injury severity score >2. J Trauma. 2011; 71(2): 396–9. PubMed Abstract \| Publisher Full Text\n\nKurtoglu M, Yanar H, Bilsel Y, et al.: Venous thromboembolism prophylaxis after head and spinal trauma: intermittent pneumatic compression devices versus low molecular weight heparin. World J Surg. 2004; 28(8): 807–11. PubMed Abstract \| Publisher Full Text\n\nScudday T, Brasel K, Webb T, et al.: Safety and efficacy of prophylactic anticoagulation in patients with traumatic brain injury. J Am Coll Surg. 2011; 213(1): 148–53. PubMed Abstract \| Publisher Full Text\n\nGersin K, Grindlinger GA, Lee V, et al.: The efficacy of sequential compression devices in multiple trauma patients with severe head injury. J Trauma. 1994; 37(2): 205–8. PubMed Abstract\n\nSaadeh Y, Gohil K, Bill C, et al.: Chemical venous thromboembolic prophylaxis is safe and effective for patients with traumatic brain injury when started 24 hours after the absence of hemorrhage progression on head CT. J Trauma Acute Care Surg. 2012; 73(2): 426–30. PubMed Abstract \| Publisher Full Text\n\nPhelan HA, Wolf SE, Norwood SH, et al.: A randomized, double-blinded, placebo-controlled pilot trial of anticoagulation in low-risk traumatic brain injury: The Delayed Versus Early Enoxaparin Prophylaxis I (DEEP I) study. J Trauma Acute Care Surg. 2012; 73(6): 1434–41. PubMed Abstract \| Publisher Full Text\n\nKim J, Gearhart MM, Zurick A, et al.: Preliminary report on the safety of heparin for deep venous thrombosis prophylaxis after severe head injury. J Trauma. 2002; 53(1): 38–42; discussion 43. PubMed Abstract \| Publisher Full Text\n\nReiff DA, Haricharan RN, Bullington NM, et al.: Traumatic brain injury is associated with the development of deep vein thrombosis independent of pharmacological prophylaxis. J Trauma. 2009; 66(5): 1436–40. PubMed Abstract \| Publisher Full Text\n\nDepew AJ, Hu CK, Nguyen AC, et al.: Thromboembolic prophylaxis in blunt traumatic intracranial hemorrhage: a retrospective review. Am Surg. 2008; 74(10): 906–11. PubMed Abstract\n\nSalottolo K, Offner P, Levy AS, et al.: Interrupted pharmocologic thromboprophylaxis increases venous thromboembolism in traumatic brain injury. J Trauma. 2011; 70(1): 19–24; discussion 25–6. PubMed Abstract \| Publisher Full Text\n\nKwiatt ME, Patel MS, Ross SE, et al.: Is low-molecular-weight heparin safe for venous thromboembolism prophylaxis in patients with traumatic brain injury? A Western Trauma Association multicenter study. J Trauma Acute Care Surg. 2012; 73(3): 625–8. PubMed Abstract \| Publisher Full Text\n\nGuidelines for the Management of Severe Traumatic Brain Injury 3rd Edition. 2012. Reference Source" }	[ { "id": "1353", "date": "07 Aug 2013", "name": "David Bar-Or", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 systematic review of Chelladurai et al. on VTE prophylaxis in patients with traumatic brain injury is an excellent review and analysis of the literature on the subject. It reinforces the urgent need for well controlled, prospective studies to assess the safety, efficacy and drug choice for thromboprophylaxis in this group of patients. Timing of intervention, severity and type of injury and associated conditions, interruption of treatment for surgical procedures, effects on the geriatric population and others are variables that would require special attention. The evidence reported in this review, as the authors conclude, supports (although weak) the use of thromboprophylaxis in this group of patients. It is probable that a subgroup may benefit more than others and it would be interesting to focus even a retrospective study to such a group.", "responses": [] }, { "id": "4154", "date": "08 Apr 2014", "name": "Ewout W. Steyerberg", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is well written and the tables and figures are clear. The conclusion is well based on the results.There is a recent review that was related to the current review. Phelan (2012) conducted a critical literature review about pharmacologic venous thromboembolism prophylaxis after traumatic brain injury. The method by Phelan was less systematic, and he only included pharmacological prophylaxis (instead of also mechanical). However, 9 of the included studies in this review were also reviewed in the study by Phelan. Overall, the current study is therefore quite similar to the review by Phelan.The main conclusion remains that further research is urgently needed in this area. Some specific comments:IntroductionThe authors state that “this population is at increased risk for VTE due to a combination of factors (i.e. the brain injury itself, other injuries, intensive care unit admission, immobilization, major surgery etc.)”. They do not mention a source here. Perhaps a subgroup of TBI patients at risk for VTE, however, not all patients will be at risk (e.g. uncomplicated mTBI patients). In the introduction I miss some information about pharmacological and mechanical prophylaxis (what is it, when is it used, examples etc). MethodsNo patient and injury characteristics are mentioned as inclusion criteria, however, GCS may be an important confounding factor in the research question. The search terms and mesh terms are not mentioned. It is not mentioned whether papers were excluded if published before a certain date. In table 1 authors mention a long list of population exclusion criteria. This seems in contrast with the ISS score > 15 in all studies, indicating multi-system trauma. Some more information is necessary here (how did the authors handle studies in which some of the patients met the exclusion criteria?). It is not clear to me what is meant by “trauma with brain injury”. Also, underweight and obesity are mentioned as exclusion criteria. How did the authors account for this? BMI is not often reported in studies examining TBI. ResultsThe injury severity score is used to indicate severity. However, this score does not account for severity of TBI. Do the studies report GCS scores? In the table with study characteristics, the study by Sadeh is not included it seems? DiscussionAn extra limitation is that the authors excluded studies that were comparing drugs not available in the US (n=26).", "responses": [] } ]	1	https://f1000research.com/articles/2-132
https://f1000research.com/articles/1-19/v1	01 Oct 12	{ "type": "Research Article", "title": "A domain sequence approach to pangenomics: applications to Escherichia coli", "authors": [ "Lars-Gustav Snipen", "David W Ussery", "David W Ussery" ], "abstract": "The study of microbial pangenomes relies on the computation of gene families, i.e. the clustering of coding sequences into groups of essentially similar genes. There is no standard approach to obtain such gene families. Ideally, the gene family computations should be robust against errors in the annotation of genes in various genomes. In an attempt to achieve this robustness, we propose to cluster sequences by their domain sequence, i.e. the ordered sequence of domains in their protein sequence. In a study of 347 genomes from Escherichia coli we find on average around 4500 proteins having hits in Pfam-A in every genome, clustering into around 2500 distinct domain sequence families in each genome. Across all genomes we find a total of 5724 such families. A binomial mixture model approach indicates this is around 95% of all domain sequences we would expect to see in E. coli in the future. A Heaps law analysis indicates the population of domain sequences is larger, but this analysis is also very sensitive to smaller changes in the computation procedure. The resolution between strains is good despite the coarse grouping obtained by domain sequence families. Clustering sequences by their ordered domain content give us domain sequence families, who are robust to errors in the gene prediction step. The computational load of the procedure scales linearly with the number of genomes, which is needed for the future explosion in the number of re-sequenced strains. The use of domain sequence families for a functional classification of strains clearly has some potential to be explored.", "keywords": [ "Microbial pangenomics has attracted interest over recent years", "stimulated by the availability of sequence data from whole-genome re-sequencing projects1–7. The pangenome of a prokaryotic species is the collection of gene families for the entire species", "as opposed to a single genome", "which is the set of genes in a functional organism. The pangenome diversity can be huge", "which is also reflected in the span of phenotypes. An example of this is found in the model organism Escherichia coli8", "9. Currently there are more than a thousand E. coli genomic projects listed10 and this number will grow in the near future", "along with genomes for many other bacteria", "it is reasonable to assume that pangenomics will attract more attention." ], "content": "Introduction\n\nMicrobial pangenomics has attracted interest over recent years, stimulated by the availability of sequence data from whole-genome re-sequencing projects1–7. The pangenome of a prokaryotic species is the collection of gene families for the entire species, as opposed to a single genome, which is the set of genes in a functional organism. The pangenome diversity can be huge, which is also reflected in the span of phenotypes. An example of this is found in the model organism Escherichia coli8,9. Currently there are more than a thousand E. coli genomic projects listed10 and this number will grow in the near future, along with genomes for many other bacteria; it is reasonable to assume that pangenomics will attract more attention.\n\nThe fundamental step in any pangenome analysis is the computation of gene families. Several approaches to computing gene families have been used in previous pangenome analyses1,11,12, but this part of the analysis has received little attention. A pangenome analysis typically involves the estimation of the size of the core and the pangenome, measured by the number of gene families, and several methods have been proposed for doing this1,11,13,14. The core is the set of gene families present in all genomes of the population. The remaining gene families are more or less abundant among the genomes. The sample pangenome size is the total number of gene families found in the currently available genomes, while the population pangenome size is the number of gene families we expect to see if every single strain was sequenced. It is the latter which is of scientific interest, but it must be estimated from the former. A fraction of the gene families will be found only in a small number of genomes, and those observed in only a single genome are called ORFans.\n\nThe first step of a gene family computation is to obtain a list of protein coding genes for each genome under study. Completed genomes will have a set of annotated genes, but the quality of these annotations may vary between projects. A pangenome analysis will often include draft genomes as well, where annotations are lacking. For these reasons it is convenient to start the analysis by a gene-finding step, treating all genomes the same way, and minimizing variability due to different annotation procedures. Even if prokaryote genes are in most cases simpler to detect than eukaryote counterparts, there are still problematic cases15. In case of the draft genomes, where the genome sequence is spread out on a (large) number of contigs, the gene-finding problem is even more difficult. Ideally, we would like to compute gene families in a way that minimizes the effect of various gene finding algorithms.\n\nThe second step is to group proteins into gene families. A gene family is basically the set of orthologs and in-paralogs collected from the various genomes. The most common approach so far is based on all-against-all alignments to compute some kind of similarity/distance between all proteins, and then finally cluster these. This approach poses some problems. First, the all-against-all approach is not computationally feasible in the long run since the number of genomes grows rapidly. Second, the clustering procedures always involve some granularity threshold implicitly defining the size/number of gene families. Some kind of thresholding seems impossible to avoid, but it would be desirable to allow it some variability over gene families, since some gene families are expected to be more conserved than others. Finally, false predicted ‘genes’ will not align well to any other, and produce singelton clusters adding, sometimes dramatically, to the total number of gene families found.\n\nWhen grouping protein coding genes, we find it natural to consider the presence of common protein domains. Domains are closely linked to gene function16. Previous work has demonstrated that the functional repertoire of a genome can be predicted by the knowledge of domain content17,18. In addition, focusing on the combination of domains in each protein, instead of the frequency of each domain separately, we come even closer to the protein function19,20. A grouping of proteins by function rather than by evolutionary distance seems like a good alternative, since pangenomics is in many cases about the study of functional diversity.\n\nThe Pfam-A database21 is a comprehensive collection of domains, curated and of high quality. Each entry is characterized by a profile hidden Markov model, and until recently it has been quite time consuming to search against such a database. However, with the launching of the HMMER3 software22,23, it is now possible to search with all proteins within a genome against the entire Pfam-A database in reasonable time (few minutes), even on a laptop. The idea of using domain information in comparative genomics is not new, and Yang et al. used genome-wide domain frequencies to reconstruct phylogenetic trees24. Their study considered diverse genomes from both prokaryotes, eukaryotes and archaea, quite opposite of pangenomics, where we focus only on genomes from the same species. An argument for using domains was that structure is more conserved than sequence, and that domain information provides a view into ancient history24. In this perspective one may question the use of domain information for pangenomics studies, since they may provide too low resolution.\n\nIn this paper we consider domain sequence families defined by the domain sequence of each protein as an alternative to gene families for pangenome studies. The domain sequence is the ordered sequence of non-overlapping domains along the protein. Clusters computed in this way will be coarse compared to previously used gene families, but are closely linked to gene function. We describe the procedure of computing such gene families, and consider the effect of gene prediction and of the use of draft genomes in the analysis. We use this approach in a pangenome study of the model organism Escherichia coli, the prokaryote species with the largest set of available genomes.\n\n\nMaterials and methods\n\nThe methods we have used for gene prediction and database scanning are all standard and free software, see below. We acknowledge that the gene finders have tuning possibilities, and that slightly different results may be obtained by using this. We have used only default settings, or setting suggested in accompanying manuals, in order to make the approach as universal as possible. The data are from the model organism Escherichia coli only, and many of the more specific results cannot be extended to other species. However, the procedures are applicable to all prokaryotes where we have genomes from many strains available.\n\nGenome sequences for Escherichia coli were downloaded from NCBI25 on June 15 2012. At that time, there were 56 completed and 291 draft genomes available as contigs.\n\nIn a pangenome study involving draft genomes some automated gene finding is needed. For each genome we predicted genes using the three popular gene finders Prodigal26, GeneMark27 and Glimmer28.\n\nThe Prodigal gene finder (v2.60) was run by the command line\n\nprodigal -i gnom.fsa -o pred.txt -q\n\nwhere gnom.fsa was a FASTA-formatted input file with the genome sequences, and pred.txt was the output file.\n\nThe GeneMark predictions were obtained by first training a Hidden Markov Model (HMM) using GeneMark.S (v4.6b):\n\ngmsn.pl –combine –clean –gm gnom.fsa\n\nwhich produced a model file GeneMark.hmm.combined.mod. Next, this was used to predict genes by GeneMark.hmm, prokaryote version 2.6:\n\ngmhmmp -m GeneMark.hmm.combined.mod -o pred.txt gnom.fsa\n\nThe Glimmer predictions were obtained by the tigr-glimmer wrapper software for linux. First, the long.orfs software was used to extract training sequences from the genome sequences:\n\ntigr-glimmer long-orfs -n -l -t 1.15 -g 30 seq.fsa coord.txt tigr-glimmer extract -t seq.fsa coord.txt >> train.fsa\n\nwhere seq.fsa was the FASTA file with a single genome sequence. This was repeated by looping through all genome sequences, producing the file train.fsa with training sequence data. Next, an Interpolated Context Model (ICM) was trained:\n\ntigr-glimmer build-icm -r glim.icm < train.fsa\n\nwhere glim.icm was the model file output. This was finally used to make gene predictions:\n\ntigr-glimmer glimmer3 -o50 -g110 -t30 –linear –extend gnom.fsa glim.icm pred.txt\n\nIn all cases the gene predictions were stored as a table with one row for each predicted gene, and with columns GenomeSequence (name of genome sequence where it is found), Strand (1 or -1), Left (smallest coordinate), Right (largest coordinate), Partial (logical indicating if the gene runs over the start/end of the genome sequence). This format makes comparison of gene predictions straightforward.\n\nWe wanted to examine the effect of various gene finders on the list of proteins obtained from each genome. For this study we only focused on the 54 completed genomes with annotated proteins in the RefSeq database29. Given the curation of the RefSeq database, and the fact that E. coli is the most well studied prokaryote genome, we expect these RefSeq annotations to be as close to the truth as one can expect for any prokaryote organism. Thus, by using the three gene finders described above we obtained four sets of proteins for each of the 54 genomes. We compared these sets using the Jaccard distance defined as\n\n\n\nwhere Sa and Sb are two sets of proteins and \|.\| indicates cardinality (number of elements in the set). A distance of 0.0 means the two sets Sa and Sb are identical, while the maximum distance of 1.0 means they are non-overlapping. In this comparison, two proteins were considered equal only if they were exactly identical (identical amino acid sequence). After extraction of domain sequences from each protein (see below) we again computed Jaccard distances. This time two proteins were considered equal if they had identical domain sequence.\n\nFor the sake of completeness all genomes, both complete and draft, were subjected to the same analysis. Since we subsequently filtered all results through the Pfam-A database (see below), we were less concerned about false positive gene predictions at this step. To obtain a comprehensive gene-list, we ran all three gene-finders on each genome, and compiled the union of the results into one long list of ORFs for each genome. In the cases where the same stop-codon had alternative starts, we always chose the longer ORF, again due to the later Pfam-A filtering. All partial ORFs (lacking start and/or stop codon) were eliminated before the analysis, but we made no attempt to resolve overlaps between different ORFs. Finally, all remaining ORFs were translated to protein.\n\nFASTA-formatted files of protein sequences were queried against the Pfam-A database version 26.021 using the HMMER 3.0 software22,23. The search was done using the command line\n\nhmmscan -o out.txt –cut_ga –noali –cpu 0 –domtblout res.txt Pfam-A.hmm prot.fsa\n\nwhere res.txt is the name of the output file, Pfam-A.hmm is the HMMER3 database and prot.fsa is the FASTA-formatted input file of protein sequences to scan. We used the option –cut_ga in order to invoke the curated inclusion thresholds inherent in the Pfam-A models. The iEvalue (independent E-value) was used as the criterion in the cases where we wanted to overrun these thresholds, using a stricter level of significance.\n\nFor every protein sequence the significant hits were listed in their order of appearance along the protein. In the cases where two or more domains were overlapping, the one with largest E-value was eliminated, and this was repeated in a recursive procedure until no more overlaps between hits were found. There is a small number of nested domains (domains inside domains) in the Pfam database, but we chose to eliminate all occurrences of overlaps. The domain sequence of a protein is the ordered list of Pfam accession numbers, and multiple hits of the same domain may occur as long as they are non-overlapping. It is a high-level alternative to the amino acid sequence representation of a protein. Proteins giving no hits in Pfam-A were discareded from the downstream analysis. This step presumably filtered out the majority of the false positive gene predictions from the gene-finding step. Finally, we grouped into domain sequence families all proteins having identical domain sequence.\n\nThe fundamental data structure for a pangenome analysis is the pan-matrix. This matrix has one row for each genome and one column for each domain sequence or gene family, and cell (i, j) holds the number of copies of domain sequence family j in genome j, alternatively just 0 or 1 to indicate absence/presence, the latter being used in this paper. Let xj be the number of genomes in which family j is present. It is natural to view xj as a random variable, the randomness mainly due to our random selection of genomes since there are obviously many genomes we could have, but have not yet, looked into. The variable xj can take any integer value from 1 to G, where G = 347 is the number of genomes in this study.\n\nEach row of the presence/absence pan-matrix is a coordinate vector indicating the location of the corresponding genome in a domain sequence space. This domain sequence space is closely related to a functional space in the sense that neighbors in this space is likely to display similar functions. This space is high-dimensional (many different functions), but in order to visualize the genomes relative location in this space we used a principal component decomposition to extract the most dominant directions. The genomes location along these dominant directions give us some indications of their relative location in the functional space.\n\nUsing a Heaps law type of regression model, Tettelin et al. made an estimate of pangenome closedness13. A closed pangenome means the gene families are sampled from a stable and finite set, as opposed to an open pangenome where gene families ‘migrate’ in and out at unknown rates. If we arrange the G = 347 genomes in some fixed order, we can define ng as the number of new families seen in genome g compared to the previous g – 1 genomes. The Heaps law says that\n\n\n\nwhere β is some intercept and α is the parameter of interest. An α below 1 indicates the number of new families does not decrease fast enough for the pangenome to be limited, i.e. if the number of genomes G grows towards infinity the number of new families observed will never reach zero. Parameters can be estimated from data, by repeatedly permuting the order of the genomes and counting ng for g = 2, …, G, after each re-ordering.\n\nAs explained above, let xj be the number of genomes in which we observe domain sequence family j. Let yg be the number of families found in g genomes (number of xj’s with value g). Then yg is also a random variable. The probability density of this variable, e.g. the one depicted in Figure 3, can be described by a K component binomial mixture model.\n\n\n\nwhere b(ρκ) is the binomial density with probability ρκ and πκ is the mixing proportion (the πκ’s sum to 1.0). The left pie of Figure 7 is a visualization of a K =12 component model, where the size of each sector is the corresponding πκ and the color of each sector indicates ρκ, for κ =1, …, 12. Summing y1, y2, …, y347 we get the number of domain sequence families seen so far, i.e. the sample pangenome size. From the binomial mixture model we can also predict y0, the number of families not yet seen, and in this way we can estimate the population pangenome size11,14. The probabilities ρk we refer to as the selection probabilities. A domain sequence family with selection probability 0.1 will on average be present (selected) in 1 out of 10 genomes.\n\nGenome diversity can also be expressed as the overlap between genomes, and for this we could use the mean Jaccard distance defined above, where Sa and Sb are two sets of domain sequences (two genomes). Genome fluidity, introduced in30, is an almost identical measure. A small Jaccard distance/genome fluidity indicates a small degree of uniqueness in each genome, i.e. large overlap. The expected overlap between two genomes can also be computed directly from a fitted binomial mixture model in an elegant way. The expected number of domain sequence families in a genome is\n\n\n\nwhere N is the population pangenome size. The expected number of domain sequences found in two independent genomes simultaneously is\n\n\n\nsince the probability of selecting a domain sequence family twice is ρκ · ρκ, given that ρκ is its selection probability. The expected overlap between two genomes can be expressed as E2/E1. Note that the pangenome size N cancels out, and the result only depends on the selection probabilities in combination with the mixing proportions. The expected overlap between 3 and more genomes can be computed along the same lines.\n\n\nResults and discussion\n\nBefore we commenced the full scale analysis, we wanted to investigate the effect of gene finding algorithms. To this end, we considered only the 54 complete E. coli genomes with annotations in the RefSeq database29 at the time of this study. In Table 1 we show how the sets of genes differ between the official RefSeq annotation and the three most commonly used prokaryote gene finders. From the upper triangle of the table we see that the Jaccard distance to the RefSeq annotations is large in all cases. Even for Prodigal, which comes closest to the RefSeq annotations, the distance is 0.26. For a list of 5000 proteins, this corresponds to around 750 proteins on each list not being found in the other. Also the differences between the gene finders are large, and there is a severe uncertainty in any list of genes obtained by any single gene finder. In the lower triangle of Table 1 we see how the differences are reduced dramatically once we only consider domain sequences. This means all proteins without Pfam-A hits are discarded, and the remaining proteins are only compared by the domain sequence. The difference in start codon predictions, causing the majority of differences in the first comparison, no longer have any impact. If two proteins have the same domains, it does not matter how they compare outside these domains.\n\nThe Jaccard distance between all pairs of sets was computed, see Method section. First, two genes were identical only if they have identical amino acid sequence. The upper triangle (above the diagonal) shows the mean Jaccard distance over the 54 genomes in this case. Next, we computed the Pfam-A domain sequence of each predicted protein, discarding all proteins without Pfam-A hits, and again computed the Jaccard distance between the sets. The lower triangle (below the diagonal) shows the mean Jaccard distance over the 54 genomes in this case.\n\nIn the RefSeq annotated genomes we found on average around 2750 unique domain sequence families (from 2555 to 2957). Using the union of the three gene finders, we found on average 2725 of the RefSeq annotated domain sequence families among these, i.e. around 25 families (1%) in each genome is not found by any of the three gene finders. On the other hand, the three gene finders find on average close to 100 extra domain sequences families in each genome. This means that either the RefSeq annotated genomes have missed quite a number of domain-containing protein sequences, or the built-in inclusion thresholds in the Pfam-A models are too liberal, resulting in a substantial number of false positive hits. To investigate the latter, we tried out stricter significance thresholds in the HMMER3 software. Even for an E-value of E = 10-10 we found around 70 extra domain sequence families per genome in the union of the three gene finders compared to the RefSeq annotation. We believe this illustrates that annotated genomes still contain several ‘holes’, which is also the conclusion of others15. The fact that gene finding in prokaryotes is simpler than in eukaryotes may have created an illusion that it is straightforward and nearly 100% reliable, when careful studies have shown that typical bacterial genomes have around 10% genes that are false positive, and another 10% of the proteins discovered by proteomics are missing31–33. Angiuoli et al. suggested an annotation procedure based on the whole-genome alignment of all genomes in a pangenome study, and found a disturbing amount of inconsistencies in public annotations34. For E. coli less than 50% of the gene-structures were consistently annotated across 30 genomes, and the majority of the inconsistencies were due to difference in start codons positions.\n\nThe input to the analysis was a set of 347 genomes of E. coli, 56 of these were completed and 291 were available as contigs, all public data downloaded from NCBI25. The left panel of Figure 1 shows the size distribution for the completed and the draft genomes. The first step was to predict genes in all genomes by three different gene finders, as explained in the Methods section. As seen in the center panel of Figure 1, this resulted in a list of roughly 7000–8000 predicted genes in each genome, which is far more than the 4500–5500 genes usually found annotated for E. coli genomes. This reflects the disagreement between the gene finders, and we expect a large proportion of false positives in this set. The second step was to scan all predicted proteins against the Pfam-A database. Protein sequences producing no hits in Pfam-A were discarded from the downstream analysis. Roughly 4000–5000 proteins from every genome produced significant hits in Pfam A, see Figure 1, right panel. We also note that the difference between completed and draft genomes is more or less nonexistent after this step. This justifies the use of draft genomes in pangenome studies like this.\n\nThe box and whisker plots illustrate the differences between completed and draft genomes in this study. The left panel shows that the 56 complete genomes are somewhat smaller in size measured in megabases. This is most likely due to unresolved overlaps between the contigs in the draft genomes. The middle panel contains the number of unique genes predicted by the three gene finders after the elimination of all partial predictions (lacking start or stop codon). Notice the large number of predicted genes in virtually all cases, annotated E. coli genomes usually have 4500–5500 genes. Among the draft genomes some genomes have very few predicted genes, seen as circles. The rightmost panel shows the number of predicted gene with at least one Pfam-A hit. Except from four draft genomes with extremely few genes, the differences between complete and draft genomes are now ignorable.\n\nConsidering all sequences from the 347 genomes, a total of 3679 unique Pfam-A domains/families produced significant hits, which is approximately 30% of the entire database. This produced a total of 5724 unique domain sequence families for the 347 E. coli genomes, with around 2500 in any single genome. This is a small number compared to the number of gene families previously found for E. coli9. The domain sequence families are expected to be fewer and larger clusters compared to the previously computed gene families. First, not all genes have domains listed in Pfam-A, and these are discarded here. This significantly shortens the list of clusters, presumably also eliminating a large majority of the false positive gene predictions. Second, some domain sequence families are large, containing proteins sharing perhaps only a single domain. In these cases a domain sequence family may contain more than one gene family.\n\nThe most common domain sequence family in E. coli is the single-domain protein containing the major facilitator superfamily (MFS) transporter with accession PF07690.11. This is found in around 50 copies in every single genome. Other very frequently occurring single-domain proteins are the binding-protein-dependent transport system inner membrane component (PF00528.17), and the ATP-binding domain of ABC-transporters (PF00005.22). Among the multi-domain proteins the sequence PF00126.22, PF03466.15 is the most common. This is a domain sequence characteristic of HTH-type transcriptional regulators, and it occurs in more than 30 copies in each of the 347 E. coli genomes investigated here. Figure 2 shows that more than 3000 of the domain sequence families are defined by a single domain, while gradually fewer families are defined by multi-domain-sequences. Single-domain proteins also clearly outnumber the multi-domain proteins in the genomes, contrary to what is sometimes claimed35. The longest domain sequence contains 25 non-overlapping Pfam-A hits in the same protein, mainly multiple copies of PF05662.9 and PF05658.9, both short repeats associated with haemaglutinins. This protein is found in 4 of the 347 genomes.\n\nThe bar chart shows how many domain sequences have 1 domain, 2 domains, … etc up to 25 domains. These are all non-overlapping hits in the protein. Single-domain proteins makes up more than half of the total number.\n\nIn Figure 3 we show the distribution (presence/absence) of domain sequences over the E. coli population. The leftmost bar is the number of ORFans, domain sequence families found in one single genome only. We found 909 ORFans when considering all 347 genomes. The rightmost bar are the 479 core domain sequence families found in at least one copy in all 347 genomes. The distribution is very similar in shape to what we usually see for pangenomes of other data sets and where gene families are computed. Hence, also the domain sequence families are distributed across genomes in this way.\n\nThe distributions of how many domain sequence families are found in 1, 2, …, 347 genomes. There are 909 ORFan families (leftmost bar), 479 core families (rightmost bar) and in total there are 5724 unique domain sequence families (sum of all bars).\n\nThe most remarkable result is the large number of ORFans. More than 15% of the domain sequence families found in E. coli are seen in only 1 out of 347 genomes. The Pfam-A models are curated and have a built-in threshold for assigning significant hits. However, given our approach, where we scan a list of presumably many false positive protein predictions, we may be extra restrictive when considering the hits. In Figure 4 we show how a systematic change in E-value cutoff changed the number of core families and ORFan families. As we lower the E-value cutoff, little happens to the number of core families, but the number of ORFans drops markedly. This demonstrates that the ORFan are, as usual, the most uncertain families. In the downstream analysis we made a parallel analysis using the E-value cutoff 10-10 as a strict alternative to the built-in thresholds.\n\nThe horizontal axis is the – log10 of the HMMER3 E-value cutoff and goes from E = 10-0 on the left to E = 10-15 on the right. The blue curve shows how the number of core families drops by stricter cutoff (going from left to right), and the red curve similar for the number of ORFan families.\n\n\n\n\n\n\n\nDomains are usually well conserved, and may show too few differences between strains within a species, i.e. all genomes contain more or less the same domain sequences. To examine this, we computed the Manhattan distance between every pair of genomes, which is simply the number of domain sequences where the two genomes differ in presence/absence status (ignoring copy numbers). Figure 5 shows a histogram of all pairwise distances. The median distance between two genomes is around 500, meaning there are 500 distinct domain sequences contained in one of the genomes but not the other. Only two genomes are identical, and this is actually the same genome, BL21(DE), sequenced at two different locations (Korea and Austria), but stored under unique accession numbers in the database. All other cases show at least 3 or more differences, and the median shows around 500 differences. Hence, even genomes of the same serotype have plenty of differences in domain sequence absence/presence. Using the strict E-value cutoff reduced the median distance to around 450, but did not change the shape of the histogram. The small ‘bump’ on the right hand side is due to a few genomes being quite different from the rest. These are all draft genomes with a large number of contigs, producing a smallish number of domain sequences.\n\nA histogram over all pairwise Manhattan distances between the genomes. The distance between two genomes is defined as the number of domain sequence families they differ in presence/absence status, i.e. a distance of 500 means there are 500 different families contained in one but not the other genome.\n\nWe may think of each domain sequence as representing a small repertoire of functions the genome can inhabit. For each genome the vector of presence/absence of the set of domain sequences will correspond to a location in a functional space, and the Manhattan distance between two genomes in this space is a functional distance. A pangenome tree can be made from these Manhattan distances, as described in36. However, any tree visualization will suffer when we have as many as 347 leaf nodes. Supplementary figure 1 illustrates a pangenome tree for the 347 E. coli genomes.\n\nThis sample of E. coli genomes contains several potential sub-samples, i.e. strains who have more in common than just being of the same species. It is natural to see where these are located in this functional space, and here we have looked at the four largest sub-samples in the data set. In37 it was found that ETEC type of strains shared some genomic features distinguishing them from other E. coli, and it is reasonable to expect that certain strain subsets will cluster within the total collection of E. coli genomes. We used a principal component analysis on the present/absent pan-matrix as described in the Methods section. The functional space is highdimensional, and plotting each genome as a dot in the first two principal component directions as in Figure 6 give us only a rather rough overview of the relations. Still, the clustering of the enterohaemmoragic serotypes (O157:H7, light blue dots and 0104:H4, dark blue dots) is clearly visible. The collection of diarrheagenic strains (red dots) are clumped at several locations, indicating a mixture of functional content. The Human Microbiome Project strains (green dots) are all located to the right hand side of Figure 6. Using the strict E-value cutoff hardly changed the picture in Figure 6 at all. This is due to the extreme stability of a multivariate analysis like PCA. Since we know from Figure 4 that this strictness reduce the number of ORFans, it means the picture in Figure 6 is marginally influenced by ORFan gene families.\n\nEach dot correspond to a genome plotted in the two first principal component directions of the E. coli functional space defined by the presence/absence of domain sequence families. There are four large subsets of genomes in the data set, and these dots are marked with colors, see figure legend. The first principal component accounts for 11% of the total data variation, and the second component 8%. Only relative positions of the genomes (dots) are important, the absolute scores on each axis lacks interpretation.\n\n\n\nIn this sample of 347 E. coli genomes we find 5724 unique domain sequences. Using the binomial mixture model suggested in14, the total pangenome size is estimated to 6040. Employing the suggested bagging procedure, we find that in 90% of the re-sampled cases the estimate is between 5998 and 6136, reflecting the uncertainty in the data. This should be regarded as a lower bound estimate, but indicates we have already seen the majority of domain sequence families in E. coli. The observed set of domain sequences covers around 95% of the predicted pangenome.\n\nWe also conducted a Heaps law analysis as suggested by13. From this it seems the population of E. coli domain sequences is open. We fitted the Heaps law model to these data, using 100 random permutation of genome ordering, and came up with the estimate 0.94 for the parameter α in the model. A value of α < 1.0 is consistent with an open population. This means the population size is unbounded when extrapolating the Heaps law trend into many more sequenced genomes. However, unbounded is a mathematical term, and an unbounded function can still grow very slowly. The model predicts that after 1 million sequenced E. coli genomes(!) we still have not seen more than 9442 domain sequences. As such, the result is not that different from the mixture model estimate.\n\nWe repeated the computations using the strict E-value cutoff in the HMMER3 software. The sample pangenome then reduces to 4745 (from 5724) observed domain sequence families, since only very significant (E < 10-10) Pfam-A hits are now considered. The binomial mixture model gives a pangenome size estimate of 4973, which still means a coverage around 95%. The Heaps law analysis, however, results in a closed population, with α = 1.01 and an estimate of its size at 4876 domain sequences. This illustrates how the choice of cutoffs in the sequence clustering may change a result completely. The Heaps law analysis is extremely sensitive to the number of ORFans in the data set.\n\nThe sample core size is 479 domain sequences, and the binomial mixture model predict the population core to be 462. However, the notion of the core is difficult, since we require a core domain sequence to be present in every single genome. Due to the uncertainties in gene prediction and computation of sequence clusters, such a crisp limitation of the core is unfortunate. From the binomial mixture model we get a much better and more robust picture by considering the estimated selection probabilities behind every mixture component. In Figure 7 we have visualized the E. coli pangenome as a pie chart, where the colors indicate the selection probabilities. Using the Bayesian Information Criterion (BIC) we found that 12 components was optimal for the current data set. This means E. coli domain sequences can be grouped into 12 distinct sectors with respect to how often they appear in the genomes. The core genes is one of these types, having selection probability 1.0 since they occur in every genome (darkest blue sector). Notice also the large sector of domain sequences with a selection probability of 0.988. Even the third darker blue sector has a selection probability of 0.966. The large number of domain sequence families in these sectors are also highly conserved, and could be seen as core families for all practical purposes.\n\nThe pie chart to the left visualizes the binomial mixture model fitted to the E. coli domain sequence family distribution. There are 12 different sectors, and the color indicates the selection probabilities. The size of the each sector show its relative contribution to the pangenome. The pangenome is dominated by domain sequences who are either a very high (darker blue sectors) or very low selection probability (pink sectors). The right hand pie chart shows the expected distribution in a single E. coli genome, where the highly conserved families (darker blue) dominates.\n\nThe coloring in Figure 7 reflects the commonly used division of the pangenome into three types of genes; the core (dark blue), the shell (light blue/green) and the cloud (orange/pink)38,39. By allowing the mixture model to have many more components, we found 12 was optimal here, we can cope with the fact that genomes are not uniformly distributed within the population. The distribution in Figure 3 is affected by this, e.g. since we have 31 genomes of serotype O157:H7 in the sample we expect there will be a small ‘bump’ in the distribution at 31 genomes, reflecting the domain sequence families common to these closely related genomes. The binomial mixture approach can be illustrated by a lunch buffet table, with each single genome as a plate to be filled with content from the pangenome (the buffet table). Some families on the buffet is always selected, and turns up on every plate. These are the core families. The other families are more or less popular, and have different probabilities of being selected. Some families are very unlikely to be selected, but if there is a large number of them, some of them will end up in almost every genome. However, these ORFans are so unlikely to appear they are never seen twice.\n\nThe right hand pie in Figure 7 shows the expected distribution of domain sequence families within an average E. coli genome. Clearly, the darker blue domain sequences makes up the majority. The size of the pangenome is largely due to the big number of ORFan domain sequences (pink sectors in left pie), but in any average genome these makes up a small minority (pink sectors in right pie). The expected overlap between two genomes is 0.90, i.e. on average 90% of the domain sequence families in one genome is also found in another. We also computed the genome fluidity to 0.11 and the mean Jaccard distance between two genomes to 0.18, both indicating a small uniqueness (lack-of-overlap) in each genome.\n\n\nConclusion\n\nAny study of pangenomes involves the clustering of sequences into gene families, and the results obtained will invariably depend on the way gene families are computed. In this paper we have proposed an alternative based on protein domains to obtain stable sequence clusters. This means we loose some information from every genome, since only proteins with known domains are considered. In this study we used only the Pfam-A database, and by extending it to also include other databases (e.g. Pfam-B, CDD, InterPro), some more hits would probably be found. Also, the resolution is on the lower end since all proteins having identical sequence of domains are clustered into the same family. However, the gain is a robustness with respect to gene prediction, eliminating many false positive ‘proteins’ from the analysis as well as the potential effect of the inconsistent annotation of start codons. Computing gene families based on the alignment of full protein sequences is more sensitive, but given the substantial uncertainty in gene prediction and annotation revealed here and by others, the domain sequence information may be a fruitful approach for pangenome studies. More attention should be given to improve gene prediction for prokaryotes, and the recent approach taken by Angiuoli et al., considering all genomes simultaneously to improve consistency, is a good idea for pangenome data.\n\nAnother advantage of the domain sequence approach is the fact that Pfam-A domains have a built-in significance threshold optimized for each individual HMM, and by using this we obtain families with variable heterogeneity reflecting the various degree of conservation of different types of proteins. The procedure for computing such domain sequence based gene families is straightforward and highly reproducible, using only publicly available software. Finally, each genome is only scanned once, which means the computational load scales linearly in the number of genomes.\n\nDespite the lower resolution in the domain sequence families, we find plenty of differences between strains of E. coli. We also find meaningfull grouping of strains, indicating that the differences are not just random ‘noise’ but have plenty of biological foundation. The use of domain sequences for classification of strains clearly has some potential, and this should be pursued further.\n\nWith as many as 347 genomes, and more than 1000 soon available, we would expect the E. coli pangenome to be fairly well covered. Using the domain sequence families, this seems to be the case. Our lower bound estimate of pangenome size indicates we have seen perhaps as much as 95% of all domain sequences ever to be found in this species. Domain databases, like Pfam-A, will still grow but the number of single domain proteins is leveling out, and the future growth will mainly be due to new domain sequence combinations40. There is an endless number of possible domain combinations even with todays databases, and for E. coli we must expect some new combinations in future sequenced genomes. However, with an overlap of 90% between any two E. coli genomes the time of big surprises is gone. The Heaps law analysis indicates the population is open, but we believe this analysis is too sensitive to a smallish number of ORFan families in the data set. Also, the entire concept seems to build on the assumption of a uniform sampling of strains, which is clearly not the case for E. coli.", "appendix": "Author contributions\n\n\n\nLS and DWU conceived of the study. LS carried out the programming. LS and DWU drafted the manuscript. Both authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was initiated when LS was a guest researcher at DWU’s group in Denmark, and this sabbatical was fully financed by the Norwegian University of Life Sciences.\n\n\nReferences\n\nTettelin H, Masignani V, Cieslewicz MJ, et al.: Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial \"pan-genome\". Proc Natl Acad Sci U S A. 2005; 102(39): 13950–13955. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLegault BA, Lopez-Lopez A, Alba-Casado JC, et al.: Environmental genomics of \"Haloquadratum walsbyi\" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species. BMC Genomics. 2006; 7(7): 171. 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BMC Bioinformatics. 2010; 11: 119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLukashin AV, Borodovsky M: GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res. 1998; 26(4): 1107–1115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelcher AL, Bratke KA, Powers EC, et al.: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007; 23(6): 673–679. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCBI RefSeq. [NCBI RefSeq]. Reference Source\n\nKislyuk AO, Haegeman B, Bergman NH, et al.: Genomic fluidity: an integrative view of gene diversity within microbial populations. BMC Genomics. 2011; 12: 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSkovgaard M, Jensen LJ, Brunak S, et al.: On the total number of genes and their length distribution in complete microbial genomes. Trends Genet. 2001; 17(8): 425–428. PubMed Abstract \| Publisher Full Text\n\nJaffe JD, Stange-Thomann N, Smith C, et al.: The Complete Genome and Proteome of Mycoplasma mobile. Genome Res. 2004; 14(8): 1447–1461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTetko IV, Brauner B, Dunger-Kaltenbach I, et al.: MIPS bacterial genomes functional annotation benchmark dataset. Bioinformatics. 2005; 21(10): 2520–2521. PubMed Abstract \| Publisher Full Text\n\nAngiuoli SV, Dunning Hotopp JC, Salzberg SL, et al.: Improving pan-genome annotation using whole genome multiple alignment. BMC Bioinformatics. 2011; 12: 272. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOchoa A, Linàs M, Singh M: Using context to improve protein domain identification. BMC Bioinformatics. 2011; 12: 90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSnipen L, Ussery DW: Standard operating procedure for computing pangenome trees. Stand Genomic Sci. 2010; 2(1): 135–141. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSahl JW, Steinsland H, Redman JC, et al.: A Comparative Genomic Analysis of Diverse Clonal Types of Enterotoxigenic Escherichia coli Reveals Pathovar-Specific Conservation. Infect Immun. 2011; 79(2): 950–960. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoonin EV, Wolf YI: Genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world. Nucleic Acids Res. 2008; 36(21): 6688–6719. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi T, Falkowski PG: Genome evolution in cyanobacteria: The stable core and the variable shell. Proc Natl Acad Sci U S A. 2008; 105(7): 2510–2515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChubb D, Jefferys BR, Sternberg MJ, et al.: Sequencing delivers diminishing returns for homology detection: implications for mapping the protein universe. Bioinformatics. 2010; 26(21): 2664–2671. PubMed Abstract \| Publisher Full Text" }	[ { "id": "466", "date": "04 Oct 2012", "name": "Frederic Bertels", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a very interesting insight into how one can reliably identify genes and classify them into gene families in fully sequenced bacterial genomes and apply this approach to 347 fully sequenced Escherichia coli genomes.They avoid annotation problems by first running a very crude gene prediction program, that returns a large number of genes with a large proportion of false positive open reading frames (ORFs). For these ORFs they then predict Pfam-A domains. All ORFs for which no domain could be predicted were discarded. They then show that the remaining set open reading frames closely resembles the number of ORFs expected from manually curated genome annotations. They then go on and analyse the domain sequence composition of all remaining ORFs and provide a comprehensive pan genome analysis. In conclusion, the paper presents a very interesting approach to solve gene classification problems in pan-genome studies, a field that rapidly gains significance as whole genome sequencing becomes cheaper and cheaper.The paper is well written and very comprehensive. The data is presented clearly and is directly downloadable from within the paper. The author even explain complex problems with interesting similes, which I thought was a nice idea and helped me to understand the presented ideas.There are only a few minor problems that should be addressed:1. One thing needs clarifying. The authors mention in paragraph two on page five that within the RefSeq annotated genomes they can identify 2725 of the 2750 unique domain sequence families for genes that were automatically identified. Also they say that they identify 100 extra unique domain sequence families in automatically identified genes.Does that mean that on average 2825 unique domain sequence families were identified? If this is so then I do not understand why in the second to last paragraph the authors write that in every single genome there are about 2500 unique sequence domain sequences. Especially since Figure 1 shows that on average there are more proteins with Pfam hits for draft genomes, and draft genomes are on average longer. Does that have to do with the fragmentation of the data that leads to splitting of domains? If so, how would that affect functional predictions?2. How do the authors deal with gene predictions where one gene is predicted on the top strand and another on the bottom strand or generally genes predicted in different reading frames by different gene prediction programs? Did the authors test whether the data that was missed by/additional to the refseq annotation were overlapping genes but in different reading frames and hence could not have been picked up by Pfam no matter what the threshold is?3. I think it would be interesting to estimate the effect that errors in gene prediction have on prediction of metabolic function etc. would have in theory. For example, what were the annotations of the unique domain sequence families that were missed by refseq or the gene finders. Were those mostly proteins of unknown function or did it include proteins which could potentially be predictive for the environment a particular bacterium could survive in?4. The paper does need some proof reading, there are quite a few grammatical mistakes.5. Throughout the paper the authors confuse the word “that” or “which” with “who”. For example in the abstract it says: “Clustering sequences by their ordered domain content give us domain sequence families, who are robust to errors in the gene prediction step.” In this case “who” refers to “domain sequence families” and hence should be substituted with “that” or “which”.6. The authors frequently use the words “eukaryote” and “prokaryote” as adjectives (e.g. third paragraph of the introduction: “Even if prokaryote genes are in most cases simpler to detect than eukaryote counterparts, there are still problematic cases.”) The proper adjective should be “eukaryotic” or “prokaryotic” (and in the example it should probably also say “their eukaryotic counterparts”).7. On page four, in the Methods part of the paper, the authors present a formula for the binomial mixture model. In this formula the running variable is k but changes to kappa in a few places (in the formula as well as in the text). This is a bit confusing and needs fixing.8. In a few places references are given in the following format: “as described in36” in those cases either the “as described in” part should be left out or it should say as described by Snipen and Ussery36″ or “as described in one of our earlier papers36“.9. There are quite a few cases where plural and singular are confused, for example in the last paragraph of page 7 it says “in the first two principal component” it should say “first two principal components”.This particular sentence is also a bit confusing and may need rephrasing. There are other cases where the authors confused “was” and “were”, for example in the last paragraph on page 9 it says “Using the Bayesian Information Criterion (BIC) we found that 12 components was optimal for the current data set.” It should either say “dividing the data set into 12 components was optimal” or “12 components were optimal”.10. The figure caption of Figure 7 requires some revision.11. In the first paragraph of page 11 the reference for Angiouli et al. is missing. The authors also mention in this sentence the recent approach of Angioula et al without explaining what this approach is and how it compares to the presented approach. After reading the manuscript again I noticed that this paper was mentioned also in the beginning of the Results and Discussion section, but I still think it would be helpful for the reader if the authors briefly outlined what Angiouli et al did to improve consistency and in particular how this could be integrated into the presented approach.12. In general the Conclusions section is harder to read and less clear than the rest of the paper, maybe it would help to spend a little bit of time revising this section.", "responses": [] }, { "id": "467", "date": "15 Oct 2012", "name": "Barry L. Wanner", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 excellent-outstanding computational analyses of the e. coli pangenome with respect to protein coding across more than 50 complete genomes and 350 complete and whole genome shotgun (incomplete) genomes.Importantly, they evaluate the proteins and protein families by recomputing/predicting families, rather than relying on GenBank/RefSeq annotations, which can vary depending upon source. Their work is well-documented and should serve as a gold standard for pangenome analyses of protein families for many bacteria.There are however, some minor improvements that can be made to this paper:1. On top right column of page 7, the authors explicitly state that “Only two genomes are identical… BL21(DE3). Since several non-identical K-12 strains are included in their Data File(MG1655, W3110, DH10A, and a couple others), authors should simply state how many K-12 strains were included in their analyses.2. It would be especially helpful to the community, if the authors can post data files of their analyses, e.g., the raw data used to generate their pie charts in Figure 7. I am sure users would more widely cite their efforts if such results can be made easily accessible.", "responses": [] } ]	1	https://f1000research.com/articles/1-19
https://f1000research.com/articles/2-70/v1	04 Mar 13	{ "type": "Case Report", "title": "Prostitution use has non sexual functions - case report of a depressed psychiatric out-patient", "authors": [ "Fátima Gysin", "François Gysin", "François Gysin" ], "abstract": "Case: A shy, depressed 30 year old male discussed his frequent ego-syntonic indoor prostitution consumption in small peer groups. Several distinctive non-sexual functions of this paid sex habit were identified.Design and method: The patient had 40 hourly psychiatric sessions in the private practice setting over 14 months. The Arizona Sexual Experience Scale was applied to compare the subjective appraisal of both paid sex and sex in a relationship.Results: The paid sex consumption functioned as a proud male life style choice to reinforce the patients fragile identity. The effect on self esteem was a release similar to his favorite past-time of kick-boxing. With paid sex asserted as a group ritual, it was practiced even with frequent erectile dysfunction and when sex with a stable romantic partner was more enjoyable and satisfying. The therapeutic attitude of the female psychiatrist, with her own ethical values, is put in to context with two opposing theories about prostitution: the ‘Sex-Work-model’ and the ‘Oppression-model’. The therapist’s reaction to the patients’ information was seen as a starting point to understanding the intrapsychic function of paid sex as a coping mechanism against depressive feelings.Conclusions: Exploring and understanding prostitution consumption patterns in young men can benefit the treatment of psychiatric disorders in the private practice setting. It is the psychiatrists task to investigate the patients hidden motives behind paid sex use to help patients achieve a greater inner and relational freedom.", "keywords": [ "paid sex use", "prostitution", "depression", "failure of genital response", "erectile disorder", "girlfriend-experience", "sex worker", "sex-work-model", "oppression-model", "Arizona Sexual Experience Scale" ], "content": "Introduction\n\nPaid sex behavior, either giving or receiving money, is a delicate matter for psychiatric patients. Patients rarely talk about this taboo subject spontaneously, and habitually psychiatrists tend not to ask about it. In contrast, prostitution use is frequent; 18% of American men paid for sex in their past, and 3% of them did so during the year before inquiry1. 2% of American women have received money for sex during their lifetime1.\n\nWe hypothesize that the personal and therapeutic attitude of health professionals towards paid sex is often ambivalent and leans towards the avoidance of the topic. There are multiple reasons for this such as the reduced importance of the topic in medical education and in psychiatry-training, a certain degree of idealizing the patient as not being prone to morally questionable behavior and, no apparent clues in the patient’s presentation and in his narrative.\n\nWe think that particularly in young men, asking actively about and understanding prostitution consumption may benefit their psychiatric and psychotherapeutic treatment. In our experience, investigating a patient’s hidden motives behind paying for sex can help patients to achieve greater inner and relational freedom.\n\n\nThe patient\n\nMr. A, a small and shy 30 year old male, was born in a north-western Portuguese village near an internationally renowned Casino-beach-resort and lived there until the age of 18. He is the only son of a working-class couple, who are both in paid employment. Mr. A attended a college in Lisbon, which was a three hour drive from his parent’s home. None of his peers from the village went to college; Mr. A was a driven individual and achieved his goal of furthering his education.\n\nAt home his parents always quarreled about his father’s infidelities, but despite this they stayed together to finance his studies. When they divorced after his graduation, he felt dejected and gradually developed a depressive state. His depressive symptoms lasted for 12 months before he came for a consultation to the F+F Gysin, Private Psychiatric Practice. As well as his parents’ divorce, he broke two toes during a kick-boxing session, which caused him to stop practicing his favorite sport. During this period he also started to date a girl who compared to him had a poor education and no fixed job. Because of this, she envied him for his higher income and thus she was unfaithful to him. The culmination of these reasons and a poor performance in his engineering job, he was encouraged by his friends and pressured by his superiors at work to see a psychiatrist, whom he selected from a health insurance list.\n\nDuring his first sessions in September 2011 he referred to having a low mood, which stemmed from being resentful for not being promoted at work, a lack of motivation, social isolation and an irrational fear of being attacked in a familiar and secure night club. He had outbursts with friends, suffered from anhedonia, a lesser sexual drive, a fear of losing his hair and gaining weight. He was also particularly dysphoric of his small height of 1,59m. He wanted help but without medication.\n\n\nHe pays for sex\n\nFor Mr. A, paid sex was not a problematic issue, nor a direct motive for his consultation. When questioned about his sex-life he was comfortable talking about his experiences, and showed a degree of consumer pride in prostitution. At the age of 22, after breaking up a four-year relationship, out of curiosity and revenge, he purchased for the first time sexual services in a sex-worker’s apartment. This use of prostitution escalated when back in his native village, where a Saturday night ritual with a group of friends started. After dinner with their girlfriends, the group would take their partners home and then all the males would leave them to visit a brothel to have a drink, fun and sometimes have paid sex with prostitutes using protection. This kind of prostitution use became a peer group standard and, for Mr. A, it seemed to be an easy victory over his shyness.\n\nMr. A became hooked and hyper-seduced2 by a specific prostitute, and he fantasized about living with her in what he described as an ‘exotic land’. However, to have or to maintain a full erection during paid sex he needed to think and imagine that he was making love to a romantic partner. The patient referred to exclusive heterosexual orientation and sexual desire, however revealed that he would suffer a loss of erection when either nervous or stressed.\n\n\nTests and scales\n\nThe Arizona Sexual Experience Scale3 is composed of five questions. As we can see in Figure 1, for Mr. A, paid sex (red circles) is less exciting and less intense compared to romantic sex (green circles).\n\nThe informal Social Atom4 is a drawing that the patient was asked to create, drawing circles to represent his most important relationships and favorite past-times (see Figure 2). The proximity to the subject (the middle circle) indicates the importance of these to him. Mr. A’s social atom showed close relationships to his parents, friends and pets and also confirmed his problematic affective intimacy with women.\n\n\nMr. A’s relationship-cycle\n\nWe can describe Mr. A’s relationship dynamics by the means of anamnestic data and through his behavior in the therapeutic setting. We followed the Operationalized Psychodynamic Diagnostic-procedure5.\n\nMr. A felt that he was not being recognized in his efforts to fulfill or please others. Often arriving late to dates with girlfriends, he not only refused to apologize, but also expected them to listen supportively to his complaints. He implicitly asked too much of others without being aware of it, and then found himself surprised by the negative responses he would receive, and found this negativity unjustified and rejecting. He felt an imbalance between giving and receiving, which reinforced his fears of intimacy (see Figure 3).\n\nWith his paid sex encounters, he tried to escape from this cycle. After sex for money, he felt that afterwards, nobody is in debt with anyone. He looked for a “girlfriend experience” free from any affective claims. His internalized couple model is characterized by infidelity, hostility and matrimonial warfare.\n\nFor Mr. A, paid sex had many simultaneous non-sexual functions, which follow Willy Pasini’s list of Non-sexual Functions of Sex6: sex as tranquilizer and antidepressive for his symptoms, as identity-support and socializer in his peer group as well as power-tool and object of exchange with women. The intrapsychic function of paid sex seems to be a narcissistic first-aid kit.\n\n\nDiagnosis and clinical evolution\n\nMr. A was diagnosed with depressive episode, moderate to severe, with mild psychotic symptoms (ICD-10: F33.2)7 and failure of genital response (episodic erectile dysfunction in a paid sex setting) (ICD-10: F52.2). Paid sex activity in general may hide the aspect of a disorder of impulse control8, however this was not present with Mr. A. Addictive and obsessive traits in his paid sex behavior were ruled out. A weekly psychotherapeutic treatment was proposed and started in monotherapy, without anti-depressive medication. The patient showed high therapy-motivation and good compliance. He accepted the therapy-program and during a period of 14 months, Mr. A attended 40 hourly sessions.\n\nThe patient gradually improved without psychotropic medication and took three weeks off work as sick-leave and three weeks off work for overdue vacations. He quickly changed to a more challenging and better rewarded job and started up kick-boxing again. Socializing better in his new job, he continued to be solitary in his private life and started to commit time to his newly adopted dog (which coincidently was his mother’s favorite activity).\n\nMoreover, he reduced his peer-group paid sexual activities but still dissipated his energy by regular night-life, drinking and paid sex consumption. His sentimental life still revolved around his problematic girlfriend, whom he had chosen when he was depressed.\n\n\nTheories of prostitution\n\nTo understand the patient’s pattern of paid sex consumption and the therapeutic attitude and reaction of the therapist, it is important to understand the actual positions and knowledge about prostitution. There is general agreement that one should distinguish clearly between street and indoor prostitution9. Street prostitution is seen as implying frequent health-risks for workers with a high degree of victimization and oppression, while indoor prostitution subject to much debate.\n\nThere are two main prostitution theories which explain and deal with indoor prostitution: the “Sex-Work model”10 and the “Oppression model”11. The first proposes legalization of prostitution as a way to earn a living, and for harm reduction, minimizing risks for sex workers and consumers. The second focuses on exploitation, victimization and abuse of women and is usually adopted by feminist movements11 and right-wing conservatives for different reasons10.\n\nPortugal does not criminalize prostitution, but linked economic activities are illegal, like renting an apartment for prostitution work. There is no regulation, and sex workers are without legal protection and do not benefit from systematic harm reduction strategies. In general, prostitution does not seem to be on sexological or political agendas and very few studies about prostitution-users are available12.\n\n\nDiscussion\n\nAlthough the patient suffered from a moderate to severe depressive episode, with mild psychotic signs, no medication was given in response to the patients’ wishes and following the tendency of psychiatrists to follow a “watchful waiting” approach. Official therapeutic recommendations in Germany and Switzerland state: “When a unique therapeutic approach is planned in patients suffering from moderate to severe acute depressive episodes, treatable in outdoor-setting, exclusive psychotherapy should have the same importance as exclusive medication when the method of treatment is chosen”13.\n\nThe attitudes of psychiatrists and psychotherapists to paid sex are rarely examined. With the clinician, a professional tolerant and neutral position may coexist with a private negative sensibility about prostitution. Unintentionally, this may result in a negative judgment of paid sex and avoidance of the patient’s narratives of paid sex behavior.\n\nFátima Gysin (the therapist of Mr. A) specialized in sexology in a sexological and psychosomatic consultation at the University of Geneva headed by Willy Pasini. In Portugal, in the private practice setting, she has treated an up-scale sex-worker for depression and anxiety unrelated to her professional activity. The patient valued her work and was determined to continue indoor, top-level prostitution. In this context, exploitation, misery and victimization are absent or less visible and sex-work appears to be frequently a free choice of profession.\n\nOne question is the degree of communication of the therapist’s personal moral, ethical or political stance to the patient. Sometimes the therapeutic process is better served by the therapist’s auto-disclosure, while generally non-disclosure is recommended14.\n\n\nConclusion\n\nAlthough it wasn’t a reason for consultation nor was it presented as a symptom, it was essential to Mr. A’s psychiatric treatment for his depressive disorder to open up his paid sex habits. The therapy helped him to make sense of and give meaning to his struggle with intimacy. The change from his depressive mood was probably facilitated by the interest given to the significance of his paid sex experience. On different levels, prostitution use can be simultaneously a symptom, a free choice and a cultural pattern. In depressed men seeking psychiatric or psychotherapeutic help, an active exploration of any existing paid sex experiences can be useful.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details was obtained from the patient. Permission has been solicited by e-mail for publishing the results of the Arizona Sexual Experience Scale (ASEX).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe paper is based on an oral presentation given during the 11th Congress of the European Federation for Sexology in Madrid, September 19–22, 2012, presented by the first author who is the patient’s psychiatrist and psychotherapist.\n\n\nReferences\n\nEdlund Lena, Korn Evelyn: A theory of prostitution. J Polit Econ. 2002; 110(1): 181–214. Publisher Full Text\n\nPasini Willy: Les armes de la séduction. Odile Jacob 2011. Reference Source\n\nMcGahuey CA, Galenberg AJ, Laukes CA, et al.: The Arizona Sexual Experience Scale (ASEX): reliability and validity. J Sex Marital Ther. 2000; 26(1): 25–40. PubMed Abstract \| Publisher Full Text\n\nSchützenberger Anne Ancelin: Psychogénéalogie. Payot, 2012. Reference Source\n\nOperationalized Psychodynamic Diagnostic. OPD-2. Springer, 2002. Reference Source\n\nPasini Willy: La Qualité des Sentiments. Payot, Paris, 1992; 295. Reference Source\n\nWHO – ICD-10 Classification of Mental and Behaviour Disorders. 1994. Reference Source\n\nAboujaoude Elias, Koran Lorrin M: Impulse Control Disorders. Cambridge University Press, 2010. Publisher Full Text\n\nWeitzer Ronald: Prostitution control in America: Rethinking public policy. Crime Law and Social Change. 1999; 32(1): 83–102. Publisher Full Text\n\nWeitzer Ronald: Legalizing Prostitution: Morality Politics in Western Australia. Br J Criminol. 2009; 49(1): 88–105. Publisher Full Text\n\nFarley Melissa: Prostitution, Trafficking, and Cultural Amnesia: What we must not know In order to keep the Business of Sexual Exploitation running smoothly. Yale J Law Fem. 2006. Reference Source\n\nBarbara G Brents, Crystal A Jackson, Kathryn Hausbeck, et al.: The State of Sex Tourism, Sex and Sin in the New American Heartland. Routledge, New York, 2009. Reference Source\n\nKüchenhoff Joachim: Psychothérapie dans la Dépression, Synthèse des recommendations S3 de la DGPPN. Forum Med Suiss. 2012; 12(12): 267–271. Reference Source\n\nGabbard GO, Roberts L, Crisp H, et al.: Professionalism in Psychiatry. APA. 2012; 169(5): 543–544. Publisher Full Text" }	[ { "id": "828", "date": "11 Mar 2013", "name": "Chiara Simonelli", "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 complete case report, where all the necessary information has been extensively provided. Overall, the report is valuable, but we have few reservations. We suggest to the Authors to:Explain more clearly some of the clinical considerations in order to facilitate the reader’s comprehension of this case. For example, they could better specify the sentence; 'which coincidentally was his mother’s favorite activity', on page 4;Within the text, if possible, comply with paragraphs’ name used in the abstract;Add in the abstract (section: Design and method) the informal Social Atom and the Operationalized Psychodynamic Diagnostic-procedure;Specify in the section 'Tests and scales' the important results emerging from Arizona Sexual Experience Scale, as it has been done for the Social Atom;Add to the ICD-10 nosography also the DSM classification: in some Countries this is more used than the other one and so it could be more comprehensible;The reference list could be improved with some recent international articles supporting your theoretical assumptions. For example we can suggest two important publications concerning different motivations to sexuality and the importance to talk about sexuality during the therapeutic process:\n\nMeston CM, Buss DM (2007), Why Humans Have Sex, Archives of Sexual Behaviour, 36: 477-507Althof SE, Rosen RC, Perelman MA, Rubio-Aurioles E (2013), Standard operating procedures for taking a sexual history, Journal of Sexual Medicine, 10: 26-35.", "responses": [] }, { "id": "897", "date": "17 Apr 2013", "name": "Antonio Pacheco Palha", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 only one clinical case and so I think it has a medium interest. Although it is present in a clear way I think the discussion should be more consistent. Anyway, it deserves to be published.", "responses": [] } ]	1	https://f1000research.com/articles/2-70
https://f1000research.com/articles/2-34/v1	07 Feb 13	{ "type": "Research Article", "title": "Verbal and novel multisensory associative learning in adults", "authors": [ "Joanne M Fifer", "Ayla Barutchu", "Mohit N Shivdasani", "Sheila G Crewther", "Joanne M Fifer", "Ayla Barutchu", "Mohit N Shivdasani" ], "abstract": "To date, few studies have focused on the behavioural differences between the learning of multisensory auditory-visual and intra-modal associations. More specifically, the relative benefits of novel auditory-visual and verbal-visual associations for learning have not been directly compared. In Experiment 1, 20 adult volunteers completed three paired associate learning tasks: non-verbal novel auditory-visual (novel-AV), verbal-visual (verbal-AV; using pseudowords), and visual-visual (shape-VV). Participants were directed to make a motor response to matching novel and arbitrarily related stimulus pairs. Feedback was provided to facilitate trial and error learning. The results of Signal Detection Theory analyses suggested a multisensory enhancement of learning, with significantly higher discriminability measures (d-prime) in both the novel-AV and verbal-AV tasks than the shape-VV task. Motor reaction times were also significantly faster during the verbal-AV task than during the non-verbal learning tasks. Experiment 2 (n = 12) used a forced-choice discrimination paradigm to assess whether a difference in unisensory stimulus discriminability could account for the learning trends in Experiment 1. Participants were significantly slower at discriminating unisensory pseudowords than the novel sounds and visual shapes, which was notable given that these stimuli produced superior learning. Together the findings suggest that verbal information has an added enhancing effect on multisensory associative learning in adults", "keywords": [ "associative learning", "multisensory integration", "audition", "vision", "verbal" ], "content": "Introduction\n\nEffective perception of our everyday environment requires that specific associations are learnt between the multiple representations of objects and events that occur across different sensory modalities. Such multisensory associative learning, particularly between the auditory and visual modalities, plays an important role in many social and cognitive processes, including object identification as well as lexical and semantic processing1–3. Multisensory processing is known to facilitate many aspects of information processing, resulting in faster motor responses4 and increased visual perceptual sensitivity 5. Indeed, Barutchu et al.6 recently demonstrated that superior auditory-visual multisensory abilities were associated with above average general intellectual abilities in children. With regard to learning, stimuli that are initially encountered as concurrent and semantically congruent auditory-visual events are more reliably remembered than those encountered separately in the auditory or visual modalities7,8. Recent studies also suggest that sound modulates visual learning9,10, and that auditory-visual training compared with visual or auditory training alone can lead to marked improvements in learning11,12. Not unexpectedly, learning by experience and prior knowledge have also been shown to influence multisensory processes at both a behavioural and neural level1,13–19.\n\nSurprisingly few studies have investigated the differences in learning patterns within and across sensory modalities. Tanabe et al.20 contrasted the learning of cross-modal and intra-modal associations in an fMRI investigation. They assessed feedback-based trial-by-trial learning of sequentially presented novel cross-modal and intra-modal pairs in a delayed match-to-sample task. In their study, no significant differences between auditory-visual and visual-visual learning were found. On the other hand, Seitz et al.12 demonstrated that auditory-visual pairs were learnt to a significantly greater degree than intra-modal (auditory-auditory and visual-visual) pairs in a statistical learning task. However, the cross-modal advantage may have resulted from the method of presentation of stimuli whereby auditory-visual pairs were presented simultaneously but intra-modal pairs were presented sequentially. This systematic variation in temporal disparity of stimulus presentation may have influenced the results.\n\nOther empirical evidence indicates that concurrently presented familiar cross-modal stimulus pairs with verbal components can influence both learning and multisensory processes1,14,15,21. Indeed, verbal stimuli have been shown to facilitate many cognitive processes, including object categorisation and localisation22,23. This raises the question of whether visual-verbal learning may lead to behavioural advantages when compared with non-verbal auditory-visual learning in adults. The association and transfer of visual and verbal information are of particular relevance, as such associations are known to play an important role in the acquisition of complex cognitive tasks, such as reading24–26. Furthermore, in infants, linguistic material such as nonsense words can enhance associations between stimuli and contribute to categorical learning27,28. However, to the best of our knowledge, no study has directly contrasted the learning of visual-verbal associations with non-verbal auditory-visual associations in adults.\n\n\nExperiment 1\n\nThe current study addressed two aspects of multisensory associative learning. Firstly, we aimed to evaluate performance differences on analogous intra-modal and multisensory learning tasks. Secondly, we further aimed to compare verbal and non-verbal multisensory associative learning. Three paired associate learning tasks were created: novel non-verbal auditory-visual (employing “novel sounds”; novel-AV), verbal-visual (verbal-AV), and visual-visual (shape-VV). We used a trial-and-error learning paradigm based on that of Tanabe et al.20, but with concurrently presented stimuli, in order to examine differences in associative learning between multisensory and intra-modal stimulus pairs under simultaneous conditions. The novel-AV and shape-VV tasks utilised non-verbal stimuli, whereas the verbal-AV task included auditory pseudowords to evaluate the impact of semantically novel, yet phonetically familiar, auditory stimuli on learning. Learning performance and task differences were evaluated by analysing changes in accuracy using signal detection theory (d-prime) and motor reaction times (MRTs).\n\n\nMaterials and methods\n\nTwenty right-handed adults (9 males), between 18 and 35 years of age (mean = 24.87 years, SD = 3.54) were recruited. All participants spoke English as a first language and were screened to ensure auditory detection thresholds and vision (distance, near and colour) were within the normal or corrected to normal ranges. Participants reported no history of neurological or psychological disorders. Estimated Full Scale IQ scores on the Wechsler Test of Adult Reading29 were above the 10th percentile for all participants except one adult, whose results were excluded from further analyses. Written informed consent was obtained from all participants. Ethics approval was obtained from the La Trobe University Human Research Ethics Committee, Bundoora, and The Royal Victorian Eye & Ear Hospital Human Research Ethics Committee, Melbourne.\n\nFour novel and arbitrarily related stimulus pairs were created for each learning task (novel-AV, verbal-AV and shape-VV). All auditory and visual stimulus pairs were presented well above threshold, and were therefore easily detectable. Visual black symbols (BS) of 3 degrees of visual angle, composed using black lines against a white background (see Figure 1: BS1, BS2, BS3 for examples), were presented at participants’ central fixation point on a 22-inch cathode-ray-tube (CRT) monitor (positioned at a distance of 1 meter from participants). These visual symbols were paired either with novel sounds that were unfamiliar and could not be vocalised (novel-AV), verbal pseudowords (verbal-AV), or other visual symbols (shape-VV) (see Figure 1 for an illustration of all stimuli used).\n\nAuditory and visual stimuli for the novel sound-visual (novel-AV), the verbal-visual (verbal-AV), and the visual-visual (shape-VV) associative learning tasks.\n\nFor the novel-AV task, novel non-speech auditory sounds were digitally created (sampling rate = 48.8 kHz, duration ~620 ms, 5 ms rise-fall time), consisting of combinations of four amplitude-modulated tones using different carrier and modulation frequencies. In the novel-AV task, for each novel sound, the carrier frequencies ranged between 400–480 Hz (novel sound 1; NS-1), 1000–1350 Hz (NS-2), 3000–3200 Hz (NS-3) and 250–4500 Hz (NS-4). Modulation frequencies were either kept at 3 Hz (NS-1), 10 Hz (NS-2), 0.5 Hz (NS-3) or 6 Hz (NS-4). In addition to the amplitude modulated (AM) tones, two pure tones of 250 Hz and 450 Hz were also added to NS-4. In the verbal-AV task, four single syllable pseudowords (matched for the duration of ~620 ms) pronounced by a young female adult were used (“jat”, “doot”, “chel” and “shoap”). Each pseudoword began with a different consonant to emphasise stimulus onset. Pseudowords were pre-recorded in a sound attenuated recording booth (sampling rate = 48.8 kHz). All verbal and novel-sounds were presented at 69 dB sound pressure level (SPL) using closed headphones. The shape-VV task employed a second visual stimulus, which was a novel solid red shape constructed by overlaying rectangular, triangular and circular shapes in different combinations (see Figure 1, shape-V). In this task the black symbol was presented superimposed and contained within the red shape such that both were presented at the participant’s central fixation point. All stimulus pairs were presented concurrently for the duration of ~620 ms with simultaneous onset and offset times.\n\nParticipants were required to learn the associations between the stimulus pairs via trial and error. Figure 2 presents a schematic diagram of the temporal sequence of a single trial. For each task, matching stimuli were four specific pairings of stimuli, out of a possible 16 stimulus combinations. The non-matching stimuli comprised the 12 other possible pairings of the stimuli. Each learning task was administered as four blocks of 32 trials (128 total trials). In 50% of the trials, matching pairs were presented, and the remaining trials consisted of non-matching stimuli. The presentation of stimulus pairs within each block was pseudorandom, such that each block consisted of 16 presentations of matching stimuli with each stimulus pair presented four times. Participants were instructed to make a button response with their right index finger when a matching stimulus pair was detected. A ‘no response’ was deemed to be an indication that the participant did not consider the stimuli to be a matched pair. Participants were allowed 3000 ms to respond, after which feedback was provided after every trial using an ascending tone burst (for a correct response) or descending tone burst (for an incorrect response), presented for a duration of 200 ms. The feedback was followed by an inter-stimulus interval, which randomly varied between 1000 to 2000 ms.\n\nTemporal sequence of a single experimental paired associate learning trial.\n\nParticipants completed the three tasks in a quiet room. Testing took place over one or two sessions, and the total test time per participant was approximately 2 hours. When two or more learning tasks were completed in a single session, a period of at least 45 minutes was implemented in-between tasks to maximise concentration for the subsequent learning task. During this time, the auditory and visual screening tasks were completed. The order in which the three learning tasks (novel-AV, verbal-AV, and shape-VV) were administered was counterbalanced across participants.\n\nA practice task was employed prior to each experimental learning task. The practice task was analogous to the proceeding experimental task; however, there were only three stimulus pairs (as opposed to four), and the stimuli were not abstract (e.g., visual stimuli were images of a square, circle and triangle rather than unfamiliar shapes). Participants were encouraged to ask questions about the practice task and up to 60 trials were administered until such time as the participant demonstrated understanding of the task requirements. In addition, immediately before the experimental tasks began, participants were presented once with each of the eight individual stimuli that made up the four stimulus pairs. This familiarisation process ensured that participants were aware of the characteristics of each stimulus, but did not receive any information regarding whether they were matching or not. Participants were instructed that during the experimental task they would be shown novel symbols, for which they would need to learn the associated pseudowords (verbal-AV), sounds (novel-AV), or shapes (shape-VV). Each of the four blocks within each task was of approximately 4 minutes duration, and participants were offered a short break of up to 1 minute between blocks.\n\nAccuracy data and motor reaction times (MRTs) were recorded. An error on a matching trial was a failure to make a motor response (i.e., miss). A motor response to a non-matching trial reflected a false alarm. Only the first 120 trials were analysed, as some participants failed to complete the entire set of 128 trials due to computer failure (only one participant on the verbal-AV condition was affected). All participants completed the first 120 trials that were analysed).\n\nTo visualise the different patterns of learning across trials for each task and stimulus pair type, “five-point moving average” analyses were employed to construct learning curves consisting of 116 overlapping windows moving in one trial steps, which were averaged across participants. Sets of five trials were used because preliminary analysis revealed that considerable learning occurred early in the experiment, and small window lengths allowed for optimal visualisation of learning effects.\n\nSignal Detection Theory (STD) and measures of discriminability (d-prime) were used to assess changes in learning trends for the three stimulus pair types. The discrimination statistic, usually known as d-prime, is a measure of an individual’s ability to distinguish true signals from noise30,31. It incorporates true responses and false alarms to minimize the effects of response bias to target and non-target stimuli. The d-prime statistic was calculated for the total 120 trials for the three stimulus pair types, and differences between each pair type were analyzed using a one-way repeated measures ANOVA. We were also interested in the ability to discriminate pairs as learning occurred across time, therefore data was subdivided into blocks of five trials each; there were a total of 24 successive blocks. Preliminary analyses revealed that d-prime values for block 8 and all subsequent blocks violated normality, thus analysis of the effects of task and stimulus pair type across the first 7 blocks were assessed using a 3 (task: novel-AV, verbal-AV, shape-AV) × 7 (block: 1, 2, 3, 4, 5, 6, 7) repeated measures ANOVA.\n\nChanges in MRTs with learning were also investigated. Based on learning trends observed in the moving average learning curves (see Figure 3), the first 20 matching trials were defined as the “learning” phase and final 20 target trials were defined as the “learnt” phase (please note that non-target stimuli did not require a response, hence could not be included in this analysis). Differences in learning between tasks were assessed using a 3 (task: novel-AV, verbal-AV, shape-VV) × 2 (phase of learning: learning, learnt) repeated measures ANOVA.\n\nMoving average of mean percentage accuracy for trials on the novel-sound auditory-visual (novel-AV), the verbal-visual (verbal-AV), and the visual-visual with red shapes (shape-VV) learning tasks. Shaded areas depict SEM along the moving average.\n\nAll significant main effects obtained in ANOVA analyses were followed up with post-hoc pairwise comparisons using Tukey corrections. For all statistical tests, alpha level was set at 0.05.\n\n\nResults\n\nParticipants demonstrated learning in all three experimental tasks (see Figure 3). It can be seen that under all conditions, initial performance was close to chance levels, and accuracy rates above 90 percent were attained on each task. The verbal-AV task was consistently performed with greater accuracy than both non-verbal tasks, with a steeper trajectory of learning and with accuracy rates reaching ceiling performance during the second half of the experiment. Of the non-verbal tasks, the novel-AV task tended to be performed with slightly greater accuracy than the shape-VV task. This novel-AV superiority was primarily evident at early learning stages; novel-AV and shape-VV accuracy rates were more similar from trial 50 onwards.\n\nSimilar effects of task were evident when d-prime measures were considered. Overall mean discriminability (d-prime) significantly differed across all stimulus conditions, (see Figure 4A), F(2, 38) = 16.77, p < 0.001. The mean d-prime value for the verbal-AV task was significantly higher than that for the novel-AV, both of which were significantly higher than the shape-VV task.\n\nA. Overall mean discriminability (d-prime) (± SEM) for the novel-sound auditory-visual (novel-AV), the verbal-visual (verbal-AV), and the visual-visual with red shapes (shape-VV) learning tasks. B. Discriminability (d-prime) first 10 blocks of trials on the novel-AV, the verbal-AV, and the shape-VV learning tasks. Each block comprised 5 consecutive trials.\n\nAnalyses of discriminability across early blocks (Figure 4B) revealed that there were no discernable differences in the patterns of learning for each task, as the interaction between task and block was not significant, F(12, 216) = 1.54, p > 0.01. However, it was found that overall discriminability measures for the three task types significantly differed across these first 7 blocks, F(2, 216) = 22.40, p < 0.001. Performance rates were significantly different between all tasks, with verbal-AV producing the greatest discriminabilty and shape-VV the poorest. In addition, with all tasks considered, mean d-prime measures significantly increased between the first two blocks, and then significant increases in discriminability occurred at block 5 before plateauing thereafter F(6, 216) = 13.45, p < 0.001.\n\nAs can be observed in Figure 3, moving averages of percent accuracy show steep learning trends within the first 20 trials with performance almost reaching ceiling levels by the end of the first 60 trials. We therefore further investigated differences in motor responses during the learning stage (first 20 trials) and the late learning stage (41–60 trials) for matching stimuli (note that non-matching stimuli did not require a response, hence, non-matching trials could not be included in the MRT analyses). A significant interaction effect between learning phase and task type was found, F(2, 36) = 3.56, p < 0.05, partial η2 = 0.17. At both learning stages, the MRTs for the verbal-AV condition were significantly faster than for the novel-AV and shape-VV tasks (see Figure 5); the MRTs for novel-AV and shape-VV tasks did not differ significantly from each other in the first 20 target trials. Furthermore, the rate of change in MRTs was significantly greater for verbal-AV trials compared with shape-VV trials. The change in MRTs for novel-AV trials did not significantly differ from either verbal-AV or shape-VV trials.\n\nAverage motor reaction times (MRTs) (± SEM) for the learning phase (Trials 1–20) and the learnt phase (Trials 41–60) in the novel-sound auditory visual (novel-AV), verbal-visual (verbal-AV), and visual-visual (shape-AV) learning tasks.\n\n\nDiscussion\n\nThe results of Experiment 1 demonstrated a significant advantage for the learning of both verbal and non-verbal auditory-visual stimulus associations over intra-modal visual-visual associations. In the verbal-visual task, associated stimuli were also identified faster. Whilst these results represent potentially important and compelling findings, it remained difficult to delineate the underlying factors causing the learning differences. The most pressing factor was the possible confounding influence of differences in stimulus discriminability at a unisensory level. A follow-up experiment evaluated whether unequal unisensory stimulus discriminability could explain differences in learning trends between the verbal-AV, novel-AV and shape-VV tasks.\n\n\nExperiment 2\n\nThe aim of Experiment 2 was to investigate whether the unisensory auditory and visual stimuli employed in the associative learning paradigms differ in discrimination difficulty. The auditory and visual stimulus sets from the learning tasks were tested in six separate forced-choice discrimination tasks (see Figure 1 for an illustration of all 6 stimulus sets). Here we investigated discrimination difficulty across the three differing task-specific stimuli: novel auditory stimuli (novel-A), verbal auditory stimuli (verbal-A), and red-shape visual stimuli (shape-V), in addition to the three sets of visual black symbols that were paired with: BS1, BS2 and BS3, respectively.\n\n\nMethod\n\nParticipants were 12 adult volunteers (6 males), aged between 18 and 35 years (M = 29.55, SD = 3.43). Participants who took part in the current control study did not participate in the learning task. All participants were right handed, had normal or corrected to normal vision, normal hearing and no prior history of neurological or psychiatric disorders.\n\nThe forced-choice discrimination tasks utilised the six stimulus sets that were employed in the three learning tasks in Experiment 1: novel sounds (novel-A), verbal pseudowords (verbal-A), red shapes (shape-V), BS1, BS2 and BS3 (see Figure 1 for an illustration of stimuli). There were four stimuli in each discrimination task, and for each task, one of the four stimuli was randomly assigned as the “target” stimulus, and the remaining three stimuli as “non-target” stimuli. The target stimuli were alternated via counterbalancing across participants such that each stimulus acted as the target stimulus on an equal number of occasions.\n\nStimuli were presented individually in the discrimination tasks. Visual stimuli were presented at central fixation on a 17-inch laptop screen at three degrees of visual angle. Auditory stimuli were presented via closed headphones. Both the auditory and visual stimuli were presented for ~620 ms duration. All other stimulus parameters for the novel sounds, verbal psuedowords, red shapes and visual black symbols (BS1, BS2 and BS3) were same as in Experiment 1.\n\nFor each task, participants were presented with 80 stimuli in two blocks of 40 trials each. The inter-stimulus interval (ISI) randomly varied between 1000–1500 ms during which a central fixation point was presented. Consistent with the learning tasks in Experiment 1, stimuli were presented in a pseudorandom order with a 0.5 target probability. The remaining stimuli consisted of non-targets with an equal probability of each type. The same stimulus was not presented for more than two consecutive trials. Participants were asked to indicate whether the stimuli were targets or non-targets by pressing buttons on a keyboard. A feedback tone pip (700 Hz, 122 ms in duration) was provided at the end of a trial if the response was incorrect.\n\nAll participants completed the six discrimination tasks. The order of task administration was counterbalanced across participants to negate any order or practice effects. Participants were instructed to press the left arrow key on a standard keyboard with their index finger in response to a target, and the right arrow key with their middle finger in response to any non-target stimuli. Participants were instructed to respond as quickly and accurately as possible. A feedback tone pip was provided when the participant’s response was incorrect; no feedback was provided for a correct response. Prior to testing, participants were given up to 20 trials as practice during which they were familiarised with the target stimulus and were able to practice performing the task.\n\nPercentage error and mean MRTs for target and non-target stimuli were calculated. All participants completed 80 trials; whilst a lower-bound response inclusion criterion of 150 ms was set to control for response anticipation, there were no responses of this nature, so no data was excluded on this basis.\n\nFor MRTs, two repeated measures ANOVAs were used to analyse differences between novel sounds, verbal pseudowords and visual shapes, and the three black symbol sets separately. Any significant effects were followed up with Tukey post-hoc tests.\n\n\nResults\n\nThe percentage error rates for all discrimination paradigms were very low, averaging below 6% error rates (see Table 1). Many participants did not make discrimination errors, leading to violations of normality. Therefore, accuracy measures were not subjected to further data analyses.\n\nThe MRTs for the novel-A, verbal-A and shape-V discrimination stimuli significantly differed from each other (see Figure 6), F(2, 22) = 26.58, p < 0.001, partial η2 = 0.71. MRTs were significantly slower for verbal auditory stimuli (verbal-A) than both novel auditory (novel-A) and visual red shape (shape-V) stimuli. However, the MRTs for novel-A and shape-V stimuli did not significantly differ from each other. No significant differences were found between MRTs for the three black symbol sets, F(2, 22) = 1.36, p < 0.05, partial η2 = 0.11.\n\nMean MRTs (+SEM) for each of the discrimination tasks: novel auditory sounds (novel-A), verbal auditory sounds (verbal-A), visual red shapes (shape-V), and the black visual symbol sets (BS1, BS2 and BS3). * p < 0.05.\n\n\nDiscussion\n\nAll auditory and visual stimuli were relatively easy to discriminate. Differences in discrimination difficulty were minimal with accuracy measures being at ceiling for all stimulus types. Although motor reaction times did not significantly differ between the black symbol sets, motor responses were significantly slower for verbal stimuli compared with the novel auditory stimuli and the red shape stimuli. This finding is consistent with prior studies suggesting that verbal stimuli require longer processing times than visual pictures19. Nevertheless, the high accuracy rates for all stimulus sets suggest that any differences in discrimination difficulty between stimulus sets are relatively small, with verbal stimuli being slower to process.\n\nIt may be argued that relative differences in the memorability of the unisensory stimuli, in addition to their discriminability, may have influenced performance in the three learning tasks in Experiment 1. However, successful performance of the current discrimination task in this experiment necessitated memory processing. An accurate and speedy response to the presented stimulus required the comparison of that stimulus to a memory trace of the “target” stimulus. Therefore, the lack of significant differences between discrimination accuracy and MRTs to the auditory and visual stimuli suggests that the stimuli did not differ in their discriminability or memoriability.\n\n\nGeneral discussion\n\nThe current results provide new evidence regarding the differences between multisensory and intra-modal learning, with more accurate performances and faster learning on the verbal-visual and novel auditory-visual tasks compared with the visual-visual task, suggesting that multisensory processing facilitates associative learning. The enhanced speed of response during the learning of verbal-visual associations compared with non-verbal stimulus associations suggest that verbal information further facilitates the associative learning process. The results of Experiment 2 suggested that the obtained task-related learning effects represented differences in associative learning rather than relative differences in stimulus discriminability between auditory and visual stimuli.\n\nAn enhancement in the learning of multisensory stimuli relative to intra-modal stimuli is not unexpected given that multisensory processing has long been known to facilitate information processing32. This facilitative effect has been shown to be substantially greater for multisensory stimulus combinations than for multiple stimuli within a single modality33. Not only do multisensory processes facilitate memory7, but they have also been shown to enhance perceptual and implicit learning, and training outcomes11,12,34–36. Consistent with these prior findings, the results of the present study indicate that this multisensory advantage extends to paired associative learning. Novel auditory-visual and verbal-visual tasks were performed with greater levels of discrimination during learning compared with the visual-visual task. Motor responses were also faster with the learning of multisensory rather than unisensory stimuli, even though the motor reaction times did not significantly differ during early stages of learning of the novel auditory-visual and unisensory visual tasks. These significant learning results were in the context of non-significant differences in motor responses between stimuli employed in the novel-AV and shape-VV tasks.\n\nThe afore-mentioned multisensory enhancement of learning contrasted with the behavioural findings of Tanabe et al.20. The differences between the results of our study and those of the fMRI study of Tanabe et al.20 may be explained by the fact that they presented stimulus pairs sequentially, 15 seconds apart, whilst we presented stimulus pairs simultaneously. The temporally concurrent presentation of multisensory stimuli would maximise the likelihood that multisensory ‘integrative’ processes are engaged. Animal studies using single-cell recordings have demonstrated that up-regulation of neural activation occurs only when stimuli are presented within a temporal window of 500 ms37. Similarly, behavioural facilitation in humans is observed only when auditory and visual stimuli are presented in close spatial and temporal proximity (reviewed in Spence and Squire38 and Wallance et al.39). An alternative explanation is that our concurrent presentation may have emphasised differences in salience and attentional resources within and across sensory modalities during dual stimulus presentations. Whilst concurrent stimulus presentations within the one modality (e.g., auditory-auditory or visual-visual) can have an interfering effect on stimulus perception, there is no such performance decrement when two concurrent stimuli are presented across different modalities (i.e., auditory-visual), even when stimuli are semantically incongruent13,40. Thus, the concurrent presentation of stimuli in the present study is likely to have facilitated information processing via modulation of either integrative or attentional processes, resulting in enhanced learning of novel multisensory stimulus pairs.\n\nWhilst both novel auditory-visual and verbal-visual tasks were associated with superior learning performances compared to the visual-visual task, additional performance gains were observed in the verbal-visual task. Both d-prime analyses indicated that greater discrimination of learned pairs was achieved in the verbal-visual task relative to the novel auditory-visual task, and the MRTs were significantly faster in the verbal-visual task in Experiment 1, yet participants were significantly slower at discriminating unisensory verbal stimuli in Experiment 2. These verbal-visual findings are noteworthy, given that the verbal stimuli used in all learning tasks in the present study were novel and therefore lacked semantic content. The only familiar or categorisable aspect of the verbal stimuli was the phonetic and verbalisable nature of the pseudowords, which allow for quicker encoding and rehearsal of the auditory stimulus component41,42. The visual-verbal learning advantage was evident despite the fact that participants were significantly slower at discriminating the individual verbal stimuli than the novel sounds and visual shapes. This finding is consistent with prior studies showing that verbal information requires longer time to processes than visual pictures19. Thus, the observed enhancements for verbal-visual associative learning cannot be attributed to a possible imbalance in discrimination difficulty between tasks; rather it appears that the processing speed advantage in the verbal-AV task is most likely related to a verbal-visual learning enhancement. This result suggests that verbal information is likely to play an important role in facilitating the accuracy and speed of human learning.\n\nFrom an early age (and throughout life) we assign arbitrary, speech-based labels to visual stimuli encountered in the environment. Even though the verbal stimuli in this study were not known words, the task reflects a common requirement of daily life, which often involves previously non-encountered words to be learnt and associated with an object or person (e.g., surnames, technical or scientific terms). Similarly, in infants, nonsense words have been shown to enhance associations between stimuli and contribute to categorical learning27,28. Verbal stimuli have also been shown to enhance other cognitive processes in adults, such as object categorization22,23. The known role of the superior temporal sulcus (STS) in performing speech and language-based functions in addition to multisensory auditory-visual integration may suggest that it plays a role in subserving this necessary skill20,43–46. Other studies have also shown greater levels of activation to multisensory stimuli involving phonetic elements in STS compared with novel auditory-visual stimuli1,47. Thus, it is likely that different neural networks underlie the processing of verbal-visual stimuli and non-verbal novel auditory-visual stimuli.\n\nIn conclusion, the present study revealed two major influences on paired associative learning. Firstly, improvements in accuracy and MRTs were demonstrated in multisensory learning tasks compared with the visual-visual learning task, and secondly, further gains in performance were obtained when auditory stimuli contained a verbal component. These results indicate that multisensory integration can facilitate associative learning, and provides new evidence that this facilitation may be further enhanced by verbal content.", "appendix": "Author contributions\n\n\n\nJMF conceptualized the project and developed the project proposal in consultation with AB, MNS and SGC. MNS programmed the experiments with input from JMF and AB. JMF collected the all the data. All authors contributed to data analysis, interpretation and the write-up of the manuscript. All authors reviewed and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests have been declared.\n\n\nGrant information\n\nWe wish to thank Neville and Di Bertalli for their financial support of this study. The Bionics Institute acknowledges the support it receives from the Victorian Government through its Operational Infrastructure Support Program.\n\n\nAcknowledgements\n\nWe would also like to thank Prof Antonio Paolini (School of Psychological Science, La Trobe University), Dr David Grayden (Electrical and Electronic Engineering, University of Melbourne) and Hamish Innes-Brown (Bionics Institute) for their support.\n\n\nReferences\n\nRaij T, Uutela K, Hari R: Audiovisual integration of letters in the human brain. Neuron. 2000; 28(2): 617–625. PubMed Abstract \| Publisher Full Text\n\nBeauchamp MS, Lee KE, Argall BD, et al.: Integration of auditory and visual information about objects in superior temporal sulcus. Neuron. 2004; 41(5): 809–823. 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PubMed Abstract \| Publisher Full Text\n\nPuce A, Epling JA, Thompson JC, et al.: Neural responses elicited to face motion and vocalization pairings. Neuropsychologia. 2007; 45(1): 93–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLupyan G, Thompson-Schill SL: The evocative power of words: activation of concepts by verbal and nonverbal means. J Exp Psychol Gen. 2012; 141(1): 170–186. PubMed Abstract \| Publisher Full Text\n\nLupyan G, Spivey MJ: Redundant spoken labels facilitate perception of multiple items. Atten Percept Psychophys. 2010; 72(8): 2236–2253. PubMed Abstract \| Publisher Full Text\n\nWindfuhr KL, Snowling MJ: The relationship between paired associate learning and phonological skills in normally developing readers. J Exp Child Psychol. 2001; 80(2): 160–173. PubMed Abstract \| Publisher Full Text\n\nThomson B, Crewther DP, Crewther SG: Wots that werd? Pseudowords (non-words) may be a misleading measure of phonological skills in young learner readers. Dyslexia. 2006; 12(4): 289–299. PubMed Abstract \| Publisher Full Text\n\nHulme C, Goetz K, Gooch D, et al.: Paired-associate learning, phoneme awareness, and learning to read. J Exp Child Psychol. 2007; 96(2): 150–166. PubMed Abstract \| Publisher Full Text\n\nWaxman SR, Markow DB: Words as invitations to form categories: evidence from 12– to 13–month-old infants. Cogn Psychol. 1995; 29(3): 257–302. PubMed Abstract \| Publisher Full Text\n\nFulkerson AL, Waxman SR: Words (but not tones) facilitate object categorization: evidence from 6– and 12–month-olds. Cognition. 2007; 105(1): 218–228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWechsler D: Wechsler Test of Adult Reading. San Antonio, TX: The Psychological Corporation 2001.\n\nMacmillan NA, Creelman CD: Detection theory: A user's guide. Cambridge: Cambridge University Press 2004. Reference Source\n\nSternberg S: Modular processes in mind and brain. Cogn Neuropsychol. 2011; 28(3–4): 156–208. PubMed Abstract \| Publisher Full Text\n\nTodd JW: Reaction to multiple stimuli. Archives of Psychology. 1912; 25: 1–65. Reference Source\n\nGingras G, Rowland BA, Stein BE: The differing impact of multisensory and unisensory integration on behavior. J Neurosci. 2009; 29(15): 4897–4902. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlais D, Cass J: Multisensory perceptual learning of temporal order: audiovisual learning transfers to vision but not audition. PLoS One. 2010; 5(6): e11283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeitz AR, Kim R, van Wassenhove V, et al.: Simultaneous and independent acquisition of multisensory and unisensory associations. Perception. 2007; 36(10): 1445–1453. PubMed Abstract \| Publisher Full Text\n\nErnst MO: Learning to integrate arbitrary signals from vision and touch. J Vis. 2007; 7(5): 7.1–14. PubMed Abstract \| Publisher Full Text\n\nWallace MT, Wilkinson LK, Stein BE: Representation and integration of multiple sensory inputs in primate superior colliculus. J Neurophysiol. 1996; 76(2): 1246–1266. PubMed Abstract\n\nSpence C, Squire S: Multisensory integration: maintaining the perception of synchrony. Curr Biol. 2003; 13(13): R519–521. PubMed Abstract \| Publisher Full Text\n\nWallace MT, Roberson GE, Hairston WD, et al.: Unifying multisensory signals across time and space. Exp Brain Res. 2004; 158(2): 252–258. PubMed Abstract \| Publisher Full Text\n\nAlais D, Morrone C, Burr D: Separate attentional resources for vision and audition. Proc Biol Sci. 2006; 273(1592): 1339–1345. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoberts LA: Modality and suffix effects in memory for melodic and harmonic musical materials. Cogn Psychol. 1986; 18(2): 123–157. PubMed Abstract \| Publisher Full Text\n\nKeller TA, Cowan N, Saults JS: Can auditory memory for tone pitch be rehearsed? J Exp Psychol Learn Mem Cogn. 1995; 21(3): 635–645. PubMed Abstract \| Publisher Full Text\n\nCalvert GA, Hansen PC, Iversen SD, et al.: Detection of audio-visual integration sites in humans by application of electrophysiological criteria to the BOLD effect. Neuroimage. 2001; 14(2): 427–438. PubMed Abstract \| Publisher Full Text\n\nWerner S, Noppeney U: Distinct functional contributions of primary sensory and association areas to audiovisual integration in object categorization. J Neurosci. 2010; 30(7): 2662–2675. PubMed Abstract \| Publisher Full Text\n\nMeyer GF, Wuerger S, Greenlee M: Interactions between Auditory and Visual Semantic Stimulus Classes: Evidence for Common Processing Networks for Speech and Body Actions. J Cogn Neurosci. 2011; 23(9): 2291–308. PubMed Abstract \| Publisher Full Text\n\nHein G, Knight RT: Superior temporal sulcus–It's my area: or is it? J Cogn Neurosci. 2008; 20(12): 2125–2136. PubMed Abstract \| Publisher Full Text\n\nvan Atteveldt N, Formisano E, Goebel R, et al.: Integration of letters and speech sounds in the human brain. Neuron. 2004; 43(2): 271–282. PubMed Abstract \| Publisher Full Text" }	[ { "id": "768", "date": "14 Feb 2013", "name": "Chris Baker", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "864", "date": "26 Mar 2013", "name": "Trevor Hine", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTwo well-run experiments: the first of which shows important differences not just in the rate of cross-modal vs uni-modal learning, but also in the processing speed involved in recognising these learned associations. The second experiment is an important control for the discriminability and processing speed for stimuli used in the first experiment. Together, this shows that the effects in the first experiment of learning simultaneously paired associations are due to genuine cross-modal facilitation.Other minor points:Page 4. Calculation of d-prime does not involve just the ‘hit’ and ‘false alarm’ frequencies, but also ‘misses’ and ‘correct rejections’ frequencies. Given this, it would be helpful if the authors made it more explicit how they calculated d-prime from your raw data.Page 7.In the sentence ‘No significant differences were found between MRTs for the three black symbol sets, F(2, 22) = 1.36, p < .05, partial η2 = . 11.’ There is a typo ‘p > .05’.", "responses": [ { "c_id": "446", "date": "29 Apr 2013", "name": "Sheila Crewther", "role": "Author Response", "response": "Dear Dr Hine, Thank you for picking up the typo and highlighting the ambiguity regarding d-prime. Indeed d-prime involves misses and correct rejection frequencies. In our case we calculated d-prime as the difference between 'hit' and 'false alarm' z scores [i.e, d-prime = Z(hit rate) - Z(false alarm rate)]. Regards Sheila Crewther. PhD" } ] } ]	1	https://f1000research.com/articles/2-34
https://f1000research.com/articles/2-29/v1	30 Jan 13	{ "type": "Case Report", "title": "Laparoscopic trocar management of a giant paraovarian cyst: a case report", "authors": [ "Mohamed Kandil", "Tarek Sayyed", "Mohamed Zakaria", "Mohamed Kandil", "Mohamed Zakaria" ], "abstract": "A 17-year-old woman had undergone exploratory laparotomy because of a huge cystic pelviabdominal mass equivalent of 36 weeks' gestation. A closed system drainage maneuver was applied via using a laparoscopic trocar to drain a revealed large left paraovarian cyst. This maneuver was found to be a simple and effective method to safely aspirate giant paraovarian cysts; thus allowing their total excision.", "keywords": [ "Paraovarian cysts occur in the broad ligament between the ovary and the tube", "predominantly arising from mesothelium covering the peritoneum (mesothelial cyst) but occasionally also from para mesonephric tissue (paramesonephric cysts or Mullerian cysts) and rarely from mesonephric remnants (mesonephric cysts or Wolffian cysts)1. Paraovarian cysts", "constitute 10–20% of all adnexal masses2. Some paraovarian cysts may reach a large size with possible complications like torsion and rupture3. These cysts are usually benign with rare incidence of malignant types4", "5. Here", "we present a case of unusually extensive proportions." ], "content": "Introduction\n\nParaovarian cysts occur in the broad ligament between the ovary and the tube, predominantly arising from mesothelium covering the peritoneum (mesothelial cyst) but occasionally also from para mesonephric tissue (paramesonephric cysts or Mullerian cysts) and rarely from mesonephric remnants (mesonephric cysts or Wolffian cysts)1. Paraovarian cysts, constitute 10–20% of all adnexal masses2. Some paraovarian cysts may reach a large size with possible complications like torsion and rupture3. These cysts are usually benign with rare incidence of malignant types4,5. Here, we present a case of unusually extensive proportions.\n\n\nCase presentation\n\nWritten informed consent for publication of the clinical details and/or clinical images was obtained from the patient.\n\nA 17 year old virgin presented with diffuse abdominal pain. History revealed a gradual increase in an abdominal swelling over the preceding 6 months. Physical examination showed a non tender tense cystic pelviabdominal mass of 36 weeks gestational size. Computerised tomography revealed 25×26 cm left ovarian simple cyst with clear contents and no septae. Serum CA125 levels were normal. Other tumor markers were not performed due to financial constraint. Through a subumbilical midline incision, a huge smooth cystic mass overlying the whole abdominal cavity was found. The cyst was isolated from its surroundings with gauze packs. A loose purse string suture was placed in the lowest accessible part of the cyst. A 5 mm laparoscopic trocar with a side track off its main sleeve was connected to a high pressure suction irrigation device via a rubber tube; the trocar was then inserted through the center of the suture which was subsequently stretched to fit around the sleeve. This created a closed system to drain the cyst. The trocar was removed leaving its sleeve in place and suction drained eight liters of clear watery fluid. The collapsed cyst was found to be left paraovarian which was exteriorized and the trocar sleeve was removed. The purse string suture was tightened to close the trocar opening. The left broad ligament was opened and the cyst wall was completely removed from the broad ligament, Figure 1. The redundant ligament peritoneum was excised and subsequently reconstructed with preservation of the tubal integrity as seen in Figure 2. The patient had an uneventful postoperative recovery.\n\nPostoperative histology reported simple benign serous cyst of mesothelial origin. The peritoneal fluid showed proteinaceous material entangling few lymphocytes and mesothelial cells with no evidence of malignancy.\n\n\nDiscussion\n\nHuge paraovarian cysts are uncommonly reported in the literature. On revising the literature, there were three case reports which had addressed comparable large paraovarian cysts but with implementation of larger incisions extending over the umbilicus for cyst extraction and excision without a policy to decrease its size before its exteriorization6–8. However, a case report for three adolescents with large paraovarian cysts had addressed decompression technique before cyst externalization and excision but in a different way9. In our case report we had dealt with such a huge cyst in a way not only to avoid morbidity of extending surgical incision but to guard against the risk of spillage of cyst contents as well. Concerning the endoscopic role, Darwish et al. reported a series of paraovarian cysts which had been excised laproscopically but were smaller in size with the largest not more than 13 cm10. However, there were two reports of large paraovarian cysts removed laparoscopically where in the first one, it was associated with acute lower abdominal pain while in the second it was associated with pregnancy11,12. We think that in all these laparoscopically operated cases, the implemented cyst decompression procedure before its removal had less control and precautions during it and in turn more risk of cyst spillage than our mentioned maneuver. It was thought that laparoscopy would be technically difficult in this case due to huge size of the cyst reaching close to xiphesternum. Direct abdominal entry with a Veress needle or trocar may have traumatized the cyst leading to risk of spillage of its content. Through laparotomy we employed a closed drainage system and safely aspirated the cyst without spillage of its content.\n\n\nConclusion\n\nOpen surgery remains the gold standard route to deal with giant paraovarian cysts. Aspiration of the cyst using a closed system followed by excision is a safe and effective treatment.", "appendix": "Author contributions\n\n\n\nMK prepared the framework of the case report, introduction, discussion and references. TS was the surgeon and prepared the presentation of the case in the manuscript. MZ assisted during the surgery and helped preparing the presentation of the case in the manuscript.\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\nReferences\n\nStenback F, Kauppila A: Development and classification of paraovarian cysts: an ultrastructural study. Gynecol Obstet Invest. 1981; 12(1): 1–10. PubMed Abstract\n\nAlpern MB, Sandler MA, Madrazo BL, et al.: Sonographic features of parovarian cysts and their complications. AJR Am J Roentgenol. 1984; 143(1): 157–160. PubMed Abstract\n\nLurie S, Golan A, Glezerman M, et al.: Adnexal torsion with a paraovarian cyst in a teenage girl. J Am Assoc Gynecol Laparosc. 2001; 8(4): 597–9. PubMed Abstract\n\nAltaras MM, Jaffe R, Corduba M, et al.: Primary parovarian cystadenocarcinoma: clinical and management aspects and literature review. Gynecol Oncol. 1990; 38(2): 268–72. PubMed Abstract \| Publisher Full Text\n\nKaur K, Gopalan S, Gupta SK, et al.: Parovarian cystadenocarcinoma: a case report. Asia Oceania J Obst Gynaecol. 1990; 16(2): 131–5. PubMed Abstract\n\nAzzena A, Quintieri F, Salmaso R: A voluminous paraovarian cyst. Case report. Clin Exp Obstet Gynecol. 1994; 21(4): 249–52. PubMed Abstract\n\nLazarov N, Lazarov L, Angelova M: Paraovarian cyst in an 18 year patient. Akush Ginekol (Sofiia). 2000; 40(4): 50. PubMed Abstract\n\nMukhopadhyay S: Giant paraovarian cyst. J Obstet Gyncol India. 2006; 56(4): 352–353. Reference Source\n\nDamle LF, Gomez-Lobo V: Giant paraovarian cysts in young adolescents: a report of three cases. J Reprod Med. 2012; 57(1–2): 65–7. PubMed Abstract\n\nDarwish AM, Amin AF, Mohammad SA: Laparoscopic management of paratubal and paraovarian cysts. JSLS. 2003; 7(2): 101–106. PubMed Abstract \| Free Full Text\n\nSindos M, Pisal N, Mellon C, et al.: Laparoscopic excision of a large paraovarian cyst presenting with acute lower abdominal pain. J Obstet Gynecol. 2004; 24(6): 717–718. PubMed Abstract \| Publisher Full Text\n\nRouzi AA: Operative laparoscopy in pregnancy for a large paraovarian cyst. Saudi Med J. 2011; 32(7): 735–7. PubMed Abstract" }	[ { "id": "794", "date": "22 Feb 2013", "name": "Sian Jones", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting case report that may help others when they come to deal with similar cases. A little more detail in the other methods alluded to in the laparoscopic cases might be helpful. The grammar and language needs editing.", "responses": [] }, { "id": "913", "date": "29 Apr 2013", "name": "Daniel Kruschinski", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 is interesting and shows the ability to manage big ovarian cysts laparoscopically. Important issues during the procedure, like the closure of the cyst incision site with sutures to prevent spillage, were correctly addressed.", "responses": [] } ]	1	https://f1000research.com/articles/2-29
https://f1000research.com/articles/2-81/v1	07 Mar 13	{ "type": "Short Research Article", "title": "Delayed ejaculation and alexithymia: what is the relationship?", "authors": [ "Paolo Maria Michetti", "Stefano Eleuteri", "Marta Giuliani", "Roberta Rossi", "Chiara Simonelli", "Paolo Maria Michetti", "Roberta Rossi", "Chiara Simonelli" ], "abstract": "Delayed Ejaculation (DE) is probably the least studied and understood of the male sexual dysfunctions (MSD). There is still little unanimity concerning its psychological/interpersonal aetiology. Previous studies found that MSD are strongly related with alexithymia, a multifaceted personality construct that describes a disturbance in the regulation of emotions.The aim of this study was to investigate the presence of alexithymia in men with DE and correlate alexithymia levels with DE severity. According to specific features of the symptoms, we hypothesized that alexithymia would not be correlated with this specific sexual disorder.54 outpatients with a diagnosis of DE assessed at the Institute of Clinical Sexology and the Urology Department of Sapienza, University in Rome were enrolled in the study. DE was diagnosed after a specialist examination and according to Diagnostic and Statistical Manual of Mental Disorders -IV-TR criteria. Participants were provided with the Toronto Alexithymia Scale (20 items; TAS-20), a self-measure of the Intravaginal Ejaculation Latency Time and an ad hoc questionnaire to collect anamnestic data.9.3% of patients could be categorized as alexithymics, 9.3% of them as borderline, while 81.4% of the sample was found to be non-alexithymic. The overall average TAS-20 score was 45.46. Results show that alexithymia is correlated neither with the presence of DE nor with its severity, in contrast to other MSDs, where this condition was found in about 30% of patients.The data presented suggest that DE, although not correlated to alexithymia, is probably related to other psychogenic features such as hypercontrol configuration. This paper can contribute to the understanding of DE, by excluding one of the possible etiological factors, previously found to be important in the onset and the maintenance of the other MSDs. More studies are needed in order to better understand DE and provide recommendations about treatment.", "keywords": [ "Delayed ejaculation", "alexithymia", "TAS" ], "content": "Introduction\n\nDelayed Ejaculation (DE) is probably the least studied and understood of the Male Sexual Dysfunctions (MSD)1. However, its negative impact is significant as it typically results in lack of sexual fulfilment for both the man and his partner; the problem is further increased when procreation is among the couple’s goals of sexual intercourse. The terms delayed, retarded, inadequate or inhibited ejaculation, idiopathic anejaculation (inability to ejaculate) and psychogenic anejaculation have all been used synonymously to describe a delay or absence of male orgasm2. The Diagnostic and Statistical Manual of Mental Disorders -IV-TR3 defines DE as:\n\n“the persistent or recurrent delay in, or absence of, orgasm after a normal sexual excitement phase during sexual activity that the clinician, taking into account the person’s age, judges to be adequate in focus, intensity, and duration. The disturbance causes marked distress or interpersonal difficulty”.\n\nIn the literature DE is reported to have an incidence rate between 3% and almost 40%, depending on age and comorbidities4–8. The essential medical causes of this problem include neurogenic, infective, endocrine and medication factors9. Psychological and interpersonal aspects are also involved in the onset and maintenance of DE, but there is little unanimity concerning these causes1.\n\nPrevious studies have found that Hypoactive Sexual Desire Disorder (HSDD), Erectile Dysfunction (ED) and Premature Ejaculation (PE) are strongly correlated with alexithymia10–14. Alexithymia (from the ancient Greek “a” for lack, “lexis” for word, and “thymos” for emotion) is a multifaceted personality construct introduced in the early 1970s by Nemiah and Sifneos15,16. The alexithymia construct describes a set of deficits in the cognitive processing of emotions, or more generally, a disturbance in the regulation of emotions17. The personality features that characterize alexithymic individuals include difficulty in identifying emotions and differentiating between emotions and the bodily sensations of emotional arousal; difficulty in communicating emotions to others; reduced imaginal and fantasy activity and an externally oriented cognitive style, a sub-factor of the alexithymia construct involving impoverished fantasy life, utilitarian thinking and a focus on external concrete data of the sensate environment18,19.\n\nA deep analysis of the literature underlines that:\n\n20–25% of subjects presenting MSDs can be classified as alexithymics10–13;\n\nthere is a significant correlation between alexithymia and the length/severity of MSD symptoms12–14;\n\nalexithymia is particularly correlated with HSDD10, ED10,12 and PE13.\n\nThe aim of this study was to investigate the presence of alexithymia in men with DE and to correlate alexithymia levels with DE severity. According to specific features of the symptoms we hypothesize that alexithymia will not be associated with this specific sexual disorder. In fact, emotional control, an important clinical feature identified in DE conceptualization20,21, has been found to be negatively associated with alexithymia22,23.\n\n\nMethods\n\n60 outpatients with a diagnosis of DE assessed at the Institute of Clinical Sexology and at the Urology Department of Sapienza, University in Rome were enrolled in the study. DE was diagnosed after a specialist examination, i.e. a careful anamnesis and a physical examination, and according to DSM-IV-TR criteria.\n\nPatients with previous and current psychotherapeutic experiences (6 patients) were not included because of the possible influence of psychotherapy on the degree of alexithymia19. Participants were informed in detail about the study, guaranteed confidentiality and anonymity and advised of their right to withdraw at any time. The ones who gave their consent to participate were provided with a research protocol including the following measures. The research protocol received institutional approval.\n\nDE severity was evaluated using a self-reported Intravaginal Ejaculation Latency Time (IELT;24) measure. The participants were given the following options to categorise the latency time: 10–20 mins, 20–30 mins, 30–40 mins, 40–50 mins and ‘never reached orgasm’.\n\nA questionnaire collecting demographic, anamnestic data and characteristics of the symptom (Lifelong or Acquired, Situational or Generalized DE) was filled by all patients. Alexithymia was measured by the 20-item Toronto Alexithymia Scale (TAS-20)25,26, a validated tool used to evaluate alexithymia levels. Even though alexithymia levels are measured on a continuous scale, different studies have established standard cut-off scores to distinguish alexithymic (TAS-20≥61) from non-alexithymic subjects (TAS-20≤51), with a borderline area for scores between 51 and 6119.\n\nUsing SPSS version 19 (IBM-SPSS Inc., 2010, Chicago, IL, USA), descriptive analyses were run in order to check sample and symptom characteristics. The relationship between alexithymia level and DE severity was investigated by Spearman’s Rho correlation coefficient.\n\n\nResults\n\nTable 1 shows the demographic characteristics of the respondents. The majority were in a relationship, employed, and had attended at least secondary school. Descriptive analysis (Table 2) revealed that the great majority of the patients presented a lifelong (73.3%) and generalized (90.6%) DE. Moreover, half of the patients declared that they had never reached orgasm, reporting that ejaculation was impossible. 81.4% of the sample was found to be non-alexithymic, with an overall average TAS-20 score of 45.46.\n\nTAS scores did not significantly correlate with the severity of DE measured by IELT (p=0.54). IELT -Intravaginal Ejaculation Latency Time.\n\n\nDiscussion\n\nProblems with “difficulty” in ejaculating may vary from a delay in the latency to ejaculation to a complete inability to ejaculate (anejaculation). According to the literature, we used the term Delayed Ejaculation (DE) to describe any and all ejaculatory disorders with either a long latency or absence of ejaculation3: our sample was composed of 50% of patients with delayed ejaculation and 50% that could not ejaculate. Furthermore, we found that 73.3% of the sample presented lifelong DE: this data is in strong contrast with the literature, where around 25% of patients suffer from lifelong DE, with the remainder reporting it as acquired7,27,28.\n\nAccording to our hypothesis, the results show that the alexithymia construct does not seem to be correlated with DE (only 9.3% of the patients could be classified as alexithymics), in contrast to other MSDs, where this condition was found in about 30% of patients, specifically it was found in 23% for suffering from HSDD, in 25% of them with PE and 34% of the ones declaring to have ED10–13.\n\nThere is some evidence supporting the inadequacy of alexithymia as an etiological factor in DE pathogenesis. From the first psychodynamic interpretations, DE has been associated with obsessive traits20,21, in which emotional control has an important role. This hyper-control configuration in men presenting DE has also been strongly confirmed in our psychotherapeutic experience for this disorder. In the clinical setting it is very common that patients with DE present a wide range of control behaviours, which affect not only their sexual experience, but also their daily activities and relations. This control is indiscriminately projected over emotions, behaviour and thoughts. The presence of this control presumes that the person recognizes his emotional responses, a trait that is typically absent in subjects with alexithymia. Indeed alexithymia and emotional control were previously found to be negatively associated22,23. This finding could explain the low rates of alexithymic traits in our sample.\n\nThe lack of difficulty in identifying and communicating emotions to others in patients with DE may also be connected to the greater social acceptance of DE than Premature Ejaculation and Erectile Dysfunction: the symptom does not seem to affect core male identity. From a somatopsychic perspective, the consequent low individual and relational distress arising from DE conditions could represent an important protective factor from creating or exacerbating high levels of alexithymia.\n\nMore research is still necessary to understand and treat DE. There is a strong need for more studies examining the precipitating and maintaining factors of this problem. Controlled and randomized outcome studies, using validated measures with sufficient follow-up periods, are necessary to determine the psychogenic variables connected with the onset and the maintenance of DE and the most efficacious methods of psychological treatment. The present studies are inadequate to make evidence-based recommendations about treatment1.\n\nThis paper can contribute to the understanding of DE by excluding one of the possible etiological factors that has been previously found to be important in the onset and the maintenance of other MSDs.\n\n\nConsent\n\nWritten informed consent was obtained from all study participants before receiving the research protocol. Each subject was advised of own right to withdraw at any time.", "appendix": "Author contributions\n\n\n\nRR, MG and SE conceived the study. CS designed the experiments. PMM, MG and SE carried out the research. RR and CS contributed to the design of experiments. MG and SE prepared the first draft of the manuscript. PMM, RR and CS contributed to the experimental design and preparation of the manuscript, supervising the study. 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 present study was funded by the Department of Clinical and Dynamic Psychology, Faculty of Medicine and Psychology, Sapienza University of Rome (Code: C26A12K7SS).\n\n\nReferences\n\nAlthof SE: Psychological interventions for delayed ejaculation/orgasm. Int J Impot Res. 2012; 24(4): 131–6. PubMed Abstract \| Publisher Full Text\n\nRowland D, McMahon CG, Abdo C, et al.: Disorders of orgasm and ejaculation in men. J Sex Med. 2010; 7(4 Pt 2): 1668–86. PubMed Abstract \| Publisher Full Text\n\nAmerican Psychiatric Association. Diagnostic and statistical manual of mental disorders, DSM-IV-TR. 4th edition, revised. American Psychiatric Association. Washington DC: 2000. Reference Source\n\nCruz N, Porst H: Ejaculatory and orgasmic disorders other then premature ejaculation. in Porst H, Reisman Y. (Eds), The ESSM Syllabus of Sexual Medicine, European Society for Sexual Medicine (ESSM) and Medix Publishers BV: 2012; 736–789.\n\nLindau ST, Schumm LP, Laumann EO, et al.: A study of sexuality and health among older adults in the United States. N Engl J Med. 2007; 357(8): 762–774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJannini EA, Lenzi A: Ejaculatory disorders: epidemiology and current approaches to definition, classification and subtyping. World J Urol. 2005; 23(2): 68–75. PubMed Abstract \| Publisher Full Text\n\nPerelman MA: Retarded Ejaculation. Curr Sex Health Rep. 2004; 1(3): 95–101. Publisher Full Text\n\nSimons JS, Carey MP: Prevalence of sexual dysfunctions: results from a decade of research. Arch Sex Behav. 2001; 30(2): 177–219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcMahon CG, Rowland D, Abdo C, et al.: Disorders of orgasm and ejaculation in men. in Montorsi F, Basson R, Adaikan G et al. (Eds). Sexual Medicine: Sexual Disorders in Men and Women Edition 2010: Paris, J Sex Med. 2010; 7(4 Pt 2): 1668–86. PubMed Abstract \| Publisher Full Text\n\nMadioni F, Mammana LA: Toronto Alexithymia Scale in outpatients with sexual disorders. Psychopathology. 2001; 34(2): 95–8. PubMed Abstract \| Publisher Full Text\n\nWise TN, Osborne C, Strand J, et al.: Alexithymia in patients attending a sexual disorders clinic. J Sex Marital Ther. 2002; 28(5): 445–50. PubMed Abstract \| Publisher Full Text\n\nMichetti PM, Rossi R, Bonanno D, et al.: Male sexuality and regulation of emotions: a study on the association between alexithymia and erectile dysfunction (ED). Int J Impot Res. 2006; 18(2): 170–4. PubMed Abstract \| Publisher Full Text\n\nSimonelli C, Bonanno D, Michetti PM, et al.: Premature ejaculation and dysregulation of emotions: research and clinical implication. Sexologies. 2008; 17(1): 18–23. Publisher Full Text\n\nReid RC, Carpenter BN, Spackman M, et al.: Alexithymia, emotional instability, and vulnerability to stress proneness in patients seeking help for hypersexual behavior. J Sex Marital Ther. 2008; 34(2): 133–149. PubMed Abstract \| Publisher Full Text\n\nNemiah JC, Sifneos PE: Affect and fantasy in patients with psychosomatic disorders. in Hill O (Ed). Modern trends in psychosomatic medicine (vol. 2). London: Butterworths; 1970; 26–43.\n\nNemiah JC, Freyberger H, Sifneos PE: Alexithymia: a view of the psychosomatic process. in Hill O (Ed). Modern trends in psychosomatic medicine (vol. 3). London: Butterworths; 1976; 430–9.\n\nTaylor GJ, Bagby RM, Parker JD: The alexithymia construct: a potential paradigm for psychosomatic medicine. Psychosomatics. 1991; 32(2): 153–164. PubMed Abstract \| Publisher Full Text\n\nTaylor GJ, Bagby RM, Ryan DP, et al.: Validation of the alexithymia construct: a measurement-based approach. Can J Psychiatry. 1990; 35(4): 290–7. PubMed Abstract\n\nTaylor GJ, Bagby RM, Parker JD: Disorders of Affect Regulation: Alexithymia in Medical and Psychiatric Illness. Cambridge: Cambridge University Press; 1997; 7(3): 240. Publisher Full Text\n\nAbraham G, Marrama P, Carani C, et al.: Psychoneuroendocrinologie du plasir. Simep s.a., Lyon-Villeurbanne: Paris; 1985.\n\nRowland DL, Keeney C, Slob AK: Sexual response in men with inhibited or retarded ejaculation. Int J Impot Res. 2004; 16(3): 270–4. PubMed Abstract \| Publisher Full Text\n\nVerissimo R, Mota-Cardoso R, Taylor G: Relationships between alexithymia, emotional control, and quality of life in patients with inflammatory bowel disease. Psychother Psychosom. 1998; 67(2): 75–80. PubMed Abstract \| Publisher Full Text\n\nVerissimo R: [Emotional intelligence: from alexithymia to emotional control]. Acta Med Port. 2003; 16(6): 407–11. PubMed Abstract\n\nWaldinger MD, Zwinderman AH, Olivier B, et al.: Proposal for a definition of lifelong premature ejaculation based on epidemiological stopwatch data. J Sex Med. 2005; 2(4): 498–507. PubMed Abstract \| Publisher Full Text\n\nBagby RM, Parker JD, Taylor GJ: The twenty-item Toronto Alexithymia Scale--I. Item selection and cross validation of the factor structure. J Psychosom Res. 1994; 38(1): 23–32. PubMed Abstract \| Publisher Full Text\n\nBressi C, Taylor G, Parker J, et al.: Cross validation of the factor structure of the 20-item Toronto Alexithymia Scale: an Italian multicentre study. J Psychosom Res. 1996; 41(6): 551–9. PubMed Abstract \| Publisher Full Text\n\nPerelman MA, Rowland DL: Retarded ejaculation. World J Urol. 2006; 24(6): 645–52. PubMed Abstract \| Publisher Full Text\n\nAbdel-Hamid IA, Saleh el-S: Primary lifelong delayed ejaculation: characteristics and response to bupropion. J Sex Med. 2011; 8(6): 1772–9. PubMed Abstract \| Publisher Full Text" }	[ { "id": "837", "date": "14 Mar 2013", "name": "Nelson Bennett", "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 seek to define the relationship of alexithymia to delayed orgasm. This is an excellent research question that needs to be answered in order to advance our knowledge psychosexual disorders. The study design and methods are appropriate. The study would be stronger if the results of the Spearman’s Rho correlation were included in the ‘Results' section. This would lend more support to the study conclusions. In reality, without the coefficient data, the data does support not the conclusion of the manuscript.", "responses": [ { "c_id": "400", "date": "20 Mar 2013", "name": "Stefano Eleuteri", "role": "Author Response", "response": "Dr. Bennett, these are the results of the Spearman's Rho correlation, to be included in the 'Results' section: ρs= - 0.086, p=0.54." } ] }, { "id": "898", "date": "17 Apr 2013", "name": "Salvatore Caruso", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research is very interesting because it focuses the attention on the relationship between emotional regulation and delayed ejaculation. The emerged results supply an appreciable suggestion for the clinical practice underlining how different male sexual disorders could be influenced by different emotional patterns. The self-report questionnaire the Authors used, beyond the total alexithymia score, provides three separate, yet conceptually related, facets of the alexithymia construct: difficulty identifying feelings and distinguishing them from the somatic sensations that accompany emotional arousal (F1), difficulty communicating feelings to other people (F2) and externally oriented thinking (F3).Despite the data revealed negative correlation between alexithymia and delayed ejaculation, the study would be stronger if the Authors could specify if the same negative trend emerges also correlating delayed ejaculation levels with the three sub-factors", "responses": [ { "c_id": "450", "date": "05 May 2013", "name": "Stefano Eleuteri", "role": "Author Response", "response": "Dr. Caruso, these are the results of the Spearman's Rho correlation for the 3 subfactors of TAS 20 , to be included in the 'Results' section: F1 (ρs= - 0.010, p=0.95), F2 (ρs= - 0.101, p=0.47), F3 (ρs= - 0.164, p=0.24)." } ] } ]	1	https://f1000research.com/articles/2-81
https://f1000research.com/articles/1-21/v1	02 Oct 12	{ "type": "Research Article", "title": "Terminal investment induced by a bacteriophage in a rhizosphere bacterium", "authors": [ "Timothée Poisot", "Thomas Bell", "Esteban Martinez", "Claire Gougat-Barbera", "Michael E Hochberg", "Thomas Bell", "Esteban Martinez", "Claire Gougat-Barbera", "Michael E Hochberg" ], "abstract": "Despite knowledge about microbial responses to abiotic stress, few studies have investigated stress responses to antagonistic species, such as competitors, predators and pathogens. While it is often assumed that interacting populations of bacteria and phage will coevolve resistance and exploitation strategies, an alternative is that individual bacteria tolerate or evade phage predation through inducible responses to phage presence. Using the microbial model Pseudomonas fluorescens SBW25 and its lytic DNA phage SBW25Φ2, we demonstrate the existence of an inducible response in the form of a transient increase in population growth rate, and found that the response was induced by phage binding. This response was accompanied by a decrease in bacterial cell size, which we propose to be an associated cost. We discuss these results in the context of bacterial ecology and phage-bacteria co-evolution.", "keywords": [ "stress / inducible response / Pseudomonas fluorescens / bacteriophages" ], "content": "Introduction\n\nPathogens are ubiquitous in natural communities1 and the antagonistic interactions they establish with their hosts are recognized as one of the main drivers of evolutionary diversification2,3. Hosts can reduce the impact of pathogens through three non-mutually exclusive processes4: (i) avoidance of either infected individuals, habitats where the pathogen is prevalent, or of the pathogen itself5, (ii) resistance to the actual infection process or post-infection immune defences6, and (iii) tolerance7. Research on these responses has generally focused on animal and plant models, but there is growing appreciation that microbes, particularly bacteria, can exhibit similar responses. For instance, bacteria can be selected for heightened levels of genetic resistance towards infection by pathogens8–10. On the other hand, although bacteria are known to display plastic responses to various types of environmental stresses11,12 and to competition13, it is unknown whether they can do so when faced with natural enemies such as bacteriophages.\n\nPlastic responses are an adaptive phenotypic change following an environmental stimulus, occurring without a concurrent change in the genotype14. They may involve behavioural, physiological or phenological changes15,16, and be triggered by direct or indirect contact with the stimulus17 or through communication with neighbouring organisms18. Phenotypic plasticity is considered to be a genetic adaptation to variable environments, but given the diversity of associated mechanisms and behaviours, it is not known to what extent different stimuli translate into different responses15,19.\n\nIndividual-level interactions between bacteria and phage may be conducive to induced responses. The first step of bacteriophage infection is the binding of phage proteins to bacterial surface proteins20, which then triggers conformational changes to both proteins21. Surface proteins used by the bacterium for signal transduction are known to be targets of bacteriophage adsorption22 and as such could trigger a response when bacteriophage binding is detected. Such a response would allow a bacterium to react to the pathogen and to eventually either evade or reduce the effects of the infection. Lytic phages are prime candidates for organisms against which bacteria may have evolved a stress response, because they typically interact with their host over short timescales, and death is inevitable once the phage has injected its DNA into a sensitive bacterial cell.\n\nIn addition, bacteriophages are widely distributed in the environment20 and interact with their hosts over relatively small spatial scales23 and throughout most of the year24,25. This could select for the expression of induced structural, physiological or behavioural responses to different enemies. Also, bacteria employ signalling pathways and have a known ability to communicate within populations26. Such pathways could induce and synchronise inducible responses before predators and pathogens are encountered, or at least before they have spread through the population, or before the point beyond which cell death is certain. All of these factors suggest that plastic stress responses to phage should be a common feature of bacterial cells and that such responses would have important repercussions for ecological and evolutionary interactions between phage and bacterial populations. Although molecular responses of bacteria to bacteriophages have been characterized27, the behavioral, ecological, and selective consequences of such responses are not known.\n\nHere we demonstrate that when confronted with phage, bacteria express transient increases in division rate at a cost to individual biomass accumulation28. Specifically, we employ the rhizosphere bacterium Pseudomonas fluorescens SBW2529 to investigate how its population growth rate is affected by exposure to inactivated populations of is lytic bacteriophage SBW25Φ230. We find that bacteria exposed to inactivated phage increase their fission rate nearly two-fold at 24 hours post-exposure. This is followed by a continual decrease in fission rate relative to the control. We also show that bacteria exposed to inactivated phage were smaller in size compared to controls. All of these effects were enhanced as the density of inactivated phage was increased. The results are consistent with a behavioural strategy that increases allocation to reproduction under stressful conditions (i.e., “terminal investment”). Terminal investment is well characterised for other host-parasite associations31, but to our knowledge has not previously been observed in bacteria and phage.\n\n\nResults\n\nBacteria exposed to UV-inactivated phage display a statistically significant higher growth rate over the first 24 hours post-exposure than non-phage controls (Kruskal-Wallis, df=3, P = 0.006; Figure 1). After this period, the estimated doubling time of exposed bacteria increased (i.e., their populations grew slower), and did so for the next 48 hours. This decrease in growth rate compared to controls is suggestive of a cost to the higher fission rate observed over the first 24 hours (Figure 1). During the fourth day post-exposure, control and treatment bacteria showed no significant differences in doubling time (KW, df=3, P > 0.05). That exposed bacteria returned to their ancestral growth rate suggests that the response over the first 24 hours was due to phenotypic plasticity and not selection on faster growing genotypes. There was a marginally significant effect on population growth for bacteria exposed to different phage concentrations (KW, df=2, P < 0.02), suggesting that the encounter rate between bacteria and phage is important in determining the population-level strength of the fission response.\n\nThis was measured for four consecutive days following four hours exposure. Bacteria exposed to phage grew significantly faster than controls over the first day, and then expressed an apparent cost in terms of smaller cell size that attenuated by the fourth day. Central points are the means of 12 replicates, and the bars are standard errors.\n\nWe hypothesized that faster doubling times would come at a cost to cell size, since cells would have less time to metabolize and convert absorbed nutrients into cell structure Twenty-four hours post-exposure, we found that phage-treated bacteria were two to three times smaller (as measured by mean cellular width) than the control (KW, df=3, P < 0.0001; Figure 2). This difference in size gradually decreased over the following 3 days, but in contrast to growth rate (Figure 1), bacteria did not attain their ancestral cell size by the end of the experiment (Figure 2). Analyses of the distribution of several flow cytometry profiles showed that a difference in cell shape is unlikely to explain this result (see data associated to this article). Finally, observations using a transmission electron microscope showed that the cells remained rod-shaped for all treatments.\n\nBacteria exposed to phage at different concentrations do not significantly differ in size. Points and bars are the same as in Figure 1.\n\nWe did not observe any difference in the impact of live phage on bacterial populations exposed to the different treatments (KW, df=2, P = 0.153), suggesting that the inducible response does not alter bacteria resistance to phage predation.\n\n\nDiscussion\n\nOur experiments reveal a previously unexplored behavioural response to bacteriophage predation: phage induce bacteria to reproduce earlier in their cell cycle. We hypothesize that this response increases the survival chances of progeny under natural conditions and demonstrate that this behaviour comes at a fitness cost of reduced size of daughter cells. Our experiments with UV-inactivated phage further demonstrate that this response is specifically due to phage binding.\n\nThese results support and extend both theoretical32 and empirical33,34 predictions that victims may lessen the fitness impact of their natural enemies through early reproduction, to cases where phenotypic responses are plastic and temporary. Increased allocation to reproduction in stressful environments–termed “fecundity compensation” or “terminal investment”31 – although never studied in bacteria-phage associations to our knowledge – has been extensively studied for other host-parasite (or organism - stressor) interactions. Terminal investment is characterized by increased reproductive rate or the earlier onset of reproduction, if the prospect of future reproduction is low35. Examples of such responses include faster host maturation36, increased oviposition rate37, and the modification of traits involved in the onset of reproduction38,39.\n\nPhenotypically plastic responses are important in that they allow individuals to cope with environmental change during their lifetimes40. As such, plasticity is expected to be favoured in variable environments when the costs of induction and phenotypic change compensate for probabilistic (expected) fitness loss41. Although it is difficult to generalize about constitutive costs of resistance across biological systems42,43, limited evidence suggests that genetically evolved, constitutive resistance in bacteria to their lytic phage could have costs of as much as 5–10% to relative fitness44.\n\nWe employed inactivated bacteriophages to evaluate how phage contact with the bacterial outer membrane mediates bacterial responses. Bacteria could be selected to exhibit an escape response in several, non-mutually exclusive ways. First, non-virulent phage may signal the presence of virulent phage in the local environment (i.e., the bacterium does not perish following initial phage contact). Senescent (inactive) phage are present in natural environments25, and many phages bind to outer membrane proteins without being infective (e.g. the bacterium is resistant;44). Moreover, it is possible that phage could detach if they sense the host to be unsuitable45. Second, when phage infect the bacterium there may be a ‘race’ between the time it takes a bacterial cell to divide (and potentially survive) and the point of no recovery associated with the maturation of phage progeny and bacterial cell lysis. Third, the response may be a consequence of lysogens competing with lytic phages for host exploitation; the latter could benefit from early host reproduction in the presence of lytic competitors. However, sequencing of the P. fluorescens SBW25 genome revealed a low abundance of prophage-like regions46.\n\nWe were not able to determine whether the bacteria or the phage benefit from faster bacterial reproduction, and the literature reports effects both of facilitation and decrease in host metabolism upon infection47. Previous theoretical work suggests that phage productivity increases in bacteria with short life-cycles48. This is supported by recent empirical study employing the same strain of P. fluorescens49. Assuming that the physiological mechanisms involved in fission rate increases are the same in the two experiments, this suggests that rapid multiplication is not adaptive for the bacterium. Upon exposure to phage, bacteria reproduce faster, but experience a persistent reduction in individual size. Smaller cells have less surface area, and assuming that the density of receptor proteins does not change with cell size, this suggests that they will have lower encounter rates with phage. One possibility is that cell division allows bacterial cells to concentrate phage in one of the daughter cells50,51, resulting in some progeny managing to escape the pathogen. Future studies should therefore focus on the possible adaptive nature of this response for both bacterium and phage.\n\n\nMethods\n\nAncestral Pseudomonas fluorescens SBW2529 were inoculated into 30 ml microcosms containing 6 mL of King’s B medium (KB), and allowed to grow under alternating rotational agitation (200 rpm for 1 minute every 30 minutes). Every 48 h following plating on solid agar, 10 CFU of the smooth morphotype were transferred into fresh KB medium. After 10 transfers, the culture was composed of smooth morphotypes only. We continued this selection procedure for another 10 transfers and then arbitrarily isolated a single CFU, which was used for all experiments described below. Experiments were conducted at 28°C in KB medium under constant rotational agitation (200 rpm).\n\nWe grew an arbitrarily selected clone of the ancestral phage SBW25Φ230 on an exponentially growing culture of fixed smooth P. fluorescens SBW25 in 3 mL of KB for 48 hours. This resulted in a culture containing approximately 108 phage per ml. The sample was then centrifuged for 3 minutes at 8000 rpm in a 1.5 ml Eppendorf tube, and the pellet discarded. Centrifugation was repeated three times to ensure all bacteria were removed (see Supplement Figure 1). Phages were then isolated by centrifuging the remaining supernatant for 8 minutes at 13000 rpm, and inoculating the pellet into fresh KB medium. The sample was thoroughly vortexed and exposed to UV light (Model 4.LC, Vilber Lourmat, Deutschland, 254 nm wavelength) at 5 cm distance for 4 hours. Extensive pilot studies demonstrated that this method was sufficient to kill all phage (see Supplement Figure 2).\n\nWe conducted a series of preliminary tests to verify how UV-inactivated phage affected bacterial hosts. First, observations under a transmission electron microscope showed that UV-inactivated phage were still intact and able to bind to their bacterial hosts. Second, we checked that bound UV-inactivated phage did not introduce phage DNA into the bacteria. This was done by inoculating 1 ml of UV-inactivated phage into 6 overnight bacterial cultures. Inactivated phage were allowed 4h to attach to the bacterial outer membrane. We separated phage and bacterial fractions by filtration using a 0.2 µm filter. We then conducted a full DNA extraction (WholeBlood NucleoSpin DNA extraction kit, Macherey-Nagel) of the filter. PCR was done using TPV1f (GATGTGAGAAAGCGATACACGG) and TPV1r (GAGAGAAGCGGGAGAGTGAA) sequences developed for this study, which selectively amplify a 550 bp fragment of the phage DNA and a 1200 bp fragment of the bacterial DNA (see Supplement Figure 1 for detailed protocols). We did not find any evidence that UV-inactivated phage was present in samples putatively containing bacteria only, thus confirming that the DNA of inactivated phage was not incorporated in the bacterial cell.\n\nWe conducted an experiment to understand how UV-inactivated phage affected bacterial behaviour. Fixed smooth SBW25 bacteria were first cultivated in 6 ml KB in 30 mL universal glass vials. 20 µL of exponentially growing bacteria (c 104 bacterial cells) were transferred into fresh KB medium with either no phage or UV-inactivated phage at ratios of 1:10, 1:2, and 1:1 (corresponding to approximately 106, 5×106, and 107 phage per ml), and then allowed to interact for 4 hours under alternating shaking (200 rpm for 1 minute every 30 minutes). Bacteria were then separated from bound phage by centrifuging (see above) and placed in fresh KB medium. 1% of each population was transferred every 24 hours into new KB medium. Each of the 4 treatments was replicated 6 times and arranged arbitrarily in a rack for incubation.\n\n\nMeasures\n\nBiomass doubling time (used as a proxy for population fitness) was measured in a Fluostar Optima spectrophotometer (28°C, constant agitation, 250 measures at 650 nm over 24 hours) each day, using the following formula:\n\n(1) Dt = [∆t ln(2)]/[ln(N) - ln(N0)]\n\nwhere N and N0 are the total biomasses (measured as optical density, OD) before and after the exponential growth phase, and ∆t is the duration of the exponential phase. Exponential phase was determined by conducting a series of windowed linear regressions over the full growth curve, and retaining the part of the curve with the largest slope (computer code given in suppl. materials part 3).\n\nIndividual cell size was measured by flow-cytometry using a FacsCantoII (BD BioSciences, San Jose, California, USA), and data (forward scatter) were analysed using the flowCore package52 in R 2.12.053. Each measure was performed on a sample of 2×105 cells without dyes.\n\nWe also estimated the sensitivity of the different treatments to live phage by measuring changes in bacterial populations. At each 24-hour transfer, 1% of the bacterial population was placed in 2 mL of fresh KB, and 20 µL of amplified phage (ca 108 viral particles) were added (a control without phage was conducted simultaneously). Bacteria CFUs were counted on solid agar after 48 hours of incubation to estimate population size.\n\nDue to non-normality of the data as assessed by a Shapiro test, we used a Kruskal-Wallis test to determine the significance of the between-treatments effects.", "appendix": "Author contributions\n\n\n\nTP, TB and MEH designed the research, TP and EM conducted the microbiology experiments, TP and CGB conducted the molecular biology experiments, TP, TB and MEH analyzed the results and wrote the paper, all authors contributed to revisions.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nTP is funded by a CNRS-Région Languedoc Roussillon doctoral grant. TB was funded by NERC. MEH is funded by ANR EvolStress (ANR-09-BLAN-099-01), by the Ec2Co “Cytrix” program, and the McDonnell Foundation (JSMF 220020294/SCS-Research Award).\n\n\nAcknowledgments\n\nWe are indebted to Cédric Mongellaz for his assistance with flow cytometry, and the Montpellier RIO Imaging center for providing technical support. We thank Michael Brockhurst and Angus Buckling for discussions.\n\n\nSupplementary material\n\nPCR cycle – 6 minutes at 95°C, then 30 cycles of 1 minute at 94°C, 1 minute at 55°C, 2 minutes at 72°C, then 10 minutes at 72°C.\n\nPCR buffer – 5 μL of buffer, 4 μL of primers at 10 pM/mL (for both TPV1f and TPV1r), 2 μL of dNTP at 5pM/mL, 1.5 μL of MgCl2 at 25mM, 5.35 μL of H20, 3 μL of sample DNA, 0.15 μL of TaqPol - conducted with a GoTaq FlexiDNA Polymerase M8301 kit from Promega.\n\nThe primers TPV1f and TPV1r yield a 1200 bp amplicon in the bacteria, and a 500 bp amplicon in phages. Our seperation method for bacteria and phage was complete, since only DNA of the intended organism was found in any given sample.\n\nWe verified the efficiency of the phage inactivation protocol by incubating the bacterial strain used for the main experiment with either live phage or phage exposed to UV for 2hrs or 4hrs. We measured the Malthusian fitness of 6 host populations near carrying capacity at low temperature (4°C, growth restrictive) over the course of 24hrs (the difference with the experiment presented in the main text is that inactivated phage were not removed over the course of this pilot study).\n\nAfter 4 hours of exposure to UV, we observed that phages do not introduce significant mortality in the bacterial population. Kruksal-Wallis test (df = 2, p = 0.02) reveals differences between treatments, with 0h and 2h being significantly different (t-test, df = 7, p < 10-5), 0h/4h being significantly different (t-test, df = 8, p < 10-5), and 2h and 4h being similar (t-test, p = 0.16) - all p-values were Bonferroni-corrected to account for multiple testing. Similarly, 2h and 4h are not significantly different from 0 (p-values of -5 -5 0.14 and 0.89 respectively, after correction).\n\ngivegrowth = function (y, x = c(1:length(y)), bw = 12)\n\n## y : optical density\n\n## x : times of the measures\n\n## bw : number of points to include in regression\n\n{\n\nlist.of.coeff- < NULL\n\nfor (i in 1:(length(x) - bw)) {\n\npart.x < - x[i:(i + bw)]\n\npart.y < - y[i:(i + bw)]\n\ncur.lm < - lm(part.y ~ part.x)$coeff[2]\n\nlist.of.coeff[i] < - cur.lm\n\n}\n\nresult < -max(list.of.coeff)\n\npos < -match(max(list.of.coeff), list.of.coeff)\n\ncoeff < -lm(y[pos:(pos + bw)] ~ x[pos:(pos + bw)])$coeff\n\nreturn(as.numeric(coeff[2]))\n\n}\n\n\nReferences\n\nLafferty KD, Dobson AP, Kuris AM: Parasites dominate food web links. Proc Natl Acad Sci U S A. 2006; 103(30): 11211–6. 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Reference Source" }	[ { "id": "457", "date": "08 Oct 2012", "name": "Britt Koskella", "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\nUnderstanding the response of bacterial populations to bacteriophage viruses is of central importance to predicting microbial dynamics. To do so requires knowledge about both the ecological and evolutionary responses of bacteria to phage in the environment.This includes possible changes in bacterial growth rate and cell size, as both have been shown to affect the rate of adsorption of phages by host cells (e.g. Hadas et al. 1997). In this paper, Poisot and coauthors investigate the inducible response of bacteria to phages by using UV-treated phages that are capable of binding to, but not infecting their host cells. They find that bacteria encountering UV-treated phages have a faster doubling time and smaller cell size than the control bacterial populations but that this response is short-lived and does not confer resistance to the phage.This is a very intriguing result that confirms work from studies using live phages (e.g. Gómez, P. and A. Buckling. 2011) and suggests that binding of phages, regardless of subsequent infection success, might play a key role in shaping bacterial population dynamics. The approach taken is a very nice way to look for inducible responses to phage and the results are, for the most part, very clear. I do, however, wonder about the independence of the results for doubling time (measured as optical density) and cell size. Surely the optical density measure is affected by the cell size? The authors state that “Analyses of the distribution of several flow cytometry profiles showed that a difference in cell shape is unlikely to explain this result (see data associated to this article)” Since this is an absolutely central result of the finding, I would find it very helpful if the authors actually presented and discussed this evidence. In fact, it was not clear to me which data were in support of this. Otherwise, I do not think that the results should be used to primarily suggest a phage-induced response of increased growth rate, with a cost of decreased cell size. Instead, perhaps it is a response of decreased cell size with a subsequent small change in doubling time? I imagine the authors have the analyses to rule out the latter possibility. Further to this, when the authors do look directly at colony forming units, rather than optical density, in their analyses of bacterial resistance to live phages they do not find a difference among the treatments. In this case, when bacteria were exposed to live phages, bacterial populations that had been exposed to inactivated phages grew to the same densities over 24 hours as those that had not been exposed to inactivated phages. I find this hard to interpret as it could suggest that a) the previous results were primarily indicative of a change in cell size, rather than growth rate, or even that b) the bacteria from the inactivated phage treatments do have a higher growth rate but were more susceptible to phages and thus had the same CFU. It would be helpful if the authors could discuss this result in more detail.I have a few additional points of clarification that I think would help readers fully understand the results.First, I wonder whether the authors could clarify their thoughts on the mechanism underlying the change to smaller cell size and/or increased doubling time of bacteria encountering inactivated phages. For example, could it be that small cell size is a response to altered numbers or activity of receptors on the bacterial cell surface? It seems that phage binding to receptors could alter their function and thus those bacterial cells with bound inactivated phages could be smaller due to decreased uptake of resources.Second, I think the finding that bacteria treated with inactivated phages show changes in growth rate and/or cell size but do not differ in terms of their resistance to live phages is quite interesting! I would be keen to know what the infection rates of the control and treated populations were, as this would help with interpretation of the result. It seems surprising that there is no change in resistance, as previous evidence suggests a strong correlation between cell size and growth rate with adsorption rate. Might this result give insight to the mechanism underlying the changes observed? The authors mention that smaller surface area would mean a lower encounter rate with phages, but this doesn’t seem to be the case when the cells are exposed to live phages.As a very minor point, I wonder whether the authors meant to say that bacteria were separated from unbound, rather than bound, phages in their methods section, as it is unclear how centrifugation would separate bacteria from bound phages.", "responses": [] }, { "id": "458", "date": "22 Oct 2012", "name": "Paul E. Turner", "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\n1. ls there a subpopulation of the UV inactivated phage that are destroyed in the process of creating them, such that addition to bacterial cultures might constitute addition of DNA that can be taken up through transformation ? If so, is it possible that this transformed DNA is being used as a nutrient by the bacteria? This might explain the slight increase in growth rate of phage-exposed bacteria in the experiments. There is precedent in other bacterial systems, but I do not know whether this provides an alternative explanation in the current study. The authors should take this possibility into account.2. It is unclear what constitutes the controls performed in this study. One choice of control would be to obtain the UV-inactivated phage, and then remove these particles via centrifugation. The particle-free supernatant would then be added to controls, so that all components (except phage presence) would be otherwise identical across treatments and controls. However, it is unclear whether this was the approach used, and therefore I am worried that the chosen control is insufficient for drawing proper conclusions in the work.Overall, the work seems very preliminary, and the data presented are not strongly supportive of the conclusions drawn.", "responses": [] } ]	1	https://f1000research.com/articles/1-21
https://f1000research.com/articles/2-127/v1	15 May 13	{ "type": "Research Article", "title": "Antisynchronous spike patterns in dye coupled GABAergic feedback neurons in the brain of the honeybee Apis mellifera", "authors": [ "Nora Vanessa de Camp" ], "abstract": "Intracellular recordings in GABAergic feedback neurons in the mushroom body of the honey bee brain revealed patterns of alternating neural activity. The recorded neurons have been identified via iontophoretic injection of Neurobiotin. The staining of groups of cells indicated dye coupling on the basis of gap junctions. The corresponding spike activity revealed spikes with different but overall comparatively low amplitudes (“spikelets”). The assumption of axo-axonal gap junctions would explain the staining of clusters of feedback neurons, alternating unit activity as well as spikelets with low amplitude. If a neuron in the electrically coupled network fires at a lower than maximal firing rate with respect to the refractory period, it will become more susceptible to laterally incoming spikes of neighbouring feedback neurons. In succession, the respective cell can be fully overtaken by neighbouring spike activity. On the one hand this proposed mechanism could lead to highly synchronous spike activity of a huge number of inhibitory synapses in the mushroom body calyces. On the other hand, the mechanism of laterally spreading activity could act like an amplifier. Additionally, the anatomical properties of Protocerebro Calycal Tract (PCT) feedback neurons could account for a reset function in order to allow for the changing activity profiles of the coupled cells. The laterally incoming potential would run in an antero- and retrograde direction. This could in principle lead to backpropagating inhibition of neighbouring PCT neurons and therefore a reset of one gating cycle. The proposed resulting oscillatory pattern of PCT activity has already been described in the literature and is confirmed by the physiological results, presented here.", "keywords": [ "Honeybee", "spike patterns", "GABA", "GAD", "protocerebro calycal tract", "spike patterns", "sychrony", "antisynchrony", "feedback neurons" ], "content": "Introduction\n\nSynchrony of spike trains in the brain has a major function for the strengthening of synaptic contacts and the formation of phenomena such as long term potentiation (LTP) (reviewed by Brown et al.1). Synchrony is one proposed mechanism for the formation of a neural syntax and inhibitory interneurons are believed to play an important role in the underlying cell assembly formation2. Gap junction-mediated electrical coupling has been shown to be a potential source of not only synchrony but also asynchrony3. Apart from mere synchrony effects, axo-axonal gap junctions in blowfly visual interneurons have been proposed to introduce a linear interpolation system between coupled neurons in order to visually extract the axis of rotation4. Gap junctions between pyramidal neurons in the rat hippocampus are suggested to contribute to sharp wave/ripple local field potentials5. In the human brain, gap junction-mediated quantum entanglement of microtubules has been proposed as a mechanism for very fast states of conscious perception6. The mushroom bodies intensively studied neuropiles of the insect brain involved with learning and memory7,8. The mushroom body in each brain hemisphere of the honeybee consists of about 170 000 intrinsic Kenyon cells9. The dentritic arborizations of these cells form the cup shaped calyces which are characterized as the main input region of the mushroom body10. Each calyx of a mushroom body is further subdivided into a lip, a collar and a basal ring neuropile11. The lip receives mainly olfactory input, the collar receives visual input and the basal ring receives mixed modality sensory input11. The Kenyon cell axons are bundled and form the two peduncles that connect the input region of the mushroom body, the two calyces, with the output regions, the alpha and beta lobes12 (Figure 1, Figure 2). The modality-specific topographic organization of Kenyon cells in the calycal regions is maintained throughout the mushroom body and causes a layered pattern of the lobes11,13. Feedback neurons are apparent in the mushroom bodies of a great variety of insect species (ant (Formica rufa, F. pratensis)14, grasshopper (Acheta domesticus)15,16, fly (Musca domestica)17 and bees (Apis mellifera)11,18).\n\nThe primary neurite runs from the lateral protocerebral lobe towards the lateral margin of the alpha lobe (alpha-exit) in one hemisphere of the bee brain. Reaching the alpha lobe, the PCT bifurcates, sending dendritic branches to both the alpha lobe and parallel axonal projections towards the calyces. The lateral and median calyces (lCal, mCal) are intensely innervated by PCT neurons, especially the lip region. The resulting cup shaped dendritic innervation areas are seen on top of the figure. Depth is color coded, the ventral and dorsal somata cluster (A3v, A3d) are superficial. Warm colors indicate a deep layer (ventral), whereas cool colors indicate a superficial (dorsal) position in the brain (blue, green, yellow, red), with blue most superficial and red for the deepest layers. Confocal scanning with a factor of 100x magnification.\n\nProtocerebro Calycal Tract (PCT) neurons are anatomically described feedback neurons in the honeybee brain, which receive inputs from the lobes and calycal regions19 and form spiny endings in the calyces which are interpreted as post-synapses10,20. It has been shown that PCT neurons do not exclusively connect corresponding regions between the calyces and lobes but also layers with different sensory modality in the mushroom body20. Due to the location of the somata in the lateral protocerebral lobe, a ventral (A3-v) and a dorsal (A3-d) cluster of feedback neurons can be distinguished13. The somata of the A3-v cluster are located in the anterior, ventral, lateral protocerebral lobe. The A3-d somata are located in the vicinity, slightly dorsal of the A3-v soma cluster13 (Figure 1). The primary neurites of both tracts run separately through the protocerebral lobe and cross half way to the alpha lobe13. Both tracts invade the alpha lobe at its lateral border, the A3-d approximately 20 µm more anterior than the A3-v13. The A3-v tract is identical to the anterior lateral protocerebral tract (a.l.p.t.), described by Mobbs11,13. The cells of the A3-v branch dichotomously in the protocerebral lobe, dorsolaterally to the alpha lobe. One branch continues in the PCT, the other branch benches ventrally and enters the alpha lobe at its lateral margin, the alpha exit point11 (Figure 1). The whole bee brain is shown in Figure 3B. The PCT neurons form layered innervation patterns in the alpha lobe. Some branches run posterior to the alpha lobe, in parallel to the Kenyon cells, towards the beta lobe (orange dendrites in Figure 2). The branch towards the beta lobe is more prominent for A3-d PCT neurons. The dendrites run via the alpha-lobe-to-beta-lobe-tract (a-b.t.13) and invade the beta lobe at its lateral margin at a depth of approximately 160 µm. The second branch of A3-v PCT neurons runs via the inner ring tract (i.r.t.11) towards the ipsilateral calyces. A3-v-PCTs innervate the entire calycal neuropile. Nevertheless, the highest density of dendritic branches of A3-v PCT neurons has been observed in a small region between the collar and basal ring (dorsal basal ring, d-br,13). The lip and the basal ring neuropiles are also common targets of A3-v innervation. In the lobe region postsynaptic spines have been described13.\n\nUnlike most PCT neurons stained in the examples beforehand, this PCT neuron has no dendrites in the calyces. The dendritic arborizations are restricted to the pedunculi (cyan and yellow). Additionally, the neuron has large dentritic trees in the alpha (magenta) and beta (orange) lobes.\n\nThe images are anatomical correlates for the recordings presented in Figure 4. Identification of PCT neurons was achieved by either full staining (panels A, C, E and F) or characteristic soma clusters (panels B and D). Panel B gives an overview of a whole bee brain, with right and left lateral protocerebral lobe (rlPL, llPL), subesophageal ganglion (SEG), alpha lobe (α-L) and calyces (Cal). The arrowheads indicate the PCT soma cluster. The arrows indicate dorsal (d) and lateral left (ll) direction. In panel A, the median and lateral Calyx (mCa, lCa) as well as the lip region (l) are indicated for one brain hemisphere. The inset in panel D corresponds to the colocalization between some intracellularly stained PCT somata of the ventral cluster (A3v). The anti-GABA staining is color coded in red, a colocalization with the intracellular staining (white) is indicated in green (200x magnification). The region of the nucleus does not contain any GABA staining and is therefore seen as small white spot (panel D, inset).\n\nThe majority of PCT neurons are GABAergic18 and in locusts, GABAergic interconnections have been shown between the lobes21. The occurrence of post- and presynaptic GABAergic terminals in the lobes has been confirmed in the bee brain22. For the A3-v PCT neurons with dendritic arborizations in the basal ring as well as pedunculus neuropile, claw-like dendritic endings have been observed. In contrast, the majority of dendritic endings for A3-v neurons that arborize in the basal ring reveal a round, bleb like character13. The physiological properties of PCT neurons are less uniform. The spontaneous firing rate is between 0 and 45 Hz20 or between 0 and 24 Hz and bursts occur frequently23. The spontaneous activity is not homogenous with respect to the spike distribution. The intracellular recorded spikes at the lateral margin of the alpha-lobe are lacking clear after-hyperpolarization \"spikelets\"23. Grünewald23 observed phasic-tonic excitatory responses (3.3–48 Hz above the respective spontaneous activity) to odorant stimulation, with a 500 ms phasic period after a latency of 75 to 128 ms. 22% of feedback neurons showed excitatory off-responses with a short duration but longer latency than on-responses23. Typically, their response to stimuli is excitatory with an extended latency between the stimulus and the increase in spike rate (25–70 ms for light stimuli, up to 400 ms for odorants)20. Nevertheless, inhibitory responses to odor stimuli have been observed20. PCT neurons respond to different sensory modalities, for example visual, olfactory or tactile stimulation but not movement20. The responses of PCT neurons to odor stimulation are more different between neurons for the same odor than in the same neuron for different odorants23. The relative spike rate is decreased after one trial odor conditioning in contrast to sensitisation23. 92% of the PCT neurons show excitatory responses after stimulation of the ipsilateral antenna with sugar solution23. Gronenberg20 observed after-effects, which are responses with latencies longer than 500 ms (up to 30 s after stimulation has been observed). Interestingly, Gronenberg20 described complex spike characteristics in an individual feedback neuron, which completely changed its response characteristics regarding latency, spike frequency and response type (inhibitory vs. excitatory) during the experiment.\n\nThis article aims to investigate the functional relationship between anatomy and physiology in PCT GABAergic feedback neurons in the mushroom body of the bee brain. The experimental results presented here point towards the occurrence of electric coupling between PCT neurons. This hypothesis will be discussed in the context of recent findings and earlier results.\n\n\nMaterials and methods\n\nNo specific permissions were required for the experiments with invertebrates (honey bees), owned by Freie Universität Berlin.\n\nForager honey bees (Apis mellifera, n=7) were caught from a winter indoor flight room with small glass vessels. The bees were chilled on ice and fixed in small plastic holders with a slit for the neck. The head was additionally stabilized towards the holder with a small piece of plastic sheet, which was adjusted with beeswax. The body was held in place with wax inside the recording chamber to prevent turning movement of the body against the head. After a recovery period of 30 minutes in a dark and moist chamber, the bees were fed to saturation with 1.25 M sucrose solution. At least one hour later the bees were operated on. The antennae were fixed with Eicosan (Sigma-Aldrich, Germany) to the head capsule. The mandibular muscles were cut and the mandibles fixed with Eicosan to the recording chamber in order to prevent movement artefacts. A trapezoid shaped cut at the clypeus between antennal joints and mandibles allowed for the removal of the oesophagus. The reference electrode was placed in the median ocellus. The head capsule was cut between the ocelli, compound eyes and antennae (without damaging the antennal nerves) in order to make the brain accessible. Trachea on the surface of the brain and mandibular glands were removed with a fine forceps. The bees’ abdomen was pressed with a piece of wax (Boxing wax sticks, Kerr Corporation, United States) in order to prevent pumping movements. A two component silicon sealant (Kwik-Sil, World Precision Instruments, Inc., United States) was used to build a fluid barrier and stabilization ring at the border of the window in the head capsule. After polymerization of the two component silicon ring, the single, fluid component A was used as surface medium on top of the brain tissue in order to prevent drying.\n\nBorosilicate glass capillaries with filament (Hilgenberg, Germany) were used for intracellular recordings (outer diameter 1 mm, length 75 mm, wall thickness 0.21 mm). A laser puller (P-2000, Sutter Instrument Co., United States) was used for capillary production. The resulting electrode resistance was between 100 and 300 Mohm, depending on the dye filling. The tip of the capillary was filled with a 5% solution of Neurobiotin Tracer (Vector Laboratories Inc., United States) in 0.2 M or 1 M K-Acetate. The electrode was inserted with a micromanipulator under visual control on the lateral margin of the alpha lobe towards the lateral protocerebral lobe neuropile at three o'clock for the left alpha lobe and nine o'clock for the right one. The onset of tissue contact to the glass capillary was indicated by the silencing of an acoustic signal (Intra767, Electrometer, World Precision Instruments, United States). From the offset of the acoustic signal for the touching point of the tissue surface, the electrode was slowly driven into the brain until a depth of 60 to 160 µm was reached. Analogue data were visualized with an oscilloscope (630, Voltcraft, United Kingdom). For data acquisition, the combination of Spike2 software and the corresponding analogue to digital converter (Micro 1401 MKII, Cambridge Electronic Design, CED) was used with a sampling rate of 20 000 Hz. Intracellular stainings (Neurobiotin) was achieved by iontophoresis with a depolarizing current of 3–4 nA for a duration of 10–20 minutes. The staining procedure was observed and interrupted in the case of unstable recordings.\n\nSpike2 was used to control a relais card (or8, bmcm, Germany) which was connected to different odor valves (The Lee Company, United States). A continuously presented air stream (1.5 m/s) without odorant was applied throughout the experiments. During the presentation of an odor the valve switched from the neutral airstream to an odorant laden airstream. The strength of the air stream was constant throughout the experiments. Each odorant supplier was filled with a small piece of filter paper, soaked with 2 µl of one odorant (orange oil, carnation oil, linalool, limonene, cineole, geraniol, hexanol, nonanol, hexanal, heptanal, octanal, 2-nonanone or 2-octanone). Blue, green, white and UV light were presented manually with an LED device. Both the olfactory and the visual stimulation took place for a duration of 4 s. Tactile stimulation was induced by a dry toothpick towards the antennae. For sugar stimulation the toothpick was soaked in 1.25 M sugar solution. The sugar stimulation consisted of one short touch towards the antennae.\n\nThe post iontophoresis circulation time in the living bee took at least 3 h or overnight in a dark and moist chamber with a temperature of 20°C. Afterwards the Kwik Sil was removed from the brain. The pre-fixed brain was dissected from the head capsule and thereafter carefully lifted from the silicon layer. The brain in the head capsule was pre-fixed in 4% paraformaldehyde (PFA, Electron Microscopy Science, United States) in PBS (NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 8 mM, KH2PO4: 1.4 mM, pH: 7.2) for 30 minutes. The brain was carefully removed from the head capsule, the silicon and surrounding tissues as well as the trachea. The brain was retained in PFA solution during this cleaning procedure. Each brain was separately fixed for an additional 4–6 h in 500 µl 4% PFA solution in a small glass vessel. The fixed brain was dehydrated in an alcohol series on a shaker at room temperature (at 50%, 70% , 90%, 99% and then 3 times at 100% ethanol and step wise back to 50% ethanol again, each step taking 10 minutes). Subsequently the brain was washed in PBS on a shaker for 10 minutes twice at room temperature and incubated in 1% TX (TritonX, Sigma, Germany) in PBS for 2 h on a shaker at room temperature. The Streptavidin conjugation (Cy5, dianova, Germany) took place over night but not longer than 20 h at 4°C on a shaker in a Streptavidin Cy5 solution 1:1000 in PBS plus 1 µl sodium acid (saturated stock solution). For confocal microscopy 1 µl of a 5% Lucifer Yellow (Invitrogen, Germany) solution in Aqua Dest (custom made) was added to achieve a background staining of the brain neuropile. From this step on the preparation was light sensitive and needed to be shielded from bleaching. Unbound Streptavidin was removed from the brain via 6 washing cycles in PBS at room temperature on a shaker (brief washing for 10 minutes, 20 minutes, two times 30 minutes and one time 60 minutes). For dehydration, an ascending alcohol series was used (50%, 70%, 90%, 99%, and twice at 100%, each step for 10 minutes on a shaker at room temperature). Afterwards the brain was stored and cleared (tissue transparency was required for confocal microscopy) either in methylsalicylate (Roth, Germany) or in a 2:1 mixture of benzylbenzoate and benzyl alcohol (Sigma, Germany). Prior to storage in methylsalicylate, the brains were washed twice for 15 minutes at room temperature on a shaker or at least twice for 30 minutes in the benzylbenzoate benzylalcohol mixture. In order to apply an antibody staining in addition to the intracellular staining, the brains were washed in a descending ethanol series, to bring them back into PBS (as described before, twice at 100%, then 99%, 90%, 70%, and 50% ethanol and PBS, each step for 10 minutes on a shaker at room temperature). The brains were dried on a piece of absorbent tissue and subsequently embedded in agarose gel (0.3 g agarose in 5 ml PBS). A Leica vibratome (VT 1000 S, Germany) was used to achieve vertical sections of 50, 80 or 100 µm thickness. The slices were washed 3 times for 30 minutes in 1% TX on a shaker at room temperature. Unspecific binding sites were blocked with 10% Normal Goat Serum (NGS, Invitrogen, Germany) in PBS 1% TX for 1 h on a shaker at room temperature. The incubation in the primary antibody (rat anti-rabbit GABA, Sigma A2052 Germany, 1:400 in PBS 1% TX plus sodium acid and NGS) took 6 days at 4°C on a shaker. Subsequently the brain was washed 6×30 minutes in PBS 1% TX and thereafter incubated in the secondary antibody (goat anti-rabbit Cy5, Cy3 or Cy2, 1:200 in PBS 1% TX plus 1 µl sodium acid) for 3 days on a shaker at 4°C. The slices were washed for 30 minutes in PBS 1% TX and 5 times for 30 minutes in PBS at room temperature on a shaker. The slices were transferred in 60% glycerine in PBS and finally in 80% glycerine in PBS for confocal microscopy.\n\nAdditionally, antibody staining against glutamic acid decarboxylase (GAD, Millipore, Chemikon, United States, AB 1511, GAD67GAD65) were conducted. The procedure was the same as described for GABA except bovine serum albumin (BSA, Sigma, Germany) was used for blocking instead of NGS.\n\nFor confocal microscopy (Leica TCS, Germany), the whole mounts were cleared in methylsalicylate or a mixture of benzyl alcohol and benzyl benzoate, as described previously and the slices in 80% glycerine in PBS. The whole mounts were embedded on metal microscope slides with a hole (diameter approximately 1 cm) in the middle. A cover slip was fixed on top of the hole with super glue on the metal surface. The resulting cavity was big enough to allow for embedding of the whole bee brain.\n\nThree lasers were used for the excitation of fluorescent dyes. Cy5 was excited with an He/Ne laser at 633 nm wavelength, Cy3 as well as micro Ruby (Invitrogen, Germany) and tetramethylrodamine (TRITC) were excited with the He/Gre laser at 543 nm, Cy2 and Lucifer yellow were excited with the Ar/Kr Laser at 488 nm wavelength. If one brain was excited with 633 nm as well as 543 nm, sequential scanning was used in order to prevent cross excitation and bleaching. The PMT (photo multiplier tubes) was chosen between 400 V and 500 V, and for intensity compensation in whole mounts, the PMT setting \"linear by gain\" was chosen. The offset was between 0 and -1 (background subtraction). For the scanning procedure, the beam expander 6, the format 1024×1024 and the frame average 2–3 was used. In the case of the 20× magnification water objective scanning steps of 1.5 to 2 µm were used, with 10× magnification water and air objectives up to 2.5 µm steps were used. Slices were scanned with 40× oil or glycerine objectives.\n\nThe gray scale of the confocal slices was reconstructed with the skeleton tree tool (Amira). Two additional algorithms allowed for a thickness adjustment and straightening on the basis of the grey scale data of the scans and the skeleton tree. Background neuropile staining with Lucifer Yellow or the (less clear) Streptavidin background were used to reconstruct the neuropiles. In accordance with the standard atlas of the honeybee brain24 the neuropiles were subdivided as follows: mushroom body with alpha lobe, beta lobe, pedunculus, medial and lateral calyx. The calyces were subdivided into lip, collar and basal ring. Additionally the protocerebrum with the central body was labelled. During the affine registration the labelled brain was coarsely adjusted to the standard brain. An affine transformation of the skeleton tree was necessary to adjust the neuron to the stretched neuropiles. During the last step, the elastic registration and transformation, a fine tuning to the standard brain was achieved.\n\nThis time consuming procedure was used for qualitatively excellent single neuron staining. In other cases the neurons have been visualized with less elaborate Amira tools, such as projection views, volrens, ortho slices or isosurfaces (Amira version 5.2).\n\nThe raw intracellular recording channels were high pass filtered (high pass 220 Hz, transition gap 150 Hz, in most cases) with the program digital filters in Spike2 (CED, United Kingdom) in order to eliminate baseline fluctuations. This procedure was necessary in order to set a continuous threshold for the spike sorting (Spike2, CED, United Kingdom). The spike sorting result was confirmed by principal component analysis (Figure 4). Only separated scatterplots in the 3D space have been used as different units. Nevertheless, errors due to the sorting procedure cannot be excluded. Taking this inevitably occurring inaccuracy into account, the results of the spike sorting were called ‘units’. For each set of units, extracted from one recording, the coefficient of correlation (Spearman’s Rho, MATLAB 2010, Simulink) was calculated. Since long term effects have been described for PCT neurons20, unbinned spike times and binned spiketimes (1 s bins) were calculated and used for the correlation measurement. The correlation measurement of original (unbinned) spike times of different unit pairs was done in order to measure simultaneous spiking on the millisecond timescale. On the other hand, the comparison on the level of 1 s bins of unit spike times includes the correlation of spike trains (bursts). Only in the case of antisynchrony of spike trains on the longer timescale (1 s bins) can the effect be seen by eye (Figure 5). Antisynchrony on the short timescale (unbinned spike times) can also be achieved by the alternating occurrence of two units (unit1, unit2, unit1, unit2...etc.). A negative coefficient of correlation on the short timescale is the rule, rather than the exception. Contrarily, synchrony on the short time scale is a strong hint for the measurement of potentials of different cells. Potentials in a single (non-electrically coupled) cell should theoretically have a low coefficient of correlation (low synchrony) for original spike times on the short timescale (original spike times) due to the refractory period. In order to achieve maximal transparency, both values were calculated and summarized in Table 1.\n\nThe result (the example here is from bee n80108 with 5 units) was used in order to estimate the quality of the sorting result. Only spatially (3D) separated clusters have been recognized as different units.\n\nThe plus and minus signs below panels indicate the on- and offset of stimulations. In panel G the stimuli have been applied manually and are encoded as red stripes in the first row. Spike frequency (Hz, 1 s bins) is color coded, as indicated in the color bar on the right side of each panel. Each panel contains 2 to 5 units. The following stimuli were applied: A [4× linalool (Lol), 2× octanone (8one), 2× heptanal (7al) and 1× hexanol (6ol)]; B [2× ipsiantennal sugar water (sipsi), contraantennal sugar water (scontra), tactile ipsiantennal stimulation (tipsi), 5× cineole (Col), 4× 8one, 2× 8one plus white light, white light, green light]; C [3× Lol and 2× Lol plus simultaneous presentation of white light]; D [2× sipsi, 1× scontra, 1× tipsi, 5× Col stimulation, 4× 8one stimulation, 2× 8one plus white light stimulation, 1× white light and 1× green light stimulation]; E [sipsi, 2× 6ol, 6ol plus green light, green light, white light, 6ol, nonanone (9one)]; F [5× Lol]; G [2× blowing, 7× Col, 4× orange, 1× Lol, 6× geraniole, 3× Lol, 2× limonene, 3× 1-nonanol, 7× clove, 2× orange, 1× blowing]. The stimulation events are shown in more detail in the peri stimulus time histograms in Figure 6, Figure 7, Figure 9 and Figure 10.\n\nN: experimental series (bees), s: short timescale, l: long timescale, u: unit, SR: Spearman’s Rho, NS: not statistically significant.\n\n\nResults\n\nDye coupling of the recorded PCT neurons occurred in every successful Neurobiotin injection (Figure 3A–F, for examples) except one anatomically exceptional PCT neuron, with no arborizations in the calycal region of the mushroom body neuropile (Figure 2). Furthermore, the recordings of such dye coupled neurons revealed different spike shapes and amplitudes, which were sorted with a spike sorting algorithm (Spike2). In comparison to the single stained PCT neuron, mentioned beforehand, the spike amplitudes of dye-coupled PCT neurons were rather low (about 10 mV, Figure 6, Figure 7) and therefore called \"spikelets\". In order to account for possible errors due to the spike sorting procedure, the resulting sorted spikes are called units. The sorting results have been checked by a principal component analysis (example in Figure 4). Only anatomically identified PCT staining, without additionally stained non-PCT neurons, have been used for further analysis. Some neurons are completely stained (Figure 3A, C, E and F), others are identified by the characteristic soma clusters (Figure 3B and D). Multiple spike or spikelet shapes as well as antisynchronous activity patterns of these units were visible in all recorded, dye coupled units. In Figure 5, the spike sorting results of single electrode intracellular recordings are visualized as color maps for 1 s bins of spike times.\n\nThe single traces 1–3 correspond to linalool stimulations, traces 4 and 5 to linalool and simultaneous white light. Neither unit1 (panel A) nor unit2 (panel B) show clear alterations in the spike rate due to the stimulation. To extract the units from the single electrode recordings, the raw data (panel C, black trace) have been high pass filtered (panel C, grey trace, high pass: 100 Hz, transition gap: 50). Spikelets of unit1 are shown in blue, unit2 spikelets in green.\n\nUnit1 (panel A), unit2 (panel B) and unit5 (panel E) show an increase in the firing rate during stimulation with linalool. Unit3 firing rate is decreased during linalool application (panel C). Unit4 has only very short activity periods (panel D). Interestingly, the unit1 response, with a phasic and a tonic component, decreases from trials 1–5 whereas the unit5 response, with an almost pure phasic activity, increases from trials 1–5. Unit2 phasic-tonic activity is oscillating, but almost constant over trials. Unit3 activity increases from trials 1–5, but no spikes occur during the first 2 seconds of linalool stimulation. The raw data of the single electrode intracellular recording (panel F, black trace) have been high pass filtered (panel F, grey trace, high pass: 100 Hz, transition gap: 50). The sorting result is shown in the colored channel.\n\nThe coefficient of correlation was calculated in order to estimate the degree of synchrony for pairs of units on two time scales (Table 1). Direct spike times (short time scale, labelled s) and 1 s binned spike occurrences (longer time scale, labelled l) were compared. The pairwise unit activity results are summarized in Table 1, including the coefficient of correlation (Spearman’s Rho, labelled SR). Antisynchrony (negative coefficient of correlation) on both timescales occurred the most often (eight unit pairs). Seven unit pairs are synchronous (positive coefficient of correlation) on the short timescale (original spike times) but antisynchronous on the larger timescale (1 s bins). Antisynchrony on the short but synchrony on the large time scale occurred in six cases. In eight cases, antisynchronous spiking for the short or long time scale is observed without a trend on the other timescale. In two cases, synchronous unit pairs were observed on both the short and the longer timescale. In one case synchrony on the longer time scale occurred with no tendency for the short time scale.\n\nMost PCT neurons can be colocalized with GABA and GAD67GAD65 immunostaining, as seen in Figure 8 (at least one intracellulary labelled cell was colocalized within each immunostaining in four out of five cases. The five dye-coupled PCT intracellular recordings revealed 23 units). In Figures 8A–G, GAD67GAD65 and GABA stainings were done simultaneously. The comparison reveals similar colocalization patterns with the intracellular staining in the somata (Figure 8A–D) as well as axonal projections (Figure 8F and G). A colocalization for GABA but not GAD67GAD65 is seen in Figure 8E. Figure 8H–L shows GABA staining of the recorded PCT neurons at different cellular locations. Some somata show a colocalization with anti-GABA antibodies (Figure 8H), others do not colocalize even though GABA stained somata are seen in near vicinity (Figure 8K). The same is true for other regions of the neuron, even in the same slice. As seen in Figure 8I and J, axonal segments, indicated by a grey arrowhead in Figure 8I, and the alpha exit point are clearly colocalized with GABA (colocalization in green), whereas dendritic regions in the alpha-lobe exhibit less colocalized regions. The white arrows in panel I point towards tiny green patches of colocalization.\n\nA colocalization between the GABA (red) and GAD67GAD65 (blue) staining with the intracellularly injected dye is indicated in green and magenta, respectively. Somata as well as axonal projections (arrows in panels E and F) in the intracellularly labelled Protocerebro Calycal Tract (PCT) neurons in the right brain hemisphere of bee n90122b are colocalized with GABA and GAD67GAD65 (panels A–G). The GABA colocalization is not homogenously distributed (panels H–L), even in the same slice (panels I and J, small patches of colocalization are highlighted by arrows). The regions for somata and axonal projections of PCT neurons within the bee brain are shown in Figure 1 and Figure 11.\n\nMultimodality, as has been previously described for PCT neurons20, can be confirmed by the data, presented here. The two units in Figure 9 are responding antagonistically towards the presentation of linalool and octanone. In the case of heptanal presentation, both units decrease their spike rate. Unit2 in panel B has a generally higher signal to noise ratio in comparison to unit1 in panel A with high spontaneous activity. Not all recorded PCT neurons responded to odor stimulation, as shown in Figure 6A and B. Unit2 in panel B increases its spontaneous spike rate continuously over stimulation trials. The original recording is shown in panel C (black trace). The high pass filtered trace is indicated in grey as well as the sorted unit1 in blue and unit2 in green.\n\nPanel A shows the frequency and single traces for unit1. In panel B the same is shown for unit2. Unit2 (B) has a lower baseline firing rate and reaches higher peak spike rates due to stimulation. The order of stimulation (trace 1 to 9 in B, trace 1 to 8 in A, because unit1 was completely silent during the last (hexanol) stimulation) is as follows: 4× linalool, 2× octanone, 2× heptanal, 1× hexanol. The response to odorants is complementary in the two recorded feedback neuron units. A decrease in the firing rate of unit2 to linalool stimulation (the first 4 trials) is accompanied by an increase in unit1. Interestingly both units change their firing rate in a similar way in response to octanone and hexanol stimulation. Oscillatory response characteristics are seen in panel B, unit2, second trace, after linalool presentation. The oscillatory pattern is characterized by undulatory spiking activity after one sensory stimulation (raster plots).\n\nFive units were extracted via spike sorting in the right brain hemisphere in bee n90127 (Figure 7F). Unit1 (Figure 7A), unit2 (Figure 7B) and unit5 (Figure 7E) increased their firing rate during the odor stimulation (5× linalool). The unit1 response, with a phasic and a tonic component, decreases from trial 1–5 (Figure 7A). Unit2 also had a phasic and tonic component but the response strength was more or less constant along trials with an additional peak in spike rate for the offset of stimulation (Figure 7B). Unit5’s response increased in strength from trial 1–5 and is purely phasic (Figure 7E). The response of unit3 to linalool stimulation is seen as a decrease in spike rate. This becomes more pronounced from trial 1–5, because the spontaneous spike rate before and after the stimulation increases (Figure 7C). Unit4 did not respond to the odor presentations (Figure 7D). Unit4 activity decreases after the onset of odor presentations and is restricted to periods where the other units show decreased spiking activity (Figure 7D). Figure 10 gives an example of the occurrence of multisensory characteristics in some PCT neurons (Figure10B). In contrast, unit1 in honeybee n90122b only responded to the first ipsiantennal sugar stimulation (Figure10B). Spontaneous activity as well as response to stimuli increased in unit1 but decreased in unit2.\n\nTraces 1–18 are the following stimuli: 2× ipsiantennal sugar water ×, contraantennal sugar water, tactile ipsiantennal stimulation, 5× cineole ×, 4× octanone ×, 2× octanone plus white light ×, white light, green light. Unit1 (A) is responding to the first ipsiantennal stimulation with sugar water. Subsequent unit1 spikes are very sparse. Unit2 (B) spike rate increases over trials, responding to light, sugar, odor and tactile stimuli. Oscillatory activity patterns are visible as well as variable latencies.\n\nIn order to estimate the spike sorting accuracy, a single stained PCT neuron, which probably had no gap junction connections, was analyzed in the same way as the other recordings. This PCT neuron did not show any dendritic arborizations in the calyces of the mushroom body. Instead, the pedunculus was innervated (color coded in yellow and cyan for the lateral and median ipsilateral mushroom body, respectively; Figure 2). The spike sorting revealed two well separated units (Figure 11A). The blue unit occurs only at the very beginning of the recording, due to tickle induced noise, which is confirmed by a principal component analysis (Figure 11B). The uniformly shaped spikes are illustrated in Figure 11C.\n\nThe spike sorting reveals 1 unit (green, top trace, panel A). The blue unit is due to noise (the bottom trace is the original recording). Tickle events for cell penetration are seen as large artefacts at the left border of panel A. In panel B, the principal component analysis for the spike sorting is shown. In panel C, original (black, bottom trace) and sorted spikes (green, top trace) are shown in order to estimate the similarity of spikes. The spikes occur regularly, despite some burst events, as seen on the right side of panel C. The spike amplitude is much higher in comparison to the spikelets in the dye coupled PCT neurons (for example Figure 7 and Figure 8).\n\n\nDiscussion\n\nThis study investigated the occurrence of dye-coupling and synchrony effects of unit activity in PCT neurons.\n\nSince single PCT neuron intracellular markings are sparse, especially with Neurobiotin (286 mw) for Neurobiotin25, it can be assumed, that electrical coupling via gap junctions occurs in PCT feedback neurons. These observations have shown that single staining is more easily achieved with Lucifer Yellow, a bigger molecule than Neurobiotin (457 mw for Lucifer Yellow25). In the following section, I will explain why gap junctions between subpopulations of PCT neurons are a parsimonious explanation for the phenomena observed in this investigation in contrast to the assumption of staining artefacts, which cannot be fully excluded. Cytoplasmic bridging seems to be unlikely, because only stable recordings have been used for analysis. Additionally, experiments with multiple neural staining other than PCT neurons have been excluded from analysis.\n\nLet us assume that neighbouring PCT neurons are firing at their maximal rate. This might be a rare scenario but stable in terms of spike propagation. The respective refractory period of the neuron makes it impossible to propagate additional axo-axonal gap junction mediated potentials from the neighbouring cells. But what happens, if the firing rate of one PCT neuron is subthreshold with respect to the refractory period? Incoming signals from neighbouring PCT neurons, which are electrically coupled in their axonal regions, become more likely in this situation, because the cell, which is firing sparsely, is now susceptible to the propagation of laterally incoming potentials. Furthermore, the gap junction potentials are spreading bidirectionally through the invaded axon. In contrast, the spike propagation in the high frequency \"potential source\"-cell is unidirectional due to the refractory period. Spike coupling via gap junctions has some important implications for spike propagation including ultra fast information transfer26. Despite the observed asynchrony on the level of competing electrically coupled feedback neurons, the axo-axonal gap junctions lead to a high level of synchrony regarding the postsynaptic sites. But without feedback inhibition, this situation might be a dead end. Since the activity pattern of dye coupled ensembles consisted of fluctuating synchrony patterns on a relatively short timescale during an intracellular recording (up to 10 minutes), it is likely that a second assumption is realized in PCT neurons, that the gap junction mediated potentials are not only forward propagated (towards the calycal region) but also retrograde (towards the lobes). If the gap junction mediated, bidirectionally spreading spikes outperform the orthodromic spike initiation27 of the respective invaded PCT neuron, a retrograde signal becomes possible. Since the observed activity in the PCT is oscillatory20, the backward-feedback signal to neighbouring, electrically coupled neurons or their input sites in the lobes can be proposed as feedback inhibition of the invading cell and therefore a break against long lasting synchronization of coupled PCT neurons.\n\nThis mechanism is a realistic scenario since GABAergic cell interconnections have been observed in the mushroom body lobes21. The precise control of synchronized spike propagation in PCT neurons might be an important step in learning and memory formation in the honeybee brain. It has been shown that synchrony-dependent processes occur in the bee brain28. Axo-axonal gap junctions are involved in the initiation of sharp wave/ripple events in the hippocampus26,29. Ripple events in turn have been shown to play an important role in the acquisition of memory in rodents30. It might be possible, that PCT neurons are a source of ripple-like local field potentials in insects. Another effect of axo-axonal gap junctions between feedback neurons might be an amplification effect regarding the output sites in the calycal region of the mushroom body, as it has been proposed to be involved in ectopic spike initiation (see Bucher and Goaillard for review31). This form of electrical connection between feedback neurons could be an alternative solution for ultrafast26, high frequency and broad band transmission to other cells without the requirement for giant fibres (as has been observed in locusts32).\n\nThe hypothesis of gap junctions between PCT neurons is further supported by the single stained exceptional PCT neuron without arborizations in the calycal region of the mushroom body, but instead has dendritic trees in the ipsilateral pedunculi (Figure 2). The spike sorting results in one unit (the second unit is clearly related to noise at the beginning of the recording, see Figure 11A). The gap junctions provide a mechanism for the coupling of subpopulations of neurons, which might be a source of synchrony for the information transferred to postsynaptic sites. Additionally, it has been shown that gap junctions can be uncoupled by rising intracellular calcium concentrations and pH changes33,34. These findings implicate that gap junction-coupled neurons cannot simply be regarded as functional units but rather that gap junctions provide complex mechanisms for neural plasticity.\n\nIn conclusion, the assumption of gap junctions between dye coupled feedback neurons in the mushroom body of bees provides an explanation for several features of these neurons, including oscillatory activity and multisensory response characteristics (Figure 6, Figure 7). The exciting question that arises is whether such oscillatory gap junction-mediated subnetworks can produce ripple-like events in insects. This is especially interesting since harp wave/ripples are associated with replay mediated consolidation of memory loads in rodents30. PCT neurons are potentially suited for the reactivation of sensory information, because they are part of complex synaptic aggregations in the mushroom body input regions, called microglomeruli19. It has been shown that Picrotoxin (a GABA A receptor blocker) injection in the honeybee brain led to reduced odor discrimination while learning ability per se was not affected. Furthermore, Picrotoxin application led to desynchronization between local field potential oscillations in the antennal lobe and the firing pattern of projection neurons35. The exact mechanisms of the output level of PCT neurons remain elusive, because they depend on the targeting of PCT neuron synapses and whether these synapses are really purely GABAergic. In order to further determine the occurrence of gap junctions in mushroom body calyces it might be interesting to perform dual intracellular recordings in the near vicinity as has been done in the rat hippocampus36, simultaneous injection of Neurobiotin and a second dye incapable of passing the gap junctions and both recordings and staining after the blockade of gap junctions with carbenoxolone36 or comparative drugs.", "appendix": "Competing interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBrown TH, Chapman PF, Kairiss EW, et al:Long-term synaptic potentiation. Science. (1988); 242: 724–8.\n\nBuzsáki G: Neural Syntax: Cell Assemblies, Synapsembles, and Readers. Neuron. 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(1978); 75: 4577–4581.\n\nSkeberdis VA, Rimkute L, Skeberdyte A, et al:pH-dependent modulation of connexin-based gap junctional uncouplers. J Physiol. (2011); 589: 3495–4506.\n\nStopfer M, Bhagavan S, Smith BH, et al:Impaired odour discrimination on desynchronization of odour-encoding neural assemblies. Nature. (1997); 390: 70–74.\n\nMercer A, Bannister AP, Thomson AM: Electrical coupling between pyramidal cells in adult cortical regions. Brain Cell Biol. (2006); 35: 13–27." }	[ { "id": "1058", "date": "12 Jul 2013", "name": "Yevgenij Yanovsky", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 data presented are of general and particular interests. All experiments have been properly done, and data were carefully analysed. However, the text in its present condition is difficult for comprehension and should be rearranged for better understanding.Title is appropriate.The Abstract should be rearranged to exclude discussion-like elements.Introduction is too long and could be shortened at least to one-half of the present size. Everything concerning figures should be excluded from Introduction and moved to Results.Minor point: Description of Fig. 3 occurred in text before that for Fig. 2.Materials and methods. This part of manuscript is also too long. It might be helpful to describe here only main or originally built methods giving commonly used as references. Again, part of the text concerning figures should be put into Results.Results. Experimental results would be better put in a more straight forward way, in support of the main line of study: morphological and immunostaining data showing die-coupling for GABA-ergic PCT neurons; spontaneous and evoked activity of these cells in aspect of de-/synchronization as an evidence for gap junction mechanisms in the network. Pharmacological tests for gap junction connectivity would be very desirable.Minor point: Fig. 2 could be combined with Fig. 11.Minor point: Conclusions are justified on the basis of results.", "responses": [] } ]	1	https://f1000research.com/articles/2-127
https://f1000research.com/articles/2-109/v1	15 Apr 13	{ "type": "Research Article", "title": "Global identification of genes and pathways regulated by Akt during activation of T helper cells", "authors": [ "Jing Cheng", "Lawrence P Kane", "Jing Cheng" ], "abstract": "We previously demonstrated that Akt differentially modulated a subset of NF-kB target genes during T cell activation. In the current study, we further explored the broader effects of Akt inhibition on T cell gene induction. Global microarray analysis was used to characterize T helper cell transcriptional responses following antigen receptor stimulation in the absence or presence of Akti1/2 (an allosteric inhibitor which targets Akt1 and Akt2), to identify novel targets dependent upon Akt and obtain a more comprehensive view of Akt-sensitive genes in T helper cells. Pathway analysis of microarray data from a CD4+ T cell line revealed effects on gene networks involving ribosomal and T cell receptor signaling pathways associated with Akti1/2 treatment. Using real-time PCR analysis, we validated differential regulation of several genes in these pathways, including Ier3, Il13, Klf6, Egr1, Ccl1 and Ccl4, among others. Additionally, transcription factor target gene (TFactS) analysis revealed that NF-kB and Myc were the most significantly enriched transcription factors among Akt-dependent genes after T cell receptor and CD28 stimulation. Akt activation elicited increases in the enrichment of NF-kB- and Myc-targeted genes. The present study has identified a diverse set of genes, and possible mechanisms for their regulation, that are dependent on Akt during T cell activation.", "keywords": [ "T helper cell", "global microarray analysis", "allosteric inhibitor", "TFactS", "CD28", "CD3", "Akt", "NF-kB" ], "content": "Introduction\n\nMapping global changes in gene expression has proven extremely useful in revealing previously unappreciated connections between groups of expressed genes and biological events, such as development and tumorigenesis. We have previously studied the role of the Akt kinase in T cells, and showed that a subset of NF-κB-dependent genes required Akt for optimal upregulation during T cell activation1. However, the gene expression program controlled by Akt signaling in activated T cells has not been elucidated. Although studies of individual transcription factors and their target genes have elucidated several aspects of Akt signaling2,3, understanding the overall program of transcriptional regulation controlled by the Akt pathway requires global expression analysis. We used global expression profiling to identify genes whose induction is dependent on Akt. A study from the Cantrell lab4 concluded that the chief role of Akt in CD8+ cytotoxic T cells is to control the transcriptional programs that direct effector versus memory cell fate. However, Akt may not have the same role in all T cell subpopulations. For example, constitutively active Akt can stimulate cell growth and survival of CD4+ T cells but not CD8+ T cells5,6.\n\nIn the current study, we tested the effects of a selective, allosteric inhibitor of Akt 1 and 2 (Akti1/2)7–10 on activated T cells and further explored potential mechanism of action of Akt, by performing network analysis of gene expression data and validating the expression changes of selected genes by real-time qPCR analysis. Our findings demonstrate that Akt inhibition by Akti1/2 significantly affects ribosomal protein expression and the cytokine-cytokine receptor interaction. Asthma and the antigen receptor signaling pathways were most affected by Akti1/2 in activated T cells. Moreover, Akt inhibition can decrease the enrichment of NF-κB- and Myc-targeted genes. These effects may contribute to the functions of dysregulated Akt activation in tumorigenesis, as well as in normal T cell activation and development6.\n\nThe importance of Akt for T cell activation and transformation led us to explore the underlying pathways and mechanisms (or target genes and downstream cellular pathways) by a genome-wide gene expression profiling approach. Therefore the aims of the present study were to (1) identify, at the genome-wide level, the genes that are differentially expressed in activated T helper cells, with or without Akt inhibition and (2) conduct bioinformatics analyses to identify the pathways and possible mechanisms involved.\n\n\nMaterials and methods\n\nBiotin-anti-mCD28 (37.51) and biotin-anti-mCD3ε (145-2C11) were obtained from BD-Biosciences (San Jose, CA). Streptavidin was obtained from Invitrogen (Carlsbad, CA) and Akti1/2 was from EMD Biosciences (San Diego, CA). rhIL-2 was obtained through the NIH AIDS Research and Reference Reagent program, Division of AIDS, NIAID, NIH (Cat.#136 from Hoffman-LaRoche, Inc.).\n\nThe D10 T cell line, a fast-growing variant of the D10.G41 murine Th2 T cell clone11–13 was maintained in RPMI 1640 media (Mediatech, Manassas, VA), supplemented with 10% heat-inactivated bovine growth serum (BGS; Hyclone, Logan, UT), 0.1 mM nonessential amino acids (Lonza, Walkersville, MD), 2 mM l-glutamine, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin (Mediatech, Manassas, VA) and 25 IU/ml rhIL-2.\n\nD10 T cells were left untreated or pretreated with 10 µM Akti1/2 for 1 h and then stimulated with biotinylated anti-mCD3/CD28 and streptavidin for 0, 2, 6 and 12 hrs. RNA extraction was performed using a commercially available kit (RNeasy, Qiagen, Frederick, MD) according to the manufacturers’ recommendations. RNA quality was confirmed based on a RNA integrity number >8 by use of the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). The microarray analysis was performed by Genomics and Proteomics Core Laboratories (GPCL) of the University of Pittsburgh, USA. An Illumina mouse RefSeq8 chip was used. Microarray data have been deposited in the GEO database and are accessible through the GEO series accession number GSE45221.\n\nTo compare the molecular characteristics between different time points, Automated Efficiency Analysis was first performed using 7 transformation methods, 9 normalization methods and 5 tests for differentially expressed genes14. A global normalization method and the J5_Quantile95_None method were applied on each time point. The differentially expressed genes were identified using caGEDA with a reasonable threshold of J5 for each time point15. To survey the spectrum of biological functions within genes, which were differentially expressed between different groups, functional classification of the genes were performed using Pathway Express (a pathway level Impact Analysis as described by Draghici et al., 200716). Pathway Express was designed to provide both statistical and biological significance in the indication of which pathways may be affected by the observed changes in gene expression. The results are summarized as Impact scores and p-values. Pathway-Express orders the affected pathways in the decreasing order of their expected importance for the given condition.\n\nQuantitative real-time PCR was performed using the ABI StepOnePlus Real-time PCR system (Applied Biosystems, Foster City, CA) as described previously1. Amplification was performed on a cDNA amount equivalent to 25 ng total RNA with 1×SYBR green universal PCR Master mix (Applied Biosystems) containing deoxyribonucleotide triphosphates, MgCl2, AmpliTaq Gold DNA polymerase, and forward and reverse primers. Specific primers for each gene were purchased from SABiosciences (Qiagen, Frederick, MD). Experimental samples and no-template controls were all run in duplicate. The PCR cycling parameters were: 95°C for 10 min, and 40 cycles of 94°C for 15s, 60°C for 1 min. The amount of cDNA in each sample was calculated by the comparative threshold (Ct) method and expressed as 2exp(Ct) using 18S RNA as an internal control. Statistical significance was determined using the Student’s T test. All statistical tests were performed using GraphPad Prism (GraphPad Prism, San Diego, CA).\n\nTFactS was used to predict the activities of transcription factors in our microarray data17. The lists of up- and down-regulated genes were compared to a list of curated target gene signatures. The nominal p-value (Pval) represents the risk of a false positive for a single test. Since the list of query genes is systematically compared to each target gene signature, a multi-testing condition is required. The e-value (Eval) represents the expected number of false positives for a given nominal value. It is computed using the formula: Eval=PvalT, where T is the number of tests.\n\n\nResults\n\nWe previously demonstrated that Akt activity was rapidly inhibited in T cells by addition of the allosteric inhibitor Akti1/2, which inhibited phosphorylation of Akt within one minute, an effect that can last as long as twelve hours1. In the present study, microarray analysis was performed at three different time points after CD3/CD28 stimulation to characterize the effects of Akt inhibition on the T cell gene activation program in helper T cells. Thus, D10 T cells (a murine Th2 T cell line) were pre-incubated with Akti1/2 or solvent, then stimulated for 2–12 hours. After stimulation, mRNA was isolated, which was converted into labeled cDNA for hybridization to Illumina chips for microarray analysis (Figure 1A). We first determined which genes were differentially modulated after T cell receptor (TCR) stimulation using caGEDA with reasonably chosen thresholds for different time points (2 h, 6 h and 12 h). Further validation and downstream analysis were then performed to confirm some of the differentially expressed genes and to extract functional information from the dataset (Figure 1B).\n\nFlowchart of experimental outline (A) and data analysis (B), as described further in the text.\n\nWe identified differentially expressed gene sets that were dependent on Akt among the three different time point groups. We compared the gene expression patterns of cells plus or minus addition of Akti1/2. First, we generated two gene lists for each time point. Gene list one represents the genes that were differentially expressed between the unstimulated and CD3/CD28 stimulated group in the absence of Akti1/2. Gene list two represents the genes that were differentially expressed between the unstimulated and CD3/CD28 group in the presence of Akti1/2. When comparing the two gene lists, three different patterns were observed:\n\n1. Genes significantly modulated by CD3/CD28 alone but not modulated in the presence of Akti1/2 (genes in this category showed Akt–dependent expression after T cell activation; first sheet in Data File 1–Data File 3 and Supplementary Figure 1)\n\n2. Genes significantly modulated by CD3/CD28 alone but less strikingly modulated in the presence of Akti1/2 (genes in this category showed some dependence on Akt; middle sheet in Data File 1–Data File 3 and Supplementary Figure 2) and\n\n3. Genes not modulated by CD3/CD28 alone but significantly modulated in the presence of Akti1/2 (genes in this category displayed Akti1/2-specific expression; last sheet in Data File 1–Data File 3 and Supplementary Figure 3).\n\n\n\n\n\n\n\nBy examining multiple time points after stimulation, we were able to obtain a kinetic picture of gene expression ± Akt inhibition. The 6 h Akti1/2 (+) and Akti1/2 (-) comparison showed the highest number of differentially expressed genes, and there were fewer differentially expressed genes after two or twelve hours of TCR/CD28 stimulation. Among these, only the genes that expressed the most consistent differences (either increased or decreased expression) were selected for further analysis. Genes with no known function were excluded.\n\nOur previous work identified several NF-κB target genes that were dependent on Akt after TCR stimulation in T helper cells, including those encoding the cytokines TNF-α, GM-CSF, and IL-10, among others1. Analysis of the microarray data confirmed the dependency of these genes on Akt activation, which inspired confidence in our results. Moreover, expression of the mRNAs encoding many secreted proteins was also decreased by Akt inhibition, including IL-13, IL-5, IL-3 and IL-4 (Figure 2). The protein products of these genes (except IL-3) were examined in our previous paper1, which confirmed similar decreases after Akt inhibition. Moreover, we found that expression of Ltb (encoding lymphotoxin β), Areg (encoding amphiregulin) and genes encoding the chemokines CCL1, CCL3 and CCL4 were also affected by Akt inhibition (Figure 2).\n\nRelative levels of expression are represented by the J5 score.\n\nOf note, several cell-surface proteins, including CD69, CD52 and CD82 were among the Akt-dependent genes after T cell activation (Figure 2). The CD82 molecule is a type III integral membrane protein with four transmembrane domains and is part of the tetra-span-transmembrane (TST) family, which also includes CD9, CD37, CD53, CD63 and CD81/TAPA-118. Interestingly, proteins of this family are involved in cell activation. For example, engagement of CD82 can deliver a co-stimulatory signal, similar to CD28, for full T cell activation, leading to strong IL-2 production19. CD52 is a small glycopeptide molecule and tethered to the outer surface of the plasma membrane by a glycosylphosphatidylinositol (GPI)-anchor20. CD52 crosslinking can also provide a co-stimulatory signal that causes the activation of normal human T lymphocytes21 and induction of CD4+ regulatory cells22. Since Akt was reported to mimic CD28 co-stimulation in a T cell line by synergizing with TCR-induced signals to increase transcription of the IL-2 promoter23, activation of these other co-stimulatory molecules might also function through this pathway.\n\nInterestingly, inhibition of Akt resulted in impaired up-regulation of the genes encoding many ribosomal proteins (Figure 3). These included: Rps6 (a major substrate of ribosomal protein kinases24); Rps8, Rps9 (reported to be activated in various tumors, such as colon cancers25); Rps10, Rps15, Rps24 (mutations in these gene result in Diamond-Blackfan anemia)26; Rpl7a (which interacts with a subclass of nuclear receptors and inhibits their ability to activate transcription)27; Rplp1 (important for elongation during protein synthesis28). All these proteins belong to either small or large subunits of ribosomes; changes in their expression may contribute to an increase in protein synthesis to accommodate numerous cellular processes involved in T cell activation, in addition to the possible connections to cancer. Importantly, the expression of 18S ribosomal genes, which was used for our qPCR housekeeping gene, did not differ between our experimental groups.\n\nRelative levels of expression are represented by the J5 score.\n\nIn order to validate the microarray data and obtain more quantitative data on specific genes, real-time PCR analysis was performed at different time points after CD3/CD28 stimulation ± Akti1/2. Akt is known to directly phosphorylate FOXO3a. Once phosphorylated, FOXO3a is excluded from the nucleus and becomes transcriptionally inactive29. Klf6 is one of the FOXO-targeted genes. Real time PCR analysis confirmed that in the Akti1/2 group, there was an up-regulation of Klf6 as expected (Figure 4). Ier3 (also named IEX-1, immediate early response gene X-1), Il13, Ccl1 and Ccl4 were selected for real-time PCR validation because their expression was greatly enhanced after CD3/CD28 stimulation and decreased in the presence of the Akt inhibitor. Of note, the gene expression of Ccl1 and Il3 was rapidly up-regulated by CD3/CD28 stimulation, while the up-regulation of Ier3 and Ccl4 expression by CD3/CD28 stimulation was delayed. Egr1 was also selected as a control gene, which showed no CD3/CD28 dependent increase in gene expression. Thus, Akt inhibition could affect different genes that showed diverse kinetics after CD3/CD28 stimulation. For all genes tested, the expression changes assessed by real-time PCR confirmed the microarray results, giving us confidence in the overall quality of the dataset.\n\nqPCR was used to validate the gene expression of Ier3, Il13, Klf6, Egr1, Ccl1 and Ccl4. The RNA samples were the same as those used for the microarray. Results are presented as relative mRNA expression, compared to the unstimulated control sample, normalized to 18S RNA expression. ** p<0.001, * p<0.05 compared with the control group.\n\nFrom our previously published array data, we found that at two, six and twelve hours after CD3/CD28 stimulation. Akti1/2 elicited a wide range of effects on expression of numerous genes, including both over- and under-expressed genes. In the present study, genes that showed changes in expression after two, six and twelve hours of stimulation in the presence of Akti1/2 were largely not overlapping and cannot be combined for subsequent analysis. There were 54, 71 and 58 genes dependent on Akt at the two, six and twelve hour time points, respectively, after CD3/CD28 stimulation (Data File 1–Data File 2 and Supplementary Figure 1).\n\nTo determine whether our gene expression signature was enriched in specific subsets of genes with known biological functions, bioinformatic functional classification analysis of the genes that were differentially expressed was carried out as described in the Methods. The functional classifications of gene sets are illustrated in Figure 5. We found that genes involved in ribosome, cytokine-cytokine receptor interaction, antigen receptor signaling pathway, hematopoietic cell lineage and asthma were significantly enriched among genes affected by Akt inhibition in the presence of CD3/CD28 stimulation.\n\nClassification enrichment was determined using Pathway Express. Significance is indicated as –Log (p-value).\n\nCo-expressed genes are often regulated by common transcription factors. Therefore, we separately analyzed the genes that showed altered expression at 2, 6 and 12 h time points after CD3/CD28 stimulation using TFactS, an algorithm that used a catalog of well-characterized transcription factor targets to predict the activity of transcription factors based on microarray data17. Enrichment of transcription factors was found at all the time points tested (Figure 6). These included NF-κB family members (RelA, Rel, NF-κB1), Myc, Jun and CREB. At the 2 h time point, Myc, NF-κB family members (NF-κB1, RelA and Rel) and Egr1 were significantly enriched. RelA, NF-κB1 and Rel, along with Myc and Egr1 were also significantly regulated after 6 h of CD3/CD28 stimulation. Moreover, other transcription factors, including Sp1, CEBPB, Ets1, Jun and Nfam1, were also enriched at the 6 h time point. At 12 h after stimulation, however, fewer transcription factors were regulated, with Myc and NF-κB1 still significantly regulated. In summary, the most significantly enriched transcription factor binding sites in the regulatory regions of Akt-dependent genes after CD3 and CD28 stimulation were those of NF-κB family members and Myc.\n\nThe list of Akt-dependent genes was submitted to TFactS (sign-less) using default settings. Significance of regulated transcription factors was determined with –log10 (e-value).\n\n\nDiscussion\n\nAkt activation impacts the expression of genes responsible for cell proliferation and survival30,31. The majority of previous global gene expression studies have investigated transcriptional programs under the combined control of both PI3K and Akt32,33. Despite much attention in recent years to the role of Akt-regulated metabolism in T cells34, a recent study provided clear evidence that Akt indeed contributes to changes in gene expression after activation of cytotoxic T cells4. We have also been interested for some time in achieving a greater level of clarity in separating the effects of PI3K and Akt during helper T cell activation35. Our recent study identified a subset of NF-κB-dependent genes that require Akt for optimal upregulation during helper T cell activation by using a targeted gene profiling approach1. In the present study, we performed a broader microarray analysis to characterize the global changes in gene expression resulting from inhibition of Akt in activated CD4+ helper T cells. Analysis of the affected genes revealed pathways that are central to the effects of Akt on helper T cell activation. Finally, analysis of the enrichment in transcription factor binding sites in our target genes further confirmed NF-κB as a regulator of these genes in response to TCR stimulation.\n\nThe generation and maintenance of memory T cells is central to the development of protective immunity, as characterized by a rapid and vigorous response after a secondary encounter with a given pathogen or antigen36,37. Recent studies have suggested that proper regulation of Akt activity is essential for the development of memory T cells. Riou et al. found that Akt plays a critical role in the phosphorylation of FOXO3 in CD4+ central memory T cells (TCM), thereby promoting TCM survival38. Abrogating the Akt survival pathway led to a greater degree of apoptosis in TCM as compared with effector memory T cells (TEM), confirming that TCM are more dependent on these pathways for their survival. Sustained and strong activation of Akt was also shown in CD8+ cytotoxic T cells (CTL) to coordinate the TCR and IL-2-induced transcriptional programs that control expression of key cytolytic effector molecules, adhesion molecules, and cytokine and chemokine receptors that distinguish effector from memory and naïve T cells4. It has been suggested39 that Akt simultaneously induces and represses expression of key genes, leading to the development of effector CTL, with the FOXO transcription factors being at the center of this process. Thus, Kim et al. found that Akt appeared to function as a cellular rheostat, controlling distinct facets of the program that governed differentiation of Ag-activated CD8+ T cells into effector cells or memory CD8+ T cells39. Myristoylated Akt transgenic mice were found to accumulate memory phenotype CD4+ T cells and to develop both tumors and autoimmunity40, effects that could, in principle, be due in part to non-metabolic outcomes of Akt activation, such as NF-κB activation41.\n\nInterestingly, the expression of Klf6, a FOXO-target gene, was increased in activated T helper cells after Akti1/2 treatment in our study. Although Klf6 did not appear in the differentially expressed gene list at any of the time points, real-time PCR showed that its expression was increased after Akt inhibition. It is known that FOXO transcription factors control regulatory T cell development and function42. Moreover, early Treg-cell-lineage commitment is regulated by Akt and FOXO family transcription factors43,44. Further studies are needed to clarify whether Akt has a similar effect on CD4+ memory cells. FOXO and NF-κB are both known targets for Akt45,46. The enrichment of multiple NF-κB family members in Akt-dependent genes confirmed our previous study emphasizing the important role of NF-κB in Akt-dependent biological processes1. FOXO-target genes were not enriched in the Akt dependent gene list after T helper cell activation, although their expression (such as that of Klf6) was increased after Akt inhibition. The Akt-dependent transcription factors that are induced by TCR stimulation in T helper cells might be different from those induced in CTL.\n\nOf note, Myc target genes were also found enriched in the subset of Akt-dependent genes after T cell activation. It is well known that Myc is associated with cell activation. However, it is now thought that Myc is not an on-off specifier of a particular transcriptional program(s) but is a universal amplifier of gene expression, increasing output at all active promoters47. Relevant for our findings, N-Myc was reported to function as a regulator of cell growth by stimulating expression of genes functioning in ribosome biogenesis and protein synthesis48. Akt was also reported to cooperate with c-Myc to increase ribosome biogenesis and cell growth, which includes the synthesis of rRNA and ribosomal proteins, processing of 45S rRNA, and assembly of functional ribosomal subunits40. In our present study, we showed that Akt inhibition decreased the expression of many ribosomal proteins, including components of both the small (40S) and large (60S) ribosomal subunits. Putting together these disparate observations, it may be that in activated T helper cells, one of the mechanisms through which Akt broadly regulates ribosomal subunit transcription is by activating Myc.\n\nAkt is also known to enhance ribosomal protein production through the mammalian target of rapamycin (mTOR) pathway. However, it was reported that direct inhibition of Akt only weakly suppressed the phosphorylation of S6 and S6K1 in CTLs4 and Akti1/2 could only weakly suppress the CD3/CD28 stimulation dependent phosphorylation of S6 in Jurkat T cells and primary T cells (unpublished data from our lab). Akt could signal through mTORC1-dependent and independent mechanisms to promote rDNA transcription in mammalian cells40. Dissecting out the mTOR-independent mechanism that Akt utilizes to regulate ribosomal biogenesis is crucial to understand the therapeutic response to Akt inhibitors in cancer.", "appendix": "Author contributions\n\n\n\nThe project was conceived by LPK. Experiments were carried out by JC. Data were analyzed by JC and LPK, in consultation with the University of Pittsburgh Genomics and Proteomics Core Laboratory (GPCL). The paper was written by JC and LPK.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by grant GM080398 from the NIH/NIGMS to LPK.\n\n\nAcknowledgements\n\nWe thank James Lyons-Weiler and Haiwen Shi of the University of Pittsburgh Genomics and Proteomics Core Laboratory (GPCL) for assistance with data analysis.\n\n\nSupplementary materials\n\nEach row represents a gene and each column shows one of the duplicates from two different groups (from left to right 0 h vs. 2 h, 0 h vs. 6 h, 0 h vs. 12 h). The red and green colors reflect high and low expression levels, respectively.\n\nEach row represents a gene and each column shows one of the duplicates from two different groups (from left to right 0 h vs. 2 h, 0 h vs. 6 h, 0 h vs. 12 h). The red and green colors reflect high and low expression levels, respectively.\n\nEach row represents a gene and each column shows one of the duplicates from two different groups (from left to right 0 h vs. 2 h, 0 h vs. 6 h, 0 h vs.12 h). The red and green colors reflect high and low expression levels, respectively.\n\n\nReferences\n\nCheng J, Phong B, Wilson DC, et al.: Akt Fine-tunes NF-κB-dependent gene expression during T cell activation. J Biol Chem. 2011; 286(41): 36076–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPande S, Browne G, Padmanabhan S, et al.: Oncogenic cooperation between PI3K/Akt signaling and transcription factor Runx2 promotes the invasive properties of metastatic breast cancer cells. J Cell Physiol. 2013; 228(8): 1784–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOuyang W, Liao W, Luo CT, et al.: Novel Foxo1-dependent transcriptional programs control T(reg) cell function. Nature. 2012; 491(7425): 554–9. 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PubMed Abstract \| Publisher Full Text\n\nDraghici S, Khatri P, Tarca AL, et al.: A systems biology approach for pathway level analysis. Genome Res. 2007; 17(10): 1537–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEssaghir A, Toffalini F, Knoops L, et al.: Transcription factor regulation can be accurately predicted from the presence of target gene signatures in microarray gene expression data. Nucleic Acids Res. 2010; 38(11): e120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShibagaki N, Hanada K, Yamaguchi S, et al.: Functional analysis of CD82 in the early phase of T cell activation: roles in cell adhesion and signal transduction. Eur J Immunol. 1998; 28(4): 1125–33. PubMed Abstract \| Publisher Full Text\n\nLebel-Binay S, Lagaudrière C, Fradelizi D, et al.: CD82, member of the tetra-span-transmembrane protein family, is a costimulatory protein for T cell activation. J Immunol. 1995; 155(1): 101–10. 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PubMed Abstract \| Publisher Full Text\n\nJastrzebski K, Hannan KM, Tchoubrieva EB, et al.: Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function. Growth Factors. 2007; 25(4): 209–26. PubMed Abstract \| Publisher Full Text\n\nBin Amer SM, Maqbool Z, Nirmal MS, et al.: Gene expression profiling in women with breast cancer in a Saudi population. Saudi Med J. 2008; 29(4): 507–13. PubMed Abstract\n\nDoherty L, Sheen MR, Vlachos A, et al.: Ribosomal protein genes RPS10 and RPS26 are commonly mutated in Diamond-Blackfan anemia. Am J Hum Genet. 2010; 86(2): 222–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZiemiecki A, Müller RG, Fu XC, et al.: Oncogenic activation of the human trk proto-oncogene by recombination with the ribosomal large subunit protein L7a. EMBO J. 1990; 9(1): 191–6. PubMed Abstract \| Free Full Text\n\nArtero-Castro A, Castellvi J, García A, et al.: Expression of the ribosomal proteins Rplp0, Rplp1, and Rplp2 in gynecologic tumors. Hum Pathol. 2011; 42(2): 194–203. PubMed Abstract \| Publisher Full Text\n\nTzivion G, Dobson M, Ramakrishnan G: FoxO transcription factors; Regulation by AKT and 14-3-3 proteins. Biochim Biophys Acta. 2011; 1813(11): 1938–45. PubMed Abstract \| Publisher Full Text\n\nHers I, Vincent EE, Tavare JM: Akt signalling in health and disease. Cell Signal. 2011; 23(10): 1515–27. PubMed Abstract \| Publisher Full Text\n\nManning BD, Cantley LC: AKT/PKB signaling: navigating downstream. Cell. 2007; 129(7): 1261–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeinonen H, Nieminen A, Saarela M, et al.: Deciphering downstream gene targets of PI3K/mTOR/p70S6K pathway in breast cancer. BMC Genomics. 2008; 9: 348. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLatres E, Amini AR, Amini AA, et al.: Insulin-like growth factor-1 (IGF-1) inversely regulates atrophy-induced genes via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. J Biol Chem. 2005; 280(4): 2737–44. PubMed Abstract \| Publisher Full Text\n\nMaciver NJ, Jacobs SR, Wieman HL, et al.: Glucose metabolism in lymphocytes is a regulated process with significant effects on immune cell function and survival. J Leukoc Biol. 2008; 84(4): 949–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKane LP, Weiss A: The PI-3 kinase/Akt pathway and T cell activation: pleiotropic pathways downstream of PIP3. Immunol Rev. 2003; 192(1): 7–20. PubMed Abstract \| Publisher Full Text\n\nObar JJ, Jellison ER, Sheridan BS, et al.: Pathogen-induced inflammatory environment controls effector and memory CD8+ T cell differentiation. J Immunol. 2011; 187(10): 4967–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKallies A: Distinct regulation of effector and memory T-cell differentiation. Immunol Cell Biol. 2008; 86(4): 325–32. PubMed Abstract \| Publisher Full Text\n\nRiou C, Yassine-Diab B, Van grevenynghe J, et al.: Convergence of TCR and cytokine signaling leads to FOXO3a phosphorylation and drives the survival of CD4+ central memory T cells. J Exp Med. 2007; 204(1): 79–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim EH, Sullivan JA, Plisch EH, et al.: Signal integration by Akt regulates CD8 T cell effector and memory differentiation. J Immunol. 2012; 188(9): 4305–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan JC, Hannan KM, Riddell K, et al.: AKT promotes rRNA synthesis and cooperates with c-MYC to stimulate ribosome biogenesis in cancer. Sci Signal. 2011; 4(188): ra56. PubMed Abstract \| Publisher Full Text\n\nKane LP, Shapiro VS, Stokoe D, et al.: Induction of NF-kappaB by the Akt/PKB kinase. Curr Biol. 1999; 9(11): 601–604. PubMed Abstract \| Publisher Full Text\n\nKerdiles YM, Stone EL, Beisner DR, et al.: Foxo transcription factors control regulatory T cell development and function. Immunity. 2010; 33(6): 890–904. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaxhinasto S, Mathis D, Benoist C: The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells. J Exp Med. 2008; 205(3): 565–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSauer S, Bruno L, Hertweck A, et al.: T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR. Proc Natl Acad Sci U S A. 2008; 105(22): 7797–802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrunet A, Bonni A, Zigmond MJ, et al.: Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "910", "date": "24 Apr 2013", "name": "David Finlay", "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 aims to determine to what extent Akt regulates the transcriptional landscape in TCR/CD28 activated CD4 T cells. While a considerable amount is known regarding Akt regulation of gene expression in activated CD8 cytotoxic T cells (McIntyre et al., 2011 Immunity 34(2) 224-236) less information exists with respect to CD4 T cells. Indeed it is true, as the authors say, that Akt may have distinct roles in different T cell subsets. Therefore, determining the role of Akt in controlling the transcriptome in CD4 T cell subsets addressed an important and relevant question. However, the data would have been of a much higher value had this study been carried out in primary T cells rather than a CD4 T cell line. There is no reason that using primary CD4 T cells would not have been feasible and I struggle to understand why this was not done. Therefore, this data needs to be viewed with this major caveat in mind.I have substantial concerns regarding the concentration of Akti1/2 that is used in this study. In CD8 T cells 1µM Akti1/2 is sufficient to completely block all Akt activity. This study uses 10µM, 10 times the dose used in CD8 T cell. When using kinase inhibitors, it is extremely important to use the lowest dose that effectively inhibits the kinase activity, as excessive doses can have increased off target effects. For example it has been shown that Akti1/2 has multiple off target effects at 10µM when screened against a panel of 80 kinases (Bain J, et al., 2007, Biochem. J. 408, 297-315). Additionally, in CD8 Cytotoxic T cells, 10µM Akti1/2 is toxic and compromises cell viability, a clear demonstration of off target effects (unpublished data). The authors should demonstrate that they are using the minimum effective dose and consider what aspect of the results that they present in this paper might be due to off target effects of the Akti1/2 compound.With respect to their data, the authors discuss the role of Foxo transcription factors as Akt substrates and there role in CD4 T cells despite the fact that they have little to no evidence that the activity of Foxo transcription factors are altered in response to Akt inhibition in this model. They state Klf6 as a Foxo target. The authors must clearly reference the work that describes Klf6 as a Foxo Target. At best Klf6 is an obscure Foxo target gene. There are numerous well defined Foxo targets in T cells, Klf2 for example, yet the absence of these Foxo target genes in the Akti1/2 regulated gene lists is not discussed at all.A minor point, in the text it is stated that CREB was one of the transcription factors predicted by TFactS to be altered in activity (bottom of page 5), yet CREB does not appear in the corresponding figure (Figure 6).", "responses": [] }, { "id": "925", "date": "03 May 2013", "name": "Ursula Bommhardt", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 publication, Cheng and Kane mapped the gene expression profile of a Th2 cell line stimulated with cross-linked CD3/CD28 Abs in the presence of Akti1/2 inhibitor using microarray and qRT-PCR analysis. They found gene expression that is not moderately or strongly affected by Akti1/2. Akt-dependent genes included cytokines (like IL-4, IL-5 and IL-13), chemokines (CCL1, CCL3 and CCL4), and a number of genes involved in the biosynthesis and function of ribosomes. Major Akt-dependent transcription factors included members of the NFkappaB family, c-myc and their target genes.The study provides interesting and novel information on Akt-dependent signalling and Akt target genes which will be very useful for the further understanding of Akt function in T cells. In this line, previous publication by Patra et al. on myrAkt expression in activated CD4+ T cells had shown increased expression of Th1 as well as Th2 cytokines, including IL-10, IL-4, IL-13 (Patra et al., JI, 2004). Thus, the data of the current study support those previous results. The authors should include this publication in their citation list as well as other publication from the groups of P. Ohashi, C. Thompson and others with regard to Akt, NFkappaB and tumorigenesis.", "responses": [] }, { "id": "957", "date": "20 May 2013", "name": "Michael Gold", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides useful information for further assessing the role of Akt signaling in the activation of CD4+ T cells. The microarray experiments that assess the effect of an Akt inhibitor compound on gene regulation by TCR/CD28 co-stimulation are well done and the accompanying bioinformatics analyses are quite sophisticated such that identified changes in gene expression needed to have passed multiple statistical tests.Although a good starting point for further studies, the data presented have several limitations. First, the experiments were carried out entirely with a T cell line. It would have been straightforward, and of significant value, to confirm some of the key gene expression changes in primary T cells stimulated with anti-CD3/anti-CD28 in the presence or absence of the Akt inhibitor. It is of interest that two of the Akt-dependent genes identified in this report are IL-13 and IL-4, suggesting that Akt is critical for the effector functions of Th2 cells. It would be worthwhile to discuss the role of Akt signaling in Th2 polarization versus Th2 function and to refer to, or propose, further studies to address this question. Second, as for any study employing chemical inhibitors, it would have been useful for the authors to briefly review selectivity of this compound as well as known off-target effects on other kinases. In this regard, confirming some of the key gene expression changes by using other commercially available Akt inhibitors with different off-target effects would help confirm the dependence on Akt. Similarly, the conclusion that many of the Akt-regulated genes may be co-regulated by NF-kB or Myc could have been tested in a limited fashion.Despite these limitations, which will likely be dealt with in follow-up studies by these authors and others, this article provides useful data that will stimulate further work and discussion.", "responses": [] } ]	1	https://f1000research.com/articles/2-109
https://f1000research.com/articles/2-124/v1	09 May 13	{ "type": "Case Report", "title": "Rhinoscleroma presenting as a nasal-palatal mass with airway obstruction", "authors": [ "Mark C Domanski", "Alexander Rivero", "David E Kardon", "Alexander Rivero", "David E Kardon" ], "abstract": "We report a case of a 45-year-old male with severe rhinoscleroma. The patient presented to the emergency room with dyspnea from a long-standing nasal-palatal mass. A tracheostomy was required for airway control. While dyspnea in the presence of an upper airway mass is typical of malignancy, consideration of non-oncological etiologies is important. We review the epidemiology, pathology, diagnosis, and treatment of rhinoscleroma.", "keywords": [ "rhinoscleroma", "Klebsiella rhinoscleramitis", "palate", "tracheostomy" ], "content": "Introduction\n\nRhinoscleroma is a chronic bacterial infection caused by Klebsiella rhinoscleromatis, a Gram-negative, non-motile, encapsulated bacillus. Due to the low infectivity of the bacteria, chronic exposure is required in order to establish infection. Rhinoscleroma is more frequent in the developing world, and is likely a secondary complication as a result of underdeveloped hygiene infrastructures, poor access to antibiotics, and overcrowded living conditions. Most cases are found in Central America, Africa and the Middle East1. The prevalence of sporadic cases outside of endemic areas is usually attributed to immigration2. Though rhinoscleroma can involve any structure of the upper respiratory tract, Klebsiella rhinoscleromatis has an affinity for nasal mucosa and thus is present in the nasal cavity in 95–100% of cases3. It can also be found in the nasopharynx (18–43%), larynx (15–40%), trachea (12%), and bronchi (2–7%)4. Here, we present a case with both nasal and palatal involvement resulting in airway obstruction.\n\n\nCase report\n\nA 45-year-old Central American male presented with a 13-year palatal mass, and new onset stridor in the background of chronic dyspnea. He denied weight loss and night sweats. He worked as a day laborer, drank socially, but never smoked. He had been unable to breathe out of his nose for at least thirty years.\n\nNasal endoscopy showed obstructed choana bilaterally. Inspection of the oral cavity showed a hard, plaque like growth involving the hard and soft palates, pharynx, and marked foreshortening of the palatoglossal folds (Figure 1). Dentition was poor. Endoscopic visualization of the larynx could only be performed transorally. The patient’s airway was tight at the level of the palatoglossal folds and base of the tongue. The vocal cords and epiglottis were uninvolved.\n\nA computed tomography (CT) scan confirmed a palatal mass, and obstructed choana. Thickening of the uvula, and hard and soft palate mucosa was noted. No palatal bony obstruction or lymphadenopathy was seen (Figure 2).\n\nHeterogeneous soft tissue is present in the nasopharynx.\n\nA local awake tracheostomy was performed to provide a secure airway. A palatal biopsy was sent for analysis and demonstrated squamous mucosa with a dense, mixed inflammatory infiltrate containing abundant plasma cells and scattered vacuolated macrophages (Mikulicz cells) (Figure 3). A Warthin-Starry stain revealed rod-shaped bacilli within the vacuolated macrophages. The bacilli were morphologically consistent with Klebsiella (Figure 4).\n\nThe patient was treated with ciprofloxacin 500 mg BID for 12 weeks. His airway symptoms improved and he was later decannulated without sequelae. He declined surgical nasal airway debridement.\n\n\nDiscussion\n\nRhinoscleroma generally progresses in three stages. The initial stage is the catarrhal or exudative phase. This is followed by the proliferative or granulomatous phase, which finally evolves into the cicatricial phase2. During the catarrhal stage, patients may have persistent rhinitis and mucopurulent discharge. In the second stage, inflamed mucosa coalesces to form granulomas. These granulomas may infiltrate other portions of the airway and then scar, giving rise to the third or cicatricial stage2. These stages usually do not exist independently. In many cases of rhinoscleroma, the presence of all three stages can be found at the time of diagnosis.\n\nRhinoscleroma is spread by person-to-person transmission. However due to the low infectivity of the pathogen, transmission requires a chronic exposure. It has also been proposed that an altered immune response along with an alteration in the CD4+ and CD8+ proportion leads to ineffective macrophage production that are susceptible to bacterial replication5.\n\nA high degree of suspicion is warranted when patients present with persistent, unremitting rhinitis or nasal obstruction unexplained by other causes. The differential diagnosis of such symptoms should include rhinoscleroma, as well as tuberculosis, syphilis, Wegener’s granulomatosis, lymphomas as well as more common carcinomas. Histopathologic evidence of rhinoscleroma includes granulomatous inflammation with large vacuolated histiocytes known as Mikulicz cells6. Canalis et al. proposed that these Mikulicz cells arise from histiocytes that migrate to areas where neutrophils have failed to contain the Klebsiella infection7. The histiocytes, however, are unable to lyse their phagocytosed Klebsiella cells, leading to the dilation of their vacuoles7. Positive culture of rhinoscleroma on MacConkey agar is diagnostic, though culture is only positive in 50–60% of patients. Thus, it is key to have high clinical suspicion in conjunction with positive histopathologic evidence to confirm the diagnosis.\n\nHistorically, treatment of rhinoscleroma was with tetracyclines and aminoglycosides such as streptomycin. However, a prospective study done in the Mayo Clinic, USA, by Andraca et al. in 1993 demonstrated the efficacy of fluoroquinolones8. Treatment with fluoroquinolones also confers the benefit of a lower side-effect profile. Dosing of the antibiotic is variable between different studies, but most agree that long-term therapy for months and sometimes years is necessary to adequately treat the infection3,7,8. Despite treatment, recurrence has been reported in up to 25% of cases at 10 years2,4. Consideration should be made when addressing whether a patient requires surgical de-bulking of the scar in rhinoscleroma formed during the cicatricial stage. Indications for surgical de-bulking include airway patency, treatment of bulky disease, and cosmesis.\n\n\nConclusion\n\nRhinoscleroma is due to chronic and indolent Klebsiella infection. Symptoms may include chronic, unremitting rhinitis or nasal obstruction that is present for years. The presenting symptom can also be more dramatic, such as airway compromise, as seen in this case. A diagnosis of rhinoscleroma is made via pathological specimens. Communication between the clinician and the pathologist as to the possibility of non-oncological processes can aid in determining the diagnosis. A Warthin-Starry stain demonstrating rod-shaped bacilli within vacuolated macrophages (Mikulicz cells) is classic for rhinoscleroma. Mainstay of treatment is long-term fluoroquinolones. Evaluation of airway patency is critical and surgical intervention may be required.\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\nMD treated the patient. DK prepared the pathological slides, which made the diagnosis. AR 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 were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMiller RH, Shulman JB, Canalis RF, et al.: Klebsiella rhinoscleromatis: a clinical and pathogenic enigma. Otolaryngol Head Neck Surg (1979). 1979; 87(2): 212–21. PubMed Abstract\n\nStiernberg CM, Clark WD: Rhinoscleroma--a diagnostic challenge. Laryngoscope. 1983; 93(7): 866–70. PubMed Abstract \| Publisher Full Text\n\nYigla M, Ben-Izhak O, Oren I, et al.: Laryngotracheobronchial involvement in a patient with nonendemic rhinoscleroma. Chest. 2000; 117(6): 1795–8. PubMed Abstract \| Publisher Full Text\n\nGaafar HA, Gaafar AH, Nour YA: Rhinoscleroma: an updated experience through the last 10 years. Acta Otolaryngol. 2011; 131(4): 440–6. PubMed Abstract \| Publisher Full Text\n\nSood N, Sood S, Arora S, et al.: Cytohistological features of rhinoscleroma. Indian J Pathol Microbiol. 2011; 54(4): 806–8. PubMed Abstract \| Publisher Full Text\n\nRobbins JB, Riedel BD, Jones T, et al.: Nasal tumor in a Peruvian man. Am J Dermatopathol. 2004; 26(3): 248. PubMed Abstract\n\nCanalis RF, Zamboni L: An interpretation of the structural changes responsible for the chronicity of rhinoscleroma. Laryngoscope. 2001; 111(6): 1020–6. PubMed Abstract \| Publisher Full Text\n\nAndraca R, Edson RS, Kern EB: Rhinoscleroma: a growing concern in the United States? Mayo Clinic experience. Mayo Clin Proc. 1993; 68(12): 1151–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "941", "date": "15 May 2013", "name": "Rakesh K Chandra", "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 manuscript is an excellent review of the topic and is well illustrated with a clinical photo, radiology, and histopathology. My only area of concern is that I wonder whether it was indeed necessary to perform a tracheotomy on this patient. If the glottis was visible transorally, and given the findings on the sagittal radiologic image shown, it looks like the patient could have been intubated. Also the lesion is too superior to cause true stridor. It could be argued that tracheotomy was advised because the patient would have had difficulty managing secretions and there was concern the lesion would swell or bleed upon biopsy, but it is difficult to imagine true stridor and that this patient couldn’t have been intubated. The report would be improved if the authors justify their decision to perform the tracheotomy in the light of these commentsTitle and Abstract are appropriate and the conclusions are otherwise balanced and justified.", "responses": [] }, { "id": "954", "date": "16 May 2013", "name": "Allen M Seiden", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describing an unusual presentation for rhinoscleroma, an infectious problem that we see rarely in the USA. It is well written, and well-organized. It would have been interesting to include patient photos post treatment. It can be a difficult diagnosis to make, so a more thorough literature review discussing potential physical findings on presentation would also have been helpful.", "responses": [] }, { "id": "979", "date": "31 May 2013", "name": "Alkis Psaltis", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an interesting case report of a condition not commonly seen in developed countries. Its unusual presentation reaffirms the need for otolaryngologists to consider infectious processes in the work up of sinonasal and nasopharyngeal masses. The paper is concise, well written and easy to follow. It provides a nice summary of rhinoscleroma particularly of the histopathophysiological features essential for diagnosis.Additional photographs demonstrating the encroachment of the mass on the pharyngeal airway (i.e. with the use of a tongue depressor), a nasal endoscopic view and a post treatment view may have been helpful in providing more visual information for the reader.I agree with the previous reviewer’s concern regarding the need for an awake tracheotomy for the management of the airway. Given the superior appearance of the mass on the sagittal CT scan and the fact that the authors state that an adequate visualization of the uninvolved vocal cords and epiglottis was obtained with transoral endoscopy, why did the authors not consider awake fiber-optic endoscopic intubation or at least mention this as an alternative for managing the airway. The discussion of the response of the mass to treatment is a little brief. I think additional information would be useful to the reader such as: Percentage decrease in the size of the mass with treatment, how the authors deemed that 12 weeks of treatment was appropriate, progress post treatment and also any scheduled future follow up.", "responses": [] } ]	1	https://f1000research.com/articles/2-124
https://f1000research.com/articles/2-123/v1	09 May 13	{ "type": "Research Article", "title": "Laryngeal mask airway insertion by classic and thumb insertion technique: a comparison", "authors": [ "Monica Goyal", "Akanksha Dutt", "Anjum S Khan Joad", "Monica Goyal", "Anjum S Khan Joad" ], "abstract": "We evaluated the efficacy of an alternative technique, for insertion of the silicone laryngeal mask airway (LMA) Classic™ in 40 American Society of Anesthesiologists grade ASA I and II patients scheduled for elective surgery. In group I (Index Finger group), the LMA was inserted by the classic index finger technique and, in group T (Thumb Insertion group), the thumb insertion technique was used. Ease of insertion, fiberoptic laryngoscopic position, cuff pressures and laryngopharyngeal morbidity were assessed in both study groups. On statistical analysis, both groups were comparable in all respects. From our study it can be concluded that thumb insertion is an effective insertion technique for the LMA Classic™.", "keywords": [ "Laryngeal mask airway", "index finger technique", "thumb insertion" ], "content": "Introduction\n\nThe thumb insertion technique offers an attractive alternative technique for insertion of a laryngeal mask airway (LMA). An LMA is conventionally inserted by the index finger insertion technique as described by Dr. Archie Brain1,2. However, there are certain conditions where LMA insertion using the index finger is difficult, as it is not possible to reach the head end of the patient, for instance during mass casualty (e.g. fire, building collapse, etc.) or in case of a patient with a stereo tactic frame3. Also, paramedical workers are often less comfortable working at the head end of the patient. In such circumstances, an alternative technique of insertion may be useful. Therefore, this study was planned to assess the thumb insertion technique.\n\nThis study was performed using the silicone LMA (LMA Classic™) to compare the two insertion techniques with respect to ease of insertion, fiberoptic laryngoscopic position, cuff pressure and patient comfort in patients for elective general anesthesia.\n\n\nMaterial and methods\n\nAfter approval from the hospital research and ethical committee, a prospective, randomized double-blind study was conducted on patients of American Society of Anesthesiologists ASA grade I & II between 18–60 years of age, undergoing elective surgery.\n\nExclusion criteria were as follows1:\n\n1. Risk of aspiration (full stomach, gastroesophageal reflux disease, pregnancy).\n\n2. Mouth opening < 2.5 cm.\n\n3. Weight < 40 kg or > 110 kg.\n\n4. Respiratory tract pathology.\n\n5. Cervical spine disease.\n\nAfter informed consent was obtained, patients were randomly allocated into two groups of 20 each. In group Index (I), the silicone LMA was inserted using the index finger insertion technique and in group Thumb (T), the silicone LMA was inserted using the thumb insertion technique. Randomization was done by opening sealed numbered envelopes.\n\nPatients were premedicated with oral diazepam 0.1 mg/kg two hours before anesthesia and intravenous Ondansetron 0.01 mg/kg just before induction of anesthesia. Intra-operative monitoring included continuous electrocardiogram (ECG), non-invasive blood pressure (NIBP), oxygen saturation (SpO2) end tidal CO2 (Et CO2) and airway pressure.\n\nFollowing preoxygenation, anesthesia was induced with intravenous fentanyl 2 mcg/kg. Two minutes after administering fentanyl, propofol 2.5 mg/kg was given intravenously4. One minute after administration of propofol, LMA insertion was attempted. During LMA insertion, if the mouth opening was inadequate, or if purposeful movement in patients was observed, further bolus doses of 10 mg propofol were given to facilitate insertion. In all patients the LMA was inserted by an experienced anesthesiologist who was well versed with both techniques of LMA insertion.\n\nIn group Index (I), the LMA was inserted from the head end of the patient after partially inflating the cuff5 (i.e. filled with half the recommended air in the cuff), and lubricating the posterior surface of the cuff with water-soluble jelly. The patient's head was supported on a firm ring with neck flexed and head extended. The tube portion of the laryngeal mask was grasped as if it were a pen; the index finger was pressed on the point where the tube adjoins the mask. The patient’s mouth was opened; the tip of the mask was placed against the inner surface of the upper incisors or gums with the aperture facing anteriorly (and the black line facing the patient's upper lip). The mask was pressed back against the hard palate to keep it flattened as it advanced into the hypopharynx, using the index finger to push upward against the palate. The tube was grasped with the other hand, straightened slightly, and then pressed down with a single, quick but gentle movement until a definite resistance was felt.\n\nIn group T, the LMA was inserted from the right side of the patient6,7, i.e. the operator stood facing the patient, in the angle made by the chest and right arm of the patient. After partially inflating the cuff (i.e. filled with half the recommended air in the cuff), the posterior surface of the cuff was lubricated with water-soluble jelly. The patient's head was supported on a firm ring with neck flexed and head extended. The tube portion of the laryngeal mask was grasped in a pen-like fashion; the thumb (instead of the index finger) was pressed on the point where the tube adjoins the mask. After opening the patient’s mouth, the tip of the mask was placed against the inner surface of the upper incisors or gums with the aperture facing anteriorly (and the black line facing the patient’s upper incisors). The mask was pressed back against the hard palate to keep it flattened as it advanced into the hypopharynx until a definite resistance was felt. In this technique, the thumb was used to apply pressure against the hard palate while advancing the LMA. The tube was grasped with the other hand while removing the thumb6.\n\nAfter insertion, cuff inflation of either device was to a \"just seal\" pressure or up to a maximum of 60 cm H2O, as measured with a simple hand-held aneroid manometer8. The volume of air used was recorded, and a larger device was substituted if leaks persisted on gentle manual ventilation.\n\nInsertion success was assessed by the following criteria:\n\n1. Establishing a clear airway.\n\n2. Rising up of device during cuff inflation.\n\n3. Anterior neck filling with device inflation.\n\n4. The device remained in midline with the black line on the posterior side of airway tube remaining in midline in line with the upper incisors.\n\nAnesthesia was maintained with isoflurane and nitrous oxide in oxygen and vecuronium bromide (0.08 mg/kg initially followed by 0.01 mg/kg every 30 minutes). Mechanical ventilation was volume controlled and time cycled with a tidal volume (5–8 ml/kg) set to maintain peak inspiratory pressure less than 20 cm of H2O, and ventilator frequency was adjusted to maintain EtCO2 at 30–38 mm of Hg with an I/E ratio of 1:2.\n\nOptimal ventilation was assessed by the following criteria9:\n\na. Adequate chest expansion.\n\nb. Stable oxygenation.\n\nc. Square wave capnograph.\n\nSoon after this, a fiberoptic bronchoscope was passed through the device and the view was graded as follows10,11:\n\na. Vocal cords fully visible.\n\nb. Vocal cords & posterior epiglottis visible.\n\nc. Vocal cords & anterior epiglottis visible.\n\nd. Vocal cords not seen but ventilation adequate.\n\nFor our study, fiberoptic view grades a. and b. were labeled good and c. and d. were labeled poor.\n\nAfter surgery, neuromuscular block was antagonized with neostigmine (0.05 mg/kg) and glycopyrrolate (0.01 mg/kg). LMA was removed after deflating the cuff when the patient regained consciousness and protective airway reflexes. The presence of blood on the LMA cuff was recorded.\n\nIn the postoperative period, patients were asked if they had a sore throat in the recovery room and 24 hours postoperatively. The responses were graded as follows.\n\na. Nil\n\nb. Mild\n\nc. Moderate\n\nd. Severe\n\nStatistical analysis was done using Chi-square test with Yates correction for qualitative analysis and analysis of variance (ANOVA) for quantitative analysis.\n\n\nResults\n\nThe mean age of first group with index finger insertion was 43.15 years whereas the mean age of second group with index finger insertion technique was 45.70 years. The mean weight of first group was 61.10 and that of second group was 55.3 years. The male: Female ratio in both groups was 1:4. There was no statistically significant difference between the two groups (Table 1). The time taken, number of attempts, cuff pressure and fiberoptic view scores were comparable in the two groups (Table 2). The insertion success of LMA & laryngopharyngeal morbidity was statistically comparable between both groups (Table 3). The time taken and number of attempts for LMA classic insertion were comparable irrespective of the technique used. No significant difference was found in the cuff pressure, fiberoptic view scores and pharyngeal morbidity in both study groups. However, the sample size was small.\n\nBoth the study groups were statistically similar with respect to age and weight. NS = Not significant.\n\nThe time taken, number of attempts, cuff pressure and fiberoptic view scores were comparable in the two groups. NS = Not significant.\n\nThe insertion success of LMA & laryngopharyngeal morbidity was statistically comparable between both groups.\n\n\n\n\nDiscussion\n\nThe manufacturers of the LMA Classic have recommended thumb insertion as one of the methods of insertion7, but there is little evidence in the literature available on its success. In this study, an attempt was made to compare the index finger and thumb insertion techniques for the LMA classic insertion. The users of LMA had a shorter learning curve compared to the endotracheal tube, and paramedical workers lacking advanced airway training easily master the skill of inserting an LMA12,13. As this group of care givers may not be as comfortable as doctors in working at the head end of the patient, the thumb insertion technique is an attractive option (this technique was demonstrated by Dr. Chandy Varghese at an airway workshop at the All India Institute of Medical Sciences, New Delhi, India in 1999, citing the reluctance of paramedical workers to work at the head end14). Also, in conditions of mass casualty e.g. fire; building collapse or earthquake, when it may not be possible to reach the head end of the patient, the thumb insertion technique would be useful. The time taken for the successful insertion of an airway device when used for airway management during anesthesia or in apneic patients during resuscitation is crucial and, therefore, has to be reasonably brief. In the present study, the time of LMA insertion was defined as the time taken from picking up the device until the time at which positive pressure ventilation was started. Even though in our study, the mean time taken in the thumb insertion group (34.00 + 17.31 sec) was longer than in the index finger group (29.00 + 28.6 sec), the difference was not statistically significant. The number of attempts required for LMA insertion using either technique was also comparable statistically.\n\nSilva and Brimacombe15 (1996) described a non-conventional (thumb) insertion technique of the LMA for general anesthesia during stereotactic implantation of fetal hypophysis in Parkinson’s disease in five patients, as the conventional approach from the patient’s head end was impeded by the stereotactic frame.\n\nThe LMA cuff pressures in both study groups were also found to be comparable. A statistically significant difference was not found in the fiberoptic view scores between the two study groups, emphasizing the fact that technique of insertion did not influence correct placement of the LMA and effective ventilation. No significant difference in the incidence of sore throat or blood on device was found in the study groups.\n\nMost of us are accustomed to efficiently placing the LMA by the index finger technique in the operation theatre, but we also need to master the thumb technique to handle difficult situations outside theatre.\n\n\nConclusion\n\nIn this patient population, the thumb insertion technique was as effective as index finger insertion technique with respect to ease of insertion and insertion success. It also provides optimal ventilation, comparable fiberoptic view scores and comparable incidence of sore throat. It is important for doctors and paramedical workers to learn both techniques, especially for situations outside the carefully controlled operating theatre environment.", "appendix": "Author contributions\n\n\n\nAnjum and Monica contributed to the conception and design of the study. Monica and Akanksha collected and analyzed the data. Akanksha wrote up the manuscript. Anjum and Akanksha both approved the manuscript.\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\nReferences\n\nJerry AD, Susan E: Dorsch \"Laryngeal mask airways\" Chapter 15 ‘Understanding Anesthesia Equipment’ 4th Edition. Editor – Sharon Zinner. Published in Pennsylvania, Williams and Wilkins 1999, 477–486.\n\nBrain A: Proper technique for insertion of the Laryngeal Mask. Anesthesiology. 1990; 73(5): 1053–4. PubMed Abstract\n\nSilva LC, Brimacombe JR: The Laryngeal mask airway for stereotactic implantation of fetal hypophysis. Anesth Analg. 1996; 82(2): 430–1. PubMed Abstract \| Publisher Full Text\n\nGoyagi T, Tanaka M, Nishikawa T: Fentanyl decreases propofol requirement for laryngeal mask airway insertion. Acta Anaesthesiol Scand. 2003; 47(6): 771–4. PubMed Abstract \| Publisher Full Text\n\nMatta BF, Marsh DS, Nevin M: Laryngeal mask airway: A more successful method of insertion. J Clin Anesth. 1995; 7(2): 132–5. PubMed Abstract \| Publisher Full Text\n\nKhan Rashid M, Bhatti TH, Ahmed Moeid S: Laryngeal mask airway and the difficult airway management. J Anaesth Clin Pharmacol. 2005; 21(3): 241–6. Reference Source\n\nupdated on 9th September 2003 access accessed on 29th May 2008. LMA North America, Inc. Reference Source\n\nBrain AI: Pressure in laryngeal mask airway cuffs. Anaesthesia. 1996; 51(6): 603. PubMed Abstract\n\nShafik MT, Bahlman BU, Hall JE, et al.: A comparison of the Soft Seal disposable and the Classic re-usable laryngeal mask airway. Anaesthesia. 2006; 61(2): 178–81. PubMed Abstract \| Publisher Full Text\n\nPayne J: The use of the Fibreoptic laryngoscope to confirm the position of the laryngeal mask. Anaesthesia. 1989; 44(10): 865. Publisher Full Text\n\nBrimacombe J, Berry A: A proposed fiber-optic scoring system to standardize the assessment of LMA position. Anesth Analg. 1993; 76(2): 457. PubMed Abstract\n\nReinhart DJ, Simmons G: Comparison of placement of the laryngeal mask airway with endotracheal tube by paramedics and respiratory therapists. Ann Emerg Med. 1994; 24(2): 260–3. PubMed Abstract \| Publisher Full Text\n\nDeakin CD, Peters R, Tomlinson P, et al.: Securing the pre-hospital airway: A comparison of laryngeal mask insertion and endotracheal intubations by UK paramedics. Emerg Med J. 2005; 22(1): 64–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlaishon R, Sotman A, Friedman A, et al.: Laryngeal mask airway insertion by anesthetists and nonanesthetists wearing unconventional protective gear: a prospective, randomized, crossover study in humans. Anesthesiology. 2004; 100(2): 267–73. PubMed Abstract \| Publisher Full Text\n\nSilva LC, Brimacombe JR: The laryngeal mask airway for stereotactic implantation of fetal hypophysis. Anesth Analg. 1996; 82(2): 430–1. PubMed Abstract \| Publisher Full Text" }	[ { "id": "939", "date": "10 May 2013", "name": "Yehuda Ginosar", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 was well designed, analyzed and presented. It is not “science” but it does address a clinically useful question and does so using an appropriate scientific methodology. The only comments that I would make require minor changes. I suggest modifying the names of the groups to “anesthesiologist at head of patient” and “anesthesiologist facing patient” – or possibly a more elegant phraseology (south-facing vs north-facing approach?) - this is really the main difference between the techniques – whether you use the thumb or index finger is a secondary difference derived from the first.This should also appear in the title. In table 3, please add a footnote explaining IS1, IS2 etc and OV 1, OV 2 etc – I know these terms appear in the text but each table should be self-explanatory.", "responses": [] }, { "id": "950", "date": "14 May 2013", "name": "Brian J Egan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/2-123
https://f1000research.com/articles/2-79/v1	06 Mar 13	{ "type": "Research Article", "title": "Internet-delivered eye movement desensitization and reprocessing (iEMDR): an open trial", "authors": [ "Jay Spence", "Nickolai Titov", "Luke Johnston", "Blake F Dear", "Bethany Wootton", "Matthew Terides", "Judy Zou", "Nickolai Titov", "Luke Johnston", "Blake F Dear", "Bethany Wootton", "Matthew Terides", "Judy Zou" ], "abstract": "Recent research indicates internet-delivered cognitive behavioural therapy (iCBT) can reduce symptoms of post traumatic stress disorder (PTSD). This study examined the efficacy of an internet-delivered treatment protocol that combined iCBT and internet-delivered eye movement desensitization and reprocessing (iEMDR), in an uncontrolled trial. Eleven of the 15 participants completed post-treatment questionnaires. Large effect sizes were found from pre-treatment to 3-month follow-up (d = 1.03 – 1.61) on clinician-assessed and self-reported measures of PTSD, anxiety and distress, with moderate effect sizes (d = 0.59 – 0.70) found on measures of depression and disability. At post-treatment, 55% of the participants no longer met criteria for PTSD and this was sustained at follow-up. Symptom worsening occurred in 3 of 15 (20%) of the sample from pre- to post-treatment; however, these participants reported overall symptom improvement by follow-up. Future research directions for iEMDR are discussed.", "keywords": [ "Results of meta-analyses indicate that both trauma-focused cognitive behavioural therapy (TF-CBT) and eye movement desensitization and reprocessing (EMDR)1 are effective in reducing PTSD symptoms. However", "barriers to accessing these treatments include stigma", "cost", "distance", "low mental health literacy", "and long waiting lists2", "3." ], "content": "Introduction\n\nResults of meta-analyses indicate that both trauma-focused cognitive behavioural therapy (TF-CBT) and eye movement desensitization and reprocessing (EMDR)1 are effective in reducing PTSD symptoms. However, barriers to accessing these treatments include stigma, cost, distance, low mental health literacy, and long waiting lists2,3.\n\nInternet-delivered psychological treatments may increase access to psychological therapy4. TF-CBT has been delivered via the internet and has shown promise in significantly reducing PTSD symptoms in military personnel5,6, university students7,8, and community samples in the U.S.7, Holland9, Iraq10, Australia11,12 and German-speakers in Europe13. For example, in a previous study12 we evaluated an internet-delivered TF-CBT (iCBT) protocol with Australian adults with a primary diagnosis of PTSD. We found large within-group effect sizes (ESs) and small-to-moderate between-group ESs on measures of PTSD symptoms, depression, anxiety and disability, in a treatment group relative to a control condition.\n\nTo our knowledge, there are no reports of studies evaluating the effectiveness of internet-delivered EMDR (iEMDR). Such a treatment may offer another model of remote treatment for PTSD. The present study aimed to explore the acceptability and efficacy of iEMDR when used in conjunction with an iCBT protocol (iCBT/iEMDR course), and evaluated using an open trial design. To increase generalizability of results, inclusion criteria were consistent with those of outpatient services. The primary hypothesis was that the iCBT/iEMDR course would be associated with significant improvements in PTSD symptoms, depression, anxiety, distress, and disability.\n\nSecondary hypotheses were that the treatment would be rated as acceptable to participants and would not be associated with adverse events.\n\n\nMethods\n\nThe study was approved by the Macquarie University Human Research Ethics Committee (HREC#: 5201100382). Participants provided informed consent. The trial is registered with the Australian and New Zealand Clinical Trials registry as ACTRN12611000151932.\n\nParticipant flow is shown in Figure 1. Participants were recruited from visitors to a research website that evaluates internet-delivered treatments (www.ecentreclinic.org). During the recruitment period, which ran over 2 weeks during June 2011, 32 individuals applied and 23 met the following inclusion criteria: (i) self-identified as having a principal complaint of PTSD as indicated by total scores above a clinical cut-off recommended to indicate probable diagnosis of PTSD14 (defined as > 44 on the PTSD Checklist (PCL-C)15) as well as a confirmed primary diagnosis of PTSD determined by clinician-administered interview using the PTSD Symptom Scale-Interview (PSS–I)16; (ii) at least one month had elapsed since the primary trauma; (iii) no psychotherapy for PTSD during the treatment period (however, supportive group and individual counselling that did not specifically target PTSD symptoms was permitted); (iv) if using psychotropic medication, no change in dosage or type of medication 1 month prior to or during treatment; (v) a resident of Australia, (vi) at least 18 years of age, (vii) had computer and internet access, (viii) not currently experiencing a psychotic mental illness, extreme current symptoms of depression (defined as a total score > 22 or responding > 2 to Question 9 (suicidal ideation) on the Patient Health Questionnaire - 9 Item (PHQ-9)17, current suicidal intent and plan, or highly dissociative (defined as a total score above 22) on the Dissociative Experiences Scale – Brief Version (DES-B)18. Sixteen participants met all the criteria and were offered treatment, and 15 subsequently began treatment and are included in analyses.\n\niEMDR: Internet-delivered eye movement desensitization and reprocessing. PHQ-9, Patient Health Questionnaire – 9 Item. MINI: MINI International Neuropsychiatric Interview. DES-B: Dissociative Experiences Scale – Brief Version.\n\nThe primary outcome measures were severity of symptoms of PTSD, measured by the PSS-I and the PCL-C. The PSS-I16 is a 17-item semi-structured clinician-administered interview based on the DSM-IV criteria for PTSD. The PCL-C15 is also a 17-item, self-report scale of PTSD symptoms based on the DSM-IV criteria for PTSD.\n\nSecondary outcomes measures included the Generalized Anxiety Disorder 7-Item Scale (GAD-7, which measures anxiety)19, the PHQ-9 (which measures depression)17, the Mini International Neuropsychiatric Interview (MINI; which was used to determine the presence of a major depressive episode, panic, agoraphobia, social phobia, obsessive compulsive disorder, and generalized anxiety disorder)20, the Kessler 10 Item21 (K10; which measures general distress), and the Sheehan Disability Scale22 (SDS, which measures impairment in psychosocial functioning). Traumatic experiences were assessed using the Life Events Checklist (LEC)23, which provides a list of traumatic events and assesses the occurrence rates of common Criterion A1 (life-threatening) traumas according to the DSM-IV. Additional outcomes included completion rates (percentage of participants who read the six online lessons of the iCBT/iEMDR course within the six weeks of the course), and treatment satisfaction (percentage who reported feeling satisfied with the program or who would recommend it to a friend).\n\nThe iCBT/iEMDR course is a six lesson online intervention utilising evidence-based principles of TF-CBT24 and EMDR25. The TF-CBT components were similar to those used in a previous internet-based CBT program for PTSD12. The course comprises text-based information and instructions and educational case stories.\n\nLesson 1 of the iCBT/IEMDR course includes information about the causes, symptomatology and neurobiology of PTSD, how cognitive, behavioural, and physical symptoms maintain PTSD, and provides instructions for physiological de-arousal strategies. Lesson 2 provides the rationale for using EMDR and detailed instructions about a self-guided iEMDR process. Lesson 3 describes cognitive restructuring strategies. Lesson 4 provides more detail on how to use cognitive restructuring for common trauma-related cognitions. Lesson 5 describes avoidance and safety behaviours and the principles of graded exposure. Lesson 6 describes the principles of relapse prevention.\n\niEMDR Intervention: The EMDR intervention follows the standard EMDR treatment protocol by Shapiro25 with the following adaptations for self-directed use via the internet: the protocol was divided into a desensitisation phase (weeks 2–4) followed by a phase aimed at anchoring the positive belief (weeks 5–6). The desensitization phase followed Shapiro’s protocol for reducing the Subjective Units of Distress (SUDS) and Validity of Cognition (VoC) rating to less than 2. Participants were instructed to anchor the positive belief in week 5 of the course only for trauma memories that were no longer distressing (VoC < 2).\n\niEMDR was conducted using a web-based EMDR tool (http://www.rapidtables.com/tool/EMDR.htm). The initial session of EMDR was conducted with the support of the therapist (JS) who guided participants by telephone through the procedure while they accessed the web-based EMDR tool. Further therapist-guided EMDR was provided as requested. Participants who reported not having used self-guided EMDR by mid-treatment were contacted and offered a second guided EMDR session. Instructions for working with blockages to processing were provided in an additional resource one week after giving the iEMDR instructions.\n\nOne Clinical Psychologist (JS) provided all clinical contact with participants, which occurred via weekly telephone calls or secure email. The clinician had received Level I and II training in EMDR by a certified EMDR instructor, and had two years experience in administering iCBT and in facilitating EMDR in face-to-face treatment. The clinician was supervised by NT.\n\nPrimary analyses were conducted using data only from questionnaire completers, defined as those who completed treatment, post-treatment or follow-up questionnaires. A secondary set of analyses was performed using an intention-to-treat (ITT) model where missing data were addressed by carrying forward the first available data (i.e. baseline-observation-carried-forward model; BOCF).\n\nPre- to post-treatment and pre-treatment to follow-up changes in questionnaire scores were analysed using paired-sample t-tests. Effect sizes (Cohen’s d)26 were calculated based on the pooled standard deviation. All analyses were performed in PASW version 18.0 (SPSS, Inc., Chicago, IL).\n\nChanges in prevalence of PTSD and comorbid disorders were calculated based on the results of telephone administered diagnostic interviews administered at pre-treatment, post-treatment and follow-up.\n\nTo measure adverse events we used Tarrier’s27 definitions of treatment worsening, defined as any increase in symptom scores greater than zero from pre- to post-treatment or follow up, and defined serious adverse events as self-reported hospitalizations, suicide attempts, or onset of substance abuse due to treatment.\n\n\nResults\n\nThe mean age of participants was 47 years (SD = 10.4), and 10/15 (66%) were women. Ten of 15 participants (67%) reported being either married or in a de facto relationship, 4/15 (27%) reported being separated or widowed and 1/15 (7%) reported being single or never married. Four of fifteen (27%) had a tertiary education, 9/15 (60%) reported having a post-high school certificate and 2/15 (13%) reported as having year 10 high school level education. One participant (7%) was in full-time employment, eight (53%) were employed part-time or studying and six (40%) reported being unemployed, retired, or disabled. Fourteen of fifteen participants (93%) reported having had previous mental health treatment and 10/15 (67%) reported taking medication related to their symptoms of anxiety or depression. One half (5/10) of the participants who completed post-treatment questionnaires reported that they were receiving individual or group supportive counselling during the treatment period that was not specifically directed at the treatment of PTSD symptoms (mean sessions = 3; SD = 2.1). Between post-treatment and follow-up, 25% (2/8) of respondents reported receiving ongoing supportive therapy (not specifically for PTSD) and 13% (1/8) commenced treatment with a psychologist specifically for PTSD (mean sessions = 4; SD = 3.5). There were no reported medication changes during the course. One quarter (2/8) of respondents reported changing their medication post-treatment. Five participants (33%) who reported not having used self-guided EMDR by mid-treatment were contacted and offered a second EMDR session guided by the therapist via telephone. None elected to participate in further EMDR, citing that EMDR had led to an increase in re-experiencing symptoms.\n\nThe most common reported primary trauma was childhood sexual abuse (9/15; 60%), followed by childhood physical abuse (2/15; 13%), domestic violence as an adult (2/15; 13%), witnessing domestic violence as a child (1/15; 7%), captivity (1/15; 7%) and life threatening illness (1/15; 7%). On average, the primary trauma had occurred 32.8 years prior (SD = 12.5). The average age at which the primary trauma occurred was 13.3 years (SD = 12.9). According to the LEC, participants reported having experienced an average of 9.2 types of trauma during their lifetime. The most common was physical assault (13/15; 87%), followed by assault with a weapon (12/15; 80%), and other unwanted or uncomfortable sexual experience (12/15; 80%).\n\nThe flow is shown in Figure 1. Eleven participants (73%) completed all six lessons. One participant completed a single lesson, two participants completed two lessons and one participant completed six lessons, but not the post-treatment assessments. There were no pre-treatment differences between completers and non-completers on the PSS-I, PCL-C or the GAD-7 at pre-treatment. Ten participants completed post-treatment questionnaires while eight completed follow-up questionnaires.\n\nPrimary outcome measures. Primary outcome scores for completers improved from pre- to post-treatment as shown in Table 1. Paired-sample t-tests revealed significant reductions on the PSS-I (t10 = 3.66, p = 0.004) and PCL-C (t10 = 2.73, p = 0.021) between pre- and post-treatment, and between pre-treatment and follow-up (PSS-I: t10 = 4.90, p = 0.001; PCL-C: t10 = 4.26, p = 0.002).\n\nNote. Intention-to-treat (ITT) model (n=15) was employed with pre-treatment scores carried forward if post-treatment or follow-up data were not available. Completer data were available for 10 participants at post-treatment and 8 at follow-up. Abbreviations: PSS-I: PTSD Symptom Scale – Interview Version; PCL-C: PTSD Checklist – Civilian Version; GAD-7: Generalised Anxiety Disorder 7-Item; PHQ-9: Patient Health Questionnaire – 9 Item; K10: Kessler 10 Item; SDS: Sheehan Disability Scale.\n\nSecondary outcome measures. Paired sample t-tests between pre- and post-treatment indicated significant reductions for completers on the PHQ-9 (t9 = 2.66, p = 0.026), GAD-7 (t9 = 2.31, p = 0.047), K10 (t9 = 2.49, p = 0.034), but not on the SDS (t9 = 1.66, p = 0.131). Significant reductions were reported between pre-treatment and follow-up on the PHQ-9 (t7 = 3.13, p = 0.017), GAD-7 (t7 = 4.16, p = 0.004), K10 (t7 = 3.95, p = 0.006), and SDS (t7 = 4.15, p = 0.004).\n\nPrimary outcome measures. A paired-sample t-test comparing pre- and post-treatment scores for the ITT sample revealed significant reductions on the PSS-I (t14 = 3.50, p = 0.004), and this was maintained at follow up (t14 = 4.59, p < 0.0001). Scores on the PCL-C did not significantly improve from pre- to post-treatment (t14 = 2.12, p = 0.053). However, at follow-up, scores on the PCL-C had significantly improved from pre-treatment (t14 = 17.76, p < 0.0001).\n\nSecondary outcome measures. Paired sample t-tests for the ITT sample revealed significant reductions between pre- and post-treatment on the K10 (t14 = 2.20, p = 0.046) but not on the PHQ-9 (t14 = 2.12, p = 0.053), GAD-7 (t14 = 2.02, p = 0.063), or SDS (t14 = 1.22, p = 0.281). There was a significant difference between pre-treatment and follow-up scores on the PHQ-9 (t14 = 2.46, p = 0.027), GAD-7 (t14 = 2.90, p = 0.012), K10 (t14 = 3.10, p = 0.008), but not on the SDS (t14 = 2.08, p = 0.056).\n\nUsing the completer analysis, large effect sizes were reported on the PSS-I, PCL-C, GAD-7, and K10 at post-treatment and a moderate effect size was reported on the PHQ-9 and SDS (Table 1). Large effect sizes were reported on all measures between pre-treatment and follow-up.\n\nUsing the ITT analysis, from pre-treatment to post-treatment a large within-group effect size was found on the PSS-I. Moderate within-group effects were found on the GAD-7, PHQ-9, and K10. A small effect size was reported on the SDS. From pre-treatment to follow-up, large effect sizes were found on the PSS-I, PCL-C, and GAD-7, and moderate effect sizes for the PHQ-9, and SDS.\n\nBased on the results of the clinician and telephone-administered PSS-I, 6/11 (55%) participants no longer met criteria for PTSD at post-treatment and 5/9 (56%) no longer had PTSD at follow-up. Based on an ITT approach with the BOCF, 5/15 (33%) no longer met criteria for PTSD at post-treatment and follow-up.\n\nWith regard to co-morbid diagnoses for completers as measured by clinician-administered MINI, the average number of co-morbid diagnoses reduced from 2.5 (SD=2.0) at intake to 1.2 (SD=1.0) at post-treatment, and further reduced to 0.6 (SD=1.6) at follow-up. According to an ITT analysis the average number of co-morbid diagnoses reduced from 2.5 (SD=1.7) at intake to 1.4 (SD=0.9) at post-treatment, and 1.1 (SD=1.1) at follow-up.\n\nThree participants reported symptom worsening as defined by Tarrier27 and no participants reported serious adverse events. Of the participants who completed post-treatment questionnaires, three participants showed symptom worsening between pre- and post-treatment on the PCL-C, and one of these had dropped out of treatment after the third lesson. All three improved between post-treatment and follow-up such that no participants worsened between pre-treatment and follow-up. No participants worsened on the PSS-I between any time points.\n\nAt post-treatment, 6/11 (55%) reported that they were very satisfied with the course, one participant (9%) was mostly satisfied, and 4/11 were neutral or somewhat satisfied. None of the participants reported being dissatisfied with the course. Nine of 11 (82%) reported they would recommend this course to a friend with PTSD.\n\n\n\n\nDiscussion\n\nThis study explored the feasibility of a combined iCBT/iEMDR course for treating PTSD in adults using an open-trial design. The results indicated significantly reduced symptoms of PTSD, depression, anxiety, distress, and disability between pre-treatment and three-month follow-up. By post-treatment, 55% of the participants no longer met criteria for PTSD, and the number of comorbid diagnoses had halved. These reductions indicate that PTSD can be treated via the internet using a combination of CBT and EMDR techniques. With respect to acceptability, this protocol was moderately tolerated, indicating that improvements would be required for further use of this intervention.\n\nCompared with ITT data from our previous trial12, the within-group effect size (ES) on the PCL-C was lower at post-treatment and follow-up. These differences may be due to changes to the protocol, the use of a patient sample composed primarily of childhood sexual abuse survivors, or due to the influence of attrition on the ITT analysis as a result of using a small sample. However, these results compare favourably to a similar study of iCBT for PTSD in an Australian sample11, as well as several face-to-face interventions for PTSD that used the PCL-C28,29, but less favourably with results of a face to face trial of TF-CBT for motor vehicle accident survivors (Blanchard, 2003 #558).\n\nIn our trial, 3/15 (20%) reported symptom worsening between pre- and post-treatment, although all three reported treatment gains by follow-up. Although no serious adverse events (e.g., hospitalizations, suicide attempts, relapse to substance use) occurred during the program, three participants reported that an increase in re-experiencing symptoms led them to discontinue. This potentially contributed to the higher attrition, moderate acceptability, and limited course and questionnaire completion rates, relative to our earlier study.\n\nThe absence of a waitlist control condition means that the improvements could have been the results of time, repeated measurement or other non-specific effects. The design did not allow determination of whether the effects were due to the iCBT or iEMDR components. The small sample size composed of a high number of multiply traumatized, childhood sexual abuse survivors may not apply to other PTSD populations.\n\n\nConclusions\n\nThe results of this small feasibility study indicate that the combined iCBT/iEMDR protocol is potentially efficacious. The magnitude of gains did not appear to be as large as our previous study, although these may have been attenuated by differences in the sample and iCBT protocol. These results indicate that future research of the relative benefits of iCBT/iEMDR is warranted.", "appendix": "Author contributions\n\n\n\nJS and NT Conceived the study and design, analysed and interpreted the data, and drafted the article. LJ, BFD, BW, MT and JZ Contributed to the design of the study and the revision of the manuscript.\n\n\nCompeting interests\n\n\n\nThere are no competing interests for any author.\n\n\nGrant information\n\nThe author(s) would like to thank the New South Wales Institute for Psychiatry (NSWIOP) for funding this research.\n\n\nReferences\n\nBisson J, Andrew M: Psychological treatment of post-traumatic stress disorder (PTSD). Cochrane Database Syst Rev. 2007; (3): CD003388. PubMed Abstract \| Publisher Full Text\n\nSpence J, Titov N, Solley K, et al.: Characteristics and treatment preferences of people with symptoms of posttraumatic stress disorder: an internet survey. PLoS One. 2011; 6(7): e21864. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrusz SG, Wagner AW, Russo J, et al.: Assessing barriers to care and readiness for cognitive behavioral therapy in early acute care PTSD interventions. Psychiatry. 2011; 74(3): 207–23. PubMed Abstract \| Publisher Full Text\n\nRuzek JI, Rosen RC: Disseminating evidence-based treatments for PTSD in organizational settings: A high priority focus area. Behav Res Ther. 2009; 47(11): 980–9. PubMed Abstract \| Publisher Full Text\n\nLitz BT, Bryant R, Williams L, et al.: A therapist-assisted internet self-help program for traumatic stress. Professional Psychology: Research and Practice. 2004; 35(6): 628–34. Publisher Full Text\n\nPossemato K, Ouimette P, Knowlton P: A brief self-guided telehealth intervention for post-traumatic stress disorder in combat veterans: a pilot study. J Telemed Telecare. 2011; 17(5): 245–50. PubMed Abstract \| Publisher Full Text\n\nHirai M, Clum GA: An internet-based self-change program for traumatic event related fear, distress, and maladaptive coping. J Trauma Stress. 2005; 18(6): 631–6. PubMed Abstract \| Publisher Full Text\n\nLittleton H, Buck K, Rosman L, et al.: From Survivor to Thriver: A Pilot Study of an Online Program for Rape Victims. Cogn Behav Pract. 2012; 19(2): 315–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLange A, Rietdijk D, Hudcovicova M, et al.: Interapy: a controlled randomized trial of the standardized treatment of posttraumatic stress through the internet. J Consult Clin Psychol. 2003; 71(5): 901–9. PubMed Abstract \| Publisher Full Text\n\nWagner B, Schulz W, Knaevelsrud C: Efficacy of an Internet-based intervention for posttraumatic stress disorder in Iraq: a pilot study. Psychiatry Res. 2012; 195(1–2): 85–8. PubMed Abstract \| Publisher Full Text\n\nKlein B, Mitchell J, Gilson K, et al.: A therapist-assisted Internet-based CBT intervention for posttraumatic stress disorder: preliminary results. Cogn Behav Ther. 2009; 38(2): 121–31. PubMed Abstract \| Publisher Full Text\n\nSpence J, Titov N, Dear BF, et al.: Randomized controlled trial of Internet-delivered cognitive behavioral therapy for posttraumatic stress disorder. Depress Anxiety. 2011; 28(7): 541–50. PubMed Abstract \| Publisher Full Text\n\nKnaevelsrud C, Maercker A: Internet-based treatment for PTSD reduces distress and facilitates the development of a strong therapeutic alliance: a randomized controlled clinical trial. BMC Psychiatry. 2007; 7: 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlanchard EB, Jones-Alexander J, Buckley TC, et al.: Psychometric properties of the PTSD checklist (PCL). Behav Res Ther. 1996; 34(8): 669–73. PubMed Abstract \| Publisher Full Text\n\nWeathers F, Litz BT, Herman D, et al.: The PTSD checklist (PCL): Reliability, validity, and diagnostic utility. Annual Conference of the International Society for Traumatic Stress Studies; San Antonio, TX 1993.\n\nFoa EB, Riggs DS, Dancu CV, et al.: Reliability and validity of a brief instrument for assessing post-traumatic stress disorder. J Trauma Stress. 1993; 6(4): 459–73. Publisher Full Text\n\nKroenke K, Spitzer RL, Williams JB: The PHQ-9: validity of a brief depression severity measure. J Gen Intern Med. 2001; 16(9): 606–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDalenberg C, Carlson E: editors. New versions of the Dissociative Experiences Scale: The DES-R (revised) and the DES-B (brief). Annual meeting of the International Society for Traumatic Stress Studies, November, Montreal, Quebec; 2010.\n\nSpitzer RL, Kroenke K, Williams JBW, et al.: A brief measure for assessing generalized anxiety disorder: the GAD-7. Arch Intern Med. 2006; 166(10): 1092–7. PubMed Abstract \| Publisher Full Text\n\nSheehan DV, Lecrubier Y, Sheehan KH, et al.: The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry. 1998; 59(Suppl 20): 22–33. PubMed Abstract\n\nKessler RC, Andrews G, Colpe LJ, et al.: Short screening scales to monitor population prevalences and trends in non-specific psychological distress. Psychol Med. 2002; 32(6): 959–76. PubMed Abstract \| Publisher Full Text\n\nSheehan DV: The Anxiety Disease. New York, NY: Scribner; 1983. Reference Source\n\nGray MJ, Litz BT, Hsu JL, et al.: Psychometric properties of the life events checklist. Assessment. 2004; 11(4): 330–41. PubMed Abstract \| Publisher Full Text\n\nFoa EB, Hembree EA, Rothbaum BO: Prolonged exposure therapy for PTSD: Emotional processing of traumatic experiences: Therapist guide. Oxford University Press, USA; 2007. Reference Source\n\nShapiro F: Eye movement desensitization and reprocessing: Basic principles, protocols, and procedures. The Guilford Press; 2001. Reference Source\n\nCohen J: A power primer. Psychol Bull. 1992; 112(1): 155–9. PubMed Abstract \| Publisher Full Text\n\nTarrier N, Pilgrim H, Sommerfield C, et al.: A randomized trial of cognitive therapy and imaginal exposure in the treatment of chronic posttraumatic stress disorder. J Consult Clin Psychol. 1999; 67(1): 13–8. PubMed Abstract \| Publisher Full Text\n\nMonson CM, Schnurr PP, Resick PA, et al.: Cognitive processing therapy for veterans with military-related posttraumatic stress disorder. J Consult Clin Psychol. 2006; 74(5): 898–907. PubMed Abstract \| Publisher Full Text\n\nSchnurr PP, Friedman MJ, Engel CC, et al.: Cognitive behavioral therapy for posttraumatic stress disorder in women: A randomized controlled trial. JAMA. 2007; 297(8): 820–30. PubMed Abstract \| Publisher Full Text" }	[ { "id": "825", "date": "11 Mar 2013", "name": "Alyssa Boasso", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, the writing is succinct and clear. The analyses are appropriate given the study design and the conclusions are justified. There are a few minor areas where further explanation or clarification is needed, these are detailed below. What is the rationale for combining the two therapies? How is iEMDR expected to add to the effectiveness of pre-existing iCBT protocols? The section on recruitment does not explain how 23 people qualified, but only 16 were enrolled. In the iEMDR Intervention subsection, the authors state, “the positive belief” but do not previously define it. Also, how many people were provided special therapist-guided EMDR? Is there conjecture about how additional sessions might have affected outcomes? In the discussion section, the authors state “these results compare favourably to a similar study...” How is the study similar? Clarifying this may help contrast your findings with the following study which used motor vehicle accident survivors. The section on worsening symptoms should address whether other similar studies have encountered the same problem? Also, is there data from the experiment that suggests which aspect of the therapy may be contributing to this issue?", "responses": [] }, { "id": "857", "date": "21 Mar 2013", "name": "Jo Abbott", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important study in that in that it is exploring the feasibility of an online intervention for PTSD that includes eye movement desensitization as well as CBT. These early stage research studies evaluating novel interventions are important prior to controlled trials and it will be interesting to see how future research can identify the benefits of each component of the intervention.There were a couple of parts of the flow chart that could be a bit clearer – I too didn't follow where the 16 participants meeting all criteria came from. I also couldn't follow in the flow chart where the following information in the Attrition section fitted: “…one participant completed six lessons, but not the post-treatment assessments”, “Ten participants completed post-treatment questionnaires while eight completed follow-up questionnaires.”Regarding reference 11 (Klein et al., 2009) – there is a more recent paper about this same trial that includes follow-up data (Klein et al., 2010) – of relevance given the comparison made to this trial.I think it is relevant to mention in the discussion that this was a telephone-assisted intervention (not entirely internet-delivered).I thought that the interpretation of the results would be facilitated if the authors made comment on which of the analyses (ITT or completer) they were placing weight on in drawing their conclusion and why. For example, in the discussion they state that “The results indicated significantly reduced symptoms of PTSD, depression, anxiety, distress, and disability between pre-treatment and three-month follow-up.”, but the disability measure was only significantly different between the groups when using completer analyses. It would also be useful if Table 1 also noted which of the mean differences were significant (rather than the reader needing to refer back to the text).I was also interested to know more about the worsening symptoms that some participants reported. Also, the Discussion states that 3/15 participants reported worsening symptoms but in the Results it reads as though five participants stopped using EMDR because they said it “led to an increase in re-experiencing symptoms”.", "responses": [ { "c_id": "423", "date": "24 Mar 2013", "name": "Jay Spence", "role": "Author Response", "response": "Dear Dr Abbott, Thank you for your feedback. We have addressed your suggestions in an updated manuscript that has recently been uploaded. We would welcome any other edits that you feel are required if they are not adequately addressed in the revised publication. Thank you again for your time in improving this paper. Kind regards, Jay Spence" } ] } ]	1	https://f1000research.com/articles/2-79
https://f1000research.com/articles/2-121/v1	03 May 13	{ "type": "Research Article", "title": "Generalized assessment of the impact of Parkinson’s disease on functional condition", "authors": [ "Olena Myalovitska", "Iryna Lobanova", "Olena Myalovitska" ], "abstract": "Individuals who develop Parkinson's disease are confronted not only with the physical manifestations of the disorder, but also with the psychosocial issues that impact on quality of life. The aim of the current work was to assess the level of psychosocial and physical functioning impairment in 95 patients with Parkinson’s disease (48 men and 47 women aged 65–79, average age 70.3±0.5, stages of Parkinson’s disease acсording to Hoehn and Yahr scale - 2–2.5). This research was carried out with the help of “Functional Limitation Profile” and “Sickness Impact Profile – 68” tests.According to the “Functional Limitation Profile” test, the patients with a diagnosis of Parkinson’s disease most commonly showed functional condition impairment in 3 categories: “walk” (92% of cases), “social interaction” (85% of cases) and “clarity of mind” (79% of cases). Less frequent condition impairments were changes in the categories “body care and motion” (72% of cases), “emotions” (65% of cases) and “work” (56% of cases). The least impaired were the categories “communication” (22% of cases), “leisure and entertainment” (24% of cases) and “food” (19% of cases). According to the “Sickness Impact Profile – 68” test, patients with Parkinson’s disease showed functional condition impairment most often in the categories “mobility control”, “social behavior” and “degree of mobility”. Patients showed impairment less frequently in the categories “somatic autonomy”, “psychic autonomy and communication”. The least impaired category was “emotional stability”. The highest percent in relation to the maximum possible point was shown in the category “mobility control”, and the lowest in the category “emotional stability”.Thus, this study demonstrates significant impact of the disease on the functional condition of these patients. Functional limits, connected with the disease appearance, are most often present in the motion and social sphere of patients’ life and less frequently in the emotional sphere.", "keywords": [ "Parkinson’s disease is a chronic degenerative neurological disorder that adversely affects an individual’s motor functioning", "which leads to disability", "and impacts on quality of life. Individuals who develop Parkinson's disease are confronted not only with the physical manifestations of the disorder", "but with the psychosocial issues that also impact on quality of life. Psychosocial aspects of Parkinson's disease may present as subtle changes with progression of the disease1–5." ], "content": "Introduction\n\nParkinson’s disease is a chronic degenerative neurological disorder that adversely affects an individual’s motor functioning, which leads to disability, and impacts on quality of life. Individuals who develop Parkinson's disease are confronted not only with the physical manifestations of the disorder, but with the psychosocial issues that also impact on quality of life. Psychosocial aspects of Parkinson's disease may present as subtle changes with progression of the disease1–5.\n\nThe purpose of the present research was to assess the level of psycho-social and physical functioning impairment in patients with Parkinson’s disease.\n\n\nMethods\n\n95 patients with Parkinson’s disease have been examined (48 men and 47 women aged 65–79, average age 70,3±0,5). An interview and complete neurologic examination, including the Hoehn and Yahr scale6, were performed on the same day. Stages of Parkinson’s disease acсording to Hoehn and Yahr scale were 2–2.5.\n\nHaving the aim to assess the impact of the disease on the everyday life of patients to the fullest extent, their interviewing included the use of the tests “Functional Limitation Profile” and “Sickness Impact Profile–68”7–15.\n\nWhile carrying out “Functional Limitation Profile” test, the change of patient’s behavior in cases of Parkinson’s disease was assessed in 12 categories of life activity: “walk”, “body care and motion”, “movement”, “household activity”, “leisure and entertainment”, “social interaction”, “emotions”, “clarity of mind”, “sleep and rest”, “food”, “communication”, “work”.\n\nWhile carrying out “Sickness Impact Profile–68” test, functional condition of patients was assessed in 6 categories of life activity. “Somatic autonomy” category reflected the extent of help that the patient requires when performing everyday activities (dressing, standing, walking, eating etc). “Mobility control” category characterized the degree of control of motion functions, including walking and actions with hands. “Psychic autonomy and communication” category described behavior, connected with mental functions and verbal communication. ”Mobility degree” category included the ways the disease limits household and professional activity. \"Social Behavior” category reflects social sphere of activity. ”Emotional stability” category reflects disease impact on the emotional sphere.\n\n\nResults\n\nWhile carrying out “Functional Limitation Profile” test, all patients showed functional conditions changes. Impairments in the category “walk” were present in 87 patients (92% of cases). Most patients noted a slowing down in walking speed (92%), shortening of the distance they could walk without rest (79%) and the need for outside assistance during walking (44%).\n\nChanges in the category \"body care and motion\" were observed in 69 patients (72% of cases). Impairments of this category were connected with slowing of movements–bradykinesia (85%), patients’ reduced ability to maintain balance (72%) and the need for outside assistance (43%).\n\nLimitations in the category \"movement\" were present in 42 patients (44% of cases) and were manifested in the inability to leave the house (15%) or room (7%), the need for support at a certain time of the day (22%).\n\nIn the category \"household activity\" changes were observed in 40 patients (42% of cases), and consisted of reducing the amount of housework (80%), increasing the time needed for rest (63%), and inability to perform hard work (63%).\n\nIn the category \"leisure and entertainment” impairments were present in 23 patients (24% of cases). The patients showed decreased period of time spent on leisure (65%) and sports (52%).\n\nChanges in the category \"social interaction\" were observed in 81 patients (85% of cases). Patients rarely participated in social activities (93%), and isolated themselves from the work team (51%).\n\nChanges in the category \"emotion\" were noticed in 62 patients (65% of cases). Patients demonstrated depressed mood background (65%), talked about the future with hopelessness (85%).\n\nImpairments in the category \"clarity of mind\" were noticed in 75 patients (79% of cases). Patients showed impairment of memory (85%) and concentration (85%), slowing of thought processes (80%).\n\nIn the category \"sleep and rest\", patients showed sleepiness in the daytime and insomnia at night (41%) and required additional time for rest because of constant fatigue (80%). Changes in this category were noted in 19 patients (38% of cases).\n\nIn the category \"food\", impairments were observed in 18 patients (19% of cases). The most common impairments included decreased appetite (55%), and need for outside assistance when eating (17%).\n\nChanges in the category \"communication\" were observed in 21 patients (22% of cases). Patients had problems with writing (93%), and difficulties in communication because of speech dysfunction (85%).\n\nImpairments in the category \"work\" were present in 53 patients (56% of cases). Some of the patients did not work at all (57%), whilst some performed some of the work at home (19%), or did less work than usual (85%), worked fewer hours (80%), performed only light work (42%), or produced lower-quality work (42%). The frequency of functional limitations is given in Table 1 and Figure 1.\n\nThe frequency of functional limitation cases in patients with Parkinson’s disease according to “Sickness Impact profile–68” test and their assessment in points are given in Table 2, Table 3 and Figure 2, Figure 3.\n\n\nConclusion\n\nThe analysis of investigation of patients with Parkinson’s disease (duration 3–5 years) according to the tests “Functional Limitation Profile” and “Sickness Impact Profile–68” showed significant changes of their functional condition connected with the occurrence of the disease.\n\nThe patients with Parkinson's disease according to the test “Functional Limitation Profile” showed functional condition impairment most often in 3 categories: “walk\", \"social interaction\" and \"clarity of mind\", while changes in the categories \"body care and motion\", \"emotion\" and \"work\" were less frequent. The least impaired were the categories \"communication\", \"food\" and \"leisure and entertainment\".\n\nThe patients with Parkinson's disease diagnosis according to the test “Sickness Impact Profile–68” showed functional condition impairment most often in the categories \"mobility control\", \"social behavior\" and \"degree of mobility\", less frequently in the categories \"somatic autonomy\" and \"psychic autonomy and communication\". The least impaired category was \"emotional stability\". The highest percent in relation to the maximum possible point was observed in the category “mobility control\", and the lowest in the category “emotional stability”.\n\nThus, according to the results of two tests, the presence of the disease affects the motion and social sphere of life in the patients with Parkinson’s disease the most and their emotional state the least.\n\nFor the most accurate assessment of the disease impact on the functional condition of the patients diagnosed with Parkinson's disease, a secondary investigation (depending on the rate of the disease progression) should be carried out. Such interrogation of disease dynamics enables detection of possible changes in the categories of life that are most affected by the disease occurrence. Assessment of the quality of life of patients diagnosed with Parkinson's disease at different stages of the disease with the help of Hoehn and Yahr Scale is a very perspective direction of future studies.", "appendix": "Author contributions\n\nO. Myalovitska and Lobanova conceived the study. Lobanova designed the experiments and carried out the research. All authors prepared the first draft of the manuscript and were involved in the revision of the graft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\nNo relevant competing interests were disclosed.\n\n\nReferences\n\nBergner M, Bobbit RA, Carter WB, et al:The Sickness impact profile: development and final revision of a health status measure. Med Care. 1981; 19(8): 787–805.\n\nBergner M, Bobbitt RA, Kressel S, et al:The sickness impact profile: conceptual formulation and methodology for the development of a health status measure. Int J Health Serv. 1976; 6(3): 393–415.\n\nBergner M, Bobbitt RA, Pollard WE, et al:The sickness impact profile: validation of a health status measure. Med Care. 1976; 14(1): 57–67.\n\nde Bruin AF, Diederiks JP, de Witte LP, et al:Assessing the responsiveness of a functional status measure: the Sickness Impact Profile versus the SIP68. J Clin Epidemiol. 1997; 50(5): 529–40.\n\nNanda U, McLendon PM, Andresen EM, et al:The SIP68: an abbreviated sickness impact profile for disability outcomes research. Qual Life Res. 2003; 12(5): 583–595.\n\nHoehn MM, Yahr MD: Parkinsonism: onset, progression and mortality. Neurology. 1967; 17: 427–442.\n\nChrischilles EA, Rubenstein LM, Voelker MD, et al:Linking clinical variables to health-related quality of life in Parkinson's disease. Parkinsonism Relat Disord. 2002; 8(3): 199–209.\n\nFindley L, Eichhorn T, Janca F: Factors impacting on quality of life in Parkinson's disease: results from an international survey. Mov Disord. 2002; 17(1): 60–67.\n\nHanna KK, Cronin-Golomb A: Impact of Anxiety on Quality of Life in Parkinson's Disease. Parkinsons Dis. 2012; 2012: 640707.\n\nKuopio AM, Marttila RJ, Helenius H, et al:The quality of life in Parkinson's disease. Mov Disord. 2000; 15(2): 216–223.\n\nSchrag A: Quality of life and depression in Parkinson’s disease. J Neurol Sci. 2006; 248: 151–157.\n\nSchrag A, Jahanshahi M, Quinn N: How does Parkinson's disease affect quality of life? A comparison with quality of life in the general population. Mov Disord. 2000; 15(6): 1112–1118.\n\nSchrag A, Jahanshahi M, Quinn N: What contributes to quality of life in patients with Parkinson’s disease? J Neurol Neurosurg Psychiatry. 2000; 69: 308–12.\n\nSchenkman M, Ellis T, Christiansen C, et al:Profile of Functional Limitations and Task Performance Among People With Early- and Middle-Stage Parkinson Disease. Phys Ther. 2011; 91(9): 1339–1354.\n\nWilliams SJ: Measuring health status? A review of the Sickness Impact and functional limitations profiles. Health Care Anal. 1996; 4(4): 273–283." }	[ { "id": "943", "date": "13 May 2013", "name": "Alberto Albanese", "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 methodology seems to be poor and is insufficiently described. There is no control group.", "responses": [ { "c_id": "585", "date": "16 Oct 2013", "name": "Iryna Lobanova", "role": "Author Response", "response": "The quality of life was assessed using “Functional Limitation Profile” and “Sickness Impact Profile - 68” tests. These allow for assessment of the physical and psychological health of patients and their social functions, as well as global self-assessment of their health. Investigating using this scale does not require a control group of virtually healthy people because the changes assessed by this scale can be observed only among sick people. We used this methodology of generalized quality of life assessment on patients with Parkinson’s disease (which is reflected in the introduction to our work)." } ] }, { "id": "1909", "date": "26 Sep 2013", "name": "Iracema Leroi", "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 an uncontrolled study using assessments tools that do not appear to have been validated in Parkinson's disease. The description of the methodology, particularly the recruitment and testing procedure, is insufficient. The literature on the topic has not been adequately reviewed. The authors have not included a Discussion of their findings. The analysis is simplistic.", "responses": [ { "c_id": "584", "date": "16 Oct 2013", "name": "Iryna Lobanova", "role": "Author Response", "response": "The quality of life was assessed using “Functional Limitation Profile” and “Sickness Impact Profile - 68” tests. These allow for assessment of the physical and psychological health of patients and their social functions, as well as global self-assessment of their health. Investigating using this scale does not require a control group of virtually healthy people because the changes assessed by this scale can be observed only among sick people. We used this methodology of generalized quality of life assessment on patients with Parkinson’s disease (which is reflected in the introduction to our work)." } ] } ]	1	https://f1000research.com/articles/2-121
https://f1000research.com/articles/2-117/v2	02 May 13	{ "type": "Web Tool", "title": "IsoCleft Finder – a web-based tool for the detection and analysis of protein binding-site geometric and chemical similarities", "authors": [ "Natalja Kurbatova", "Matthieu Chartier", "María Inés Zylber", "Rafael Najmanovich", "Natalja Kurbatova", "Matthieu Chartier", "María Inés Zylber" ], "abstract": "IsoCleft Finder is a web-based tool for the detection of local geometric and chemical similarities between potential small-molecule binding cavities and a non-redundant dataset of ligand-bound known small-molecule binding-sites. The non-redundant dataset developed as part of this study is composed of 7339 entries representing unique Pfam/PDB-ligand (hetero group code) combinations with known levels of cognate ligand similarity. The query cavity can be uploaded by the user or detected automatically by the system using existing PDB entries as well as user-provided structures in PDB format. In all cases, the user can refine the definition of the cavity interactively via a browser-based Jmol 3D molecular visualization interface. Furthermore, users can restrict the search to a subset of the dataset using a cognate-similarity threshold. Local structural similarities are detected using the IsoCleft software and ranked according to two criteria (number of atoms in common and Tanimoto score of local structural similarity) and the associated Z-score and p-value measures of statistical significance. The results, including predicted ligands, target proteins, similarity scores, number of atoms in common, etc., are shown in a powerful interactive graphical interface. This interface permits the visualization of target ligands superimposed on the query cavity and additionally provides a table of pairwise ligand topological similarities. Similarities between top scoring ligands serve as an additional tool to judge the quality of the results obtained. We present several examples where IsoCleft Finder provides useful functional information. IsoCleft Finder results are complementary to existing approaches for the prediction of protein function from structure, rational drug design and x-ray crystallography. IsoCleft Finder can be found at: http://bcb.med.usherbrooke.ca/isocleftfinder.", "keywords": [ "Ligand", "Tanimoto", "protein structure", "Pfam", "PDB" ], "content": "Introduction\n\nCurrent computational methods for the prediction of protein function from structure are restricted to the transfer of functional annotation based on similarities. In instances where global sequence or structural similarities do not provide clues about protein function, one alternative is to detect binding-site similarities. Such similarities may help predict potential small-molecules that bind to the protein, thus providing valuable functional information. Binding-site similarities may also help recognize atoms involved in non-bonded interactions thus aiding in the rational design of inhibitors.\n\nMethods for the detection of local structural similarities vary primarily in the type of representation (generally simplified) and search method, usually via the detection of sub-graph isomorphism1 or geometric hashing2. However, due to the time-consuming nature of the detection of sub-graph isomorphism, methods have resorted mostly to simplifications in the form of pseudo-atoms3,4 or simplified surface patches5. Shulman-Peleg et al.6,7 combined the simplified representation, graph-matching-based method of Schmitt et al.4 with a geometric hashing pre-screening step. With the exception of IsoCleft8, methods that make use of full atomic representation, i.e. utilizing the coordinates of all non-hydrogen atoms, are few and only applicable in limited cases. The method of Kobayashi & Go9 requires the superimposition of bound ligands. Brakoulias & Jackson10 use a geometric method to compare a large dataset of molecular environments of phosphate groups that also require the pre-definition of the molecular environments from the position of the PO4 groups. A more thorough review of methods for the detection of local structural similarities can be found in Najmanovich et al.11 and references therein. The IsoCleft program, developed for the detection of binding-site 3D local atomic similarities8 uses an all atom (non-simplified) representation, in a two-stage graph-matching process. IsoCleft can compare large binding sites in a timely manner and does not need any information regarding bound ligands.\n\nThis section recapitulates the description of the IsoCleft program, the engine behind the IsoCleft Finder tool, as presented previously8. IsoCleft utilizes graphs to conceptually represent binding-sites in order to take advantage of the well-developed graph-matching techniques for the detection of similarities between graphs (sub-graph isomorphism). A graph is defined by a set of nodes and edges where each edge connects a pair of nodes through a specific property. Graphs can be used to represent any entity (real objects, ideas, etc.) composed of smaller parts (nodes) and their (pairwise) relationships to each other (edges). Given two sets of atoms defining the query cleft and a target binding-site under comparison, the question that needs to be answered is: what is the largest subset of atoms in both clefts in direct correspondence with each other geometrically as well as chemically?\n\nIn the representation of a binding site as a graph, nodes represent atoms Aim (atom i out of a total of Nm in molecule m) while an edge represents the Euclidian distance between any two atoms. In other words, in this representation of a binding-site all atoms define nodes and all nodes are interconnected by edges. Both nodes and edges are assigned properties (referred as colorings in graph theory). Nodes (atoms) are assigned an atom type property that determines the types of molecular interactions they may participate in. IsoCleft employs the atom type scheme of Sobolev et al.12, in which atoms are classified into eight types: hydrophilic, hydrogen bond (HB) acceptor, HB donor, hydrophobic, aromatic, neutral, neutral-donor and neutral-acceptor. Edges are assigned a (coloring) value dc, the Euclidean distance between the atoms they connect in the 3D binding site structure. Viewing each cleft as a graph, the task is to find the largest common sub-graph isomorphism. This is done through the construction of a second graph, known as an association or correspondence graph, derived from the two initial cleft graphs under comparison.\n\nThe construction of the association graph requires the definition of nodes and edges. Nodes represent chemical similarity while edges represent geometric similarity. Each node k = {i,t} defines a possible correspondence between a pair of atoms Aim and Atn of identical type, one from each cleft under comparison (clefts m and n). This condition assures that the final subset of atoms in common between the two clefts corresponds pairwise to the same type. An association graph edge is created between two association graph nodes if the binding-site graph edges connecting the corresponding pairs of atoms in each binding-site graph are similar. In other words, if we have two nodes k = {i,t} and l = {j,s} in the association graph, an edge will be created if:\n\n\n\nThe above condition of geometric similarity, used when creating edges in the association graph, is such that a clique (a subset of nodes fully connected by edges) corresponds to a subset of atoms in each cleft in which all pairwise distances between atoms in one cleft are satisfied by the corresponding atoms in the other cleft. The parameter Dnode implicitly permits to accommodate the effect side-chain flexibility to a certain extent. In IsoCleft Finder, this parameter is set at Dnode = 4 Å.\n\nThe objective of the graph matching procedure is the detection of the largest clique, i.e. the largest subset of atoms of identical type in equivalent spatial positions between the two clefts. This set of atoms can be used to superimpose both clefts under comparison (Figure 1).\n\nDetected common binding-site atoms are displayed as spheres. While the superimposition was performed using binding-site atoms, the RMSD of the equivalent ligand atoms is 1.1 Å.\n\nThe combinatorial nature of association graphs can lead to exponentially large graphs, both in terms of number of nodes and density of edges. This is a major drawback when employing association graphs to detect common sub-graph isomorphisms since the computational cost of clique detection algorithms increases very rapidly with the size of the association graph. IsoCleft introduces two innovations that allow us to overcome this common problem associated with graph matching.\n\nThe first innovation is to perform the graph matching in two stages. In the first stage, an initial superimposition of the two binding-sites under comparison is performed via the detection of the largest clique in an association graph constructed using only Cα atoms of equivalent residues in the two clefts. The minimum rank-order (r) of each residue's JTT substitution matrix probabilities13 is used to set the level of allowed residue similarity (association graph node coloring). Edges in the first-stage association graph represent, as described above, Euclidean distance differences (Equation 1) but this time between Cα atoms, with Dnode = 3.5 Å. Once the largest Cα clique is obtained, its transformation matrix and translation vectors are used to superimpose all atoms in both clefts using the least square method of Arun et al.14. In other words, the first stage performs a Cα-based superimposition of all atoms in the clefts under comparison based on the detection of the largest subset of similar residues in equivalent spatial positions. The residue-similarity JTT minimum rank-order threshold parameter is set at r = 5.\n\nIn the second graph matching stage, all non-hydrogen atoms are used. Association graph nodes are created with the requirement that two atoms, one from each cleft, be of the same type, as described earlier, and that their spatial distance after the first stage superimposition be within the default value n = 4 Å. This distance threshold is used to decrease the size of the association graph and it is the reason why the initial graph matching stage is performed. In effect, a pair of atoms, one from each cleft and of identical type that would otherwise define an association graph node, will be too distant to do so after the first-stage superimposition. The Ca atoms artificially included in the set of cleft atoms for the first stage are not considered in the second stage and thus do not contribute directly to the detection or measurement of similarity. IsoCleft utilizes the Bron & Kerbosch algorithm15 to detect the largest clique in the association graphs on both stages of the graph matching process.\n\nThe second innovation introduced in IsoCleft is to exploit the fact, noted by Bron & Kerbosch15, that their algorithm has the tendency to produce the cliques in decreasing size order. IsoCleft implements what we call “Approximate Bron & Kerbosch” (ABK). In ABK, the first clique is selected as the solution (and the search procedure is stopped) rather than detecting all cliques in order to find the largest. Utilization of ABK allows us to obtain an optimal or nearly optimal solution in a fraction of the time that would be needed using the original algorithm, without any noticeable effect on the results. The fractional loss in accuracy when using ABK compared to the original Bron & Kerbosch algorithm is likely to be minor compared to the effect of intrinsic noise inherent to biological systems (in part as a consequence of flexibility) in addition to the noise introduced with the choice of empirical parameters (e.g., in the definition of the association graph).\n\nThe stand-alone version of the IsoCleft program (freely available for academic users upon request) requires a number of user-defined parameters. In IsoCleft Finder, these parameters are fixed with default values previously obtained through an extensive heuristic search in parameter space. The default values maximize the average area under the Receiver-Operator Characteristic (ROC) curve (AUC)8 in predictions of ligand-binding classes based on binding-site similarities across non-homologous protein families that convergently evolved to bind similar ligands.\n\n\nResults\n\nThis article describes: 1. The implementation of the non-redundant dataset of target binding-sites with known levels of cognate similarity (ICFDB v. 1.6), 2. The IsoCleft Finder web-interface for the IsoCleft program with which to determine the level of binding-site similarity between query binding-sites and those in the ICFDB 1.6 dataset, 3. A set of visual and computational post-search analysis tools that help judge the quality of the detected hits, and 4. The results for several cases in which IsoCleft Finder provides useful functional information. All together, IsoCleft Finder is a unique web-based tool that provides direct and convenient access to the detection of atomic level binding-site similarities to research groups without bioinformatics expertise or the necessary computational resources required for large-scale database searches.\n\nA non-redundant dataset of target binding-sites, called ICFDB (current version 1.6), was developed based on the Procognate database16. Procognate provides cognate-similarity levels for a large subset of proteins in the PDB database. A cognate ligand is a natural ligand, that is, a substrate or cofactor of the given protein (as recorded in the KEGG database17). In other words, in a complex between a protein and a cognate ligand, the structural determinants of molecular recognition were subjected to natural selection. While statistically significant binding-site similarities are interesting irrespective of what ligand the target protein is bound to, if that ligand is highly similar to a cognate ligand, these similarities are more informative regarding the potential biological function of the query protein. Cognate similarities in Procognate can vary from zero, where there is no similarity between the bound ligand and the cognate ligands, to one, when a cognate ligand is the one present in the solved structure. The choice of a cognate similarity threshold is subjective and it is therefore impossible to determine a single particular threshold appropriate for all purposes and query types as the number of atoms in the ligand has an effect on this threshold. A statistically relevant hit to a binding site bound to a large ligand with smaller cognate similarity might offer equal or greater relevant functional clues as a hit to a smaller ligand with greater cognate similarity.\n\nOur dataset contains a subset of unique Pfam18 ligand combinations from Procognate with associated levels of cognate-ligand similarity. The dataset contains 7339 entries of which 970 have a coefficient of cognate-ligand similarity equal or larger than 0.95 (Figure 2). While any statistically relevant match may offer potential clues regarding the function of a protein, matches to complexes involving ligands with high levels of cognate similarity may in addition reflect distant evolutionary relationships. The dataset contains 6110 unique PDB entries (with resolution 2.16 ± 0.45 Å and 27 NMR structures), representing 6093 different Uniprot entries19 belonging to 997 Pfam families and containing 29905 unique gene ontology (GO) molecular function terms20. In terms of ligands, ICFDB v. 1.6 contains 3833 different small-molecules. The dataset is available for browsing at http://bcb.med.usherbrooke.ca/files/ICFDB1_6.txt, can be downloaded as a zip file through a link in the IsoCleft Finder web page http://bcb.med.usherbrooke.ca/isocleftfinder or downloaded below.\n\nThe majority of ligands display low cognate similarity. The peak at the extreme right of the distribution represents those cases where the bound ligands are nearly identical to the cognate ligands.\n\nThe IsoCleft Finder tool consists of three pages: input, cleft definition and output. Help is provided in the form of notes and hovering tips.\n\nThe input page (Figure 3) allows a user to provide the 4-letter code of an existing PDB entry or upload a file in PDB format containing an entire structure or only the atoms defining a cleft. A cleft file must contain four or more atoms and include the alpha carbon atom of any residues represented. When an entire structure is used, the three largest clefts are defined automatically using our own implementation of the Surfnet algorithm21. Furthermore, the user may provide some parameters to better define the clefts, such as a chain or residue/bound-ligand identifier. The former avoids clefts defined at the interface between chains while the latter helps detect a cleft that is not among the largest three. To identify a residue or bound ligand we utilize the residue ID in RESNUMCA format, where RES represents the three-letter PDB residue code, NUM represents the residue number, C represents the chain identifier and A represents the alternative location code. A dash is used in cases where C and/or A are blank characters in the coordinates file (e.g., BTN300-- or ASN94A-). If, for example, only the RES code is given (particularly useful in the case of ligands), we utilize a basic pattern matching procedure to detect all possible residue ID matches within the PDB file and the user then selects the appropriate match to proceed. The user may provide an email address, in which case a link with the results is sent via email. Otherwise, the user must save the link of the results page upon job submission or leave the browser window open.\n\nThe input page offers the possibility to output the top hits belonging to different Pfam families. This option permits the inspection of the results from a broader perspective from the protein point of view in the sense that high scoring matches of similar binding-sites will in effect represent cases of divergent evolution with more remote homologues or cases of convergent evolution.\n\nFinally, a threshold value for the minimum level of cognate similarity can be chosen. Higher values decrease the size of the target dataset as per the distribution in Figure 2.\n\nThe cleft definition page (Figure 4) contains consecutive panels, one for each cleft. The user may deselect those clefts that are not relevant. Each cleft is shown in an interactive 3D Jmol (http://www.jmol.org) applet. All residues that contribute at least one atom to the cleft are shown in red. The right panel can be used interactively to deselect any unnecessary residues. Any ligands present in the cleft are also colored.\n\nThe user is able to de-select residues initially included in the cleft(s).\n\nRunning times vary depending on input values and system load. In particular, the threshold level of cognate similarity defines what subset of the binding-site target dataset is used in the search and affects considerably the running time as, for example, with a value of 0.95 only 970 pairwise comparisons will be required while a value of 0.0 will imply 7339 pairwise comparisons.\n\nThe output page (Figure 5) returns the top scoring (most similar) binding-sites in the form of a table as well as a Jmol applet in which the ligands bound to the target binding-sites are superimposed on the query binding-site based on the detected atomic similarities. For each hit, the number of atoms in common (NC) is presented as well as a Tanimoto Score of Similarity8:\n\n\n\nSee http://bit.ly/Xz0Qm.\n\nwhere NA and NB represent the number of atoms in the query and target binding-sites, respectively. Two measures of statistical significance, Z-score and p-value, are calculated for the number of atoms in common and the Tanimoto Score of Similarity. The mean (µ) and standard deviation (σ) values are calculated and used to define the Z-score, z = (T–µ)/σ. For the purpose of calculating p-values, data points with z < –3 or z > 7 are removed followed by recalculation of µ and σ. This process is repeated up to five times. The p-value is derived from the final Z-scores using an extreme value distribution as follows22:\n\n\n\nwhere Γ is the gamma function and Γ’(1) = –0.5772157.\n\nIt is important to note that as the size of the target binding-sites varies, one may find hits that when compared to each other contain larger numbers of atoms in common to the query but smaller Tanimoto Scores of Similarity. Several such cases can be found in Table 1–Table 3. For a given number of atoms in common, as the size of a target binding-site increases, the statistical significance of its Tanimoto Score of Similarity decreases. As it is not possible to decide if a large number of atoms in common is more desirable than a large fraction of atoms in common, both measures should be used to determine the functional relevance of the hits obtainer.\n\n1 Number of atoms in common.\n\n2 Tanimoto Score of Similarity.\n\n1 Number of atoms in common.\n\n2 Tanimoto Score of Similarity.\n\n1 Number of atoms in common.\n\n2 Tanimoto Score of Similarity.\n\nSeveral links are provided to download the results and to access further information regarding the matches. These include links to the list of atomic correspondences and superimposed coordinates as well as to external sources such as Pfam18 and PDBsum23.\n\nFinally, a second table shows the pairwise topological similarity24 between the target ligands present in the top scoring target binding-sites. Similarities between top scoring ligands represent an independent source of evidence in support of the biological relevance of the detected binding-site similarities. A large number of equivalent ligand atoms in equivalent positions in space (as a consequence of the binding-site superposition produced by IsoCleft) may point to binding-site atoms in the query protein that are important from a molecular recognition point of view8. In the case shown in Figure 5, we used the adenosine monophosphate (AMP) bound cleft of Escherichia coli aspargine synthase (PDB code 12as) as query. L-aspargine (ANS), the product of the reaction, is also bound. The bound AMP and ASN molecules define two independent binding-sites, both of which are found within the top scoring hits. The second and third hits (ATP and ADP) represent different Pfam families and folds but, as can be seen in Figure 5, their adenine and ribose moieties superimpose very well compared to the bound AMP molecule.\n\nThe strength of the IsoCleft and the IsoCleft Finder interface can be seen with the Human mitotic spindle checkpoint kinase Bub1 (PDB code 3e7e) query protein in Figure 6. Among the found hits (Table 1), the six top hits represent protein kinase (PF00069), tyrosine kinase (PF07714) and alpha-kinase (PF02816) Pfam families. The seventh hit is PDB code 2aqx, a phosphotransferase (inositol polyphosphate-PF03770) that has a distinct domain fold from the typical kinase domain, but one that has independently evolved to bind ATP. The eighth hit is PDB code 1d7l (FAD binding domain-PF01494) bound to riboflavin (RFL). The superimposition of the ligands based on the similarities (Figure 7) shows that IsoCleft was able to identify similar arrangement of conserved residues able to bind the common scaffolds of ATP and RFL. Crystal structure 2bl4 represents a group III iron-activated dehydrogenase (alcohol dehydrogenase-PF00465). This protein binds NAD, a common co-factor that shares a common scaffold with ATP. Again the superimposition of the ligands, as seen in the Jmol window (Figure 6), confirms that the similarities identified relate to residues that evolved to bind common moieties.\n\nThe riboflavin (RFL) ligand from PDB entry 1d7l (in cyan) is superimposed based on the similarities identified by IsoCleft. This result suggests that the two proteins have a similar arrangement or residues that were conserved throughout evolution to bind the common moieties of ATP and RFL.\n\nThese results clearly show that IsoCleft Finder has the ability to predict potential ligands by identifying similarities across domains of dissimilar folds. The potential for detecting binding-site similarities across fold families is clearly interesting from a rational drug design point of view. Such similarities can be used to determine potential cross-reactivity or polypharmacological targets to be integrated into the drug design process. In this scenario detected similarities can determine what kind of potential interactions should be prevented or prioritized in the case of cross-reactivity or polypharmacology respectively.\n\nThe core engine behind the IsoCleft Finder tool, the IsoCleft program, was previously tested for the detection of binding-site similarities across non-homologous protein families8. IsoCleft has also been applied to study the similarities within histone methyltransferase cofactor binding-sites25 as well as within the human cytosolic sulfotransferase family26,27. In this section we present four functional predictions performed with IsoCleft Finder to demonstrate its applicability in real scenarios for the prediction of protein function from structure.\n\nThe first case is that of a hypothetical protein (cgd2_2020) from Cryptosporidium parvum (PDB code 2pd0), solved by the Structural Genomics Consortium (SGC). It is worth noting that this example is one for which all methods currently available as part of the Profunc meta-server for function prediction28 did not provide functional clues (data not shown). These include, among others, global structural (fold) similarity, various sequence and sequence-profile based similarity methods, reverse templates and genome co-localization.\n\nWhile the bound 2-(n-morpholino)-ethanesulfonic acid (MES) is part of the crystallization buffer, it may have serendipitously found its niche in a biologically relevant binding site. For that reason, we used the bound MES to define the binding site. The top two results obtained with IsoCleft Finder are the human purine nucleoside phosphorylase [PDB code 1v2h bound to guanine (GUN; cognate similarity of 0.81) with 21 atoms in common, Tanimoto Score of Similarity 0.404, Z-score 4.10 and p-value 9.20E-03] and the E. coli homologue of the same protein [PDB code 1pke bound to 5-(6-amino-2-fluoro-purin-9-yl)-2-hydroxymethyl-tetrahydro-furan-3-ol [2-fluoro-2'-deoxyadenosine] (2FD; cognate similarity of 0.85) with 22 atoms in common, Tanimoto Score of Similarity 0.386, Z-score 3.78 and p-value 1.38E-02. The atoms in common between the 2pd0 cleft and those in 1v2h and 2pke superimpose with RMSD of 1.75 Å and 2.08 Å respectively. Figure 8 shows the target ligands superimposed on the query protein based on the superimposition of the atoms in common. While the superimposition is performed using the detected binding-site atoms in common, the purine rings of the target ligands superimpose quite well with each other as well as with the aromatic ring of the solvent molecule. The fact that the two top hits represent the same reaction in two different organisms (Homo sapiens and E. coli) gives further support to the functional prediction.\n\nThe query structure 2pd0 (in orange) is used as reference in which the target binding site ligands guanine (GUN; from PDB entry 1v2h in red) and 5-(6-Amino-2-Fluoro-Purin-9-Yl)-2-Hydroxymethyl-Tetrahydro-Furan-3-Ol [2-Fluoro-2'-Deoxyadenosine] (2FD; from PDB entry 1pke, in green) are superimposed based on their binding site similarities to the query cleft defined by the bound ligand 2-(N-Morpholino)-Ethanesulfonic acid (MES, in blue). A good quality superimposition of the aromatic rings of the different ligands is obtained as a consequence of the superimposition of the binding sites performed by IsoCleft.\n\nThe second example is that of the conserved protein of unknown function ca_c3497 from Clostridium acetobutylicum atcc 824, whose structure was recently solved by the Midwest Center for Structural Genomics (MCSG) with PDB code 3d0j. As in the previous example, the Profunc server failed to give any statistically significant functional clues. Again in this case, a molecule from the crystallization buffer (glycerol, ligand ID: GOL142A-) seems to have serendipitously detected the binding-site (Figure 9). In this case when looking at the top hits based on Tanimoto Score of Similarity (TSS) we find D-glucose (GLC, TSS Z-score 3.74) and D-Glucose-1-Phosphate (G1P, TSS Z-score 3.44) bound to two proteins from different Pfam families (PDB codes 1k1w and 1nt4 respectively). Alternatively, when looking at number of atoms in common we find 27 atoms in common on the top two hits (Z-score 3.17), again to two different Pfam families (PDB codes 1vh3 and 2vfz) bound to two different molecules both containing sugar a moiety in similar positions in space (see http://goo.gl/7QhjD). Taken together, the IsoCleft Finder results suggest a function related to binding sugar moieties.\n\nThe ligand GOL142A- (in red) is used to define the binding-site (in blue) with the subset of atoms in its surface (in yellow) used as query against the ICFDB subset with cognate similarity level equal to 0.9.\n\nThe third and fourth examples show the results of IsoCleft Finder from our collaboration with the Joint Center for Structural Genomics that were used to support the functional annotation of the proteins in question. The third example is that of the binding-site of the first structure of a member of Pfam family PF06474, a new fold of unknown function from Pseudomonas aeruginosa. The IsoCleft Finder analysis identified similarities between the hydrophobic groove along the cleft entrance and dimerization interface of the query protein (PDB code 2h1t), and the lipid-binding site in Candida rugosa lipase (PDB code 1lpn; 31 atoms in common, Tanimoto Score of Similarity 0.200, Z-score 3.98, p-value 1.08E-02) suggesting involvement in glycolipid metabolism29.\n\nFourth, also in collaboration with the Joint Center for Structural Genomics, is the case of two proteins (the product of genes SP00140 and Sbal_2486), the first representatives of PFAM family PF06938 (DUF 1285) from Silicibacter pomeroyi (PDB code 2re3) and Shewanella baltica (PDB code 2ra9), respectively. The IsoCleft Finder analysis of 2re3 (Table 2) identified shared features between the inter-domain cleft of 2re3 and sugar, phosphate and purine-binding proteins (PDB codes 1pwh, 1dqa, 1dm3, 1gpe, 1v0j, 1hwy, 2vfs and 1q6p)30.\n\nSimilar hits (adenosylcobalamin, heme, dideoxy sugars, NAD, thiamine diphosphate) were obtained for 2ra9 (Table 3). These similarities, suggest that a nucleotide-based ligand may bind these proteins30 with a possible involvement in signal transduction.\n\nThe independent assignment of similar potential ligands to distinct members of the family adds strength to each individual assignment and contributes to the elucidation of the function of the family as a whole.\n\n\nConclusions\n\nIn recent years, with the advent of structural genomics projects, a number of proteins with known structure but without functional annotation came to light. In these cases, the detection of binding-site similarities may provide useful functional information complementary to those of existing methods.\n\nIn this work we introduce the IsoCleft Finder. IsoCleft Finder is a web-tool for the detection of binding-site similarities between putative ligand-interacting clefts and an associated dataset of target binding-sites with defined levels of cognate-ligand similarity. The ICFDB dataset v.1.6 is useful to study ligand-protein interactions and we encourage its download and use. IsoCleft Finder offers a powerful web-interface to define clefts and to visualize and analyze the obtained results of binding-site similarities.\n\nWe demonstrate the use of IsoCleft Finder with cases for which other methods did not provide functional clues. As such, these predictions remain to be validated experimentally. However, in all cases, multiple independent hits obtained with IsoCleft Finder point to similar ligands.\n\nIsoCleft Finder has other applications, such as the detection of potential cross-reactivity targets and small-molecule binders. In the first case, binding-site similarities to unrelated proteins might indicate potential unexpected cross-reactivity targets that would not have been detected otherwise. In the second case, clefts derived from high quality homology models may be used as input to detect potential binding small-molecules that may help produce protein crystals (of the complex) for proteins that would not crystallize in the absence of a stabilizing ligand.\n\nIsoCleft Finder gives access to a broad range of users, including experimental groups without in house computational expertise as well as bioinformatics groups without the necessary resources to perform the computationally intensive calculations involved in the detection of binding-site chemical and structural similarities.", "appendix": "Author contributions\n\n\n\nRN devised the work, developed the ICFDB dataset and created the search scripts. NK developed the web interface. MC tested the system. RN, NK, MC and MIZ 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\nReferences\n\nKreher DL, Robert Stinson D: Combinatorial Algorithms: Generation, Enumeration, and Search. 1998; 344. Reference Source\n\nRigoutsos I, Wolfson HJ: Geometric hashing: an overview. IEEE Computational Science & Engineering. 1997; 4(4): 10–21. Publisher Full Text\n\nWeskamp N, Kuhn D, Hullermeier E, et al.: Efficient similarity search in protein structure databases by k-clique hashing. Bioinformatics. 2004; 20(10): 1522–1526. PubMed Abstract \| Publisher Full Text\n\nSchmitt S, Kuhn D, Klebe G: A new method to detect related function among proteins independent of sequence and fold homology. J Mol Biol. 2002; 323(2): 387–406. 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PubMed Abstract \| Publisher Full Text\n\nJones DT, Taylor WR, Thornton JM: The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992; 8(3): 275–282. PubMed Abstract \| Publisher Full Text\n\nArun KS, Huang TS, Blostein SD: Least-squares fitting of two 3–D point sets. IEEE Trans Pattern Anal Mach Intell. 1987; 9(5): 698–700. PubMed Abstract \| Publisher Full Text\n\nBron C, Kerbosch J: Algorithm 457: Finding all cliques of an undirected graph. Commun ACM. 1973; 16(9): 575–577. Publisher Full Text\n\nBashton M, Nobeli I, Thornton JM: PROCOGNATE: a cognate ligand domain mapping for enzymes. Nucleic Acids Res. 2008; 36(Database issue): D618–D622. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M, Araki M, Goto S, et al.: KEGG for linking genomes to life and the environment. Nucleic Acids Res. 2008; 36(Database issue): D480–D484. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBateman A, Coin L, Durbin R, et al.: The Pfam protein families database. Nucleic Acids Res. 2004; 32(Database issue): D138–D141. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniProt Consortium: The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res. 2010; 38(Database issue): D142–D148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCamon E, Magrane M, Barrell D, et al.: The Gene Ontology Annotation (GOA) Database: sharing knowledge in Uniprot with Gene Ontology. Nucleic Acids Res. 2004; 32(Database issue): D262–D266. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaskowski RA: Surfnet: A program for visualizing molecular-surfaces, cavities, and intermolecular interactions. J Mol Graph. 1995; 13(5): 323–330. PubMed Abstract \| Publisher Full Text\n\nLaskowski RA, Watson JD, Thornton JM: Protein function prediction using local 3D templates. J Mol Biol. 2005; 351(3): 614–626. PubMed Abstract \| Publisher Full Text\n\nLaskowski RA, Chistyakov VV, Thornton JM: PDBsum more: new summaries and analyses of the known 3D structures of proteins and nucleic acids. Nucleic Acids Res. 2005; 33(Database issue): D266–D268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman SA, Bashton M, Holliday GL, et al.: Small Molecule Subgraph Detector (SMSD) toolkit. J Cheminform. 2009; 1(1): 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampagna-Slater V, Mok MW, Nguyen KT, et al.: Structural chemistry of the histone methyltransferases cofactor binding site. J Chem Inf Model. 2011; 51(3): 612–623. PubMed Abstract \| Publisher Full Text\n\nNajmanovich RJ, Allali-Hassani A, Morris RJ, et al.: Analysis of binding site similarity, small-molecule similarity and experimental binding profiles in the human cytosolic sulfotransferase family. Bioinformatics. 2007; 23(2): e104–e109. PubMed Abstract \| Publisher Full Text\n\nAllali-Hassani A, Pan PW, Dombrovski L, et al.: Structural and chemical profiling of the human cytosolic sulfotransferases. PLoS Biol. 2007; 5(5): e97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaskowski RA, Watson JD, Thornton JM: ProFunc: a server for predicting protein function from 3D structure. Nucleic Acids Res. 2005; 33(Web Server issue): W89–W93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBakolitsa C, Kumar A, McMullan D, et al.: The structure of the first representative of Pfam family PF06475 reveals a new fold with possible involvement in glycolipid metabolism. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010; 66(Pt 10): 1211–1217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan GW, Bakolitsa C, Miller MD, et al.: Structures of the first representatives of Pfam family PF06938 (DUF1285) reveal a new fold with repeated structural motifs and possible involvement in signal transduction. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010; 66(Pt 10): 1218–1225. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "945", "date": "13 May 2013", "name": "Andrew J Bordner", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 IsoCleft Finder web server described in this article can detect and align ligand binding sites in protein structures sharing similar chemical and geometrical properties to the query, even if they do not share homology that is detectable by sequence similarity. The server also provides p-values, which are useful for assessing the confidence of each prediction. The authors discuss a timely application of their method, predicting cognate ligands for the many functionally uncharacterized proteins with experimental structures solved by structural genomics projects. Based on some examples that I ran, the web server returns the results quickly (less than 1 minute). Overall, this paper clearly describes the IsoCleft method and web interface, which should be a useful tool for structural biologists.A few minor corrections/comments:The ICFDB data set contains some small non-biological ligands, like SO4 and glycerol, which may bind in ligand binding pockets but not share chemical similarity with the cognate ligand. There should be a brief discussion of why these were included.The web server results page currently shows an all-atom wire representation of the entire protein, which makes it difficult to find one’s way around the molecule. I would recommend using a simpler cartoon representation of the protein while keeping the wire representation of site residuesThere is a typo “obtainer” on page 7.", "responses": [] }, { "id": "990", "date": "07 Jun 2013", "name": "Andras Fiser", "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 web interface is presented to a protein structure based small molecule binding site prediction algorithm that was published before.The algorithm is based on matching binding sites alone with graph matching tools using an annotated set of 7339 structures. The actual method was published before and tested in various conditions. Here, a nicely designed interface is provided that works well. I would like to see more options on the JMOL interface, options to switch between different molecular presentations, as the wire presentation is hard to follow in more complicated instances.My major reservation is that certain parts, sentences and sometimes almost full paragraphs are identical to those in the authors’ earlier published paper in Bioinformatics that describes the method. Although the authors do mention that they are reiterating the description of the Isocleft program, these sections should be rewritten. It might be better if the current paper focused on the web interface and functionalities and did not try to repeat all the methodological details.", "responses": [] }, { "id": "1139", "date": "18 Jul 2013", "name": "John P. Overington", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 prediction of function is a major challenge from the output of structural genomics programs, and the ability to do this via binding site similarities is an obvious and potentially powerful way to assign function. A further application of protein site similarity approaches is in the area of polypharmacology - the binding of drugs to multiple targets, especially those involving anticipation of adverse drug reactions (ADRs) - a nice example of this sort of similarity is in the recent reporting of the molecular target for the side effects of many sulfa drugs reported by Haruki et al. (2013). As such, IsoCleft Finder is a potentially useful tool for a range of different researchers. The method focusses on the analysis and prediction of small molecule binding, and as such would miss prediction of protein-protein interactions, and we know that much of biology and disease is composed of protein-peptide and protein-protein interactions. The method in the article is clearly explained, in almost enough detail to allow reimplementation, and there are improvements into the computational efficiency of IsoCleft Finder - the two stage filtering, and the approximate Bron and Kerbosch heuristics. I have a few comments that to me were not clear, or are worthy of clarification:How were protonation states and tautomers treated in the analysis? These will have key roles in defining the similarity of the site features. How does the method deal with the possibility of chiraly related sites? The distances between features will be the same, but in real space they will be different. In practice, the real space fit of Ca positions will screen these out, but there could be a set of interesting similarities that are overlooked?Can atoms belong to multiple types - hydrophilic, hydrogen bond acceptor, etc? To me it sounds like they could, and if so, what are the equivalence sets, or the priority rules to assign the most important features?How does the method score similar sites - same ligand, different asymmetric unit, same ligand orthologous protein, same ligand, different crystal form etc? Is the method stable enough, or are the differences seen enough to calibrate the likelihood of more distant relationships being identified being real?Can the method deal with cases where the 'target' is actually composed of a protein and group like a haem? Many enzymes have cofactors, and are obligated to correct site formation. Many methods struggle to handle this simply, so if IsoCleft Finder could handle these it would be a significant improvement over other methods.MES doesn't have an aromatic ring, whereas the purine rings do - in the cgd2_2020 case. Fixing the typo here to just reflect ring overlap would fix this.", "responses": [] } ]	2	https://f1000research.com/articles/2-117
https://f1000research.com/articles/2-119/v1	01 May 13	{ "type": "Research Article", "title": "Expectant fathers’ knowledge of maternal morbidity: a Sri Lankan experience", "authors": [ "Amaya Weekrakkody", "Gihan M Weerasinghe", "Mayumi P Weerasinghe", "Gayan L Weerasekara", "Suneth B Agampodi", "Amaya Weekrakkody", "Gihan M Weerasinghe", "Mayumi P Weerasinghe", "Gayan L Weerasekara" ], "abstract": "Background: Male partners play an important and vital role in the decision-making process regarding pregnant women’s health. The purpose of the present study was to assess the knowledge and awareness of expectant fathers about Gestational Diabetes Mellitus (GDM), Pregnancy Induced Hypertension (PIH), and anaemia during pregnancy.Methods: A cross sectional descriptive study was carried out among expectant fathers whose partners were attending antenatal clinics at the Anuradhapura Teaching Hospital, Sri Lanka. All consenting participants were interviewed by investigators using an interviewer administered questionnaire to collect data on knowledge of risk factors, symptoms, complications and their control. Statistical analysis was performed using the Kruskal Wallis test. Results: Of the 246 expectant fathers studied, 192 (78%) were aware of GDM, 183 (74.4%) and 154 (62.6%) were aware of PIH and anaemia during pregnancy, respectively. The total number of answers provided by expectant fathers ranged from 0 to 33 (of 41 questions). There were 44 fathers who could not answer even a single question. For GDM, anaemia, and PIH, the percentages of expectant fathers who failed to provide at least a single correct answer were 24.8%, 40.2%, and 31.3%, respectively. The median number of total correct answers provided increased steadily along with the average income (chi-square 31.24, p<0.001) and educational level (chi-square 33.57, p<0.001). Expectant fathers in the 25-34 age group had significantly higher scores, compared to younger and older fathers (chi-square 15.11, p=0.001). Fathers experiencing the second pregnancy of their spouses also had higher scores.Conclusions: Expectant father’s knowledge of the selected morbidities was limited. To improve maternal health, any health promotional programmes should include expectant fathers.", "keywords": [ "The important role of men as the primary stakeholders of women’s reproductive and sexual health has been widely accepted. In 1994", "20", "000 United Nations delegates with member status pressed for men’s involvement in reproductive health. The programme of action of the International Conference on Population and Development (ICPD) stressed men’s shared responsibility for women’s health1. Although the primary focus was on reducing violence against women and children", "the resolution clearly declared that men should take responsibility for the empowerment of women." ], "content": "Introduction\n\nThe important role of men as the primary stakeholders of women’s reproductive and sexual health has been widely accepted. In 1994, 20,000 United Nations delegates with member status pressed for men’s involvement in reproductive health. The programme of action of the International Conference on Population and Development (ICPD) stressed men’s shared responsibility for women’s health1. Although the primary focus was on reducing violence against women and children, the resolution clearly declared that men should take responsibility for the empowerment of women.\n\nStudies on family planning2,3, HIV/AIDS4, abortions4 and breastfeeding5 all provide clear evidence for the important and vital role of male partners in the decision-making process regarding women’s health. Interventional studies on reproductive health programmes targeting either males or both sexes, compared to programmes targeting only females, have shown a significant effect on the reproductive health outcome of women. With this growing body of evidence, the concept of men as “gatekeepers” of women’s health has been developed into a positive approach that includes men as partners in improving women’s health.\n\nNevertheless, these involvements are often driven by the gender power relationships given to male partners in various cultures6. While reproductive health education and health promotion programmes that target adolescent boys and young men are acceptable in most parts of the world, shared responsibility for pregnancy-related matters heavily depends on the nature of gender roles within the society. In addition, fear of losing respect from peers, lack of communication skills, lack of knowledge, and perceptions of masculinity are also shown to have a major effect on this involvement. Despite having a lack of knowledge and involvement, men often dominate the decision-making processes related to pregnancy, especially in male-dominated South Asian cultures.\n\nStudies in maternal health have shown that early interventions during the antenatal period have a major protective effect for medical conditions complicating pregnancy and direct pregnancy-related acute complications such as haemorrhage, rupture of the uterus, and obstructed labour. These interventions include early identification, early treatment and behaviour modifications.\n\nHowever, screening tests and procedures for these conditions require resources, and early detection is not currently at an optimal level in most developing countries. We hypothesized that this could be partly due to a lack of awareness about maternal morbidity among pregnant women, and specifically among their partners who play a major role in decision-making. The knowledge of expectant fathers is vital to ensure that healthcare is sought early to prevent complications due to these conditions. The purpose of the present study was to assess the knowledge and awareness of expectant fathers about Gestational Diabetes Mellitus (GDM), Pregnancy Induced Hypertension (PIH), and anaemia during pregnancy.\n\n\nMethods\n\nThis study conformed to the Helsinki Declaration and to local legislation. All participants gave informed consent to participate in this study. Ethical clearance was obtained from the Research and Ethics Committee, Faulty of Medicine and Allied Sciences, Rajarata University of Sri Lanka.\n\nThe study was conducted in Anuradhapura, Sri Lanka, during November to December 2010. Anuradhapura district is situated in the North Central Province of Sri Lanka. The resident population is around 830,0007. Over the five years preceding this study the annual number of births reported from the district was around 16,000. The crude birth rate is 20.7/1000 population. Infant and neonatal mortality rates of Anuradhapura district (17.2/1000 live births (LB) and 11/1000 LB) are significantly higher than those reported in Sri Lankan national data, which are 11/1000 LB and 8.7/1000 LB, respectively8.\n\nThe study population for the present study included expectant fathers whose partners were attending antenatal clinics at the Anuradhapura Teaching Hospital. The study sample included the expectant fathers that were accompanying pregnant women to the antenatal clinics. The participants included fathers who were waiting during the clinic hours as well as the fathers who came to the clinic just to drop off the pregnant women at the clinic (this was a common practice and usually they returned around four hours later). The authors visited the antenatal clinics at the starting time and introduced themselves to expectant fathers, explained the objectives of the study, and handed out the self-administered questionnaires. The study sample for the present study included all expectant fathers visiting selected clinics during the study period. Since the study followed a non-probability sampling procedure, the optimal sample size was not calculated. However, it was hypothesized that if the 80% of the expectant fathers are aware of the selected morbidities and a simple random sampling procedure was followed, a minimum of 246 expectant fathers were required with 95% confidence limits and 5% absolute precision.\n\nVariables for the study included basic socio-demographic variables and knowledge about GDM, PIH and anaemia. For each condition selected, expectant fathers were first asked about their awareness of the existence of certain disease conditions, for example, ‘Are you aware that pregnant women could develop a condition called gestational diabetes mellitus?’. Then a selected set of questions on risk factors, clinical features, complications and preventative measures (primary and secondary) was included for all selected conditions (e.g., ‘Which of the following could be risk factors for developing GDM?’). Only true risk factors, features and preventative measures were provided in the questionnaire. This approach was followed as a way of including health education and to avoid the possibility of giving incorrect messages through the survey. The variables were selected after discussion with medical, obstetric, and community medicine experts. The questionnaire was developed in English and translated into Sinhalese.\n\nAll demographic variables were analyzed as categorical variables. Answers to individual questions were presented as percentages. The total number of correct answers provided by participants was also reported as a percentage. The knowledge distribution was expected to have a skewed distribution because of the distribution of educational status in this population is also skewed, and non-parametric tests were carried out for significance testing.\n\n\nResults\n\nThe total number of expectant fathers studied was 246. The mean age of the participants was 30.2 years (standard deviation (SD) of 6.2 years) and the mean age of the pregnant mothers was 26.6 years (SD of 5.6 years). Table 1 shows the demographic characteristics of the study sample.\n\nGCE O/L-General certificate in Education Ordinary Level.\n\nGCE A/L-General certificate in Education Advanced Level.\n\nThe prevalence of GDM, PIH and anaemia during the present or previous pregnancies as reported by the fathers was 6.9% (n=17), 4.5% (n=11) and 11.4% (n=28), respectively. Of the 246 expectant fathers studied, 192 (78%) were aware of GDM, and 183 (74.4%) and 154 (62.6%) were aware of PIH and anaemia during pregnancy, respectively.\n\nOf the risk factors listed in the questionnaire, a family history of GDM/diabetes mellitus (DM) was recognized by 48.8% of the study sample (Table 2). Of the respondents, 135 (54.9%) knew that increased frequency of urination was a common clinical presentation for GDM/DM. The probability of developing noninsulin-dependent diabetes mellitus (NIDDM) in later life as a complication of GDM was known to 98 (39.8%) respondents. Screening for GDM if risk factors were present was indicated as an appropriate intervention to control GDM by 30.5% (n=75) of participants.\n\nIncreasing maternal age was the risk factor most commonly recognized among expectant fathers as a risk factor for developing anaemia during pregnancy, which was reported by 26.8% (n=66) of the fathers. All other risk factors were known to less than 15% of the respondents. However 92 fathers (37.4%) reported headache as a common clinical presentation of anaemia, and 65 (26.4%) were aware of the increased risk of postpartum haemorrhage. Half of the study sample responded that adding iron-rich food to the diet would help to prevent anaemia. Dizziness was identified as a probable symptom of PIH by 58.1% (n=143) participants. The most well known complication of PIH was stillbirth (13.4%). Nearly half of the participants agreed that PIH needed pharmaceutical intervention for proper control.\n\nThe total number of answers provided by the expectant fathers ranged from zero to 33 (of 41 questions). The distribution of the total scores showed a highly-skewed distribution with majority aggregating towards low scores. There were 44 fathers who could not answer even a single question. For GDM, anaemia, and PIH, the percentage of expectant fathers who failed to provide at least a single correct answer was 24.8%, 40.2%, and 31.3%, respectively.\n\nThe distribution of score totals by socio-demographic characteristics is illustrated in Figure 1–Figure 5. Median scores increased steadily along with the average income and educational level. Expectant fathers in the 25–34 age group had significantly higher scores, compared to younger and older fathers. Fathers experiencing the second pregnancy of their spouses also had higher scores. Ethnic group was not associated with the scores obtained in this study sample.\n\n(The box covers the Interquartile range (IQR); whiskers extend 1.5 IQR from the box; circles shows outliers lying between 1.5 to 3 IQR from box and the asterisk shows extremes more than 3 IQR from box. Numbers denotes the identification number for the study unit).\n\n(The box covers the Interquartile range (IQR); whiskers extend 1.5 IQR from the box; circles shows outliers lying between 1.5 to 3 IQR from box and the asterisk shows extremes more than 3 IQR from box. Numbers denotes the identification number for the study unit).\n\n(The box covers the Interquartile range (IQR); whiskers extend 1.5 IQR from the box; circles shows outliers lying between 1.5 to 3 IQR from box and the asterisk shows extremes more than 3 IQR from box. Numbers denotes the identification number for the study unit).\n\n(The box covers the Interquartile range (IQR); whiskers extend 1.5 IQR from the box; circles shows outliers lying between 1.5 to 3 IQR from box and the asterisk shows extremes more than 3 IQR from box. Numbers denotes the identification number for the study unit).\n\n(The box covers the Interquartile range (IQR); whiskers extend 1.5 IQR from the box; circles shows outliers lying between 1.5 to 3 IQR from box and the asterisk shows extremes more than 3 IQR from box. Numbers denotes the identification number for the study unit).\n\n\nDiscussion\n\nPregnancy and childbirth have long been considered as a women’s domain9 by communities in which the involvement of male partners is minimal. Some studies even suggest that the number of expectant fathers seeking healthcare for their spouse’s problems is lower than that of non-expectant fathers10. The reason for this could be due to the fact that women are more likely to persuade men to access healthcare rather than men being able to persuade women to seek help11. However, the intentions of both partners have shown to be a better predictor of health behavior12; thus the father’s knowledge is vital in health-seeking behavior during pregnancy. The present study provides evidence to show that the expectant fathers have a limited knowledge of the main maternal morbidities. As this study showed that there was very poor knowledge, the results could not be interpreted, but there is definitely room for improvement.\n\nAnemia was assumed to be a well-known condition among Sri Lankan people due to several factors. It is a major determinant in maternal health, and the public health programme in Sri Lanka has conducted endless campaigns to prevent this condition over the last few decades. In a study done in 2009–10 period, the prevalence of anemia among pregnant women in Anuradhapura was reported as 14%13. However, the present study shows that in Anuradhapura, 37.4% of fathers were not even aware that anemia could be a problem during pregnancy. This raises a major concern regarding the effectiveness of the present health education programmes. Either these programmes are only directed at pregnant mothers, or the programmes have failed to convey their essential messages.\n\nKnowledge of GDM was slightly better than for other conditions, most likely due to the increasing community prevalence of diabetes in Sri Lanka, because the knowledge of the direct complications in pregnancy associated with GDM was poor. Prevalence of GDM in Sri Lanka is increasing, and a community-based study showed that around 10.3% of pregnant mothers in Sri Lanka experience GDM14. The present screening programme for GDM is not functioning well, due to a lack of facilities. If the community, especially expectant fathers, can be made aware of this condition, it will change care-seeking behavior and more cases will be detected and treated, thus preventing severe complications to both mother and child.\n\nEclampsia and PIH are listed as the second leading cause of maternal deaths in Sri Lanka. The condition accounts for a considerable proportion of hospitalizations, and the disease burden is very high. Routine blood pressure and urine albumin measurements are carried out in antenatal clinics as screening tests for PIH. In this study sample, risk factors and complications of PIH were known to a limited number of expectant fathers. However, dizziness, which is a non-specific sign of PIH, was known to more than 50% of the study sample as a clinical manifestation.\n\nOne major observation related to all three conditions was lack of knowledge about the complications of selected conditions. This lack of knowledge reduces the perception of risk due to these conditions. Risk perception is a core concept of health behavior, which is largely based on health literacy15. The study results suggest that the Sri Lankan maternal health programme should change their strategies in order to improve knowledge about these conditions.\n\nExploratory models have shown that health risk perception which is the basis for care-seeking depends on social class16, socioeconomic determinants, and the structure and practices of the health system17. Among pregnant mothers, knowledge about pregnancy risk factors18 and GDM has been shown to be associated with educational status19. We also observed that level of education and income are positively associated with expectant fathers’ knowledge about maternal morbidities.\n\nSuch inequalities in social factors seriously affect heath and underpin all other determinants of health. Sri Lanka has shown remarkable success in overcoming these social inequalities in reducing maternal deaths. However, the maternal morbidity pattern still reveals a high level of inequality, and poor knowledge among expectant fathers could be one major determinant of the problem.\n\n\nLimitations\n\nThe study sample consisted of expectant fathers who were visiting clinics. These fathers may be more health conscious and not representative of the total population of expectant fathers. The knowledge observed may be higher than the actual prevailing knowledge. The majority of the individuals in the study sample were visiting antenatal clinics at the teaching hospital in Anuradhapura. These fathers might represent a group of fathers whose socioeconomic background was different from fathers living in rural areas. Some of the questions included in the questionnaire were testing a very high level of health literacy. However, the percentage of correct answers was only used as a subjective score in the interpretation.\n\n\nConclusions\n\nThe limited level of knowledge among expectant fathers clearly shows room for the improvement of maternal health programmes. We suggest that safe motherhood programmes should have practical and operational strategies to include expectant fathers in maternal health promotion programmes.\n\n\nConsent\n\nAll participants gave informed consent to participate in this study. Ethical clearance was obtained from the Research and Ethics Committee, Faulty of Medicine and Allied Sciences, Rajarata University of Sri Lanka.", "appendix": "Author contributions\n\n\n\nAW, GLW, MPW and GMW are second year medical undergraduates. They conceptualized the study and carried out data collection, processing and analysis. SBA is the supervisor of the study, guided the other authors and prepared the manuscript.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nPart of this study was funded through a grant from Maternal Health Task Force of Engender Health (Grant number: GMH-106-01).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to acknowledge all the expectant fathers that participated in this study.\n\n\nReferences\n\nSummary of the International Conference on Population and Development, 5–13 September 1994. Earth Negot Bull. 1994; 6(39): 1. PubMed Abstract\n\nFisek NH, Sumbuloglu K: The effects of husband and wife education on family planning in rural Turkey. Stud Fam Plann. 1978; 9(10–11): 280–5. PubMed Abstract\n\nKim CH, Lee SJ: Role of husband in family planning behavior. Psychol Stud Popul Fam Plan. 1973; 1(5): 2–23. PubMed Abstract\n\nDudgeon MR, Inhorn MC: Men’s influences on women’s reproductive health: medical anthropological perspectives. Soc Sci Med. 2004; 59(7): 1379–95. PubMed Abstract \| Publisher Full Text\n\nRempel LA, Rempel JK: The breastfeeding team: the role of involved fathers in the breastfeeding family. J Hum Lact. 2011; 27(2): 115–21. PubMed Abstract \| Publisher Full Text\n\nSingh A, Ram F: Men’s Involvement during Pregnancy and Childbirth: Evidence from Rural Ahmadnagar, India. Popul Rev. 2009; 48(1). Publisher Full Text\n\nDepartment of Census and Statistics Sri Lanka. Estimated mid year population by sex and district. 2010. Reference Source\n\nFamily Health Bureau Sri Lanka. Statistics, Anuradhapura district. 2011. Reference Source\n\nBarua A, Pande RP, MacQuarrie K, et al.: Caring Men? Husbands’ Involvement in Maternal Care of Young Wives. Econ Polit Wkly. 2004; 39(52): 5661–5668. Reference Source\n\nQuill TE, Lipkin M, Lamb GS: Health-care seeking by men in their spouse’s pregnancy. Psychosom Med. 1984; 46(3): 277–83. PubMed Abstract\n\nNorcross WA, Ramirez C, Palinkas LA: The influence of women on the health care-seeking behavior of men. J Fam Pract. 1996; 43(5): 475–80. PubMed Abstract\n\nBecker S: Couples and reproductive health: a review of couple studies. Stud Fam Plann. 1996; 27(6): 291–306. PubMed Abstract \| Publisher Full Text\n\nChathurani U, Dharshika I, Galgamuwa D, et al.: Anaemia in pregnancy in the district of Anuradhapura, Sri Lanka--need for updating prevalence data and screening strategies. Ceylon Med J. 2012; 57(3): 101–6. PubMed Abstract \| Publisher Full Text\n\nGinige S, Wijewardhena K, Wijeyaratne CN: Prevalence of gestational diabetes mellitus in Homagama divisional director of health services area. J Coll Community Physicians Sri Lanka. 2004; 9: 40–42.\n\nBrewer NT, Chapman GB, Gibbons FX, et al.: Meta-analysis of the relationship between risk perception and health behavior: the example of vaccination. Health Psychol. 2007; 26(2): 136–45. PubMed Abstract \| Publisher Full Text\n\nFarber N: Perceptions of pregnancy risk: a comparison by class and race. Am J Orthopsychiatry. 1994; 64(3): 479–84. PubMed Abstract \| Publisher Full Text\n\nAtkinson SJ, Farias MF: Perceptions of risk during pregnancy amongst urban women in northeast Brazil. Soc Sci Med. 1995; 41(11): 1577–86. PubMed Abstract \| Publisher Full Text\n\nOdimegwu C, Adewuyi A, Odebiyi T, et al.: Men’s role in emergency obstetric care in Osun State of Nigeria. Afr J Reprod Health. 2005; 9(3): 59–71. PubMed Abstract \| Publisher Full Text\n\nCarolan M, Steele C, Margetts H: Attitudes towards gestational diabetes among a multiethnic cohort in Australia. J Clin Nurs. 2010; 19(17–18): 2446–53. PubMed Abstract \| Publisher Full Text\n\n\n\n\n" }	[ { "id": "1033", "date": "01 Jul 2013", "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 manuscript, which addresses the awareness of expectant fathers about health issues in expectant mothers. Clearly, health issues in pre-pregnant state and during pregnancy contribute towards compromised maternal and neonatal-infant health including mortality and such information is not commonly addressed in the literature. The authors have mentioned that this issue was addressed at an international conference, which was focused at reducing maternal and perinatal-infant morbidity and mortality. Suggested revisions: Although, the authors describe and analyze these data at great length, the manuscript is conspicuous by the absence of similar data about the expectant mothers from the same group. If they do not have such information or have not collected the same, it would be advisable to include historical data of this population or similar population, as collected by them or others in the literature.The authors have also reported that the neonatal and infant mortality was higher in the area, where the study was conducted, compared to the rest of Sri Lanka. Does this information address this disparity?It is not clear whether the fathers were aware of, or given information as to how this knowledge and these health issues would affect outcomes of these pregnancies, health of other members of their families and future health of their children - the authors should specify this. Do authors have any knowledge about the number or percentage of patients that attended these clinics in their pregnancies?In limitation of this study authors have addressed the issue of fathers who attend the clinic and those who do not. For those who do not, is it because they have limited resources and less knowledge?The authors have addressed three important issues: anemia, diabetes and eclampsia. How do they propose to use this information and implement changes if necessary, to educate the population by various measures including social awareness, impact on society, country and thus improve maternal and child health? They indeed mention that the Sri Lankan maternal health program study should change their strategy in order to improve knowledge about these conditions - this strategy should be a part of multi-prong approach.", "responses": [] }, { "id": "1119", "date": "18 Jul 2013", "name": "Tabassum Firoz", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important body of work as men's participation is critical in achieving good maternal health outcomes.I would caution the authors from making a statement that men are the 'primary stakeholders' or 'gatekeepers' in women's health. While you make the point that is certainly true in many societies, it important to keep in mind that how women access care is more complex. The root causes which are economic and social go beyond men.A minor point about semantics: I would avoid using PIH- either use gestational hypertension (or pre-eclampsia- I could not tell from the questions what you were targeting as these two entities are very different while being on the same spectrum). Secondly, instead of 'developing countries', I would suggest 'low income countries'.I am concerned that the language and the level of knowledge required to understand the questionnaire is indeed very high. You do rightfully point this out. Can you explain why you chose such a sophisticated level? How does this relate back to your objective of assessing knowledge around morbidity? The questions are at the level a health care professional might answer. A small percentage of your sample didn't even have any education! Most questionnaires should be targeted to a grade 6 (by North American standards) level.From a public health perspective, the key messages should be to recognize that a woman is sick (perhaps even have a certain condition in mind) and to know what actions should be taken to prevent and/or treat the condition. Most certainly, the key is to seek care as soon as illness is recognized. I don't think the questions reflect this.Secondly, some of the choices are wrong- for example, anemia does not present with severe headache. In fact, many serious conditions in pregnancy present with headache and this gives the impression that a less severe condition like anemia can present like this. Heel edema/ankle edema may be present in normal pregnant women and therefore, it is not a good choice. Bed rest is actually not recommended for the hypertensive disorders of pregnancy.It would be interesting to know how men whose partners previously had these conditions scored compared to men whose partners did not have these conditions.I think the authors need to revise the discussion section with more limitations and as well propose how this knowledge can be used.", "responses": [] }, { "id": "4139", "date": "17 Mar 2014", "name": "Shahirose Premji", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI wish to extend my appreciation for the invitation to referee this important study which examines expectant fathers’ knowledge and awareness about pregnancy complications – gestational diabetes mellitus, pregnancy-induced hypertension, and anaemia – that may impact health outcomes of both the mother and baby. The relevance of the work undertaken is well articulated in the introduction and the authors used a non-experimental descriptive research design to assess expectant fathers’ knowledge and awareness about selected morbidities. Fathers were recruited from antenatal clinics at the Anuradhapura Teaching Hospital. Eighteen percent (44 of 246) of expectant fathers were unable to answer a single question (awareness and knowledge). The remaining fathers (82%) demonstrated varied levels of awareness about gestational diabetes mellitus (78%), pregnancy-induced hypertension (74%), and anaemia (63%) and knowledge regarding risk factors, signs and symptoms, associated complications, and management strategies. The findings have implications for Sri Lankan maternal health programme intent to improve health literacy of the population and strategies to promote health seeking behaviors.\n\nMajor CommentsIntroduction:Hypothesis: The statement leading to the hypothesis emphasizes health inequities (i.e. lack of resources related to screening and managing morbidities); however this is not the focus of the study. Moreover, the hypothesis would suggest that the authors wish to explain why some expectant mothers and fathers seek care while other don’t; asserting that lack of knowledge and awareness may be one factor. The way in which the hypothesis is stated would suggest that the study examines both expectant mothers’ and expectant fathers’ knowledge and awareness about selected morbidities. The descriptive nature of the study would suggest that you want to determine whether expectant fathers whose partners are seeking care have increased knowledge and awareness given their role in decision making about seeking care. Design and Methods:Setting: Provide a more detailed description of the study setting to determine how representative the sample is of the population seeking care at this hospital. For example, whether it is common practice for expectant fathers to accompany their partner (i.e. wait with then or drop them off) to the antenatal clinic, and the average number of women who attend the clinic per day, to give perspective for the short duration of recruitment (November to December 2010). Recruitment: How did you enroll the expectant fathers who dropped off their partners at the clinic? Your demographic description should indicate the numbers of expectant fathers who were waiting with their partners and the number who were dropping their partner off. Provide more detail related to the recruitment process (e.g. the number who refused to participate, language use, if unable to sign how was this managed, what were potential risks/harms and how did you manage these?). Questionnaire: Provide more details. The English version of the questionnaire uses medical terms (e.g., gestational diabetes mellitus, Polyphagia) which participants may not be familiar with. For example, in some cultures diabetes is referred to as sugar in the blood. When translating the English to Sinhalese, was the language appropriately changed? What was the level of readability in the Sinhalese language? Was the questionnaire back-translated to English to ensure language equivalency? Was the questionnaire piloted? Given the variability in literacy (i.e., levels of education) within the community, how did participants complete the questionnaire? For instance, did the researcher or research assistant assist by using an interview format? On page 3 it is stated that “The total number of answers provided by the expectant fathers ranged from zero to 33 (of 41 questions)”. The questionnaire shared in Appendix 1 only has 23 questions. Explain how each of the questions, particularly those with multiple components (e.g., risk factors) was presented and how responses were elicited.\n\nAnalysis:You indicate “All demographic variables were analysed as categorical variables”; however report the mean age of participants and their partners. Were data normally distributed to report means? Revise this statement accordingly. Results:First paragraph: provide a brief description of your sample by highlighted distinct characteristics. For instance, for most women this was their first pregnancy. In the discussion comment on how your sample compares to the general population (i.e., how representative is it). Is it mostly first time mothers who access antenatal clinics? Did all expectant fathers provide demographic information? If so, include n of the sample in the table. You provide data on the prevalence of gestational diabetes mellitus, pregnancy induced hypertension, and anaemia as reported by fathers (page 3 second paragraph of results). There are two issues with this statement: (1) prevalence refers to the proportion of a population. Your study used a convenience sample. (2) The estimate was based on fathers’ report but many of the fathers were not able to recognize these morbidities. Please clarify. Findings: need to be presented more clearly. The missing data needs to be accounted for in the tables and explanations provided in the text. Please explain how the percentages were calculated. There are instances when the total percent count is over 100% (e.g. GDM risk factors) and in some instance well below 100% (e.g. GDM complications). Did all participants answer questions related to demographic characteristics? If so, an important consideration would be describing how similar or different the demographic characteristics were between respondents and non-respondents. Examination of the distribution of responses based on specific demographic variables: Explain what you mean by “score totals”m and how this score was determined? Given the multiple comparisons being made, you are likely to find a significant difference by chance (i.e., Type 1 error). Consider the p value adjustments required for these multiple comparisons. These findings should be interpreted with caution given the nature of the questionnaire (i.e., medical terms used), the type of design, and number of participants who were able to respond. Discussion:The first paragraph discusses expectant fathers seeking health care and non-expectant fathers seeking health care, which is not the focus of your study. Delete. Needs more depth and breadth by addressing: Findings regarding awareness: was this what you expected given the policy changes (i.e., campaigns in the country) over the last 5 years? Why may the findings not be in keeping with what you expected? Lack of knowledge may reduce risk appraisal which guides health behaviour (i.e. seeking care); hence how do you explain the health care seeking behaviour of these couples? What else may be guiding their health seeking behaviors? The World Health Organization recommends 4 antenatal clinic visits – what is the potential implication of the finding of your study with regarding to compliance to this recommendation. Knowledge about selected morbidities: discuss every aspect evaluated (e.g. risk factors, signs and symptoms, etc.). What can be gleaned from these findings? Are similarities and differences? Would you expect the population have knowledge about management? Discuss the role of parity with reference to health seeking behaviors and knowledge or health literacy. Be careful with the inferences you are making. For example, improving knowledge will improve health seeking behavior. You findings suggest that expectant fathers seek health care for their pregnant spouse even though they don’t have the knowledge. Refocus the discussion with emphasis on the findings and implications for practice, future research and policy. Limitations:Consider revising after you have addressed the above points. For instance, if medical terms were used, this may explain why so many respondents were not able to complete even one question. Quality of written English:Results and Discussion: a few sentences which are not clear. Page 3, last paragraph has a couple of sentences which are not clear. Page 7, discussion, first paragraph, second sentence and last sentence in the paragraph. Page 7, last paragraph beginning knowledge of GDM delete “because the knowledge of the direct complications in pregnancy associated with GDM was poor” as this does not explain the finding. Was there another point you were trying to make? Please clarify. Page 8, limitations, last sentence in the paragraph – not sure what you mean. Minor Comments:Title: insert “selected” after knowledge of. Abstract: The results section may need to be modified after you have addressed the methodological issues. Language: change developing country to lower middle income (The World Bank income level categorization for Sri Lanka). Tables and Figures: Ensure all abbreviations are written out in full at the bottom. Figures 1-5: present in the order in which they are discussed in the text.", "responses": [] } ]	1	https://f1000research.com/articles/2-119
https://f1000research.com/articles/2-118/v1	01 May 13	{ "type": "Opinion Article", "title": "Morgellons: a novel dermatological perspective as the multisystem infective disease borreliosis", "authors": [ "Peter Mayne", "John S English", "Edward J Kilbane", "Jennie M Burke", "Marianne J Middelveen", "Raphael B Stricker", "John S English", "Edward J Kilbane", "Jennie M Burke", "Marianne J Middelveen", "Raphael B Stricker" ], "abstract": "Morgellons disease (MD) is a term that has been used in the last decade to describe filaments that can be found in human epidermis. It is the subject of considerable debate within the medical profession and is often labeled as delusions of parasitosis or dermatitis artefacta. This view is challenged by recent published scientific data put forward between 2011-2013 identifying the filaments found in MD as keratin and collagen based and furthermore associated with spirochetal infection. The novel model of the dermopathy put forward by those authors is further described and, in particular, presented as a dermal manifestation of the multi-system disease complex borreliosis otherwise called Lyme disease. A differential diagnosis is drawn from a dermatological perspective. The requirements for a diagnosis of delusional disorder from a psychiatric perspective are clarified and the psychological or psychiatric co-morbidity that can be found with MD cases is presented. A concurrent case incidence is also included. Management of the multisystem disease complex is discussed both in general and from a dermatological perspective. Finally replacement of the term ‘Morgellons’ by ‘borrelial dermatitis’ is proposed within the profession.", "keywords": [ "Morgellons", "Lyme disease", "filaments", "delusional parasitosis", "dermatitis" ], "content": "Introduction\n\nThere has been considerable debate about the disorder referred to as Morgellons disease (MD), which has no general medical acceptance as a disease entity. Rather it has been labeled ‘delusions of parasitosis’ or ‘dermatitis artefacta’ with enormous stigma attached to these terms and patients have been inappropriately medicated as discussed later. The dermopathy is characterized notably by filaments found under or protruding from the skin on microscopic examination from any area of anatomy without predilection for any particular site. Analysis has shown these to be monofilamentous exhibiting multiple colors including black, red, orange, blue, purple, white and clear1,2. They also fluoresce under UV light (except for the black ones)1,2. In 2011 similarity between MD and bovine digital dermatitis (BDD), a known spirochetal disease of cattle, was identified and published3. In 2013 Morgellons tissue was demonstrated to contain keratin and collagen filaments by several staining methods4. Spirochetal loads were found by light microscopy at 1000x magnification with silver nitrate staining and also by immunofluorescent staining with polyclonal anti-borrelial antibodies at 400x and 1000x magnification4,5. Further analysis using real time PCR demonstrated that the spirochetes were borrelial species5. This article firstly analyses the position of these findings in dermatology with comparative and differential analysis at the dermal level but then also highlights the characteristics that distinguish the dermopathy as part of a multisystem disease that principally involves arthritic, cardiac, neurological and psychological or psychiatric components. Further it is proposed that the diagnosis of delusions of parasitosis should be replaced with a novel described dermopathy as a feature of a multisytem disease.\n\n\nHistorical matters from an organic viewpoint\n\nA PubMed search using the term \"Morgellons\" on the 28th January 2013 returned 42 articles. From a historical perspective, after filtering these results for an organic rather than delusional or psychiatric causation the following milestones are noted. The term Morgellons was reported by Kellett in 1935 as being coined in 1674 by Sir Thomas Browne in his monograph entitled \"De vermiculis capillaribus infantium\"6. The affected child described experienced critical break outs of hair-like extrusions from the back, which upon occurring relieved the child from \"coughs and convulsions\". This information was re-reported and further elucidated historically by Emslie-Smith in the British Medical Journal in 19467. The terminology was reapplied around 2002 to describe the phenomenon of fine filaments found in skin using at least 60x magnification1. Mary Leaitao had found the terminology while researching her son’s dermopathy thus popularizing the term in a modern context. In 2010 Savely and Stricker described a population with microscopically confirmed filaments2. In 2011 Middelveen and Stricker published a novel perspective demonstrating marked similarity between MD and BDD3. The latter affects the skin above the hooves of cattle causing sores and lameness with associated economic loss and has been known and accepted as a spirochetal disease in the veterinary profession since 19748.\n\nIn 2012 Pearson et al. published a long awaited Centre of Disease Control (CDC) sponsored study on the possible causes of MD9. The key findings of this study of only 115 patients were: median age 52, 77% female, 77% Caucasian, 70% chronic fatigue symptoms, 54% reported poor state of health, 59% were found to have cognitive defects on testing, 63% had somatic complaints, 50% had detectable drugs in hair analysis and 78% reported exposure to solvents. Psychological issues spanned poor attention and memory, highly significant somatic disorders, depression and 24% had significant alcohol and drug use. Histological examination showed 57% solar elastosis or otherwise features consistent with arthropod bites or chronic excoriation. No parasites or mycobacterium were detected. Finally materials harvested were cotton or silicon. In the last paragraph of that paper it states under the discussion:\n\n\"We were not able to conclude based on this study whether this unexplained dermopathy represents a new condition, as has been proposed by those who use the term Morgellons, or wider recognition of an existing condition such as delusional infestation, with which it shares a number of clinical and epidemiologic features\".\n\nHowever in view of the prevalence of psychological issues found in the study the authors make the final recommendation:\n\n\"In the absence of an established cause or treatment, patients with this unexplained dermopathy may benefit from receipt of standard therapies for co-existing medical conditions and/or those recommended for similar conditions such delusions infestation\".\n\nOne can interpret an open finding from this study, though the difficulty of showing an absence of an organic cause is acknowledged. In the study, matters pertaining to skin biopsy, coexisting multisystem disease and psychiatric co-morbid conditions are presented in a manner that aligns with the content of this review, as will be discussed. Also, it is noteworthy that this study used symptomatology exceeding three months duration as an exclusion criterion, which is not presented until the discussion on page nineteen, paragraph two of the article. This would have impacted markedly on incidence/prevalence figures and also the detection of multisystem disease in longer standing cases.\n\nIn contrast most of the results from the PubMed search mentioned at the beginning of this section, are dedicated to the diagnosis ‘delusions of parasitosis’. A further search using the term \"delusions parasitosis\" on the same date returns 320 findings all of which present non-organic illnessness. It is not possible to find a single paper amongst these with substantiated psychiatrically diagnosed delusional disorder in line with the discussion in the ‘Clinical features–psychiatric’section of this article.\n\n\nPrevalence\n\nSavely and Stricker found an association between MD and tick-borne diseases and hypothyroidism, a high prevalence of the condition in middle-aged white women and an increased prevalence of both smoking and substance abuse2. Although depression was noted in MD a pre-existing delusional disease was not reported. A retrospective study on delusions of parasitosis by Foster et al. analyzing Mayo Clinic, Rochester, Minnesota records from 2000 to 2007 gives a valuable insight as to whether these people in fact had MD, and shows a 75% female prevalence10. Symptom duration averaged 2.3 years with a range of 2 weeks to 23 years and more than 80% of patients were older than 50 years10.\n\n\nClinical features–dermatological\n\nThe symptoms described in this section are those noted by the first and last authors in their clinical practice. It is fair to say that much of the citable literature, as referred to above, describes this same symptomatology.\n\nPatients present with severe itching, pain, crawling or stinging sensations which may be localized, and may describe symptoms similar to formication or something growing in and extruding from the skin. There may be no visual dermopathy in early presentation. Patients may appear obsessed or even distressed with their symptoms and may describe finding filaments or tiny dark specks (some of which glitter) on or in the skin (Figure 1). Often patients have used magnification devices and may present with a container that demonstrates the offending particle or bug, which is usually dismissed as eschar debris by the professional and has been termed the ‘matchbox sign’11. Sometimes there is a belief that a bug or mite is growing in the skin and some will report that they have seen them. Questioned on this the patient may state they see little insects that fly past their eyes. It is plausible that these insects are attracted to the detritus of the damaged surface. In other cases several people (particularly familial, of which a case is briefly discussed hereunder) may develop the disorder at the same time and in this scenario the patient often believes they have transmitted the condition to family or friends. Sometimes patients exhibit highly compulsive behavior with excessive cleaning of the home in the fixed belief that the environment is infective. Temporary marital separation for the good health of the partner is not uncommon.\n\nIn these scenarios it becomes important to the patient to find evidence of filament disease, if they have not already done so, by magnification of itchy skin or evident dermopathy. Examination with a high power magnification of at least 60x is essential in the search for the presence of filaments under unbroken skin1–3. It is important not to fall into the trap of mistaking cotton and synthetic filaments for Morgellons filaments (the latter are typically smaller) and anyone familiar with a dermoscope knows all too well that the former are frequently seen loose on the skin or adhered to a damaged or ulcerated surface. Figure 2 shows cotton fibers adhered to sebaceous hyperplasia on the face of a 65 year old man using dermoscopy as well as a dermoscopic comparison between a MD wound ball filament and an overlying piece of cotton. Note that the single fiber strands on the side of the twisted cotton thread are closer in size to that of the MD fiber depicted here. The fact that the CDC study only used a dermoscope in the search for filaments may explain why only cotton fibers were found in that study, and so the opportunity to find the smaller filaments found in MD was missed9.\n\nA) Dermoscopy view of cotton fibers adhered to sebaceous hyperplasia. B) Dermoscopic comparison to a Morgellons wound ball filament with an overlying piece of cotton (blue).\n\nIn more advanced forms of the disease there are widespread chronic elevated lesions, sometimes papular but more usually a plaque, ranging in size from 5 mm to 2 cm or more, and generally with recurrent surface eschar. Rarely the plaques can be huge (up to the length of the thigh). There may be ulcers and considerable eschars at more advanced stages.\n\nIf the disorder was not chronic, it could easily be mistaken for impetigo or other infective presentations such as staph folliculitis. Persistence for many weeks raises the differential diagnosis of scabies but this can be ruled out if careful observation shows that every wound is persistent and cycling over a 3–4 week period, and no mites or track marks are found.\n\nNail dystrophy may be present, somewhat similar to a psoriatic rather than fungal appearance, though the distinction can at times be difficult. Mucosal surfaces may be involved12,13. Patients have also described sudden deterioration of dental health. This is so far unreported in the literature. Of note is that treponema are considered normal oral flora but a recent study by Dabu et al. implicates a treponemal role in periodontal disease14.\n\nThe condition may also be compared to the dermal manifestation of syphilis in its secondary and tertiary stages where pruritus can be a dominant feature, and mucous membrane as well as nail dystrophy changes may be present15–20. It is also important to distinguish MD from intravenous drug abuse where formication can be a feature.\n\nLichenification may be present from the continual scratching and distinguishing MD from eczema by the detection of MD filaments is thus important. Post inflammatory hyper and hypo-pigmentation, depending on Fitzpatrick skin type, is a long standing feature of MD (Figure 3). There may be changes of lichen simplex chronicus in an older lesion and eventually ulceration. A widespread nodular appearance could be mistaken for nodular prurigo (Figure 4). There can be widespread pruritus without skin inflammation or restricted areas of inflammation.\n\nThe dermal presentation of MD is therefore a confusing picture though it appears that the inflammation and/or lesions are caused by dermal mechanical stress and irritation. It is proposed the mechanical stress is both internal and external from the filaments and scratching respectively. It is very important to note that the patient will report that they are scratching and damaging the skin surface, unlike the denial that is known to occur in factitial dermatitis21.\n\n\nClinical features–systemic\n\nThe authors propose that MD is a manifestation of Lyme disease (LD) which is otherwise called borreliosis, an infection vectored to humans by ticks. This section will therefore focus on LD, as will other sections in this manuscript.\n\nAlong with the previously described dermatological presentation it is necessary for the practitioner to consider the presence of symptoms or signs of multisystem disease. Attention must be given to evidence of arthritic (large joint mono or pauci arthritis), cardiac (AV blocks, carditis, pericarditis and pancarditis) and neurologic disorder22–24. In a recent review Biesieda has covered the scope of these presentations25. Gastrointestinal, genitourinary, gestational, musculoskeletal, nephritic and ophthalmic problems may occur26–29. Symptomatic history can extend for years. Pediatric LD has extensive similarities to adult forms of the condition (except for meningopolyradiculoneuritis and acrodermatitis chronica atrophicans, which are typically not seen in children) as noted in a comparative review by Esposito and co-authors30. A very comprehensive set of guidelines for LD diagnosis and management (2010) is available from Deutsche Borreliose-Gesellschaft, the German Borreliose Society and adequately sets out the spectrum of the disease and its management. It is available at http://www.borreliose-gesellschaft.de/Texte/guidelines.pdf. Dermatologists are well versed in the knowledge of conditions requiring urgent intravenous antibiotic therapy. In a 2012 review Sambrano and co-authors set out recent recommendations on therapy and dosages for a range of diseases with dosages including LD with its variant presentations and the necessities of oral versus intra venous therapy31.\n\nAppropriate referral may also be considered as the dermatologist may restrict his interest in the patient to dermatological symptoms. In the past the dermatologist in a MD scenario has been diagnosing a psychiatric disorder and furthermore offering and suggesting psychotropics in a manner beyond and outside his sphere of expertise as discussed in the next section.\n\n\nClinical features–psychiatric\n\nIn addition to the dermatological, constitutional and systemic symptoms described, there are usually components of psychiatric morbidity in patients with MD. The first, third and last authors have encountered a range of illness across the depressive/anxiety spectrum in their clinical encounters with MD. Unfortunately, non-psychiatric medical practitioners or un-informed psychiatrists often label these patients as delusional, specifically with a well circumscribed somatic delusion which includes belief in infestation with insects or imagined inanimate objects such as filaments and particles. While these patients do suffer from psychiatric morbidity it is most often in the depressive and anxiety disorders spectrum and not likely a primary psychotic process.\n\nA confounding factor may be that the pathophysiology of MD itself somehow impacts the peripheral or central nervous system in ways that create psychiatric symptoms (disorientation, visual changes or hallucinations) or amplifies other stimuli (tactile etc.) and results in a misperception of a real stimulus to be perceived as something else altogether. Of note, the case of misperception of a real stimulus does not qualify as a psychotic symptom as in fact the symptom is based in reality, or on real stimuli. According to the Diagnostic and Statistical Manual of Mental Disorders fourth text revision (DSM-IV) TR32, delusional disorder is an illness characterized by the presence of non-bizarre delusions in the absence of other mood or psychotic symptoms. Delusions are false beliefs based on incorrect inference about external reality that persist despite the evidence to the contrary. These beliefs are not ordinarily accepted by other members of the person's culture. The subtypes of delusion in the DSM-IV TR include: erotomanic, grandiose, jealous, persecutory, somatic, mixed, and unspecified.\n\nInterestingly, patients with MD are diagnosed with delusions of parasitosis or infestation, neither of which exists as a clinical entity in the American classification of psychiatric disorder. Neither do these diagnoses exist in the World Health Organisation International Classification of Diseases33 where the closest diagnosis would be factitious dermatitis. From a psychiatric viewpoint the closest diagnostic category in the DSM-IV TR would be the subtype of somatic delusions, referring to the belief held by an individual that they have a physical defect or general medical condition that has yet to be identified. The medical profession must be careful when making a diagnosis of somatic delusion for certain specific reasons. First, history has proven that the medical establishment has often misconceived a collection of symptoms, or syndrome, to be psychological in origin (or psychologically mediated) rather than physiological. A prominent example is the aetiology of Peptic Ulcer Disease, once believed to be stress induced but now known to be caused by helicobacter infection34. The consequences of such misdiagnosis include delay in appropriate treatment, worsening of the actual disease process, and punitive labelling of the patient as \"psychiatric\", which in itself can have several adverse outcomes such as psychtropic medication side effects.\n\nSecond, the diagnostic criteria for delusional disorder, as outlined below, are quite specific in guiding clinicians (specifically mental health professionals) on how to arrive at this \"rule out\" or diagnosis of exclusion. The most recent criteria for a delusional disease, which are not manifest in MD patients, include:\n\nA: Nonbizarre delusions occurring for at least 1 month's duration involving situations that occur in real life, such as being followed, poisoned, infected, loved at distance, deceived by spouse or lover, or having a disease.\n\nB: DSM-IV-TR Criteria A for schizophrenia has never been met. Patients do not have simultaneous hallucinations, disorganized speech, negative symptoms such as affective flattening, or grossly disorganized behavior. Note: Tactile and olfactory hallucinations may be present in delusional disorder if they are related to the delusional theme.\n\nC: Apart from the impact of the delusion(s) or its ramifications, functioning is not markedly impaired and behavior is not obviously odd or bizarre.\n\nD: If mood episodes have occurred concurrently with delusions, their total duration has been brief relative to the duration of the delusional periods.\n\nE: The disturbance is not due to the direct physiological effects of a substance, either a drug of abuse or a medication, or a general medical condition.\n\nThe \"matchbox sign\" and \"meme\" are oft quoted reasons supporting a diagnosis of psychotic beliefs in patients presenting with symptoms of MD. The \"matchbox sign\" is the term applied to the scenario where a patient brings in samples of what they believe to be foreign material from their own bodies to their physician for inspection. This behavior was first described by Perrin in 1896 but the term \"matchbox sign\" was not coined until a 1983 case presentation in The Lancet in which the author noted the use of the matchbox as a device to store and present a specimen11. The \"meme\" is the concept of an idea or behavior that spreads from person to person within a culture; it acts as a unit for carrying cultural ideas which can be transmitted from one mind to another through writing, speech, rituals or other forms (i.e. internet) of communication. In 2009 the paper \"Morgellons Disease as Internet Meme\" was published in the psychiatric journal Psychosomatics, further spreading the belief that MD is a fabrication of man’s psyche, a fabrication that can be transmitted to others as easily as its symptoms are reported on various websites35.\n\nInterestingly, various fields of medicine hold the randomized, controlled, double blinded study to be the gold standard by which to measure efficacy or statistical associations in diagnosis, treatment, and prognosis. The authors are unaware of any studies that use a standardized and methodological approach to prove the relationship between bringing samples of perceived infesting specimens to a physician and the appropriate diagnosis of delusional disorder. In the same vein they are also unaware of studies that can demonstrate that access to the internet or to knowledge obtained on the internet about MD acts as the inciting factor or as a propagating vector for the spread of a delusional disorder. If this were the case, and given the explosion of media forms (radio, television, internet, mobile devices etc.), one might expect a far more drastic rise in the reported cases of delusional disorders of all types over the past 100 years.\n\n\nDifferential diagnosis\n\nAn appropriate comparison grouping would be arthropod bites, dermatitis artefacta, drug reaction, eczema including nummular and discoid types, impetigo, lichen planus, lichen simplex chronicus, nodular prurigo, scabies including Norwegian scabies, tinea corporis, toxoplasmosis (cutaneous) and severe pruritus from systemic disease e.g. lymphoma and syphilis (secondary and tertiary). The tropical diseases cutaneous larva migrans, leishmaniasis, gnathostomiasis and schistosomiasis may have to be considered if there is a positive travel history but these should be specifically diagnosable. The perforating dermatoses have been described and compared to MD by Savely, Leitao and Stricker1. These include acquired perforating dermatosis, elastosis perforans serpiginosa, Kyrle disease, perforating folliculitis and reactive perforating collagenosis. The banishment of the concept of delusions of parasitosis as accepted medical terminology is proposed and it should be noted that there is no such listing as a recognised entity in the DSM-IV (TR) or the upcoming DSM-5 as discussed above.\n\n\nDiagnosis\n\nMD has been defined as the presence of varying coloured filaments visible with light microscopy at a magnification of at least 60x in or protruding from skin lesions or under unbroken skin2. Detecting the presence of spirochetes may be possible by light microscopy, but now the detection of borrelial species by culture, staining, immunofluorescent staining, electron microscopy or PCR is an equally valid proof in the presence of MD symptoms5. However there is also the aspect of describing the character of the dermis as affected by these filaments, and this is discussed below showing a multivariate presentation and histology confirming dermatitis. The presence of itch and inflammation makes this condition a dermatitis as distinct from a dermatosis, though as discussed, pruritus can be a leading symptom with no skin change evident. The reverse scenario of a patient with borrelial infection describing a coexistent skin condition with a similar time line requires close consideration of the possibility of MD. Also of note is the fact that erythema migrans (EM) lesions may be described by patients as itchy and painful, and generalized pruritus has been noted in some patients with EM, the implication being that MD can have an instantaneous onset, which is further discussed in the next section36. Finally, typical features of MD involve persistence, recurrence, varying sites and cyclical dermal involvement.\n\n\nConcurrent case incidences\n\nOften two or more members of a family can be affected simultaneously. The lead author can report multiple instances in his practice; however in one scenario a family of three were infested with ticks after an Indian myna bird, Acridotheres tristis, was trapped in their house on the east coast of Australia for two days while they were away. The young wife and twelve month old child proceeded to develop symptomatology of MD. At first clinical presentation to the lead author six months later, the wife presented symptoms of MD together with some evidence of LD, reporting brain fog, poor concentration and memory, dental pain, depression, anxiety, panic, insomnia, irritability and right sided facial pain in the distribution of the second and third division trigeminal nerve. All symptoms were new and commenced with the MD. No neurological deficit was demonstrable. A diagnosis of Morgellons and neurological Lyme was made, confirmed with positive real-time PCR for Borrelia sp. on whole blood at Australian Biologics in Sydney, Australia. The method is as previously described5 and is also presented below. Blood was also forwarded to IGenex California USA for western blot analysis which was positive for LD. IgM was evident at bands 18+, 31+, 41+ and 58++ kDa. The IgG showed indeterminate bands at 31, 34 and 39 kDa and positive at bands 41++, 45+ and 58+ kDa. She presented with photos of a fed nymph tick removed from her body (which at the time was not formally analysed) and also a black speck from her skin for my analysis. She had examined the speck under 200x magnification and found a black ball of twisted filament. That speck is presented at 500x magnification showing a ball of firmly wound filament which was red at the higher magnification (Figure 5).\n\n\nSkin microscopy\n\nDermoscopic scanning for anything smaller than a textile filament may be helpful but it is very difficult at this 10x resolution to identify Morgellons filaments with certainty. Higher magnification is required. Savely and Stricker recommended a minimum of 60x2. An eschar specimen from a female Morgellons patient in her late forties was obtained and bisected. The dermoscopic view is shown in this case and does show filaments at 10x magnification (Figure 6). See Figure 7 for a light microscopy image of the same material at 500x. See Figure 8 for Scanning Electron Microscopy (SEM) images of the same material (note the frond like appearance).\n\nIn a different male patient in his late forties with a shaved scalp, blue filaments appearing to arise from the infundibulum of a hair shaft were imaged (Figure 9). This image was obtained by direct observation of skin using a 500x digital microscope. It appears that the filaments are perpendicular to the hair and leaving the hair follicle at the level of the infundibulum. The smallest is approximately 2.5 microns in diameter. The filaments appear to originate from the outer sheath of the hair apparatus and the source could be anywhere from above the sebaceous gland on the acute angle side or on the obtuse side above the erector pilii muscle and then up to the superficial flattened areas of the epidermis. Clinically the patient also had neuroborreliosis for the same time interval as his skin disease. Please refer to the endpoint polymerase chain reaction (PCR) analysis of this patient’s whole blood in the Endpoint section.\n\n\nHistopathology and pathogenesis\n\nSkin histology may show acanthosis and spongiosis particularly in an early lesion. Other descriptors found are psoriasiform hyperplasia, thickened granular layer, compact hyperkeratosis and parakeratosis thus giving a rather confusing picture. Sometimes the filament will be seen and reported as extraneous material. However protein-specific staining has shown the filaments may be composed of keratin or collagen4. Middleveen and Stricker drew an association between Morgellons and bovine digital dermatitis as previously discussed3. In 2006 Savely and Stricker drew an association between Morgellons and LD, also a spirochetal infection2. Middleveen et al. showed spirochetal evidence in Morgellons tissue by microscopy, culture, immunostaining, scanning and transmission electron microscopy and showed the presence of Borrelia species with PCR evidence5. In MD the filaments, as well as growing outwards and in the horizontal plane, can grow through the dermis and into subdermal tissues with papilla formation (these papillae encase helically coiled filaments). See the under surface of an eschar from a diagnosed MD patient (Figure 10). Finally in biopsy reports from the skin of patients believed to have delusions of parasitosis, Hylwa describes dermatitis features on histological examination in 61% of patients studied, including 33 cases of chronic dermatitis, 10 cases of subacute dermatitis and 6 cases of lichen simplex chronicus37.\n\n\nPCR testing\n\nSpirochetal presence in the skin of diagnosed Morgellons patients has previously been demonstrated, including PCR detection of borrelial pathogens5. In this segment PCR validation of systemic borrelial infection with diagnosed MD is addressed.\n\nBorrelial real time PCR testing was carried out on the aforementioned female patient (concurrent case presentation) at Australian Biologics by the fourth author in August 2012. B. burgdorferi was detected using the Eco™ Real-Time PCR system with software version 3.0.16.0. The sample was analysed with positive and negative controls using primers and a probe for the borrelial 16S gene target. The primers were AB-Bor1 (proprietary to Australian Biologics). The thermal profile for the analysis involved incubation for 2 mins at 50°C, polymerase activation for 10 mins at 95°C then PCR cycling for 40 cycles of 10 secs at 95°C dropping to 60°C sustained for 45 secs. Subject material showed a positive quantitative cycle Cq result of 36.39. Please see amplification plot where there are also two positive controls to the top and 2 negative controls at the base and Cq table to the right (Figure 11).\n\nBorrelial PCR testing (endpoint) was carried out on the aforesaid male patient (discussed in the skin microscopy section) at Australian Biologics by the fourth author in April 2011. The method is tabulated viz:\n\nDNA was prepared by blood collection (7 ml) into 1 ACD tube and centrifuged at 1200 rpm for 10 minutes. The buffy coat layer was removed by taking a small amount of red blood cells and plasma with the leukocytes then 500 µl of buffy coat cells were pipetted into a sterile 1.5 ml Eppendorf tube and 500 µl of Qiagen AL buffer added. Qiagen was sourced from their Australian distributor in Chadstone, Victoria. DNA was extracted using the Qiagen QIAamp® DNA Blood Mini Kit, according to the manufacturer’s instructions, and eluted in 75 μl of Qiagen AE buffer. Then primers targeting the Borrelia burgdorferi rpoC gene were used. The rpoC gene was amplified using Forward Primer 5’-AAATGGCTAAAGTAAGCGGAATTGTAC-3’ and Reverse Primer 5’-CAGAAATTCTGTAAACTAATCCCACC-3’, as described by Adelson M. E. et al38. In a 25 µl reaction consisting of 12.5 µl of Promega’s GoTaq® DNA Polymerase (Promega, Australia) 7.5 pmoles of each primer, 4.5 µl H2O and template of 5 µl. Product size 231 bp. Cycling conditions were initial denaturation at 95º for 45s followed by a 10 cycle Touchdown of 15s@95º, 45s@65º–56º and 45s@72º then 50 cycles 15s@95º, 45s@55º, 45s@72º with final extension of 2mins@72º. PCR products were analysed by standard 2.5% agarose gel. Product of the PCR amplification was purified using the Marligen Rapid PCR Purification Kit (Marligen Biosciences, Australia) following manufacturer’s instructions. The product was eluted with 50 µl of warmed TE buffer (Promega, Australia) and forwarded to Australian Genome Research Facility (AGRF) in Sydney, Australia for sequencing.\n\nSerum endpoint PCR gave a positive result for borrelial species. The sequence produced was 5’-CGGGGGGCCGAGGGATCGTTGAAAGGTAAAGGCTTATTATATTTTAGATGAGTATGGGGTTGAC-3’. The post edited sequence was 5’-TTATTATATTTTAGATGAGTATGGGGTTGAC-3’. This sequence was then submitted for Basic Local Alignment Search Tool (BLAST) inquiry as previously described39,40. The results show the top two findings to be Borrelia garinii. Significant findings are presented in Table 1 and the top two alignments with two matches in Figure 12.\n\n\nManagement and prognosis\n\nThere has been considerable debate about MD treatments. To date, an exhaustive list of home remedies can be found on the internet, but evidence supporting the efficacy of these treatments is lacking. Segments of the profession have tried therapy with antihelminthics including ivermectin, mebendazole, praziquantel and albendazole, but this approach as sole therapy is by and large ineffective and false conclusions as to their efficacy can be drawn by the cycling nature of the dermopathy. No published material is available supporting such use. The lead author has found that adequate treatment of B. burgdorferi will suppress and control the disorder, more easily so in earlier disease (unpublished observations). Such treatment requires simultaneous antibiotic polypharmacy. Cell wall drugs are useful for the extracellular spirochete forms, tetracyclines or macrolides are useful for the intracellular forms, while imidazole compounds such as metronidazole and tinidazole are useful for the cystic forms of B. burgdorferi. In conjunction with LD treatment, the myriad associated coinfections need to be addressed including Anaplasma, Babesia, Bartonella, Ehrlichia and Rickettsia. With advancing knowledge of cell-wall deficient forms of the Lyme spirochete, the mechanisms associated with chronic protracted LD are being understood, albeit slowly41,42. This knowledge also helps us to understand why skin examination looking only for spirochetes can be unrewarding as given the right conditions both the intracellular and cystic forms can morph back to a spirochetal form. A recent study highlighted the significant burden of protracted LD patient care on healthcare systems43, and delayed diagnosis of MD and LD adds to that burden. We do not know if skin co-infections can be excluded at this juncture, nor can we rule out the possibility of co-factors playing a crucial role in filament formation. These are areas for ongoing research as is the exact mechanism of filament production. To date the long-term prognosis for protracted LD illness that includes MD remains unsettled.\n\n\nNomenclature\n\nLD or Lyme borreliosis as it is sometimes called, is borreliosis in the strictest sense. The dermatology profession now has to consider that in the same manner, the more correct terminology for MD would be ‘borrelial dermatitis’. ‘Spirochetal dermatitis’ could be considered as a suitable term but in the authors opinion is not specific enough as the pathogens appear to specifically belong to members of the genus Borrelia.\n\n\nDiscussion\n\nBorrelial dermatitis or MD is characterized by the progressive onset of skin irritation due to epidermal formation of filaments known to be keratin or collagen4 in nature and arising from the base of the epidermis and can arise from a disrupted hair follicle as demonstrated in Figure 9. Filaments are typically 5–50 microns in diameter. Extrusion through skin and entrapment under the skin leads to pruritus, formication, other tactile disturbances and subsequent skin damage, both internal and external, which at the extreme can manifest as large chronic ulcerations. The clinical appearance of skin can range from an eczema-like appearance through to that of nodular prurigo. Mucosal surfaces may similarly be affected and nail dystrophy may be present. Evidence of multi systemic LD (borreliosis) can be found. Spirochetal presence can be demonstrated and borrelial infection implicated5. Although it is not the authors’ belief that MD is primarily a psychotic disorder, it is evident that psychiatric comorbidity is often present in this patient group. Whether this is due to a pre-existing psychiatric diagnosis or one which develops such as acute stress reaction, adjustment disorder or post-traumatic stress disorder during the course of emerging MD symptoms and subsequent attempts at treatment, it is important to recognize psychiatric comorbidity. Timely identification and efficacious treatment of psychiatric symptoms can improve overall psychological well-being, possibly reduce the chance that patients avail themselves of dangerous and idiosyncratic treatments for MD symptoms, increase rapport and follow up with physicians in multiple specialities, and preserve the dignity and respect which is due to the patient. Hylwa and co-authors reported a significant load of psychiatric burden in a study of Mayo clinic patients with \"delusional infestation\" but psychiatric delusional conditions were not reported44. We argue that Morgellons disease, a disorder by and large dismissed by the profession as delusions of parasitosis, in particular by dermatologists and primary care physicians, and supposedly highly vectored by internet transmission should now be called borrelial dermatitis within the profession and acknowledged as a presentation of a multisystem infective disease (LD) and treated appropriately as such35. Research needs to be directed at identifying the fundamental flaw in filament production at the cellular level, which may involve RNA/DNA dysfunction, in the hope of addressing further treatment.", "appendix": "Consent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patients.\n\n\nAuthor contributions\n\nPM conceived the report and developed the dermatological aspects with JE. EK contributed all psychiatric components. JB carried out all PCR testing and with PM examined BLAST alignments. MM contributed to microbiology components. RS reviewed Morgellons features. PM and RS developed systemic LD presentation. PM prepared the first draft of the manuscript except for that component specifically made by EK. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\nPJM, EJK and RBS serve without compensation on the medical advisory panel of the Charles E. Holman Foundation. MJM serves without compensation on the scientific advisory panel of the Charles E. Holman Foundation. Each otherwise has no relevant competing interests to declare. JSE and JMB also have no conflicts to declare.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe lead author wishes to acknowledge the DPD course of the Department of Dermatology at Cardiff University and the DCPDS annual meeting in 2011 where he was given the opportunity to present a short talk on Morgellons which became the formative vessel for this paper in collaboration with the second author. The authors thank Dr Robert Allan for manuscript review.\n\n\nReferences\n\nSavely V, Leitao M, Stricker R: The mystery of Morgellons disease: infection or delusion?. Am J Clin Dermatol. 2006; 7(1): 1–5.\n\nSavely VR, Stricker RB: Morgellons disease: Analysis of a population with clinically confirmed microscopic subcutaneousfibers of unknown etiology. Clin Cosmet Investig Dermatol. 2010; 3: 67–78.\n\nMiddelveen MJ, Stricker RB: Filament formation associated with spirochetal infection: a comparative approach to Morgellons disease. Clin Cosmet Investig Dermatol. 2011; 4: 167–77.\n\nMiddelveen MJ, Mayne PJ, Kahn DG, et al:Characterization and evolution of dermal filaments from patients with Morgellons disease. Clin Cosmet Investig Dermatol. 2013; 6: 1–21.\n\nMiddleveen MJ, Burugu D, Poruri A, et al:Association of spirochetal infection with Morgellons disease [v1; ref status: indexed, http://f1000r.es/8g] F1000 Research. 2013; 2(25).\n\nKellett CE: Sir Thomas Browne and the disease called the Morgellons. Ann Med Hist. 1935; 7: 467–79.\n\nEmslie-Smith AH: Myiasis, fillan, and the Morgellons. Br Med J. 1946; 1: 962.\n\nCheli R, Mortellaro CM: Digital dermatitis in cattle. Proc 8th Int Meet Dis Cattle; Milan, Italy. 1974; pp. 208–213.\n\nPearson ML, Selby JV, Katz KA, et al:Clinical, epidemiologic, histopathologic and molecular features of an unexplained dermopathy. PLoS One. 2012; 7(1): e29908.\n\nFoster AA, Hylwa SA, Bury JE, et al:Delusional infestation: clinical presentation in 147 patients seen at Mayo Clinic. J Am Acad Dermatol. 2012; 67(4): 673.e1–10.\n\nLee WR: Matchbox sign. Lancet. 1983; 2(8347): 457–8.\n\nGrosskopf C, Desai B, Stoopler ET: An oral ulceration associated with Morgellons disease: a case report. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011; 112(2): e19–23.\n\nDovigi AJ: Intraoral Morgellons disease or delusional parasitosis: a first case report. Am J Dermatopathol. 2010; 32(6): 603–5.\n\nDabu B, Mironiuc-Cureu M, Jardan D, et al:Identification of four Treponema species in subgingival samples by nested-PCR and their correlation with clinical diagnosis. Roum Arch Microbiol Immunol. 2012; 71(1): 43–7.\n\nAlessi E, Innocenti M, Ragusa G: Secondary syphilis. Clinical morphology and histopathology. Am J Dermatol. 1983; 5: 11–17.\n\nMacLeod JM: Syphilitic onychia. Proc R Soc Med. 1910; 3: 59–60.\n\nCormia F: Syphilitic onychia. Arch Derm Syphilol. 1938; 38: 432–433.\n\nChapel TA: The signs and symptoms of secondary syphilis. Sex Transm Dis. 1980; 7(4): 161–4.\n\nCole GW, Amon RB, Russell PS: Secondary syphilis presenting as a pruritic dermatosis. Arch Dermatol. 1977; 113(4): 489–90.\n\nAlergant CD: The seven-year itch. Br J Vener Dis. 1961; 37: 200–1.\n\nNielsen K, Jeppesen M, Simmelsgaard L, et al:Self-inflicted skin diseases. A retrospective analysis of 57 patients with dermatitis artefacta seen in a dermatology department. Acta Derm Venereol. 2005; 85(6): 512–5.\n\nWormser GP, Dattwyler RJ, Shapiro ED, et al:The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2006; 43: 1089–134.\n\nBacon RM, Kugeler KJ, Mead PS: Centers of Disease Control and Prevention (CDC) Surveillance for Lyme disease – United States, 1992–2006. MMWR Surveill Summ. 2008; 57: 1–9.\n\nBartůnĕk P, Gorican K, Veiser T, et al:Significance of Borrelia infection in development of dilated cardiomyopathy (a pilot study). Prague Med Rep. 2007; 108: 339–47.\n\nBiesiada G, Czepiel J, Lesniak MR, et al:Lyme disease: review. Arch Med Sci. 2012; 8(6): 978–82.\n\nReisinger EC, Fritzsche C, Krause R, et al:Diarrhea caused by primarily non-gastrointestinal infections. Nat Clin Pract Gastroenterol Hepatol. 2005; 2(5): 216–22.\n\nMacDonald AB: Gestational Lyme borreliosis. Implications for the fetus. Rheum Dis Clin North Am. 1989; 15(4): 657–77.\n\nLittman MP: Lyme nephritis. J Vet Emerg Crit Care (San Antonio). 2013; 23(2): 163–73.\n\nMc Causland FR, Niedermaier S, Bijol V, et al:Lyme disease-associated glomerulonephritis. Nephrol Dial Transplant. 2011; 26(9): 3054–6.\n\nEsposito S, Bosis S, Sabatini C, et al:Borrelia burgdorferi infection and Lyme disease in children. Int J Infect Dis. 2013; 17(3): e153–e158.\n\nSambrano B, Gordon R, Mays R, et al:Intravenous antibiotics used in dermatology. Dermatol Ther. 2012; 25(1): 70–81.\n\nAmerican Psychiatric Association Diagnostic and statistical manual of mental disorders. (4th ed., text rev.) (2000).\n\nWorld Health Organization ICD-10: International statistical classification of diseases and related health problems. (10th rev. ed.). New York, NY. (2008).\n\nWarren JR, Marshall B: Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983; 1(8336): 1273–5.\n\nLustig A, Mackay S, Strauss J: Morgellons disease as internet meme. Psychosomatics. 2009; 50(1): 90.\n\nBerger BW: Dermatological manifestation of Lyme Disease. Rev Infect dis. 1989; 11(Supp 6): S1475–81.\n\nHylwa SA, Bury JE, Davis MD, et al:Delusional infestation, including delusions of parasitosis: results of histologic examination of skin biopsy and patient-provided skin specimens. Arch Dermatol. 2011; 147(9): 1041–5.\n\nAdelson ME, Rao RV, Tilton RC, et al:Prevalence of Borrelia burgdorferi, Bartonella spp., Babesia microti, and Anaplasma phagocytophila in Ixodes scapularis ticks collected in Northern New Jersey. J Clin Microbiol. 2004; 42(6): 2799–801.\n\nNational Institutes of Health, National Center for Biotechnology Information, Basic Alignment Search Tool [Bethesda, MD, USA] Available from http://blast.ncbi.nlm.nih.gov/.\n\nMayne PJ: Investigation of Borrelia burgdorferi genotypes in Australia obtained from erythema migrans tissue. Clin Cosmet Investig Dermatol. 2012; 5: 69–78.\n\nStricker RB, Johnson L: Lyme disease: the next decade. Infect Drug Resist. 2011; 4: 1–9.\n\nStricker RB, Johnson L: Lyme disease diagnosis and treatment: lessons from the AIDS epidemic. Minerva Med. 2010; 101(6): 419–25.\n\nJohnson L, Aylward A, Stricker RB: Healthcare access and burden of care for patients with Lyme disease: a large United States survey. Health Policy. 2011; 102(1): 64–71.\n\nHylwa SA, Foster AA, Bury JE, et al:Delusional infestation is typically comorbid with other psychiatric diagnoses: review of 54 patients receiving psychiatric evaluation at Mayo Clinic. Psychosomatics. 2012; 53(3): 258–65." }	[ { "id": "993", "date": "07 Jun 2013", "name": "Alan B MacDonald", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe abstract is concise, cogent and informative. The methods and analytical procedures used are clear and acceptable. The authors have provided sufficient information for the work to be replicated. The conclusions of the article are sensible and balanced.", "responses": [] }, { "id": "1576", "date": "03 Sep 2013", "name": "Natalino H Yoshinari", "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 conclude that Morgellons is a distinct skin disease related to Borrelia infection. However, some questions deserve comments:This is a descriptive manuscript, based on the authors' research and case reports. Unfortunately, it is lacking a large panel of Morgellons cases with presentations of clinical symptoms, laboratory findings (including additional Borrelia infection research), biopsies with histological and immunohistological studies for spirochetal identification and skin filament examination (including anti-collagen type I and III antibodies, since the authors believe that the filaments are collagens and keratin) and PCR for Borrelia in all collected skin samples. Previous studies carried out by the authors suggest that spirochetes were found in Morgellons disease patients, and also in bovine digital dermatitis (BDD). In fact, BDD is a real spirochetosis causing disease in animals. However, the finding of spirochetes in a small number of Morgellons cases by Mayne et al. could be contested. Spirochete-like structures (artifacts) have been previously reported following blood or skin cultures in BSK medium1. These structures are spiral and also stained by silver nitrate. These artifacts appear as a consequence of bacteria death (for any microorganisms with flagella and not necessarily Borrelia)2. Additionally, spirochetes are common microorganisms found in the human mouth and bowel. Therefore, eventual contamination of open wounds by these bacteria is a real possibility. Are fatigue, arthritis, myalgia, psychiatric symptoms, and cognitive disturbances part of Morgellons disease as cited in the paper? These and other symptoms can be observed in chronic fatigue syndrome (CFS) or myalgic encephalomyelitis (ME), which is a very severe acquired disease caused by many infectious agents (including Borrelia), environmental conditions/toxins and physical or emotional trauma. Patients with ME show fatigue, headache, muscle/joint pain, psychiatric abnormalities, immune/allergic disturbance, and autonomic and cardiovascular disturbances. Instead of considering Morgellons as distinct disease responsible for the emergence of such symptoms, it may be more judicious to accept that Morgellons cases should, in fact, be classed as additional clinical manifestations of ME. In this respect, Morgellons should not be considered to be a true distinct clinical disorder, but instead an additional symptom of ME.It is interesting to note that patients with Brazilian Lyme disease imitator Borreliosis (Baggio-Yoshinari syndrome) (BYS) exhibit a high frequency of myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) symptoms. For this reason, confirmation of ME/CFS in suspected patients is taken into account when diagnosing BYS3. The presence of such symptoms creates enormous confusion in patients with late manifestations of Brazilian Borreliosis, since the distinction between clinical manifestations of true BYS relapse (generally treatable) and ME symptoms (barely treatable) is very difficult. Additionally, recent studies have also suggested Borrelia infections as one of the etiological agents of ME/CFS4.In conclusion, the paper written by Peter Mayne et al. must be approved with reservations. It is necessary for there to be a larger bank of cases and the inclusion of additional scientific methodologies in order to provide definite proof for the presence of Borrelia in skin tissues. Biochemical and immunological procedures should also be employed, to confirm the existence of collagen and keratin in the filaments.", "responses": [] }, { "id": "2472", "date": "18 Nov 2013", "name": "Peter Lepping", "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 article by Mayne et al. argues that Morgellons Disease should be seen as a form of Lyme Disease. The authors completely dismiss the possibility of Morgellons being a form of Delusional Infestation. Two of the main authors (Middelveen and Stricker) have a history of publications that argue for Morgellons to be seen as a valid disease entity. The authors summarise very few cases where they claim that patients with self-declared Morgellons Disease have tested positive for Lyme Disease. However, they completely omit the fact that in the recent article by Pearson et al., which they do mention, Lyme Disease was not found in any significant number in the >100 cases examined. If Lyme Disease was in any way a scientifically relevant entity in Morgellons Disease, that study would have shown it. In contrast there was no evidence in the Morgellons patients examined that Lyme Disease is relevant to their presentation. Therefore any individual cases put forward by the authors themselves should be dismissed as case reports which have not been confirmed by bigger studies. The authors also completely dismiss the overwhelming evidence in people with Delusional Infestation that they respond to antipsychotic medication. Instead the authors repeat their well-rehearsed arguments from previous papers that a diagnosis of Delusional Infestation is allegedly stigmatising, wrong and any symptoms of depression or anxiety found in their patients are secondary to a filament infestation. They try to counteract the argument that most of these patients will have Delusional Infestation by quoting DSM guidelines for the diagnosis of a delusional disorder. The authors claim that there is no specific entity of delusional infestation in ICD-10 or DSM-4 and it therefore cannot be reliably diagnosed. However, they accept the existence of Monodelusional Disorders in both DSM and ICD. Freudenmann and Lepping have pointed out in their Clinical Microbiology Reviews publication that Delusional Infestation is easily diagnosable within DSM and ICD as a Monodelusional Disorder. In essence, we have here an article of well-rehearsed arguments about why Morgellons Disease should not be classified as Delusional Infestation by authors who have a history of writing similar articles. They do not offer any new evidence at all and deliberately omit significant evidence against their assertions. The authors have dismissed the possibility of Morgellons being a form of Delusional Infestation in contrast to the vast majority of experts, without offering any reasonable evidence for their opinion.", "responses": [] }, { "id": "2208", "date": "25 Nov 2013", "name": "Roland Freudenmann", "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 an opinion article based on a research article from the same group in this journal (Middelveen et al., 2013). In their work(s) the authors associate “Morgellons Disease” (MD) with Lyme disease/spirochetal infections.The U.S. Centers for Disease Control and Prevention (CDC) studied “MD” cases (called “Unexplained dermopathy”), but did not find any evidence for a real infection (Pearson et al., 2012). This corroborated pre-existing large consensus in the medical community that patients with “MD” suffer from a psychiatric, not an infectious disorder, and that it is justified to view MD as a disorder unknown to medical science.The two papers in question do not provide reliable new data that justifies any divergent conclusions, because:The data basis is very poor. The methods used have been qualified as doubtful by microbiology and genetics experts on this website (as far as I can tell, as a psychiatrist/neuroscientist). The findings have not been replicated, especially not by groups without personal connection to the (former) “Morgellons Research Foundation/MRF” (for background information see Freudenmann & Lepping, 2009). Assessment: The authors over-interpret their data and suggest using a new name for something old, which is misleading. Based on the two papers, and contrary to the authors’ conclusion, there is no need to make any changes in the way that the medical profession approaches patients with self-diagnosed “Morgellons” (i.e. delusional infestation) and borreliosis /positive borrelia burgdorferia serologies, as long as \"MD\" is not accepted as an existing (infectious) illness by the CDC or by any responsible federal authority worldwide. Existing treatment guidelines for (neuro-)borreliosis provide excellent information for the physician on which patient needs antibiotic therapy and who does not.The authors use a flawed line of reasoning to argue that patients with “MD” do not meet criteria of a psychiatric disorder simply because the name “delusional infestation” is not mentioned in the DSM-5 delusional disorder chapter.", "responses": [] } ]	1	https://f1000research.com/articles/2-118
https://f1000research.com/articles/1-62/v1	07 Dec 12	{ "type": "Research Article", "title": "F1FoATP synthase a-subunit of Stenotrophomonas sp. DL18 from Indian Soda Lake, Lonar: a brief report", "authors": [ "Devendra Lingojwar", "Ravikant Jadhav", "Kachru Gawai", "Devendra Lingojwar", "Ravikant Jadhav" ], "abstract": "Lonar Lake, an Indian Soda Lake, is well known for its biodiversity of extremophiles including alkaliphiles. Most of the molecular studies on Lonar Lake alkaliphiles are based on molecular identification by 16S ribosomal RNA along with numerous applications in the biotechnology industry. However, molecular basis of adaptation of these alkaliphiles to high alkaline conditions is incompletely understood. Attempts were made to isolate and identify alkaliphiles from their naturally occurring original habitat, i.e. Lonar Lake, India with high alkaline conditions of pH 10.5. One facultative alkaliphile, Stenotrophomonas species DL18, was studied for F1FoATP synthase a-subunit with reference to alkaliphile-specific domains. Although the a-subunit of Stenotrophomonas DL18 showed significant similarity with neutrophiles, the isolated bacterium is an alkaliphile and optimally grows at pH 10.5.", "keywords": [ "ATP is the molecular currency for a living cell", "which is not only merely growing and dividing but also continuously responding to external environmental stimuli. To sustain life in extreme conditions", "microorganisms devise specific mechanisms for their adaptation. Along with other transporter proteins", "ATP synthase is widely considered as one of the key molecules for adaptation at alkaline conditions." ], "content": "Introduction\n\nATP is the molecular currency for a living cell, which is not only merely growing and dividing but also continuously responding to external environmental stimuli. To sustain life in extreme conditions, microorganisms devise specific mechanisms for their adaptation. Along with other transporter proteins, ATP synthase is widely considered as one of the key molecules for adaptation at alkaline conditions.\n\nHydrolysis of nucleoside tri-phosphates, specifically ATP, provides the chemical energy to drive a wide variety of cellular reactions. ATP synthases are central to ATP production during oxidative phosphorylation. These are energy-coupling factors and hence called F1Fo-ATP synthases. The Fo integral membrane protein complex (the subscript ‘o’ denotes its inhibition by the drug oligomycin) provides a transmembrane pore for protons, whereas the peripheral protein F1 (the subscript ‘1’ indicates that it was the first of several factors isolated from mitochondria) is involved in catalysis1. F1 consists of five subunits α3β3γ1δ1ε1, with a ring of α- and β-subunits alternating around a single γ-subunit2. Fo is a membrane embedded domain with subunits ab2 c10–15. Out of these subunits, the a-subunit is a stator and c-ring is a rotor ring through which ions (H+ or Na+) are translocated3–6. Each c-chain from the ring consists of two α-helices traversing the membrane, and the polar loop extends out of the membrane to interact with the γ-and ε-subunits. A cytoplasmic F1 catalytic domain is connected with a membrane-embedded Fo domain by a central (γε) and peripheral (b2δ) stalk2–6.\n\nDownhill ion translocation across the membrane through Fo causes rotation of the c-ring, which induces conformational changes in the catalytic β-subunit and results in ATP synthesis. The c11 ring from Ilyobacter tartaricus has Na+ ion binding specificity, while the c13 ring from Bacillus pseudofirmus OF4 and the c15 ring from Spirulina platensis has H+ ion binding specificity6. Ion coordination geometry and distances determine the ion specificity. Translocated ions bind to conserved carboxylate of aspartate or glutamate (D/E) in the outer α-helices of c-rings5,6. However, ions are further coordinated by a network of residues. The inner and outer α-helices are in a staggered position. Out of these, the inner helices are hydrophobic in nature and in contact with the phospholipids, while outer helices are hydrophilic with a-subunit interaction for ion translocation2,6. The transmembrane electric potential controls the kinetics of rotary motion, which seems to be independent of the ionic gradient5. However, alkaliphiles grow at high environmental pH, which poses the thermodynamic problem of synthesizing ATP with ATP synthase. For this, there are some crucial amino acid residue adaptations in the a- and c-subunit solving the problem of proton capture from an alkaline environment and subsequent translocation to the binding sites on the c-ring6.\n\nVarious studies based on the mechanism of proton binding7,8, through hydronium ion proton retention and transportation for ATP synthesis in the bacterial system including alkaliphiles9,10 suggest the presence of alkaliphile specific conserved amino acid motifs in transmembrane helix-4 (TMH-4) and TMH-5 of the a-subunit and the inner and the outer helix of the c-subunit3,11–14 of the ATP synthase Fo subunit as well as Na+/H+ antiporter15 and other cation binding proton transporters including multiple drug transporters6,16. The presence of the above mentioned alkaliphile specific sequences of ATP synthase Fo subunit along with Na+/H+ antiporters and other multiple drug transporters are the major strategies for pH homeostasis of extremely alkaliphilic species.\n\nMost studies have focused on the proton translocation channel in the a-subunit of ATP synthase. The arginine residue of the a-subunit, which transfers protons to the c-subunit, is conserved in almost all bacterial species3. Recent developments in molecular studies of facultative alkaliphiles suggests the presence of a highly conserved AXAXAXA motif in the amino terminal helix and a PXXEXXP motif in the carboxy terminal helix of the ATP synthase c-subunit in Bacillus pseudofirmus OF4, an established facultative alkaliphile6,17,18. However, similar experimental evidence from other geographic locations such as highly alkaline soda lakes need further exploration to understand pH homeostasis in facultative alkaliphiles. This study explores the comparison of the ATP synthase a-subunit of facultative alkaliphilic aerobes isolated from Lonar Lake with established and reported alkaliphiles. The present study deals with the isolation, identification and analysis of alkaliphile specific amino acid motifs in the a-subunit of ATP synthase.\n\n\nMaterials and methods\n\nUnderwater sediment soil samples were collected from 350 meters away from Kamalaja Devi Temple end, Lonar Lake, Buldhana, Maharashtra, India. The initial screening was performed at pH 9.5. After mix culture was obtained by the spread plate method, pure culture of each type of colony was maintained for further studies. Then isolates were further studied in the range of pH 7 to pH 12.\n\nBacterial genomic DNA was isolated by the DNAzol method19. DNA quantization and quality control for protein contamination was carried out by spectrophotometric absorbance at A260 and A280. The small subunit ribosomal RNA (16S rRNA) PCR for identification of bacterium was performed with forward primer (16S20F: 5’ATGTTGATCATGGCTCA3’) and reverse primer (16S1540R: 5’AAGGAGGTGATCCAACCGCA 3’)20. Briefly, master mix was prepared for 16S rRNA PCR: 10x PCR Rxn Buffer without MgCl2 (Invitrogen, P/N Y02028B Lot no. WK1B1b, USA), 1mM MgCl2 (Invitrogen, P/N Y02016B Lot no. WK2B1a, USA), 200 µM dNTP mix (Merck, India), 100 picomoles of each reverse and forward primers (Integrated DNA technologies, USA), 2.5U of Taq Polymerase enzyme (Invitrogen, USA, 11615-010 Lot No. VKRB1E) and nuclease free water (Merck, India) was added to make up a final volume of 100 µl. Following thermal cycling conditions were used for PCR: Initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min and primer extension at 72°C for 2 min. Thirty cycles of PCR were followed by final extension at 72°C for 5 min followed by cooling at 4°C.\n\nATP synthase Fo amplification primers were designed based on S. maltophilia K279a as follows: forward primer Steno atp1F: 5’CCTGGCGGATCCTTAGATCTCCG 3’ and reverse primer Steno atp1R: 5’CAGTGAGGATCCTTAGATCTCCGAGGCCAGCT 3’. Briefly, PCR reaction mixture of 100 µl was prepared as 10x PCR Rxn Buffer (Invitrogen), 50mM MgCl2 (Invitrogen) 1.8 µl, 10 mM dNTP mix 3.0 µl (Merck), 100 picomoles of each forward and reverse primers (Integrated DNA technologies, USA), 5U Taq DNA polymerase (Invitrogen) with pfu (Chromous biotech, India) and bacterial genomic DNA templates 200 ng, with remaining nuclease free water (Merck, India). The thermal cycling conditions were: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30s, primer annealing at 55°C for 30s and primer extension at 72°C for 3 min. Final extension was carried out at 72°C for 5 min and stored at 4°C. Results of 16S rRNA and ATP synthase Fo amplicons were visualized on 1% agarose gel with 200 ng/ml ethidium bromide (sd fine, India) and results were observed and analyzed with the help of Bio-Rad gel documentation system XR with Bio-Rad Quantity-One 4.6.5 software.\n\nThe 16S rRNA PCR product sequencing was carried out by modified Sanger’s dideoxy chain termination cycle sequencing method21. Electropherogram was read by an automated DNA sequencer (Applied Biosystems ABI3500 XL Genetic Analyzer, Big Dye Terminator version 3.1 Cycle sequencing kit) for 1.5 kb amplicon of the isolate. The resulting final DNA sequence of isolate was subjected to BLAST analysis on the NCBI web server. The phylogenetic tree of 16S rRNA of the isolate was constructed by using a neighbor-joining (NJ) method with 1000 replicates of bootstrap in MEGA4.1 software22. The bootstrap consensus tree inferred from 1000 replicates was selected to represent the evolutionary history of the taxa for 16S rRNA sequence analysis.\n\nFurther, the DNA sequencing of the ATP Fo subunit of selected isolates was performed by primer walking method. The NCBI Blast server was used for the identification of the ATP synthase a-subunit and the phylogenetic analysis of the a-subunit was carried out with the help of MEGA4.1. The blastx was used to get the amino acid sequence of the a-subunit. The a-subunit of isolate was compared with that of established alkaliphiles, acidophiles and neutrophiles with the help of ClustalW.\n\n\nResults and discussion\n\nAcross the pH gradient, growth of morphologically different bacterial colonies was observed. Out of these, some bacterial colonies were found with pink and orange pigmentation along with no pigmentation i.e. white colonies. The orange pigmented bacterium was identified as Stenotrophomonas species based on BLAST analysis of 16S rRNA gene sequence and titled as Stenotrophomonas species DL18 (GenBank Accession number: JN995612). Stenotrophomonas species DL18 optimally grow at the pH 9.0 to 10.0. However, the pH range of growth was pH 7.0 to 12.0 (Supplementary figure 1). The Stenotrophomonas species DL18 is known to be an aerobic, facultative alkaliphilic curved rod (Supplementary figure 2).\n\nLeft panel 50,000 times magnification, right panel 100,000 times magnification.\n\nBLAST analysis of the ATP synthase a-subunit of the Stenotrophomonas species DL 18 suggests maximum identity at the amino acid level (259 identical amino acids from a total of 266 amino acids of Stenotrophomonas species SKA14; GenBank Accession number: ZP_05136035). The amino acid residue arginine, which was found to be conserved in almost all bacterial species in the a-subunit, was observed at position 200 (Arg200) (Supplementary figure 3). Moreover, other amino acids that were conserved in most of the bacteria include Leu207, Arg210, Leu211, Gly213, Asn214, Gly218, Gln252, Ala253, Phe255 (E. coli numbering system for ATP synthase a-subunit) in TMH-4 and TMH-5 and the corresponding amino acids were also found in the Stenotrophomonas species DL 18 in alignment (Figure 1, Figure 2).\n\nFurther, the homology modeling was carried out by using SWISS MODEL using respective reference PDB structure and the modeled structure was visualized by Pymol.\n\nThe most conserved amino acid residues are shown as bold and underlined.\n\nConserved sequences are underlined and significant variant residues for channel formation are shown in bold. These residues are mainly from TMH-4 and TMH-5.\n\nBacillus megaterium DSM 319 (GenBank Accession number: YP_003600289) grows strictly in neutral pH environments. However, Bacillus clausii KSM K-16 (GenBank Accession number: YP_177349) is a reported alkaliphile.\n\nIt was observed that the most conserved arginine residue of the a-subunit (Arg200 of Stenotrophomonas species DL18) was aligned with the expected position of the facultative alkaliphile Bacillus pseudofirmus OF4 (i.e. Arg172; GenBank Accession number: YP_003426326). This positively charged Arg200 plays an elemental role in the function of the Fo rotor3. Amino acid Lys180 of Bacillus pseudofirmus OF4 was replaced by Gly208 in the Stenotrophomonas species DL 18. In addition, a glycine residue was observed at the same position in other alkaliphiles, T. cyclicum (Gly206; GenBank Accession number: YP_004537849), and same amino acid family Ala230 in Theoalkalivibrio species K90mix (GenBank Accession number: YP_003461818) as shown in Figure 2. On the other hand, glycine was also at the same corresponding position in alignment for the acidophiles, A. ferrooxidans (GenBank Accession number: YP_002221206), and A. cryptum (GenBank Accession number: YP_001233541) (Figure 3). Lys180 and corresponding amino acids were located in transmembrane helix-4 (aTMH-4) in Bacillus pseudofirmus OF4 and other alkaliphiles23,24. In the Stenotrophomonas species DL 18, a histidine residue, which is conserved in other reference species of the same genus (S. maltophilia K279a GenBank Accession number: YP_001973793; and S. sp. SKA14) and other alkaliphiles T. cyclicum (His244), and Theoalkalivibrio species K90mix (His262), was present at position 240 (His240) (Figure 2). However, E. coli K12 DH10B (GenBank Accession number: YP_001732559), considered as neutrophile, can adapt to slightly alkaline conditions i.e. up to pH 8.0 and this may be due to the presence of His245. It was proposed that Gly120 and Lys180 form a channel residing within the proton uptake pathway of the a-subunit through which protons pass onto the neighboring c-subunit in Bacillus pseudofirmus OF418.\n\nConserved sequences from TMH-4 and TMH-5 are underlined and significant variant residues for channel formation are shown in bold.\n\nHowever, Ala198 from the Stenotrophomonas species DL 18 and Ala196 from T. cyclicum were found to correspond with Gly208 in E. coli and Gly170 in Bacillus pseudofirmus OF4 in alignment of the a-subunit as shown in Figure 2. In a similar way, Gly207 was observed in Stenotrophomonas species DL 18, while in this respective position alanine was found in alkaliphiles and neutrophiles. But both amino acids correspond from the same amino acid family. However, one of the studies reported the His245 along with the Glu219 positioning plays a critical role in ion translocation in E. coli25, while in Stenotrophomonas species DL 18 these were at His240 and Glu209 as shown in Figure 2.\n\nMcMillan et al25 showed the importance of the residue with a basic side chain along with its pKa value in ATP synthesis linked with alkaline environment. However, these studies were carried out with a mutation study in the Bacillus pseudofirmus OF4 a-subunit, specifically with the position of the residue at 180. These studies included mutations at Lys180 position as Gly180, His180 and Arg180. These mutations showed the ATP synthesis at neutral, neutral to alkaline, and only at alkaline conditions (>pH 8.5), respectively i.e. residue with a strong base, such as lysine or arginine, was ideally appropriate to function at alkaline pH. Since, it was particularly marked for histidine, which has a pKa in a neutral range, it showed significant ATP synthesis activity at pH 9.0. However, exchange mutations at Gly120 and Lys180 to Lys120 and Gly180 showed ATP synthase activity in Bacillus pseudofirmus OF4. Similar studies in E. coli showed that the positions Gly218 and His245 along with G1u219 had a critical interaction with Fo function26,27.\n\nAfter comparison with acidophiles, neutrophiles and alkaliphiles, Leu197 of the Stenotrophomonas species DL 18 was found to be conserved in TMH-4 (Figure 3). From alignment, Glu209 of the Stenotrophomonas species DL 18 was conserved in alkaliphiles, neutrophiles and some acidophiles except His186Acidiphilium cryptum. In addition, the position of Gly212 in Bacillus pseudofirmus OF4 corresponds to Ser212 in E. hirae (neutrophile; GenBank Accession number: YP_006487510), His245 in E. coli and His240 in the Stenotrophomonas species DL 18 while glutamate in acidophiles, Glu222 in A. ferrooxidans and Glu225 in A. cryptum as shown in Figure 3. This showed that the channel formation may involve a glycine residue along with other residues, specifically acidic, basic and neutral side chain, which play vital roles in ATP synthesis in acidophile, alkaliphiles and neutrophiles. Hence, these residues are found to be critical in channel formation. In the same scenario, the Gly208 and His240 may be form the proton translocation channel in Stenotrophomonas species DL 18. Thus the basic side chain residue His240 and other amino acid residues may be responsible for growth of the Stenotrophomonas species DL 18 at high alkaline pH.", "appendix": "Author contributions\n\nDevendra Lingojwar contributed to the conception and design of the project, acquired, analyzed and interpreted the data, review of literature, and prepared the manuscript. Ravikant Jadhav helped prepare the manuscript, contributed to data collection and conducted bioinformatic analysis. Kachru Gawai provided overall guidance for the project, helped prepared the manuscript and revised it critically for intellectual content. All authors approved the final version of the article.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSpecific facilities related to molecular biology were kindly provided by ATG LAB. No specific grant was available for this project. The entire project was carried out with the support from the ATG LAB as an intramural project.\n\n\nAcknowledgements\n\nAuthors are thankful to the Department of Chemistry, University of Pune, India for providing the place of research and infrastructure and ATG LAB for providing laboratory facilities whenever needed. The authors are also thankful to Mrs. Sarita Lingojwar, Admin Head and Laboratory Manager, ATG LAB, for providing kind help and support for laboratory facilities during this project.\n\n\nReferences\n\nMcCarty RE: A plant biochemist's view of H+ -ATPases and ATP synthases. J Exp Biol., 1992, 172, 431–441.\n\nStock D, Leslie A, Walker JE, et al:Molecular architecture of the rotary motor in ATP synthase. Science, 1999, 286, 1700–1705.\n\nFujisawa M, Fackelmayer O, Liu J, et al:The ATP synthase a-subunit of extreme alkaliphiles is a distinct variant mutation in the critical alkaliphile-specific residue Lys-180 and other residues that support alkaliphile OXPHOS. J Biol Chem, 2010, 285(42): 32105–15.\n\nCain BD, Simoni RD: Proton translocation by F1Fo ATPase of Escherichia coli. J Biol Chem., 1989, 264(6), 3292–3300.\n\nDimroth P, Ballmoos C, Meier T, et al:Electrical power fuels rotary ATP synthase. Structure, 2003, 11, 1469–1473.\n\nPreiss L, Yildiz O, Hicks DB, et al:A new type of proton coordination in an F1Fo-ATP synthase rotor ring. PLoS Biol, 2010, 8, e1000443.\n\nKrah A, Pogoryelov D, Langer JD, et al:Structural and energetic basis for H+ versus Na+ binding selectivity in ATP synthase Fo rotors. Biochem Biophys Acta, 2010, 1797, 763–772.\n\nBallmoos CV, Dimroth P: Two distinct proton binding sites in the ATP synthase family. Biochemistry, 2007, 46, 11800–11809.\n\nBallmoos CV: Alternate proton binding mode in ATP synthesis. J Bioenergy Biomembr., 2007, 39, 441–445.\n\nDimroth P, Ballmoos CV, Meier T, et al:Catalytic and mechanical cycles in F-ATP synthases. EMBO Rep, 2006, 7(3), 276–282.\n\nLiu J, Fackelmayer OJ, Hicks DB, et al:Mutations in a helix-1 motif of the ATP synthase c-subunit of Bacillus pseudofirmus OF4 cause functional deficits and changes in the c-ring stability and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry, 2011, 50, 5497–5506.\n\nMatthies D, Preiss L, Klyszejko AL, et al:The c13 ring from a thermoalkaliphilic ATP synthase reveals an extended diameter due to a special structural region. J Mol Biol., 2009, 388, 611– 618.\n\nFillingame RH, Dmitriev OY: Structural model of the transmembrane Fo rotary sector of H+-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ. Biochimica et Biophysica Acta., 2002, 1565, 232–245.\n\nRivera-Torres IO, Krueger-Koplin RD, Hicks DB, et al:pKa of the essential Glu-54 and backbone conformation for subunit c from the H+-coupled F1Fo ATP synthase from an alkaliphilic Bacillus. FEBS Lett, 2004, 575, 131–135.\n\nKrulwich TA, Sachs G, Padan E, et al:Molecular aspects of bacterial pH sensing and homeostasis. Nat Rev, 2011, 9, 330–343.\n\nJanto B, Ahmed A, Ito M, et al:Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.5. Environ. Microbiol., 2011, 13(12): 3289–309.\n\nLiu J, Fujisawa M, Hicks DB, et al:Characterization of the functionally critical motifs of the ATP synthase c-subunit from an alkaliphilic Bacillus. J Biol Chem., 2009, 284(13), 8714–8725.\n\nWang ZX, Hicks DB, Guffanti AA, et al:Replacement of amino acid sequence features of a- and c-subunits of ATP synthase of alkaliphilic Bacillus with the Bacillus consensus sequence results in defective oxidative phosphorylation and non fermentative growth at pH 10.0. J Biol Chem., 2004, 279(25), 26546–26554.\n\nChomczynski P, Mackey K, Drews R, et al:DNAzol: A reagent for the rapid isolation of genomic DNA. BioTechniques, 1997, 22, 550–553.\n\nBrosius J, Palmer JL, Kennedy JP, et al:Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci, 197875, 4801–4805.\n\nSanger F, Coulson AR: A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol., 1975 May 25; 94(3), 441–8.\n\nTamura K, Dudley J, Nei M, et al:MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol., 2007, 24, 1596–1599.\n\nIvey DM, Krulwich TA: Two unrelated alkaliphilic Bacillus species possess identical deviation in sequence from those of other prokaryotes in regions of Fo proposed to be involved in proton translocation through the ATP synthase. Res Microbiol., 1992, 143, 467–470.\n\nIvey DM, Krulwich TA: Organisation and nucleotide sequence of ATP genes coding the ATP synthase from alkaliphilic Bacillus firmus OF4. Mol Gen Genet., 1991, 229, 292–300.\n\nMcMillan DGG, Keis S, Dimroth P, et al:A specific adaptation in the a-subunit of thermoalkaliphilic F1Fo-ATP synthase enables ATP synthesis at high pH but not at neutral pH values. J Biol Chem., 2007, 282(24), 17395–17404.\n\nHartzog PE, Cain BD: Second-site suppressor mutations at glycine 218 and histidine 245 in the a-subunit of F1Fo ATP synthase in Escherichia coli. J Biol Chem., 269(51), 32313–32317.\n\nCain BD, Simoni RD: Interaction between Glu-219 and His-245 within the a-subunit of F1Fo-ATPase in Escherichia coli. J Biol Chem., 1988, 263(14), 6606–6612." }	[ { "id": "654", "date": "09 Jan 2013", "name": "Thomas Meier", "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- The overall content of this work is very poor. It contains: growth of bacteria in medium, PCR, sequencing, sequence alignment and a homology model. It is what is done today in a student practicum.- The homology model (structure of subunit α, Supplementary Figure 3) is a homology model based on a model (this was NO real structure), which basically also means, one could just take 4 random alpha helices and put them together in a bundle. - The authors should mention in the methods section which genes from Fo were amplified by PCR.- Supplementary material figure 1: The window ledge and the arm of the photographer is visible on the picture.- Supplementary material figure 2: The quality of the left EM micrograph is poor, the picture is not in focus. The picture on the right side is distorted, I cannot see anything there.- Page 5 line 1: The Cook lab did not use Bacillus pseudofirmus OF4 but rather Bacillus TA2.A1- Figure 2 and Figure 3: The alignment should contain Bacillus TA2 because the authors refer to it in the text on Reference 25 (McMillan et al.). - Figures 2 and 3 appear to be very similar. It may have made more sense to include all the information in one figure to avoid redundancies and save space.The scientific value of this work is questionable. It is a good start to investigate the α-subunit from Stenotrophomonas but it is too early to publish as such.", "responses": [] }, { "id": "712", "date": "18 Jan 2013", "name": "Gregory Cook", "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\nI don't see this study has much relevance to the field as it adds nothing to the literature in this area and is not great science e.g. the homology model is not based on a structure but just a model. The authors should have also included a c subunit analysis to check for the presence/absence of Na+-binding motifs. Overall the work is not of a standard in which others in the field can benefit.", "responses": [] } ]	1	https://f1000research.com/articles/1-62
https://f1000research.com/articles/2-58/v1	22 Feb 13	{ "type": "Research Article", "title": "The Binding Ring Illusion: assimilation affects the perceived size of a circular array", "authors": [ "J Daniel McCarthy", "Colin Kupitz", "Gideon P Caplovitz", "J Daniel McCarthy", "Colin Kupitz" ], "abstract": "Our perception of an object’s size arises from the integration of multiple sources of visual information including retinal size, perceived distance and its size relative to other objects in the visual field. This constructive process is revealed through a number of classic size illusions such as the Delboeuf Illusion, the Ebbinghaus Illusion and others illustrating size constancy. Here we present a novel variant of the Delbouef and Ebbinghaus size illusions that we have named the Binding Ring Illusion. The illusion is such that the perceived size of a circular array of elements is underestimated when superimposed by a circular contour – a binding ring – and overestimated when the binding ring slightly exceeds the overall size of the array. Here we characterize the stimulus conditions that lead to the illusion, and the perceptual principles that underlie it. Our findings indicate that the perceived size of an array is susceptible to the assimilation of an explicitly defined superimposed contour. Our results also indicate that the assimilation process takes place at a relatively high level in the visual processing stream, after different spatial frequencies have been integrated and global shape has been constructed. We hypothesize that the Binding Ring Illusion arises due to the fact that the size of an array of elements is not explicitly defined and therefore can be influenced (through a process of assimilation) by the presence of a superimposed object that does have an explicit size.", "keywords": [ "Size Illusions", "Size Perception", "Visual Illusions" ], "content": "Introduction\n\nCorrectly perceiving the size of objects is essential to successfully interact with the world around us. Due to the fact that we sense the 3D visual world through an analysis of 2D retinal images, the process of size perception is intrinsically ambiguous. As a result, the perception of an object’s size arises from the integration of multiple sources of visual information including the size of its retinal image, its perceived distance1, and its size relative to other objects in the visual scene2,3. These constructive processes are revealed through a number of classic size illusions such as the Ebbinghaus Illusion4 (Figure 1A) and the Delboeuf Illusion5,6 (Figure 1B). In each of these illusions, the perceived size of an explicitly defined object (the inner circle) is influenced by the context in which it is presented. Here we address the question of how we perceive the size of an implicitly defined object–an array of elements–by introducing a novel variant of the Ebbinghaus illusion that we have named the Binding Ring Illusion. We describe the illusion and investigate the underlying mechanisms that lead to misperceived size.\n\nNotable size illusions: A) Ebbinghaus illusion, B) Delboeuf illusion.\n\nA basic stimulus that elicits perception of the Binding Ring Illusion is composed of a circular array of small circles onto which a larger circle is superimposed as shown in Figure 2A. The superimposed circle leads to an underestimation of the perceived radius of the circular array relative to an equally sized array without the binding ring (Figure 2B). In the following experiments, we investigate the magnitude of this illusory decrease in size and attempt to study the components of the stimulus that are responsible for the observed effect.\n\nWhich array of circles looks bigger? A) The test stimulus used in experiment 1 or B) The reference stimulus used in experiment 1.\n\nIn experiment 1, we demonstrate that the effect of the binding ring can be quantified. In experiment 2 we demonstrate that the illusion arises due to assimilation toward the binding ring7,8. In experiment 3, we investigated the effect of spatial frequency. Finally, in experiments 4a and 4b we investigated the roles of local configural features in producing the illusion.\n\n\nGeneral methods\n\nFive observers (3 men, 2 women; age range: 18–25; mean age = 20.2) participated in experiment 1, ten observers participated in experiment 2 (4 men, 6 women; age range: 19–28; mean age = 22.4), twelve observers participated in experiment 3 (6 men, 6 women; age range: 18–27; M = 21.75) and five observers participated in experiment 4 (3 men, 2 women; age range: 18–25; M = 20.6). All participants were naïve to the aims of the experiments, reported normal or corrected-to-normal vision and received course credit for their participation. Prior to participating, each observer provided informed consent according to the guidelines of the Department of Psychology and the IRB of the University of Nevada, Reno.\n\nStimuli were presented on a Dell Trinitron P991 monitor (19 inches, 1024 × 768) with an 85 Hz refresh rate. The stimulus computer was a 2.4 GHz Mac Mini with an NVIDIA GeForce 320M graphics processor (256MB of DDR3 SDRAM). Stimuli were created and presented with the Psychophysics Toolbox9 for MATLAB (Mathworks Inc., Natick, MA). In experiments 1, 2 and 4, the stimuli were white (120 cd/m2) presented on a black (0.06 cd/m2) background. In experiment 3, the stimuli were either low- or high-pass filtered and presented on a grey background (20.5 cd/m2). Luminance values were measured with a Photo Research PR655 spectroradiometer. Participants placed their head on a chin rest and viewed the stimuli binocularly from a distance of 57cm.\n\n\nExperiment 1\n\nThe goal of experiment 1 was to establish a ‘standard’ configuration and quantify the magnitude of the illusion in order to serve as a baseline for further testing. Specifically, we sought to measure the perceived reduction in size of an array superimposed with a binding ring compared to an unbound array of equal size.\n\nThe basic paradigm is illustrated in Figure 3. On a given trial, participants were presented with a small central fixation point (0.35º) for 500ms, followed by the additional simultaneous presentation of two circular arrays (a reference and a test) for 500ms at which point the stimuli were removed from the screen and replaced by a random noise mask displayed for 500ms to discourage afterimages. Participants indicated by pressing one of two buttons (two-alternative forced choice; AFC), which of the two stimuli had appeared larger. Each array consisted of 12 small equally spaced circles with radii of 0.05º visual angle. On every trial, the reference array had a fixed radius of 3º visual angle (from the center of the array to the center of any circular element). Using the method of constant stimuli10, we investigated the perceived size of each of two test arrays compared to the reference array. The test array either had a binding ring superimposed or did not (the lack of a binding ring served as a control condition). In non-control conditions the binding ring had a radius selected to match the radius of the test circular array that was measured from the center of the array to the center of one of the smaller component circles. Because the control array did not have a superimposed binding ring it was identical to the reference array in all ways except its trial-by-trial size. As such, it was used to determine A) how accurately observers were able to perform the task (discriminate the sizes of the arrays) and B) to serve as a point of comparison for determining the size of the illusory effect. On each trial, the radius of the test array was selected from the following list: 2.5º, 2.6º, 2.8º, 2.9º, 3.0º, 3.1º, 3.3º, 3.4º and 3.5º.\n\nA fixation point appeared for 500ms followed by the presentation of the reference and test array for 500ms at which point the stimuli were removed from the screen and replaced by a noise mask for another 500ms to prevent the formation of afterimages. The screen then remained blank until participants indicated which array appeared larger via a key press.\n\nOn each trial, the centers of the two arrays were randomly positioned within a circular radius of 1.16º of visual angle centered 6.75º along the horizontal axis to the left or right of the central fixation point. This positional-jitter was used to prevent observers from basing their judgments on horizontal matching or distance comparisons with the edges of the monitor. Participants were instructed to maintain fixation on the center of the screen throughout the experiment. The sides on which the two arrays were presented were randomly determined on each trial. In total there were 18 trial types: nine test radii for both the test and control array types. Trials were pseudo-randomly ordered such that 20 of each trial type were presented in random order for a total of 360 trials. Prior to the experiment, participants were trained on 20 trials of the largest and smallest test array sizes that were not included in the analyses.\n\nFor each array size, we computed the percentage of times the test or control array was perceived to be larger than the reference. Thus, for the test (bound) and control (unbound) arrays, nine values (one for each radius) were calculated. Because the 2AFC task has two categorical responses, the following sigmoidal-shaped binomial-logit function was then fit to the corresponding data for each of the two test arrays using the MATLAB (glmfit() command)11:\n\n\n\nThe points of subjective equality (PSE) were determined for each subject by interpolating the 50% chance level from the function fit to the data (x = –b1/b2). The PSE indicates the size the test array needs to be in order to be perceived as equal in size to the reference. The resulting curves plotted for the mean responses across participants are shown in Figure 4. The clear and steep-sloped sigmoidal shaped psychometric curve derived from the control condition in which neither the reference nor control array have a binding ring confirm that participants were able to perform the task and accurately report their perceptions. Specifically, participants were at chance performance when the two arrays were indeed the same size. The rightward shift of the other psychometric curve, derived from the test (binding ring) condition, demonstrates that the size of the array was underestimated when the binding ring was present. The inset of Figure 4 illustrates the mean points of subjective equality across subjects for the test and control arrays.\n\nPsychometric curves indicate the mean fit of the data averaged across all participants. The inset of the figure illustrates the mean points of subjective equality (PSEs) for the test and reference stimuli. The black curve indicates that participants were accurately judging the radius of an unbound array. The grey curve shows that the array radius must increase by ~0.18º to be perceived as being the same size as an unbound array. Thick curves with error bars indicate the mean response across participants for each array radius. Thin curves indicate the function fitted to the data. Error bars represent the standard error of the mean PSE computed across subject.\n\nA paired t-test between the PSEs of the test and control arrays revealed that the addition of the binding ring significantly (t(4) = 7.71, p < 0.01) reduced the perceived size of the test array. The mean difference of the PSEs between the test and control arrays was ~ 0.18º of visual angle or 6% of the overall radius of the array. Thus, an array superimposed with a binding ring of radius 3.18º was perceived as having the same size as a no-binding ring array with a radius of 3º. This is comparable in magnitude to underestimation effects observed in the classic Ebbinghaus and Delbeouf Illusions12,13.\n\n\n\n\nExperiment 2\n\nHaving quantified the magnitude of the illusion in experiment 1, we explored possible mechanisms contributing to the underestimation of perceived size. Size illusions such as the Delboeuf and Ebbinghaus illusions have been explained on the basis of size assimilation (when the size of the element of interest is biased toward the reference component) and contrast (when the size of the element of interest is biased away from the reference component)7,8,14,15. Because the size of an array is not explicitly defined, it remains unclear whether the illusion arises due to assimilation or contrast with the binding ring. In order to determine if the illusion is mediated by assimilation or contrast we investigated the effect of changing the size of the binding ring (bottom of Figure 5). If assimilation is responsible for the effect, we would predict that the magnitude of the illusion should increase as the binding ring gets smaller. Alternatively, if the illusion arises due to contrast, the magnitude of the illusion should increase as the binding ring gets larger.\n\nThe radius of the binding rings from left to right: 2º, 2.67º, 3º, 3.33º and 4º; circular array radius was consistently 3º. The graph illustrates the PSEs for each stimulus type: lines and asterisks below the bars indicate significant (p < 0.05) differences, based on post-hoc paired t-tests, between the three overlapping binding ring conditions. Superimposed asterisks indicate significant changes in perceived size induced by the presence of the binding ring compared to an array without a binding ring (observed for all five conditions). A positive shift on the ordinate axis indicates that the array must increase in size to be perceived as having the same radius as the reference and vice versa. Error bars indicate standard error of the mean.\n\nThe basic stimuli were the same as those used in experiment 1; however, due to the larger number of configurations tested, we used staircase procedures16 rather than the method of constant stimuli. The individual trials within the staircases again consisted of a reference and test array simultaneously presented for 500ms. As in the previous experiment, the reference array had a fixed radius of 3º; however, unlike the previous experiment, the reference contained a binding ring and the test array did not. This was done so as to be able to compare the magnitude of the illusion for a fixed size array across binding-ring sizes. Separate staircases were run for five distinct reference conditions defined by the size of the binding ring with radii chosen from the following list: 2º, 2.67º, 3º, 3.33º and 4º (see Figure 5). Because the radius of each circular element was 0.5º, the binding ring did not physically overlap with the circular array in two of the five conditions and was either entirely inside (2º) or outside (4º) the array. Four staircases were run for each trial type–two in which the initial test or control array was larger than the reference array (descending) and two in which it was initially smaller (ascending). The starting radius for the test or control array was randomly selected to be 0.5º to 1º larger or smaller than the radius of the reference array. On each trial, participants completed a 2AFC task indicating which stimulus had appeared larger. According to standard staircase procedures, the size of the test array was adjusted by a step size ranging randomly on each iteration from 2 to 5 pixels (0.07º to 0.18º) in the direction opposite of the participant’s response. The staircase finished when four reversals were recorded. In total, each participant completed 20 staircases presented in pseudorandom order.\n\nThe mean of the four reversal points was calculated for each staircase and the PSE for each reference condition was obtained by averaging these results across the corresponding four staircases. Although we used a different experimental paradigm than in experiment 1, the size of the measured effect when the binding ring had a radius of 3.0º (same as in experiment 1) is comparable to that observed in experiment 1 (0.16º vs. 0.18º). The results illustrated on the top of Figure 5 indicate:\n\nA) The size of the binding ring had a significant influence on the perceived size of the array (main effect of binding ring size - repeated measures ANOVA: F(4, 36) = 31.22, p < 0.001).\n\nB) In each of the five conditions, the perceived size of the array was significantly influenced by the presence of the binding ring (one sample two-tailed, t-tests vs. zero: all p < 0.05 uncorrected).\n\nC) The binding ring array was perceived as being smaller than an array without a binding ring in each of the four conditions in which the radius of the binding ring was less than the radius of the exterior portion of the array.\n\nD) In the condition where the binding ring completely encompassed the array, the perceived size of the array was larger than when no binding ring was present. This is the reverse effect of the other four conditions.\n\nE) The magnitude of the effect is greatest when the binding ring is superimposed on the array. A follow-up planned comparison of the three overlapping conditions with the two non-overlapping conditions revealed that the magnitude of the effect is significantly larger when the binding ring intersects the array compared to when it does not intersect the array (F(1, 9) = 50.87, p < 0.001).\n\nF) A second repeated measures ANOVA examining just the three superimposed conditions revealed that the size of the binding ring (within this range) significantly influences the magnitude of the illusion (F(2, 18) = 3.84, p < 0.05). As can be seen in Figure 5, the magnitude of the illusion increases as the size of the binding ring is reduced for the three superimposed conditions. However, once the binding ring becomes too small, such that it no longer overlaps the array, the magnitude of the illusion is greatly decreased. That said, even in this innermost binding ring condition, the perceived size of the array is underestimated. It is noteworthy that this particular stimulus condition is quite similar to that typically used in the Ebbinghaus Illusion (see right side of Figure 1A). Here we demonstrate that there are in fact two illusory effects revealed in the Ebbinghaus Illusion, one operating on the inner circle (making it appear larger) and one operating on the outer array (making it appear smaller). Taken together, these observed effects are consistent with the assimilation hypothesis and inconsistent with the contrast hypothesis. Furthermore, these results appear to indicate that the outer edge of the circular array is serving as a boundary for the assimilation. When the radius of the binding ring exceeds this boundary, the assimilation bias is to increase the perceived size of the array. Similarly, if the radius of the binding ring is within this boundary, the assimilation bias is to decrease the perceived size of the array.\n\n\n\n\nExperiment 3\n\nIn this and the following experiment, we attempt to identify specific stimulus factors that may influence the Binding Ring Illusion. It is suggested that some visual illusions, including the the Müller-Lyer17, are mediated by differential processing of low- and high-spatial frequency information18–22. Here, we investigated the effect of spatial frequency filtering on the magnitude of the Binding Ring Illusion (Figure 6A).\n\nA) Low- (top left) and high-pass (top right) versions of the binding ring stimuli. B) Results of experiment 3. The points of subjective equality (PSE) of both high (HSF) and low spatial frequency (LSF) conditions are plotted for both the control and binding ring conditions. Error bars represent standard error of the mean. Asterisks indicate significance at p < 0.05.\n\nThe stimuli and procedures used in experiment 3 were analogous to those used in experiment 1 except that the stimuli were either high- or low-pass filtered. The high-pass cutoff was set at (2 cpd) and the low-pass cutoff was (0.5 cpd). Stimuli were presented on a gray (20.5 cd/m2) background. In each case, similarly filtered stimuli were compared to each other (i.e. a high spatial frequency (HSF) reference was compared to a HSF test or control). In total there were 36 trial types: nine test radii (same as in experiment 1) for the test and control array types (with and without binding ring) in both HSF and low spatial frequency (LSF) conditions. Trials were pseudorandomly ordered so that 20 of each trial type were presented for a total of 720 trials.\n\nAfter fitting the curves using the same procedures described above, we conducted a 2 (binding ring vs. control) × 2 (HSF vs. LSF) repeated measures ANOVA on the derived PSE for each of the four conditions. This analysis revealed a main effect of the binding ring on perceived size (F(1, 11) = 27.05, p < 0.01), a main effect of spatial frequency on perceived size (F(1, 11) = 12.30, p < 0.01) and a significant interaction between the binding ring and spatial frequency (F(1, 11) = 5.57, p < 0.05). As can be seen in Figure 6B, for both low- and high-pass stimuli, the array containing the binding ring was perceived to be smaller than when no binding ring is present. As reflected in the significant interaction, this effect is greater when the stimuli are low- as compared to high-pass filtered. Although the illusion was observed in both the LSF and HSF configurations, the size of the effect observed for the LSF condition (~0.3º) is substantially larger than that observed in the previous experiments that range from 0.16º to 0.18º. In contrast, when the stimuli were high-pass filtered, the resultant ~0.2º reduction in perceived size is comparable to that observed in the previous experiments.\n\n\n\n\nExperiment 4\n\nLastly we investigated the role of the local configural features of the stimulus. A number of visual illusions can be attributed to the processes of local configural features23. In these two closely related experiments, we investigated the impact of presenting only parts of the binding ring on the presence or magnitude of the Binding Ring Illusion. In doing so we address the question of whether the Binding Ring Illusion is mediated by the processing of specific local features, or perhaps on the basis of more global representations of the objects present in the image.\n\nIn the first part of this experiment, the binding ring only connected the array of elements so that it was not visible in the interiors (Figure 7A). In the second part of the experiment, the binding ring was present solely within the interiors of the array elements (Figure 7B) leaving them unconnected from each other.\n\nA) The test stimulus used in experiment 4a. B) The test stimulus used in experiment 4b. C) The results of experiment 4a. D) The results of experiment 4b. There was no significant difference (n.s.) in perceived size between the control and binding ring tests in either condition.\n\nThe stimuli and procedures used in experiments 4a and 4b were identical to those used in experiment 1 except that in the test condition, the binding ring only connected the array of elements as if viewing a chain of pearls (experiment 4a) or the binding ring was only visible in the interiors of the array elements (experiment 4b).\n\nThe data shown in Figure 7C were analyzed using the same curve fitting method described in the results of experiment 1. A paired samples t-test between the PSEs of the test and control arrays revealed no significant difference in perceived size (t(4) = 2.05, ns).\n\nThe data shown in Figure 7D were analyzed using the same curve fitting method described in the previous experiments. A paired t-test between the PSEs of the test and control arrays again revealed no significant difference in perceived size (t(4) = 1.93, ns).\n\nThe Binding Ring Illusion was not observed in either of the partial binding ring configurations tested here. As such, we can conclude that processing of the preserved local features does not underlie the illusion. Alternatively, these results suggest that mechanisms within a higher, more global stage of processing may underlie the illusion.\n\n\n\n\nGeneral discussion\n\nIn this manuscript we introduced a new size illusion that we call the Binding Ring Illusion. The Binding Ring Illusion is a variant of the Delboeuf and Ebbinghaus Illusions and demonstrates that the perceived size of an array is subject to effects of assimilation. Specifically, the perceived size of a circular array of elements is underestimated when a ‘binding ring’ is superimposed on the array. The purpose of the above experiments was to demonstrate that the illusion can be quantified, to investigate possible explanations for its occurrence and to begin to characterize the stimulus factors that lead to misperceived size.\n\nA number of observations can be made based on the results of these experiments. Firstly, the Binding Ring Illusion can indeed be quantified. Using both methods of constant stimuli and adaptive staircases we were able to measure significant differences in the perceived size of an array as a function of the presence and size of a binding ring. Secondly, the size of the binding ring significantly influences the magnitude of the illusion (experiment 2). Specifically, in order to produce the greatest effect, the binding ring has to superimpose the array and furthermore, as the size of the superimposed ring decreases, the magnitude of the illusion increases. These findings are consistent with existing assimilation theories of similar size illusions such as the Delboeuf and Ebbinghaus illusions7,8,13,15. In addition, the perceived size of the array was slightly increased only when the binding ring was large enough to completely encompass the array. This suggests that the outer radius of the array serves as the reference point for the assimilation process. It has been argued that assimilation is largely influenced by the perceived unification of the components as a single object15,24. This is consistent with our results such that the strongest assimilation effects occurred when the circle was superimposed on the array and could be easily perceived as a unified figure. In these conditions we observe a significantly larger magnitude of size illusion than when the binding ring was not superimposed on the array.\n\nThe Delboeuf and Ebbinghaus Illusions both demonstrate that the perceived size of an interior object can be influenced by the presence of a surrounding stimulus. The Binding Ring Illusion, on the other hand, provides a complimentary observation that the perceived size of a surrounding stimulus can be influenced by the presence of an interior stimulus. Indeed, the smallest binding ring condition in experiment 2 is an identical configuration to that commonly used to demonstrate the Ebbinghaus illusion.\n\nSecondly, the magnitude of the illusion is greater when the stimuli are low- compared to high-pass filtered (experiment 3). However, in both cases the magnitude of the illusion is comparable if not greater than that observed with full spectrum stimuli. As such, we can conclude that: either processes within distinct spatial frequency channels can independently lead to the illusion, or the illusion is mediated by mechanisms at a later stage of processing that follows the integration of high- and low-spatial frequencies. In the latter case, one may conjecture that the initial LSF bias is attenuated once spatial frequency information has been integrated in object recognition areas such as those located in the inferotemporal cortex (IT)25. One hypothesis for why the illusion is greater in the LSF condition is that due to blur, the individual array elements in the LSF condition are perceptually larger than the HSF and full-spectrum conditions. Because the elements appear larger, the perceived distance between the outer radius of the array and the binding ring is increased. The results of experiment 2 suggest that this may lead to an increased assimilation effect. Alternatively, the blurring of the image increases the thickness of the binding ring and it could be the case that this increases its effect on the perceived size of the array. Further research will be necessary to fully determine why the blurred stimulus increased the magnitude of the illusion.\n\nThirdly, the illusion does not manifest when only the local configural features of the binding ring are present. This was true even when the binding ring only connects the array elements (experiment 4a). This result is intriguing because past work has demonstrated that elements that are perceptually grouped into a common object will be perceived to be closer together than those that are not. Specifically, if a series of dots are arranged to form a dotted contour, the distance between any two adjacent dots will be perceived as being shorter than the distance between any one of them and another equally-distanced dot that does not lie along the contour26. One possible extension of this observation is that an object formed out of the grouping of individual elements may appear smaller on the basis of the elements appearing closer together. Based on this assumption we thought it possible that the partial binding ring of experiment 4a may serve as an additional cue that the individual elements belong to a common object and therefore lead to it appearing smaller. The configuration of experiment 4a explicitly links the elements of the array; however, this does not lead to the illusory reduction in perceived size. This may be explained by the observed effects of the Oppel-Kundt Illusion27,28 that demonstrates that the distance between two points is overestimated when it is filled with a number of tick marks compared to two equally spaced points of an undivided extent; however, as the density of these divisions increases, the effect diminishes28. Therefore, connecting the interior elements should lead to a more veridical perception as observed here. As such, it is unlikely that the Binding Ring Illusion arises due to a misperception of the perceived distance between array elements.\n\n\nConclusion\n\nAlthough we readily perceive a circular array of elements as a circle, there are many possible alternate perceptions that could be formed. For example, the circles could be grouped into pairs symmetrical about the vertical axis, or perceived as ellipses arranged in an elliptical array that is receding in depth. That we perceive such a stimulus as a circle reflects the constructive processes that are embodied in the functional and structural architecture of the visual system. Importantly, the circle that we perceive does not explicitly exist in the retinal image and must therefore be constructed. As such, the size of the circle that we perceive must be itself constructed as well. The Binding Ring Illusion demonstrates that this constructive process includes the assimilation of other co-occurring stimuli, particularly those that spatially overlap the array.", "appendix": "Author contributions\n\n\n\nDan McCarthy: contributed to the theoretical foundation, experimental design, data analysis and writing of the manuscript Colin Kupitz: contributed to the experimental design, implementation, data collection, data analysis and writing of the manuscript Gideon Caplovitz: contributed to the theoretical foundation, experimental design and writing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nInstitutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 1P20GM103650-01 and a grant from the National Eye Institute of the National Institutes of Health: 1R15EY022775.\n\n\nReferences\n\nEmmert E: Grossenverhaeltnisse der Nachbilder. Klin Monbl Augenheilkd. 1881; 19: 443–450.\n\nRoberts B, Harris MG, Yates TA: The roles of inducer size and distance in the Ebbinghaus illusion (Titchener circles). Perception. 2005; 34(7): 847–856. PubMed Abstract \| Publisher Full Text\n\nRoss HE, Plug C: The history of size constancy and size illusions. In: Walsh V, Kulikowski J, editors. Perceptual Constancy: Why Things Look the Way They Do. Cambridge: Cambridge University Press, 1998; 499–528. Reference Source\n\nEbbinghaus H, Ernst D: Grundzüge der Psychologie. vols. I and II: Veit; 1902. Reference Source\n\nDelboeuf J: Seconde note sur de nouvelles illusions d'optique: Essai d'une théorie psychophysique de la manière dont l'oeil apprécie les grandeurs. Bulletins de l'Académie Royale des Sciences, Lettres et Beaux-arts de Belgique. 1865; 20: 70–97. Reference Source\n\nDelboeuf JLR: Sur une nouvelle illusion d'optique. Academie Royale des Sciences, des Lettres et des Beaux Arts de Belgique. Bulletins. 1892; 24: 545–558.\n\nCoren S, Girgus JS: Seeing is deceiving: the psychology of visual illusions. Hillsdale: Lawrence Erlbaum Associates; 1978; 255. Reference Source\n\nRobinson JO: The psychology of visual illusion. London: Hutchinson, 1972; 288. Reference Source\n\nBrainard DH: The Psychophysics Toolbox. Spat Vis. 1997; 10(4): 433–436. PubMed Abstract \| Publisher Full Text\n\nLaming D, Laming J: F. Hegelmaier: On memory for the length of a line. Psychol Res. 1992; 54(4): 233–239. PubMed Abstract \| Publisher Full Text\n\nWichmann FA, Hill NJ: The psychometric function: I. Fitting, sampling, and goodness of fit. Percept Psychophys. 2001; 63(8): 1293–1313. PubMed Abstract \| Publisher Full Text\n\nGirgus JS, Coren S, Agdern M: The interrelationship between the Ebbinghaus and Delboeuf illusions. J Exp Psychol. 1972; 95(2): 453–455. PubMed Abstract \| Publisher Full Text\n\nOyama T: Japanese studies on the so-called geometrical-optical illusions. Psychologia. 1960; 3: 7–20. Reference Source\n\nGirgus JS, Coren S: Assimilation and contrast illusions: differences in plasticity. Percept Psychophys. 1982; 32(6): 555–561. PubMed Abstract \| Publisher Full Text\n\nGoto T, Uchiyama I, Imai A, et al.: Assimilation and contrast in optical illusions. Jpn Psychol Res. 2007; 49(1): 33–44. Publisher Full Text\n\nGescheider G: Chapter 3: The Classical Psychophysical Methods. In: Psychophysics: the fundamentals. 3rd ed. Hillsdale: Lawrence Erlbaum Associates; 1997; 45. Reference Source\n\nCarrasco M, Figueroa JG, Willen JD: A test of the spatial- frequency explanation of the Müller-Lyer illusion. Perception. 1986; 15(5): 553–562. PubMed Abstract \| Publisher Full Text\n\nGinsburg AP: Psychological correlates of a model of the human visual system. DTIC Document, 1971; 119. Reference Source\n\nGinsburg AP: Is the illusory triangle physical or imaginary? Nature. 1975; 257(5523): 219–220. PubMed Abstract \| Publisher Full Text\n\nGinsburg AP: Visual information processing based on spatial filters constrained by biological data. DITC Document, 1978; 338. Reference Source\n\nGinsburg AP: Visual form perception based on biological filtering. Sensory experience, adaptation and perception. 1984; 53–72. Reference Source\n\nGinsburg AP, Evans DW: Predicting visual illusions from filtered images based upon biological data (A). J Opt Soc Am. 1979; 69: 1443. Reference Source\n\nZöllner F: Ueber eine neue Art von Pseudoskopie und ihre Beziehungen zu den von Plateau und Oppel beschrieben Bewegungsphaenomenen. Annalen Physik Chemie. 1860; 186(7): 500–523. Publisher Full Text\n\nGoto T, Uchiyama I, Imai A, et al.: Assimilation and contrast. Handbook of the science of illusion. 2005; 164–176. Reference Source\n\nKveraga K, Ghuman AS, Bar M: Top-down predictions in the cognitive brain. Brain Cogn. 2007; 65(2): 145–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoren S, Girgus JS: Principles of perceptual organization and spatial distortion: the gestalt illusions. J Exp Psychol Hum Percept Perform. 1980; 6(3): 404–412. PubMed Abstract \| Publisher Full Text\n\nKundt A: Untersuchungen über Augenmass und optische Täuschungen. Poggendorff Analle. 1863; 120: 118–158.\n\nOppel JJ: Ueber geometrisch-optische Täuschungen. Jahresberichte des physikalischen Vereins zu Frankfurt. 1854/1855; 37–47. Reference Source" }	[ { "id": "849", "date": "18 Mar 2013", "name": "Ming Meng", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paper presents a novel variant of visual size illusion that is named the Binding Ring Illusion by the authors. The illusory effect is quite strong as one could easily experience the illusion him/herself by looking at figure 2. The authors further tested several stimulus conditions to investigate why the Binding Ring Illusion occurs. Their results suggest that size assimilation at a relatively high level in the visual processing stream may underlie the illusion. I think this is a clearly written paper, the results are clear-cut, and the conclusion of this paper is well supported by the results.", "responses": [] }, { "id": "855", "date": "21 Mar 2013", "name": "Dejan Todorović", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a study of a size perception illusion related to the Ebbinghaus illusion and the Delboeuf illusion. The study includes quantification of the basic phenomenon and some relevant parameters. It is well designed and executed, and contributes to our knowledge of this class of illusions. Size illusions of this class have been known for a long time and are still lacking a generally accepted explanation, but have not been studied much in recent years.My only substantial criticism concerns the authors’ suggestion that the effect they studied is based on a high-level mechanism, late in the processing stream. I don’t find that they have offered enough evidence for such a conclusion. Note that recent research on the Ebbinghaus illusion (Schwarzkopf et al 2010, Nature Neuroscience, 14(1), 28-30), not cited by the authors, indicated that the strength of the illusion correlates with the size of V1, suggesting contributions to the illusion fairly early in the visual stream.As a minor comment, it is commendable that the authors have cited the early research on size illusions, but some of the references, although often cited in that form in the recent literature, are in fact incorrect. For example, Oppel did not publish about the the illusion named after him in 1854/55 but in 1860/61, and in that paper he did not report about the dependence of the illusion on the number of tickmarks. For more details, see Wackermann J. & Kastner K. (2010), Acta Neurobiol Exp, 70: 423–434. Also, Ebbinghaus did not publish the illusion named after him in the reference cited in the paper, and in fact seems to have never published it. For details, see Burton, G. (2001), History of Psychology, 4, 228-244.", "responses": [] }, { "id": "873", "date": "03 Apr 2013", "name": "Simone Gori", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 presented manuscript is very interesting and well written. The experiments are very well conducted and it is a pleasure to read. The authors introduce a new size illusion and they investigate it using a very good methodology, providing clear results. I have some suggestions that in my opinion could improve the manuscript even more, but my general opinion is extremely positive.Please see my suggestions below:1. The introduction is really short, and it doesn't cover the topic of size illusion properly. Several other interesting examples of size illusions should be cited and discussed to provide a better understanding of the topic. Firstly, I would suggest discussing the literature on the illusions that are cited by the authors which are very similar to the one that they introduced here. For example, several interesting papers have been produced on The Ebbinghaus Illusion (which should maybe be called the Ebbinghaus-Titchener illusion, referring also to the 1902 Titchener book that made it popular) that maybe should be discussed (just few examples, without pretending to be complete at all: Roberts et al. 2005; Nemati, 2009; Doherty et al., 2010; Schwarzkopf and Rees, 2013). The same can be said for the related Delboeuf illusion, which is suggested to be caused by the same brain mechanisms by Roberts et al. (2005). A few examples of recent works that may be worth checking are: Zanuttini and Daneyko (2010); Jaeger and Long (2007). Moreover, other interesting size illusions could be mentioned in the introduction, for example: The Muller-Lyer Illusion (Zeman et al. 2013; Plewan et al. 2012; Proulx and Green, 2011), the Oppel-Kundt Illusion (Wackermann, 2012a,b; Savazzi et al., 2012) and the related filled area illusion (Giora and Gori, 2011); the Breathing Light Illusion (Gori and Stubbs, 2006; Anstis et al. 2007; Gori et al. 2010), StarTrek Illusion (Qian and Petrov, 2012), Accordion Grating Illusion (Gori et al., 2011; 2013; Yazdanbakhsh and Gori, 2011) and the Fat Face Illusion (Thompson and Wilson, 2012; Sun et al. 2012), to name just a few.2. Moreover, it would be nice if the authors could explain in more detail why studying this new illusion is relevant. In other words I would like to know which characteristics separate this new configuration from the ones already known. 3. In the introduction of Experiment 3, it should be noted that the filled area illusion (Giora and Gori, 2010) is influenced by spatial frequency and a similar procedure to the one used in this study was employed. As both are size illusions, it would be interesting to discuss the similarities between the two.4. When the authors state that several illusions are based on local configuration features, as well as citing the 1860’s work, they should also cite some new literature to provide more up to date references.5. In the discussion, the difference between perceiving a difference and perceiving a group (Gori and Spillmann, 2010) could be interesting to discuss in relation to the last experiment.6. The link with the Oppel-Kundt Illusion could be discussed in more detail, for example, by also referring to the work done by Wackermann.7. In the discussion, some references to the potential brain mechanisms involved would be the icing on the cake in my opinion. Visual illusions are open windows on how the brain works and they are interesting tools to investigate it in a non-invasive manner. It would be nice to read about the hypothetical brain mechanisms underlying specific illusions.ReferencesAnstis S, Gori S, Wehrhahn C. Afterimages and the breathing light illusion. Perception. 2007;36(5):791-4.Doherty MJ, Campbell NM, Tsuji H, Phillips WA. The Ebbinghaus illusion deceives adults but not young children. Dev Sci. 2010 Sep 1;13(5):714-21.Giora E, Gori S. The perceptual expansion of a filled area depends on textural characteristics. Vision Res. 2010 Nov 23;50(23):2466-75.Gori S, Giora E, Agostini T. Measuring the Breathing Light Illusion by means of induced simultaneous contrast. Perception. 2010;39(1):5-12.Gori S, Spillmann L. Detection vs. grouping thresholds for elements differing in spacing, size and luminance. An alternative approach towards the psychophysics of Gestalten. Vision Res. 2010 Jun 11;50(12):1194-202.Gori S, Stubbs DA. A new set of illusions--the dynamic luminance-gradient illusion and the breathing light illusion. Perception. 2006;35(11):1573-7.Gori, S., Giora, E., Yazdanbakhsh, A. and Mingolla, E. (2011) ‘A new motion illusion based on competition between two kinds of motion processing units: The Accordion Grating’, Neural Networks, 24: 1082-1092.Gori, S., Giora, E., Yazdanbakhsh, A. and Mingolla, E. (2013) ‘The novelty of the “Accordion Grating Illusion”’, Neural Networks, 2013 Mar;39:52.Jaeger T, Long S. Effects of contour proximity and lightness on Delboeuf illusions created by circumscribed letters. Percept Mot Skills. 2007 Aug;105(1):253-60.Nemati F. Size and direction of distortion in geometric-optical illusions: conciliation between the Müller-Lyer and Titchener configurations. Perception. 2009;38(11):1585-1600.Plewan T, Weidner R, Eickhoff SB, Fink GR. Ventral and dorsal stream interactions during the perception of the Müller-Lyer illusion: evidence derived from fMRI and dynamic causal modeling. J Cogn Neurosci. 2012 Oct;24(10):2015-29.Proulx MJ, Green M. Does apparent size capture attention in visual search? Evidence from the Muller-Lyer illusion. J Vis. 2011 Nov 23;11(13).Qian J, Petrov Y. StarTrek illusion--general object constancy phenomenon? J Vis. 2012 Feb 16;12(2):15. Roberts B, Harris MG, Yates TA. (2005). \"The roles of inducer size and distance in the Ebbinghaus illusion (Titchener circles).\". Perception. 34 (7): 847–56.Savazzi S, Emanuele B, Scalf P, Beck D. Reaction times and perceptual adjustments are sensitive to the illusory distortion of space. Exp Brain Res. 2012 Apr;218(1):119-28.Schwarzkopf DS, Rees G. Subjective Size Perception Depends on Central Visual Cortical Magnification in Human V1. PLoS One. 2013;8(3):e60550.Sun YH, Ge L, Quinn PC, Wang Z, Xiao NG, Pascalis O, Tanaka J, Lee K. A new \"fat face\" illusion. Perception. 2012;41(1):117-20.Thompson P, Wilson J. Why do most faces look thinner upside down? Iperception. 2012;3(10):765-74. Titchener, E.B. (1902). Experimental psychology: A manual of laboratory practice. New York, NY: MacMillan & Co., Ltd.Wackermann J. Determinants of filled/empty optical illusion: Influence of luminance contrast and polarity. Acta Neurobiol Exp (Wars). 2012;72(4):412-20.Wackermann J. Determinants of filled/empty optical illusion: differential effects of patterning. Acta Neurobiol Exp (Wars). 2012;72(1):89-94.Yazdanbakhsh A, Gori S. Mathematical analysis of the accordion grating illusion: a differential geometry approach to introduce the 3D aperture problem. Neural Netw. 2011;24(10):1093-101.Zanuttini L, Daneyko O. Illusory lightness in the Delboeuf figure. Percept Mot Skills. 2010 Dec;111(3):799-804.Zeman A, Obst O, Brooks KR, Rich AN. The müller-lyer illusion in a computational model of biological object recognition. PLoS One. 2013;8(2):e56126.", "responses": [] } ]	1	https://f1000research.com/articles/2-58
https://f1000research.com/articles/2-9/v1	10 Jan 13	{ "type": "Research Article", "title": "II. Capsular vaso-mimicry formed by transgenic mammary tumor spheroids implanted ectopically into mouse dorsal skin fold: cellular mechanisms of metastasis", "authors": [ "Halina Witkiewicz", "Phil Oh", "Jan E Schnitzer", "Phil Oh", "Jan E Schnitzer" ], "abstract": "Most cancer patients die of metastatic disease, not primary tumors, while biological mechanisms leading to metastases remain unclear and effective therapies are missing. Using a mouse dorsal skin chamber model we had observed that tumor growth and vasculature formation could be influenced by the way in vitro cultured (avascular) spheroids of N202 breast tumor cells were implanted; co-implantation of lactating breast tissue created stimulating microenvironment, whereas the absence of the graft resulted in temporary tumor dormancy. This report addressed the issue of cellular mechanisms of the vasculogenic switch that ended the dormancy. In situ ultrastructural analysis revealed that the tumors survived in ectopic microenvironment until some of host and tumor stem cells evolved independently into cells initiating the vasculogenic switch. The tumor cells that survived and proliferated under hypoxic conditions for three weeks were supported by erythrogenic autophagy of others. However, the host microenvironment first responded as it would to non-immunogenic foreign bodies, i.e., by encapsulating the tumor spheroids with collagen-producing fibroblasts. That led to a form of vaso-mimicry consisting of tumor cells amid tumor-derived erythrosomes (synonym of erythrocytes), megakaryocytes and platelets, and encapsulating them all, the host fibroblasts. Such capsular vaso-mimicry could potentially facilitate metastasis by fusing with morphologically similar lymphatic vessels or veins. Once incorporated into the host circulatory system, tumor cells could be carried away passively by blood flow, regardless of their genetic heterogeneity. The fake vascular segment would have permeability properties different from genuine vascular endothelium. The capsular vaso-mimicry was different from vasculogenic mimicry earlier observed in metastases-associated malignant tumors where channels formed by tumor cells were said to contain circulating blood. Structures similar to the vasculogenic mimicry were seen here as well but contained non-circulating erythrosomes formed between tumor nodules. The host’s response to the implantation included coordinated formation of new vessels and peripheral nerves.", "keywords": [ "\"Discovery consists of seeing what everybody has seen and thinking what nobody has thought\"." ], "content": "\n\n\"Discovery consists of seeing what everybody has seen and thinking what nobody has thought\".\n\n– Albert Szent-Gyorgyi\n\n\nIntroduction\n\nTwo main problems persisting in oncology are related; they are (1) the incomplete understanding of the mechanism by which tumors spread from primary locations to multiple organs and (2) the lack of selective anti-cancer treatments. A developmental regulatory program involved in embryo implantation, referred to as the \"epithelial-mesenchymal transition\" (EMT)1–4 was adopted to explain how transformed epithelial cells could acquire the ability to metastasize, i.e., to invade surrounding nonmalignant tissues and to disseminate, in a multistep process including entering and leaving the circulatory system5,6. However, no satisfactory mechanism for the spread of non-epithelial tumors to secondary locations was proposed. Therefore, an alternative to EMT regulatory programs playing a role in invasiveness of carcinoma cells should be considered, as pointed out elsewhere7.\n\nAttempts made to elucidate the cellular mechanism of metastasis-initiating events have included retrospective extrapolation from the distribution of established metastases, namely the preference of specific tumors to metastasize in certain organs but not in others. One of the earliest studies addressed the issue by injecting fixed and stained tumor cells into the left side of the heart in rabbits and determining their subsequent distribution in tissue sections of several organs. The results supported the hypothesis that the distribution of metastases was determined by the mechanics of circulation and the consequent location of embolic tumor cells but they did not exclude a role of local \"soil\" factors8. Other studies, by making a connection with genetic diversity of tumors, suggested that metastases might have a clonal origin and the dissociated representatives of particular subpopulations could be directed to their related tissues. However, generating biological diversity continued among different metastatic foci9. Metastases of particularly aggressive cancers of different types (not only melanoma10) were associated with patterned vasculogenic mimicry, i.e., a network of periodic acid Schiff-stained (glycoproteins containing11) \"loops\" that represented blood-containing micro-vascular \"channels\", generated by the aggressive tumor cells without participation of endothelial cells (ECs) and independently of angiogenesis12,13. How these structures facilitated metastasis was not clear14,15. Elevated incidence of metastasis was also correlated with autophagy of internal organelles in tumor cells, although the mechanism behind this was unclear16. Reports based on a variety of experiments have suggested that, depending on the context, autophagy could either stimulate or prevent cancer17. Thus, the question regarding the way in which autophagy influences metastasis has remained unanswered18. Two other intriguing issues were the inefficiency of tumor formation in experimental settings and the targeting of a selected sub-population of tumor cells by an anticancer drug. (1) Theoretically, a single cell could be capable of establishing the tumor but large numbers and a latent period were actually required19,20. (2) A selective uptake of the endoradiotherapeutic 6-[211 At-astato-2-methyl-1,4-naphtoquinol bis(diphosphate) drug only by those tumor cell nuclei that contained alkaline phosphatase isoenzyme was demonstrated21. Those observations, together with the heterogeneity of tumors known for a long time but not fully understood20,22, suggest the existence of cancer stem cells (CSCs) in spite of the undifferentiated phenotype of the malignant cells. The definitive proof of the CSCs was lacking23.\n\nThe problem with nonspecific side-effects causing activities of anti-cancer drugs was to be circumvented by targeted drug delivery. That hope was based on the observation that endothelial surfaces had variable, organ-specific properties24–29. However, how molecules cross the endothelial barrier and successfully reach the intended vascular destination has turned out to be another problem. The targeted destruction of established tumor vessels themselves resulted only in reduction of the tumor growth30 (as expected by J. Folkman31) because tumors could regenerate their vasculature. The approach did appear effective in some non-neoplastic diseases32,33. The issue of the tumor vessels’ permeability is rather perplexing. On the one hand, the vessels prevent anticancer drugs from penetrating the tumors, while on the other hand, they are known to be abnormally leaky34,35.\n\nWe had observed earlier that in our model, formation of the tumor vasculature (vessels and blood) could be accelerated by availability of homologous tissue stem cells (TSCs) from a co-implanted graft36. Without them, the process was relatively slow and the growth of implanted avascular tumor spheroids was limited, yet eventually the vasculogenic switch did happen. That raised the following questions: (1) how did the tumor survive the lag period (about three weeks) without vasculature and (2) how was the problem solved eventually? In addition to providing answers to those questions, the results shown below suggest a new cellular mechanism for initiating metastasis. We use the term TSCs according to the 2002 functional definition by M. Loeffler and I. Roeder37,38. It refers to the stemness as a capability of a system rather than individual cell lineages.\n\nWe present a new type of vaso-mimicry in the murine model of breast tumor that was morphologically different from the vasculogenic mimicry previously described and postulate its role in facilitating the passive transport of tumor cell clusters to secondary locations and in determining the increased permeability of such fake vessels. Understanding the process is critical from the clinical point of view. If correct, it would bring the focus of future studies to the energy metabolism-related initial steps (as discussed elsewhere36) and could result in the identification of new methods of inhibiting some of these steps before the angiogenic switch has had a chance to evolve, therefore, potentially preventing the metastases.\n\n\nMaterials and methods\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\nTwo recipient mice of the 5 used in the two accompanying articles were assessed in this report. The same numbering system was used in all three articles. The experimental design is summarized in Table 1.\n\nThe host mice were 8–9 weeks old athymic nude females purchased from Harlan. 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 N202.1A+H2B-GFP cell line (generated by stable transfection of the parental murine breast cancer cell line, N202.1A39 to express GFP under histone H2B promoter40) was obtained from Drs. J. Lustgarten and P. Borgstrom and used to form tumor spheroids by culturing 2 × 105 cells per well for 2–3 days prior to implantation. A week after establishment of mouse dorsal skin chambers, the tumor spheroids were implanted directly on skin (ectopically)41. The tumors were incubated in the chambers for three weeks (Table 1). Their final size was about 1–3 mm in diameter. The GFP-specific rabbit polyclonal IgG (ab290) was from Abcam; 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 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 plain 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 citrate42 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 the antigenic epitops (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 Tokuyasu43. 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 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. We used the graphics editing program, Adobe PhotoShop, to add cell type-specific color-coding shown in the twin set of images included in the Supplement.\n\n\nResults\n\nThree weeks after the ectopic implantation of tumor spheroids, the vasculature formation, i.e., formation of tumor-supporting blood and vessels was evidently retarded in comparison to pseudo-orthotopicly implanted tumors described elsewhere36. However, the host response to the surgical injury was well advanced (Figure 1 & Figure S1). A multi-cellular layer of connective tissue was growing between the tumor and the glass wall of the chamber, therefore, it was also generating its own vasculature ([A] in Figure 1 & Figure S1). Here, the term \"vasculature\" includes vessels and blood and \"erythrosome\" is used as a synonym for the \"erythrocyte\", because the latter is not a cell36. Acellular areas of collagen matrix contained erythrosomes that were vessel-free, although not extravasated. Those areas were not necrotic. Occasionally some blood cells were in close contact with supporting nucleated cells ([B & C] in Figure 1 & Figure S1). Outside the tumor capsule, a primitive forming vessel morphologically resembled some of those seen around pseudo-orthotopicly implanted tumors after only five days ([D] in Figure 1 & Figure S1). Fibroblasts also encapsulated small tumor nodules. The population of tumor cells inside the encapsulated nodules was heterogeneous ([E–G] in Figure 1 & Figure S1). Evidently the tumor cells were also capable of converting into erythrosomes and by doing so in a non-synchronized fashion, they could enable survival of other tumor cells. However, the tumor’s ability to generate the genuine vessels was limited at that stage; therefore, the tumor could not grow. Some tumor cells (CSCs?) did however begin regenerating their vasculogenic potential that had been dormant during the years of in vitro culturing ([F & G] in Figure 1 & Figure S1). Thus the vasculogenic switch did occur in the absence of the homologous tissue stem cells (TSCs) from the graft but only after a considerable delay (about two weeks). Until that time, some tumor cells survived at the expense of others.\n\nThree weeks after implantation, the tumor environment consisted of single migrating cells that were secreting ECM components, mainly collagen and others converting into erythrosomes [A]. The erythroblast [B] and the eosinophil [C] were each in contact with a nucleated supporting cell on one side. A small primitive vessel located next to the tumor [D] resembled those early ones seen in the pseudo-orthotopic environment five days after implantation (Figure 2 in36). Tumor cells were encapsulated by fibroblasts and the cell population inside the capsules appeared heterogeneous. There were erythrosomes [E], and the nuclei of some cells had sinuses [F & G] similar to those in ECs of a forming artery (Figure 11 in36). Featured in [H] is a part of a larger elongated capsule containing a cluster of tumor cells in the center and multiple erythrosomes around it, all covered with typical flat, collagen-producing fibroblasts, whereas a similar structure in [I] contained mainly erythrosomes and platelets. Both were remarkably similar to a vein.\n\nMost tumor cells displayed ultrastructural features characteristic of hypoxia, i.e., mitochondrial changes and dilated endoplasmic reticulum (ER) cisternae without ribosomes. In some locations, hypoxic tumor nodules were breaking apart via prominent anoikis (loss of attachment between cells44) with abundant nano-tentacles ([A–C] in Figure 2 & Figure S2). Commonly, cells located next to each other had mitochondria changing in opposing ways. They were either loosing their internal cristae without shrinking and thus generating electron-lucent vacuoles (seemingly empty or containing whorled membranes that might be intermediate stages of the internal membranes degradation) or becoming smaller and electron dense. The first type of the morphological changes of mitochondria had been shown to occur as a result of genetically simulated hypoxia followed by necrosis45. The second type at first resembled the appearance of mitochondria during mitosis and later, they were indistinguishable from the dark granules in erythroblasts ([D–F] in Figure 2 & Figure S2) and consistent with published images of peroxisomes46–49. Such opposing changes occurring simultaneously in cells sharing the same microenvironment suggested different fates for them. The one with initiated necrosis could potentially recover when the other had completed its conversion into erythrosome(s). That is because erythrosomes are capable of secreting anaerobicly generated ATP50. Oxygen is not critical for erythrosomes themselves because they do not have mitochondria to use it. Initially, electron-dense regions of the tumor cell nucleus contained chromatin in both cases. However, that changed with the progression of the erythrogenic conversion when detecting histone H2B simultaneously exposed electron dense regions of the nucleus that did not contain chromatin ([E] in Figure 2 & Figure S2). Iron accumulation could be a good alternative reason for such increased electron density not attributable to chromatin condensation. Chromatin degradation products contained all the elements needed to make hemoglobin except iron. Therefore, a cellular conversion into erythrosomes during the neo-hematopoiesis would require soliciting iron from outside the tumor. Indeed, in tumor bearing mice the accumulation of iron was reported to shift from the spleen and liver to the tumor site51. The fibroblasts that encapsulated the tumor were of host origin, similar to the host connective tissue \"membrane\" encapsulating orthopedic implants52. The GFP-labeled mitotic chromosomes identified the tumor cell whereas the unlabeled fibroblast, on the other side of collagen layer separating the two, must have been of host origin ([F] in Figure 2 & Figure S2). Together, the host fibroblasts and the encapsulated tumor-derived blood elements created the capsular vaso-mimicry that morphologically resembled veins ([G–I] in Figure 1 & Figure S1). The tumor cell population was clearly heterogeneous; therefore, it could survive by some cells feeding on others, initially via lactic acid secretion53 and then via erythrogenic autophagy36. In this way, the metabolic requirements of the encapsulated tumor nodules appeared to be responsible for initiating the capsular vaso-mimicry.\n\nHypoxia in tumor cells was manifested by structural changes of mitochondria resulting in their condensation into smaller and more electron dense (darker) forms or in degradation of cristae (left and right, respectively) [A], occasionally leading to autophagic vacuoles with whorls of membranes (arrows in [B & C]) or to peroxisomes (arrows in [D]). Partial separation of the tumor cells from one another (anoikis44) with emergence of meandering nanotentacles (stars in [A & C]) created intercellular passages and greatly increased the cell surface. Immunocytochemical detection of GFP-labeled histone H2B in tumor cells is shown in [E & F]. The tumor cell in [E] had a nucleus with electron-dense regions that did not contain H2B, except for a small upper region, demonstrating incomplete chromatin degradation (arrow). The upper left corner of [F] corresponds to the boxed area of the insert and showed H2B-specific label in a mitotic chromosome of the tumor cell. A layer of collagen fibers (cf) separated that cell from the elongated fibroblast with unlabeled nucleus (upper right corner) of [F]. The arrow in the insert points to a condensed mitochondrion.\n\nNot all tumor nodules were successfully encapsulated at the time of tissue harvest. In some regions, the fibroblasts appeared trapped between possibly faster-growing tumor nodules and, commonly, such fibroblasts were undergoing the erythrogenic autophagy as well. Typically, their cytoplasmic remnants were still present between the erythrosomes and tumor cells. The elongated cells like the one shown between the tumor nodules (Figure 3 & Figure S3) had large nuclei undergoing conversion into erythrosomes and a sparse, metabolically active cytoplasm generating energy and synthesizing protein. What appeared in the two-dimensional image as a single file of erythrosomes between the tumor nodules was not a rouleau of circulating erythrocytes.\n\nConsecutive splitting of an erythroschizont (red-splitting-body) into erythromers (erythrosomes); one fragment completely separated and the rest in the process of splitting, all surrounded by leftover cytoplasm of the cell producing them [A]. Four enlarged regions of that cell [B–E]. A distinct region of the erythroschizont indicated the location of the next, already initiated, split (arrows) [A & C]. Vesicles on both sides of the mitochondrion contained collagen fibers (arrows) [E].\n\nThe non-malignant tissue repair included formation of new blood vessels and peripheral nerves (Figure 4 & Figure S4). Developing vessels and nerves arranged in heterotypic pairs suggested a coordinated regulation of their morphogenesis (Figure 4 & Figure S4). In some regions, ECs converting into erythrosomes (hemogenic endothelium) were also seen (Figure 8 in54).\n\nTwo neuro-vascular pairs. The upper pair were less mature; the boxed area of the nerve in [A] (enlarged in [B]) showed incomplete myelination of the neuron (arrows). The pair located next to the muscle cells (MC) [C] appeared more mature; the myelination of one neuron was completed (arrow). Morphology of the erythrosomes surrounded by endothelium was also more mature in [C]. The boxed area, enlarged in [D], featured abundant caveolae (arrows) of the multilayered neurothelium and a difference in thickness of the collagen fibers (cf) between those in endoneurium (thinner) and in surrounding ECM (thicker) [D].\n\n\nDiscussion\n\nSurvival of the ectopically implanted breast tumor cells for three weeks without the support of a host circulatory system was possible due to the erythrogenic autophagy36. The ability of cells to undergo such nucleo-cytoplasmic conversion was not tumor-specific. The initial host response to tumor nodules by encapsulating them with fibroblasts was simultaneous with the response to surgical injury caused by the implantation. During the repair process, as the layer of connective tissue grew thicker beneath the glass wall of the chamber, it too was forming new vasculature. That finding showed similarity between the cellular mechanisms of vasculature morphogenesis in growing malignant and non-malignant tissues. Hypoxia, the common metabolic denominator, could force erythrogenesis upon different types of cells, including differentiated ones for example ECs54, if the condition persisted.\n\nThe tumor ecological niche resembled a perpetuum mobile in its ability to survive without blood vessels. There was a turnover of tumor cells; they kept proliferating and succumbing to erythrogenic autophagy. The system was not really isolated because it used metabolites from the microenvironment, but depending solely on diffusion for that purpose, the tumor could not increase its size. Non-vasculogenic tumors do not grow over several weeks although the tumor cells keep proliferating at a rate similar to that of vasculogenic tumors; \"They have no or non-functional vessels\"55. Those \"non-functional\" vessels could have been non-circulating erythrosomes, most likely derived from the tumor cells. That was, in fact, an experimental result demonstrating that some tumor cells could survive at the expense of others. Such postnatal extramedullar erythropoiesis at a location other than the bone marrow (medulla ossea) was not unprecedented; spleen56–59 and adipocytic tissues60 have that potential as well.\n\nThe relevance of the variable metabolism within a single tumor nodule was that tumor-derived erythrosomes might indeed extend viability of adjacent tumor cells by supplying them with vital energy in the absence of vasculature. Hemoglobin has evolved to bind oxygen cooperatively, i.e., most efficiently when it is abundant (in lungs where the oxygen concentration is about 100 torr) and gradually less and less efficiently as erythrosomes move through arteries and veins (in peripheral tissues, the oxygen concentration is about 20 torr)61. In tumors experiencing hypoxia, one would expect the binding of oxygen to hemoglobin to be least efficient, so the erythrosomes would not compete for oxygen with the tumor cells.\n\nThe ability to convert into erythrosomes was not limited to erythropoietic lineage derived from myeloid precursors. Understandably so, because the inducing factor, hypoxia, affected the most fundamental function of all living cells, i.e., the respiration, generating vital energy aerobically. Under hypoxia, they all had only one alternative to extend their existence, namely, anaerobic generation of energy indispensable to sustain life. That metabolic pathway being shared by all cells experiencing hypoxia imposed the formation of similar ultrastructural features on all of them (convergence). Therefore, knowing what the cells do and how they do it regardless of cell lineages is important to control tumor growth. Unfortunately anaerobic metabolism is not unique to tumor cells; others are neutrophils and muscle cells, precursors of erythrosomes in bone marrow, and any dividing cells at mitosis54.\n\nThe microenvironment determined the fate of tumor cells in a way similar to controlling the fate of other cells. The interactions were mutual and ever changing. The unrelated cells could become similar enough to act as \"relatives\". In other words, the heterologous environment did not kill the transplanted tumor but gradually the exogenous tissue acquired the ability to engage in the paracrine dialog with local TSCs (perhaps by acquiring proper cell adhesion molecules62), or the tumor activated its own SCs (CSCs). Trans-differentiation of tumor SCs into ECs was also reported earlier in glioblastoma63–66. When that happened, the dormant tumor underwent a vasculogenic switch67. If the process was slow enough, it might not be completed within the life span of the host and such tumors would be unnoticed due to their small size, limited by 100–200 µm oxygen diffusion range68. Reported dormant tumors had < 1 mm diameter, possibly including fibroblastic coats and necrotic centers67.\n\nTwo cellular mechanisms normally beneficial to the organism when acting independently, one involved in tissue nourishment and the other in healing, i.e., erythrogenesis and scar formation (or foreign body encapsulation) respectively, became deleterious by creating the capsular vaso-mimicry when they coincided in the ectopically implanted tumor. The newly emerged vessel-imitating structures contained cells of tumor and host origin. They did not contain circulating blood initially, but could potentially fuse with the morphologically similar host lymphatic vessels or veins. If the conversion of the tumor cell population into erythrosomes were incomplete at the time of the merger, the fusion would facilitate metastasis. The anastomosis with lymphatic vessels might be more likely than with blood vessels (particularly arteries) because the former are comprised of a single endothelial cell layer, have no pericytes and only incomplete basement membrane. That would be consistent with the observation that metastases of most cancers occurred initially through the lymphatics69. The dissociation of tumor cells from one another, i.e., anoikis70, commonly seen in necrotic regions, might be due to hypoxic stress and starvation. That way each cell would gain direct access to interstitial fluid and the cell surface would greatly increase through multiple, meandering nano-tentacles that appeared to be an early sign of stress. At the same time, the loss of attachment to other cells could facilitate their dissemination by breaking the tumor tissue into small cell clusters or single cells that could be carried away by blood flow. Whereas converting a fraction of the growing tumor population into erythrosomes solved the immediate energy metabolism problem for the rest of the population temporarily, the capsular vaso-mimicry could assure a future steady supply of new energy resources in the end.\n\nConcerning the prospect of controlling initiation of metastasis, the cellular mechanism presented here appeared more manageable because it did not depend on great biological diversity of primary tumors. The initiation of capsular vaso-mimicry was governed by metabolic requirements rather than the genetic repertoire of the tumor. Clusters of primary tumor cells could be passively carried to different tissues by blood flow and become immobilized when they reached vessels narrower than their own dimensions, in a tissue non-specific manner. However, the fate of such randomly dispersed metastatic tumor \"seeds\" would depend on their phenotypic compatibility with the local \"soil\"71. Thus, the vasculogenic switch could occur in the secondary locations either immediately or after a variable period of latency depending on the initial degree of relevant similarities. Liver being formed relatively early during embryogenesis and later maintaining primitive vasculature might be most compatible with tumors for that reason and therefore most prone to metastases, as observed clinically. If the metastasized tumors did not adjust their properties as needed to establish paracrine dialog with local TSCs, they would stay dormant. The dormancy would not necessarily be permanent because surviving via erythrogenic autophagy was accompanied by proliferation ([F] in Figure 2 & Figure S2) and therefore equipped the tumor with a source of the biological diversity. The concept of the distant niche anticipating an invader and getting ready for it72,73 could be adopted as follows. Dissemination of tumor cells through circulating blood could occur due to capsular vaso-mimicry targeting all organs but a successful metastasis would only develop in tissues somewhat similar to the one where the primary tumor developed. This would be consistent with the seed and soil theory71,74,75. On the other hand, if the tumor was large enough to produce meaningful levels of cytokines and growth factors in circulating blood, the effect on un-invaded homologous tissues should be comparable to that caused by a smaller number of tumor cells that have metastasized. That is how anatomically distant but phenotypicly compatible tissue could become activated by the tumor before metastasizing cells got there.\n\nThe structures shown in Figure 3 & Figure S3 and the earlier reported vasculogenic mimicry10 could be of the same nature. The remnants of cells that produced erythrosomes could be responsible for PAS staining due to their glyco-lipid components and, more importantly, for fusion with capillaries of the main circulatory system, at stages later than analyzed here. Our tumors were significantly smaller (diameter of < 1 mm) than those described in the literature (1 cm or more). A lack of hierarchy in the network pattern of the aggressive tumors suggested a lack of blood circulation. However, small molecules used to study intra-tumoral microcirculation by injecting a dye into a vessel located close to it could rapidly diffuse through such spaces12,13. If blood were circulating through the vascular mimicry, there should be no problem with drug delivery to such tumors. Therefore, that kind of mimicry probably is a form of fibroblastic autophagy associated with the presence of metastatic tumors. The metastases could have been initiated via capsular vaso-mimicry earlier, when the primary tumor nodules were small.\n\nThe new understanding of the cellular mechanisms involved in the tumor neovasculature formation provokes some additional retrospective thoughts on earlier published results regarding vasculature-related issues that were also based on the model of breast tumors grown in the mouse skin fold chamber. For example, abnormal microvascular oxygen transport demonstrated by spectral imaging of hemoglobin saturation76,77. Anastomoses between vessels with significantly different oxygenations could be explained by the fusion of hypoxic capsular vaso-mimicry with a vein containing circulating blood; the direction of the flow initiated that way would be expected to be the same as in the vein involved in the fusion, as observed (Figure 8 in77). The resulting larger hybrid vessel would initially have a flattened profile, as seen in malignant neurilemmoma grown in a hamster cheek pouch chamber78. What looked like \"acute local stoppage of blood flow\" could correspond to the lack of the flow in the vascular vaso-mimicry before the anastomosis77. Shunting of inspired oxygen into tumor venules, presumed to occur due to arterio-venous anastomoses (Figure 10 in76) could alternatively be explained by stable saturation of hemoglobin located in non-circulating erythrosomes within the tumor capsule, as well as within the regions mimicking vessels (Figure 1 & Figure S1). The oxygen could have remained bound to hemoglobin if the surrounding cells did not have structurally sound (functional) mitochondria. We have shown that mitochondria of tumor cells in the microenvironment of such stagnant erythrosomes were structurally impaired (converted into peroxisomes or necrotized) and accompanied by calmyrites. Such ultrastructural features are consistent with anaerobic metabolism. In the absence of functional mitochondria, the tumor cells would have no use for the abundant oxygen. Consequently, it shouldn’t be surprising that increased oxygenation of breast adenocarcinoma by treatment with, for example, darbepoetin alpha, had no desirable effect on the tumor’s responsiveness to radiotherapy79. Oxygenation was probably increased in the erythrosomal component of the tumor, not in the tumor cells (a distinction impossible to make by clinical radiology).\n\nThe distance between capillaries in tissue sections suggested that, within the 100–200 µm zones, cell membranes did not present a barrier for diffusion of nutrients or for oxygen. On the other hand, toxic metabolic products can be sequestered into intracellular vesicles to protect the cytoplasm. Although indistinguishable by TEM, the bi-layer lipid membranes vary with regard to their molecular composition. The leaky outer cell membranes permit passage of small molecules whereas the more selective inner vacuolar membranes provide a mechanism for intracellular compartmentalization. Considering what we now know about cytoevolution leading to ECs differentiation36, one could make a premise that the luminal surface of the polarized endothelial cell was a functional equivalent of the inner membrane. Therefore, it could present a barrier preventing uncontrolled diffusion of some molecules. Caveolae would serve as a compensation for such an indiscriminate barrier and would provide a structural basis for selective (controlled) transport across the membrane. That might be why ECs have them in great numbers and the absence of caveolae in brain endothelium correlates with the functional blood-brain barrier80. Vascular lumen in that context would be a functional equivalent of an intracellular vesicle. One could conclude that host fibroblasts encapsulating the tumor and creating the capsular vaso-mimicry by positioning themselves around erythrosomes should not present a barrier for the diffusion process because they did not go through the process of cytoevolution resulting in polarization of the outer cell membrane into luminal and abluminal. Morphologically, the tumor capsular vaso-mimicry resembled lymphatic vessel or vein, however it was neither. It had fibroblasts in place of ECs, therefore diffusion across the walls of the capsular vaso-mimicry (and further) would not be restricted. That could be a new explanation for the leakiness of the tumor pseudo-vessels, whereas in genuine tumor-supporting vessels, control of permeability would remain tight.\n\n\nConclusions\n\nThe results demonstrated that a balance between tumor growth and formation of its own vasculature could shift reversibly as dictated by a changing microenvironment. In vitro, where proper atmosphere and nutrients were available, tumor cells did not need vasculature and none formed. That changed when they found themselves back in the live mice but not connected to the host vasculature. Hypoxia forced some tumor cells to change their energy metabolism to an anaerobic pathway. That way, they could salvage the remaining tumor cells: by secreting lactic acid53 or ATP50 (similar to muscle cells and erythrocytes, respectively), and by initiating the vaso-mimicry. Time gained by the metabolic switch allowed for triggering the genuine vasculogenic switch and exposed self-organizing potential of the malignant tissue (limited to formation of its own vasculature). Establishing the metastatic tumors by creating the capsular vaso-mimicry required sufficient numbers of cancer cells in the initiating nodule. While some tumor cells were evolving into erythroblasts and megakaryocytes and inducing differentiation of ECs, others kept proliferating. Such activation of differentiation in some cancer cells was consistent with the organoblasts concept36. By definition, they could be referred to as cancer stem cells (CSCs). However, alkaline phosphatase cannot be used as a marker specific for CSCs because it was detected in erythroblasts81. If the enzyme plays a role in degradation of chromatin during the erythrogenic conversion of erythroblasts it could be associated with growth of any tissue, not only malignant tissue.\n\nEventually, the heterologous host TSCs also engaged in paracrine dialog with the tumor (via cytokines and growth factors72). The distinction between the two sources of SCs was based here on the location where early stages of vasculature formation were seen - within the tumor capsule or next to it (Figure 1 & Figure S1). Whereas the existence of somewhat controversial CSCs23,82 was exposed in the ectopic environment, it was masked by availability of homologous TSCs in the pseudo-orthotopic one36. Such interpretation regarding the role of homologous TSCs in neo-vasculature morphogenesis was consistent with earlier reports stating that it was not bone marrow-derived EC precursors83,84 but rather TSCs from the tumor microenvironment differentiated into vasculature that supported the tumor growth85. They were also shown to support vascular nonmalignant engraftment86.\n\nOn the other hand, after pseudo-orthotopic implantation, TSCs from grafted breast tissue formed vasculature for the breast tumor because malignant tissues maintained some characteristics of their tissue of origin. The two related cell types cooperated in executing the tissue self-organizing potential36. The source of SCs that generated tumor vasculature under different circumstances (tumor, host or grafted homologous tissue) mattered with regard to how soon the vasculature formation could begin. However, in each case, hematopoiesis supporting the growing tissues was extramedullar. That observation was new and suggested a physiological role for the aerobic glycolysis (the Warburg phenomenon) in tissue morphogenesis, as addressed elsewhere54.", "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\nThis work was supported by National Institutes of Health Grants (to JES): R01CA119378, PO1CA104898 and R01CA083989.\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\nThree weeks after implantation, the tumor environment consisted of single migrating cells that were secreting ECM components, mainly collagen (yellow) and others converting into erythrosomes (red) [A]. The erythroblast [B] and the eosinophil [C] were each in contact with a nucleated supporting cell on one side (green). A small primitive vessel, constituted mainly by erythrosomes (red) held together with megakaryocytes and platelets (purple), was located close to the tumor (brown) [D]. It resembled those early ones seen in the pseudo-orthotopic environment five days after implantation (Figure 2 in36). Tumor cells were encapsulated by fibroblasts (yellow) and the cell population inside the capsules appeared heterogeneous [E]. There were erythrosomes (red) and the nuclei of some cells had sinuses (green) [F & G] similar to those in ECs of a forming artery (Figure 11 in36). Featured in [H] is a part of a larger elongated capsule containing a cluster of tumor cells (brown) in the center and multiple erythrosomes around it, all covered with typical flat, collagen-producing fibroblasts, whereas a similar structure in [I] contained mainly erythrosomes and platelets (purple). Both were remarkably similar to a vein.\n\nHypoxia in tumor cells was manifested by structural changes of mitochondria resulting in their condensation into smaller and more electron dense (darker) forms or in degradation of cristae (left and right from the red line, respectively) [A], occasionally leading to autophagic vacuoles with whorls of membranes (arrows in [B & C]) or to peroxisomes (arrows in [D]). Partial separation of the tumor cells from one another (anoikis44) with emergence of meandering nanotentacles (stars in [A & C], no color added) created intercellular passages and greatly increased the cell surface. Immunocytochemical detection of GFP-labeled histone H2B in tumor cells is shown in [E & F]. The tumor cell in [E, light red] had a nucleus with electron-dense regions that did not contain H2B, except for a small upper region, demonstrating incomplete chromatin degradation (arrow). The upper left corner of [F] corresponds to the boxed area of the insert and showed H2B-specific label in a mitotic chromosome of the tumor cell (brown). A layer of collagen fibers (cf) separated that cell from the elongated fibroblast (yellow) with unlabeled nucleus (upper right corner) of [F]. The arrow in the insert points to a condensed mitochondrion.\n\nConsecutive splitting of an erythroschizont into erythrosomes (red); one completely separated and the rest in the process of splitting, all surrounded by leftover cytoplasm of the cell producing them (yellow) [A]. Four enlarged regions of that cell [B–E]. A distinct region of the erythroschizont indicated the location of the next, already initiated, split (arrows) [A & C]. Vesicles on both sides of the mitochondrion (orange) contained collagen fibers (arrows) [E].\n\nTwo neuro-vascular pairs. 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Acta Haematol. 1984; 72(5): 303–308. PubMed Abstract \| Publisher Full Text\n\nMaenhaut C, Dumont JE, Roger PP, et al.: Cancer stem cells: a reality, a myth, a fuzzy concept or a misnomer? An analysis. Carcinogenesis. 2010; 31(2): 149–158. PubMed Abstract \| Publisher Full Text\n\nPurhonen S, Palm J, Rossi D, et al.: Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth. Proc Natl Acad Sci U S A. 2008; 105(18): 6620–6625. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLarrivee B, Niessen K, Pollet I, et al.: Minimal contribution of marrow-derived endothelial precursors to tumor vasculature. J Immunol. 2005; 175(5): 2890–2899. PubMed Abstract\n\nSun L, Hui AM, Su Q, et al.: Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain. Cancer Cell. 2006; 9(4): 287–300. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "713", "date": "21 Jan 2013", "name": "Ygal Haupt", "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 report here on how tumour cells survive in an ectopic environment by implantation of spheroids of N202 breast cancer cells into a mouse dorsal skin chamber. Their major finding is that the implanted tumour spheroids survived for 3 weeks without any support of the host circulatory system.They propose that vasculogenic switch and erythrogenic authophagy of some tumour cells are the key process driving the survival and growth of the tumour cells. They find that tumour cells secrete ECM component and can generate their vasculature by converting into erythrosomes thereby supporting other tumour cells. The authors propose that this process is governed by anaerobic metabolism. The limitations of this study are: 1) the study is based on in situ ultra structural analysis of tumour growth and survival. There was no metastatic response or circulating tumours in their model, hence no cellular mechanism of metastasis as the title implies.Further, the study is based on one mouse model and one breast tumour cell line. While the study highlights the importance of anaerobic metabolism in metastasis, this concept has been discussed in the literature.", "responses": [ { "c_id": "436", "date": "23 Apr 2013", "name": "Halina Witkiewicz", "role": "Author Response", "response": "In situ ultrastructural analysis The issues addressed in our study are central in tumor biology and have been approached in many ways. However, without the type of validation possible by the in situ analysis of a particular living system at high resolution, the published work left many questions unanswered and created multiple controversies. Yet, our in depth analysis of the TEM images would not have been possible without the other approaches. We drew freely from the abundant experimental data accumulated in the literature regardless of the method used to generate them. The visual information from our work inspired logical connections between various kinds of earlier published data, often derived from various sources. Therefore, our conclusions and hypotheses do not rely solely on TEM. Our approach fills up the essential void; it “connects the dots”. The approach and the model we used are the strength of our study (as discussed in article III). The tissue ultrastructural analysis in situ brings together aspects of tumor biology traditionally treated independently: hematopoiesis, vascular biology, tumor metastasis and metabolism. Such exposure of the interdependence among those aspects introduces the tissue level into the general conversation on tumor biology at cellular & molecular levels. What a single experimental approach is depends on the definition. One can base the criteria on instruments used or on targets analyzed. Bridging the tissue and molecular levels, we used TEM to characterize tissues, cells, organelles and specific proteins in situ (immunocytochemistry). We used genetically engineered, GFP-labeled, cell line and the graft-donor mice. Intra-vital light and fluorescent microscopy helped to monitor the rate of tumor growth in vivo. The future belongs to this sort of combination of methods, i.e., the use of ultrastructural analyses in situ to validate other methods. No metastatic response In the title of the amended article we have added the word “implications” to reflect the distinction between the new results and their implications more precisely. In other parts of the article that distinction is clear. The original findings (including the blood elements and tumor cells inside the vessel-resembling structure, i.e., the capsular vaso-mimicry) are described in the Results section whereas the implications derived from those data and their significance are placed in the Discussion section. For the reader’s work such implications may be most useful when they represent new ideas and envision new directions for the future research. Without hinting in the second part of the title on significance of the new findings some readers could miss the article despite its relevance to their studies. One model Even though our initial observations have been limited to the breast cancer model, there are some indications that the capsular vaso-mimicry is not unique to that tumor type. As discussed in the article, the structure related to the capsular vaso-mimicry (Figure 3) was of the same nature as the vascular mimicry first seen in melanoma [PM:10487832]. Such mimicry was subsequently found in tumors of ovary [PM:11290546; PM:11839501], prostate [PM:11813211; PM:12359755] and in glioblastoma [PM:20375132]. Reporting studies based on one tumor type in one animal model is not unusual. The phenomenon of encapsulating tumors with fibroblasts is common among various types of tumors, although not every encapsulation forms the capsular vaso-mimicry. The prominent fibrous capsule associated with non-invasive insulinoma in the mouse model of pancreatic islet tumorigenesis did not coincide with the invasive tumor type either [PM:12086849]. Unlike the monolayer, a thick layer of fibroblasts encapsulating tumor did not resemble a vascular wall. Figure 5 in the amended version of the article shows examples of both. The concept The capsular vaso-mimicry has not been discussed in the literature and the issue of leakiness of the tumor vessels remains unclear. No mechanism was proposed to make logical connections between the molecular events and the intercellular relations leading to the metastasis via the erythrogenic autophagy. Previously reported correlations between anaerobic metabolism and metastasis were used to predict clinical outcome." } ] }, { "id": "762", "date": "24 Apr 2013", "name": "Maria Vinci", "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 an interesting model that involves the ectopic implantation of tumour spheroids (N202 breast tumour cell line) into the mouse skin fold chamber. By using high quality ultra structure images, the authors describe tumour survival to hypoxia and formation of capsular vaso-mimicry as the event that determine the vasculogenic switch, 3 weeks after implantation and that facilitate metastasis. Although the work is valuable for the novelty of the process described, on the other side, it also presents some issues, for which I have reservations. The work is based only on one tumour model, the number of animals used for some of the observations is too low, the analysis is performed only by in situ ultrastructure and no quantitative data are provided as well as no information on the number of fields of view/slide analysed. Also the images shown are all in very high power magnification. It would be useful to include images at lower power magnification to have a better overview of the surrounding tissue. Also, the authors do not provide any evidence of tumour metastasis, or tumour dissemination occurring from the original implantation, but imply that the observed vaso-mimicry process could facilitate metastasis. The authors should have looked at the process at different time points: earlier and later than the 3 weeks time point selected for the study. Minor comments: the article is in general too wordy with the results often containing discussion points. Typos: in the title \"ectopicly\" should be \"ectopically\" and on page 9, \"phenotipicly\" should be \"phenotipically\".", "responses": [ { "c_id": "438", "date": "23 Apr 2013", "name": "Halina Witkiewicz", "role": "Author Response", "response": "Magnification The article has been amended to include examples of images taken at lower power magnification. Figure 5 shows how the overall topography of each section was examined before focusing on the most interesting regions. Implications for tumor metastasisIn the title of the amended article we have added the word “implications” to reflect the distinction between the new results and their implications more precisely. In other parts of the article that distinction is clear. The original findings (including the blood elements and tumor cells inside the vessel-resembling structure, i.e., the capsular vaso-mimicry) are described in the Results section whereas the implications derived from those data and their significance are placed in the Discussion section. For the reader’s work such implications may be most useful when they represent new ideas and envision new directions for the future research. Without hinting in the second part of the title on significance of the new findings some readers could miss the article despite its relevance to their studies. In situ ultrastructure only The issues addressed in our study are central in tumor biology and have been approached in many ways. However, without the type of validation possible by the in situ analysis of a particular living system at high resolution, the published work left many questions unanswered and created multiple controversies. Yet, our in depth analysis of the TEM images would not have been possible without the other approaches. We drew freely from the abundant experimental data accumulated in the literature regardless of the method used to generate them. The visual information from our work inspired logical connections between various kinds of earlier published data, often derived from various sources. Therefore, our conclusions and hypotheses do not rely solely on TEM. Our approach fills up the essential void; it “connects the dots”. The approach and the model we used are the strength of our study (as discussed in article III). The tissue ultrastructural analysis in situ brings together aspects of tumor biology traditionally treated independently: hematopoiesis, vascular biology, tumor metastasis and metabolism. Such exposure of the interdependence among those aspects introduces the tissue level into the general conversation on tumor biology at cellular & molecular levels. What a single experimental approach is depends on the definition. One can base the criteria on instruments used or on targets analyzed. Bridging the tissue and molecular levels, we used TEM to characterize tissues, cells, organelles and specific proteins in situ (immunocytochemistry). We used a genetically engineered, GFP-labeled cell line and the graft-donor mice. Intra-vital light and fluorescent microscopy helped to monitor the rate of tumor growth in vivo. The future belongs to this sort of combination of methods, i.e., the use of ultrastructural analyses in situ to validate other methods. One tumor model Even though our initial observations have been limited to the breast cancer model, there are some indications that the capsular vaso-mimicry is not unique to that tumor type. As discussed in the article, the structure related to the capsular vasomimicry (Figure 3) was of the same nature as the vascular mimicry first seen in melanoma [PM:10487832]. Such mimicry was subsequently found in tumors of ovary [PM:11290546; PM:11839501], prostate [PM:11813211; PM:12359755] and in glioblastoma [PM:20375132]. Reporting studies based on one tumor type in one animal model is not unusual. The phenomenon of encapsulating tumors with fibroblasts is common among various types of tumors, although not every encapsulation forms the capsular vasomimicry. The prominent fibrous capsule associated with non-invasive insulinoma in the mouse model of pancreatic islet tumorigenesis did not coincide with the invasive tumor type either [PM:12086849]. Unlike the monolayer, a thick layer of fibroblasts encapsulating the tumor did not resemble a vascular wall. Figure 5 in the amended version of the article shows examples of both. Quantitative data & time points Our goal was accomplished by generating unprecedented photographic documentation of spontaneous intercellular relations leading to formation of the capsular vaso-mimicry. The nature of the study was exploratory and observational therefore the results are qualitative. The images represented raw data, i.e. a direct demonstration of the phenomena that had actually occurred in vivo. The animals were not subjected to any kind of treatment except implantation of the tumor. The conventional thinking in terms of comparing treated and control groups over a period of time to measure an anticipated effect and to evaluate its statistical significance was not applicable here. The time progression was not critical either, as opposed to the embryonal growth and development, because the tumor never matures. In vivo the cells were responding to locally variable microenvironment therefore they were not functionally synchronized. Consequently a range of different stages of the observed processes could be found simultaneously within single sections. To illustrate our conclusions we selected representative images after careful analysis of many (several hundreds per mouse at various magnifications). One should keep in mind that the tumors were about 1 mm in “diameter” when viewed through a dissecting microscope. The photographed fields were selected, not randomly picked; therefore stating how many they were would be meaningless. We stopped the analysis when the findings became redundant and we understood the observed phenomena. In the literature the number of studied animals tends to be inversely proportional to complexity of assays. For example, one study had animal groups of variable size: n=18 for flow cytometry and n=6 for histology (PM:15883207). For electron microscopy studies it is common to use samples from small number of individuals (PM:20439620). In all five mice that we used (in this and the accompanying articles) the fundamental cellular mechanism of initiating the vasculature formation turned out to be the same. Thus, the number of animals was adequate for the goal of this particular study. Increasing the number of sacrificed animals above the necessary minimum, just for the number sake, would be superfluous and against the animal welfare rules. Minor comments Dealing with images as raw data makes it difficult to find the right balance between describing what they show (and what the readers can see) and what they mean. We have tried our best to justify the interpretation by indicating what criteria were used to identify the presented structures. That often involved including literature references. Some improvements have been made in the amended version of the article. Both forms of the adverbs “ectopicly” & “ectopically” as well as \"phenotypicly\" & \"phenotypically\" are used by different sources. Another example of such inconsistency is the pair of words “tumor” & “tumour”. We leave the choices to the editor." } ] } ]	1	https://f1000research.com/articles/2-9
https://f1000research.com/articles/2-116/v1	23 Apr 13	{ "type": "Research Article", "title": "Self-organization of signal transduction", "authors": [ "Gabriele Scheler" ], "abstract": "We propose a model of parameter learning for signal transduction, where the objective function is defined by signal transmission efficiency. We apply this to learn kinetic rates as a form of evolutionary learning, and look for parameters which satisfy the objective. This is a novel approach compared to the usual technique of adjusting parameters only on the basis of experimental data. The resulting model is self-organizing, i.e. perturbations in protein concentrations or changes in extracellular signaling will automatically lead to adaptation. We systematically perturb protein concentrations and observe the response of the system. We find compensatory or co-regulation of protein expression levels. In a novel experiment, we alter the distribution of extracellular signaling, and observe adaptation based on optimizing signal transmission. We also discuss the relationship between signaling with and without transients. Signaling by transients may involve maximization of signal transmission efficiency for the peak response, but a minimization in steady-state responses. With an appropriate objective function, this can also be achieved by concentration adjustment. Self-organizing systems may be predictive of unwanted drug interference effects, since they aim to mimic complex cellular adaptation in a unified way.", "keywords": [ "Signal transduction systems are often modeled as networks of biochemical kinetic equations implemented as continuous-time dynamical models using differential equations1", "2. If we regard a subset of species as inputs", "and make sure that the system always converges to equilibrium values by using weakly reversible equations3–5", "we may transform these models into a set of matrices fulfilling the role of input-output transfer functions", "i.e. a mapping from sustained input signal levels to steady-state concentrations for all target species6. Protein signaling functions (psfs) are a systemic generalization of individual dose-response functions", "which are usually described by Hill equations7. In contrast to Hill equations", "which are not available for enzymatic reactions", "which only calculate relative concentrations", "and which only work for one reaction in isolation", "the psf system calculates enzymatic and complex formation reactions in a complex systemic environment using absolute concentrations6. In addition", "the reaction times to equilibrium are calculated as delay values", "and the dynamic shape (“transients”) is also available for further analysis." ], "content": "Introduction\n\nSignal transduction systems are often modeled as networks of biochemical kinetic equations implemented as continuous-time dynamical models using differential equations1,2. If we regard a subset of species as inputs, and make sure that the system always converges to equilibrium values by using weakly reversible equations3–5, we may transform these models into a set of matrices fulfilling the role of input-output transfer functions, i.e. a mapping from sustained input signal levels to steady-state concentrations for all target species6. Protein signaling functions (psfs) are a systemic generalization of individual dose-response functions, which are usually described by Hill equations7. In contrast to Hill equations, which are not available for enzymatic reactions, which only calculate relative concentrations, and which only work for one reaction in isolation, the psf system calculates enzymatic and complex formation reactions in a complex systemic environment using absolute concentrations6. In addition, the reaction times to equilibrium are calculated as delay values, and the dynamic shape (“transients”) is also available for further analysis.\n\nIn this paper, we want to ask the question of optimality of signal transduction. From an evolutionary standpoint, we assume that any biological signal transduction system is constructed with optimized efficiency of signal transmission. Furthermore, we assume that cells have the ability to adapt to perturbations of protein concentrations and changes in extracellular signaling by reinstating signal transmission efficiency. In the following, we investigate this question using a biologically realistic system – beta-adrenergic signaling in a submembrane compartment of a mouse embryonic fibroblast for a single input scenario, focusing on a selected target species as relevant output or actuator of the system (Figure 1A).\n\nA. Biochemical Reaction System with Selected Input (red) and Output (blue) B. (Top) ISO-pVASP transfer function (with RGS KO, red) in the MEF model11 (Bottom) Experimental Response to RGS KO for GPCR signal-response in a yeast model10 C. (Top) Distribution of Extracellular Signals Used to Calculate Signal Transmission. From a baseline signal S0 [10nM–900nM] the extracellular signaling level St rises to 110%,150% and 200%, but not above 1µM. (Bottom) Transfer Functions for ISO → pVASP. Shown is the original model, optimization by delay, by RC,S, and by both.\n\nExperimental analysis of signal transduction systems has shown fold-change responses to changes in input8,9. Accordingly, input-output transfer functions usually follow the shape of hyperbolic (saturating) curves, which are equivalent to sigmoids for logarithmically scaled input6. In Figure 1B we show the effect of a knock-out (KO) for a RGS protein in an experimental assay in yeast10, and compare this with the effect in the model system11. We see that the effects of the RGS KO on dose-response signaling efficiency are robust across very different cellular systems.\n\nUsually, when we use a computational model to investigate perturbations, we only study the effects as reflected in the simulation. By utilizing optimization in terms of signaling efficiency, we can make the system itself adjust to the perturbation. In this way, we are studying signal transduction as a self-organizing system, which uses objective functions to adapt. This basic idea is extremely powerful, and could be used with different kinds of constraints on parameters, reaction times, etc. and with different, multiple input scenarios for larger systems. To explore this question further is of significant importance in assessing cellular health and functioning.\n\n\nMethods\n\nFigure 1A shows the example system, a submembrane compartment with a GPCR (G-protein coupled receptor) pathway from a mouse embryonic fibroblast, with ISO as input (extracellular ligand to β(2)-adrenergic receptors) and the phosphorylation of a protein VASP as output. This model was implemented as an ODE model with 23 reactions and 27 molecular species, derived from 12 initial concentrations (cf. Table 1, Table 2). The parameters were adapted to experimental biological data (not shown11). In this subsystem, the central cAMP response often follows a plateau curve, i.e. a rise to steady-state, but cAMP transients which are typically observed in cytoplasm may also occur11. The dose-response transfer functions were derived as in6.\n\nA biological signal transduction system is defined by its state variable vector x, the set of all kinetic rate and initial concentration parameters.\n\nWe hypothesize that an efficient signal transmission would maximize the response coefficient RC,S (the response of species C to input S) defined as\n\n\n\nwith concentration change of target C and input S from baseline (t=0) to signal time t. For RC,S, values < 1 show signal loss, with 1 for perfect transmission, and values > 1 showing signal amplification. We may also optimize for the slope s of the sigmoid at half-maximum concentration. This is equivalent to maximizing RC,S, provided that the input signal remains entirely between the upper and lower boundaries of the sigmoid (Figure 1C). By optimizing for s, additional to RC,S, we may force the system to implement a switch-like function instead of a more linear function. However, shifting the sigmoid function to the left or to the right is more important as the slope in our models.\n\nIn addition, we optimize for reaction time (delay to steady-state). Steady-state is defined, pragmatically, as relative change of less than 2% over 100s. The delay (dS) is computed for 90% (EC90) of steady-state. We may now define an aggregate objective function:\n\n\n\nto select the system state variables that minimize delay and maximize response.\n\nIn addition to signal transmission from extracellular concentration changes onto steady-state concentrations, such as they typically occur for temporally integrating proteins like transcription factors, we also look at signal transmission by transients, i.e. peak concentration, in response to extracellular signals. In this case, we minimize the delay to peak value, maximize the response at peak value, and minimize the response at steady-state value.\n\n\n\n\nResults\n\nThe computation of signal transmission efficiency will be explained here for a single input-output pathway of cAMP/PKA-mediated transmission in a cellular membrane compartment. It is clear that a complex signaling system may have several inputs, and a large number of outputs or target proteins, and this is especially the case for cAMP-mediated signaling. Nonetheless we will focus on the simple case here to explain the basic principle. The input is a membrane receptor, a G-protein coupled receptor (GPCR), β(2)AR, which is activated by an extracellular pharmacological agonist ISO (isoproterenol); the output is a membrane protein, VASP, which promotes actin filament elongation. Activation of the receptor selectively inhibits VASP by phosphorylating VASP to pVASP via protein kinase A (PKA) activation. In Figure 1B the original ISO/pVASP transfer function is shown, which we use here for further optimization. The system was trained for a signal distribution between 10nM and 1 µM ISO adjusting both kinetic rate and concentration parameters (Figure 1A). The optimization method used is a simplex algorithm12. Figure 1B shows the transfer function before and after adjustment, maximizing for RC,S, dS or the combined function f. The results are summarized in Table 3. We see that optimizing for the response coefficient alone shifts the function to the right to better cover the input range. Optimizing for the delay alone shifts it to the left, speeding up signals in the lower range (which are slower). Both objectives together (equally weighted) are fairly close to the original, biologically validated curve, with an improved RC,S.\n\nIn general, biochemical reactions are faster at higher substrate concentrations, but the relative concentration change in response to an increase in the enzyme or the binding partner is less. This is a fundamental trade-off between delay and transmission efficiency that may define an optimal operating range for a signaling system and be of relevance in disease processes6. The results obtained with this experiment are simple, intuitive and encourage continuing to explore the basic idea.\n\nIn principle, we may use all parameters in a system, concentrations or kinetic rates, to maximize signal transmission. But the evolution of protein structure and interactions shows that it is fine-tuning of molecular kinetic parameters which is subject to evolutionary learning, while concentrations are often regulated adaptively in each cell. Mass-action kinetics approximate molecular kinetic parameters, even though there are significant sources of uncertainty, such as the stochastics of molecular interactions. In the following, we explore the idea that kinetic rate learning operates on evolutionary time-scales, and that biologically attested signal transduction pathways contain reaction rates which are optimal in terms of signal transmission efficiency. We use known concentration ranges, specific by cell type, together with kinetic rate optimization.\n\nTo explore the parameter space, we drew 1662 values for all kinetic rate parameters (kon, koff, kcat) from a distribution of 20% to 500% of the original parameters, and calculated RC,S and dS for the corresponding models, relative to an improved signal distribution from 1nM to 1µM (Figure 2A). We find that there are parameter combinations which greatly improve efficiency of the signal transmission function. The basic distribution of a uniform low value of RC,S for fast dS and a wide variability in RC,S above a certain threshold in dS is robust against different types of signaling input (cf. also Figure 3A). This may, however, be highly dependent on the reaction network that underlies the transfer function. We have not further explored this question. To test for robustness of these systems against variability of concentration, we repeated experiments for 100 systems with 20% variation of original concentrations, which corresponds to generally accepted noise levels (cf.13,14). As expected, this low variation did not significantly affect the quality of a set of kinetic rates (Supplementary table).\n\nA. Distribution of systems according to R and d values. ~ 1600 different systems states were randomly generated with k-parameter variations (20–500%), RC,S and d values were measured with the signal set shown on top. B. Kd and KM parameters for high efficiency (red, f < 2) and low efficiency (green, f > 5) systems. C. Cross-correlation of Kd values.\n\nA. (top) Dynamics of selected systems with low (ftransient < 3, blue) or high (ftransient > 10, red) propensity for a transient response. (bottom) Distribution of concentration parameters according to dp, RC,Sp (grey) or RC,S, d (red). B. Concentration changes in response to optimizing for transients, with plateau signaling (left) and strong transients (right), sorted by ftransient. C. Concentration shifts (orange=high, green=low) in the biochemical reaction network for a high transient system, as averaged from B. Conforming to intuition, we see that the earlier, driver complex (G-protein) is high for transients, which increase the on-slope for cAMP production.\n\nWe further analysed the parameter combinations with different signal transmission efficiency. In Figure 2B and C, we distinguish low and high efficiency signal transmission. Interestingly, we find, with respect to signal efficiency of the transfer function, that all parameters are “sloppy” (allow a wide variation), there are no “stiff” (low variation) parameters15. Standard deviation for all parameters in our case is similar (Figure 2B). Since it has been argued that optimization to experimental data yields reactions which allow more variance than others, as an indication of their influence on the signal transmission pathway, this analysis seems to contradict this effect. Possibly, these results pertain mostly to parameter variation that results from matching a networked system with many species to selected time-series data for only few species, which may behave differently from general optimization.\n\nCo-regulation of protein expression in cellular systems is important in disease progression and often a problem in targeted interventions. Here we are exploring the question of self-organization of protein concentration after a perturbation that reduces one protein to only 10% of its previous concentration. Keeping kinetic rates fixed, all concentrations in the system are allowed to adjust until optimality of signal transmission and delay is reinstated. There is a number of interesting observations here (cf. Figure 4A), which relate to the biological reality. For instance, reducing PDE4B causes much regulation in other proteins, but it is almost never targeted. In contrast, reducing PP1 has little effect, but PP1 is frequently responsive to other proteins. Reducing PKA, RGS and AC6 leads to widespread down-regulations, to maintain sensitivity of signaling, but reducing the receptor beta-2 leads to up-regulation.\n\nA. Concentration changes in response to total protein concentration reduction. Selected concentrations were reduced to 10% (shown on the x-axis). Learning was applied until f values were improved (shown on top for each experiment). Colors show the relative adjustment of all concentrations on the y-axis. B. Transfer function response to shifts in input signal range (shown on top). C. Concentrations changes in response to extracellular signal shift. Matrix is constructed as in A.\n\nThere are many individual adaptations which can be interpreted to maintain the sensitivity and responsivity of the small molecule cAMP. The results show that protein regulation is highly sensitive to positions and roles of individual proteins in the reaction network in transmitting the signal. This problem may also be amenable to a more principled mathematical analysis16. Another idea would be to rank reaction systems defined by kinetic parameters as in Figure 2 by how well they adapt to perturbations.\n\nIn our example, the quality of the readjustment is not always the same, but the simple optimization scheme that we use may easily produce suboptimal solutions (local minima). Concentration changes may be caused by genetic up- or down-regulation, secretion and re-uptake, increased degradation, RNA interference, etc. and are therefore not easy to model from the standpoint of mechanistic biological modeling, which would need modules for all biologically attested processes. A unified perspective by a set of constraints and a set of objectives, such as has been envisaged here, may lead to better predictive results and may also be used as a guiding principle in constructing mechanistic models. Since there are intricate biological processes of adjusting concentrations, we may assume that in the cell optimal solutions are more easily found.\n\nFrom the standpoint of disease modeling, an unusual protein concentration may be an adaptive response where a still functioning cell in a dysfunctional external signaling environment struggles to keep signal transmission efficient to support cellular function. In such a case, targeting this protein by pharmacological intervention will lead to co-regulation on other proteins. In general, as well as in real biological signaling systems, it is the localization of the input signaling range and the distribution of signals that the system transmits which are important for optimization. If we select the signal set that we optimize for in the right way, the input range will move towards the loglinear range, i.e. between the lower and upper input boundaries (Figure 4B). As a result, a number of internal concentrations will change. Figure 4C shows the relative concentration changes that result from a shift in input range. Interestingly, the low shift requires a strong reduction in RGS, and upregulation for PDE4B, among other effects. GsaGDP (the activating G protein) is strongly increased, and GiGDP (the inhibitory G protein) is decreased. In the high shift, GiGDP and PP1 (the phosphatase which decreases pVASP) are stronger, and the beta-2 receptor density is much increased. In the future, it will be interesting not only to calculate these effects based on different optimization measures, and in multiple input-output scenarios, but also to compare this with attested cases of biological adaptation. By reverse engineering, we may infer an optimization measure from a sufficient number of attested co-regulations. For instance in addiction, protein co-regulation as a form of sensitization is well-attested17, but also in cancer where intercellular signaling (e.g. by cytokines18) is affected. Gene expression data may then be mined not only for evidence of the mechanics of genetic regulatory pathways but also for evidence of shifts in extracellular signaling, which cause altered protein expression.\n\nThe appearance of a transient vs. a plateau signal (or even a dampened oscillation) in response to sustained signaling depends on the construction of the biochemical reaction network (negative feedback interactions) and its parameters. We show that we can also train a system for the appearance of a transient response to a sustained signal. This means to search for high response at peak, low delay to peak, but also a low response at steady-state (see Section “Objective function” and Figure 3A). This requirement of invariance for the steady-state is sometimes considered a form of “robustness” or “homeostatic regulation” of the cellular response19,20, and signaling by transients is widely regarded as an important mechanism. Here we found that concentration adjustment is quite sufficient to acquire a switch from transient to plateau response, with a high variability of response shape dependent on concentration, but also on the size of the signal (Figure 3A). In Figure 3B we show the distribution of concentrations that achieve high or low propensity for transients, as indicated by ftransient. We focus on concentrations with fairly uniform up- or down-regulation to create an experimental graph in Figure 3C. The simplicity of Figure 3C undermines the notion, as proven by the random search optimization, that deviations from up- or down-regulation for individual concentration may not be irrelevant noise, but rather part of a guided adaptation process that has many different solutions. We may have to consider this complexity to understand concentration adjustments in biological cells.\n\nBy using different objective functions for each target, it will be quite possible to combine different goals for targets in a multiple-output signaling system. Interestingly, a self-organizing cellular signaling system may also incorporate adaptive controls as objective functions, in particular when ion channels or membrane transporters are targeted. In this case, the goal of the system is to respond to an input (e.g. ISO at beta-2 receptors) such that the magnitude of another input such as calcium (ion channels as targets) is controlled. Signal transmission efficiency for such a target is not to be optimized to a maximum value, but instead to the appropriate ratio between the inputs. We have not followed up on this idea, but it may be worth further investigation as an example of natural computation for adaptive control.\n\n\nDiscussion\n\nThe idea to look for parametric optimization as the basis of realistic cellular properties has been applied with success to metabolic fluxes21. In that case, optimization of growth is usually regarded as the single objective function. Here we use another objective function, signal transmission efficiency, to study the adaptive response of a signaling system to perturbation in concentrations. This equals maximization of concentration change in a target species in response to input signal. Optimization of growth and optimization of signal transmission are therefore related.\n\nSignal transmission in a cellular system may have multiple functions: transcription factor activation, which may be related to the cell cycle, to morphological change or to adjustment of protein concentrations, cytosolic kinase/phosphatase activation with multiple cellular targets, membrane protein activation such as ion channels or receptors, etc. We assume that signal transduction has been optimized by evolution, and that concentration adjustment exists to maintain effective signal transmission. We have shown how to optimize a single-input single-output system for both speed and signal efficiency due to the basic properties of kinetic equations6. It is easy to extend the present discussion to optimize for multiple outputs in parallel, and a system could also be optimized for a number of I/O functions. In that case, other measures, such as mutual information, may also be employed as objectives. We used optimization in a two-step process: (a) in evolutionary learning, in order to find kinetic rates for estimated concentrations (b) in cellular adaptation, in order to re-calibrate the model in response to perturbations in concentrations, changes in extracellular signaling, or to effect a transient vs. plateau-like response. This also means that we have transitioned from a biologically defined system that is built bottom-up from available parameters and data to a self-organizing, learning system that adjusts to changes on evolutionary or individual time-scales.\n\nMatching models to experimental time-series data is an under-constrained problem that often yields large ranges of suitable parameters15,22. Analysing signals and targets to find optimal transmission parameters may be used to further constrain and investigate the parametric space. Signal transmission efficiency in a biological system can be measured directly23,24 and be compared to what is theoretically possible given a set of equations. During evolution, new protein subtypes develop with a different set of interactions and regulations. This corresponds to an adjustment of the available set of reactions, a type of structural learning to overcome the bottlenecks that are a result of tightly specified molecular kinetics and limited adjustment of concentrations.", "appendix": "Competing interests\n\n\n\nNo relevant competing interests disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary table\n\nShown are the original, the mean and std values.\n\n\nReferences\n\nBhalla US, Iyengar R: Emergent properties of networks of biological signaling pathways. Science. 1999; 283(5400): 381–387. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "2347", "date": "06 Nov 2013", "name": "Reimer Kühn", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 studies optimization of parameters in a network of bio-chemical reactions for signal-transduction in a one-input-one-output setting, seeking either to maximize a stationary response coefficient, or to maximize the slope at half-maximum of the (sigmoid) stationary response function, or to minimize the delay to steady state. Combinations of these individual criteria are also looked at. Optimization is performed over the space of kinetic rates and the initial concentrations of reactants of the set of kinetic equations making up the signal transduction system.The paper is very interesting and addresses a valid problem. It takes a well tested tenet as its starting point, viz. that signal transduction - like many other biological processes - “has been optimized by evolution, and that concentration adjustment exists to maintain effective signal transmission.”Thus parameters are sought which would optimize a given signal transduction system. The example studied in the present paper consists of a set of 23 chemical reactions involving 27 molecular species, and one or a combination of several criteria as mentioned above are used to drive the optimization. This paradigm, it is argued would naturally give rise to a notion of self-organization of said system, as well as support adaptability under changing external conditions.While I find this an attractive proposition in principle, and one that could carry quite far in further attempts to understand cellular processes, I am not entirely convinced that the way in which it is implemented in the present paper is the most appropriate.My main concern is related to the fact that, kinetic coefficients are included in the space of parameters over which optimization of response coefficients is performed. Kinetic coefficients are, however, largely given by the chemistry, or rather the physical properties of reactants involved. There is thus only a restricted scope for varying these parameters; they may be varied to be sure (though not independently!) by changing temperature, or the pH or the salinity of the solute in which these reactions occur. Since neither of these conditions would change widely under natural in-vivo conditions, there is thus a question whether this form of optimization is actually a good model of the one chosen by nature in the evolutionary process. Differences between optimal and original values of kinetic coefficients are not reported in the paper. It would be interesting to know whether they are small (hence whether nature has exploited the given reactions to the full).Indeed, using minor modifications of the present setup, which would allow to describe the co-variation of kinetic coefficients with temperature, pH and salinity, one might thus conceivably be able to verify that - given the set of reactions describing the signalling system - nature is indeed running them under optimal conditions.An altogether more ambitious project would consist in implementing a more appropriate model of the optimization process likely to have been implemented by nature to find precisely these 23 reactions. This form of optimization would require something of a more combinatorial nature (changing or modifying reac-tants thus reactions themselves) rather than parameters describing given reactions. Genetic algorithms, in some sense also implemented by nature, could be the method of choice to go about it.", "responses": [ { "c_id": "861", "date": "14 Jun 2014", "name": "Gabriele Scheler", "role": "Author Response", "response": "Concerning kinetic coefficients: Often many different variants of a protein, or an enzyme, exist, with different kinetic properties. Especially in the G-protein coupled pathway, variants for adenylate cyclase, phosphodiesterase, RGS proteins, G-coupled receptors are on the order of dozens or more (including splice variants). Accordingly, the actual kinetic rate e.g. for cAMP degradation can be fine-tuned by the expression level of the different PDE's involved. These protein variants also often have differentiated expression by cellular compartment and different regulatory interactions. Still, I believe the potential for kinetic rate learning by evolutionary evolvement of enzymes into subtypes is present.I agree that learning of a set of reactions would be a more ambitious and worthwhile project." } ] }, { "id": "3996", "date": "03 Jun 2014", "name": "Eugene V. Koonin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article reports a new approach to modeling biological signal transduction systems based on the simple but important assumption that such systems have been evolutionarily optimized for maximum signal transmission efficiency. By embedding evolutionary optimization into the equations, the author obtains a self-organizing, evolving model. The approach is not fundamentally novel as the idea comes from the well-developed field of flux balance analysis. Apparently, however, this methodology has not been previously applied to signal transduction.The current article is somewhat too abstract to judge on the ultimate utility of the approach but certainly, this is a step in an interesting direction.", "responses": [] } ]	1	https://f1000research.com/articles/2-116
https://f1000research.com/articles/2-57/v1	21 Feb 13	{ "type": "Research Article", "title": "Parasitic wasp females are attracted to blends of host-induced plant volatiles: do qualitative and quantitative differences in the blend matter?", "authors": [ "Masayoshi Uefune", "Soichi Kugimiya", "Rika Ozawa", "Junji Takabayashi", "Masayoshi Uefune", "Soichi Kugimiya", "Rika Ozawa" ], "abstract": "Naïve Cotesia vestalis wasps, parasitoids of diamondback moth (DBM) larvae, are attracted to a synthetic blend (Blend A) of host-induced plant volatiles composed of sabinene, n-heptanal, α-pinene, and (Z)-3-hexenyl acetate, in a ratio of 1.8:1.3:2.0:3.0. We studied whether qualitative (adding (R)-limonene: Blend B) or quantitative changes (changing ratios: Blend C) to Blend A affected the olfactory response of C. vestalis in the background of intact komatsuna plant volatiles. Naïve wasps showed equal preference to Blends A and B and Blends A and C in two-choice tests. Wasps with oviposition experience in the presence of Blend B preferred Blend B over Blend A, while wasps that had oviposited without a volatile blend showed no preference between the two. Likewise, wasps that had starvation experience in the presence of Blend B preferred Blend A over Blend B, while wasps that had starved without a volatile blend showed no preference between the two. Wasps that had oviposition experience either with or without Blend A showed equal preferences between Blends C and A. However, wasps that had starvation experience in the presence of Blend A preferred Blend C over Blend A, while those that starved without a volatile blend showed equal preferences between the two. By manipulating quality and quantity of the synthetic attractants, we showed to what extent C. vestalis could discriminate/learn slight differences between blends that were all, in principle, attractive.", "keywords": [ "Plants infested by herbivorous insects release volatiles called herbivore-induced plant volatiles (HIPVs)", "which attract carnivorous natural enemies such as parasitic wasps and predators1–3. Blends of HIPVs differ from those of volatiles emitted by intact or artificially damaged plants and are specific to plant species", "cultivars and developmental stage", "as well as to herbivore species and developmental stage1–3. Natural enemies facilitate this specificity to find their victims. For example", "parasitic wasps can distinguish between a blend of suitable host-induced plant volatiles and one of unsuitable host- or nonhost-induced plant volatiles to find a host4–6. Discrimination by the predatory mite between volatiles from plants infested with the prey and plants infested with the nonprey was also reported7. These plant-specific responses by carnivores may be due to innate olfactory preferences or to olfactory learning of prey-infested plant volatiles4–15." ], "content": "Introduction\n\nPlants infested by herbivorous insects release volatiles called herbivore-induced plant volatiles (HIPVs), which attract carnivorous natural enemies such as parasitic wasps and predators1–3. Blends of HIPVs differ from those of volatiles emitted by intact or artificially damaged plants and are specific to plant species, cultivars and developmental stage, as well as to herbivore species and developmental stage1–3. Natural enemies facilitate this specificity to find their victims. For example, parasitic wasps can distinguish between a blend of suitable host-induced plant volatiles and one of unsuitable host- or nonhost-induced plant volatiles to find a host4–6. Discrimination by the predatory mite between volatiles from plants infested with the prey and plants infested with the nonprey was also reported7. These plant-specific responses by carnivores may be due to innate olfactory preferences or to olfactory learning of prey-infested plant volatiles4–15.\n\nLearning is widespread among insects, and is relied on for all major activities16. Parasitic wasps are well-established model systems for research on insect learning17. Adult wasps can learn specific blends of HIPVs7–13,15. For example, Fukushima et al. (2002) reported that Cotesia kariyai (Watanabe) (Hymenoptera: Braconidae), a parasitoid of Mythimna separata larvae (Lepidoptera: Noctuidae), that were preconditioned by simultaneous exposure to infested maize volatiles and host feces showed increased responses to a synthetic blend of five HIPVs of low specificity (i.e., induced by both artificial and host damage)8. Takemoto et al. (2009) reported that Aphidius ervi, an aphid parasitoid, were not attracted to volatiles from host-infested broad bean plants over intact plant volatiles when they had emerged in clean Petri dishes, but when artificially exposed to infested-plant volatiles during emergence, the wasps showed a significant preference for infested-plant volatiles12.\n\nCotesia vestalis is a specialist parasitoid of diamondback moth (DBM) (Plutella xylostella) larvae, which feed on crucifer plants. We previously reported that naive C. vestalis were preferentially attracted to crucifer plants with DBM larval damage over artificially-damaged or nonhost (Pieris rapae larvae)-infested plants6. More recently, we reported that a blend of four compounds in the headspaces of DBM-infested cabbage plants attracted naive C. vestalis14. The synthetic mixture was composed of sabinene, n-heptanal, α-pinene, and (Z)-3-hexenyl acetate at a ratio of 1.8:1.3:2.0:3.0. The attractiveness of the synthetic blend was confirmed both under laboratory14,18 and field conditions18.\n\nAn intriguing question about HIPV-mediated interactions between host-infested plants and natural enemies is to what extent natural enemies can distinguish qualitative and/or quantitative differences between two attractive volatile blends in combination with learning. To answer this question, we qualitatively or quantitatively manipulated the synthetic blend of volatiles that attracts C. vestalis to DBM-infested cabbage plants. By changing the ratio or adding another host-induced component to the artificial volatile blend, we tested whether these differences affected the olfactory responses of C. vestalis via learning of plant volatiles associated with hosts or foods. To our knowledge, this is the first report to show the extent to which wasps can recognize/learn qualitative or quantitative differences in volatile blends by using a synthetic blend of HIPVs attractive to C. vestalis.\n\n\nMaterials and methods\n\nUnless specified otherwise, all procedures described below were conducted in a climate-controlled room (25±3°C, 60±10% relative humidity [RH], 16-hour light: 8-hour dark [16L:8D] lighting schedule). Komatsuna (Brassica rapa var. perviridis L. cv. Rakuten) plants (Takii & Co., Ltd., Kyoto Japan) were cultivated in soil (Ikubyou-baido: Takii & Co., Ltd., Kyoto Japan) in individual plastic pots (diameter: 90 mm, depth: 70 mm) for 4–5 weeks.\n\nDBM were collected in a field near Kyoto, Japan, and mass-reared on potted komatsuna plants in the climate-controlled room. Eggs were collected every a few days, and hatched larvae were reared on cut plants in small cages (25 × 15 × 10 cm high).\n\nCotesia vestalis were obtained from parasitized DBM larvae collected in the same field. Newly-emerged adults were maintained in acryl cages (35 × 25 × 30 cm high) with 50% aqueous honey as food for 3 d to ensure mating. Female wasps were then individually transferred to glass tubes (2 cm diameter, 13 cm long) containing 50% aqueous honey as food and kept in a dark climate-controlled chamber (18±3°C, 60±10% RH, 24D) to suppress flight and prolong lifespan before use. Females were never kept for more than 6 d. At least 1 h before the start of each experiment, naive female wasps were transferred to the experimental chamber (25±3°C, 60±10% RH).\n\nThe synthetic mixture of HIPVs was prepared as follows. Pure compounds of (Z)-3-hexenyl acetate (Wako Pure Chemical Industries, Ltd., Osaka, Japan), n-heptanal (Wako Pure Chemical Industries, Ltd., Osaka, Japan), α-pinene (Tokyo Chemical Industry Co. Ltd., Tokyo Japan, sabinene (Soda Aromatic Co. Ltd. Tokyo, Japan) and (R)-limonene (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were dissolved in triethyl citrate (TEC) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) (0.01% (w/w) TEC solution) to achieve slow volatilization. Using gas chromatography–mass spectroscopy (GCMS; Agilent 6890N/5973MSD System, Agilent, Santa Clara, CA, USA) with a TC-Wax capillary column (GL Sciences, Tokyo, Japan), the ratios of compounds in synthetic mixtures were adjusted to match those released by an infested cabbage plant14. The gas chromatograph oven temperature was programmed to rise from 65°C to 120°C at 3°C/min, and then from 120°C to 245°C at 5°C/min. The synthetic mixture (Blend A) was composed of sabinene, n-heptanal, α-pinene, and (Z)-3-hexenyl acetate at a ratio of 1.8:1.3:2.0:3.014. This blend was called Blend A.\n\nWe already reported that Blend A did not become more attractive to C. vestalis by adding (R)-limonene, which was found in higher amounts in the headspaces of DBM-larvae-infested cabbage plants than intact ones14. Thus, to test whether C. vestalis discriminated qualitative differences in volatile blends, we added limonene to make Blend B. The ratios of sabinene, n-heptanal, α-pinene, (Z)-3-hexenyl acetate, and (R)-limonene were adjusted to be 1.8:1.3:2.0:3.0:1.0 by GCMS. Next, to test whether female wasps could discriminate the ratios of the four compounds in the blend, we prepared a third blend (Blend C) of sabinene, n-heptanal, α-pinene, and (Z)-3-hexenyl acetate at a ratio of 1.0:1.0:1.0:1.0. In two-choice tests, we compared either Blends A vs. B or Blends A vs. C.\n\nFemale wasps that had experienced oviposition or starvation were prepared as follows. To generate oviposition-experienced C. vestalis, we confined a second stadium DBM larva in a glass vial (2 cm in diameter, 5 cm long), and then introduced a female C. vestalis. In most cases, oviposition occurred within 5 min. Wasps that oviposited on the larvae were used for bioassays. Starvation-experienced C. vestalis were then confined in a glass vial (2 cm in diam., 5 cm long) for 2 h without food.\n\nTo prepare wasps that had been exposed to volatiles during oviposition or starvation experience, we placed a piece of filter paper (1.5 × 1.5 cm) impregnated with 10 μL of a TEC solution of a volatile blend (see below) into the tube before confining a host larva and a wasp. For control (unexposed) experiments, we used a blank piece of filter paper of the same size. To compare the effects of added limonene to those of the original blend (Blend A), we used Blend B for the oviposition and starvation experiments. In the comparison of quantitatively-different blends (Blends A and C), we used Blend A for the oviposition and starvation experiments.\n\nFemale C. vestalis were tested for their flight responses toward two potted intact komatsuna plants with a different blend. Two potted plants were placed in an acrylic cage (25 × 30 × 35 cm with three nylon-gauze–covered windows and one door) in a climate room at 25±2°C, 50–70% RH, and with continuous fluorescent light (20W, 3000 lux) without directed airflow6. We placed a piece of filter paper (2 × 2 cm) impregnated with a 0.2 g blend in a Petri dish (diameter: 3.5 cm) at the base of the potted plants. Wasps were released individually from a glass tube positioned halfway between the plants. Each repeatedly hovered over the plants inside the cage, and when it first visited a plant (landed and initiated ambulatory search), it was removed with an aspirator. The plant visited was scored as its choice. For each replicate, usually 10 wasps were tested sequentially using the same pair of potted seedlings. Each treatment had three or four independent replicates. Female C. vestalis were used only once. New plants and blends were used for each replicate.\n\nTwo-choice data were analyzed using a replicated G-test19. Wasps that chose neither plant were discarded from this analysis.\n\n\nResults\n\nWe first confirmed that oviposition-inexperienced (naïve) female wasps showed no significant preference between Blends A and B in the choice chamber (G-test, Gt = 5.9925, P = 0.1997; heterogeneity among samples: Gh = 3.9711, P = 0.2646; pooled effect of treatment: Gp = 2.0213, P = 0.1551) (Figure 1a). When the female wasps had prior oviposition experience on hosts in the absence of synthetic blends, they showed an equal distribution between Blends A and B (Gt = 1.2421, P = 0.7429; Gh = 1.2050, P = 0.5474; Gp = 0.0370, P = 0.8474) (Figure 1b). However, when the female wasps had prior oviposition experience in the presence of Blend B, they showed a significant preference for Blend B over Blend A (Gt = 7.9547, P = 0.0470; Gh = 2.6415, P = 0.2669; Gp = 5.3131, P = 0.0212) (Figure 1c).\n\nEach bar showed % of females that chose either of the blends (x axis). Numbers within bars indicate the numbers of females that landed on each plant. (a) Response of naïve C. vestalis. (b) Response of C. vestalis with oviposition experience. (c) Response of C. vestalis with oviposition experience in the presence of Blend B. (d) Response of C. vestalis with starvation experience. (e) Response of C. vestalis with starvation experience in the presence of Blend B. : 0.05 > P > 0.01, ns: not significantly different by G-test.\n\nWhen the female wasps had experienced starvation in the absence of synthetic blends, they distributed equally between Blends A and B in the choice chamber (Gt = 3.0677, P = 0.3813; Gh = 2.2013, P = 0.3326; Gp = 0.8664, P = 0.3520) (Figure 1d). However, when they had been starved in the presence of Blend B, they significantly preferred Blend A over Blend B (Gt = 9.3013, P = 0.0255; Gh = 0.6395, P = 0.7263; Gp = 8.6618, P = 0.0032) (Figure 1e). Thus, both oviposition and starvation experience affected wasp preferences for Blend A or Blend B.\n\nWe first confirmed that naïve female wasps showed no significant preference between Blends A and C in the choice chamber (Gt = 3.6264, P = 0.4589; Gh = 0.8118, P = 0.8466; Gp = 2.8147, P = 0.0934) (Figure 2a). When the female wasps had previously oviposited in the absence of any synthetic blend, they distributed equally between Blends A and C in the choice chamber (Gt = 1.7570, P = 0.6243; Gh = 0.7502, P = 0.6872; Gp = 1.0068, P = 0.3157) (Figure 2b). Even when female wasps had oviposited in the presence of Blend A, they still showed no preference between Blends A and C in the choice chamber (Gt = 6.0272, P = 0.1103; Gh = 5.987, P = 0.0501; Gp = 0.0400, P = 0.8415) (Figure 2c).\n\nEach bar showed % of females that chose either of the blends (x axis). Numbers within bars indicate the numbers of females that landed on each plant. (a) Response of naïve C. vestalis. (b) Response of C. vestalis with oviposition experience. (c) Response of C. vestalis with oviposition experience in the presence of Blend B. (d) Response of C. vestalis with starvation experience. (e) Response of C. vestalis with starvation experience in the presence of Blend B. : 0.05 > P > 0.01, ns: not significantly different by G-test.\n\nWhen the female wasps had previously experienced starvation in the absence of any synthetic blend, they also distributed equally between Blends A and C in the choice chamber (Gt = 1.4221, P = 0.7004; Gh = 1.3876, P = 0.4997; Gp = 0.0344, P = 0.8527) (Figure 2d). However, when they experienced starvation in the presence of Blend A, they significantly preferred Blend C over Blend A (Gt = 6.9032, P = 0.0750; Gh = 0.3765, P = 0.8284; Gp = 6.5268, P = 0.0106) (Figure 2e). Thus, only starvation experience affected the choice between Blends A and C.\n\n\n\n\nDiscussion\n\nWe already reported the flight preference of C. vestalis to pure and mixed synthetic chemicals (Blend A)14. When offered alone against pure solvent, none of the pure compounds elicited a significant preference in naive females of the parasitoid. However, when offered in mixtures against pure solvent, Blend A stands out as eliciting a significant preference. This mixture did not become more attractive by adding myrcene, camphor or limonene (compounds found to increase significantly in response to herbivory), and was just not significantly different in attractiveness from DBM-induced cabbage odor. Thus, Blend A triggered innate response in naive parasitoids, whereas the individual compounds did not. It was suggested that predatory mites, Phytoseiulus persimilis, did not recognize attractive HIPV in odor mixture but perceived odors as a synthetic whole11,20. Here, we showed to what extent parasitic wasps, C. vestalis, could recognize artificially created blend variation.\n\nWe manipulated the quality (Blend B) and quantity (Blend C) of the normal attractive synthetic blend (Blend A). The qualitative difference between Blends A and B was the presence in Blend B of (R)-limonene, which is not attractive to C. vestalis14, and the quantitative difference between Blends A and C was the ratio of the four compounds (1.8:1.3:2.0:3.0 vs. 1.0:1.0:1.0:1.0, respectively). Naïve wasps did not distinguish either qualitative or quantitative differences, suggesting that this wasp may have relatively broad responses to blends that contain these four essential compounds. By contrast, when the wasps had a positive experience (oviposition success) with a volatile blend, they could distinguish quality, i.e., they preferred the modified Blend B, but not quantitative differences, i.e., they preferred neither Blend A nor Blend C. Furthermore, when the wasps had a negative experience (starvation) in the presence of a volatile blend, they could distinguish both qualitative and quantitative differences. We believe this is the first study to show the extent to which parasitic wasps can identify qualitative and quantitative volatile differences by comparing naïve and tuned responses via associative learning.\n\nIn this study, the wasps experienced oviposition or starvation with or without volatile blends in glass tubes. Under natural conditions, parasitic wasps on host-infested plants would encounter not only HIPVs, but also host by-products such as feces. Fukushima et al. (2001) reported the ability of C. kariyai females to learn plant volatiles together with feces in a wind tunnel7. The females experiencing host by-products together with the volatiles extracted from infested leaves for the first time showed an increased olfactory response. However, such behavioral changes were not observed in females that experienced only the host by-products or the volatiles. Whether multiple experiences of C. vestalis with host by-products together with HIPVs affects their olfactory responses to qualitatively and/or quantitatively different blends of HIPVs must be studied to clarify the mechanisms involved in plant-specific responses by parasitic wasps to HIPVs.", "appendix": "Author contributions\n\n\n\nMU, SK and JT conceived the study and designed the experiments. MU, RO and SK carried out the research. MU, SK, and JT wrote the paper. All authors read and improved the manuscript and 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 partly supported by a Grant-in-Aid for Scientific Research (S) (No. 19101009) from the Global Centre of Excellence Program “Formation of a Strategic Base for Biodiversity and Evolutionary Research: from Genome to Ecosystem” of Kyoto University, Japan, and by the Japan Society for the Promotion of Science (JSPS): Core-to-Core project “Studies on ecological interaction networks that promote biodiversity — From gene to ecosystem”.\n\n\nAcknowledgements\n\nWe are grateful to the members of the Center for Ecological Research, Kyoto University, Japan, for valuable discussions.\n\n\nReferences\n\nTakabayashi J, Dicke M: Plant—carnivore mutualism through herbivore-induced carnivore attractants. Trends Plant Sci. 1996; 1(4): 109–113. Publisher Full Text\n\nArimura G, Matsui K, Takabayashi J: Chemical and Molecular Ecology of Herbivore-Induced Plant Volatiles: Proximate Factors and Their Ultimate Functions. Plant Cell Physiol. 2009; 50(5): 911–923. PubMed Abstract \| Publisher Full Text\n\nDicke M, van Loon JJ, Soler R: Chemical complexity of volatiles from plants induced by multiple attack. Nat Chem Biol. 2009; 5(5): 317–324. PubMed Abstract \| Publisher Full Text\n\nTakabayashi J, Takahashi S, Dicke M, et al.: Developmental stage of herbivore Pseudaletia separata affects production of herbivore-induced synomone by corn plants. J Chem Ecol. 1995; 21(3): 273–287. Publisher Full Text\n\nDe Moraes CM, Lewis WJ, Paré PW, et al.: Herbivore-infested plants selectively attract parasitoids. Nature. 1998; 393: 570–573. Publisher Full Text\n\nShiojiri K, Takabayashi J, Yano S, et al.: Flight response of parasitoids toward plant-herbivore complexes: A comparative study of two parasitoid-herbivore systems on cabbage plants. Appl Entomol Zool. 2000; 35(1): 87–92. Publisher Full Text\n\nFukushima J, Kainoh Y, Honda H, et al.: Learning of host-infested plant volatiles in the larval parasitoid Cotesia kariyai. Entomol Exp Appl. 2001; 99(3): 341–346. Publisher Full Text\n\nFukushima J, Kainoh Y, Honda H, et al.: Learning of herbivore-induced and nonspecific plant volatiles by a parasitoid Cotesia kariyai. J Chem Ecol. 2002; 28(3): 579–586. PubMed Abstract \| Publisher Full Text\n\nDe Boer JG, Posthumus MA, Dicke M: Identification of volatiles that are used in discrimination between plants infested with prey or nonprey herbivores by a predatory mite. J Chem Ecol. 2004; 30(11): 2215–2230. PubMed Abstract \| Publisher Full Text\n\nDe Boer JG, Snoeren TAL, Dicke M: Predatory mites learn to discriminate between plant volatiles induced by prey and nonprey herbivores. Animal Behav. 2005; 69(4): 869–879. Publisher Full Text\n\nvan Wijk M, de Bruijn PJ, Sabelis MW: Predatory mite attraction to herbivore-induced plant odors is not a consequence of attraction to individual herbivore-induced plant volatiles. J Chem Ecol. 2008; 34(6): 791–803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakemoto H, Powell W, Pickett J, et al.: Learning is involved in the response of parasitic wasps Aphidius ervi (Haliday) (Hymenoptera: Braconidae) to volatiles from a broad bean plant, Vicia faba (Fabaceae), infested by aphids Acyrthosiphon pisum (Harris) (Homoptera: Aphididae). Appl Entomol Zool. 2009; 44(1): 23–28. Publisher Full Text\n\nCosta A, Ricard I, Davison AC, et al.: Effects of rewarding and unrewarding experiences on the response to host-induced plant odors of the generalist parasitoid Cotesia marginiventris (Hymenoptera: Braconidae). J Insect Behav. 2010; 23(4): 303–318. Publisher Full Text\n\nShiojiri K, Ozawa R, Kugimiya S, et al.: Herbivore-specific, density-dependent induction of plant volatiles: Honest or \"cry wolf\" signals? PLoS One. 2010; 5(8): e12161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakemoto H, Powell W, Pickett J, et al.: Two-step learning involved in acquiring olfactory preferences for plant volatiles by parasitic wasps. Animal Behav. 2012; 83(6): 1491–1496. Publisher Full Text\n\nPapaj DR, Lewis AC: Insect learning. Chapman & Hall, New York 1993; 398. Publisher Full Text\n\nDukas R: Evolutionary biology of insect learning. Annu Rev Entomol. 2008; 53: 145–60. PubMed Abstract \| Publisher Full Text\n\nUefune M, Choh Y, Abe J, et al.: Application of synthetic herbivore-induced plant volatiles causes increased parasitism of herbivores in the field. J Appl Entomol. 2012; 136(8): 561–567. Publisher Full Text\n\nSokal RR, Rohlf FJ: Biometry. 3rd Edition. Freeman WH and Company, New York. 1995. Reference Source\n\nvan Wijk M, de Bruijn PJ, Sabelis MW: Complex odor from plants under attack: Herbivore's enemies react to the whole, not its parts. PLoS One. 2011; 6(7): e2174. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "807", "date": "04 Mar 2013", "name": "Jarmo K. Holopainen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUefune et al. have studied if qualitative or quantitative changes in the synthetic blend of the herbivore-induced plant volatiles will affect the olfactory response of Cotesia vestalis, a parasitoid of Diamondback moth. Their experiments are clearly described, carefully designed and conducted. The results of this study show the significance of associative learning for the behaviour this wasp species. The results are also giving valuable new information of the extent to which parasitic wasps can identify qualitative and quantitative differences in herbivore-induced plant volatiles. When we know how to fine tune the associative learning in parasitic wasps that will improve the efficiency of the use of C. vestalis in biological control.", "responses": [] }, { "id": "851", "date": "18 Mar 2013", "name": "Yonggen Lou", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 written by Uefune et al. studied the effect of qualitative and quantitative changes in a synthetic blend on the behavioural response of Cotesia vestalis, a specialist parasitoid of diamondback moth. The experimental design and methods are correct, and have been clearly described. By bioassay, the authors found that the wasps could distinguish qualitative changes when they had a positive experience (oviposition success) with the volatile blend; while when they had a negative experience (starvation) with the blend, the wasps could distinguish both qualitative and quantitative differences. The result provides new insights into the role of associative learning in the host searching behavior of wasps, and also new information of the extent to which qualitative and quantitative changes can influence the behavioral responses of the parasitoid. Minor concerns: It would be better to regard “ratio change” as a qualitative change; For both quantitative and qualitative changes, only one case was measured. It would be better to investigate more cases.", "responses": [] } ]	1	https://f1000research.com/articles/2-57
https://f1000research.com/articles/2-115/v1	22 Apr 13	{ "type": "Research Article", "title": "Variation in candidate genes CLOCK and ADCYAP1 does not consistently predict differences in migratory behavior in the songbird genus Junco", "authors": [ "Mark P Peterson", "Mikus Abolins-Abols", "Jonathan W Atwell", "Rebecca J Rice", "Borja Milá", "Ellen D Ketterson", "Mikus Abolins-Abols", "Jonathan W Atwell", "Rebecca J Rice", "Borja Milá", "Ellen D Ketterson" ], "abstract": "Recent studies exploring the molecular genetic basis for migratory variation in animals have identified polymorphisms in two genes (CLOCK and ADCYAP1) that are linked to circadian rhythms and correlate with migratory propensity and phenology among individuals and populations. Results from these initial studies are mixed, however, and additional data are needed to assess the generality and diversity of the molecular mechanisms that regulate the biology of migration. We sequenced CLOCK and ADCYAP1 in 15 populations across the two species of the avian genus Junco, a North American lineage in which multiple recently diverged subspecies and populations range from sedentary to long-distance migrants. We found no consistent associations between allele length and migratory status across the genus for either CLOCK or ADCYAP1. However, within two subspecies groups, populations that migrate longer distances have longer CLOCK alleles on average. Additionally, there was a positive relationship between ADCYAP1 allele length and migratory restlessness (zugunruhe) among individuals within one of two captive populations studied—a result similar to those reported previously within captive blackcaps (Sylvia atricapilla). We conclude that, while both ADCYAP1 and CLOCK may correlate with migratory propensity within or among certain populations or species, previously identified relationships between migratory behavior and sequence variants cannot be easily generalized across taxa.", "keywords": [ "candidate genes", "migration", "birds", "evolution", "population differences" ], "content": "Introduction\n\nEvery year billions of birds make round-trip flights from their breeding grounds to suitable winter climes and back again1. This seasonal migration requires immense coordination of different systems including fattening, locomotion, orientation, and activity2. Several aspects of migratory behavior including onset, duration, intensity, and orientation are known to be heritable and to respond quickly to artificial and natural selection – indicating a degree of genetic control3–7. However, other studies indicate that both learning and developmental environments can also shape migratory phenotypes2,8,9.\n\nThe propensity to migrate appears extremely labile on an evolutionary scale, with shifts between migratory and sedentary status independently arising, often repeatedly, in multiple lineages10–12. In recent decades, many populations and species are reducing the distance or frequency of migrations, and others are ceasing to migrate altogether, presumably in response to changing environments world-wide7,13–15. Understanding the molecular genetic mechanisms that regulate migration biology is imperative both for 1) understanding the evolutionary processes of behavioral adaptation and diversification, and 2) conserving and managing the phenomenon of animal migrations in the face of ongoing environmental change. However, relatively few studies have addressed the molecular genetic mechanisms of migration16. Our goal in this study was to build upon previous candidate gene studies to determine whether or not similar genetic mechanisms explain variation in migratory propensity at the level of individuals, populations, and species across taxa.\n\nPrevious studies have identified a number of candidate genes that may be involved in regulating migration, with particular focus on circadian rhythm genes. For example, the gene CLOCK controls several aspects of the circadian rhythm in mice17, and in the blue tit (Cyanistes caeruleus) it varies across latitudinal clines18, and with timing of breeding19. In salmon (Oncorhynchus spp.), CLOCK microsatellite repeat length also varies geographically and predicts migratory timing20–22. Monarch butterflies (Danaus plexippus) use CLOCK as part of the circadian pathway that guides their annual migration23, suggesting that CLOCK may play a role in the timing of migration across vertebrate and invertebrate lineages. However, blackcaps (Sylvia atricapilla24) bluethroats (Luscinia svecica18), and swallows of the genus Tachycineta25 do not show such correlations between this CLOCK polymorphism and migratory behavior or geographic location, despite possessing genetic variation at the locus. Furthermore, barn swallows (Hirundo rustica) show substantial variation in migratory phenotype, but very little variation at this CLOCK polymorphism26.\n\nIn another gene, adenylate cyclase-activating polypeptide 1 (ADCYAP1), length of a microsatellite repeat predicts migratory propensity of both populations and individuals in blackcaps24. ADCYAP1 is expressed throughout the brain and body of vertebrates27 and encodes pituitary adenylate cyclase-activating polypeptide (PACAP) (reviewed in28). PACAP is involved in many diverse behavioral and physiological phenotypes, including the circadian system (reviewed by Vaudry et al.28). Specifically, in chickens (Gallus gallus), PACAP directly activates CLOCK and other circadian genes in the pineal gland29. Further, PACAP stimulates release of melatonin30, and is a key component in entraining the circadian system to the light-dark cycle31, suggesting that it may be involved in circannual rhythms that rely on sensing day length, such as migration. Additionally, PACAP is involved in a large number of physiological and behavioral effects, from feeding behavior to breathing28, many of which may be related to physiological changes involved in migration24.\n\nThe body of work to date thus indicates that certain genes are associated with migratory phenotype in some way across phyla and within classes. However, these findings also warrant further study of the genetics of this complex phenotype at multiple levels in independent systems to determine whether selection is using conserved genetic machinery to arrive at similar phenotypic outcomes (e.g., Korsten et al.32).\n\nHere, we examined allelic variation in both CLOCK and ADCYAP1 in the genus Junco at the levels of species, populations, and individuals. The junco species group is currently comprised of the yellow-eyed junco (J. phaeonotus) and the dark-eyed junco (J. hyemalis), each of which contains multiple subspecies and populations showing a wide range of migratory behaviors33–35. Several of these subspecies have been identified as distinct species in the past (e.g. Miller35) and investigations into the genetic structure and systematics of the group are ongoing (e.g. Milá et al.36, McCormack et al.37, Rasner et al.38 and Whittaker et al.39).\n\nThe various dark-eyed junco subspecies offer an especially exciting opportunity to investigate migratory differences between closely related populations. There are 15 subspecies comprising the species34, and genetic similarity indicates that these closely related forms may have radiated just within the last 10,000 years–spreading across North America following the most recent glacial maximum36. This rapid diversification has limited the ability to identify clear phylogenetic patterns within the species36. These subspecies and populations differ markedly in migratory phenotype, ranging from completely sedentary to those that migrate thousands of kilometers (Table 1), with several populations also exhibiting individual variation in migratory behavior in which some individuals migrate and others do not12,34,35,40. Further, a coastal population of the Oregon junco (J. h. thurberi) has diverged from a montane altitudinal migrant population in the last 30 years and become sedentary in a milder environment on the campus of the University of California in San Diego41,42.\n\nGroup categories follow generally from distinct groups of subspecies or unique forms as discussed by Milá et al. (2007)36 or Nolan et al. (2002)34. Subspecies designations follow Miller (1941)35 as summarized by Nolan et al. (2002)34 and Sullivan et al. (1999)33; dark-eyed subspecies are noted as J. h. spp. and yellow-eyed subspecies as J. p. spp. Migratory Behavior codes indicate the range of migratory behaviors inferred from breeding and wintering distributions, as follows: LD2 = long distance II, at minimum 1600 km up to 5600 km, depending on migratory connectivity; LD1 = long distance I, likely 400–700 km, possibly up to 5000 km, depending on migratory connectivity; R = regional, typically greater than 200 km; A = altitudinal, typically less than 200 km; P = apparent partial migration documented; F = apparent facultative migration documented; S = sedentary (see text for references). Migratory score ordinally ranks population migratory propensity, similar to previous published accounts24.\n\nThe diversity of migratory behavior along with the close genetic relationships among divergent subspecies and populations make the junco species an excellent system in which to further examine the role of candidate genes linked to migration. Based on earlier studies of CLOCK and ADCYAP1 variation18,24,43, we predicted that junco populations that migrate longer distances would possess longer microsatellite repeat-length alleles of both CLOCK and ADCYAP1 when compared with those that migrate shorter distances including altitudinal migrants and sedentary populations. We also predicted that allele length would positively covary with individual variation in migratory restlessness within populations.\n\n\nMethods\n\nPopulations of juncos were classified according to a scale of migratory distance based on published reports in the literature33–35,40–42,44,45, personal observation. Each population was ranked on a scale from 1 (sedentary) to 6 (long-distance migrant) based on the average distance migrated by individuals in that population (Table 1). Distance migrated also varies within populations. For example, in the Eastern United States, females46 and adults47 generally migrate farther from their breeding ranges than males and yearlings, respectively. Some junco populations also exhibit partial migration, in which a subset of individuals remains on the breeding grounds while others depart34,35. The classification system employed here broadly categorizes the general migratory phenotype of each population and was organized to resemble previous reports for other species (e.g. blackcaps24).\n\nIndividuals from two populations of J. h. thurberi residing in southern California were captured as recently independent offspring and held in captivity under identical conditions in a ‘common garden’. Individuals were held individually (0.61 m × 0.61 m × 0.61 m cages) in a climate controlled indoor aviary with ad libitum access to food and water. Individuals were neither visually nor acoustically isolated, and light regimes were changed bi-weekly to match photoperiod at their home latitude. All methods were reviewed and approved by the IACUC at Indiana University (protocol #09-037). To reduce the probability of capturing siblings and pseudoreplication, individuals were captured from multiple locations, and one individual from each identified sibship (one in each population) was randomly included in all analyses, as in previous studies24. Additional studies of these captive common garden populations are reported elsewhere (48; Atwell et al. in review).\n\nBriefly, spring nocturnal restlessness behavior was scored from 3 March to 1 August 2010 by recording intervals of night-time perch-hopping activity of birds housed indoors in individual cages equipped with microswitched perches. This method is considered effective to capture 95% of migratory behavior observed in infrared video and ultrasound methods49, and even non-traditional songbird migrations tend to occur at night50,51. Juncos typically migrate at night34, and this sampling period overlapped with the typical spring migration as well as breeding seasons of dark-eyed juncos34,41. As was found in prior studies of migratory restlessness in the junco (e.g., Ketterson and Nolan52) and other species, migratory restlessness extended into the typical breeding period, perhaps due to caged birds’ inability to perform reproductive activities53. Nocturnal hops were recorded starting 30 minutes after dusk until 30 minutes before dawn under a daylight regime that simulated the photoperiod at their native latitude. GraphPad Prism 3.0 (GraphPad, USA) was used to quantify the seasonal intensity of individual spring migratory restlessness by calculating the area under each fitted seasonal profile curve, which provided a total migratory propensity score for each bird (n = 3614; Atwell et al. in review).\n\nAnalysis of population differences in genotype was conducted using DNA collected between 1996–2010 from adult juncos that were captured from breeding or wintering populations as a part of other studies. Less than 100 μL of whole blood was collected via capilary tube from a small puncture in the alar vein for future extraction. Both males and females were included, and in all populations in which sexes were known, the sexes did not differ in allele length for either locus (t-test, all p > 0.1). Birds sampled in their winter range were captured from December through February, after juncos are thought to have arrived at their wintering grounds and before they have departed in the spring34,54. All individuals from breeding locations were captured as adults during the breeding season of that population. No known siblings were included in these analyses.\n\nWe extracted DNA from whole blood from 15 populations spanning the range of migratory phenotypes in the genus Junco (Table 1). We developed primers surrounding a microsatellite repeat of interest based on previously published reports for CLOCK18,43 and ADCYAP124,43 in birds, and we modified the primers to more closely match the junco sequence based on the junco transcriptome (55; See Table 2 for primer details). The primers were used to amplify the respective polymorphisms in a single multiplex reaction using an amplification kit and following the manufacturer’s directions (Qiagen Inc, Valencia, California, USA). An initial 15´ heat activation cycle (95°C) was followed by 35 cycles of 94°C denaturation for 30”, 60°C annealing for 90”, and 72°C extension for 60”, and a final elongation of 10´. PCR products were then diluted 1:200 in ddH20 with LIZladder (Applied Biosystems, Carlsbad, California, USA), denatured at 95°C for 5´, and placed on ice. Samples were analyzed on an ABI 3730 using Peak Scanner v1.0 (Applied Biosystems, Carlsbad, California, USA).\n\nBoth primers are based on Steinmeyer et al.43, and CLOCK was modified to match junco sequences55.\n\nIn order to assess the relationship between allele length and migratory behavior among populations for both loci, we performed Spearman's correlations between population mean allele length and population migratory status. Qualitatively similar results are obtained with major allele count methods that have been applied in previous studies18,24 and when only dark-eyed subspecies were considered (data not shown). Within the sub-species groups for which we sampled multiple populations (Oregon juncos and slate-colored juncos, see Table 1), we additionally performed a nested ANOVA on allele lengths with population as a grouping factor within each subspecies to determine whether mean allele length differed significantly between populations or these subspecies.\n\nTo relate individual variation in allele length of ADCYAP1 and CLOCK to migratory restlessness, we fit linear models with migratory restlessness as the dependent variable and all combinations of population, mean allele length of the individual, sex, and each pairwise interaction as predictors. The resulting models were compared against each other using Akaike's Information Criterion. When the best fit model yielded a significant interaction term, we followed with a separate linear model for each population56.\n\nAll statistical analyses were performed in R version 2.15.257. Hardy-Weinberg equilibrium and heterozygosity were calculated with the package genetics58. All reported p-values are two-tailed, and hence conservative with respect to a priori directional hypotheses.\n\n\nResults\n\nWe identified 16 alleles at the ADCYAP1 locus ranging in length from 154 to 181 base pairs (bp), and all populations contained between five and ten alleles (Table 3). Allele lengths of 161 or 163 bp were most common in all populations. Nearly all of the identified alleles differed by multiples of two bases from these common alleles. The exceptions were represented in only 12 individuals, including five individuals from the Laguna Mountain population that shared an allele 164 bp long. All populations, both separately and combined, were in Hardy-Weinberg equilibrium (all p > 0.73).\n\nTable lists the number of individuals from each population possessing each of the identified ADCYAP1 alleles. Number of individuals genotyped is indicated as “n” for each population. Het = calculated heterozygosity for the population. Other abbreviations follow from Table 1.\n\nThere was no correlation between the population mean allele length of ADCYAP1 and population migratory status (Spearman's correlation, rho = 0.022; p = 0.938; Figure 1). Within the sub-species groups sampled at multiple locations the length of the ADCYAP1 allele did not differ between subspecies, or within subspecies (nested ANOVA, subspecies difference: F1,296 = 0.233, p = 0.629; population within subspecies: F4,296 = 0.852, p = 0.493; Figure 2).\n\nNo correlation was found between allele length and migratory score (Spearman's correlation, p > 0.05).\n\nADCYAP1 allele length did not differ between populations within the sub-species for which we have multiple sampling locations. Shown are means and standard errors for Oregon juncos listed by breeding grounds (panel A) and slate-colored juncos listed by wintering grounds (panel B). Population migration scores are shown in parentheses with higher numbers indicating populations that migrate longer distances (see Table 1 for more details).\n\nWe identified eight alleles at the CLOCK locus that varied in length from 267 to 285 bp, and all but one population (J. h. insularis from Guadalupe island) contained between two and four alleles (Table 4). A single allele (276 bp) was the most common in all populations except J. p. alticola from Guatemala where the 279 bp allele was most common. All but one of the additional alleles (present in a single individual) differed from the most common allele by multiples of three base pairs. All populations, both separately and combined, were in Hardy-Weinberg equilibrium (all p > 0.05).\n\nTable lists the number of individuals, from each population, possessing each of the identified CLOCK alleles. Number of individuals genotyped is indicated as “n” for each population. Het = calculated heterozygosity for the population. Other abbreviations follow from Table 1.\n\nPopulation mean allele length of CLOCK did not correlate with migratory status (Spearman's correlation, rho = -0.38, p = 0.159; Figure 3) when all populations were compared. Among the sampled populations of Oregon juncos (J. h. oreganus and J. h. thurberi) and slate-colored juncos (J. h. hyemalis) there was a significant effect of population within subspecies on CLOCK allele length, but not a difference in CLOCK allele length between populations (nested ANOVA, subspecies difference: F1,300 = 1.235, p = 0.267; population within subspecies: F4,300 = 2.468, p = 0.045; Figure 4). In both groups, populations that migrated longer distances possessed longer alleles at the CLOCK locus.\n\nNo correlation was found between population mean CLOCK allele length and migratory status (Spearman's correlation, p > 0.05).\n\nWithin each subspecies group for which we have multiple sampling locations, there is a trend for populations that migrate a longer distance to have longer CLOCK alleles. Figure shows mean and standard error for Oregon juncos listed by breeding grounds (panel A; ANOVA, p = 0.056) and slate-colored juncos listed by wintering grounds (panel B; ANOVA, p = 0.18; see text for details). Population migration scores are shown in parentheses with higher numbers indicating populations that migrate longer distances (see Table 1 for more details).\n\nWe tested for an association between mean ADCYAP1 allele length and level of migratory restlessness within the two captive populations of Oregon juncos (J. h. thurberi, Laguna Mountain and UC-San Diego). The most informative linear model described individual migratory restlessness in relation to population, mean ADCYAP1 allele length, and the population by allele length interaction (with Laguna Mountain as reference: effect of allele length = 712.9, p = 0.029; effect of population = 117,459.8, p = 0.056; population*allele length effect = -732.3, p = 0.053; model R2 = 0.225, p = 0.036). This interaction model performed better than all other tested models (Akaike's Information Criterion = 669.3; all other models AIC > 670.8; see Methods for list of models). Because the interaction term indicates a different effect of allele length on migratory behavior in each population, we ran separate linear models for each population56 which confirmed that longer individual mean ADCYAP1 allele length marginally predicted greater migratory restlessness in the migratory Laguna Mountain population (effect of allele length = 712.9, R2 = 0.169, p = 0.090; Figure 5, Supplementary Table 1), but not in the sedentary UC-San Diego population (effect of allele length = -19.36, R2 = 0.001, p = 0.844; Figure 5).\n\nPlot shows mean allele length of ADCYAP1 against individual migratory restlessness score for Oregon juncos from the migratory population at Laguna Mountain, CA, USA (solid circles, solid line) and the sedentary population in San Diego, CA, USA (open circles, dashed line). Mean allele length marginally predicts migratory behavior in the Laguna Mountain, but not San Diego, populations.\n\nThere was no relationship between mean CLOCK allele length and individual migratory restlessness behavior (Figure 6, Supplementary Table 1). The most informative linear model described only a population difference: the Laguna Mountain population displayed more restlessness than the population from UC-San Diego (with Laguna Mountain as reference: effect of population = -1299.4, p = 0.054; R2 = 0.102). For a more thorough treatment of this population difference, see Atwell et al. (in review). This population-only model performed better than all other tested models (Akaike's Information Criterion = 670.8; AIC for all other models > 671.32).\n\nPlot shows individual migratory restlessness score for Oregon juncos against the mean length of the CLOCK alleles from the migratory population at Laguna Mountain, CA, USA (solid circles) and the sedentary population in San Diego, CA, USA (open circles). Points are slightly jittered for each population to improve visualization. There is no significant relationship between CLOCK length and migratory restlessness.\n\n\nDiscussion\n\nPrevious studies have related migratory behavior in birds and fish to allelic variation in CLOCK and ADCYAP1. This study examined whether variation in these genes also relates to migratory behavior in the avian genus Junco. At the level of populations, we found that longer CLOCK alleles were associated with longer migratory distance within two sub-specific groups, J. h. oreganus and J. h. hyemalis. CLOCK was not, however, significantly associated with migratory distance across the genus as a whole or with individual intensity of migratory restlessness. In the case of ADCYAP1, longer alleles were not associated with migratory distance among populations within subspecies groups or across the genus. However, longer alleles were associated with higher levels of individual migratory restlessness in one of two populations studied. Together these findings suggest that allelic variation in both CLOCK and ADCYAP1 may contribute to differences in migratory behavior at some levels of analysis (within-subspecies or within-populations) but not reliably across the genus.\n\nThe lack of consistent relationships between molecular genetic variation and migratory variation suggested by our data are overall consistent with previous research on both CLOCK and ADCYAP1, as these genes are related to migratory and breeding behavior in only some of the species investigated. Within birds, CLOCK is associated with migratory and breeding behavior in blue tits19, but not black caps, bluethroats, great tits, or swallows18,24,25,59. Similarly, a relationship between ADCYAP1 and migratory behavior has been found only in blackcaps24, but has yet to be reported in other species. Despite these conflicting results, allelic variation in each of these genes is potentially consistent with a direct relationship with migratory behavior (see below), suggesting that these genes may indeed be important to the evolution of migratory and breeding behavior.\n\nThere are at least three reasons why the associations reported here might differ according to which populations or species were investigated: 1) variation in other, related genes (i.e., genetic background); 2) variation in the degree of linkage between the allelic variation we measured and functional genetic differences; or 3) variation in the environment under which the association was sought. These explanations have been suggested for other genes with variable patterns of association with phenotype (e.g., Korsten et al.32). We consider how each of these three explanations for variation in relationship applies to migratory behavior and CLOCK or ADCYAP1, and we suggest that population differences in genetic background are the most likely explanation. If this is indeed the case, then future studies of candidate genes will benefit from expanding the number of target genes examined to include related genes that may contribute to differences in genetic background.\n\nCLOCK alleles differ by multiples of three base pairs (Table 4), consistent with previous results in other avian species (e.g., Johnsen et al.18, Steinmeyer et al.43) and with the location of the repeat within the coding region of CLOCK43. It has previously been suggested that presence in the coding region might make these polymorphisms functional18, particularly because the repeat is responsible for a polyglutamine tract expansion in a region known to control the rate at which the CLOCK protein activates the downstream circadian pathway60. Polyglutamine expansions control the activity of many genes (e.g., Chamberlain et al.61). Thus, variation in allele length could play a direct role in modifying migratory behavior by altering the threshold day length signal to activate or suspend migratory behavior, or by modifying its downstream effects.\n\nThe ADCYAP1 alleles varied primarily by 2 base pairs (Table 3), consistent with previous results and the fact that this microsatellite polymorphism falls within the 3´ untranslated region (UTR) of the ADCYAP1 gene24,43. The mechanism by which this variation may relate to behavioral differences remains unknown. Di-nucleotide repeats in the regulatory regions of genes, such as 3´ UTR, can modify the expression of a gene62. This polymorphism may simply be linked with another causative mutation in the coding region24. The amino acid code of ADCYAP1, however, is 97% conserved from humans to chickens28, making 3´ UTR based variation in expression far more likely than a polymorphic variation in protein sequence. Studies of in vivo expression of various alleles in multiple tissues would be necessary to confirm a role for this mutation in modifying the expression of ADCYAP1 in living animals.\n\nMigratory behavior is a complex phenotype consisting of changes to movement, orientation, feeding, fattening, and sleep patterns53, and therefore may be modulated by many genes. The fact that CLOCK and ADCYAP1 are related to migration in several systems suggests that they may be some of the key regulators. However, the large component of migratory behavior that is not explained by these genes may be related to other genes in related pathways. Changes in these other, related genes may thus reduce or modify the effect of CLOCK and ADCYAP1 on migratory behavior, and explain the pattern of relationships identified in both previous studies18,24,25,59 (described above) and this work. Similar patterns have been found for other complex traits such as response to stress, including in experimental evolution of yeast63. Despite similar repeated phenotypes, independently evolved lineages did not duplicate the same transcriptional (and therefore genetic) responses to a novel stressor63. This suggests that divergent Junco populations may have utilized unique genetic mechanisms to achieve the same phenotypic end, thus masking the role of any single candidate gene across the genus.\n\nDifferences in genetic background may explain the lack of genus-wide patterns of relationship between migratory phenotype and CLOCK or ADCYAP1 and the pattern of relationship found in previous studies. If other, as yet unidentified, genes also contribute to divergence in migratory and breeding behavior, then the relationships identified between phenotype and genotype would be limited to the groups or species in which CLOCK or ADCYAP1 are responsible for the divergence. In contrast, those species and populations that do not demonstrate a relationship between migratory phenotype and CLOCK or ADCYAP1 may have diverged due to changes in other genes. These genes might be involved in the circadian pathway, such as BMAL1 or Period 264; the sensing of photoperiod changes, such as thyroid hormone deiodinases65 or vertebrate ancient opsin66; or transcriptional regulation of these systems67.\n\nDifferences in genetic background may also explain the details of the relationships found between migratory phenotype and CLOCK or ADCYAP1. The Oregon and slate-colored subspecies groups both demonstrate a positive relationship between CLOCK allele length and migratory behavior. Importantly, the population mean CLOCK allele lengths are not different between the subspecies groups, and all of the slate-colored juncos migrate longer distances than the Oregon juncos. Similarly, allele length of ADCYAP1 predicts migratory restlessness in the altitudinal migrant Laguna Mountain population, but not in the sedentary San Diego population despite the fact that both populations exhibit variation at the locus and in migratory restlessness. While sedentary populations are known to exhibit seasonal migratory restlessness68, it is possible that changes in other genes inhibit migration in this sedentary population sufficiently to overwhelm the variation in restlessness contributed by ADCYAP1. Together, these results suggest that variation in other genes across the genus account for some of the shifts in migratory phenotype, but that CLOCK and ADCYAP1 may still play important roles in further modification of migratory behavior.\n\nAn alternative explanation for the finding that allele length of both CLOCK and ADCYAP1 were related to migratory behavior at only some levels (within subspecies or population) of analysis is that the alleles we assessed are not functional, but are instead genetically linked to functional differences. If so, this linkage may be distorted in some populations due to recombination or mutation. For those populations, the alleles we measured would no longer be informative as indicators of migratory behavior. The presence of the relationship in multiple independently derived lineages could be explained by independent events that link the allele at the causal locus to the same microsatellite alleles. For example, in great tits (Parus major), only one of four populations studied, demonstrated a relationship between a single nucleotide polymorphism (SNP) in dopamine receptor 4 (DRD4) and exploratory behavior32, perhaps due to the breaking of a genetic linkage69.\n\nIn the case of CLOCK and ADCYAP1, however, several pieces of evidence point against disrupted linkage as the source of variation in the genotype-phenotype relationship. Similarly to previous studies that found a significant relationship between CLOCK or ADCYAP1 and migratory behavior (described above), the alleles associated with increased migratory behavior in this study were always longer. If linkage were responsible for the association, there is no reason to predict that longer alleles would repeatedly be linked to increased migratory propensity. This does not, by itself, rule out linkage–it is possible that this directional pattern is coincidental and will be reversed in another species yet to be studied. In addition, unlike the length polymorphisms studied in CLOCK, the SNP in DRD4 related to exploratory behavior is a synonymous mutation69–that is, it causes no change in the protein that is encoded by the gene. Similarly, the variation in ADCYAP1 is predicted to affect expression43, while the SNP in DRD4 is unlikely to do so (but see Duan et al.70). Given the existence of a known mechanism that could relate allelic variation in CLOCK and ADCYAP1 to functional consequences (detailed above), disrupted linkage seems less likely to account for variable strength of association than other possible causes.\n\nA final potential explanation for the lack of a consistent relationship between allelic variation and migratory phenotype is that the roles of CLOCK and ADCYAP1 are modulated by the different environments that populations and subspecies experience. In three-spine sticklebacks (Gasterosteus aculeatus), for example, environmental variability in the presence of predators masks genetic variation related to several behavioral traits71. Similarly, many environmental cues, such as temperature and precipitation, modulate migratory behavior9; such environmental modulators may account for some of the differences between junco populations and may mask the effects of variation in CLOCK and ADCYAP1.\n\nEnvironmental differences do not, however, appear to be consistent with the pattern of relationships found between migratory phenotype and CLOCK or ADCYAP1. First, the positive relationship between individual variation in migratory restlessness and length of ADCYAP1 was found in only one of two tested populations (Laguna Mountain), despite the fact that they were tested in an identical common garden experiment48, and winter in the same environment41. Second, the within subspecies pattern seen between CLOCK allele length and migratory distance is seen in both Oregon and slate-colored juncos, and these two subspecies differ in the amount of environmental divergence between populations. The sampled Oregon populations breed in divergent climates and winter in similar environments, while the slate-colored populations all breed in a similar environments, but migrate different distances to divergent wintering grounds34,41. Together, the findings in CLOCK and ADCYAP1 suggest that environmental differences between populations, while potentially important, are not sufficient to explain the pattern of associations that we found.\n\n\nConclusions\n\nWe found no evidence for a predictable relationship between migratory behavior and the lengths of CLOCK or ADCYAP1 alleles across the genus Junco. However, we found some support for a role of CLOCK allele length within subspecies groups, and ADCYAP1 allele length within a population, in modifying migratory behavior. The lack of consistent detectable associations between allelic variation and behavioral variation among populations and individuals as reported in our study, adds an important layer in further understanding the potential roles of candidate genes in complex ecological and evolutionary processes. To explore this layer further, future sequencing projects may wish to similarly consider both between-group and within-group variability in genotype-phenotype relationships when conducting analyses of other candidate genes.\n\nFocusing on variation in a small number of genes with predicted functional significance holds the potential to identify major functional hubs in complex phenotypes across the animal kingdom. The redundancy of the genome and complexity of functional genetic, regulatory, and signaling networks, however, means that over evolutionary time scales, candidate loci may gain or lose functional significance in the presence of an alternative environment, linkage, or genetic background. Our results highlight the fact that different mechanisms may contribute to variation in complex phenotypes when examining populations or species. This does not mean that candidate gene approaches should be abandoned altogether. Rather, with the increasing availability of next-generation sequencing, it may become more efficient to expand the scope of candidate gene sequencing projects to include other genes in the same pathway, as this breadth may provide insight into other, as yet unidentified, genetic mechanisms.", "appendix": "Author contributions\n\n\n\nMPP analyzed and collected data and drafted the manuscript; MAA analyzed and collected genotype data; JWA analyzed and collected migratory data; RJR collected migratory data; BM collected DNA samples and migratory data; EDK conceived of the study and guided the researchers. All authors contributed to final analyses and editing of this manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the National Science Foundation (IOS-0820055 to EDK, DGE-0504627 to MPP, IOS-1209564 to EDK and MPP); the National Institutes of Health (T32-HD049336 to EDK and MPP), and Indiana University (Internal Research Funding to MPP).\n\n\nAcknowledgements\n\nThe authors thank Dustin Reichard, Christine Bergeon Burns, Dawn O'Neal, and Pau Aleixandre along with past members of the Ketterson and Milá research groups for their work in collecting the DNA samples used in this project. Sampling on Guadalupe Island was made possible by Alfonso Aguirre and Julio Hernández Montoya at the Grupo de Ecología y Conservación de Islas (GECI).\n\n\nSupplementary material\n\nAllele calls for both CLOCK and ADCYAP1 are provided for each individual. 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PubMed Abstract \| Publisher Full Text\n\nKorsten P, Mueller JC, Hermannstadter C, et al.: Association between DRD4 gene polymorphism and personality variation in great tits: a test across four wild populations. Mol Ecol. 2010; 19(4): 832–843. PubMed Abstract \| Publisher Full Text\n\nSullivan KA: Yellow-eyed junco. In Birds of North America. Edited by Poole A. Cornell Lab of Ornithology, 1999; 464: 1–23.\n\nNolan V Jr, Ketterson ED, Cristol DA, et al.: Dark-eyed Junco: Junco hyemalis. In The birds of north america. Edited by Poole A. Cornell Lab of Ornithology, 2002; 716: 1–44.\n\nMiller AH: Speciation in the avian species Junco. Univ Calif Publ Zool. 1941; 44(3): 173–434.\n\nMilá B, McCormack JE, Castaneda G, et al.: Recent postglacial range expansion drives the rapid diversification of a songbird lineage in the genus Junco. Proc Biol Sci. 2007; 274(1626): 2653–2660. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCormack JE, Maley JM, Hird SM, et al.: Next-generation sequencing reveals phylogeographic structure and a species tree for recent bird divergences. Mol Phylogenet Evol. 2012; 62(1): 397–406. PubMed Abstract \| Publisher Full Text\n\nRasner CA, Yeh P, Eggert LS, et al.: Genetic and morphological evolution following a founder event in the dark-eyed junco, Junco hyemalis thurberi. Mol Ecol. 2004; 13(3): 671–681. PubMed Abstract \| Publisher Full Text\n\nWhittaker DJ, Dapper AL, Peterson MP, et al.: Maintenance of MHC Class IIB diversity in a recently established songbird population. J Avian Biol. 2012; 43(2): 109–118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtwell JW, ONeal DM, Ketterson ED: Animal migration as a moving target for conservation: intra-species variation and responses to environmental change, as illustrated in a sometimes migratory songbird. 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Proceedings of the San Diego Society of Natural History. 2005; 39: i–vii, 1–645.\n\nKetterson ED, Nolan V: Geographic variation and its climatic correlates in sex-ratio of eastern-wintering dark-eyed juncos (Junco-hyemalis hyemalis). Ecology. 1976; 57(4): 679–693. Publisher Full Text\n\nKetterson ED, Nolan V: The evolution of differential bird migration. In Current Ornithology. Edited by Johnston RF. Plenum Press; 1983; 1: 357–402.\n\nAtwell JW, Cardoso GC, Whittaker DJ, et al.: Boldness behavior and stress physiology in a novel urban environment suggest rapid correlated evolutionary adaptation. Behav Ecol. 2012; 23(5): 960–969. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerthold P, Fiedler W, Querner U: Migratory restlessness or Zugunruhe in birds - a description based on video recordings under infrared illumination. J Ornithol. 2000; 141(3): 285–299.\n\nMukhin A, Grinkevich V, Helm B: Under Cover of Darkness: Nocturnal Life of Diurnal Birds. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChamberlain NL, Driver ED, Miesfeld RL: The length and location of CAG trinucleotide repeats in the androgen receptor n-terminal domain affect transactivation function. Nucleic Acids Res. 1994; 22(15): 3181–3186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiley DE, Krieger JN: UTR dinucleotide simple sequence repeat evolution exhibits recurring patterns including regulatory sequence motif replacements. Gene. 2009; 429(1–2): 80–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStern S, Dror T, Stolovicki E, et al.: Genome-wide transcriptional plasticity underlies cellular adaptation to novel challenge. Mol Syst Biol. 2007; 3: 106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFidler AE, Gwinner E: Comparative analysis of Avian BMALI and CLOCK protein sequences: a search for features associated with owl nocturnal behaviour. Comp Biochem Physiol B Biochem Mol Biol. 2003; 136(4): 861–874. PubMed Abstract \| Publisher Full Text\n\nNakao N: Genome-wide Analysis of Seasonal Reproduction in Birds. Journal of Poultry Science. 2009; 46(2): 81–86. Publisher Full Text\n\nHalford S, Pires SS, Turton M, et al.: VA Opsin-Based Photoreceptors in the Hypothalamus of Birds. Curr Biol. 2009; 19(16): 1396–1402. PubMed Abstract \| Publisher Full Text\n\nWray GA: The evolutionary significance of cis-regulatory mutations. Nat Rev Genet. 2007; 8(3): 206–216. PubMed Abstract \| Publisher Full Text\n\nHelm B, Gwinner E: Migratory restlessness in an equatorial nonmigratory bird. PLoS Biol. 2006; 4(4): e110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFidler AE, van Oers K, Drent PJ, et al.: Drd4 gene polymorphisms are associated with personality variation in a passerine bird. Proc Biol Sci. 2007; 274(1619): 1685–1691. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuan J, Wainwright MS, Comeron JM, et al.: Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. Hum Mol Genet. 2003; 12(3): 205–216. PubMed Abstract \| Publisher Full Text\n\nDingemanse NJ, Van der Plas F, Wright J, et al.: Individual experience and evolutionary history of predation affect expression of heritable variation in fish personality and morphology. Proc Biol Sci. 2009; 276(1660): 1285–1293. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "915", "date": "30 Apr 2013", "name": "Miriam Liedvogel", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPeterson and coworkers investigate phenotypic correlates of genetic variation in two species of the bird genus Junco at the level of individuals, populations and species across taxa, which is a valuable contribution to our understanding of the genetic basis of the complex behavioural phenotype of migration. The methodological approach the authors have chosen is a candidate gene approach at two loci (Clock and ADCYAP1), which have previously been analysed in the context of their putative involvement in shaping migratory phenotypes in different taxa. The study is a direct follow up from the study by Müller et al. 2010, and a very welcomed attempt to repeat previous findings in another system to obtain a better understanding of associations between genetic variation and behaviour, as well as to assess their generality. This is much needed since associations between polymorphisms in candidate genes and behavioural traits have often proven to be difficult to repeat across populations or species, and replication is indeed a valuable tool to infer the generality of association patterns.The difficulty and my primary concern with the study as well as the precursor study is that candidate genes (defined as genes that have been studied in one (model) organisms and there have been found to influence the expression of one focal phenotype) for migratory traits are probably not know to date, as none of the model organisms show a migratory phenotype. The genes in focus here are candidate genes for candidate traits, probably involved in shaping the migratory phenotype (i.e. the circadian phenotype involved in timing of migration). Thus, conclusions drawn upon these data based on correlative evidence should be evaluated with great caution. This study is useful in the way that it adds data to the pool of studies that have explored candidate genes of candidate traits for the migratory phenotype, which are difficult to interpret and don’t allow drawing any clear conclusions upon. The study is well presented and the additional data confirm the apparent inconsistency and the difficulty to interpret these data, probably highlighting the weakness of the candidate gene approach in this field of migratory research, and I support their publication. With candidate gene studies per se we do not expect to get huge effects, and in the area of migration genetics, I suggest that future research should avoid using a candidate gene approach, as this is unlikely to yield clear results - at least unless clear candidate genes for the migratory phenotype have been identified de novo.", "responses": [] }, { "id": "934", "date": "08 May 2013", "name": "Francisco Pulido", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "944", "date": "13 May 2013", "name": "Roi Dor", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPeterson and co-authors have investigated the association between genotypes of two candidate genes (Clock and ADCYAP1) and migratory behaviour in the avian genus Junco. They examined this association among subspecies, populations and individuals. Previous studies have found contradicting results regarding the association between phenotypic variation and genotype of these genes; therefore, more similar studies are required in order to determine the generality of this relationship and the role of these genes.The authors have sampled and analysed a large number of individuals and populations and analysed the data adequately. Unfortunately, the results are mostly negative and prevent from reaching clear-cut conclusions regarding Clock or ADCYAP1 and migratory propensity. However, studying the relationship between genotypes and phenotypes on several levels (subspecies, population, and individuals) enabled performing interesting comparisons and detecting some positive patterns.The authors have properly discussed the results and potential explanations and mechanisms for the differences found among the levels examined.I agree with the authors that correlating genomes with phenotypic variation may be the next step to identify potential candidate genes that may have a role in complicated behaviours such as migration.Despite the mostly negative results, I believe that this nicely-written paper provides a fine contribution to the literature.One technical comment – it would be interesting to learn the nature of the ADCYAP1 alleles that are just one base-pair apart.", "responses": [] } ]	1	https://f1000research.com/articles/2-115
https://f1000research.com/articles/2-114/v1	19 Apr 13	{ "type": "Research Article", "title": "Comparison of arterial and venous blood biomarker levels in chronic obstructive pulmonary disease", "authors": [ "Emer Kelly", "Caroline A Owen", "Amadeus Abraham", "David L Knowlton", "Bartolome R Celli", "Victor Pinto-Plata", "Caroline A Owen", "Amadeus Abraham", "David L Knowlton", "Bartolome R Celli", "Victor Pinto-Plata" ], "abstract": "Purpose: The development of novel biomarkers is an unmet need in chronic obstructive pulmonary disease (COPD). Arterial blood comes directly from the lung and venous blood drains capillary beds of the organ or tissue supplied. We hypothesized that there would be a difference in levels of the biomarkers metalloproteinase 9 (MMP-9), vascular endothelial growth factor A (VEGF-A) and interleukin 6 (IL-6) in arterial compared with venous blood. Methods: Radial artery and brachial vein blood samples were taken simultaneously in each of 12 patients with COPD and seven controls with normal lung function. Circulating immunoreactive MMP-9, VEGF-A and IL-6 levels in serum were measured using quantitative enzyme-linked immunosorbent assays. Results were compared using a Student’s paired t test. The study was powered to determine whether significant differences in cytokine levels were present between paired arterial and venous blood samples. Results: In the 12 patients with COPD, four were female, and age ranged 53-85 years, mean age 69 years. Three patients in the control group were female, with age range 46-84 years, mean age 64.7 years. In the COPD group, three patients had mild, five moderate and four severe COPD. No significant difference was found between arterial and venous levels of MMP-9, VEGF-A or IL-6. Conclusions: In this pilot study, levels of the measured biomarkers in arterial compared with venous blood in both COPD patients and healthy controls did not differ. This suggests that as we continue to chase the elusive biomarker in COPD as a potential tool to measure disease activity, we should focus on venous blood for this purpose.", "keywords": [ "Arterial", "biomarkers", "interleukin 6 (IL-6)", "metalloproteinase 9 (MMP-9)", "vascular endothelial growth factor A (VEGF-A)" ], "content": "Introduction\n\nChronic obstructive pulmonary disease (COPD) is a major public health issue, predicted to become the 3rd leading cause of mortality in the United States1,2. At present, the Global Initiative for Obstructive Lung Diseases (GOLD) divides patients into categories of mild, moderate, severe and very severe based on the forced expiratory volume in 1 second (FEV1). This classification has been shown to predict outcome and has been pivotal in guiding treatment of the disease3.\n\nThe progression of COPD has classically been determined by the change of the FEV1 over time, usually measured over years. Indeed, the duration of the large trials that have evaluated the effect of pharmacological or surgical benefits in patients with COPD have ranged from 2 to 4 years4–7. It follows that defining disease activity or response to therapy with validated biomarkers that reflect disease progression would make potential future interventions easier to evaluate. Several studies have reviewed the roles of different potential biomarkers in COPD8. Our group has recently shown that serum levels of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF alpha) were higher and matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) lower in patients with severe and very severe COPD compared with smoker and non-smoker controls without airflow obstruction9. Furthermore, the levels correlated with several phenotypic expressions of the disease including exercise capacity, quality of life, exacerbation and mortality9. Data from the ECLIPSE investigators, who studied a larger cohort of COPD patients, showed that several biomarkers correlated with baseline FEV1 but only one correlated with rate of decline in FEV1, the Clara-Cell Protein 16 (CC-16)10. While there is no question that a biomarker would be of great importance in COPD, the sample site having the greatest yield has not been clearly defined11.\n\nBlood from the systemic circulation returns to the lung for gas exchange and, theoretically, the presence of biomarkers could be modified during the transient time blood spends in the pulmonary vessels. Arterial blood should provide a more direct window to events occurring in the lungs than venous blood, which is perhaps more reflective of events happening in the capillary bed of the organ/tissues that it supplies. This is most evident in the significant differences that exist between arterial and venous blood gas measurements.\n\nWe conducted this pilot study to test the hypothesis that in patients with COPD (a primary disease of the lungs) there is a difference in serum concentrations of the biomarkers MMP-9, VEGF-A and IL-6 between simultaneously collected arterial and venous blood. Further, we compared the results with similar samples from patients without airflow obstruction that served as controls.\n\n\nMaterials and methods\n\nThis was a prospective pilot study. Samples were collected from patients attending the pulmonary clinic at St. Elizabeth’s Medical Center in Boston, Massachusetts. The study was approved by the Human Institutional Review Board of the institution and all patients signed the informed consent. The patients in the COPD group had smoking history ≥ 20 pack-years (1 pack year is equivalent to 1 year at 20 cigarettes per day) and had post-bronchodilator FEV1/FVC (forced vital capacity) ratio, < 0.7 after 400 μg of inhaled salbutamol. Patients had stable COPD and were not included if they had a history of an exacerbation in the last 3 months. The controls were patients attending the pulmonary clinic at the same institution with no history of COPD and normal lung function.\n\nAge, gender, smoking history and body mass index (BMI) were recorded for every participant. All subjects performed a spirometry according to the American Thoracic Society (ATS) recommendations and standard references and severity of COPD was categorized using GOLD staging. The BODE score is a composite score of BMI, degree of obstruction as recorded by FEV1, dyspnoea as quantified by the modified Medical Research Council dyspnoea scale and exercise tolerance, as measured with a 6 minute walk test12.\n\nSimultaneous radial artery and antecubital venous samples were obtained in each participant. The blood samples were centrifuged immediately at 2500 rpm for 10 minutes and serum stored at -80ºC. Circulating immunoreactive MMP-9, VEGF-A and IL-6 levels in the serum were measured using commercially available quantitative enzyme-linked immunosorbent assays (ELISA) (Human IL-6 ELISA Ready-Set-Go! eBioscience, San Diego, CA; Human total MMP-9 DuoSet, R&D Systems, Inc. Minneapolis, MN; Human VEGF-A Platinum ELISA from eBioscience, San Diego, CA).\n\nWe worked out a power/sample size calculation based on the premise that this was a study of a continuous response variable from matched pairs of study subjects. Prior data indicated that the difference in the response of matched pairs is normally distributed with standard deviation of 0.3 in the case of MMP-9 and VEGF-A, and 0.4 for IL-613. If the true difference in the mean response of matched pairs is 0.3, we would need to study 10 pairs of subjects to reject the null hypothesis with probability (power) of 0.8. If the true difference in the mean response of matched pairs is 0.4, we would need to study 12 pairs of subjects to be able to reject the null hypothesis with a probability (power) of 0.8. The Type I error probability associated with this test is 0.05.\n\nGroups were compared using the Student’s t test for normally distributed variables and Mann-Whitney U test for variables not normally distributed. p ≤ 0.05 was considered statistically significant. Spearman’s rank order correlation coefficient was also used for non-parametric data. Statistical analysis was performed using a commercial statistical package (Sigma Stat, Sigma Plot).\n\n\nResults\n\nThe characteristics of all participants are shown in Table 1. The twelve patients in the COPD group (4 female) ranged in age between 53 and 85 years. Three had mild, five had moderate and four had severe COPD by the GOLD classification14. The seven patients (three females) included in the control group had normal lung function and their mean age range was similar to the COPD group (46–84 years). The majority of subjects in both groups were ex-smokers, with only 3 patients in the control group being never-smokers.\n\nMMP-9 (Figure 1): Serum MMP-9 levels were not normally distributed in all of the subjects (n =19). The median venous MMP-9 level was 40,706 pg/ml, (interquartile range 23,659–79,595 pg/ml). The median arterial level was 37,653 pg/ml (interquartile range 21,833–64,351 pg/ml). There was no significant difference between the venous and arterial levels, p = 0.812. There was no difference between COPD and control levels of venous or arterial MMP-9.\n\nNo difference was found (p = 0.812). Pairing of the two groups was shown to be effective with rs (Spearman, Approximation) 0.8782 and p<0.0001.\n\nVEGF-A (Figure 2): In the 19 subjects, the VEGF-A levels in venous blood had a median value of 67.24 pg/ml, with interquartile range 45.24 to 268.90 pg/ml whereas in arterial blood the median value was 89.88 pg/ml, with interquartile range 58.90 to 239.38 pg/ml. There was no statistically significant difference between arterial and venous samples (p = 0.249). No significant difference was observed between patients with COPD and controls.\n\nNo difference was found (p = 0.550). Pairing of the two groups was shown to be effective with rs (Spearman, Approximation) 0.7526 and p = 0.0001.\n\nIL-6 (Figure 3): IL-6 levels were undetectable in many of the subjects. The median venous level of IL-6 was 0, with an interquartile range of 0 to 4.52 pg/ml and the median arterial level was also 0 pg/ml with an interquartile range of 0 to 3.61 pg/ml. There was no statistically significant difference (p = 0.986) between venous and arterial levels of this cytokine. As levels of IL-6 were undetectable in many of the patients, it was not possible to determine if a significant difference existed between those with COPD and the control group.\n\nNo difference was found (p = 0.986). Pairing of the two groups was shown to be effective with rs (Spearman, Approximation) 0.9966 and p<0.0001.\n\n\nDiscussion\n\nThis pilot study showed no difference in serum levels of MMP-9, VEGF-A or IL-6 between arterial and venous blood samples in a group of patients with COPD and controls without airflow obstruction. Our results indicate no advantage of obtaining arterial over venous samples for the determination of these biomarker levels in patients with COPD.\n\nAt present, the most important determinant of COPD severity and progression is the FEV1 value and its progression over time, usually measured in years. This approach has its limitations and fails to account for the so-called activity of the disease15. As more treatments for COPD are emerging, the importance of biomarkers that reflect disease activity becomes paramount. A biomarker, by definition, is “any molecule or material (e.g. cells and tissues) that reflects the disease process”16. Because of its ease of accessibility, work has focused on peripheral blood biomarkers. Many have been investigated with initial work showing C reactive protein (CRP) levels to be associated with degree of airway obstruction, although subsequent work in patients with moderate to severe COPD found no relationship between CRP levels and reduced survival17–19. Other promising biomarker candidates previously studied include circulating levels of Clara cell secretory protein-16 (CC-16)20, surfactant protein (SP)-D21 and serum amyloid A (SAA)22. Serum levels of CC-16, a marker of Clara cell toxicity, are reduced in patients with COPD10, while SP-D is increased in smokers with and without COPD21, and SAA may be a potential biomarker of COPD exacerbation22.\n\nThere are no prior published results on cytokine measurement in arterial blood in COPD patients. Studies where arterial vessels are accessed for cardiopulmonary bypass or for extracorporeal liver support have measured arterial blood levels of various cytokines but only within narrow inclusion criteria, and arterial and venous level comparisons were not published23,24. In a study of the effect of moderate hypothermia on systemic and internal jugular plasma IL-6 levels after traumatic brain injury in humans, IL-6 was found to be significantly higher in internal jugular venous blood than in arterial plasma25. In the setting of recent neuro-trauma this is understandable. Having stressed the importance of suitable potential biomarkers in COPD, we hypothesized that determining the optimal source of the sample could be of potential value. Our most important finding was that for the biomarkers studied, levels did not differ between arterial and venous samples. This was not due to the nature of the biomarkers selected because the markers we investigated in this study were chosen based on prior work demonstrating the level of IL-6 was higher and MMP-9 and VEGF levels lower in patients with more advanced COPD compared with controls9,26,27. Although the host response to insult is, to a large extent, compartmentalized to the affected lung, cytokine spillover into the systemic circulation has been shown to occur10,28 and be measurable in the systemic circulation10,28. We did not find differences in serum biomarker levels between patients with COPD and those without airflow obstruction; however, the study was not powered to explore this hypothesis.\n\nOur pilot study has some limitations. First, the number of patients recruited could be considered small, but it was powered to address whether there was a difference between arterial and venous levels of MMP-9, VEGF-A and IL-6. The use of matched samples allowed for accurate interpretation of the results with these subject numbers. Second, the blood samples were drawn from the radial artery and from a peripheral antecubital vein. Possibly, to obtain more accurate sampling of blood immediately leaving the lung, pulmonary arterial sampling would have been optimal. However, sampling of central venous blood is invasive and would not offer any practical advantage. Third, it is possible that the 3 selected analytes are not \"exclusively\" produced in the lung (as it is the case for SPD and CC16) and represent the systemic compartment and not just the lung milieu. This possibility requires the simultaneous measurement of these biomarkers in future studies.\n\nIn summary, this pilot study shows that there was no difference in levels of MMP-9, VEGF-A or IL-6 when measured in blood samples from the radial artery compared with peripheral venous samples. This suggests that as we continue to chase the optimal biomarker in COPD as a potential tool to measure disease activity, focusing on venous blood for this purpose remains a valid option.", "appendix": "Author contributions\n\n\n\nEK and CAO were responsible for sample analysis. AA, DLK, BRC and VPP were involved in patient recruitment and data collection. All authors were involved in manuscript 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\nAcknowledgements\n\nWe wish to acknowledge gratefully the patients who participated in this study.\n\n\nReferences\n\nMurray CJ, Lopez AD: Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study. Lancet. 1997; 349(9064): 1498–1504. PubMed Abstract \| Publisher Full Text\n\nBuist AS, McBurnie MA, Vollmer WM, et al.: International variation in the prevalence of COPD (the BOLD Study): a population-based prevalence study. Lancet. 2007; 370(9589): 741–750. PubMed Abstract \| Publisher Full Text\n\nGlobal Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management and prevention of COPD: updated 2010. 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PubMed Abstract \| Publisher Full Text\n\nCelli BR, Cote CG, Marin JM, et al.: The body-mass index, airflow obstruction, dyspnea, and exercise capacity index in chronic obstructive pulmonary disease. N Engl J Med. 2004; 350(10): 1005–1012. PubMed Abstract \| Publisher Full Text\n\nLiu SF, Chin CH, Wang CC, et al.: Correlation between serum biomarkers and BODE index in patients with stable COPD. Respirology. 2009; 14(7): 999–1004. PubMed Abstract \| Publisher Full Text\n\nRabe KF, Hurd S, Anzueto A, et al.: Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med. 2007; 176(6): 532–555. PubMed Abstract \| Publisher Full Text\n\nVestbo J, Rennard S: Chronic obstructive pulmonary disease biomarker(s) for disease activity needed--urgently. Am J Respir Crit Care Med. 2010; 182(7): 863–864. PubMed Abstract \| Publisher Full Text\n\nCazzola M, MacNee W, Martinez FJ, et al.: Outcomes for COPD pharmacological trials: from lung function to biomarkers. Eur Respir J. 2008; 31(2): 416–469. PubMed Abstract \| Publisher Full Text\n\nPinto-Plata VM, Mullerova H, Toso JF, et al.: C-reactive protein in patients with COPD, control smokers and non-smokers. Thorax. 2006; 61(1): 23–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMannino DM, Ford ES, Redd SC: Obstructive and restrictive lung disease and markers of inflammation: data from the Third National Health and Nutrition Examination. Am J Med. 2003; 114(9): 758–762. PubMed Abstract \| Publisher Full Text\n\nde Torres JP, Pinto-Plata V, Casanova C, et al.: C-reactive protein levels and survival in patients with moderate to very severe COPD. Chest. 2008; 133(6): 1336–1343. PubMed Abstract \| Publisher Full Text\n\nLomas DA, Silverman EK, Edwards LD, et al.: Evaluation of serum CC-16 as a biomarker for COPD in the ECLIPSE cohort. Thorax. 2008; 63(12): 1058–1063. PubMed Abstract \| Publisher Full Text\n\nLomas DA, Silverman EK, Edwards LD, et al.: Serum surfactant protein D is steroid sensitive and associated with exacerbations of COPD. Eur Respir J. 2009; 34(1): 95–102. PubMed Abstract \| Publisher Full Text\n\nBozinovski S, Hutchinson A, Thompson M, et al.: Serum amyloid a is a biomarker of acute exacerbations of chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2008; 177(3): 269–278. PubMed Abstract \| Publisher Full Text\n\nStadlbauer V, Krisper P, Aigner R, et al.: Effect of extracorporeal liver support by MARS and Prometheus on serum cytokines in acute-on-chronic liver failure. Crit Care. 2006; 10(6): R169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIskesen I, Saribulbul O, Cerrahoglu M, et al.: Pentoxifylline affects cytokine reaction in cardiopulmonary bypass. Heart Surg Forum. 2006; 9(6): E883–887. PubMed Abstract \| Publisher Full Text\n\nAibiki M, Maekawa S, Ogura S, et al.: Effect of moderate hypothermia on systemic and internal jugular plasma IL-6 levels after traumatic brain injury in humans. J Neurotrauma. 1999; 16(3): 225–232. PubMed Abstract \| Publisher Full Text\n\nPinto-Plata V, Toso J, Lee K, et al.: Profiling serum biomarkers in patients with COPD: associations with clinical parameters. Thorax. 2007; 62(7): 595–601. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinto Plata V: Stability and validation of serum biomarkers in COPD, association to outcomes using a novel method. In submission. 2011.\n\nSin DD, Leung R, Gan WQ, et al.: Circulating surfactant protein D as a potential lung-specific biomarker of health outcomes in COPD: a pilot study. BMC Pulm Med. 2007; 7: 13. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "911", "date": "25 Apr 2013", "name": "Prof RA Stockley", "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 the article is generally well written and the methods and results are likely to be correct, there is one major flaw that really should have been addressed before embarking on this negative project; Trans lung changes in mediators means you absolutely have to obtain pulmonary arterial and peripheral arterial blood. Even then differences could reflect a “cleaning” of the blood which is a non respiratory function of the lung and is most notable in the clotting differences. Peripheral arterial and venous blood will if anything reflect changes in the local limb capillary circulation and clearly misses changes to more systemic venous blood that is fully mixed in the pulmonary artery. This concept was recognised over 30 years ago and although the Authors mention it in discussion as a potential limitation I think this is a definite limitation. The study also fails to include the likelihood of some shunt in COPD lungs. Finally it is not clear whether the blood was allowed to clot but as serum is mentioned the assumptionis that it was and this would have the effect of activating local cells during the clotting process so ideally plasma should be studied.", "responses": [ { "c_id": "545", "date": "04 Sep 2013", "name": "Emer Kelly", "role": "Author Response", "response": "Thank you for taking the time to review our work. We acknowledge the limitations of this work and agree that there would be advantages to taking pulmonary arterial samples to give more physiological data. We share your concerns that the peripheral arterial sample may better reflect the occurrences in the distal capillary bed and we have raised these issues in the discussion. We were endeavouring to investigate the site of biomarker testing that could be used in real-life clinical scenarios and therefore chose radial arterial sampling. Serum was the tested sample in this study. Future work could give answers for these remaining physiological questions." } ] }, { "id": "935", "date": "08 May 2013", "name": "Ravi Kalhan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 pilot study the authors evaluate whether there are differences in arterial versus venous blood in 3 potential biomarkers of COPD. Since the bulk of prior work on COPD biomarkers has been focused on venous blood and there are potential limitations to not using arterial blood. This is an appropriate study question.The methods are appropriate, statistical analyses are sound. There is the limitation of sampling pulmonary arterial and venous blood to understand the “direct” lung effects on blood markers, but this is likely neither feasible nor practical in biomarker discovery as its clinical use would be limited.", "responses": [ { "c_id": "544", "date": "04 Sep 2013", "name": "Emer Kelly", "role": "Author Response", "response": "Thank you for your comments." } ] }, { "id": "988", "date": "07 Jun 2013", "name": "Hajime Takizawa", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 pilot study, the authors compared 3 potential biomarkers between arterial and venous blood samples among normal and COPD patients. Although the methods as well as statistical analyses are sound, there exist some limitations, as discussed by the authors; such as, small number of samples, unclarified differences between pulmonary arterial and venous blood, and limited number of biomarkers. I believe this study becomes a milestone for the future larger-scaled study for these important topics.", "responses": [ { "c_id": "543", "date": "04 Sep 2013", "name": "Emer Kelly", "role": "Author Response", "response": "Thank you for your comments." } ] } ]	1	https://f1000research.com/articles/2-114
https://f1000research.com/articles/2-113/v1	19 Apr 13	{ "type": "Case Report", "title": "Post-orgasmic illness syndrome: a case report", "authors": [ "Abdalla M Attia", "Hossam A Yasien", "Magda H Al-Ziny", "Hossam A Yasien", "Magda H Al-Ziny" ], "abstract": "Post orgasmic illness syndrome (POIS) is a newly described syndrome. Manifestations of this syndrome may be physical, cognitive or both. Many theories have been proposed to explain the causes of this syndrome including allergy to seminal components, allergy to unknown proteins released during ejaculation or a psychosomatic etiology. We present a case of POIS with a manifestation of atopy that may be consistent with the allergy hypothesis.", "keywords": [ "Post orgasmic illness syndrome", "allergy", "ejaculation." ], "content": "Introduction\n\nPost-orgasmic illness syndrome (POIS) was first reported and named by Waldinger and Schweitzer in 20021. This recently described syndrome may be more prevalent than one might expect, but has not received much attention and, we think, many cases may be misdiagnosed.\n\nTo the best of our knowledge, aside from two cases reported by Waldinger and Schweitzer1, two by Ashby and Goldmeier2, and one each by Dean (personal communication), Mullhal (personal communication) and Ashworth (personal communication), and the self reported cases on the site of the Naked Scientist’s discussion forum (www.thenakedscientists.com/forum), no more scientific discussion on POIS exists in textbooks, medical journals, or scientific meetings or congresses.\n\nPOIS appears to be principally a male orgasmic disorder, as most of the reported cases are males. Its manifestations start within seconds after orgasm and may continue for 4–7 days. These manifestations differ in their severities but, in most cases they are severe enough to make the patient abstain from the sexual activities; especially ejaculation and orgasm. They are not uniform for each patient and can be grouped based on having a specific cluster of symptoms1,2.\n\nThe most commonly reported manifestations are cognitive disorders and flu-like symptoms. The former is described by the patient as including brain fog, with inability to focus, communicate or process information. Some patients may suffer from temporary aphasia, irritability, anxiety, inability to relax and social phobias. The flu-like manifestations are; fever, sore throat, headache, chill, over-sweating with severe muscular, and bone and joint pains to the extent of severe exhaustion and fatigue1. One patient reported transient memory loss after each orgasm2.\n\nNothing is currently known regarding the underlying etiopathology of POIS, but the presence of manifestations in symptom clusters and absence of uniformity may point to different etiologies of the disease. It has been theorized that an allergic reaction could be responsible1. Waldinger and Schweitzer stated that during ejaculation and orgasm, many chemicals are released in the blood and an allergic reaction may occur in response to one (or more) of them, causing POIS manifestations. Alternatively, a psychosomatic disorder theory has been proposed by Krishnamurti and Ashoor (personal communications), who stated that these manifestations occur in individuals who believe that loss of vital fluid from the body (i.e. semen) causes weakness. Hypothyroidism (Dean, personal communication), hyperglycemia, hypertension, cortisol depletion, decreased (Dehydroepiandrosterone) DHEA, decreased testosterone, elevated prolactin and disorders of the CNS including alterations in serotonin, catecholamine and endorphin activity, are other suggested theories for this syndrome1 (Dean, personal communication; Ashworth, personal communication).\n\nThere is currently no effective treatment for POIS. Strong analgesics, such as NSAIDs, tramadol HCl and selective serotonin re-uptake inhibitors, taken one hour pre-coital may help some patients but are of no benefit in others1,2.\n\n\nCase\n\nHere we present a 45 year old Egyptian engineer who had been in a stable marriage for 10 years and had 3 children. Shortly post-orgasm (within 4–5 seconds), he feels severe fatigue, tiredness and exhaustion with severe muscular, bone and joint pains so that opening his hands becomes very painful. The condition is accompanied by headache, a pale face, eye irritation, low concentration, anxiety and dizziness with severe itching. The patient reported that these manifestations started early with puberty and increased in severity with age and occur with all orgasms whatever the type of sexual activity; night emission, masturbation or vaginal ejaculation. These manifestations are so severe that during the first 2 days post-orgasm he can't go to work, though they gradually fade and disappear by the 5th day. The patient abstains from sexual activity, although he has a strong desire and rigid erections. He has no history of chronic diseases, operations or drug intake except for life-long atopic manifestations of bronchial asthma, allergic rhinitis and neurodermatitis and occasionally uses symptomatic treatment to treat these manifestations.\n\nOn examination the patient had fair general health, was well built and had complete secondary sex characters. His weight was 97 kg, height was 177 cm and blood pressure was 125/85 mm/Hg.\n\nThe results of routine laboratory tests (complete blood picture, renal function, blood sugar and prostatic smear) were all normal. The results of other laboratory tests are shown in Table 1.\n\n* These elevations are due to fatty liver with no viral cause (as determined by PCR & liver ultra-sonography).\n\nDHEA-S: Dehydroepiandrosterone sulfate.\n\nT3: Tri-iodothyronine.\n\nT4: Tetra-iodothyronine.\n\nTSH: Thyroid stimulating hormone.\n\nALT: Alanine transaminase.\n\nAST: Aspartate transaminase.\n\nThe patient received strong analgesics in the form of Ibubrofen (400 mg on demand) and tramadol (50 mg one hour pre-coitally) but there was no reported benefit. A selective serotonin re-uptake inhibitor (escitalopram 10 mg daily at bedtime for 3 months) was also tried with no benefit.\n\n\nDiscussion\n\nThe exact etiopathology of POIS is currently unknown. The presentation of symptoms appear in clusters and the differences from one patient to another suggests that there may be more than one cause for this syndrome. Hyperglycemia1, low cortisol, low testosterone, elevated prolactin (Ashworth, personal communication), hypothyroidism (Dean, personal communication) and low DHEA1 have all been proposed to explain the etiopathology of POIS. All of these parameters were assessed in this patient and proved to be normal. We believe that the psychosomatic theory, where the belief that loss of vital fluid (i.e. semen) causes weakness, is not applicable to this patient as he is highly educated, successful in his job, has an intact personality and a stable marital life. The elevated liver enzymes in this patient are not related to his problem as his POIS manifestations have been present since puberty.\n\nWhat should be considered in this patient is his life-long atopy, including neurodermatitis. He reported severe itching after each orgasm as one of his POIS manifestations. We believe this factor to be very interesting, as it may point to and support an allergic etiology1 in this patient. Previously reported cases did not inquire about or evaluate allergic reactions1,2. As such, we believe that it is very important to re-evaluate these cases and any forthcoming reported ones for any allergic and hypersensitive reactions.\n\n\nConclusion\n\nMuch more attention to POIS is necessary to avoid misdiagnosis, to determine its exact etiopathology and to identify an effective treatment. A possible association with different allergic reactions is worthy of further investigation and evaluation.", "appendix": "Consent\n\nWritten informed consent for publication of clinical details was obtained from the patient.\n\n\nAuthor contributions\n\nAMA: performed clinical description, clinical analysis, theory proposal and final revision. HAY: wrote and revised the manuscript. MHA: performed chemical analysis for the blood samples and final revision. All authors agreed to the publication of the manuscript.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nReferences\n\nWaldinger MD, Schweitzer DH: Postorgasmic Illness Syndrome: two cases. J Sex Marital Ther. 2002; 28(3): 251–5.\n\nAshby J, Goldmeier D: Postorgasm Illness Syndrome-A spectrum of Illness. J Sex Med. 2010; 7(5): 1976–1981." }	[ { "id": "909", "date": "24 Apr 2013", "name": "Selim Cellek", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an interesting case of post-orgasmic severe fatigue and allergy-like reactions. Although several similar cases have been reported in the literature or professional discussion forums, we are still far away from understanding the pathophysiology of this phenomenon or how to manage it clinically. In this reviewer’s opinion, one of the main reasons for not being able to understand the pathophysiology is the lack of robust clinical data. To my knowledge, no one has collected blood samples from patients before and after sexual activity to determine the changes in patients’ chemistry. One obvious and simple thing to measure is the neutrophil, basophil counts and Ig levels to assess whether this is indeed an allergic reaction and what type of allergic reaction it might be. Others may be other biomarkers of allergy or inflammation. The field is looking forward to such a study.", "responses": [ { "c_id": "442", "date": "27 Apr 2013", "name": "abdalla attia", "role": "Author Response", "response": "Dear Dr Cellek, Thanks for your comment. We think that this case, as you mentioned, needs further study in order to clarify the pathogenesis that we proposed. The allergic theory against semen components, published by Waldinger and his colleagues, may be consistent with ours, although we have some reservations. The hypothesized atopy in these patients needs to be investigated well before and after orgasm. In our case, we recommend studying IgE levels and you have recommended measuring basophil and neutrophil counts before and after. If evidence of atopy could be found, these patients may respond to systemic steroids, as we tried to show with our patient. Survey and screening for these patients is the way to discover more about the pathogenesis of this condition and we are ready to cooperate with others to study such cases. Thanks, Abdalla Attia The author of the case" } ] }, { "id": "924", "date": "03 May 2013", "name": "Michael Fraser", "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 find this case report interesting, but no more than that. The clinical entity which has acquired the acronym POIS is intriguing and for that reason alone worthy of appearing in print to increase awareness of its possible existence. I do feel that a cluster of symptoms does not make for greater interest. I would be fascinated to see someone consider neuroradiologic imaging (PET/MRI) in these subjects.", "responses": [ { "c_id": "449", "date": "03 May 2013", "name": "abdalla attia", "role": "Author Response", "response": "Dear Dr Fraser, Thanks for your comment. Here in our case, we propose an allergic theory that may correlate between POIS and Atopy. So, we think that PET/MRI may be just wasting money, especially if the patient has these signs and symptoms just after each orgasm, apart, he is completely normal or has signs and symptoms of atopy. Otherwise, these patients may need to be investigated in regard to the issue of atopy to prove or deny our theory. Thanks,Abdalla Attia The author of the case" } ] }, { "id": "946", "date": "14 May 2013", "name": "David Goldmeier", "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\nPOIS may start up to a couple of hours after orgasm.What is the evidence for an allergic cause in this man- allergy history is very common in the general population?If allergy is the cause, have you tried him on high dose levocetirazine prior to orgasm?Naked Scientists assert Niacin helpful- did he try it?Does he also have chronic fatigue syndrome?Why not try a strong non steroidal e.g. diclofenac?Does he have POIS if he has sex but doesn’t orgasm?", "responses": [ { "c_id": "457", "date": "15 May 2013", "name": "abdalla attia", "role": "Author Response", "response": "Dear David Goldmeier, Thanks for your participation. The following are the explanations for your reservations;1- Yes, POIS may start within a couple of hours after orgasm, however our case and the reported cases mostly start just after orgasm. 2- In our case, severe itching and eye irritation, which are some of the atopic manifestations, started just after orgasm each time it occured. 3- Although levocetrizine use is a good idea and we did not try it, it alone seems to be insufficient in relieving atopic symptoms and we estimate the results may be not satisfactory. We tried to use systemic steroids but the patient had refused. 4- Niacin did not reach our knowledge at that time to use it, but we think it is worthy enough to try. 5- No he has not suffered from chronic fatigue syndrome. 6- He tried ibuprofen 400 mg before and after sexual activity, tramadol hydrochloride as we mentioned but with no response. 7- POIS manifestations started in this patient only after orgasm and not sex without orgasm" } ] }, { "id": "964", "date": "23 May 2013", "name": "Marcel D Waldinger", "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\nWe would like to note that we encourage the publication of case reports on Post Orgasmic Illness Syndrome (POIS). POIS was previously recognized and reported by Waldinger et al.1,2,3 who proposed 5 preliminary criteria, which were extracted from a large study of 45 Dutch males with POIS1. Moreover, in 2011 Waldinger et al. postulated that POIS is caused by an immunological reaction against a man’s own semen. This concept was based on a placebo-controlled study amongst 33 men who underwent skin-prick tests with diluted auto-semen1. The skin prick reaction appeared to be positive in 88%1. In addition, hyposensibilisation with auto-semen showed to ameliorate POIS symptoms in two men who consented with hyposensibilisation2. Notably, Waldinger et al. also showed that of all 45 men with POIS 58% had an atopic constitution, suggesting a relationship between an allergic constitution and POIS1. Apart from being incomplete in their references (references 1 and 2 are not mentioned in the article of Attia et al.), the authors of the current case report quoted the first publication of Waldinger and Schweitzer erratically by mentioning that analgesics, such as NSAIDs, tramadol and SSRIs taken precoital “may help some patients” suggesting that these drugs may be effective in some way to treat POIS. However, in the original description of POIS in 2002, none of these drugs were suggested as being clinically effective to reduce POIS symptomatology. Existence of atopic or allergic conditions remains a cornerstone of POIS and was clearly previously reported in the aforementioned original studies. Therefore, it is ironical and also flawed when Attia et al. stated that “previous reported cases did not inquire about or evaluate allergic reactions”. We feel obliged to clarify these wrong statements since scientific prudence must prevail particularly in case of claims of new disease concepts.", "responses": [ { "c_id": "487", "date": "13 Jun 2013", "name": "abdalla attia", "role": "Author Response", "response": "Dear Prof Waldinger, Thank you for your review of this paper. Although we respect your opinion, we are disappointed and surprised at your comments on the article, particularly as you state that you would encourage further publications on POIS. We discovered our case of POIS at the end of 2009. At this point, there seemed to be only one similar publication (Waldinger & Schweitzer, 2002) on POIS but this did not refer to atopy. We found that our patient was atopic. In addition to the cognitive and body pains he feels post orgasm, his atopy flares up, producing eye irritation and severe body itching. After preparing our first report in December 2009, and before publication, we did try to contact you for your opinion as the sole other reporter of this syndrome but received no response from repeated attempts.In 2010, we shared this case report at the ISSM forum. To our knowledge, this was before any other published report of atopy in relation to POIS. Many of our colleagues who are ISSM members commented and discussed the case at this forum, one of them being Prof. David Goldmeier. This case report was also presented as a poster at the 20th World Congress for Sexual Health, held in June 12-16, 2011, in Glasgow, UK, and was published in the conference proceedings. On the basis of this history, we suggest that we may have been the first to suggest that atopy may be a precipitating factor for POIS and that this condition should be checked for in any POIS cases.We would also like to respond to your other comment regarding the use of NSAIDs, tramadol and SSRIs in this condition. The reference to Waldinger et al. 2002 in this section is related to the sentence ‘There is currently no effective treatment for POIS’ and we apologise if this is unclear. We agree that this reference should be corrected so that it is attached to the correct statement. Our patient did not get any benefits from trying these drugs in contrast to the results of Ashby & Goldmeier (2010). In regards to testing for allergic reactions, we would like to ask whether you think that the skin prick test is reliable as a diagnostic test for allergy. Is it valid to conclude that POIS patients are allergic to their own semen on the basis of this test and suggest that this is the cause of POIS? We would suggest that skin prick tests can lead to many false positive and negative results. As andrologists we know (and there is a body of evidence for this), that semen is regarded as foreign by the body and the immune system. Immune tolerance to semen is not present. Semen is separated from the immune system by a very competent blood–testis barrier that is formed by the highly efficient Sertoli–Sertoli cell junctional complex. We would suggest that this is not a ‘hypothetical membrane’. In certain known pathological conditions this barrier may be broken. If this occurs, auto-antibodies can form against semen. Thus, if a subject’s own semen is then injected intradermally, a reaction may take place as it is recognized as a foreign antigen. We would suggest that many people would get a positive reaction on the basis of such a prick test even though they do not suffer from POIS. If allergy to the patient’s own semen is a suspected cause of POIS, it will be necessary to measure serum and seminal plasma anti-sperm antibodies; IgA, IgG and IgM, to conduct immuno bead and MAR testing and to report on the patient’s seminogram changes. This might also suggest that POIS patients would be mostly infertile due to formation of anti sperm antibodies. Given these concerns regarding prick testing, we do not believe that the cause of POIS is allergy to one’s own semen and also have doubts about the use of hyposensitization as a possible treatment." } ] } ]	1	https://f1000research.com/articles/2-113
https://f1000research.com/articles/2-112/v1	18 Apr 13	{ "type": "Case Report", "title": "Giant aneurysm of the basilar artery in an 86 year old woman", "authors": [ "Yong peng Yu", "Hong qin Zhao", "Wei feng Ren", "Xiang lin Chi", "Hong qin Zhao", "Wei feng Ren", "Xiang lin Chi" ], "abstract": "In this article we present an 80 year old female patient with an unruptured giant aneurysm of the basilar artery presenting with posterior circulation ischemic symptoms. Angiography and CT revealed giant basilar aneurysmal dilatation with severe and wide intracranial arteriosclerosis. We described the uniqueness of this case. Giant basilar aneurysm is associated with various complications particularly brain stem infarction. It is emphasized that arteriosclerosis plays an important role in the formation of giant basilar aneurysms.", "keywords": [ "Aneurysm", "Ischemic", "Artery", "Arteriosclerosis" ], "content": "Introduction\n\nGiant basilar aneurysm is a rare condition with elevated mortality within a few days of onset if untreated. On the basis of clinical course, a giant aneurysm may be categorized as a chronic type which grows relatively slowly, and may lead to serious complications such as cerebral ischemia or subarachnoid hemorrhage1. We report a case of an 80-year-old woman with a surgically untreated giant basilar aneurysm. The patient presented ischemic events involving posterior circulation without aneurysmal rupture or bleeding.\n\n\nCase report\n\nAn 86-year-old woman who had a 10-year history of hypertension was admitted to the hospital with slurred speech, diplopia, vomiting and left limb paralysis for one hour. On physical examination she was afebrile, her heart rate was 72 beats per minute, her blood pressure 170/110 mmHg, and her oxygen saturation 96%. The neurological state at acceptance was: dysarthria, right conjugate gaze palsy, pseudobulbar palsy, positive bilateral Babinski sign and grade zero (Oxford Scale) in muscle strength of the left limbs. Computed tomography (CT) of the brain showed an area of high density in the front of the pons (Figure 1A) which was very similar to a pontine hemorrhage. Magnetic resonance imaging (MRI) scans of the brain revealed acute brainstem infarction (Figure 1B), CT angiography of the head showed a giant aneurysm of the basilar artery (27 mm × 10 mm) (Figure 1C and D). On the basis of imaging studies, the patient received a diagnosis of brainstem infarction and giant aneurysm of the basilar artery, most likely secondary to severe atherosclerotic arterial disease. The patient’s family declined to undergo further investigation, surgical management or endovascular intervention, citing the expense, the size of the aneurysm and the risks associated with surgical intervention as reasons. The patient instead received conservative treatment with enalapril to control blood pressure and aspirin for anti-blood platelet aggregation. Her condition improved and became stable.\n\n(A) CT scan of the brain showing an area of high density in the front of the pons (arrow) similar to a pontine hemorrhage. (B) Brain MRI revealed acute brainstem infarction (arrow). (C, D) CT angiography of the head showed a giant aneurysm of the basilar artery (27 mm by 10 mm) (arrow).\n\n\nDiscussion\n\nIntracranial aneurysms are vascular abnormalities that are most commonly seen in elderly patients with severe atherosclerosis. They can occur between 35–65 years, particularly in populations with a mean age over 502. 60% of ruptured aneurysms occur in women3. Basilar aneurysms can be graded according to their diameter into small, (<12 mm), large (12–25 mm), and giant (>25 mm)4,5. Small aneurysms are usually asymptomatic, while large ones may cause distal embolism or occlusion of the perforating arteries. 60% are found in anterior circulation and 40% in posterior circulation with a predilection for the vertebrobasilar arteries6. However, the cause of giant aneurysms remains elusive7. Several mechanisms have been proposed for the formation of a giant aneurysm, including the effect of prolonged hemodynamic stress, roles and relationships of anatomic location, hemodynamic and degenerative factors, physical exertions and emotional stress, congenital abnormality, mechanical injury resulting from poststenotic turbulence, inflammatory vasculopathy, severe reticular fiber deficiency in the muscle layer, and intimal disruption from arterial dissection7,8. Clinically, a giant aneurysm usually presents as a space-occupying lesion with a compressive effect on posterior fossa structures, or causes posterior circulation infarction due to the occlusion of penetrating vessels, or distal embolism from the thrombus in the aneurysm lumen1. Alternative mechanisms including compression, vasospasm and hemodynamic mechanisms are more sensitive to orthostatic hypotension, in cases of the poor cerebral collateral circulation9,10.\n\nThis patient was unique in several ways. First, the patient presented with a giant basilar aneurysm without a history of ischemic stroke, and had extensive and severe intracranial arterial atherosclerosis. Secondly, posterior circulation symptoms and signs such as vomiting, diplopia, dysarthria and right conjugate gaze palsy were the initial clinical manifestations with high density as showed in the crainal CT (Figure 1A). Cranial MRI displayed brainstem infarction and computer angiography revealed a giant basilar artery aneurysm, which was not the typical spindle shape, with wide caliber in the proximal centre and more slender at the extremities. Thirdly, a freshly formed thrombus was not found in the giant basilar aneurysm as is normally the case. Aside from brainstem infarction, there were no acute ischemic lesions in other brain parenchyma. Brainstem infarction may result from occlusion of the pontine perforators due to hypertensive arteriosclerosis, which may lead to perforating artery disease11. A possibility is that extrinsic compression by the expanding giant aneurysm led to occlusion of the pontine perforators, although artery-to artery embolism occurs relatively often in patients with giant basilar aneurysms12.\n\nIf a cranial CT scan shows high density in the brainstem, and there is suspicion of a brainstem hemorrhage, angiography should be performed quickly to check for the presence of a giant basilar artery aneurysm. Our case is rare and emblematic because of the patient’s advanced age and the progressive growth of the untreated chronic giant aneurysm presenting with ischemic events but without history of ischemic stroke, rupture or bleeding. Severe and wide intracranial arteriosclerosis may play a pivitol role in the formation of giant aneurysms of the basilar artery.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient’s next of kin.", "appendix": "Author contributions\n\n\n\nHong qin Zhao and Yong peng Yu: involved in the diagnosis, treatment and revision of the manuscript for content. Wei feng Ren and Xiang lin Chi: drafted the manuscript for content. All the authors agreed to final publication of this manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCappellari M, Tomelleri G, Piovan E, et al.: Chronic fusiform aneurysm evolving into giant aneurysm in the basilar artery. Neurol Sci. 2012; 33(1): 111–115. PubMed Abstract \| Publisher Full Text\n\nVlak MH, Algra A, Brandenburg R, et al.: Prevalence of unruptured intracranial aneurysms, with emphasis on sex, age, comorbidity, country, and time period: a systematic review and meta-analysis. Lancet Neurol. 2011; 10(7): 626–36. PubMed Abstract \| Publisher Full Text\n\nSapina L, Lojen G, Janjetovic Z, et al.: Giant aneurysm of basilar artery. Coll Antropol. 2011; 35(2): 607–609. PubMed Abstract\n\nYasui T, Komiyama M, Iwai Y, et al.: Evolution of incidentally-discovered fusiform aneurysms of the vertebrobasilar arterial system: neuroimaging features suggesting progressive aneurysm growth. Neurol Med Chir (Tokyo). 2001; 41(11): 523–7. PubMed Abstract \| Publisher Full Text\n\nWakhloo AK, Mandell J, Gounis MJ, et al.: Stent-assisted reconstructive endovascular repair of cranial fusiform atherosclerotic and dissecting aneurysms: long-term clinical and angiographic follow-up. Stroke. 2008; 39(12): 3288–3296. PubMed Abstract \| Publisher Full Text\n\nAminoff MJ, Daroff RB: Encyclopedia of the neurological sciences. 2003; 1: 161. Reference Source\n\nNakayama Y, Tanaka A, Kumate S, et al.: Giant fusiform aneurysm of the basilar artery: consideration of its pathogenesis. Surg Neurol. 1999; 51(2): 140–145. PubMed Abstract \| Publisher Full Text\n\nAminoff MJ, Daroff RB: Encyclopedia of the neurological sciences. 2003; 1: 161. Reference Source\n\nNakayama Y, Tanaka A, Kumate S, et al.: Giant fusiform aneurysm of the basilar artery: consideration of its pathogenesis. Surg Neurol. 1999; 51(2): 140–145. PubMed Abstract \| Publisher Full Text\n\nCappellari M, Tomelleri G, Piovan E, et al.: Chronic fusiform aneurysm evolving into giant aneurysm in the basilar artery. Neurol Sci. 2012; 33(1): 111–115. PubMed Abstract \| Publisher Full Text\n\nGallego Cullere J, Erro Aguirre ME: Basilar branch occlusion. Curr Treat Options Cardiovasc Med. 2011; 13(3): 247–260. PubMed Abstract \| Publisher Full Text\n\nde Oliveira Rde M, Cardeal JO, Lima JG: [Basilar ectasia and stroke: clinical aspects of 21 cases]. Arq Neuropsiquiatr. 1997; 55(3B): 558–562. PubMed Abstract \| Publisher Full Text" }	[ { "id": "901", "date": "19 Apr 2013", "name": "Jennifer N Slim", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Authors discuss hypertensive arteriosclerosis and hypothesize on extrinsic compressive forces from the expanding aneurysm as contributing to the acute ischemic changes noted in the brainstem. It would be interesting to learn more of the clinical course with improvement in blood pressure control and particularly if there were any significant reductions in aneurismal size on subsequent imaging.", "responses": [] }, { "id": "923", "date": "03 May 2013", "name": "Azize Esra Gürsoy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/2-112
https://f1000research.com/articles/2-110/v1	17 Apr 13	{ "type": "Data Article", "title": "Cocaine self-administration in rats lacking a functional trpc4 gene", "authors": [ "Kristin C Rasmus", "Casey E O'Neill", "Ryan K Bachtell", "Donald C Cooper", "Kristin C Rasmus", "Casey E O'Neill", "Ryan K Bachtell" ], "abstract": "The canonical transient receptor potential (TRPC) family of Ca2+ permeable, non-selective cation channels is abundantly expressed throughout the brain, and plays a pivotal role in modulating cellular excitability. Unlike other TRPC channels, TRPC4 subtype expression in the adult rodent brain is restricted to a network of structures that receive dopaminergic innervation, suggesting an association with motivation- and reward-related behaviors. We hypothesized that these channels may play a critical role in dopamine-dependent drug-seeking behaviors. Here, we gathered data testing trpc4 knockout (KO) rats and wild-type (WT) littermates in the acquisition of a natural sucrose reward (10 days), and cocaine self-administration (13 days) at 0.5 mg/kg/infusion. Rats lacking the trpc4 gene (trpc4-KO) learned to lever press for sucrose to a similar degree as their WT controls. However, when they were switched to cocaine, the trpc4-KO rats had substantially reduced cocaine-paired lever pressing compared to WT controls. No obvious group differences in inactive lever pressing were observed, for any time, during cocaine self-administration.", "keywords": [ "Canonical transient receptor potential (TRPC) channels are a group of non-selective cation channels that have recently gained more attention due to their involvement in neuronal excitability1. This family of channels consists of 7 members (TRPC1-7) that can be turned on in response to the activation of the Gq alpha subunit of G-protein-coupled receptors2. Stimulation of Gq alpha protein-coupled receptors activates phospholipase C Beta (PLCβ) producing elevations in inositol triphosphate (IP3) and intracellular Ca2+ 3. TRPC channels contain three calmodulin sites and an IP3 site on the C-terminus of each subunit4", "5. Thus", "intracellular signaling resulting from Gq alpha protein-coupled receptors can enhance the activity of TRPC channels2. These unique properties allow these channels to play a pivotal role in responding to intracellular Ca2+ signaling", "thereby affecting neuronal excitability. The TRPC4 channel is one of the two most abundant TRPC channel subtypes found in the adult mammalian brain6. We have shown that TRPC4 channels are highly expressed in corticolimbic regions including the lateral septum", "hippocampus", "prefrontal cortex (PFC)", "and the amygdala6. The expression pattern of the TRPC4 channels within brain reward areas along with its ability to regulate neuronal excitability raised the interesting possibility that TRPC4 channels are important for motivated behaviors and may play a role in drug reinforcement. In the present study", "we explored cocaine self-administration in trpc4 KO rats and their WT littermates to test the hypothesis that TRPC4 channels play a role in natural (sucrose) and drug (cocaine) reward-related behaviors." ], "content": "Introduction\n\nCanonical transient receptor potential (TRPC) channels are a group of non-selective cation channels that have recently gained more attention due to their involvement in neuronal excitability1. This family of channels consists of 7 members (TRPC1-7) that can be turned on in response to the activation of the Gq alpha subunit of G-protein-coupled receptors2. Stimulation of Gq alpha protein-coupled receptors activates phospholipase C Beta (PLCβ) producing elevations in inositol triphosphate (IP3) and intracellular Ca2+ 3. TRPC channels contain three calmodulin sites and an IP3 site on the C-terminus of each subunit4,5. Thus, intracellular signaling resulting from Gq alpha protein-coupled receptors can enhance the activity of TRPC channels2. These unique properties allow these channels to play a pivotal role in responding to intracellular Ca2+ signaling, thereby affecting neuronal excitability. The TRPC4 channel is one of the two most abundant TRPC channel subtypes found in the adult mammalian brain6. We have shown that TRPC4 channels are highly expressed in corticolimbic regions including the lateral septum, hippocampus, prefrontal cortex (PFC), and the amygdala6. The expression pattern of the TRPC4 channels within brain reward areas along with its ability to regulate neuronal excitability raised the interesting possibility that TRPC4 channels are important for motivated behaviors and may play a role in drug reinforcement. In the present study, we explored cocaine self-administration in trpc4 KO rats and their WT littermates to test the hypothesis that TRPC4 channels play a role in natural (sucrose) and drug (cocaine) reward-related behaviors.\n\n\nMaterials and methods\n\nAll experiments were conducted in accordance with guidelines of the ‘Institutional Animal Care and Use Committees’ at the University of Colorado at Boulder. Eight-week-old (250–300 grams) male trpc4-KO rats and their wild-type Fischer 344 littermates (Transposagen, Lexington, KY, USA) in these studies were housed separately. The trpc4-KO rats were generated using the Sleeping Beauty transposon system6. We have previously reported some phenotypic characteristics of trpc4-KO rats that indicate a reduction in social interaction and normal learning on simple and complex tasks7–9. In the sucrose self-administration studies, nine trpc4-KO, nine WT and 7 heterozygote rats were used, while 7 animals from each strain were used in all cocaine self-administration experiments.\n\nThree polymerase chain reaction (PCR) primers were designed and used as 20–24 oligonucleotide sequences (Eurofins MWG Operon, Ebersberg, Germany) to genotype rats. Reactions were carried out using Choice-Taq Blue DNA polymerase (Denville Scientific Inc., Metuchen, NJ, USA) in a Techne Touchgene thermal cycler (Techne, Minneapolis, MN, USA). Ethidium bromide-stained 1.5% agarose gels (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA) were photographed with a Kodak Gel Logic 200 UV transilluminator imager (Carestream Health Inc., Rochester, NY, USA) using a 100 bp DNA ladder in the first lane of the gel as reference (New England BioLabs, Ipswich, MA, USA).\n\nAll self-administration procedures were performed in operant conditioning chambers (Med-Associates, St. Albans, VT, USA) equipped with two response levers, a sucrose hopper, and an infusion pump system. Animals were food-deprived to 85% free feeding weight and trained to lever-press for sucrose pellets on a fixed ratio 1 (FR1) reinforcement schedule for 5 days/week. A discriminative cue light stimulus was illuminated above the lever paired with sucrose delivery. A correct lever response resulted in the delivery of a sucrose pellet (45 mg). With the termination of the cue light and a 20 second time out period (TO 20s), responding produced no consequence. Inactive lever responses produced no consequence throughout the session. The session was complete when the animal had administered 50 sucrose pellets and the latency (in minutes) to acquire 50 pellets was recorded as the dependent variable. Failure to reach criteria (50 pellets) within 120 minutes resulted in termination of the session. Animals completed 10 sucrose self-administration sessions, which was sufficient to establish stable baseline sucrose responding in all groups.\n\nFollowing sucrose self-administration, rats were given ad libitum food. After 24–48 hours of free feeding, catheters were implanted into the jugular vein under halothane anesthesia (1–2.5%). During recovery, catheters were flushed daily with 0.1 ml of 0.9% heparinized saline to maintain patency. Rats were allowed 4–7 days to recover in their home cage before experimental procedures began.\n\nFollowing at least 4–7 days recovery from surgery, animals were trained to self-administer intravenous cocaine (0.5 mg/kg/100 μl injection) on an FR1 schedule in daily 2-hour sessions for 5 days/week. Cocaine injections were delivered over 5 seconds and were concurrent with the illumination of a cue light above the active lever. Drug and cue delivery were followed by a 15 second time out period, where the house light remained off and responding produced no consequence. Inactive lever responses produced no consequence throughout testing. No differences were observed between groups on the inactive lever (see data set).\n\nCatheter patency was tested at the completion of the experiment to ensure that differences in self-administration were not due to catheter failure. Fatal Plus cocktail (390 mg/ml pentobarbital sodium, 0.01 mg/ml propylene glycol, 0.29 mg/ml ethyl alcohol, 0.2 mg/ml benzyl alcohol) was administered through the animal’s catheter at 0.2 ml/kg. Catheter patency was confirmed if the animal responded immediately (within 1 second) with muscle atonia and lethality following Fatal Plus administration. Animals with faulty catheters (n=4) were excluded from these studies (see data set).\n\nFatal Plus was obtained from Vortech Pharmaceuticals, LTD (Dearborn, MI, USA). Cocaine hydrochloride was obtained from Sigma-Aldrich (St. Louis, MO, USA). Cocaine was dissolved in sterile-filtered physiological saline (0.9%).\n\n\nResults\n\nThe Sleeping Beauty (SB) gene-trap transposon method was used to create the trpc4-KO animals7. The SB method uses cut-and-paste transposable elements to generate heritable loss-of-function mutations. Figure 1A shows the location of the trpc4 gene on the rat genome and where the transposon was inserted. By inserting the SB transposon into the first intron of the trpc4 gene, the full-length protein product can be deleted. Using primers for the trpc4-KO and WT alleles, we were able to confirm the deletion using PCR and gel electrophoresis (Figure 1B).\n\nA. Schema of the trpc4 gene in the rat genome and the Sleeping Beauty (SB) gene knock-out system. The trpc4 gene is located on chromosome 2 of the rat genome, between 143.35 Mb and 143.49 Mb. The SB transposon was inserted into the first intron of trpc4, therefore creating a complete knock-out of the coding sequence. B. Ethidium bromide-stained agarose gel visualizing the 905 bp marker for the wild-type allele and the 510 bp marker for the trpc4 KO allele. To genotype the animals, 1.5% agarose gel electrophoresis was used.\n\nAs shown in Figure 2A, both the trpc4-KO rats and their WT littermates acquired 50 sucrose pellets at similar levels during the first 3 days and last 3 days of sucrose training. By day 7, all groups reached stable baseline, responding averaged between 16 and 26 seconds to acquire 50 pellets. Thus, it appears that trpc4-KOs do not differ in their ability to learn to self-administer a natural reward, sucrose, on a FR-1 schedule (see data set). The submitted data set contains responding on the active and inactive levers and the number of sucrose pellets for all three treatment groups across ten days of sucrose training.\n\nA) The average latency in seconds for the trpc4 knock-out (KO), wild-type (WT) and heterozygote (HET) rats to acquire 50 sucrose pellets in the allotted time period (120 minutes) during the first 3 days and the last 3 days is shown. The animals learned to lever-press for sucrose pellets on a fixed-ratio 1 (FR1) reinforcement schedule for 5 days/week. All self-administration chambers were equipped with an active and inactive lever. The active lever was paired with a discriminative cue light stimulus. A correct lever response resulted in the sucrose reward, the termination of the cue light, and a 20 second time-out (TO 20s) period where responding produced no consequences. B) The average self-administered cocaine for WT, HET and KO rats during daily 2 hour sessions on a FR1 reinforcement schedule for the first 3 days and last 3 days of acquisition is shown.\n\nWe next sought to determine whether trpc4-KO rats would differ in cocaine reinforced behavior. Cocaine self-administration summary results are presented in the trpc4-KO, WT and trpc4 heterozygote (HET) rats on an FR1 schedule of reinforcement (Figure 2). Figure 2B illustrates a reduction in responding to cocaine in the trpc4-KO group over the first 3 days and last 3 days (Figure 2B). The submitted data set contains responding on the active and inactive levers and the number of cocaine infusions for all three treatment groups across thirteen days of cocaine self-administration training.\n\n\nSummary\n\nData is presented showing acquisition of sucrose and cocaine self-administration infusions in trpc4 WT, HET and KO rats. The summary data show that for the first and last 3 days, rats lacking a functional trpc4 gene have normal sucrose mediated reward, and reduced acquisition of cocaine self-administration compared to WT rats.\n\n\n\nRaw data: Sucrose and cocaine self-administration data set\n\nThis data set includes raw data for sucrose intake and cocaine self-administration experiments for all three rat strains used in our study – trpc4-KO, wild-type and heterozygous animals. The sucrose intake data is over a 10-day period, while the cocaine self-administration data is 13 days. Both data sets include averages for the first 3 days and last three days of experiments, also shown in Figure 2. Catheter patency tests and lever press data is also included. Catheter patency was tested and recorded daily. Lever press data shows the total lever presses (inactive and active) for each animal, which will differ from number of drug infusions due to the time-out period between infusions.", "appendix": "Author contributions\n\n\n\nKR, RKB and DCC designed the experiments, assembled and summarized the data and wrote the paper. CEO carried out the experiments.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by National Institute on Drug Abuse (NIDA) grant R01 DA24040 (DCC).\n\n\nReferences\n\nPutney JW: Physiological mechanisms of TRPC activation. Pflugers Arch. 2005; 451(1): 29–34. PubMed Abstract \| Publisher Full Text\n\nSchaefer M, Plant TD, Obukhov AG, et al.: Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5. J Biol Chem. 2000; 275(23): 17517–17526. PubMed Abstract \| Publisher Full Text\n\nZhu X, Jiang M, Peyton M, et al.: trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry. Cell. 1996; 85(5): 661–671. PubMed Abstract \| Publisher Full Text\n\nClapham DE, Runnels LW, Strubing C: The TRP ion channel family. Nat Rev Neurosci. 2001; 2(6): 387–396. PubMed Abstract \| Publisher Full Text\n\nOrdaz B, Tang J, Xiao R, et al.: Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation. J Biol Chem. 2005; 280(35): 30788–30796. PubMed Abstract \| Publisher 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\nGeurts AM, Wilber A, Carlson CM, et al.: Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon. BMC Biotechnol. 2006; 6: 30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlipec WD, Nguyen P, Deeney B, et al.: Deletion of the trpc4 gene and its role in simple and complex strategic learning. Nat Prec. 2012. Publisher Full Text\n\nRasmus KC, Wang JG, Varnell AL, et al.: Sociability is decreased following deletion of the trpc4 gene. Nat Prec. 2011. Publisher Full Text" }	[ { "id": "904", "date": "10 May 2013", "name": "Xiu-Ti Hu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 purpose of this study was to determine whether cocaine self-administration of rats was altered by trpc4 knockout (KO). The title is appropriate and the Abstract is a suitable summary for this article. The article is well-constructed and clear. Outcomes from this study were important, suggesting a potential role of TRPC4 channels in neurons that mediate cocaine self-administration in the related structures of the rat brain. The study was well-designed and sensible data are obtained using a cutting-edge technology. Providing the methods and statistical results will further strengthen this article.", "responses": [] }, { "id": "948", "date": "14 May 2013", "name": "Eleanor Simpson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addresses if loss of TRPC4 affects self administration of sucrose and cocaine. The title is appropriate and the abstract is a suitable summary of the article. The article is clearly written, the experiments are properly conducted and the methods are sufficiently described. The data file provided is clearly laid out, and most of the relevant data is included. It would be useful for the authors to also include lever press data for the sucrose self administration task, in case there is a strain difference in responding on the active lever during the 20s timeout periods between trials. Statistical analysis of both lever press data as well as # cocaine infusions across sessions will determine if the authors claim that acquisition of cocaine self administration is reduced in TRPC4 KO rats is statistically significant.", "responses": [] } ]	1	https://f1000research.com/articles/2-110
https://f1000research.com/articles/2-90/v1	14 Mar 13	{ "type": "Short Research Article", "title": "Paging Doctor Google! Heuristics vs. technology", "authors": [ "Kenar D Jhaveri", "Peter B Schrier", "Joseph Mattana", "Peter B Schrier", "Joseph Mattana" ], "abstract": "The most dramatic development in medical decision-making technology has been the advent of the Internet. This has had an impact not only on clinicians, but has also become an important resource for patients who often approach their doctors with medical information they have obtained from the Internet. Increasingly, medical students, residents and attending physicians have been using the Internet as a tool for diagnosing and treating disease. Internet-based resources that are available take various forms, including informational websites, online journals and textbooks, and social media. Search engines such as Google have been increasingly used to help in making diagnoses of disease entities. Do these search methods fare better than experienced heuristic methods? In a small study, we examined the comparative role of heuristics versus the 'Google' mode of thinking. Internal medicine residents were asked to “google” key words to come up with a diagnosis. Their results were compared to experienced nephrology faculty and fellows in training using heuristics and no additional help of internet. Overall, with the aid of Google, the novices (internal medicine residents) correctly diagnosed renal diseases less often than the experts (the attendings) but with the same frequency as the intermediates (nephrology fellows). However, in a subgroup analysis of both common diseases and rare diseases, the novices correctly diagnosed renal diseases less often than the experts but more often than the intermediates in each analysis. The novices correctly diagnosed renal diseases with the same frequency as nephrology fellows in training.", "keywords": [ "google", "Dr.Google", "diagnostic skills", "residency", "nephrology" ], "content": "Introduction\n\nIn medical problem solving and decision-making, experts often use heuristics, or methods of problem solving for which no formula exists, but are instead based on informal methods or experience1. Heuristics help generate accurate decisions in an economical manner for both time and cost. In a sense, expert strategies are immensely adaptive1. While invaluable in helping the experienced clinician arrive at a diagnosis faster, the use of heuristics is associated with biases inherent in efficient decision making and, therefore, can lead to specific patterns of error2. The use of technology employs an algorithmic, rather than a heuristic, approach to medical problem solving and at speeds much greater than human capacity. Various technologies have been experimented with in medicine for years. Past efforts have included computer programs specifically designed to help clinicians make medical decisions and diagnose conditions more efficiently and accurately1,3. Electronic medical records and information technology have improved access to and ease of use of patient data. Technology does not merely facilitate or augment decision-making, but it reorganizes decision-making practices1.\n\n\nEnter “Dr. Google”\n\nThe most dramatic development in medical decision technology has been the advent of the Internet. Use of social media tools such as Facebook and Twitter allow for sharing of information and getting information at a much faster rate than previously thought. Search engines have slowly emerged as useful tools to get data regarding medical knowledge. Clinicians can utilize search engines to help them with decision-making. Search engines, the most popular of which is Google3, allow for the algorithmic surveying of all available information in an attempt to provide the most meaningful and useful information to the end user. It is plausible that the use of search engines could substantially aid the clinician, especially when dealing with diagnostic or therapeutic challenges involving great complexity and multiple variables, but the effectiveness of search engines as an aid to the clinician is incompletely defined, as suggested by a recent study by Krause et al.4.\n\nAs technology infiltrates everyday medicine, the debate about the appropriate role for information technology within medicine has intensified5,6. Early on, concern was raised regarding the utility of search engines to direct patients and clinicians to relevant sources7. More recently, there is mounting anecdotal evidence of miraculous or fantastic accounts of patients and physicians-in-training “googling” the answer to a medical question that had experts stumped8. There have been several small studies looking at the ability of doctors at various levels of training and experience to correctly diagnose a disease using Google based on case presentations from the New England Journal of Medicine (NEJM). Falagas et al. did a head-to-head comparison of three learners (two medical students and one “trainee doctor”) in which the learners first provided their diagnoses to NEJM cases without help, and then repeated the exercise with the help of Google and Pubmed9. While the findings did not reach statistical significance, the study suggested that use of Google and Pubmed may be helpful in generating a differential diagnosis9. Tang and Ng took 26 cases, also from the case records series in the New England Journal of Medicine, and selected 3–5 search terms for each case and entered them into Google10. Using this approach, the Google search provided the correct diagnosis in 58% of the cases10. The conclusions of the studies were essentially the same: Google (and probably other search engines and algorithmic technologies) appears to be a viable clinical tool to aid in physician diagnosis and learning.\n\n\nComparison\n\nDoes “googling” a diagnosis replace an experienced physician’s clinical acumen? “Googling” a clinical question may be especially useful in the case of rare or syndromic diseases, but may be less likely to be useful in diagnosing more common diseases. To assess this possibility, we reviewed and analyzed the use of Google as a diagnostic tool in renal diseases and compared it to the experience of fellows and attending staff. A total of 21 members participated in the study (7 novices, 7 intermediate levels- fellows and 7 experts -attendings). We created 103 pairings of common and uncommon renal diseases with keywords related to the features of the disease using a standard renal textbook as a guide (Appendix 1). The diseases were then categorized as common or rare based upon the consensus of the investigators. This association was not indicated on the worksheets given to the participants. The order of the questions was then randomized and worksheets were made with approximately fifteen keyword pairings per page. Experts (nephrology attendings) and intermediates (nephrology fellows) were given the entire list of keywords (one page at a time) and asked to identify the associated diseases without any aid. Novices (first- and second-year internal medicine residents) were given approximately three pages at random and asked to use Google to identify the renal disease associated with the keywords. The novices were given standardized instructions requiring that they only use the first ten results (first page of results) returned from a Google search. They were then only permitted to use the first page of each of the ten results that appear on the first Google search page. A detailed instruction sheet is attached for reference (Appendix 2). The residents were instructed to use any or all of the keywords, as they saw fit, and they were allowed to try different iterations of the keywords if their original search did not yield a diagnosis they were satisfied with. The residents were supervised/proctored by one of the investigators; questions were limited to explanations of the rules. The percent of diagnoses correctly identified from the keywords was identified for each test-taking group, and the groups were compared with each other two at a time. The diseases were then categorized as common or rare based upon consensus of the investigators. Worksheets were created with keywords groupings for each disease listed and space provided for a study participant to record the suspected diagnosis. The association of common versus rare was not indicated on the worksheets given to the participants. The participants were asked to complete as many pages as they were willing to complete. All participating experts answered a total of 229 questions. All participating intermediates answered a total of 254 questions. All participating novices answered a total of 230 questions.\n\nThe percent of diagnoses correctly identified from the keywords was identified for each test-taking group and the groups were compared with each other two at a time. A t-test was calculated for each pairing; p-values were calculated using Microsoft Excel. A subgroup analysis was also conducted for common diseases and for rare diseases. Table 1 and Table 2 show examples of the common and rare diseases chosen, and the keywords and their associated diseases, respectively.\n\n\nIs “Dr. Google” better than experience?\n\nOverall, with the aid of Google, the novices (internal medicine residents) correctly diagnosed renal diseases less often than the experts (nephrology attendings) (72.2% vs. 84.7%, p<0.001), but with the same frequency as the intermediates (nephrology fellows) (72.2% vs. 71.5%, p=0.795). In a subgroup analysis of common diseases, the novices correctly diagnosed renal diseases less often than the experts (76.6% vs. 90.5%, p<0.001) and intermediates (76.6% vs. 82.3%, p=0.031). However, in a subgroup analysis of rare diseases, the novices correctly diagnosed renal diseases less often than the experts (65.2% vs. 76.1%, p=0.014), but more often than the intermediates (65.2% vs. 56.2%, p=0.029). This study is unique, in that it directly compares heuristic and algorithmic problem solving, using the dominant technology of our time: the Internet via Google. It also addresses which types of problems are best solved using the heuristics of an experienced clinician and which problems benefit most from algorithmic problem solving with the aid of a search engine. Limitations of the short research include single center study, investigator bias and limited number of participants. While this question will require further study, our findings suggest that for uncommon clinical entities, the use of search engine technology may be able to increase the diagnostic performance of a novice to an intermediate level.\n\n\nWould you use Google to help diagnose your patient?\n\nCan the computer really out think the doctor in making a diagnosis? A recent editorial in The New York Times11 begs this question as well and suggests that in rare diseases, and in many instances, a computer software program would have saved many lives. This might be true for rarely encountered conditions but perhaps not for common diseases. Rare diseases are often not diagnosed at the first encounter with a physician, and hence the term “rare”. A computer-based query, as used in Google, might help diagnose a rare illness faster, but cannot substitute for the heuristic thinking process of a physician and the matching of patterns facilitated by a physician’s experience. Hence, while search engines and diagnostic programs will likely continue to evolve as diagnostic tools, they can aid, but cannot replace the thought processes of the experienced clinician.", "appendix": "Author contributions\n\n\n\nKDJ and PBS designed the idea. The concept and experiment were IRB exempt at NSLIJ Health System. KDJ and JM wrote the manuscript.\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\nPart of this work was presented at the national American Society of Nephrology Annual Renal Week Nov 2011, Philadelphia, PA, USA.\n\n\nReferences\n\nPatel VL, Kaufman DR, Arocha JF: Emerging paradigms of cognition in medical decision-making. J Biomed Inform. 2002; 35(1): 52–75. PubMed Abstract \| Publisher Full Text\n\nMarewski JN, Gigerenzer G: Heuristic decision making in medicine. Dialogues Clin Neurosci. 2012; 14(1): 77–89. PubMed Abstract \| Free Full Text\n\nHenderson J: Google Scholar: A source for clinicians? CMAJ. 2005; 172(12): 1549–1550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrause R, Moscati R, Halpern S, et al.: Can emergency medicine residents reliably use the internet to answer clinical questions? West J Emerg Med. 2011; 12(4): 442–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWyatt JC, Sullivan F: eHealth and the future: promise or peril? BMJ. 2005; 331(7529): 1391–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiustini D: How Google is changing medicine. BMJ. 2005; 331(7531): 1487–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLindberg DA, Humphreys BL: Medicine and health on the Internet: the good, the bad, and the ugly. JAMA. 1998; 280(15): 1303–4. PubMed Abstract \| Publisher Full Text\n\nGreenwald R: . . . And a diagnostic test was performed. N Engl J Med. 2005; 353(19): 2089–2090. PubMed Abstract \| Publisher Full Text\n\nFalagas ME, Ntziora F, Makris GC, et al.: Do PubMed and Google searches help medical students and young doctors reach the correct diagnosis? A pilot study. Eur J Intern Med. 2009; 20(8): 788–90. PubMed Abstract \| Publisher Full Text\n\nTang H, Ng JH: Googling for a diagnosis--use of Google as a diagnostic aid: internet based study. BMJ. 2006; 333(7579): 1143–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHafner K: For second opinion, consult a computer? New York Times on the web. 2012. Reference Source\n\n\n\n\n" }	[ { "id": "852", "date": "19 Mar 2013", "name": "Rudy Bilous", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an intriguing report but the approach is a bit simplistic. Atypical presentations of common conditions are more frequently encountered than typical presentations of rare ones. Thus it is really hard to test the hypothesis fully. The internet is more likely to throw up rare options that may result in unnecessary and perhaps dangerous and costly investigations. The fellows are on a journey of understanding and without the experience are likely to score slightly less well than Dr Google. The authors have also made many comparisons and do not appear to have adjusted the level of statistical significance.", "responses": [] }, { "id": "839", "date": "19 Mar 2013", "name": "Melanie Hoenig", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 enjoyed the article and was particularly pleased that the authors provided sufficient examples of the keywords used in queries. The commentary was balanced. It is worth noting that while 'Dr. Google' may be helpful in identifying the diagnosis once provided with the keywords or search term, it takes a clinician to be able to sort through the detailed history, physical examination and laboratory data to determine which aspects of the presentation are worth using for a “search.”", "responses": [] } ]	1	https://f1000research.com/articles/2-90
https://f1000research.com/articles/2-107/v1	09 Apr 13	{ "type": "Commentary", "title": "An estimation of the prevalence of nonmelanoma skin cancer in the U.S.", "authors": [ "Eshini Perera", "Rodney Sinclair", "Rodney Sinclair" ], "abstract": "Nonmelanoma skin cancer (NMSC) is the most commonly diagnosed cancer in Australia and has a significant impact on the cost of and use of healthcare resources. Current estimates of NMSC in the USA are 3.5 million cases in 2010 compared to 1.63 million cases of all other cancers combined. However, we believe that this figure significantly underestimates the prevalence of NMSC in the USA. We calculated that melanoma is diagnosed 5.7 times more in the USA than in Australia. In Australia, in 2010, there were 767,000 NMSC diagnoses. If the ratio of melanoma: NMSC is constant in both Australia and the USA, then there should be 5.7 times the number of NMSC in the USA or 4.3 million cases. The assumptions that underlie this calculation are discussed.", "keywords": [ "Nonmelanoma skin cancer (NMSC) includes basal cell carcinoma and squamous cell carcinoma of the skin", "and is common in both Australia and the USA. In fact", "in both Australia and the USA", "NMSC is more common than all other forms of cancer combined1–3. NMSCs are predominately managed privately in an outpatient setting. NMSC is not a reportable cancer and NMSC data are not recorded on national cancer registries in either Australia or the USA." ], "content": "Introduction\n\nNonmelanoma skin cancer (NMSC) includes basal cell carcinoma and squamous cell carcinoma of the skin, and is common in both Australia and the USA. In fact, in both Australia and the USA, NMSC is more common than all other forms of cancer combined1–3. NMSCs are predominately managed privately in an outpatient setting. NMSC is not a reportable cancer and NMSC data are not recorded on national cancer registries in either Australia or the USA.\n\nEstimates of the incidence of NMSC in both Australia and the USA are obtained by periodic surveys. Incidence data record an individual’s first episode of skin cancer within a calendar year. As NMSC patients commonly develop multiple primary cancers, incidence data are likely to significantly underestimate the burden of disease.\n\nIn order to calculate NMSC prevalence in Australia, data from more than 8 million skin cancers treated between 1 January 1997 and 31 December 20104 were evaluated by Fransen et al. Data from the study were obtained from Medicare Australia. The population of Australia grew 23% from 18.3 million in 1997 to 22.6 million in 20105. Roughly 95% of the population or 21,470000 people are of European or Anglo-Saxon descent. Medicare Australia is a Commonwealth Government-funded universal health insurance scheme. Medicare Australia data captures 98% of all skin cancer treatments in Australia. Fewer than 2% of NMSC lesions are treated in public hospitals that record data independently6. All skin cancer treatments in Australia, except those provided in a public hospital, are individually itemized and recorded by Medicare Australia for the purpose of patient reimbursement. Item numbers for the excision, cryotherapy, curettage or laser treatment of skin cancers require histological confirmation of the diagnosis or clinical diagnosis by a registered dermatologist prior to Medicare coding.\n\nThe total number of NMSC treatments increased by 186% from 412,493 in 1997 to 767,347 in 20104. The number of treatments are estimated to increase to 938,991 (95% CI, 901,047–976,934) by 20154.\n\nSo, what is the prevalence of NMSC in the USA? Rogers et al. estimated that in 2010 more than 3.5 million skin cancers were diagnosed in over 2 million people2. The number of practicing dermatologists in USA in 2010 was 95987. This figure suggests on average that each dermatologist treats 364 NMSCs a year, or one a day. Anecdotally, this figure seems low, even accounting for differences of skin cancer prevalence in each state.\n\nAnother way to estimate the burden of NMSC in USA is to compare the incidence of melanoma in the USA to that in Australia. Melanoma is a cancer that is reportable in both the USA and Australia. Reporting is mandatory and data on melanoma prevalence are likely to be more complete compared to data on NMSC.\n\nIn 2010, it was predicted that 68,130 melanomas would be recorded on the cancer registry in the USA3 and 11,900 were predicted for the national cancer registry in Australia in 20101. This amounts to 7.1 melanomas per dermatologist per year in the USA and 21.6 melanomas per dermatologist in Australia in 2010.\n\nNational registries data indicate that 5.7 times the number of melanomas were diagnosed in the USA among a population that is 14 times larger than Australia. One model6 assumes that the climate, ethnicity, sun exposure behaviour and population differences that lead to the higher incidence of melanoma in Australia do not affect the ratio of melanoma to NMSC. We also assume that melanoma is recorded with equal accuracy and completeness in both the USA and Australia. Based on these assumptions, the number of NMSCs treated in the USA in 2010 would also be 5.7 times the number of NMSCs treated in Australia. As 767,000 NMSCs were treated in Australia in 2010, we estimate that 4.3 million NMSC were treated in USA.\n\nThe population of the USA was 308.7 million in 2010 and 72.4% were White or European American8, 12.6% were black or African American8. The remaining 15% comprised people of different ethnicities including Hispanic or Latino, Asian, Native American, Hawaiian and Pacific Islanders8. The skin cancer with the least ethnic variation in incidence is acral lentiginous melanoma (ALM). ALM only represents 2–3% of all melanoma diagnosed in the USA9. If ALM were excluded from the total number of melanomas diagnosed in the USA in 2010, that reduces our estimate of NMSC in USA from 4.3 to 4.21 million cases.\n\nThis is roughly an extra 700,000 cases on top of the current estimations of NMSC by Rogers et al2. This now equates to approximately 1.2 NMSCs per US dermatologist per day. Anecdotal experience would suggest this figure still underestimates the true burden of NMSC in USA. A prospective registry may be required to better estimate the full burden of NMSC and to ensure that there are adequate physical resources and manpower to treat NMSC patients in the USA.", "appendix": "Author contributions\n\n\n\nRS conceived the article. EP and RS prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nAustralian Institute of Health and Welfare & Australasian Association of Cancer Registries. Cancer in Australia: an overview 2010. (Cancer series no 60). AIHW Cat. No 56. Canberra: Australian Institute of Health and Welfare; 2010. Reference Source\n\nRogers HW, Weinstock MA, Harris AR, et al.: Incidence Estimate of Nonmelanoma Skin Cancer in the United States, 2006. Arch Dermatol. 2010; 146(3): 283–287. PubMed Abstract \| Publisher Full Text\n\nAmerican Cancer Society. Cancer facts and figures 2010. Atlanta: American Cancer Society; 2010. Reference Source\n\nFansen M, Karahalios A, Sharma N, et al.: Non-melanoma skin cancer in Australia. Med J Aust. 2012; 197(10): 565–568. PubMed Abstract \| Publisher Full Text\n\nAustralian Bureau of Statistics. Population projections, Australia, 2006 to 2101. (Cat. No. 3222.0). Canberra: Australian Bureau of Statistics; 2008. Reference Source\n\nSinclair R: Nonmelanoma skin cancer in Australia. Br J Dermatol. 2013; 168(1): 1–2. PubMed Abstract \| Publisher Full Text\n\nYoo JY, Rigel DS: Trends in Dermatology: Geographic Density of US Dermatologists. Arch Dermatol. 2010; 146(7): 779. PubMed Abstract \| Publisher Full Text\n\nHumes K, Jones N, Ramirez R: Overview of Race and Hispanic Origin: 2010. Washington DC. U.S. Census Bureau; 2011. Reference Source\n\nBradford PT, Goldstein AM, McMaster ML, et al.: Acral lentiginous melanoma: incidence and survival patterns in the United States, 1986–2005. Arch Dermatol. 2009; 145(4): 427–34. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "893", "date": "15 Apr 2013", "name": "Gabriella Fabbrocini", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 can be considered very interesting for the new epidemiological insights that it provides. The study is interesting for the evaluation of relationship between melanoma and non-melanoma skin cancers (NMSC) in Australia and in USA; it highlights the underestimation of NMSC prevalence in USA, so it opens new inputs for a better demographic evaluation of these tumors.The title is simple and appropriate for the content of the article and the abstract can be considered a good summary of the work. The design of the study, and relative materials and methods are clearly explained and appropriate for this study. All data are clearly specified, clearly exposed and well schematized. The conclusions respect the basis of the study results.", "responses": [] }, { "id": "890", "date": "15 Apr 2013", "name": "Kamran Ghoreschi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of this commentary are based on the estimations and calculations made by the authors of the work. The commentary itself is interesting, and helps provoke the idea that there is a higher prevalence of non-melanoma skin cancer (NMSC) in the US. However, the authors quite rightly demonstrate the potential weaknesses in the epidemiology of the disease; it is still difficult to collect data on the epidemiology of skin cancer even in Western countries. The authors do encourage prospective registries to help estimate NMSC, however as discussed, sun exposure and other factors have to be considered too in order to provide a more definitive answer for the prevalence of NMSC in the US.", "responses": [] } ]	1	https://f1000research.com/articles/2-107
https://f1000research.com/articles/2-106/v1	08 Apr 13	{ "type": "Research Article", "title": "Rifampicin induced virulence determinants increase Candida albicans biofilm formation", "authors": [ "Miriam Vogel", "Martin Köberle", "Holger Schäffler", "Monika Treiber", "Ingo B Autenrieth", "Ulrike K Schumacher", "Miriam Vogel", "Martin Köberle", "Holger Schäffler", "Monika Treiber", "Ingo B Autenrieth" ], "abstract": "Increased intravenous catheter use has been paralleled by increased bacterial and yeast bloodstream infection. Biofilm formation, which is associated with the cell surface hydrophobicity (CSH) phenotype, represents a major pathogenicity strategy of Candida albicans, becoming especially important in the colonization of intravascular medical devices. Increasing evidence shows the induction of virulence factors in C. albicans by diverse substances. Therefore, we investigated whether rifampicin, an antibiotic shown to be capable of inducing MDR1 expression in C. albicans may also promote the formation of a pathogenic biofilm. In response to 40 µg/mL rifampicin, an enhanced retention of C. albicans SC5314 cells on polystyrene culture plates was observed by measuring increased metabolic activity by XTT assay, indicating induction of biofilm formation. Rifampicin treatment also induced fibronectin binding, cell hydrophobicity and germ tube formation. Furthermore, increased RNA and protein expression of CSH1p, a major mediator of CSH, was demonstrated. We conclude that exposure to rifampicin may result in upregulation of key Candida virulence determinants, potentially boosting pathogenicity and supporting biofilm formation. This finding gains clinical significance from the increasing popularity of rifampicin-coated catheters, which might provide an advantageous gateway for Candida bloodstream infections.", "keywords": [ "Candida albicans is an opportunistic yeast pathogen that has been established as a persistent and growing threat for critical ill patients over the last three decades. Candida species are the most common fungal species to cause invasive infections and at least one half of candidemia cases in non-neutropenic patients occur in an ICU or surgical ward1. Invasive candidiasis is associated with poor prognosis", "with mortality rates of up to 30%2", "3. Above all", "C. albicans ranks as the fourth most common cause of bloodstream infection", "carrying one of the worst prognoses3–5. The increase of Candida infections in the ICU comes at the same time as medical progress with the development of broad spectrum anti-bacterial therapy and immunosuppressive therapy1. There is also a close connection between the increased use of intravascular medical devices and the advent of bloodstream infections among the most frequent and potentially lethal nosocomial infections5–7. Catheter implantation frequently provides a gateway for the systemic entry of Candida and other pathogens from diverse sources. Additionally", "hydrophobic catheter surfaces are a favorable habitat for pathogens that are able to form biofilms. They provide pathogens with a reservoir that is difficult to eradicate even by high dose antimycotic therapy", "as resistance to antifungals is increased 10 to 1000 times compared to planktonic cells8", "9. Known mechanisms of antifungal resistance under biofilm conditions are the upregulation of drug efflux pumps", "sequestration of antifungals by matrix glucans and the development of a persister cell subpopulation10–12. Therefore", "hydrophobic attachment to tissues and artificial surfaces can be regarded as a key virulence determinant of Candida spp. Adhesion of Candida and other fungi to polymeric materials correlates with cell surface hydrophobicity (CSH) phenotype13", "though tissue and plastic binding are also mediated by adhesins14. The CSH1 gene product has been shown to be one of the mediators of CSH phenotype", "localizing on the yeast cell surface and enhancing cell hydrophobicity as well as fibronectin binding15–17. It is thought to be induced by germ tube formation of single attaching yeast cells", "subsequently evolving into a dense network of hyphae and pseudohyphae18. Quorum sensing of the densely packed cells regulates transcription of glucans that form a polymeric intercellular matrix19", "20. We and others have recently shown that substances of diverse structure and origin may induce Candida pathogenicity factors. 17-β-estradiol increased growth and germ tube formation mediated increased temperature resistance by induction of the Hsp90 chaperon and elevated expression of the multidrug transporters CDR1 and CDR2. As coumarin and phenol also upregulated Hsp90 and CDR1", "the authors of this study concluded that the response to estrogen might be rather unspecific21", "22. Cigarette smoke induced the expression of histolytic enzymes and increased candidal adhesion in vitro23. Rifampicin", "a common antibiotic", "induces MDR-1 expression by Candida albicans that in turn can lead to modestly elevated minimal inhibitory concentrations (MIC) for fluconazole by some isolates24", "25. Rifampicin-impregnated central-venous catheters have been increasingly used in clinical trials and one of these showed significantly increased Candida colonization26. To investigate whether rifampicin used in antibacterial catheter coatings to control bacterial catheter-related bloodstream infections may boost Candida virulence on these devices", "we examined the possible induction of C. albicans SC5314 virulence factors involved in surface adhesion and biofilm formation by this drug." ], "content": "Introduction\n\nCandida albicans is an opportunistic yeast pathogen that has been established as a persistent and growing threat for critical ill patients over the last three decades. Candida species are the most common fungal species to cause invasive infections and at least one half of candidemia cases in non-neutropenic patients occur in an ICU or surgical ward1. Invasive candidiasis is associated with poor prognosis, with mortality rates of up to 30%2,3. Above all, C. albicans ranks as the fourth most common cause of bloodstream infection, carrying one of the worst prognoses3–5. The increase of Candida infections in the ICU comes at the same time as medical progress with the development of broad spectrum anti-bacterial therapy and immunosuppressive therapy1. There is also a close connection between the increased use of intravascular medical devices and the advent of bloodstream infections among the most frequent and potentially lethal nosocomial infections5–7. Catheter implantation frequently provides a gateway for the systemic entry of Candida and other pathogens from diverse sources. Additionally, hydrophobic catheter surfaces are a favorable habitat for pathogens that are able to form biofilms. They provide pathogens with a reservoir that is difficult to eradicate even by high dose antimycotic therapy, as resistance to antifungals is increased 10 to 1000 times compared to planktonic cells8,9. Known mechanisms of antifungal resistance under biofilm conditions are the upregulation of drug efflux pumps, sequestration of antifungals by matrix glucans and the development of a persister cell subpopulation10–12. Therefore, hydrophobic attachment to tissues and artificial surfaces can be regarded as a key virulence determinant of Candida spp. Adhesion of Candida and other fungi to polymeric materials correlates with cell surface hydrophobicity (CSH) phenotype13, though tissue and plastic binding are also mediated by adhesins14. The CSH1 gene product has been shown to be one of the mediators of CSH phenotype, localizing on the yeast cell surface and enhancing cell hydrophobicity as well as fibronectin binding15–17. It is thought to be induced by germ tube formation of single attaching yeast cells, subsequently evolving into a dense network of hyphae and pseudohyphae18. Quorum sensing of the densely packed cells regulates transcription of glucans that form a polymeric intercellular matrix19,20. We and others have recently shown that substances of diverse structure and origin may induce Candida pathogenicity factors. 17-β-estradiol increased growth and germ tube formation mediated increased temperature resistance by induction of the Hsp90 chaperon and elevated expression of the multidrug transporters CDR1 and CDR2. As coumarin and phenol also upregulated Hsp90 and CDR1, the authors of this study concluded that the response to estrogen might be rather unspecific21,22. Cigarette smoke induced the expression of histolytic enzymes and increased candidal adhesion in vitro23. Rifampicin, a common antibiotic, induces MDR-1 expression by Candida albicans that in turn can lead to modestly elevated minimal inhibitory concentrations (MIC) for fluconazole by some isolates24,25. Rifampicin-impregnated central-venous catheters have been increasingly used in clinical trials and one of these showed significantly increased Candida colonization26. To investigate whether rifampicin used in antibacterial catheter coatings to control bacterial catheter-related bloodstream infections may boost Candida virulence on these devices, we examined the possible induction of C. albicans SC5314 virulence factors involved in surface adhesion and biofilm formation by this drug.\n\n\nMethods\n\nC. albicans wild-type strain SC531427 was kindly provided by J. Morschhäuser, Institute for Molecular Infection Biology, Würzburg, Germany. The strain was kept as a frozen stock in glycerol at -80°C. For experiments, frozen cells were streaked on yeast-dextrose agar plates (5 g of yeast extract, 10 g of peptone, 20 g of dextrose, 15 g of agar, 40 mg gentamicin per litre), incubated at 30°C overnight and sub-cultured in YNB liquid medium (0.67% Yeast Nitrogen Base, 0.5% dextrose) at 30°C. Cultures were diluted in 25 mL YNB medium to an optical density at 600 nm (OD600) of 1 and were incubated at 30°C for 4 h with gentle shaking. For induction of the rifampicin response, 40 µg/mL rifampicin was used if not indicated otherwise.\n\nUnless stated otherwise, all reagents have been purchased at Sigma-Aldrich (Taufkirchen, Germany). Compounds were dissolved in acetone (menadione), DMSO (rifampicin) or water (all others).\n\nBiofilm formation of SC5314 was quantified by the metabolic activity retained on a 96-well polystyrene plate (Falcon; BD Labware, Franklin Lakes, NJ) as described by Rammage and co-workers28. In brief, 105 cells /well were allowed to adhere to the plate for 48 h. After three times washing thoroughly with phosphate-buffered saline (PBS) (Gibco; Invitrogen, Carlsbad, CA), 2 h incubation with 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) substrate (0.5 mg/mL + 1 µM menadione in Ringer’s solution) was performed in the dark. Absorbance of reduced XTT was measured in a microtiter plate reader (Tecan, Maennedorf, Switzerland) at 490 nm.\n\nTo assay Candida cell hydrophobicity, the number of adherent micro particles (Serva, Heidelberg, Germany) per cell was quantified as described by Hazen & Hazen29. 100 µL of a 2×106 cells/mL SC5314 suspension in ice cold PBS were mixed with 100 µL microsphere solution (~ 8,4×108 particles/mL) in acid-washed glass vessels. Two minutes of incubation at room temperature was followed by 30 s of rigorous mixing. Samples of this mixture were subjected to phase contrast microscopy (Axiolab / Axiovert 200 + Axiocam HRc microscope; Carl Zeiss Microimaging, Esslingen, Germany). Relative hydrophobicity was determined as the fraction size of cells with three or more adhering particles.\n\nExtraction of Candida cells from an aqueous suspension with xylene was performed by a protocol adapted from Rosenberg and co-workers30. In brief, 4 mL of SC5314 suspension were vortexed with 1 mL of xylene for 2 min in glass tubes. After 15 min incubation at 37°C, tubes were cooled to room temperature and the OD600 of the aqueous phase was determined (Biophotometer, Eppendorf, Hamburg, Germany). Samples without xylene treatment served as controls. Relative hydrophobicity was calculated as the ratio of control samples to xylene treated samples.\n\nFor the detection of fibronectin binding, Candida cells were sub-cultured in medium supplemented with 0.001% or 0.0001% human fibronectin (Sigma-Aldrich) for 1 h. Subsequently, cells were examined microscopically or washed 3x with PBS and subjected to immunoblot analysis.\n\nTo induce germ tube formation, 1×106 C. albicans yeasts/mL were transferred into the cell culture medium RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 10% (v/v) (Sigma-Aldrich) FCS, 2 mM L-glutamine, penicillin (100 U/mL) and streptomycin (100 µg/mL) (Biochrom KG) and seeded into a 24-well plate. After 1 h, pictures were taken by phase contrast microscopy and germ tube length was determined with the Photoshop 6 measure tool (Adobe, San Jose, CA).\n\nTotal RNAs were extracted from C. albicans cells by use of the MasterPure™ Yeast RNA Purification Kit (EPICENTRE, Madison, Wisconsin). Contaminating DNA was removed by the TURBO DNA-freeTM Kit (Ambion Inc, Austin, Texas). Briefly, RNA was incubated at 37°C for 30 min with 2 U TURBO DNase per 10 µg of RNA. DNase was inactivated by adding DNase inactivation reagent for 2 min at room temperature. Total RNA concentrations were spectrophotometrically quantified. RNA samples were stored at -80°C or were used immediately. cDNA was synthesized with oligo(dT)-primers (New England Biolabs, Frankfurt, Germany) and Superscript™ III reverse transcriptase (Invitrogen, Karlsruhe, Germany), using 1 µg of total RNA. Controls examined C. albicans elongation factor 1 (EFB1) housekeeping gene, which contains an intron of 365 bp. Absence of genomic DNA was verified by a single intronless PCR product of EFB1. PCR were performed according standard protocols with 0.5 U Taq DNA polymerase (Roche, Mannheim, Germany). To amplify CSH1 or EFB1 the primers AGT AGA AAG CAT ATC TTA GCC G (fwd) and GCT TGT TGT CTA AGA ACT GC (rev) or AGT CAT TGA ACG AAT TCT TGG C (fwd) and ATC AAC TTC ATC ATC AGA ACC G (rev) were used. Cycling conditions were 94°C for 30 s, 63°C for 30 s and 72°C for 60 s for CSH1 and 94°C for 30 s, 60°C for 30 s and 72°C for 60 s for EFB1. Only samples from the exponential phase of PCR amplification were examined. Equivalent volumes of PCR product were analysed on 1.8% agarose gels stained with ethidium bromide.\n\nProteins were isolated by the method of Hiller et al31. Equal protein amounts as determined by Bradford assay were separated on a 10% SDS gel and transferred to nitrocellulose membranes by wet-blot. Further membrane protein binding was blocked by incubation in 5% skimmed milk powder containing wash buffer. CSH1p or fibronectin were detected by subsequent incubation with mouse anti-CSH1 (1:10,000; kindly provided by D. Singleton) or rabbit anti-human fibronectin (1:4000; Sigma-Aldrich) primary antibodies and horseradish peroxidase (HRP) labeled rabbit anti-mouse or swine anti-rabbit IgG secondary antibodies (both 1:1000; DAKO, Glostrup, Denmark), respectively. Chemiluminescence of Amersham ECL reagent (GE Healthcare, Waukesha, WI), was detected on CL-XPosure Film (Thermo, Rockford, IL).\n\nUnless indicated otherwise, data shown are representative of at least two independent experiments. Differences between mean values were analyzed using two tailed Student’s t test. P < 0.05 was considered statistically significant.\n\n\nResults\n\nThe cell surface hydrophobicity (CSH) phenotype was identified as a major factor conferring virulence to Candida species by many means, first of all by enhancing attachment to tissues and foreign materials, thus being a prerequisite for efficient biofilm formation13,32. To examine whether rifampicin might increase C. albicans CSH phenotype expression, we quantified the adhesion of microspheres to SC5314 with or without rifampicin treatment. Microsphere adherence to SC5314 cells robustly doubled in response to rifampicin treatment, showing increased hydrophobicity and suggesting CSH phenotype induction (Figure 1A and 1B). Another method established for the measurement of microbial surface hydrophobicity is the measurement of extraction from an aqueous suspension with an organic solvent30. After rifampicin treatment, extraction of SC5314 was significantly increased, providing additional support for increased hydrophobicity (Figure 1C).\n\n(A) Microsphere adherence to SC5314 after treatment with 40 µg/mL rifampicin. Representative phase-contrast picture, arrows indicate adherent microspheres. (B) Relative hydrophobicity; means + SD of three independent microsphere adherence experiments. (C) Rifampicin enhances extraction by the organic solvent xylene. Mean relative hydrophobicity normalized on untreated controls calculated from four independent extraction experiments + SEM. Significant difference (p < 0.05). (D) Detection of fibronectin retention. Representative phase-contrast picture showing the formation of fibronectin fibers thickly coated by clustered Candida cells (condition with fibronectin and rifampicin). (E) Western blot of SC5314 with or without rifampicin and fibronectin (0.001%, 0.0001%) pre-treatment for 1 h.\n\nThe affinity of Candida for fibronectin has been known for a long time and has been regarded as a virulence property enabling tissue adhesion by extracellular matrix binding, thus promoting initiation as well as dissemination of candidiasis15. Strikingly, microscopic observation revealed dense clustering to long, bottle brush like aggregates (Figure 1D). Western blot analysis of SC5314 cultured in medium supplemented with human plasma fibronectin in the presence or absence of rifampicin proved strongly enhanced fibronectin retention of rifampicin treated cells after thoroughly washing (Figure 1E).\n\nBiofilm formation by Candida spp. on tissues or implanted materials is a process with high clinical impact, as it mediates increased resistance to antifungal agents and protection from host defenses. Additionally, biofilms act as pathogen reservoirs, boosting persistent infection9. To get a quantitative idea of how rifampicin affects Candida retention on plastic materials, we assayed XTT reduction of adhering C. albicans SC5314 after culture in polystyrene wells for 48 h, untreated or treated with 40 µg/mL rifampicin. We observed an 1.6-fold significant increase in metabolic activity retained after thoroughly washing on the culture plate induced by rifampicin treatment, indicating that rifampicin enhances biofilm formation by C. albicans (Figure 2A).\n\n(A) Biofilm formation of SC5314 after treatment with 40 µg/mL rifampicin. Mean and SD of XTT absorption normalized on untreated controls of three independent experiments; difference is statistically significant (p < 0.05). (B) Rifampicin-induced germ tube formation, representative phase-contrast microscopy. (C) Germ tube length in µm; mean + SD of three independent experiments. *Significant difference (p < 0.005).\n\nThe morphological differentiation ability of C. albicans plays an important role in biofilm maturation that follows hydrophobic attachment. Mutants deficient in the yeast phenotype show reduced adhesion to tissues and polymers and are more easily removed under high-salt assay conditions, indicating improved anchoring of the biofilm by yeast phenotype cells. However, the dimorphic phenotype enables the formation of thicker biofilms with higher cell numbers than yeast-only strains that may display superior resistance to chemical and mechanical stress, respectively33. In our experiments, rifampicin treatment turned out to profoundly induce germ tube formation, accelerating germ tube growth leading to increased hyphae to yeast cells ratio. Average germ tube length was doubled 4 h after induction (Figure 2B and 2C). Interestingly, our data are paralleled by the observations of other authors who report enhanced filamentous growth induced by tetracycline and 17-β-estradiol21,34.\n\nCSH1 gene expression may be a mediator of the CSH phenotype15–17. Upon rifampicin treatment, an immediate increase in CSH1 transcription could be detected in SC5314, as well as sustained high level expression of CSH1 mRNA until 4 h after induction (Figure 3A). Induction of CSH1 transcription is matched by elevated CSH1p protein levels in SC5314 lysates (Figure 3B).\n\n(A) Kinetic of rifampicin (20 µg/mL) induced CSH1 mRNA expression in SC5314. Samples were collected at indicated time points during rifampicin exposition. (B) Csh1p expression in SC5314. Western blot of equal protein amounts after rifampicin-induction (40 µg/mL, 4 h).\n\n\nDiscussion\n\nIn this work we addressed the question of whether rifampicin might promote Candida albicans biofilm formation, thus potentially aggravating the threat of catheter-related and other infections by this pathogen. We could show increased SC5314 cell hydrophobicity, indicating a shift to a CSH phenotype. It has to be taken into account that a recent report failed to correlate the hydrophobicity of 50 clinical C. albicans isolates with their adhesiveness to polystyrene35. Other reports however, have shown hydrophobicity not only to correlate with Candida adhesiveness but to be part of concerted pathogenicity factor expression23,36. Fibronectin binding was also strongly increased after rifampicin treatment, showing the induction of an additional trait contributing to increased adherence. In line with these findings we could demonstrate that rifampicin treatment induces enhanced Candida retention on plastic by metabolic activity assay. Additionally, rifampicin enhanced germ tube formation, known to promote an exploratory and invasive lifestyle. Reports showing germ tube formation to be induced a few hours after CSH increase or surface attachment strongly argue it to be part of the biofilm program29. Germ tubes also enhance biofilm compression strength and mediate phagocyte escape, suggesting a key role in biofilm pathogenesis18,37. Csh1p is an aldo-keto reductase and is homologous to aryl-alcohol dehydrogenases in Saccharomyces cerevisae17. CSH1 transcription is activated by Zap1, a negative regulator of the matrix component soluble β-1,3 glucan and thought to mediate the Zap1 effect by intercellular signalling20. To answer the question of whether CSH1 expression might be part of the CSH phenotype induced by rifampicin, we analysed CSH1 mRNA and protein levels in C. albicans SC5314. We found CSH1 mRNA upregulation and an increase in CSH1p protein levels.\n\nInterestingly, CSH1p deficiency has been shown to drastically reduce the fibronectin binding properties of C. albicans, thus opening the possibility that increased fibronectin binding results from CSH1p upregulation, though host fibronectin binding seems to be mediated by multiple Candida surface proteins15,16. Previously we demonstrated that rifampicin upregulated C. albicans MDR1 expression24. Co-induction of CSH1 and MDR1 has also been shown for a fluconazole-resistant C. albicans patient isolate38. Moreover, Mdr1 has been shown to be upregulated immediately after adhesion39, and both CSH1 (orf19.4477) and MDR1 (orf19.5604) transcription is mediated by the multidrug resistance regulator Mrr1p40. This raises the possibility that Mrr1p may be a target of rifampicin in C. albicans. Taken together, the results of this study show a significant upregulation of Candida virulence determinants that promote pathogenic biofilm behaviour by the antibiotic rifampicin. Thus, antibacterial rifampicin coatings of intravascular medical devices could potentially oppose efforts to diminish their microbial colonization. This effect could also be suspected to interfere with the promising effects of some recent antimycotics in inhibiting biofilm growth41. Furthermore, the data shown here contribute to the growing evidence showing that miscellaneous structurally unrelated substances that are xenobiotic to yeasts are capable of inducing mediators of Candida virulence and drug resistance. Eventually, enhanced induction by cooperation of several of these substances cannot be excluded (e.g. tetracycline + rifampicin coated catheter in combination with estrogen)21,26,34.", "appendix": "Acknowledgements\n\nWe thank David Singleton for kindly providing the CSH1p antibody and Tarun Mehra for proofreading the manuscript. Parts of this work were presented at the 8th ASM conference on Candida and Candidiasis, Denver, USA 2006.\n\n\nAuthors' contributions\n\nMV performed the experiments and analyzed the data. HS, MT and MK participated in performing experiments and data analysis. MK drafted the manuscript. UKS designed and coordinated the study, analyzed the data, supervised MV, HS and MT and together with MV participated in finalizing the manuscript. IBA participated in study design and executed critical readings of the manuscript. All authors read and approved the final manuscript.\n\n\nGrant information\n\nThis work has been funded by a research grant from Landesstiftung Baden-Württemberg to UKS.\n\n\nCompeting interests\n\nUKS has participated as an investigator in multicenter studies for GSK, Pfizer and Wyeth Pharmaceuticals and has received speaker’s honoraria from BioMérieux, Becton Dickinson and Pfizer.\n\n\nReferences\n\nPappas PG, Kauffman CA, Andes D, et al:Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis. (2009); 48: 503–535.\n\nPfaller MA, Diekema DJ: Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev. (2007); 20: 133–163.\n\nZilberberg MD, Shorr AF: Fungal infections in the ICU. Infect Dis Clin North Am. (2009); 23: 625–642.\n\nPfaller MA, Diekema DJ: Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev. (2007); 20: 133–163.\n\nKumamoto CA, Vinces MD: Alternative Candida albicans lifestyles: growth on surfaces. Annu Rev Microbiol. (2005); 59: 113–133.\n\nKojic EM, Darouiche RO: Candida infections of medical devices. Clin Microbiol Rev. (2004); 17: 255–267.\n\nSitges-Serra A, Girvent M: Catheter-related bloodstream infections. World J Surg. (1999); 23: 589–595.\n\nDouglas LJ: Candida biofilms and their role in infection. Trends Microbiol. (2003); 11: 30–36.\n\nRamage G, Martinez JP, Lopez-Ribot JL: Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS Yeast Res. (2006); 6: 979–986.\n\nMukherjee PK, Chandra J, Kuhn DM, et al:Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols. Infect Immun. (2003); 71: 4333–4340.\n\nLaFleur MD, Kumamoto CA, Lewis K: Candida albicans biofilms produce antifungal-tolerant persister cells. Antimicrob Agents Chemother. (2006); 50: 3839–3846.\n\nNett JE, Crawford K, Marchillo K, et al:Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene. Antimicrob Agents Chemother. (2010); 54: 3505–3508.\n\nLi X, Yan Z, Xu J: Quantitative variation of biofilms among strains in natural populations of Candida albicans. Microbiology. (2003); 149: 353–362.\n\nLi F, Palecek SP: Distinct domains of the Candida albicans adhesin Eap1p mediate cell-cell and cell-substrate interactions. Microbiology. (2008); 154: 1193–1203.\n\nSingleton DR, Fidel PL Jr, Wozniak KL, et al:Contribution of cell surface hydrophobicity protein 1 (Csh1p) to virulence of hydrophobic Candida albicans serotype A cells. FEMS Microbiol Lett. (2005); 244: 373–377.\n\nSingleton DR, Masuoka J, Hazen KC: Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity. J Bacteriol. (2001); 183: 3582–3588.\n\nSingleton DR, Hazen KC: Differential surface localization and temperature-dependent expression of the Candida albicans CSH1 protein. Microbiology. (2004); 150: 285–292.\n\nHawser SP, Douglas LJ: Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect Immun. (1994); 62: 915–921.\n\nNett JE, Sanchez H, Cain MT, et al:Interface of Candida albicans Biofilm Matrix-Associated Drug Resistance and Cell Wall Integrity Regulation. Eukaryot Cell. (2011); 10: 1660–1669.\n\nNobile CJ, Nett JE, Hernday AD, et al:Biofilm matrix regulation by Candida albicans Zap1. PLoS Biol. (2009); 7: e1000133.\n\nCheng G, Yeater KM, Hoyer LL: Cellular and molecular biology of Candida albicans estrogen response. Eukaryot Cell. (2006); 5: 180–191.\n\nZhang X, Essmann M, Burt ET, et al:Estrogen effects on Candida albicans: a potential virulence-regulating mechanism. J Infect Dis. (2000); 181: 1441–1446.\n\nBaboni FB, Barp D, Izidoro AC, et al:Enhancement of Candida albicans virulence after exposition to cigarette mainstream smoke. Mycopathologia. (2009); 168: 227–235.\n\nVogel M, Hartmann T, Koberle M, et al:Rifampicin induces MDR1 expression in Candida albicans. J Antimicrob Chemother. (2008); 61: 541–547.\n\nWu T, Wright K, Hurst SF, et al:Enhanced extracellular production of aspartyl proteinase, a virulence factor, by Candida albicans isolates following growth in subinhibitory concentrations of fluconazole. Antimicrob Agents Chemother. (2000); 44: 1200–1208.\n\nLeon C, Ruiz-Santana S, Rello J, et al:Benefits of minocycline and rifampin-impregnated central venous catheters. A prospective, randomized, double-blind, controlled, multicenter trial. Intensive Care Med. (2004); 30: 1891–1899.\n\nGillum AM, Tsay EY, Kirsch DR: Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet. (1984); 198: 179–182.\n\nRamage G, Vande WK, Wickes BL, et al:Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother. (2001); 45: 2475–2479.\n\nHazen BW, Hazen KC: Dynamic expression of cell surface hydrophobicity during initial yeast cell growth and before germ tube formation of Candida albicans. Infect Immun. (1988); 56: 2521–2525.\n\nRosenberg M, Judes H, Weiss E: Cell surface hydrophobicity of dental plaque microorganisms in situ. Infect Immun. (1983); 42: 831–834.\n\nHiller D, Sanglard D, Morschhauser J: Overexpression of the MDR1 gene is sufficient to confer increased resistance to toxic compounds in Candida albicans. Antimicrob Agents Chemother. (2006); 50: 1365–1371.\n\nHazen KC, Brawner DL, Riesselman MH, et al:Differential adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse tissues. Infect Immun. (1991); 59: 907–912.\n\nBaillie GS, Douglas LJ: Role of dimorphism in the development of Candida albicans biofilms. J Med Microbiol. (1999); 48: 671–679.\n\nMcCool L, Mai H, Essmann M, et al:Tetracycline effects on Candida albicans virulence factors. Infect Dis Obstet Gynecol. (2008); 2008: 493508.\n\nRaut J, Rathod V, Karuppayil SM: Cell surface hydrophobicity and adhesion: a study on fifty clinical isolates of Candida albicans. Nihon Ishinkin Gakkai Zasshi. (2010); 51: 131–136.\n\nBlanco MT, Sacristan B, Lucio L, et al:[Cell surface hydrophobicity as an indicator of other virulence factors in Candida albicans]. Rev Iberoam Micol. (2010); 27: 195–199.\n\nParamonova E, Krom BP, van der Mei HC, et al:Hyphal content determines the compression strength of Candida albicans biofilms. Microbiology. (2009); 155: 1997–2003.\n\nSchulz B, Kai W, Schmidt A, et al:Difference in virulence between fluconazole-susceptible and fluconazole-resistant Candida albicans in a mouse model. Mycoses. (2011); 54: e522–e530.\n\nMateus C, Crow SA Jr, Ahearn DG: Adherence of Candida albicans to silicone induces immediate enhanced tolerance to fluconazole. Antimicrob Agents Chemother. (2004); 48: 3358–3366.\n\nMorschhauser J, Barker KS, Liu TT, et al:The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog. (2007); 3: e164.\n\nCao Y, Dai B, Wang Y, et al:In vitro activity of baicalein against Candida albicans biofilms. Int J Antimicrob Agents. (2008); 32: 73–77." }	[ { "id": "894", "date": "15 Apr 2013", "name": "Malcolm Whiteway", "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 logic of this paper is reasonable – if rifampicin enhances biofilm formation in C. albicans, then treating medical implants with rifampicin could have unexpected negative consequences. The experiments themselves are straightforward and in general support the idea that rifampicin influences surface hydrophobicity in C. albicans, and ultimately has a moderate effect on biofilm formation assayed through essentially XTT detected activity on a plastic surface. Considerably more sophisticated assays of biofilms are possible, and a number of genes have been identified that are critical for standard biofilm formation. To really probe the impact of the drug on Candida biofilms, the authors could assess the impact of mutants on the process under study, do ultrastructural assessment of the biofilms, and do analysis of the effect of rifampicin on a variety of biofilm models. Thus, the impact of the paper would be considerably enhanced if the sophistication of the biofilm assay was greater.", "responses": [] }, { "id": "951", "date": "14 May 2013", "name": "Guilhem Janbon", "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 effect of rifampicin on adhesion and hydrophobicity of Candida albicans. This is an important issue because (1) this antibiotic is commonly used and (2) Candida albicans is prone to develop biolfilms that are resistant to a number of antifungal drugs. The authors convincingly show that cells treated with rifampicin make more germ tubes, change their hydrophobicity and become more adherent to fibronectin. They also demonstrate that these phenotypic changes are associated with an increased expression of the gene CSH1.However, it remains to be proved that these phenotypes and the increased expression are mechanistically linked. The analysis of CSH1∆ strain phenotype in presence of rifampicin could provide information concerning this matter.", "responses": [] }, { "id": "3209", "date": "28 Jan 2014", "name": "Dipshikha Chakravortty", "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 by Vogel et al. raises the very interesting and crucial problem of rifampicin coated catheters posing a threat due to Candida growth. Rifampicin by itself is not active against Candida, however various studies have shown that its combination with amphotericin B may enhance the anti-Candida activity.CommentsFig. 1D: Detection of fibronectin retention - This is very difficult to conclude from the given photograph. The photograph depicts crowded accumulation of Candida. Where are the \"Fibronectin fibres\"? Please provide high resolution photos, and provide evidence for the fibronectic fibres. Fig. 1E: \"Western blot of SC5314\"- this statement is not correct. Western blot or immunoblot should be detecting the protein, though in this figure the detection is of fibronectin - the readers will get confused as the sentence is not correct. Please provide the fibronectin addition as μg/ml rather than the percentage. It would be beneficial if the authors look into the status of fibronectic binding proteins in Candida's response to rifampicin. Fig. 3A: Densitometric analysis is required to appreciate the induction of CSH1 gene in response to rifampicin. Fig. 3B: For the western blot, housekeeping genes from Candida should be used as a loading control, and the densitometric analysis should be provided. The dose dependent response to rifampicin is required. How does rifampicin induce the CSH expression? What is the mechanism? The discussion should be strengthened by providing the probable mechanism. Is this phenomenon specific to rifampicin?", "responses": [] } ]	1	https://f1000research.com/articles/2-106
https://f1000research.com/articles/2-105/v1	08 Apr 13	{ "type": "Review", "title": "Advances in systemic therapy for advanced pancreatobiliary malignancies", "authors": [ "Thorvardur R Halfdanarson", "Sigurdis Haraldsdottir", "Mitesh J Borad", "Sigurdis Haraldsdottir", "Mitesh J Borad" ], "abstract": "Pancreatobiliary malignancies are relatively uncommon and the overall prognosis is poor. Treatment options for advanced disease are limited to systemic therapy for metastatic disease and a combination of systemic therapy and radiation therapy for locally advanced but unresectable tumors. There have been significant advances in the treatment of pancreatobiliary cancers in recent years but the prognosis for patient survival remains disappointingly poor. We review the current treatment options for locally advanced pancreatobiliary malignancies and highlight recent advances in systemic therapy, including novel approaches using targeted treatments.", "keywords": [ "Pancreatobiliary cancer", "unresectable tumors", "systemic therpay" ], "content": "Introduction\n\nPancreatobiliary malignancies are relatively uncommon malignancies that generally have a poor prognosis (Figure 1). In 2012, almost 42,000 new cases of pancreatic cancer and 10,000 new cases of gallbladder and bile duct cancer were expected in the USA1. The prognosis of patients with pancreatic cancer and intrahepatic cholangiocarcinoma is poor, with an estimated 5-year overall survival of 2–5%. Patients with extrahepatic bile duct cancer and gallbladder cancer have a slightly better survival, but the overall 5-year survival is still only 12–15%2. Worldwide, the mortality rates for bile duct cancer seem to have decreased slightly over recent decades, a trend that may in part be due to improved diagnostic modalities and more widespread use of the surgical removal of the gallbladder (cholecystectomy) for gallstones (these being a known cause of gallbladder cancer)3. Despite the observed improvements in prognosis, the majority of patients with pancreatobiliary carcinoma still present at an advanced stage where resection is not feasible2. Of all patients with newly diagnosed pancreatic cancer, almost half have metastatic disease at diagnosis, with an additional 22% having either node-positive disease or a large tumor invading adjacent organs (known as a T4 lesion)2. Bile duct carcinomas tend to be less advanced at presentation than pancreatic cancer, which probably explains the better prognosis to some extent. Other factors, such as differences in the genetic basis of these cancers, may provide further insight into the differences in outcomes. Further therapy following resection (adjuvant therapy) has been shown to improve the outcome of patients with pancreatic cancer. The best studied adjuvant therapies are systemic therapy for 6 months with gemcitabine and post-operative concurrent chemotherapy with gemcitabine and 5-fluorouracil but the optimal adjuvant therapy remains undefined. Although adjuvant chemotherapy or chemoradiotherapy for resected pancreatic cancer has been shown to be beneficial, most patients who undergo resection eventually succumb to the disease4–6. The role of adjuvant therapy for resected bile duct cancer is less certain and there is a dearth of well-conducted prospective studies on the subject. A recent phase III trial did not show conclusive evidence for the benefit of adjuvant chemotherapy following resection of periampullary adenocarcinoma7. After adjusting for other prognostic factors, a benefit of adjuvant therapy was observed. Multiple retrospective studies do, however, support the role of radiotherapy or chemoradiotherapy, although the benefits seem modest8–11. Two recent meta-analyses have also suggested that there may be benefit of adjuvant therapy12,13. The majority of patients will at some point be diagnosed with advanced disease, either at the time of first diagnosis or at a later stage once the cancer recurs. There is thus a great need for improvements in advanced therapy for these malignancies. This article will discuss palliative treatment options for pancreatobiliary malignancies from the standpoint of medical and radiation oncology, focusing on chemotherapy, radiotherapy or both. A discussion of the treatment of the symptoms of advanced pancreatobiliary malignancies such as pain management and treatment of biliary obstruction is outside the scope of this review14,15.\n\n\nPancreatic carcinoma\n\nMany patients with pancreatic cancer present with unresectable cancer and, in fact, only 10–20% of patients are deemed to be operative candidates16. For the remainder of patients, the outcome is bleak, with nearly all patients succumbing to their disease within 2 years of diagnosis. Patients with advanced locoregional (i.e. localized, nonmetastatic) disease have a median survival of 9–10 months, which is only a few months better than in patients with metastatic disease17. The optimal therapy for locally advanced pancreatic cancer is not known, but chemotherapy, radiation therapy and a combination thereof is frequently used. A small randomized trial reported improved survival and better quality of life (QOL) in patients treated with a combination of the DNA synthesis inhibitor 5-fluorouracil (5-FU) and radiation therapy18. Chemotherapy alone has also been shown to improve survival in patients with advanced pancreatic cancer when compared with the best supportive care19. Two studies evaluating the benefits of adding chemotherapy to radiation therapy yielded conflicting results, but a pooled analysis suggested a benefit from concurrent chemoradiotherapy compared with radiotherapy alone20–22. Two recent trials compared chemoradiotherapy with chemotherapy alone and came to a different conclusion, with one suggesting a benefit of adding radiotherapy and the other not23,24. The use of concurrent chemoradiation therapy for locally advanced pancreatic cancer is also supported by phase II studies25,26. Chemoradiotherapy was not found to be superior to chemotherapy alone in a recent systematic review, but the heterogeneity and small size of the included studies makes comparisons difficult27. It is worth mentioning that the prematurely closed Eastern Cooperative Oncology Group (ECOG) study 4201, which compared gemcitabine monotherapy with chemoradiation therapy using gemcitabine as a radiosensitizer followed by gemcitabine monotherapy, suggested a modest benefit of the combination therapy24. It seems that not all patients may benefit from the addition of radiotherapy, and the challenge is how best to identify those who may be helped with combination therapy.\n\nAn increasingly used approach is to initiate chemotherapy (induction therapy), and if there is no evidence of progression with new liver metastases after 2–3 months as visualized by CT scanning, patients are considered for concurrent chemoradiotherapy. The rationale for this approach is that a substantial proportion of patients will progress within this timeframe while on chemotherapy and the site of progression is frequently in the liver or elsewhere outside of the conventional radiation field. These patients would probably not have benefited from the addition of radiotherapy. It is likely that induction therapy selects out those patients who would be more likely to benefit from the addition of radiotherapy. This approach is supported by two recent retrospective studies and is increasingly being used in the USA28,29.\n\nIn summary, concurrent chemoradiation either upfront or preceded by 2–3 months of chemotherapy seems to be an appropriate standard for the management of locally advanced pancreatic cancer. The role of chemotherapy alone without radiation is less certain – but certainly a viable option for patients who either choose not to receive radiation therapy or have a contra-indication to such therapy. Recent studies have shown that complete loss of expression of the signal transduction protein SMAD4 is associated with a higher incidence of distant metastases and that tumors that retain SMAD4 expression are less likely to metastasize30,31. Determination of SMAD4 expression may have a role in guiding therapy, in which patients with tumors expressing SMAD4 could be considered for incorporation of locoregional therapy into the treatment plan.\n\nMetastatic pancreatic carcinoma is a uniformly fatal disease, and systemic chemotherapy is only modestly effective in prolonging survival and maintaining quality of life. Pancreatic cancer most commonly metastasizes to the liver (Figure 2). The results of several key phase III trials in first-line therapy for pancreatic cancer are reported in Table 1. Gemcitabine, which is a nucleoside analog, either alone or in combination, has been the mainstay of therapy for more than a decade. Gemcitabine was reported to be more effective than 5-fluorouracil in a landmark trial published in 1997 that established gemcitabine as the chemotherapeutic agent of choice for advanced pancreatic cancer32. Although gemcitabine only modestly prolonged survival (median survival 5.65 vs. 4.41 months), the effect on the clinical benefit response, a composite of measurements of pain, performance status, and weight loss was more marked. Since then, numerous trials have explored the addition of other drugs in combination with gemcitabine, generally with unimpressive results. Individual trials and meta-analyses have shown benefits of gemcitabine-based combinations over gemcitabine monotherapy, but the magnitude in terms of clinical improvements is small and in some cases of questionable clinical significance19,33. The benefits from combination therapy may be more pronounced in patients with good performance status33,34. Performance status reflects the physical activity of patients and their ability to care for themselves and is commonly graded on a scale of 0 to 4 (the Eastern Cooperative Oncology Group scale) where patients with a performance score of 0 have no restrictions from their malignancy and patients with a score of 4 are completely disabled, bedbound and unable to carry out any self care. Gemcitabine monotherapy remains an acceptable treatment option, especially for patients with impaired performance status.\n\nMultiple small liver metastases (yellow arrows) and a larger metastasis in the left lobe of the liver (red arrow).\n\n*Statistically significant with p<0.05\n\nTwo commonly used agents added to gemcitabine are capecitabine and erlotinib. Capecitabine, an oral fluoropyrimidine, when added to gemcitabine (GEM-CAP) was shown to improve progression-free survival with a nonsignificant trend towards improvement in overall survival compared with use of gemcitabine alone in a meta-analysis and is a reasonable combination for patients with good performance status35. As ~90% of pancreatic cancers have an activating mutation in the GTPase KRAS protein36, significant effort has been put towards targeting the RAS–RAF–MEK–ERK pathway along with other pathways. The addition of erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR), to gemcitabine, resulted in a minimal but statistically significant improvement in overall survival by 2 weeks (median overall survival 6.2 vs. 5.9 months), although at the cost of increased toxicity37. The observed survival increase with the addition of erlotinib is of questionable clinical significance, but the trial is remarkable for the fact that it is the first and only phase III trial to show a benefit from adding a targeted agent to gemcitabine. The gemcitabine–erlotinib combination was subsequently approved for use in the USA but not in Europe. Other recent studies did not, however, show any benefit of adding bevacizumab, sorafenib, axitinib or cetuximab to gemcitabine38–41. Farnesyltransferase inhibitors targeting the Ras pathway have not proven to be successful in management either42. A recent phase I/II study of gemcitabine combined with the mitotic inhibitor nab-paclitaxel yielded promising results, where patients with increased levels of stromal ‘secreted protein acidic and rich in cysteine’ (SPARC) had a greater degree of benefit compared with those patients who had lower stromal SPARC (overall survival of 17.8 vs. 8.1 months, P=0.0431)43. The results of a larger phase III trial comparing this combination with gemcitabine monotherapy were presented at the Gastrointestinal Cancers Symposium in January 201344. In this trial 861 patients were randomized and received either weekly nab-paclitaxel with gemcitabine or gemcitabine alone. Overall response rates (23% vs. 7%), progression-free survival, PFS (5.5 vs. 3.7 months) and overall survival, OS (8.5 vs. 6.7 months) were all significantly improved in the combination arm. Grade 3 or more adverse events more commonly seen in the combination arm included neutropenia (38% vs. 27%), fatigue (17% vs. 7%) and neuropathy (17% vs. 1%) but overall the combination was well tolerated. This combination is therefore likely to get approval by the FDA in the coming months. The role of SPARC in patient selection will be further elucidated from the phase III data. The results of a phase III trial with the combination of masitinib (multityrosine kinase inhibitor) and gemcitabine are also expected in 2013, where, according to a press release from the pharmaceutical company AB Science, two subgroup populations had increased overall survival by 6 and 2.7 months, characterized by a genetic biomarker and patients with cancer pain, but not in the overall patient population (http://www.ab-science.com/file_bdd/1351622639_abscienceresultph3pancreasvdefuk.pdf).\n\nThe microenvironment within pancreatic cancers is frequently hypoxic relative to normal pancreatic tissue and the hypoxic properties of pancreatic cancers are being exploited in clinical trials45. A recent randomized phase II study of the hypoxia-targeted prodrug TH-302 with gemcitabine in previously untreated patients showed an improvement in progression-free survival (PFS) with the combination when compared with gemcitabine alone46. These results will need to be confirmed in a larger trial.\n\nWhole-genome exome sequencing of pancreatic adenocarcinomas has recently been completed and should equip us with more drug targets in the future47,48.\n\nThe role of gemcitabine as an essential component of the chemotherapy for pancreatic cancer has recently been called into question. A phase III trial compared a multi-agent regimen of 5-fluorouracil, leucovorin, irinotecan and the DNA synthesis inhibitor oxaliplatin (FOLFIRINOX) with gemcitabine alone. Overall survival was markedly improved in the FOLFIRINOX group compared with the gemcitabine group. The median overall survival of patients receiving FOLFIRINOX was 11.1 months vs. 6.8 in the gemcitabine group. FOLFIRINOX was more likely to result in adverse events, and febrile neutropenia was seen in 5.4% of patients. Despite higher toxicity, fewer patients in the FOLFIRINOX group had deterioration of their quality of life at 6 months (66% vs. 31%). Fewer than 40% of patients in the trial had tumors located in the head of the pancreas compared with 60–70% of all patients presenting with pancreas cancer. It is therefore unclear whether the results are applicable to most patients with pancreatic cancer, and biliary obstruction with jaundice would certainly preclude giving irinotecan in many cases. However, a subgroup analysis indicated a similar benefit to patients with tumors outside the head of the pancreas. Subgroup analyses also showed that patients older than 65 years and patients with an ECOG performance status of 1 also benefited from more aggressive therapy. Furthermore, patients receiving FOLFIRINOX were less likely to experience a decline in quality of life compared with patients on gemcitabine. The encouraging results have led to the acceptance of FOLFIRINOX chemotherapy for patients with metastatic pancreatic cancer, especially those with good performance status.\n\nThe prognosis for patients progressing after first-line (i.e. initial) therapy is very poor, and no standard treatment approach exists. These patients should be considered for enrollment on clinical trials whenever possible. In patients previously treated with gemcitabine, subsequent second-line therapy with oxaliplatin, 5-fluorouracil and leucovorin (OFF) has been shown to improve overall survival modestly when compared with best supportive care alone49. FOLFIRINOX may be an option for younger patients with good performance status who have not received such therapy previously, but prospective studies are lacking50. Other agents such as taxanes and irinotecan may have activity in pretreated patients and can be considered in selected patients51–53. No prospective data exist regarding therapy for patients progressing after first-line FOLFIRINOX, but gemcitabine, either alone or in combination with other agents such as capecitabine or nab-paclitaxel may be used if performance status allows.\n\n\nBiliary carcinoma (intrahepatic and extrahepatic cholangiocarcinoma and gallbladder carcinoma)\n\nCarcinomas of the biliary system are uncommon malignancies and frequently unresectable at the time of diagnosis54. Given the relative rarity of these cancers, very few large treatment trials have been performed and much of the evidence guiding treatment decisions stems from retrospective and epidemiological studies.\n\nPatients with locally advanced biliary carcinoma may benefit from concurrent chemoradiation therapy for palliative purposes, and such treatment is currently suggested as one of the treatment options in published guidelines55,56. Fluoropyrimidines such as 5-fluorouracil or capecitabine are frequently used as radiosensitizing agents. Other locoregional treatment options for locally advanced but unresectable bile duct cancer include radiation brachytherapy (in which the radiation source is placed at very close proximity within the tumor) and photodynamic therapy (in which light and photosensitizing agents are used to kill the cancer cells)57. Despite aggressive local therapy, locoregional failures are frequent58. For those patients who do not desire to have radiation therapy or where such therapy is contraindicated, chemotherapy alone is recommended. Patients with intrahepatic cholangiocarcinoma limited to the liver may be considered for liver-directed therapy such as hepatic artery chemoembolization or radioembolization59,60. Best supportive care remains an option for patients with poor performance status and for patients who do not want any anti-cancer therapy. Such patients may be significantly helped using procedures aimed at improving or preserving quality of life, including biliary drainage and aggressive pain and symptom control.\n\nSystemic therapy (chemotherapy) is frequently used in the management of metastatic biliary carcinoma, but again there is a dearth of well-conducted randomized trials owing to the relative rarity of this malignancy. A small study of patients with either pancreatic cancer or biliary cancer showed that chemotherapy resulted in improved survival and quality of life compared with using no such therapy61. A similar study showed that the combination of gemcitabine and oxaliplatin (GEMOX) was superior to best supportive care in terms of overall and progression-free survival in patients with advanced carcinoma of the gall bladder62. Gemcitabine, either alone or in combination with other chemotherapeutic agents, is commonly used for patients who wish to receive chemotherapy, but multiple other regimens not containing gemcitabine do exist63–70. Gemcitabine has been shown to be both safe and effective, even in elderly patients65. Gemcitabine has been used in combination with platinum agents and fluoropyrimidines in multiple small phase II trials, but it was not until 2009 that a regimen that can be considered a standard emerged. The ABC-02 trial was a multicenter randomized phase III trial conducted in the United Kingdom that compared gemcitabine monotherapy with gemcitabine combined with low-dose cisplatin71. In this trial, 410 patients with locally advanced or metastatic biliary cancer were randomized to receive either cisplatin (25 mg/m2) with gemcitabine (1000 mg/m2) on days 1 and 8, every 3 weeks for eight cycles or gemcitabine alone (1000 mg/m2) on days 1, 8, and 15, every 4 weeks for six cycles for up to 24 weeks. Patients on the combination therapy arm had both longer overall survival (11.7 vs. 8.1 months) and progression-free survival (8 vs. 5 months). Both regimens had acceptable toxicities. The results from a smaller Japanese phase III study support the superiority of the gemcitabine–cisplatin combination compared with gemcitabine alone70. The combination of gemcitabine and cisplatin can thus be considered a reasonable standard for first-line therapy of metastatic biliary cancer in patients with good performance.\n\nTrials evaluating targeted agents (such as key receptors and signaling proteins) in advanced hepatobiliary carcinoma have largely been disappointing and, to this date, no clear role for such therapy exists.\n\nVascular endothelial growth factor (VEGF, a signaling protein that regulates blood vessel growth) has been found to be overexpressed in cholangiocarcinoma as well as many other tumors72. Other mutations such as activating mutations in BRAF (22%) and KRAS (45%) have been reported73, and these findings have led to several phase I/II clinical trials with targeted agents. The angiogenesis inhibitor bevacizumab was given with the EGFR inhibitor erlotinib in a phase II trial where 9 of 53 patients had partial responses but only 6 (12%) were sustained, and the duration of best response was similar to that seen with chemotherapy (average of 7.6 months)74. A phase II trial where bevacizumab was given with GEMOX found it to be safe and effective (41% with partial response)75. GEMOX paired with cetuximab in a phase II trial produced similar results (63% with objective response)76.\n\nDespite promising phase II trials, targeted agents have not produced any breakthrough results, and phase III trials are needed to evaluate whether combinations including targeted drugs are superior to standard chemotherapy. GEMOX with and without erlotinib was compared in a phase II trial and, although erlotinib did improve response rates (30% vs. 16%), it did not significantly affect progression-free survival (median 5.8 vs. 2 months), which was the primary endpoint73. In an unplanned analysis of cholangiocarcinomas only (the trial included gallbladder and ampullary cancers as well), the progression-free survival was significantly better in the erlotinib group (5.9 vs. 3 months), but the erlotinib treatment had more toxicities77. Other targeted agents that have been evaluated in advanced biliary cancers include the tyrosine kinase inhibitor sunitinib and the MEK 1/2 (MAP kinase kinase 1/2) inhibitor selumetinib78,79. The role of the newer agents needs to be defined in larger trials.\n\nSecond-line therapy is commonly offered to patients progressing after initial chemotherapy, but the data supporting such therapy are very limited. A recent retrospective study suggested that there are modest benefits of second-line therapy, with a possible advantage of doublet therapy (in which two agents are co-administered) compared with single-agent therapy80. It is reasonable to offer patients with preserved performance status a second-line systemic therapy, and an attempt should be made to choose agents from a class different from that used in the first-line setting given that the cancer cells may have become resistant to the previously used class of drugs. All such patients should be considered for participation in a clinical trial if available.\n\n\nConcluding remarks\n\nAdvanced pancreatic and biliary cancer remain difficult to treat, and responses are usually short-lived and the prognosis poor. Combination therapies for metastatic pancreatic cancer such as using the DNA synthesis inhibitors FOLFIRINOX or gemcitabine with the cell-division inhibitor nab-paclitaxel appears more effective than gemcitabine alone, but single-agent gemcitabine remains an appropriate option for the elderly and for patients with impaired performance status. Second-line therapy for advanced pancreatic cancers is not very effective, and better treatment options are clearly needed. Using gemcitabine and cisplatin is a reasonable first-line therapy for advanced biliary cancer, but, as with pancreatic cancer, second-line therapy has yielded disappointing results, and the prognosis remains poor. It is unlikely that further refinements of cytotoxic chemotherapy regimens will result in substantial improvement of prognosis. There is an unmet need for better treatment options and future improvements are likely going to be secondary to new targeted agents and not cytotoxic chemotherapy.", "appendix": "Author contributions\n\n\n\nThe initial drafting of the manuscript was done by TRH. All authors were involved in the design, identifying and retrieving pertinent literature and revision of the draft manuscript. All authors have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSiegel R, Naishadham D, Jemal A: Cancer statistics, 2012. CA Cancer J Clin. 2012; 62: 10–29. PubMed Abstract \| Publisher Full Text\n\nRies LAG, Young JL, Keel GE, et al.: SEER Survival Monograph: Cancer Survival Among Adults: U.S. SEER Program, 1988–2001, Patient and Tumor Characteristics. National Cancer Institute, SEER Program, NIH Pub. No. 07–6215, Bethesda, MD, 2007. Reference Source\n\nRandi G, Malvezzi M, Levi F, et al.: Epidemiology of biliary tract cancers: an update. Ann Oncol. 2009; 20: 146–159. 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Jpn J Clin Oncol. 2012; 42(11): 1043–1053. PubMed Abstract \| Publisher Full Text\n\nRiechelmann RP, Townsley CA, Chin SN, et al.: Expanded phase II trial of gemcitabine and capecitabine for advanced biliary cancer. Cancer. 2007; 110(6): 1307–1312. PubMed Abstract \| Publisher Full Text\n\nKnox JJ, Hedley D, Oza A, et al.: Combining gemcitabine and capecitabine in patients with advanced biliary cancer: a phase II trial. J Clin Oncol. 2005; 23(10): 2332–2338. PubMed Abstract \| Publisher Full Text\n\nAndre T, Reyes-Vidal JM, Fartoux L, et al.: Gemcitabine and oxaliplatin in advanced biliary tract carcinoma: a phase II study. Br J Cancer. 2008; 99(6): 862–867. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNehls O, Oettle H, Hartmann JT, et al.: Capecitabine plus oxaliplatin as first-line treatment in patients with advanced biliary system adenocarcinoma: a prospective multicentre phase II trial. Br J Cancer. 2008; 98(2): 309–315. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOkusaka T, Nakachi K, Fukutomi A, et al.: Gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer: a comparative multicentre study in Japan. Br J Cancer. 2010; 103(4): 469–474.PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValle J, Wasan H, Palmer DH, et al.: Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010; 362(14): 1273–1281. PubMed Abstract \| Publisher Full Text\n\nPark BK, Paik YH, Park JY, et al.: The clinicopathologic significance of the expression of vascular endothelial growth factor-C in intrahepatic cholangiocarcinoma. Am J Clin Oncol. 2006; 29(2): 138–142. PubMed Abstract \| Publisher Full Text\n\nTannapfel A, Sommerer F, Benicke M, et al.: Mutations of the BRAF gene in cholangiocarcinoma but not in hepatocellular carcinoma. Gut. 2003; 52(5): 706–712. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLubner SJ, Mahoney MR, Kolesar JL, et al.: Report of a multicenter phase II trial testing a combination of biweekly bevacizumab and daily erlotinib in patients with unresectable biliary cancer: a phase II Consortium study. J Clin Oncol. 2010; 28(21): 3491–3497. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu AX, Meyerhardt JA, Blaszkowsky LS, et al.: Efficacy and safety of gemcitabine, oxaliplatin, and bevacizumab in advanced biliary-tract cancers and correlation of changes in 18-fluorodeoxyglucose PET with clinical outcome: a phase 2 study. Lancet Oncol. 2010; 11(1): 48–54. PubMed Abstract \| Publisher Full Text\n\nGruenberger B, Schueller J, Heubrandtner U, et al.: Cetuximab, gemcitabine, and oxaliplatin in patients with unresectable advanced or metastatic biliary tract cancer: a phase 2 study. Lancet Oncol. 2010; 11(12): 1142–1148. PubMed Abstract \| Publisher Full Text\n\nLee J, Park SH, Chang HM, et al.: Gemcitabine and oxaliplatin with or without erlotinib in advanced biliary-tract cancer: a multicentre, open-label, randomised, phase 3 study. Lancet Oncol. 2012; 13(2): 181–188. PubMed Abstract \| Publisher Full Text\n\nBekaii-Saab T, Phelps MA, Li X, et al.: Multi-institutional phase II study of selumetinib in patients with metastatic biliary cancers. J Clin Oncol. 2011; 29(17): 2357–2363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYi JH, Thongprasert S, Lee J, et al.: A phase II study of sunitinib as a second-line treatment in advanced biliary tract carcinoma: a multicentre, multinational study. Eur J Cancer. 2012; 48(2): 196–201. PubMed Abstract \| Publisher Full Text\n\nWalter T, Horgan AM, McNamara M, et al.: Feasibility and benefits of second-line chemotherapy in advanced biliary tract cancer: A large retrospective study. Eur J Cancer. 2013; 49(2): 329–35. PubMed Abstract \| Publisher Full Text" }	[ { "id": "889", "date": "15 Apr 2013", "name": "Madappa Kundranda", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: Succinct. Content: Very good compilation of the latest and most appropriate studies including the recently presented MPACT study in advanced pancreatic cancer.Conclusions: Balanced.", "responses": [] }, { "id": "902", "date": "22 Apr 2013", "name": "Christina Wu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 concise and appropriate for the content. The abstract summarizes the paper well. The review covers the phase II and III clinical trials for advanced hepatobiliary cancers. The conclusions are well balanced and support the findings of the paper.", "responses": [] } ]	1	https://f1000research.com/articles/2-105
https://f1000research.com/articles/2-28/v1	30 Jan 13	{ "type": "Clinical Practice Article", "title": "Management of acute and post-operative pain in chronic kidney disease", "authors": [ "Malvinder S Parmar", "Kamalpreet S Parmar", "Kamalpreet S Parmar" ], "abstract": "Chronic kidney disease is common and patients with many co-morbid conditions frequently have to undergo surgical procedures and, therefore, require effective pain management. The pharmacokinetics of various analgesic agents are not well studied in patients with chronic kidney disease and the risk of accumulation of the main drug or their metabolites, resulting in serious adverse events, is a common scenario on medical and surgical wards. It is common for these patients to be cared for by 'non-nephrologists' who often prescribe the standard dose of the commonly used analgesics, without taking into consideration the patient's kidney function. It is important to recognize the problems and complications associated with the use of standard doses of analgesics, and highlight the importance of adjusting analgesic dosage based on kidney function to avoid complications while still providing adequate pain relief.", "keywords": [ "Illustrative clinical scenario:- an example to illustrate the problem." ], "content": "\n\nIllustrative clinical scenario:- an example to illustrate the problem.\n\nA 63-year-old man with diabetes and end stage renal disease on maintenance continuous ambulatory peritoneal dialysis was admitted to the hospital with gangrene of the left foot and underwent left below-knee amputation under spinal anesthesia. Postoperatively, he was given meperidine (Demerol) 50–75 mg intramuscularly every 3 hr, as required, by the orthopedic surgeon, but this was found to be ineffective in controlling pain. The anesthetist ordered morphine 10–15 mg intramuscularly or subcutaneously every 3 hr as required (pro re nata) in the evening. He received 325 mg of meperidine in the first 24 hr and additional 200 mg of meperidine next day (total 525 mg) and 55 mg of morphine within the first 48 hr after surgery. 48 hr after surgery, he was found to be confused and somnolent. On examination the patient was drowsy and somnolent, but arousable with twitching of arms, and complained of mild pain at the stump site when awakened. He had labored breathing with a respiratory rate of 8 breaths per minute. An arterial blood gas showed pH of 7.19, pCO2 of 60, pO2 of 86 and oxygen saturation of 95%. His mental state improved transiently with intravenous naloxone, confirming narcotic effect. How could this situation have been prevented while effectively providing pain relief?\n\nChronic kidney disease (CKD) is common1 and is regularly accompanied with various co-morbidities. Often these patients with various co-morbidities undergo operative procedures and require analgesics. The most-responsible physician providing the care to these individuals during their hospitalization is often a non-nephrologist. Opioids are commonly used in the management of post-operative pain, and often a standard dose is used by the health-care providers without taking into consideration kidney function2. In North America, morphine and meperidine (Demerol) still remain the commonly used parenteral agents, and acetaminophen with codeine (Tylenol #3) a commonly used oral agent for management of post-operative pain in patients with3 and without renal failure. Prolonged narcotic effects and ventilatory depression due to morphine and other opioids have been known for over a century in patients with kidney failure4–10 and the various contributing factors are outlined in Table 1. Despite the increased potential for respiratory depression, especially in patients with CKD, life-threatening cases of opiate toxicity continue to occur not only in patients with kidney disease11, but also in patients without kidney disease12,13. It is important to highlight the issue of central nervous system (CNS) and respiratory depression with opioid analgesics in patients with CKD and formulate strategies to prevent these complications, while providing effective pain relief.\n\n* A118G polymorphism protects against M6G-related opioid toxicity28.\n\n§ Routes that use first pass metabolism results in higher production of metabolites than those that bypass it.\n\n¶ P-glycoprotein inhibitors increase uptake of M6G across blood brain barrier16,17.\n\nº Concomitant antibiotics by altering bacterial flora reduces bacterial glucuronidase, resulting in reabsorption of M6G.\n\n@ Extensive 2D6 metabolism may occur in up to 1/3rd of patients of east African heritage – increasing the risk of opioid overdose from codeine.\n\nIn addition to the post-operative pain, patients with CKD may have underlying chronic pain that is often multi-factorial14 i.e., ischemic pain from peripheral vascular disease, neuropathic pain of polyneuropathy (diabetes), bone pain from osteoporosis or dialysis-associated amyloidosis, and musculoskeletal pain that may or may not be related to kidney disease. Post-operative pain is often nociceptive, whereas chronic pain may be nociceptive, neuropathic or mixed. It is also important to recognize that depression is common in CKD15 and that it may complicate the management and patient’s ability to cope with pain16.\n\n\nAssessment of pain\n\nAssessment of pain requires detailed history to elucidate the cause of pain, its location, nature (acute, chronic or acute and chronic), intensity and its impact on physical, social and emotional functioning17.\n\n\nSpectrum of pain\n\nAcute pain is a protective and an adaptive response to injury, whereas chronic pain or the persistence of pain beyond the healing phase is maladaptive18. Acute pain results from direct stimulation of sensory neurons, called nociceptors. Nociceptors have two types of axons, the rapidly conducting thinly myelinated Aδ fibers that mediate the initial phase of acute pain that is extremely sharp pain, and the more slowly conducting unmyelinated C fibers mediate the second phase of acute pain that is more prolonged and less intense following injury. Nociceptors can be stimulated by mechanical, thermal, chemical and inflammatory stimuli18.\n\nPain lasting longer than 3 months or beyond the duration required for complete tissue healing is called chronic pain. It may be nociceptive, resulting from prolonged tissue injury with persistent activation of nociceptors, neuropathic resulting from a lesion or a process affecting the somatosensory system or mixed processes.\n\n\nIntensity of pain\n\nPain intensity may be measured by one of the major pain rating scales including verbal, numerical and visual analogue scales. However, a numerical rating scale comprising of a range of numbers from 0 to 10, is the most widely used system in clinical practice and is generally based on a subjective 10-point scoring system, where 0 denotes the absence of pain and 10 the worst pain imaginable19.\n\n\nPain management\n\nEffective pain management requires a multifaceted approach with an understanding of the type (nociceptive, neuropathic or mixed), underlying cause, assessment of intensity and duration of pain17. In addition, assessment of concomitant medications and co-morbidities is important when selecting analgesic agent(s). Adjuvant analgesics and non-pharmacological methods of pain control are important to consider in such circumstances17. We review the strategies to manage acute pain in CKD with main focus on the use of opioids since most of the challenges occur in the use of opioids in CKD.\n\nIn 2005, a modified World Health Organization (WHO) step-wise approach was proposed for pain management in patients with CKD20 (Figure 1) and this approach was found to be effective in acute pain control in >90% of hemodialysis patients, at least short-term, in a 4-week study21. This approach is important to follow to achieve ‘balanced analgesia’ by effectively utilizing ‘co-analgesics’ (Box 1).\n\n*Non-steroidal anti-inflammatory agents, including cyclo-oxygenase-2 inhibitors should be avoided, except for acute pain with close clinical observation.\n\nOpiate: Naturally occurring alkaloid i.e., morphine or codeine.\n\nOpioid: Any natural or synthetic compound that has morphine-like properties.\n\nBalanced analgesia: Principle of combining different classes of analgesics to optimize efficacy while minimizing side-effects.\n\nCo-analgesic: A drug administered alongside opioids enabling a reduction in opioid dose.\n\nAdjuvant: A substance that helps and enhances the pharmacologic effects of an agent (analgesic).\n\n\nNon-opioid analgesics\n\nAcetaminophen: Has potent analgesic and antipyretic properties and is effective in various acute and chronic painful syndromes. It is the analgesic of choice in the elderly and in patients with kidney disease22 and should be considered in all patients (except in patients with hepatic insufficiency) experiencing pain, regardless of pain level, until pain is relieved or up to a maximum dose of 3,000 mg a day23 or 2.6 g/d in high-risk patients24 (malnourished or alcoholic). It often does not require dose adjustment in CKD but some authors recommend increasing dosing interval from every six to eight hours when GFR is <10 mL per minute25. Acetaminophen is underutilized in the peri-operative period and when prescribed it is often in an \"on demand\" basis rather than \"by the clock\". It’s important to prescribe at least half of total daily dose \"by the clock\"17 e.g., Acetaminophen 500 mg four times a day and remaining dose could be given \"on demand\" or in combination with other agents.\n\nAnti-inflammatory agents: Nonsteroidal anti-inflammatory drugs (NSAIDs) or cyclo-oxygenase (COX-2) inhibitors block prostaglandin synthesis and, when used as co-analgesic, reduce opioid need by 30–50%. However, because of their gastro-intestinal and cardio-renal side effects, they should be avoided for prolonged use in CKD, as these agents may decrease renal blood flow and may precipitate acute renal failure and can cause life-threatening hyperkalemia. However, if used, it should be for short term management (3 to 7 days) of acute pain and consider short-acting agents26 with careful monitoring of renal function and serum potassium.\n\n\nAdjuvant analgesics\n\nAdjuvant analgesics are medications with a primary indication other than pain that may possess analgesic activity or modify pain response. These are often helpful for management of chronic neuropathic pain. Examples include cabamezapine, gabapentin, and pregabalin. These agents are not required for management of acute pain. However, CKD patients may have associated underlying chronic neuropathic pain that could affect acute pain response or management. Therefore, some patients may already be taking these agents for control of their 'chronic pain’ and these agents may be stopped inadvertently in the peri-operative period. If this is the case, adjuvant analgesics should be resumed when possible, with considerations being taken for the possible drug-drug interactions when prescribing opioid analgesics for acute pain.\n\n\nOpioids\n\nThe relative potency of commonly used opioids is shown in Figure 1. As most opioids or their metabolites are excreted by the kidneys, dosage adjustment is often required when estimated glomerular filtration rate (eGFR) falls below 50 ml/min or during stages 3b, 4 and 5 of CKD, and for patients on dialysis (Table 2)27,28. In addition to CNS and respiratory depression, constipation is a common side effect that is important to identify and manage, especially in patients on peritoneal dialysis, as constipation is the common cause of catheter malfunction29, and appropriate laxatives should be used in conjunction with treatment.\n\nMorphine: Morphine is the most studied opiate in kidney disease. It is primarily an agonist at the µ-receptors with pain relief at µ1-receptors, and causes respiratory depression and constipation through its agonist action at µ2-receptors14. Morphine is metabolized in the liver to several metabolites30. Of these diamorphine and morphine-3-glucuronide (M3G) do not bind to opiate receptors, whereas normorphine and morphine-6-glucuronide (M6G) do, with M6G being approximately 10-fold more potent than morphine31. All metabolites are excreted in the urine along with about 10% of the parent compound. M6G accumulates in kidney failure and causes CNS and respiratory depression32. M6G crosses the blood brain barrier (BBB) slowly, and once it crosses the BBB, the CNS effects may be prolonged and may persist after stopping morphine or after hemodialysis, as the M6G slowly re-equilibrates across the BBB back into systemic circulation6. Transport of M6G across the BBB is an active process mediated by 170-kd P-glycoprotein. P-glycoprotein is an ATP-dependent carrier system that acts as an efflux pump to transport chemicals or metabolites from endothelial cells to vascular compartment and inhibition of this results in an increase of M6G in the brain33,34. The analgesic and respiratory depressant effects are related to serum concentrations of both morphine and M6G31. A dosage reduction of 25%, 50% and 75% is recommended in patients with mild (stage 3), moderate (stage 4) and severe kidney failure (stage 5) respectively. However, 20–25% of patients may tolerate the full dose of morphine without ill-effects because of A118G polymorphism35,36.\n\nMeperidine: Normeperidine, an active metabolite of meperidine, is neurotoxic and accumulates in renal failure and may cause a range of neuroexcitatory effects ranging from irritability to agitation to seizures37. Although normeperidine is removed by hemodialysis38, its regular administration is contraindicated in patients with any degree of renal failure39.\n\n(Dextro)Propoxyphene: It is no longer available in North America and its metabolite norpropoxyphene is excreted by the kidneys and accumulates in renal failure39. This results in CNS and respiratory depression, cardiac conduction disturbances and hypoglycemia. Both the parent drug and its metabolite are not removed by hemodialysis and is contraindicated in patients with renal failure39.\n\nHydromorphone: Hydromorphone is an analogue of morphine with shorter duration of action and with excellent efficacy in moderate to severe pain. It is five to seven times potent than morphine. It is eliminated by both hepatic (60%) and renal routes40. Hydromorphone doesn’t accumulate in renal failure because of its rapid conversion to its less-potent metabolite hydromorphone-3-glucuronide (H3G) that accumulates in renal failure but is effectively removed by hemodialysis41. However, typical opioid adverse effects have been reported in patients with renal failure42, but experience suggests efficacy without excess toxicity, if monitored carefully43.\n\nCodeine/Dihydrocodeine: Codeine is metabolized by the liver to a variety of active metabolites that are renally excreted44. The half-life of codeine is prolonged 5-fold in hemodialysis patients45. Codeine and its metabolites accumulate in renal failure and can cause hypotension and CNS and respiratory depression46,47. A similar elimination pathway is proposed for elimination of dihydrocodeine, although this has not been fully evaluated39. Dihydrocodeine can cause prolonged narcosis after therapeutic doses in patients with acute48 or chronic renal failure49. Therefore, these agents should be used with caution in patients with renal failure. A reduction in dose by 50% is suggested for codeine in renal failure39,45 and chronic use should be avoided.\n\nOxycodone: Oxycodone is a strong opioid with a higher oral bioavailability. It is metabolized by the liver to active metabolites – noroxycodone and oxymorphone50. Approximately 19% of the drug is excreted unchanged in the urine. The clearance of oxycodone and its metabolites is reduced in renal failure50 and results in an increase in plasma concentration by 50% and prolongation of its half-life by an hour50. There is lack of consensus of its use in CKD because of varied case reports of toxicity and good tolerance. When required, use cautiously and start with a lower dose51.\n\nBuprenorphine: Buprenorphine is a long-acting semi-synthetic partial agonist with the advantage of having less respiratory depression and hypotension52,53 but has a ceiling effect54,55. There is no significant difference in the clearance of the drug in patients with normal or impaired renal function. It has limited oral bioavailability because of an extensive first-pass metabolism. Sublingual, transdermal or parenteral administration is required. It is extensively metabolized by the liver with less than 30% renal excretion. The metabolite norbuprenorphine may accumulate in renal failure that has minor analgesic activity. Buprenorphine is 96% protein bound so is not dialyzable56.\n\nFentanyl, Alfentanil, and Sufentanil: Fentanyl, Alfentanil and Sufentanil are potent opioid receptor agonists, with a rapid onset of action and shorter duration of action. Fentanyl is rapidly metabolized in liver to inactive metabolites. Less than 10% of the parent drug is excreted in urine with no significant accumulation in CKD, but there is considerable inter-patient variability in fentanyl pharmacokinetics when given as a continuous infusion or via transdermal route. However, no dosage modification is necessary in patients with renal failure when fentanyl is given as a bolus. However, prolonged sedation is observed in critically ill patients when fentanyl is given as a continuous infusion, as its half-life increases to 25 hr39 due to saturation of its distribution sites, independent of renal function. Accumulation of fentanyl may occur when given by infusion39 or transdermal patch. Its clearance may be altered in advanced kidney disease57 and prolonged sedation and ventilatory depression have been observed in patients with end stage renal disease following fentanyl infusion39. Fentanyl is highly protein bound (80–86%), has a high volume of distribution, and removal by hemodialysis is negligible58,59. Opioid-naïve patients should not be initiated on transdermal fentanyl because of its variable absorption.\n\nTramadol: Tramadol is an opioid analgesic that also inhibits serotonin and noradrenaline reuptake. It is extensively metabolized by the liver and 30% of the parent drug and 60% of active metabolites are excreted in the urine60. It is effective for both nociceptive and neuropathic pain and has the advantage of less sedation and respiratory depression compared to other opiates. A common side effect is nausea. A rare but important side effect is seizures, especially in patients taking medications that lower the seizure threshold, like selective serotonin reuptake inhibitors (SSRI). In addition, tramadol may precipitate serotonin syndrome61 (a potentially life-threatening drug-interaction manifested by excess serotonin effect resulting in cognitive, autonomic and somatic effects) in patients taking SSRI62. In advanced CKD, the elimination half-life of tramadol may double60 and the dose should be decreased to 100 mg every 12 hr in patients with eGFR of 30 ml/min and 50 mg every 12 hr when eGFR is <10 ml/min. Tramadol is significantly removed by dialysis and should be administered after hemodialysis on dialysis days63.\n\nMethadone: Methadone is a synthetic opioid with five to 10 times the potency of morphine, is highly protein bound (70–87%) and has a half-life of 30 hr. Approximately 20% after a single dose is excreted unchanged and about 30% as inactive metabolites in the urine39. There is a 3-fold increase in the excretion of metabolites on chronic dosing, suggesting enhanced metabolism64. The removal by hemodialysis is insignificant65. Although in 2 patients with CKD the plasma concentrations were no higher than patients with normal renal function66, there is a well-described risk of accumulation and toxicity with methadone, even in patients with normal renal function, so experienced specialist supervision is warranted. A dose reduction of 50% to 75% is suggested in patients with renal failure, but it is difficult to use in patients with CKD because of its long half-life and wide inter-individual variation in clearance65. Methadone requires a specialist in pain management with a special license to prescribe it, and is often not a practical choice for acute pain management.\n\n\nSummary\n\nPatients with CKD have a high burden of co-morbidities that modulate pain response, and when these individuals undergo operative procedures, standard orders for pain control can be written without taking into consideration the patient's kidney function. Unfortunately, the data on the effects and tolerability of various analgesic agents are limited in such individuals and, given the lack of data, the recommendations are mainly based on expert opinion17,67 in patients with CKD with68 and without dialysis43. The general principles of pain assessment and management should be adopted and analgesia should be prescribed incorporating the modified WHO analgesic ladder20 in patients with CKD, with close and frequent monitoring for side effects of drug or its metabolite accumulation. The nephrologist and a pharmacist should be involved in the management of these patients. The non-opioid analgesics should be used early and ‘by the clock’, rather than ‘on-demand’, and the need for opioid analgesics should be re-evaluated every 24–48 hr, as most patients may tolerate the prescribed initial dose of the opioid analgesics, with toxic effects often emerging after 48 hr, when the parent drug and its metabolites starts accumulating. Vigilance in nursing is also required to monitor for early effects of opiate toxicity. Box 2 provides recommendations and practical points for acute pain management in CKD based on the available evidence.\n\nBefore prescribing analgesics, consider kidney function to avoid toxicity that may occur from accumulation of the parent drug or their metabolites.\n\nThe general principles of pain assessment and management should be adopted and analgesia should be prescribed incorporating the modified WHO analgesic ladder.\n\nCo-analgesics like acetaminophen should be considered “by the clock” rather than “by demand”.\n\nDo not write standing (or long-term) orders for opioids. Reassess the need and the dose of opioids every 24–48 hr.\n\nAvoid fentanyl transdermal patch in opioid-naïve patients.", "appendix": "Author contributions\n\n\n\nMSP conceived the idea. KSP performed literature review and prepared part of the initial draft. Both authors contributed equally to modifications and approve the current version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCoresh J, Byrd-Holt D, Astor BC, et al.: Chronic kidney disease awareness, prevalence, and trends among U.S. adults, 1999 to 2000. J Am Soc Nephrol. 2005; 16(1): 180–8. PubMed Abstract \| Publisher Full Text\n\nParmar MS: Safe drug prescribing. CMAJ. 2002; 166(13): 1651; author reply 1651. PubMed Abstract \| Free Full Text\n\nDiskin CJ, Thomas S: Intravenous analgesic use in dialysis patients. Nephrol Dial Transplant. 1999; 14(10): 2530–2. PubMed Abstract \| Publisher Full Text\n\nToogood F: The use of morphine in cardiac disease. Lancet. 1898; 2: 1393–4. Publisher Full Text\n\nYeo I: On the use of opium in acute and chronic diseases. 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A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in acute and chronic pain states. Drugs. 1993; 46(2): 313–40. PubMed Abstract\n\nBoyer EW, Shannon M: The serotonin syndrome. N Engl J Med. 2005; 352(11): 1112–20. PubMed Abstract \| Publisher Full Text\n\nGillman PK: Monoamine oxidase inhibitors, opioid analgesics and serotonin toxicity. Br J Anaesth. 2005; 95(4): 434–41. PubMed Abstract \| Publisher Full Text\n\nIzzedine H, Launay-Vacher V, Abbara C, et al.: Pharmacokinetics of tramadol in a hemodialysis patient. Nephron. 2002; 92(3): 755–6. PubMed Abstract \| Publisher Full Text\n\nBerkowitz BA: The relationship of pharmacokinetics to pharmacological activity: morphine, methadone and naloxone. Clin Pharmacokinet. 1976; 1(3): 219–30. PubMed Abstract\n\nFurlan V, Hafi A, Dessalles MC, et al.: Methadone is poorly removed by haemodialysis. Nephrol Dial Transplant. 1999; 14(1): 254–5. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "785", "date": "19 Feb 2013", "name": "Howard S Smith", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 this article. However, on table 3 and figure 1, oxycodone with acetaminophen is considered step 2 of the WHO analgesic ladder but oxycodone by itself is considered step 3. I would change this.", "responses": [] }, { "id": "789", "date": "20 Feb 2013", "name": "Paul E Carns", "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 first clinical scenario includes meperidine as a drug used in the patient. The use of meperidine has significantly been reduced in the USA and is monitored as a negative finding when used in patients over 60. Thus hospitals have made efforts to reduce or eliminate its use except in treatment of rigors and given in low doses only. I am not sure if this would be the best scenario to start the article, although its issues with renal insufficiency are substantial.I would refrain from the use of the word 'narcotic' in your discussion in favor of the word opioid.In the discussion regarding 'adjuvant medications' there is no mention that nearly all these medications require dose adjustments in renal insufficiency. Since the article focuses on this topic, I believe it should be included beyond just mentioning that some patients may be using these medications.The last sentence in the 'Acetaminophen' section gives specific dosing recommendations to give half of the daily dose followed by round the clock dosing. Is this opinion or should a reference be included?Since propoxyphene is no longer available, I would consider eliminating it from the discussion. I am unsure if it is still being used in other countries. If so, I guess I would leave it in.The Codeine/Dihydrocodeine paragraph makes no mention of the active metabolite morphine from codeine. Discussion of other drugs in this section refer to actual names of metabolites. Given that morphine was discussed earlier in the document, including this information is important in my view.The chart indicating % amounts in the column headed by eGFR/ml rates does not really say that the percentages indicate a percent dose reduction. It should be made more clear even though obvious to me. The methadone section says that it requires a special license to prescribe. This is not entirely true. The special license is only required when treating opioid addiction. If methadone is used for pain control, no special license is needed. I would also include that methadone is not to be used in opioid naïve patients as you do with the fentanyl patch.", "responses": [] } ]	1	https://f1000research.com/articles/2-28
https://f1000research.com/articles/2-48/v1	13 Feb 13	{ "type": "Research Article", "title": "Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Nav1.5 expressed in HEK293 cells", "authors": [ "Katja Reinhard", "Jean-Sébastien Rougier", "Jakob Ogrodnik", "Hugues Abriel", "Katja Reinhard", "Jean-Sébastien Rougier", "Jakob Ogrodnik" ], "abstract": "Background: The pore-forming subunit of the cardiac sodium channel, Nav1.5, has been previously found to be mutated in genetically determined arrhythmias. Nav1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Nav1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Nav1.5 can be generated.Methods: In this short study, we (1) compared the biophysical properties of the sodium current (INa) generated by the mouse Nav1.5 (mNav1.5) and human Nav1.5 (hNav1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Nav1.5 construct that was modified with specific epitopes.Results: The biophysical properties of mNav1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNav1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of INa and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNav1.5.Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNav1.5.", "keywords": [ "Cardiac sodium channel", "Nav1.5", "HEK293 cells", "electrophysiology" ], "content": "Introduction\n\nThe voltage-gated cardiac sodium channel Nav1.5 is responsible for the initial phase of the cardiac action potential and plays a central role in cardiac impulse propagation1. Its role in human disorders has been underlined by the findings of several hundred mutations in its gene, SCN5A, that are linked to inherited cardiac electrical disorders such as congenital long QT syndrome and Brugada syndrome2. In recent years, it has been demonstrated that Nav1.5 interacts with and is regulated by different proteins (recently reviewed by Shy et al.3). Many of these interacting proteins were also found to be mutated in patients with genetically-determined cardiac arrhythmias4. The generation of genetically-modified mouse models, harbouring mutations in the Scn5a gene, has proven to be a very informative approach to investigate the various human phenotypes that are linked to the genetic variants of this gene5. Since Nav1.5 interacts with many proteins during its life cycle in cardiac cells, it would be of great interest to generate a mouse model that permits the biochemical purification of Nav1.5 with high efficiency, hence allowing the co-purification of interacting proteins. The identity of these co-purified proteins may then be determined by using mass spectrometry-based technologies. In order to do this, one needs to first generate a knock-in mouse model, where a high-affinity epitope would be added to the mouse Scn5a gene that codes for Nav1.5.\n\nThe goals of this short study were (1) to compare the biophysical properties of the sodium current (INa) generated by mouse Nav1.5 and human Nav1.5 constructs expressed in HEK293 cells, and (2) to investigate the possible alterations of the biophysical properties of human Nav1.5 constructs that were modified with specific epitopes. We used the common fluorescent GFP and YFP proteins as epitopes, which provide the advantage of being detectable without the use of antibodies. However, these tags can only be added to the N- and C-termini, which are both intracellular in Nav1.5, and which are thus, not easily accessible. Therefore, we additionally chose the FLAG-epitope (Sigma-Aldrich), which consists of a short sequence that can be inserted into the extracellular loops of Nav1.5. The results of these studies will have to be taken into account when planning the generation of a mouse line bearing an epitope-tagged Nav1.5 channel.\n\n\nMethods\n\nHEK293 cells (Robert S Kass laboratory, Columbia University, New York) were transfected by Lipofectamine LTX (Invitrogen) according to the manufacturer‘s instructions. The plasmids used were the 2019 amino acid isoform of the mouse voltage-gated sodium channel (pcDNA3-mNav1.5; a gift from Thomas Zimmer, University of Jena, Germany6), human Nav1.5 (pcDNA3.1-hNav1.5), and three differently tagged hNav1.5 (pcDNA3.1-hNav1.5-GFP-N-terminal, pEYFP-hNav1.5, and pcDNA3.1-FLAG(299/300)-hNav1.5). The FLAG-tag is an eight amino acid-long epitope (DYKDDDDK) that was inserted into the extracellular loop linking the transmembrane segments S5 to S6 of domain I, between the residues Leu-299 and Val-300; GFP and YFP were previously added by T. Zimmer to the N-terminal7. For wild-type and tagged hNav1.5, 1 µg of one of the listed plasmids, 1 µg of empty pcDNA3.1 (Invitrogen), and 0.4 µg of DNA coding for CD8 (Robert S Kass laboratory) was used for transfection. In order to measure currents of comparable size, 0.01–1 µg of mNav1.5 was co-transfected with 1 µg of empty pcDNA3.1, and 0.4 µg of DNA coding for CD8. Transfected HEK293 cells were then grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% calf serum (Gibco), 0.2% glutamine (Sigma), and 20 mg/ml gentamycin (Gibco), and incubated at 37°C with 95%O2/5%CO2.\n\nAll experiments were performed in the whole-cell voltage-clamp mode. The extracellular solution contained (in mM): 50 NaCl, 80 NMDG-Cl, 5 CsCl, 2 CaCl2, 1.2 MgCl2, 10 HEPES, 5 Glucose, adjusted to pH 7.4 with CsOH, and with an osmolality of 280–290 mOsm. The internal solution consisted of (in mM): 70 CsAsp, 60 CsCl, 1 CaCl2, 1 MgCl2, 10 HEPES; 11 Cs2EGTA, 5 Na2ATP, adjusted to pH 7.2 with CsOH, and with an osmolality of 297 mOsm. Recordings were performed at room temperature (20–22°C) using a VE-2 amplifier (Alembic Instruments, Montreal, Canada). Data was acquired by Clampex 10.2 (Axon Instruments, Union City, Canada). Membrane resistance was ≥ 1 GΩ and access resistance ≤ 6.1 MΩ. Transfected cells were recognized by the addition of 1 µl/ml Dynabeads CD8 (Invitrogen) into the extracellular solution. Current-voltage (I/V) curves` were assessed by depolarising cells from a holding potential of -100 mV to voltages of between -80 and 40 mV during 20 ms. Steady-state inactivation properties were measured by the following protocol: the cells were kept at a holding potential of -100 mV and then hyper- and depolarised during 500 ms to voltages of between -120 and 0 mV in steps of 5 mV, followed by 20 ms at the voltage that elicited the maximal response during the I/V-protocol. Voltage-dependent activation was read either from the I/V- or the steady-state inactivation-protocol. To characterise the recovery from inactivation, the cells were depolarised from a holding potential of -100 mV for 100 ms, repolarised to -100 mV at a recovery time of 0.25–3000 ms, and depolarised again for 25 ms. By varying the time of the first depolarisation step from 3 to 3000 ms followed by 25 ms of repolarisation, the onset of slow inactivation was determined (see insets of Figure 2 and Figure 4).\n\nPeak values for all protocols were detected and measured by Clampfit 10.2 and I/V-relationships were fitted using KaleidaGraph 3.5 (Synergy Software, Reading, USA). Values were normalised to membrane capacitance. The following formula was used to fit I/V-curves and to calculate reversal potentials: INa = (Gmax(V-Vrev,Na))/(1+eV-V0.5/K) with INa = sodium current in pA, Gmax = max. conductance = 60 Ω-1, Vrev,Na = reversal potential = 40 mV, K = (-zδF)/FR = equilibrium constant = -5, V0.5 = voltage for 50% of maximum current = -20 mV. Activation and inactivation curves were fitted with the Boltzmann equation f0 = 1/(1+ eV-V0.5/K) with f0 = fraction of open channels/total available channels. Statistical analyses were performed using two-tailed Student's t-tests. A p value <0.05 was considered statistically significant.\n\n\nResults\n\nTo compare the biophysical properties of the cardiac sodium channel Nav1.5 from the human (hNav1.5) or the mouse sequence (mNav1.5), we measured the electrophysiological properties of hNav1.5 and mNav1.5, transiently expressed in HEK293 cells. Representative INa recordings are shown in Figure 1. The responses to all applied protocols revealed similar characteristics for both channels, except for the reversal potential and the slope of steady-state inactivation (Figure 2 and Table 1). The peak currents from the I/V-protocol were at -15 mV for both channels (Figure 2A). Furthermore, activation and inactivation of 50% of the channels occurred for both channels at ~-28 mV and ~-71 mV, respectively. In addition, the slopes of the activation curve were comparable for both channels (6.00 mV/e-fold in human and 6.24 mV/e-fold in mouse). Significant differences could be detected in the reversal potential Vrev (51.0 mV and 56.6 mV, P<0.01) and in the slope of the inactivation curve (5.95 mV/e-fold and 6.67 mV/e-fold, P<0.01) (Figure 2B). In addition, mNav1.5 had a tendency to recover faster from inactivation (Figure 2C). The fraction of channels entering into a slow inactivation state was similar for both channel types (Figure 2D).\n\n(A) Voltage-dependent currents measured for hNav1.5 expressed in a HEK293 cell. (B) Data from the same protocol for mNav1.5.\n\nThe voltage-clamp protocols used are shown in the corresponding insets. For B–D, the voltage x was adjusted to the voltage that elicited maximum current during the current voltage (I/V)-protocol (-10 to -30 mV). (A) I/V-protocol for assessment of reversal potentials. Peak currents were measured for both channels at -15 mV. Calculated reversal potentials are marked with square data points. (B) Voltage-dependence of activation and steady-state inactivation (SSI). The data was fitted with the Boltzmann formula. Only the slope of the inactivation curve differs between mouse and human sodium channels (shallower in mNav1.5). (C) Recovery from inactivation. The duration between the depolarising steps was varied from 0.25 to 3000 ms. mNav1.5 had a slight tendency to recover faster than hNav1.5. (D) Onset of slow inactivation. The duration of the first step was varied from 0.25 to 3000 ms. The relative number of channels entering slow inactivation is similar for both types. (A–B) n(hNav1.5) = 22, n(mNav1.5) = 17. (C–D) n(hNav1.5) = 9, n(mNav1.5) = 7. P<0.01 obtained by two-tailed Student's t-tests; error bars indicate standard errors.\n\nData was obtained with current voltage (I/V)- and steady-state inactivation protocols. Mean values and standard errors are shown. P<0.01 obtained by two-tailed Student's t-tests.\n\n\n\nThe second set of experiments addressed the effects of adding epitopes to Nav1.5 on its biophysical properties. To do this, we assessed the influence of these epitopes on INa by expressing differently tagged hNav1.5 in HEK293 cells and performing whole-cell voltage-clamp experiments similar to those described above. YFP- and GFP-tags were added to the N-terminus; the FLAG-tag was inserted into the extracellular loop linking S5 to S6 of domain I, between residues Leu-299 and Val-300. Representative INa recordings for all transfected constructs are shown in Figure 3 and the data is summarised in Table 2. With the exception of the GFP-tagged construct, tagging of hNav1.5 led to a significant decrease in peak current Imax (Figure 4A) compared to the control WT hNav1.5 (FLAG: 57 pA/pF with P<0.01, YFP: 120 pA/pF with P<0.05, WT hNav1.5: 240 pA/pF). Adding GFP did not affect any of the biophysical properties of the human sodium channel, while a shallower activation slope (6.87 vs. 5.91 mV/e-fold, P<0.05, Figure 4B and Table 2) was observed for the YFP-tagged channel. The most pronounced effects were observed for the FLAG-tagged hNav1.5. The activation slope was significantly shallower (6.96 vs. 5.91 mV/e-fold, Figure 4B and Table 2), indicating that the activation of this channel is less sensitive to voltage changes. In addition, the V1/2 of activation was shifted towards more positive voltages by about 5 mV, with -23.9 mV in FLAG-hNav1.5, compared to -28.9 mV in untagged hNav1.5. Finally, the reversal potential was decreased in the FLAG-hNav1.5 (FLAG 39.3 mV and untagged 51.8 mV, Figure 4B). Recovery from inactivation (Figure 4C) and onset of slow inactivation (Figure 4D) were comparable for all channels.\n\n(A) Voltage-dependent currents measured for hNav1.5 expressed in a HEK293 cell. The same data for (B) FLAG-hNav1.5 (C) YFP-hNav1.5, and (D) GFP-hNav1.5.\n\nData was obtained with current-voltage (I/V)-, and steady-state inactivation protocols. Mean values and standard errors are shown. P<0.05, P<0.01 obtained by two-tailed Student's t-tests (all statistics were calculated with untagged hNav1.5 channel as a reference).\n\nThe voltage-clamp protocols used are shown in the corresponding insets. For B–D, the voltage x was adjusted to the voltage that elicited maximum current during the current voltage (I/V)-protocol. (A) I/V-protocol for assessment of reversal potentials. Tagging with N-terminal YFP and FLAG (L299/V300) significantly decreases peak currents. Calculated reversal potentials are marked with square data points. (B) Voltage-dependence of activation and steady-state inactivation. The data was fitted with the Boltzmann formula. The activation slope of FLAG- and YFP-tagged channels is shallower compared to the untagged hNav1.5. V1/2 is shifted by 5 mV for FLAG-hNav1.5. (C) Recovery from inactivation. The duration between the depolarising steps was varied from 0.25 to 3000 ms. No differences between the different channels could be detected. (D) Onset of slow inactivation. The duration of the first step was varied from 0.25 to 3000 ms. The relative number of channels entering slow inactivation is similar for all four channel types. (A–B): n(untagged) = 7, n(FLAG) = 11, n(YFP) = 11, n(GFP) = 8. (C): n(untagged) = 12, n(FLAG) = 5, n(YFP) = 11, n(GFP) = 8. (D): n(untagged) = 10, n(FLAG) = 8, n(YFP) = 10, n(GFP) = 8. *P<0.01 obtained by two-tailed Student's t-tests; error bars indicate standard errors.\n\n\n\n\nDiscussion\n\nThe present study demonstrates (1) that the biophysical properties of mouse Nav1.5 are essentially similar to the human homolog when expressed in HEK293 cells, and (2) that adding epitopes either upstream of the N-terminus of human Nav1.5 or in one of the extracellular loops reduces the amount of INa and alters some of its biophysical properties. Interestingly, GFP in the N-terminus was the only epitope that did not modify any of the measured biophysical properties of hNav1.5. The most pronounced effects could be observed by the insertion of the FLAG-tag in an extracellular loop. In this construct, not only was the amount of INa drastically decreased, but also the activation properties of the sodium channel were altered. The smaller changes found in the properties of YFP-hNav1.5 might be partially linked to the different vector used for this epitope, especially since no alterations could be observed for GFP-hNav1.5.\n\nThese findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the mouse Nav1.5 gene. Different combinations of epitopes and insertion sites might reveal better candidates for in-vivo approaches. Furthermore, additional studies should be performed in HEK293 cells co-expressing other subunits and regulating proteins, and in native cardiomyocytes in order to assess the effects of added epitopes on the interactions with these proteins.", "appendix": "Author contributions\n\n\n\nKR, JSR, JO, and HA designed the experiments. KR performed the experiments and analysed the data. KR and HA wrote the manuscript. JSR, JO, and HA supervised the project. All authors commented on the manuscript and approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a grant from the Swiss National Science Foundation to HA (310030B_135693).\n\n\nAcknowledgments\n\nWe would like to thank D. Shy for her helpful comments on this manuscript.\n\n\nReferences\n\nKleber AG, Rudy Y: Basic Mechanisms of Cardiac Impulse Propagation and Associated Arrhythmias. Physiol Rev. 2004; 84(2): 431–88. PubMed Abstract \| Publisher Full Text\n\nWilde AA, Brugada R: Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac Sodium Channel. Circ Res. 2011; 108(7): 884–97. PubMed Abstract \| Publisher Full Text\n\nShy D, Gillet L, Abriel H: Cardiac Sodium Channel Nav1.5 Distribution in Myocytes via Interacting Proteins: the Multiple Pool Model. Biochim Biophys Acta. 2013; 1833(4): 886–94. PubMed Abstract \| Publisher Full Text\n\nAbriel H: Cardiac sodium channel Na(v)1.5 and interacting proteins: Physiology and pathophysiology. J Mol Cell Cardiol. 2010; 48(1): 2–11. PubMed Abstract \| Publisher Full Text\n\nDerangeon M, Montnach J, Baro I, et al.: Mouse Models of SCN5A-Related Cardiac Arrhythmias. Front Physiol. 2012; 3: 210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimmer T, Bollensdorff C, Haufe V, et al.: Mouse heart Na+ channels: primary structure and function of two isoforms and alternatively spliced variants. Am J Physiol Heart Circ Physiol. 2002; 282(3): H1007–H1017. PubMed Abstract\n\nZimmer T, Biskup C, Dugarmaa S, et al.: Functional expression of GFP-linked human heart sodium channel (hH1) and subcellular localization of the a subunit in HEK293 cells and dog cardiac myocytes. J Membr Biol. 2002; 186(1): 1–12. PubMed Abstract \| Publisher Full Text" }	[ { "id": "777", "date": "18 Feb 2013", "name": "Céline Fiset", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "798", "date": "25 Feb 2013", "name": "Jamie Vandenberg", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript from Reinhard and colleagues describes the electrophysiology of epitope tagged sodium channels expressed in mammalian cell lines. This system is commonly used for the characterizing the electrophysiological and/or trafficking phenotypes of clinically occurring mutants. Some of the constructs described in this study have been reported before (see e.g. reference 7 in the manuscript: Zimmer T, Biskup C, Dugarmaa S, et al.: Functional expression of GFP-linked human heart sodium channel (hH1) and subcellular localization of the a subunit in HEK293 cells and dog cardiac myocytes. J Membr Biol. 2002; 186 (1): 1–12) but to my knowledge the construct with the insertion of a FLAG-tag into the extracellular loop linking S5 to S6 of domain I, between residues Leu-299 and Val-300, is novel. Whilst it is unfortunate that this construct has altered electrophysiological properties, it should be useful for live cell trafficking assays as the extracellular epitope can be recognized without having to lyse the cells. Reagents such as those described in this study have been extremely useful and will continue to be so. In this context it is useful to have proper baseline characterization of their properties, which can be used as a reference point for other users. The manuscript however could have benefited from a more thorough analysis of the trafficking properties of the tagged channels and a broader discussion of the advantages and limitations of the use of the mammalian expression system for characterizing mutant sodium channels. One particularly pertinent recent example of the limitations of the heterologous expression system is that of the D1275N cardiac sodium channel which causes minimal perturbation to gating when expressed in vitro but has a marked loss of function when expressed in vivo in gene-targeted mice (see Watanabe H et al., Striking In Vivo Phenotype of a Disease-Associated Human SCN5A Mutation Producing Minimal Changes in Vitro, Circulation. 2011; 124:1001-1011.)", "responses": [] } ]	1	https://f1000research.com/articles/2-48
https://f1000research.com/articles/2-104/v1	03 Apr 13	{ "type": "Data Article", "title": "The effects of behavioral control over stress on GABAergic spontaneous inhibitory postsynaptic currents in prefrontal cortical pyramidal neurons", "authors": [ "Juan A Varela", "Jungang Wang", "Donald C Cooper", "Juan A Varela", "Jungang Wang" ], "abstract": "Traumatic events may lead to anxiety, depression and post-traumatic stress disorder (PTSD). However, the majority of individuals exposed to trauma do not develop these disorders. The stressor controllability paradigm has been widely used as a model for understanding the neurobiology underlying factors that confer vulnerability and resilience to the outcome of traumatic events. In this paradigm rats receive a series of tail shocks: one group of rats have control over the termination of the shock by means of turning a wheel (escapable shock, ES), while the other “yoked” group of rats receive physically identical shocks but have no control over shock termination (inescapable shock, IS). In subsequent behavioral tests that model components of anxiety and depression, IS rats without control show increased signs of behavioral depression, while ES rats that have control over the shock behave as naïve home caged (HC) rats. We have previously reported that individual deep layer pyramidal neurons from the ventral medial prefrontal cortex (vmPFC) exhibit changes in their intrinsic excitability following ES. To examine if there is a corresponding reduction in synaptic inhibition, we tested IS, ES and HC deep layer pyramidal neurons under identical conditions. Collecting such electrophysiological data from pyramidal neurons after exposure to stress is a technical challenge, yet very useful for conductance-based neural simulations and computational modeling. Here we present a data set of spontaneous inhibitory postsynaptic currents (sIPSCs) gathered from whole-cell patch-clamp recordings of individual prefrontal cortical deep layer neurons from adult rats (60-70 days old) after exposure to ES, IS or HC. In order to analyze the data, we provide our script used for the detection of synaptic events written for the scientific/engineering program Igor Pro that allows users to define their own event detection parameters.", "keywords": [ "Lack of control over stressors may be a predisposing factor for anxiety disorders", "such as post-traumatic stress disorder (PTSD)", "depression", "and drug dependence. However", "not all individuals who experience stress develop these disorders suggesting a mechanism for resilience1. To explore this relationship at a neurobiological level", "we utilized the stressor controllability paradigm2", "3. Behavioral control over tail shock in rats prevents the physiological and behavioral consequences of equivalent uncontrollable shock as measured by anxiety tests (social interaction", "shuttle box escape times", "etc)4. Moreover", "a week after escapable shock (ES) treatment", "if the rat is subjected to an uncontrollable stress situation", "such as social defeat or inescapable shock (IS) treatment", "the rat will still behave as a naïve home caged (HC) rat on anxiety tests5", "a process termed “behavioral immunization”6. The ventral medial prefrontal cortex (vmPFC) has been implicated in the protective effects of ES through a series of pharmacology experiments controlling cortical excitability. Inactivation of the vmPFC by GABA agonists during ES treatment results in a loss of the typical protective effects", "conversely", "pharmacological activation of the vmPFC with GABA antagonists during the IS treatment results in a gain of the protective effects7", "8. We have previously shown that after two hours post-treatment", "the intrinsic excitability of the deep layer vmPFC pyramidal neurons is increased in rats that received ES but not IS treatment compared to naïve HC rats9. However", "the balance of excitation/inhibition is what ultimately determines the overall excitability", "we therefore set out to gather data on the overall state of the inhibitory circuit by recording spontaneous inhibitory postsynaptic currents (sIPSCs) from the layer 5/6 pyramidal neurons. This in turn will allow us (and other groups) to build detailed computer simulations to better understand the cortical circuitry", "and how inhibitory plastic changes occur shortly after stressful situations." ], "content": "Introduction\n\nLack of control over stressors may be a predisposing factor for anxiety disorders, such as post-traumatic stress disorder (PTSD), depression, and drug dependence. However, not all individuals who experience stress develop these disorders suggesting a mechanism for resilience1. To explore this relationship at a neurobiological level, we utilized the stressor controllability paradigm2,3. Behavioral control over tail shock in rats prevents the physiological and behavioral consequences of equivalent uncontrollable shock as measured by anxiety tests (social interaction, shuttle box escape times, etc)4. Moreover, a week after escapable shock (ES) treatment, if the rat is subjected to an uncontrollable stress situation, such as social defeat or inescapable shock (IS) treatment, the rat will still behave as a naïve home caged (HC) rat on anxiety tests5, a process termed “behavioral immunization”6. The ventral medial prefrontal cortex (vmPFC) has been implicated in the protective effects of ES through a series of pharmacology experiments controlling cortical excitability. Inactivation of the vmPFC by GABA agonists during ES treatment results in a loss of the typical protective effects; conversely, pharmacological activation of the vmPFC with GABA antagonists during the IS treatment results in a gain of the protective effects7,8. We have previously shown that after two hours post-treatment, the intrinsic excitability of the deep layer vmPFC pyramidal neurons is increased in rats that received ES but not IS treatment compared to naïve HC rats9. However, the balance of excitation/inhibition is what ultimately determines the overall excitability, we therefore set out to gather data on the overall state of the inhibitory circuit by recording spontaneous inhibitory postsynaptic currents (sIPSCs) from the layer 5/6 pyramidal neurons. This in turn will allow us (and other groups) to build detailed computer simulations to better understand the cortical circuitry, and how inhibitory plastic changes occur shortly after stressful situations.\n\n\nMaterials and methods\n\nTwenty seven adult (60–70 days old and weighing 275–350 g at the time of testing) male Sprague-Dawley derived rats were bred and reared at the University of Colorado, Boulder, USA. Rats were housed with free access to food and water in groups of two or three in a vivarium with a 12-hour light/dark cycle. Stress exposure occurred in the first four hours of the light phase. All behavioral procedures were approved by the University of Colorado Institutional Animal Care and Use Committee. Eleven rats received ES treatment, 7 received IS treatment and 9 rats remained in their home-caged environment.\n\nRats received ES, IS or remained naive. One hundred electric tail-shocks were administered through copper electrodes augmented with electrode paste by a Precision Regulated Animal Shocker (Coulbourn Instruments, Allentown, PA, USA) to rats restrained in 14 × 11 × 17 cm (length × width × height) acrylic boxes with a wheel 7 cm wide and 9.5 cm in diameter located on the wall opposite the tail. Each box was enclosed in a sound-attenuating chamber. Tail-shocks were presented on a variable interval-60 second schedule (VI-60). For rats that received ES, turning the wheel at the front of the chamber terminated each tail-shock after ¼ turn of the wheel. If the response was performed within 5 seconds of shock onset, the response requirement doubled for the next trial and proceeded until a maximum of 4 wheel-turns was reached. If a response was made after 20 seconds the requirement was reduced by half; if no response was made by 30 seconds the shock was terminated and the requirement was reset to ¼ turn. In order to maintain escape behavior the shock intensity was 1.0 mA for the first 33 trials, 1.3 mA for the following 33 trials and 1.6 mA for the remaining 34 trials. Since only one rat per day could be used in electrophysiology studies, we chose to deliver IS on a schedule generated from archival ES escape latency data8. On a trial-by-trial basis, the mean escapable latency, the standard deviation, and the maximum latency and minimum latency were computed from a random sample of 10 archival ES subjects. Each rat in the IS group received a unique series of 100 shocks. Shock durations were computed by taking a random interval from between the average minimum and the trial mean ± standard deviation. If the maximum shock length was less than the mean + standard deviation, then the maximum shock length served as the range maximum. The algorithm produced a series of shocks akin to those generated by the ES rats included in the patch clamp studies. Animals in the HC control group remained in their cages.\n\nStandard artificial cerebrospinal fluid (aCSF) and recording solutions were used; aCSF composition was (in mM) NaCl 124, KCl 2.5, NaHCO3 26, MgCl2 2, CaCl2 2 and glucose 10; pH = 7.4; 310 mOsm/l and the recording solution (in mM) CsCl 115, HEPES 10, KCl 20, MgCl2 2, Na2ATP 3, Na2GTP 0.3, biocytin 0.1%, pH = 7.3, 280 mOsm. 0.3% Biocytin (Sigma) was added to the recording solution to visualize the recorded cell type and location.\n\nTwo hours after shocking, ES and IS rats were anesthetized with isofluorane (MWI, Boise, ID, USA), perfused with aCSF (4°C) and their brains were extracted. Coronal slices (300 µm) were taken from the prefrontal cortex using a vibratome (VT-1000P, Leica Microsystems, Nussloch, Germany). Slices were placed in oxygenated aCSF (95% O2) at 35°C for 30 minutes and then kept at room temperature until slices were removed for electrophysiological recordings.\n\nWhole-cell voltage-clamp recordings were obtained at 33 ± 2°C. Patch-clamp electrodes were pulled (Flaming/Brown P-97, Sutter Instruments, CA, USA) from 1.5 mm outer diameter borosilicate glass (Sutter Instruments, CA, USA) and filled with a CsCl-based intracellular solution. Electrode resistance was 3–5 MΩ in the bath and series resistance was less than 30 MΩ during the recordings. Slices were visualized using a 40× water immersion objective (Zeiss, Germany) mounted on an Infinity-tube FM-100 (Infinity Photo-Optical Co, Boulder, CO, USA). Voltage-clamp recordings were obtained with a BVC-700 amplifier (Dagan, Minneapolis, MN, USA), with no compensation. Data acquisition and analysis were performed using custom software written for Igor Pro (Wavemetrics Inc., Lake Oswego, OR, USA). Inhibitory postsynaptic current (IPSC) analysis was performed using a custom analysis program developed in the Cooper laboratory as described below.\n\nAfter recording, the slice was removed from the recording chamber and placed in 4% paraformaldehyde. Recorded neurons were visualized by incubation with the Vectastain PK-6100 ABC reagent (Vector labs, Burlingame, CA, USA) and di-amino benzidine (DAB, Sigma-Aldrich) solution (1 tablet/ 5 ml). Slices were floated onto glass slides and cover-slipped with DPX mounting solution (Sigma-Aldrich). Only neurons with a pyramidal morphology and soma in prelimbic (PL) layers 5/6 were included for analysis.\n\n\nResults\n\nIn order to analyze the data we developed a new script for the scientific/engineering program Igor Pro. The advantages of such a script are as follows: 1) It allows for multiple events analysis. This is particularly important for analyzing sIPSCs, since a large number of interneurons in the cortex are fast spiking or bursting10 and this results in a large number of “multiple” events. 2) It incorporates seamlessly with Igor Pro, a program used by many groups to collect and analyze electrophysiological data. This allows the program to analyze the collected traces within the experiment file and allows us to take advantage of the included mathematical and graphical functions in Igor Pro. 3) It is fully modifiable, any user knowledgeable in Igor Pro programming can add or remove any features that are needed for their particular purposes. 4) During the user-controlled portion of the program, it allows the user to manually define events that were not detected automatically, and finally, 5) The script is available at no charge.\n\nThe events are detected by analyzing the first and second derivative of the raw data trace. The raw data is smoothed using a binomial (Gaussian filter) method11 using a 500 point sliding window and the point-to-point derivatives are calculated using the functions included in Igor Pro (Euler method12). The first derivative represents the change in the trace results in peaks that are at the beginning of the event (Figure 1), while the second derivative maximums are where the local minimums are located. A simple algorithm checks if the putative peak is the lowest (highest for outward events) point in a 5 ms region and corrects the event detection accordingly. During the user portion of the program, the user can accept detected events, add events or reject events. The user is also able to correct the baseline and peak of the current analyzed event. Alternatively, the user may opt not to check the proposed analysis and accept the given solution by the program without checking. The program output consists of probability histograms for amplitude, inter-event interval (IEI) and frequency of events, their respective cumulative distribution curves and an average event trace from where the decay time constant is calculated. The user at this point is able to place constraints to the analyzed data by rejecting events less than a certain amplitude, for example, if smaller events are within the noise level. All data are then summarized and presented in a single page layout.\n\nA. Sample traces from the three different groups: Home caged (black), escapable shock (red) and inescapable shock (blue). B. ISPC analysis, from the raw data (red) the first (green) and second derivative (blue) are taken. The peaks of the first derivative indicate the fast change in current in the raw data and therefore indicate the event initiation, the maximum in the second derivative indicates the local minima or the peak of the event; utilizing this information, the baseline (green circle) and peak (blue circle) are calculated. Afterwards, the program allows for the acceptance or rejection of the proposed events. Inset: Expanded event and derivatives (black bar).\n\nQuantifying inhibitory postsynaptic currents (IPSCs) following controllable and uncontrollable stress in the prelimbic (PL) region of the prefrontal cortex (vmPFC): In order to understand the overall state of the inhibitory circuit in the PL we studied sIPSCs frequency and amplitude from pyramidal neurons in the deep layers of the cortex (Figure 2). The sIPSCs from home cage (HC, n=11), IS (n=7) and ES (n=9) groups were measured using the detection method described above (also see data set). Descriptive cumulative histograms of the amplitude and frequency of sIPSC events show an increase in the sIPSC frequency, but not amplitude under IS conditions. The increased sIPSC event frequency appears to be reduced in the ES group.\n\nA. sIPSCs amplitude in pico amperes (pA) cumulative distributions for the different treatments, home caged (HC, black), escapable shock (ES, red) and inescapable shock (IS, blue). B. sIPSCs semi-log amplitude histograms, HC (11 rats, 14 recorded neurons), ES (9 rats, 14 recorded neurons) and IS (7 rats, 11 recorded neurons). C. sIPSC inter-event interval (IEI) cumulative distributions showing the three treatments overlaid. D. sIPSCs semi-log IEI histograms showing the three treatment groups overlaid.\n\n\nSummary\n\nHere we present whole-cell patch clamp recording data sets from IS, ES and HC rat pyramidal neurons and an analysis program. Preliminary analysis suggests that control over stress appears to blunt the enhanced inhibition of the vmPFC that results from lack of control. Further detailed statistical analysis of sIPSC frequency and amplitude is necessary to indicate a pre- versus post-synaptic mechanism. We have made this data available together with a software program to allow users to adjust event thresholds and incorporate the results into computational modeling simulations of prefrontal cortical networks.", "appendix": "Author contributions\n\nDCC and JAV conceived the study and designed the experiments. JAV and WJ carried out the sIPSC recordings. JAV wrote the IGOR Pro script. DCC and JAV prepared 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\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by National Institute on Drug Abuse grant R01-DA24040 (to DCC).\n\n\nAcknowledgments\n\nThe authors would like to thank Steve Maier and John Christianson for their conversation and support, and Matthew Pomrenze for his help in caring for the animals.\n\n\nReferences\n\nMancini AD, Bonanno GA: Predictors and parameters of resilience to loss: toward an individual differences model. J Pers. 2009; 77(6): 1805–32.\n\nWeiss JM: Effects of coping responses on stress. J Comp Physiol Psychol. 1968; 65(2): 251–60.\n\nMaier SF, Seligman ME: Learned Helplessness: Theory & Evidence. J Exp Psychol. 1976; 105; 3–46.\n\nMaier SF, Watkins LR: Role of the medial prefrontal cortex in coping and resilience. Brain Res. 2010; 1355: 52–60.\n\nAmat J, Aleksejev RM, Paul E, et al: Behavioral control over shock blocks behavioral and neurochemical effects of later social defeat. Neuroscience. 2010; 165(4): 1031–8.\n\nWilliams Jon L, Maier Steven F: Transituational immunization and therapy of learned helplessness in the rat. J Exp Psychol Anim Behav Process. 1977; 3(3): 240–252.\n\nAmat J, Baratta MV, Paul E, et al: Medial prefrontal cortex determines how stressor controllability affects behavior and dorsal raphe nucleus. Nat Neurosci. 2005; 8(3): 365–71.\n\nAmat J, Paul E, Watkins LR, et al: Activation of the ventral medial prefrontal cortex during an uncontrollable stressor reproduces both the immediate and long-term protective effects of behavioral control. Neuroscience. 2008; 154(4): 1178–86.\n\nVarela JA, Wang J, Christianson JP, et al: Control over stress, but not stress per se increases prefrontal cortical pyramidal neuron excitability. J Neurosci. 2012; 32(37): 12848–53.\n\nMarkram H, Toledo-Rodriguez M, Wang Y, et al: Interneurons of the neocortical inhibitory system. Nat Rev Neurosci. 2004; 5(10): 793–807.\n\nHaralick R, Shapiro L: Computer and Robot Vision, Addison-Wesley Publishing Company.1992; 1, pp 346–351.\n\nAtkinson KE: An Introduction to Numerical Analysis (2nd ed.). New York: John Wiley & Sons, (1989)." }	[ { "id": "892", "date": "15 Apr 2013", "name": "Sayamwong Hammack", "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 preliminary report from Varela et al., investigating spontaneous inhibitory postsynaptic currents in the ventral medial prefrontal cortex of rats exposed to inescapable shock, escapable shock, or no shock. Their data suggest that inescapable shock increases the frequency of spontaneous inhibitory currents, and that this effect is attenuated when shock is escapable. The methodology of the report was sound, and the data are important. Due to the preliminary nature of this report, both the rationale and the summary/discussion were extremely short; however, they seemed too short. The relevance of these data to the current ideas regarding the role of the vmPFC in mediating the effects of IS/ES could be discussed at greater length, as these data seem to suggest that inescapable shock actually enhances inhibition on the vmPFC. Beyond this, I only recommend the following clarifications:The first paragraph seems to suggest that depression and drug dependence are anxiety disorders, and that shuttle box escape time is a test of anxiety. This should be clarified.The authors should justify why neurons were recorded from layer 5/6.Given the vagaries of electrophysiology, it is understandable that the typical escape/yoke paradigm wasn't used for this study; however, an analysis showing that the average amount of shock received between the two shocked groups did not differ would strengthen these data considerably.The strategy of increasing shock intensity to maintain escape behavior does not seem standard. This is not necessarily a problem, but the authors should explicitly state that inescapably shocked rats received the same treatment.Do the authors think that the increased vmPFC inhibition is related to serotonin release from the dorsal raphe nucleus? Again, some discussion seems warranted.", "responses": [] }, { "id": "940", "date": "10 May 2013", "name": "Andre Der-Avakian", "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 report, Varela et al. used electrophysiology to determine that exposure to uncontrollable stress, which leads to behavioral deficits relating to depression and anxiety, increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in layer 5/6 pyramidal neurons of the prelimbic area of the medial prefrontal cortex (mPFC). Exposure to controllable stress, which does not produce the behavioral deficits described above, produced a more modest increase in sIPSC frequency.These data provide a clear and logical extension of previously published data from this group indicating that controllable stress increases the excitability of mPFC pyramidal neurons. Thus, the authors may further discuss the relationship between both data sets. Do the results presented here confirm the authors’ views that controllable stress has an overall excitatory effect on mPFC pyramidal neurons relative to uncontrollable stress? Along these lines, can the authors speculate as to whether control over stress directly excites mPFC pyramidal neurons, or rather prevents the enhanced inhibition of these neurons during stress exposure per se, or both?Since the standard yoked stress procedure was not used in this study, in addition to verifying that the physical properties of the controllable and uncontrollable stressors were similar as mentioned by another reviewer, discussion of previous studies from this group confirming the behavioral effectiveness of the stress procedure reported here would strengthen the interpretation of the electrophysiology data.Also, please verify the correct sample sizes as they are reported differently in the Methods and Results sections.", "responses": [] } ]	1	https://f1000research.com/articles/2-104
https://f1000research.com/articles/2-103/v1	03 Apr 13	{ "type": "Research Article", "title": "Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors", "authors": [ "Thomas B Kepler" ], "abstract": "One of the key phenomena in the adaptive immune response to infection and immunization is affinity maturation, during which antibody genes are mutated and selected, typically resulting in a substantial increase in binding affinity to the eliciting antigen. Advances in technology on several fronts have made it possible to clone large numbers of heavy-chain light-chain pairs from individual B cells and thereby identify whole sets of clonally related antibodies. These collections could provide the information necessary to reconstruct their own history - the sequence of changes introduced into the lineage during the development of the clone - and to study affinity maturation in detail. But the success of such a program depends entirely on accurately inferring the founding ancestor and the other unobserved intermediates. Given a set of clonally related immunoglobulin V-region genes, the method described here allows one to compute the posterior distribution over their possible ancestors, thereby giving a thorough accounting of the uncertainty inherent in the reconstruction.I demonstrate the application of this method on heavy-chain and light-chain clones, assess the reliability of the inference, and discuss the sources of uncertainty.", "keywords": [ "B-cell", "statistical inference", "unobserved ancestor", "unmutated ancestor", "v-region" ], "content": "Background\n\nDuring the course of an infection, the host's immune system produces antibody molecules that bind to molecular determinants (antigens) on the infectious agent, thereby neutralizing the agent and targeting it for removal by additional antimicrobial effectors. The heavy and light chain immunoglobulin (Ig) genes that encode the components of the antibody molecule result initially from the stochastic intrachromosomal rearrangement of gene segments arrayed in libraries of such gene segments1. These genes are further modified after the activation of the B cells that possess them through somatic hypermutation targeted to the rearranged Ig genes2. Those B cells whose Ig genes encode molecules with greater affinity for the eliciting antigen gain a proliferative and survival advantage. In this way, the overall affinity of the pool of serum antibodies increases, sometimes by two or more orders of magnitude. This affinity maturation3 is an essential component of the establishment of humoral immunity, the basis for the large majority of successful vaccines4.\n\nA great deal has been learned about affinity maturation, particularly with regard to the mechanism of somatic hypermutation5 and the dynamic organization of the cellular environment in which affinity maturation takes place6,7 (see the recent review by Shlomchik and Weisel8), but the mechanism underlying the selective aspects of affinity maturation remains poorly understood. There is increasing interest in the manipulation of affinity maturation pathways in vaccinology9 and thus in comparing the biophysical properties of mature antibodies to those of their inferred unmutated ancestors (UA)10–18. Little attention has been paid, however, to the uncertainties inherent in the inference of these UAs. Given the sensitive dependence of antibody-antigen interactions on single amino acid changes19, estimating these uncertainties is essential. Under some circumstances, there may be more than one history consistent with prior knowledge that is supported by the data; having the means to determine these cases and provide a set of alternative UAs that as an ensemble cover a significant posterior probability could be valuable, as was shown by Alam et al. in a study of the affinity maturation of a broadly neutralizing anti-HIV-1 antibody14.\n\nThe inference of ancestral rearrangements involves the alignment of two (light chain) or three (heavy chain) gene segments in tandem to the target mature Ig gene. The identities of the gene segments are not known in advance. Instead, there is a library of gene segments from which each segment is drawn stochastically; the identity of each segment is part of the inference. The problem is complicated by randomness in the location of the recombination points, where each gene segment begins or ends, because this condition implies that the alignments are not independent. Further challenges are encountered by the presence of nontemplated (N-) nucleotides added at random to the junctions between gene segments, and of course, by point mutations.\n\nThere is a well-developed literature on ancestor reconstruction in phylogenetics (see, for example, Pagel et al., 200420). This line of research has informed the development of my methods, but the problem at hand requires tools beyond those that have been developed by its practitioners. The difference between the previous phylogenetic methods and the method described here is that the former do not take into account the complex process through which the Ig ancestor is constructed. This process places a strong statistical constraint on what ancestral states are permissible. My method owes a great deal to this prior work but does not aim to improve upon it fundamentally. It simply extends a small part of its methods to a new domain of application.\n\nIndependent of this previous work from phylogenetics there are applied methods developed by computational immunologists. Indeed, computational methods developed to address the problem have been used for some time21. There are several different approaches and corresponding programs available online for carrying out these analyses, including iHMMune22, V-Quest23, Joinsolver24, and SoDA and SoDA225,26. None of these applications, however, provides either of two features essential for the systematic reconstruction of clonal histories. First, one must be able to use all of the information available in a set of clonally related Ig genes in a statistically principled manner. All currently available Ig alignment tools work with one sequence at a time. Second, one needs systematic uncertainty estimates on the UA. In order to say anything of interest about the UA and the clonal history, there must be some level of certainty that the inferred sequence really is the actual UA.\n\nThe method described here provides these features. It is based on a hierarchical model of Ig gene development that produces an analysis of the clonal history and posterior probabilities on the UA. The method uses the information available across all members of a clone in a consistent and powerful manner.\n\n\nMethods\n\nOne starts with a query set Q of observed Ig variable-region gene sequences assumed to share descent from a common ancestor α. The task is to estimate the DNA sequence α or, more generally, a posterior probability on α. There are two distinct stochastic processes that together give rise to Q. The stochastic intrachromosomal rearrangement process transforms the germline configuration to the unmutated (naïve) ancestor. Somatic mutation transforms the naïve ancestor to the mature (mutated) antibodies that are observed. To each of these stochastic processes there corresponds a probability function, each of which, in turn, has a natural interpretation within the framework of Bayesian inference. The rearrangement process generates a distribution P0 (α) on unmutated ancestors. For each unmutated ancestor a, somatic mutation then generates the likelihood function P(Q \| α) relating the ancestor to the observed query sequences. Once these functions are computed, Bayes' Theorem is used to compute the posterior probability on α given Q,\n\n\n\nTo avoid unnecessary complication, light chain sequences will be used for illustration. The extension to heavy chains is straightforward, but even for the simpler light chains the notation becomes clumsy and obscures the intuition behind the method. Heavy chain rearrangements involve an additional gene segment (DH) and two junctions rather than the one that light chain rearrangements have. Figure 1 illustrates the parameterization of a heavy-chain rearrangement and provides a guide applicable to both heavy and light-chain rearrangements.\n\nLabelled vertical arrows indicate the positions of the recombination sites: 1) RV = 1; 2) RD1 = 5; 3) RD2 = 7; 4) RJ = 3. The dashed arrow 2a indicates a possible alternative recombination site: RD1 = 3. Lower-case letters in the gene-segment sequences indicate mismatches between the observed sequence and the gene segment. The last line shows N nucleotide sequences consistent with the observed sequence.\n\nA light-chain rearrangement results from the selection of a V-gene segment V, the selection of a J-gene segment J, the specification of the recombination point in both of these segments RV, RJ, and the sequence n of the N nucleotides randomly added to the junction between the gene segments. These elements are regarded as parameters in a statistical model: V and J are categorical parameters naming specific gene segments, RV and RJ are integers, and n is a DNA sequence. RV is defined as the position of the 3' - most V nucleotide included in the rearrangement; RJ is the position of the 5' - most J nucleotide included. The DNA sequence n may have length zero (meaning that the V and J segments are directly joined and no N nucleotides occur).\n\nEach combination of parameter values generates a specific DNA sequence, although a given sequence may be generated by more than one set of parameter values. One computes the posterior distribution on these parameters, and uses it to generate posteriors probabilities on the quantities of interest, such as the nucleotides at each position of the founder gene.\n\nLet S(V, J, RV, RJ, n) be the sequence generated by indicated arguments. Then the distribution on unmutated rearrangements is\n\n\n\nwhere I is the Boolean indicator: I [true] = 1, I [false] = 0 and π is the prior probability on rearrangement parameters.\n\nV and J are taken to be independent and π(V, J, RV, RJ, n) = π(V, RV) π(J, RJ) π(n). Although this assumption is not strictly true–there are small correlations among V, D, and J gene segments27, the inclusion of these correlation would have very small effects on the resulting inference at the cost of substantial computational effort.\n\nFor the analyses in this paper I use gene-segment libraries derived from the IMGT reference libraries28. These libraries contain multiple alleles for each gene segment locus. Priors are assigned to the gene segments such that each gene segment locus has the same prior probability, regardless of the number of allelic variants present. Within a gene-segment locus, the distribution on alleles is uniform. When more prior information is available–for example, if one knows the allelic frequencies in the relevant population or knows precisely which alleles are carried by the subject–this information is easily accommodated in the prior probabilities.\n\nThe recombination sites are also assigned prior probabilities uniformly across their assumed range. The largest allowed value for RV corresponds to the position just 3' of the codon encoding the second invariant cysteine residue. The largest allowed value of RJ corresponds to the position just 5' of the codon encoding the invariant tryptophan residue. For all gene segments, the smallest allowed value of the recombination points is -4, corresponding to four P nucleotides29.\n\nFor N-nucleotide sequences, an improper prior is used, formally assigning a uniform distribution over all sequence lengths. The use of this uninformative prior is computationally convenient and has little consequence in practice, since ancestral sequences that have excessively long N regions will be judged very unlikely to give rise to the observed sequences and will not contribute substantially to inferences. The mechanics of this phenomenon will become clearer when I describe the computation of the likelihood and sequence alignment.\n\nThe second probability function required is the likelihood, describing the probability that the query sequences Q arose from a given ancestor α by somatic mutation. The likelihood function depends implicitly on the multiple sequence alignment used as well as on the assumed phylogenetic tree. It is computationally infeasible to account completely for these additional sources of uncertainty. Indeed, it remains a significant challenge in the general case30. Fortunately, somatic hypermutation only infrequently creates insertions or deletions31, which are the major cause of uncertainty in multiple sequence alignment. Rather than sum over many multiple alignments, for each gene segment I use the alignment with the maximum score as detailed below.\n\nI assume that the complete multiple alignment AC can be decomposed into a multiple sequence alignment AQ among the query sequences in Q and the alignment A between AQ and the UA. AQ is estimated in advance and treated as given in subsequent computations. Then for each gene segment, the maximum likelihood alignment between it and AQ is computed.\n\nEvery tree T can be represented by a tree T1 with unit average branch length and a mutation rate μ taken to multiply each branch of T1 to yield T. Although the estimated ancestor is insensitive to variation in the assumed tree32, the estimate of uncertainty is clearly sensitive to the assumed overall mutation rate, i.e., to the overall scaling of the branch lengths.\n\nThe procedure is to iteratively estimate T1 given the UA and the UA given T1, integrating over μ at each stage. One starts with a simple tree T1 invariant under permutations of the gene assignments to tips (a palm tree, with a branch from the root to the last common ancestor of Q and branches of equal length from the last common ancestor to each member of Q). Then, given T1, estimate the posterior on the rearrangement parameters (integrating over μ), find the UA with maximum posterior likelihood, use this sequence at the root to re-estimate T1, and continue iteratively until convergence is reached.\n\nAlthough the pairwise alignments AV, AD, and AJ of the V, D and J gene segments to Q are not independent, they are conditionally independent given the recombination points. Therefore, the likelihood factorizes into components corresponding to gene segments as follows, using the light chain for the example,\n\n\n\nThe last function contains the dependence among the gene segment pairwise alignments. f(RV, RJ, AV, AJ) = 1 when the position of RJ in AQ is 3' of the position of RV in AQ, that is, when the gene segments do not overlap. Otherwise, it is zero.\n\nThe positions in the ancestor are assumed to evolve independently. For a single query sequence q, one has\n\n\n\nwhere qi is the nucleotide at position i in the query, L is the length of q, αi is the nucleotide at position i in the ancestor, and λ is the product of time and mutation rate, or branch length. The function M represents the substitution model. For this paper, I use the simple Jukes-Cantor form33.\n\nWithin each component of the likelihood, the substitution model allows the computation of the likelihood for any sequence α placed at the root of T, conditional on T. Since the columns of the individual gene segment alignments are independent, the overall likelihood is the product of the likelihoods for each column in the alignment, each of which is given by taking the product of the likelihoods along each branch in T and summing over all combinations of nucleotides at the interior nodes34.\n\nGiven a pair of nucleotide sequences with one taken to be derived from the other, an alignment between them is equivalent to an accounting of the mutations via which the derivation occurred. Given a substitution model, there is an alignment scoring scheme that corresponds to that substitution model, so that the score for any alignment is the log of the likelihood of the corresponding set of substitutions.\n\nIt is straightforward to generalize these observations to the alignment of a nucleotide sequence against a set of sequences, the multiple sequence alignment among which is presumed given. Let the set of nucleotides at position i in the alignment be denoted qi and the nucleotide in the ancestor at position i be denoted αi. The following pairwise alignment scoring scheme is obtained.\n\nMatch score–aligning the jth position in the ancestor against the ith position in the derived gene:\n\n\n\nInsertion score–aligning a gap in the ancestor against the ith position of the derived sequence:\n\n\n\nDeletion score–placing a gap at any position in the derived sequence:\n\n\n\nwhere x is any nucleotide. To account for long deletions or insertions one could use an affine gap score, but in this paper just the simple gap penalties above are used.\n\nIn addition to the standard scoring elements for pairwise alignment, the alignment of rearranging antigen receptors requires an additional scoring element for the treatment of N nucleotides. The score for the assignment of a given nucleotide to a generic N nucleotide rather than to a specific N nucleotide state (A,G,T,C) is computed. Denoting by πN (x) the prior probability for a random N nucleotide to have state x, the score corresponding to the assertion that position i in the query sequence alignment is encoded by an N nucleotide is\n\n\n\nFor the analyses conducted in this paper, πN (x) = 1/4 for all nucleotides x, though, again, the use of informative priors is straightforward.\n\nWith all the components of the scoring function in place, one is able to use dynamic programming to find the alignment that maximizes the alignment score.\n\n\n\nThe algorithm is schematized as follows.\n\n(Preparation)\n\nAlign Q using multiple sequence alignment to give AQ.\n\nAssume an initial unit-length palm tree, T1.\n\nWhile not converged:\n\n{\n\nEstimate rearrangement parameters given T1.\n\nFor each discretized value of μ\n\n{\n\nCompute the likelihood for each αi ∈ {A, C, G, T} at each position i of AQ.\n\nAlign each gene segment V, (D), J in the gene segment library to AQ, using Eqs. (5–8), computing the likelihood for the relevant parameters in each alignment.\n\nCompute the posterior on α conditional on μ using Eqs. (1,2).\n\n}\n\nCompute the posterior on μ.\n\nMarginalize the posterior on α over μ.\n\nAdd the modal (maximum posterior probability) UA\n\nα* to Q.\n\nEstimate new tree T1' with α* at root.\n\nIf T1' == T1 converged = true\n\nElse T1 = T1'\n\n}\n\nBecause of N nucleotides and increased uncertainty estimating DH gene segments, the complementarity determining region 3 (CDR3) is typically the region of lowest confidence. In addition, all three CDRs accumulate mutations more rapidly than the framework regions in both selected and unselected genes27. For these reasons, CDR3 is susceptible to having its true mutation rate underestimated. The heuristic employed here is to assume a mutation frequency two-fold higher in CDR3 than in the remainder of the gene. This value is consistent with the enhancement of mutation frequency measured in CDR1 and CDR2 where there is much greater confidence in the counting of mutations35.\n\nThe foregoing method was implemented using CLUSTALW36 within BioEdit to compute AQ, PHYLIP's dnaml37 for clonal tree estimation, and software I developed, ARPP UAI, for all other computations.\n\n\n\n\nResults\n\nTo examine the reliability of error estimation for the method, I identified two relatively large sets of clonally-related genes for testing. The first, Clone H, is a set of 84 heavy-chain genes38 of common length 376 nucleotides (nt), with an average (± standard deviation) pairwise difference of 30.4 ± 9.4 nt and a maximum pairwise distance of 61 nt. Figure 2 shows the clonal phylogram for this set of sequences. The second, Clone K, is a set of 12 kappa-chain sequences16 of length 299, with an average of 12.2 ± 4.8 nt differences and a maximum pairwise distance of 21 nt.\n\nThe scale bar shows evolutionary distance, or expected number of mutations per position.\n\nI applied the inference procedure to Clone H and found that the VH gene segments with the greatest posterior probabilities are VH4-3401 and VH4-3403, with nearly identical posterior probabilities of 0.49 each. These two alleles differ from each other in two places. The majority of sequences in the alignment matches one of the alleles at one of these two informative sites but matches the other allele at the other informative site. The modal DH gene segment is DH6-601 with posterior probability 0.94. The modal JH gene segment is JH6-102 with posterior probability greater than 0.99. The most likely rearrangement has VH using as many as 7 p-nucleotides, no N nucleotides in the V-D junction, and 14 N nucleotides in the D-J junction (Figure 3). The observed sequences have an average mutation frequency of 8.0% compared to the UA.\n\nNucleotide alignment of observed heavy-chain sequences, inferred unmutated ancestor, and modal gene segments, with the probable error (below), illustrating the influence of N nucleotides, junctions, and allelic ambiguity on uncertainty. The large probable error at position 273 is due to allelic ambiguity. A second position in FR1 has similar probable error due to allelic ambiguity (not shown). HCDR3 is indicated. The 84 sequences at the top of the alignment are fragments of the observed members of Clone H (naming is arbitrary). The 4 sequences at the bottom of the alignment are the modal unmutated ancestor (UA), and the modal gene segments. A dot in the sequence indicates a match to the UA at that position.\n\nThe UA of Clone K is inferred to have been rearranged using VK1-391 with probability greater than 0.999 and to the JK11 with probability 0.98. No N nucleotides are required for the rearrangement. The observed sequences have an average mutation frequency of 5.6% compared to UA.\n\nThe inference procedure produces a posterior marginal probability mass function over nucleotides at each position of the UA. The probable error at each position is defined as one minus the maximum value of the posterior probability at that position. The total probable error is the sum of the probable errors over positions, and gives the expected number of mismatches between the inferred modal UA and the true UA.\n\nTo examine the reliability of the estimated probable error, I subsampled the sequence sets and performed the inference on each of the subsamples. For Clone H, ten pseudorandom samples for each size 1, 3, 9, and 27 were generated. For Clone K, UAs were estimated using each of the individual sequences alone. The resulting modal UAs for all sets were compared to the modal UAs inferred from the complete set.\n\nFor Clone H, the total probable error for the UA inferred from the complete set is 2.0. Figure 4 shows the results of these analyses for Clone H. The observed number of mismatches for each subsample is plotted against the total probable error for that subset. The distribution of probable error by nucleotide position shows that some uncertainty is attributable to uncertainty in the allele used in the ancestral rearrangement (Figure 3, position 273) and some is attributable to uncertainty in the N nucleotides and junctions (Figure 3, HCDR3).\n\nThe number of mismatches between the modal unmutated ancestor (UA) for each subsample compared to the UA for the Clone H complete set vs. the estimated error summed over all positions for each Clone H subsample UA. Symbol color indicates subsample size as shown in the legend. The larger symbols indicate the means; the half-widths of the error bars are the standard errors of the means. The dashed vertical line indicates the total probable error using the complete 84-sequence set.\n\nFor Clone K, the total probable error for UA inferred from the whole set is 0.07. For the 12 UAs obtained from individual sequences, the mean total probable error is 0.14 ± 0.05. There were no mismatches among the light-chain UAs.\n\nTo quantify the impact of the prior distributions on the inference, I performed the inference using the same sequence sets, but with a simple uniform prior on nucleotides at each position rather than the prior based on knowledge of the rearrangement mechanism and gene segments. Under this model, the modal UA differs from that of the full rearrangement-based model in 11 positions for the heavy-chain clone, and in 10 positions for the light-chain clone. The total probable error for the heavy chains and light chains is 8.5 and 11.5, respectively for the model with uniform priors.\n\n\nDiscussion\n\nI have developed a method for the inference of clonal history in sets of affinity-matured clonally-related immunoglobulin genes. The method allows one to compute posterior distributions on the rearrangement parameters, and hence marginal distributions on several elements, including the nucleotide sequence of the unmutated ancestor.\n\nThe probable error is strongly dependent on the interplay of N nucleotides and mutation frequency. This phenomenon occurs because nucleotides near the recombination junction are ambiguous with regard to their origin. A nucleotide that does not match the relevant gene segment at a position near the unknown recombination junction may have been encoded by the gene segment and mutated. Alternatively, it may have been encoded by an N nucleotide. The relative probabilities of these alternatives depend on the mutation frequency. If there are few mutations elsewhere in the gene (where they can be determined more reliably) the likelihood of a mismatch in the junction being due to a mutation is small.\n\nThe second major source of uncertainty is allelic diversity. It is often the case, as it is with Clone H, that mutation has destroyed the information required to distinguish which of two or more alleles was used. The greater part of the total uncertainty will be due to one of these two phenomena (Figure 3). This state of affairs also implies that the errors may be correlated, and the distribution of the total number of mismatches overdispersed, as is evident in Figure 4.\n\nOne expects the total uncertainty to be proportional to the distance from the root to the most recent common ancestor of the observed sequences (as long as that distance is not too large). Adding related sequences to a clonal set improves the inference to the extent that they push back the time of the most recent ancestor.\n\nWhere there are few N nucleotides and allelic polymorphism either not present or not obscured by mutations, the UA can be inferred with great precision, even in the presence of significant levels of mutation, as is the case with Clone K.\n\n\nConclusions\n\nTechnology now provides immunologists with the means to reconstruct clonal histories, synthesize the unobserved ancestors, and retrace the steps of affinity maturation to provide deeper insight into the humoral immune response in general and into vaccine design in particular. But the value of the information obtained in this way is wholly dependent on the reliability of the inferential part of the reconstruction. If the ancestors and intermediates are misinferred, the reconstructed history will be potentially misleading.\n\nThe methods outlined here are intended to ensure reliable inference and to indicate when multiple histories must be considered.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by NIH/NIAID research contract HHSN272201000053C to (TBK, PI) and a Vaccine Development Center grant in the Collaboration for AIDS Vaccine Discovery Program from the Bill and Melinda Gates Foundation (B. Haynes, PI).\n\n\nAcknowledgements\n\nI thank Grace Kepler, Barton Haynes, Larry Liao and the members of the Duke Human Vaccine Institute Antibodyome group for stimulating discussions.\n\n\nReferences\n\nTonegawa S: Somatic generation of antibody diversity. Nature. 1983; 302(5909): 575–581. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "884", "date": "09 Apr 2013", "name": "Deborah Dunn-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 is a really useful tool for immunoglobulin affinity maturation studies – particularly now that technology enables us to generate large clonal families. A minor point, in the file downloads information you state “The program requires a Windows operating system to run.” It would be useful to be more specific – 32 or 64 bit? Which version of Windows?", "responses": [] }, { "id": "903", "date": "22 Apr 2013", "name": "Austin Hughes", "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 method presented in this paper depends on the assumption that reconstructing somatic rearrangement events occurring in the ancestors of B cell clones can be improved by using phylogenetic methods to infer the unmutated ancestor (UA) of mature antibodies. The methods used to reconstruct the UA are standard phylogenetic methods, but the statistical approach taken ignores biological complexities. First, the process of affinity maturation of antibodies is a selective process, not just the accumulation of mutations. Certain mutations, which increase affinity, are selectively favoured, while those that decrease affinity are eliminated. Thus, the author’s assumption of the independence of nucleotide positions in the sequence seems unjustified, as does the use of the simple Jukes-Cantor model. In addition, phylogenetic reconstruction in this case (involving short sequences subject to positive selection, resulting in terminal branches that are very long in comparison to internal branches) is likely to be unreliable. The author cites one study suggesting that ancestral sequence reconstruction may not be highly sensitive to tree topology, but the sequences used in that study may not be directly comparable to the present case. It would have been nice to see some quantitative evidence regarding the influence of tree topology on UA reconstruction with these data. Furthermore, if tree topology doesn’t matter for the results, why go through the whole elaborate process of phylogenetic tree reconstruction?In spite of these reservations, the author is to be congratulated for drawing attention to the potential value of using the information in clonally related sequences for inference of ancestral rearrangement.", "responses": [ { "c_id": "1297", "date": "14 Apr 2015", "name": "Thomas Kepler", "role": "Author Response", "response": "I thank Professor Hughes for his careful reading of the manuscript and his informed and helpful criticisms. I agree with him that more investigation is required to determine how best to use statistical phylogenetics to analyze antibody affinity maturation. As we are working on how best to incorporate greater biological realism into these methods, including taking account of selective forces and the dependence of local nucleotide sequence on the intrinsic mutation rate--tasks that are far from trivial--I want to emphasize that what we have done, as recognized by Prof. Hughes, is to adapt phylogenetic methods to a problem area in which they were not being applied previously. This required combining, for the first time, a stochastic model for VDJ rearrangement with a model of molecular evolution into a hierarchical composite model. We are continuing to refine both components and the methods overall.We believe that even this initial step, taken with simplified models for both processes, realizes a substantial improvement over previous work. Furthermore, this method points the way, as suggested, to better methods based on the same idea.Regarding variability in the estimated tree, what I claim is that the estimated tree provides a better basis for inference than does a procedure that uses no tree at all, or even one that uses a star or other simplified topology. It's not that the tree does not matter at all, it's that the inference obtained by averaging over multiple trees with high likelihood is not expected to differ a great deal from that obtained using just the ML tree, though both would differ from that obtained \"treelessly\". This claim, though partially supported by previous findings, requires further investigation." } ] }, { "id": "908", "date": "23 Apr 2013", "name": "James Crowe Jr", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCurrent high throughput DNA sequencing technologies, including those for amplicons such as PCR products of antibody gene transcripts, allow for the production of millions or billions of nucleotide sequence files. An intriguing finding that has emerged recently is that B cells of apparent clonal families encoding highly related antibody transcripts, representing what appear to be somatic variants, circulate in the peripheral blood and other tissues. After sequence alignment, it seems very intuitive to infer that highly related sequences actually derived in vivo from single B cell clones. However, as the author points out, there is uncertainty in the inferences made by alignment or conventional phylogenetic tools because of the nontemplated regions of recombined antibody genes, and the high frequency of somatically mutated residues in clones from memory B cells. Current computational tools for identification of likely germline gene segments used in the original recombination are reasonable, but currently there are not adequate tools to determine the likelihood as to whether particular recombined and somatically mutated sequences derived biologically from another less mutated sequence in the repertoire from a sample. This is the gap that the author attempts to fill with the tool described. This tool likely has significant limitations, but it is important that such tools be developed and tested, with comparison to biological experiments. As sequencing technologies become ever more efficient, it is likely that increased sequencing depth will allow experimentalists to ‘fill in the gaps’ of these types of proposed phylogenies, offering some level of verification of the accuracy of the inferences. Expression and testing of binding of antibodies in intermediate nodes of these phylogenies could be used to experimentally validate the relevance of the inferences. This type of work is already ongoing in several laboratories aimed at rational vaccine and antibody design. I am not a mathematician, so I cannot comment as to whether the statistical methods are really appropriate in this work. I can comment however that there are a number of limitations that arise from biological particulars of antibody gene repertoires that likely need to be accounted for in later iterations of this tool. The possible number and diversity of nontemplated junctional nucleotides is theoretically nearly infinite and position independent, but structural constraints limit the length and type of residues encoded in junctions. In fact, canonical structural configurations of the necks of the hypervariable loops (CDRs) likely limit the sequence diversity that can be observed in peripheral blood expressed antibodies after selection. I am not certain if these structural constraints can be used to constrain the inferred phylogenies generated, but that would be very helpful, since somatic variants are unlikely to violate the common structural determinants of the antibody paratopes in antigen-specific repertoires. Antibody genes contain more mutable codons than many other genes encoding proteins, so the likelihood of coding changes may need to be accommodated. Insertions and deletions occur with reasonable frequency in these genes during the process of somatic hypermutation. Some sequences that arise from somatic hypermutation stimulated by a foreign antigen may be eliminated due to autoreactivity or other selective pressures. I also did not see any methods for dealing with sequencing errors, which are vexing in this context. All of these biologic phenomena affecting antibody repertoires make inference of antibody gene phylogenies especially challenging.Nevertheless, I find it encouraging that new tools like this are being developed that can be tested, evolved and validated in this area. The sequencing technologies present the practical problem of inferring relationships between observed transcripts already, and laboratory experimentalists need practical tools like this for establishing limited sets of candidate genes to synthesize and study. As larger repertoires from more diverse sets of individuals are obtained, the relevance of these tools will become clear. It is especially intriguing to think that, with sufficient sequencing efforts, we may be able to define all possible commonly expressed antibodies and their phylogenies, not just within individuals but across populations.", "responses": [] } ]	1	https://f1000research.com/articles/2-103
https://f1000research.com/articles/2-102/v1	03 Apr 13	{ "type": "Commentary", "title": "Cancer surgery induces inflammation, immunosuppression and neo-angiogenesis, but is it influenced by analgesics?", "authors": [ "Patrice Forget", "Olivier Simonet", "Marc De Kock", "Olivier Simonet", "Marc De Kock" ], "abstract": "Surgery remains a main part of the treatment of most solid tumors. Paradoxically, rapid disease progression may be a consequence of surgery in patients presenting with a dysregulated inflammatory response, and increased angiogenesis consequent to a suppressed antitumoral immune response. Physicians taking care of cancer patients should be aware of the important findings that indicate that analgesic techniques could play a role in these phenomena.", "keywords": [ "The natural history of cancer is a complex and rapidly evolving field. For example", "in breast cancer", "the growth of the primary tumor and the dissemination of neoplastic cells are linked", "at least in part", "to inflammation leading to immune dysfunction and an/or increased angiogenesis1", "2. For breast cancer", "as well as for most solid tumors", "surgery remains a main part of the treatment. However", "paradoxically", "the surgical period", "and the associated inflammatory reaction", "is itself a high risk factor for the development of metastases and this phenomenon may be explained by the rapid release of inducers of angiogenesis concomitant to a profound immunosuppression1", "2. One example", "sometimes observed", "is the rapid postoperative development of additional tumors and metastasis when a primary tumor is surgically removed2." ], "content": "Introduction\n\nThe natural history of cancer is a complex and rapidly evolving field. For example, in breast cancer, the growth of the primary tumor and the dissemination of neoplastic cells are linked, at least in part, to inflammation leading to immune dysfunction and an/or increased angiogenesis1,2. For breast cancer, as well as for most solid tumors, surgery remains a main part of the treatment. However, paradoxically, the surgical period, and the associated inflammatory reaction, is itself a high risk factor for the development of metastases and this phenomenon may be explained by the rapid release of inducers of angiogenesis concomitant to a profound immunosuppression1,2. One example, sometimes observed, is the rapid postoperative development of additional tumors and metastasis when a primary tumor is surgically removed2.\n\nOther factors can accentuate this phenomenon, including the metabolic and hormonal changes that occur and are determined by the inflammatory/catecholaminergic \"stress reaction\" to surgery3. To counteract these effects, perioperative physicians, including anesthesiologists, surgeons and oncologists, must help the patient to maintain homeostasis against the consequences of both cancer and tissular attrition. Anesthetic and analgesic techniques are one part of this strategy, but their effects, however important, are different and not well understood. Indeed, these drugs may influence immunity and tumor development, either directly by interfering with cellular mechanisms (e.g. cell apoptosis) or indirectly by interactions with the endocrine and sympathetic systems.\n\nIn this paper, we discuss the consequences of perioperative inflammation in cancer surgery on immunity and angiogenesis. Secondly, we describe why analgesic techniques may play a role in these phenomena.\n\n\nPerioperative inflammation-related immunosuppression and neoangiogenesis are seen in cancer patients at risk of relapse\n\nThe early existence of dormant metastasis is matter of debate2. One argument is the kinetics observed in cancer recurrence after breast cancer surgery. Indeed, when analyzing the timing of the relapse of patients under endocrine therapy, recurrences occur gradually over the first 10 to 15 years. In contrast, in women not treated with endocrine therapy (i.e. with estrogen-receptor-negative tumors), the majority of cancer relapses occur in the first two years4. This suggests that these tumor cells have been maintained in a \"dormant\" state in the first group of women, whereas they may be present early in both groups5.\n\nThe risk for these patients in undergoing surgery is that rapid growth of these cells could be induced through perioperative inflammation. Indeed, following a surgical trauma, a great, but short-lasting, inflammation is correlated with a potent immune response that precedes a longer duration of immunosuppression1. The role of this immunosuppression is probably to minimize the intensity of the proinflammatory response, and reduce the risks of autoimmune disorders and/or necrosis of tissue6,7. Locally and throughout the body, the release of cytokines underlies the initiation, maintenance and regulation of the inflammatory response. After tissue injury, monocytes and macrophages rapidly release interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-α). IL-1 stimulates and maintains the secretion of other cytokines such as interleukin 6 (IL-6) that are major mediators of the systemic effects of the stress response. The production of adrenocorticotropic hormone (ACTH) and cortisol are stimulated and are regulated by a negative feedback system8.\n\nThe tissue damage caused by surgery is also likely to generate pain. This causes the secretion of endogenous opioids that provide analgesia of short duration by their peripheral and central effects9. Peripherally, the presence of opioid receptors on immune cells allows β-endorphins to have a direct effect on the proliferation, migration and cytotoxicity of these cells9. Some neurotransmitters, including substance P, can interact with the pain pathways. At the central level, active pain regulatory mechanisms act via the periaqueductal gray matter of the midbrain and through the β-endorphin and catecholaminergic pathways. Norepinephrine inhibits natural killer (NK) cell activity via the β-2 receptor. The cholinergic system and the vagus nerve play a role that is often opposite to that of the sympathetic system10.\n\nIn turn, the brain monitors and controls these loops of inflammation. The hypothalamic-pituitary, cortex and cerebellum are involved in the control of lymphoid organs, especially the spleen and many adrenergic receptors are present on B and T lymphocytes, macrophages, neutrophils and NK cells. Finally, the endocrine system interacts with the brain, immune cells and most organs6,7. Glucocorticoids, for example, have anti-inflammatory properties but are essential for normal immune response. Prostaglandins and prostacyclin, particularly PGE2, are produced, among others, by dendritic cells and macrophages. They have a major role in the regulation of immunity in general and NK activity in particular. In addition, PGE2 plays a major role in the stimulation of epithelial cell proliferation, inhibition of apoptosis, production of mutagens and stimulation of angiogenesis11. This may explain the high incidence of overexpression of COX-2 in breast cancer, as in other cancers, and higher aggressiveness of these tumors11.\n\nNeo-angiogenesis is probably a major step in the induction of growth of a dormant metastasis. Indeed, it appears that solid tumors cannot grow larger than 2–3 mm in diameter unless they induce their own blood supply12. The expression of the angiogenic phenotype, physiologically to promote wound healing, is a complex process that depends on a number of cellular and molecular events, including degradation of the surrounding basement membrane, migration of endothelial cells, cell proliferation, the formation of tube-like structures and the maturation of these endothelial-lined tubes into new blood vessels13.\n\nAs a consequence, most of the modifications in the immune system and the angiogenesis phenotype are linked to the degree of tissue injury and its consequent inflammation. The logical strategy is then to promote minimally invasive surgery that is designed to limit these impacts, to maintain homeostasis14 and reduce the stress response8. These arguments have led some authors to consider that minimally invasive procedures might be favorable in terms of immunity when compared with invasive procedures due to the need to manage other changes driven by the \"secondary aggressions\", i.e., hypothermia, hemorrhage, and psychological factors such as anxiety1.\n\n\nEffect of analgesics on inflammation, anticancer immunity and angiogenesis\n\nA possible way to influence the perioperative inflammatory reaction and their consequences for immune cells and angiogenesis is by modifying analgesic techniques. This could have an important impact on patient outcome following surgery.\n\nMacrophages, T lymphocytes, NK T and other cells and many cytokines are involved in the defense mechanisms of nonspecific immunity. Of these, the role of NK cells in defense against infection and the development of tumor cells1,15–17 has been largely demonstrated. The study of animal models has helped us to understand their role in the mechanisms behind perioperative anti-metastatic protection, but also their vulnerability16–18. In humans, a significant correlation between NK activity and patient prognosis is related to the development of metastases19. The strong depression of the cytotoxicity of the NK cells in the perioperative period may significantly alter the defense mechanisms of patients1.\n\nAnalgesic techniques that reduce the inflammatory response may be favorable in terms of immunity8. Recent data suggest that the analgesic techniques (intravenous opioids, non-steroidal anti-inflammatory drugs (NSAIDs) and locoregional analgesia) could have an impact on the long-term prognosis after cancer surgery20,21, including for breast cancer22,23. Opioids are the drugs most studied in the context of the perioperative immune response. In the absence of pain, morphine induces a decrease in NK activity25. It is not clear whether it is the opioids, but possibly also the withdrawal of opioid therapy, that is responsible for opioid-induced immunosuppression24. Nevertheless, pain itself induces a significant immune response. Because this response leads to a significant degree of immunosuppression, postoperative analgesia is, by itself, immunoprotective1. Morphine therefore allows the maintenance of NK activity and protection against metastasis in animal pain models25. In humans, similar arguments can be used to offer the lowest effective dose of opioid24. Data on synthetic opioids have revealed a similar phenomenon with fentanyl and sufentanil, probably mediated via the µ-opioid receptor. Nevertheless, differences linked to selective µ1-activation by synthetic opioids, in contrast to µ3-activation by morphine, could explain the more favorable profile of morphine and may merit further investigation25. Opioids could also affect angiogenesis and the growth of tumors. In the perioperative period and after tissue injury, important angiogenic signals, including epithelial growth factors (VEGF), act via receptor tyrosine kinases and G-protein-coupled receptor (GPCR). This may have a major importance as morphine can transactivate these GPCR, increasing neo-angiogenesis13.\n\nProstaglandins are another major perioperative influence on immunity and angiogenesis and the promotion of tumor growth26. The data concerning the maintenance of NK activity by NSAIDs are encouraging, in some, but only a few human clinical studies. In a retrospective study, we showed that receiving ketorolac, a NSAID, just before surgery, was an independent factor associated with longer recurrence-free survival after breast cancer surgery22. These results may be explained by the fact that prostaglandin E2 (PGE2), released by monocytes and dendritic cells in order to regulate the inflammatory cascade, profoundly depresses cellular antitumoral immunity, i.e. NK activity27. This suppression of NK activity, and possibly an initial flare-up of angiogenesis, dissipates quickly after the removal of the prostaglandins. There may then be a short therapeutic window when NSAIDs may have a potent impact on the oncological outcome22.\n\nIt seems clear that regional anesthesia, particularly central blocks, are associated with anti-inflammatory effects, and allow the protection of anticancer immunity, including NK activity, after major surgery28, but this effect has not been demonstrated after minor surgery29. It is possible that this effect is responsible for a lower incidence of recurrence after surgery for breast, colon or prostate cancers, however this is an unresolved debate due to poor methodology and lack of perioperative immune monitoring20,21,23,30–33.\n\nAnesthesiologists are using many other drugs that are not necessarily associated with analgesic effects. Data are sparse and mostly inconclusive concerning barbiturates, halogenated gases, propofol and etomidate. It seems likely that ketamine, a widely used co-analgesic, has a dose-dependent effect, being protective at low doses during a painful stimulus and potentially harmful at high doses in the absence of surgery24. One interesting topic is the potential protective role of alpha-2-agonists, such as clonidine, typically used as co-analgesic during surgery on perioperative NK activity24.\n\n\nIs it time to change our pain management practices?\n\nA goal of optimal control of pain may appear to be obvious, but it still remains a major concern for perioperative physicians. The quality of analgesia is facilitated by combinations of molecules (NSAIDs and anti-hyperalgesics such as ketamine) and techniques (intravenous, locoregional with local anesthetics). At this time, it is not clear whether these techniques may improve oncological outcome alone, indirectly by the avoidance of opioids, or both. Nevertheless, they improve pain management, postoperative rehabilitation and the prognosis for patients. These reasons remain the main argument to recommend such analgesic approaches before definitive conclusions on their influence on the oncological outcome8,14.\n\nMoreover, it appears that high doses of opioids are carriers of long-term side effects that are probably underestimated (including opioid-induced hyperalgesia). The study of the role opioids play in the increase in angiogenesis and postoperative immunosuppression is of particular importance in oncological surgery, knowing that NK activity could be a prognostic criterion13,19. Taking into account that they improve pain management, it is appropriate to propose opioid-sparing strategies, such as locoregional techniques and sympathetic modulation using intravenous α-2 agonists24.\n\n\nConclusions\n\nUnderstanding cancer development mechanisms is a major goal of clinical research and may lead to the development of strategies to counter the multiple factors involved in cancer pathology. It is very important to study all aspects of the problem, permitting the development of strategies associated with a higher long term survival after cancer surgery. The data available already affirm that the immune system and the angiogenesis phenotype play key roles in anticancer defenses, that the potential influence of surgery-related inflammation is large and that the effects of drugs and the techniques of analgesia may be important. Perioperative physicians must be aware that an optimal analgesic strategy may have a long-term impact on patient outcome.", "appendix": "Author contributions\n\n\n\nPF and MDK contributed to the design, the selection of the literature and the redaction of the manuscript. OS contributed to the redaction of the manuscript and was the scientific/medical advisor.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nShakhar G, Ben-Eliyahu S: Potential prophylactic measures against postoperative immunosuppression: Could they reduce recurrence rates in oncological patients? Ann Surg Oncol. 2003; 10(8): 972–92. PubMed Abstract \| Publisher Full Text\n\nRetsky M, Demicheli R, Hrushesky W, et al.: Surgery triggers outgrowth of latent distant disease in breast cancer: an inconvenient truth? Cancers. 2010; 2(2): 305–337. Publisher Full Text\n\nWrona D: Neural-immune interactions: An integrative view of the bidirectional relationship between the brain and immune systems. J Neuroimmunol. 2006; 172(1–2): 38–58. PubMed Abstract \| Publisher Full Text\n\nStanojević-Bakić N, Vucković-Dekić L, Radomirović S, et al.: The influence of surgery and anesthesia on lymphocyte functions in breast cancer patients: in vitro effects of indomethacin. Neoplasma. 1999; 46(1): 54–60. PubMed Abstract\n\nMansell J, Monypenny IJ, Skene AI, et al.: Patterns and predictors of early recurrence in postmenopausal women with estrogen receptor-positive early breast cancer. Breast Cancer Res Treat. 2009; 117(1): 91–98. PubMed Abstract \| Publisher Full Text\n\nElenkov IJ, Wilders RL, Chrousos GP, et al.: The sympathetic nerve – An integrative interface between two supersystems: The brain and the immune system. Pharmacol Rev. 2000; 52(4): 595–638. 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PubMed Abstract \| Free Full Text\n\nSingleton PA, Lingen MW, Fekete MJ, et al.: Methylnaltrexone inhibits opiate and VEGF-induced angiogenesis: role of receptor transactivation. Microvasc Res. 2006; 72(1–2): 3–11. PubMed Abstract \| Publisher Full Text\n\nKehlet H: Multimodal approach to control postoperative pathophysiology and rehabilitation. Br J Anaesth. 1997; 78(5): 606–17. PubMed Abstract \| Publisher Full Text\n\nCurtis JL, Punturieri A: Enhancing antitumor immunity perioperatively: a matter of timing, cooperation, and specificity. Am J Respir Cell Mol Biol. 2003; 28(5): 541–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nForget P, Collet V, Lavand'homme P, et al.: Does analgesia and condition influence immunity after surgery? Effects of fentanyl, ketamine and clonidine on natural killer activity at different ages. Eur J Anaesthesiol. 2010; 27(3): 233–40. PubMed Abstract \| Publisher Full Text\n\nPage GG, McDonald JS, Ben-Eliyahu S: Pre-operative versus postoperative administration of morphine: impact on the neuroendocrine, behavioural, and metastatic-enhancing effects of surgery. Br J Anaesth. 1998; 81(2): 216–23. PubMed Abstract \| Publisher Full Text\n\nBar-Yosef S, Melamed R, Page GG, et al.: Attenuation of the tumor-promoting effect of surgery by spinal blockade in rats. Anesthesiology. 2001; 94(6): 1066–73. PubMed Abstract \| Publisher Full Text\n\nLiljefors M, Nilsson B, Hjelm Skog AL, et al.: Natural killer (NK) cell function is a strong prognostic factor in colorectal carcinoma patients treated with the monoclonal antibody 17–1A. Int J Cancer. 2003; 105(5): 717–23. PubMed Abstract \| Publisher Full Text\n\nBiki B, Mascha E, Moriarty DC, et al.: Anesthetic technique for radical prostatectomy surgery affects cancer recurrence: a retrospective analysis. Anesthesiology. 2008; 109(2): 180–7. PubMed Abstract \| Publisher Full Text\n\nChristopherson R, James KE, Tableman M, et al.: Long-term survival after colon cancer surgery: a variation associated with choice of anesthesia. Anesth Analg. 2008; 107(1): 325–32. PubMed Abstract \| Publisher Full Text\n\nForget P, Vandenhende J, Berliere M, et al.: Do intraoperative analgesics influence breast cancer recurrence after mastectomy? A retrospective analysis. Anesth Analg. 2010; 110(6): 1630–1635. PubMed Abstract \| Publisher Full Text\n\nExadaktylos AK, Buggy DJ, Moriarty DC, et al.: Can anesthetic technique for primary breast cancer surgery affect recurrence or metastasis? Anesthesiology. 2006; 105(4): 660–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nForget P, De Kock M: [Could anaesthesia, analgesia and sympathetic modulation affect neoplasic recurrence after surgery ? A systematic review centred over the modulation of natural killer cells activity]. Ann Fr Anesth Reanim. 2009; 28(9): 751–68. PubMed Abstract \| Publisher Full Text\n\nStefano GB, Kream RM, Mantione KJ, et al.: Endogenous morphine/nitric oxide-coupled regulation of cellular physiology and gene expression: implications for cancer biology. Semin Cancer Biol. 2008; 18(3): 199–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalker W, Rotondo D: Prostaglandin E2 is a potent regulator of interleukin-12 and interleukin-18-induced natural killer cell interferon-gamma synthesis. Immunology. 2004; 111(3): 298–305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYakar I, Melamed R, Shakhar G, et al.: Prostaglandin e(2) suppresses NK activity in vivo and promotes postoperative tumor metastasis in rats. Ann Surg Oncol. 2003; 10(4): 469–79. PubMed Abstract \| Publisher Full Text\n\nKoltun WA, Bloomer MM, Tilberg AF, et al.: Awake epidural anesthesia is associated with improved natural killer cell cytotoxicity and a reduced stress response. Am J Surg. 1996; 171(1): 68–72. PubMed Abstract \| Publisher Full Text\n\nTøonnesen E, Huttel MS, Christensen NJ: Natural killer cell activity in patients undergoing minor gynaecological surgery. Eur J Anaesthesiol. 1987; 4(2): 119–25. PubMed Abstract\n\nGottschalk A, Ford JG, Regelin CC, et al.: Association between epidural analgesia and cancer recurrence after colorectal cancer surgery. Anesthesiology. 2010; 113(1): 27–34. PubMed Abstract \| Publisher Full Text\n\nTsui BC, Rashiq S, Schopflocher D, et al.: Epidural anesthesia and cancer recurrence rates after radical prostatectomy. Can J Anaesth. 2010; 57(2): 107–12. PubMed Abstract \| Publisher Full Text\n\nForget P, Leonard D, Kartheuser A, et al.: Choice of Endpoint and Not Reporting All the Analgesics Used May Render Inconclusive Studies on Oncological Outcome. Anesthesiology. 2011; 114(3): 717. Publisher Full Text\n\nForget P, Tombal B, Scholtès JL, et al.: Do intraoperative analgesics influence oncological outcomes after radical prostatectomy for prostate cancer? Eur J Anaesthesiol. 2011; 28(12): 830–5. PubMed Abstract" }	[ { "id": "879", "date": "08 Apr 2013", "name": "Alain Borgeat", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 more and more clues that inflammation, immune system and cancer recurrence may be directly associated. Therefore, this manuscript is very actual. It is well constructed and raises basic questions concerning the expected future adaptations of the management of the perioperative period.", "responses": [] }, { "id": "914", "date": "07 May 2013", "name": "Erxi Wu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 research topic. It describes that cancer surgery induces inflammation, immunosuppression, and angiogenesis. Also these phenomena can be influenced by analgesics. As we know, Galen (Aelius Galenus) removed some tumours surgically, but he generally believed that cancer was best left untreated. The underlying mechanisms were not clear then. The research community can benefit from this well-written paper as this paper has updated the information for this topic.This paper can be improved if the mechanisms would have been discussed in depth. After cancer surgery, HIF-1alpha and MMPs may also be up-regulated. This paper has not mentioned them. Thus, it would be helpful if authors search and study more literature. I have simply searched some literature, I think the following papers should be cited:Tavare AN et al. Cancer recurrence after surgery: direct and indirect effects of anesthetic agents. Int J Cancer. 2012 doi: 10.1002/ijc.26448 Retsky M et al. NSAID analgesic ketorolac used perioperatively may suppress early breast cancer relapse: particular relevance to triple negative subgroup. Breast Cancer Res Treat. 2012 doi: 10.1007/s10549-012-2094-5Deegan CA et al. Anesthetic technique and the cytokine and matrix metalloproteinase response to primary breast cancer surgery. Reg Anesth Pain Med. 2010 doi: 10.1097/AAP.0b013e3181ef4d05Thaker PH et al. Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma. Nat Med. 2006 doi: 10.1038/nm1447", "responses": [] }, { "id": "938", "date": "09 May 2013", "name": "Dan Benhamou", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTo the Editor,First, note that it is extremely difficult to write detailed comments as lines are not numbered. Please modify the text to facilitate reviewers’ work.This is a nice review of the role of analgesia on postoperative outcome after cancer surgery. As stated by the authors, we still need confirmatory data and the present status of our knowledge only suggests a speculative effect, even if most studies concur to suggest a beneficial effect of a well-performed postoperative analgesia. It would also be important to note that all additional factors of a well-performed anesthesia (i.e. avoidance of hypotension or of hypothermia, treatment of anemia, prevention of hyperglycemia…) may also be factors that could improve postoperative outcome after cancer surgery.That said, the reviewer has several minor comments to add:Page 2, 2nd column, L12 of 1st paragraph: is instead of are (the subject of the sentence is “The production of…”)Page 2, 2nd column, L2 of 3rd paragraph: hypothalamic-hypopituitary complex/system/unit/axis… (choose one word)Page 3, 1st column, L9 of 2nd paragraph: add “reduced”? (…with invasive procedures due to the reduced need…)Page 3, 1st column, L1 of 5th paragraph: in this sentence and all along the text, use “regional” instead of “locoregional”Page 3, 1st column, L10 and following lines of 5th paragraph: paragraph unclear: reorganize sentences to present first the beneficial effects of opioids (reduced pain) and then their harmful effects to facilitate readingPage 3, 2nd column, L3 of 3rd paragraph: what do the authors mean by “protection of anticancer immunity”?Page 4, 1st column, L10 of 2nd paragraph: it appears somewhat surprising that alpha-2 agonists are promoted with some apparent strength given the fact that our knowledge on these drugs in this context are not better that for other agents. Please modify the sentence to attenuate the strength of this recommendation.", "responses": [] } ]	1	https://f1000research.com/articles/2-102
https://f1000research.com/articles/2-101/v1	02 Apr 13	{ "type": "Opinion Article", "title": "Energy efficiency as a unifying principle for human, environmental, and global health", "authors": [ "Luigi Fontana", "Vincenzo Atella", "Daniel M Kammen" ], "abstract": "A strong analogy exists between over/under consumption of energy at the level of the human body and of the industrial metabolism of humanity. Both forms of energy consumption have profound implications for human, environmental, and global health. Globally, excessive fossil-fuel consumption, and individually, excessive food energy consumption are both responsible for a series of interrelated detrimental effects, including global warming, extreme weather conditions, damage to ecosystems, loss of biodiversity, widespread pollution, obesity, cancer, chronic respiratory disease, and other lethal chronic diseases. In contrast, data show that the efficient use of energy—in the form of food as well as fossil fuels and other resources—is vital for promoting human, environmental, and planetary health and sustainable economic development. While it is not new to highlight how efficient use of energy and food can address some of the key problems our world is facing, little research and no unifying framework exists to harmonize these concepts of sustainable system management across diverse scientific fields into a single theoretical body. Insights beyond reductionist views of efficiency are needed to encourage integrated changes in the use of the world’s natural resources, with the aim of achieving a wiser use of energy, better farming systems, and healthier dietary habits. This perspective highlights a range of scientific-based opportunities for cost-effective pro-growth and pro-health policies while using less energy and natural resources.", "keywords": [ "Energy efficiency", "obesity", "climate change", "calories", "fossil fuels" ], "content": "Introduction\n\nSeveral interrelated challenges now face the world, including (1) providing adequate food, clean drinking water, and non-renewable energy resources, which exist in finite supplies, to an exponentially growing population; (2) creating a sustainable global economy that does not destroy the environment or compromise human health; and (3) limiting the detrimental socioeconomic and health effects of the worldwide epidemic of unhealthy nutrition and obesity. How can we handle these challenges? We believe that today the right answer to many of these problems is a more efficient and wise use of energy, food, water, and other natural resources. The current economic model is unsustainable. As we will discuss in this paper, reliance on technology to produce more food and energy to drive economic growth can be successful in the short term, but it has long-lasting, seriously detrimental consequences for human and environmental health and, eventually, for societal well-being. In contrast, energy efficiency can play a key role in promoting human, environmental, and planetary health and sustainable economic development.\n\nThe term “energy efficiency” usually refers to devices or engineered systems that provide the same level of output or benefit with less energy consumption. However, accumulating scientific evidence indicates that energy efficiency is also an important principle for optimizing physiological functions within organisms, both simple and complex, including mammals. Hundreds of studies have shown that moderately energy-restricted animals live much longer and in better health than animals that have free access to food energy1–3. A similar outcome is seen when the activity of energy-sensing cellular pathways (i.e. the insulin and insulin-like growth factor 1 signaling pathway and the mammalian target of rapamycin pathway) is reduced by genetic manipulation or chemical inhibitors2.\n\nSimilarly, and in a way that is not just analogical but organically and causally linked, energy and resource efficiency is vital at the level of society and the biosphere. Essential for achieving environmental health and sustainable economic development, reducing the consumption of non-renewable energy and other natural resources has profound implications for human health as well. While it is clear that a lack of access to energy and resources, or their uneven distribution, chokes economic development, excess energy consumption from fossil-fuel sources promotes extensive pollution and global warming, and is a sign of economic and public ill-health and inequality4. Squandering energy resources, even if carbon-free, has collateral impacts–such as potential excessive exposure to cadmium or other toxic compounds, excessive mining and demand for rare earths and other precious materials, water depletion, and resource waste, which degrades well-being5. More generally, energy and other resource waste is a critical sign of a system that is not providing for basic needs or supporting innovation, and is ultimately damaging the biosphere and human health as well6.\n\nCorrecting excessive dietary energy intake to achieve optimal body weight and health, and deploying more energy-efficient buildings, vehicles, appliances and industrial equipment, fit into a continuum of actions that hold the potential to reduce the world’s projected energy needs by more than half, and to become the prime movers of cost-effective control of pollution and global greenhouse gas emissions7,8. In contrast, producing and consuming more fossil-fuel and food energy causes a vicious cycle by increasing unhealthy emissions, global warming, floods, droughts, land desertification, water shortages, and ultimately, reduced crop harvests9,10.\n\nWe propose the concept that a deep parallel connection exists between over/under consumption of energy at the level of the human body and at the level of the biosphere, and that this connection has profound implications for human and environmental health. While it is not new to highlight how efficient use of energy and other resources can address some of the key problems our world is facing, little or no unifying framework exists to combine and harmonize these concepts of sustainable system management across diverse fields (i.e. biology, medicine, ecology, economics, engineering, information technology, etc.) into a single theoretical body. Clearly, there is a need for new strategies and effective policies that encourage integrated changes in the use of the world’s natural resources, with the aim of achieving a wiser use of energy, better farming systems, and healthier dietary habits. We believe it is necessary to develop more complex models of analysis based on a multi-objective set of constraints.\n\n\nEnergy efficiency and human health\n\nLife expectancy at birth has almost doubled in most developed countries over the last century, with the oldest group (aged >65 years) being the most rapidly growing segment of the population11. However, the overall increase in average life span is far greater than that of healthy lifespan. Globally, about 80% of older adults are affected by at least one chronic disease, and 50% have two or more chronic diseases (e.g. cardiovascular disease, stroke, cancer or type 2 diabetes)12. These chronic diseases, which according to the World Health Organization (WHO) are largely preventable13, are the leading cause of morbidity and mortality, as well as major contributors to economic losses and a driver of social burdens12,13. These problems are exacerbated by the current epidemic of excess weight and obesity, in which excessive adiposity is causally associated with an increased risk of developing type II diabetes, cardiovascular disease, cancer, and disability3,12,13. Accordingly, our (unpublished) data, derived from a very large dataset of Italian patients seen by general practitioners through the National Health Search Network, show that excessive body weight is associated with a striking increase in health-care costs that could very likely lead to the bankruptcy of the health care system (Figure 1). Clearly, Italy is not an exception in the industrialized world.\n\nAge-adjusted outpatient health care costs (e.g. pharmaceutical, diagnostic and specialist visit expenditure) are shown per capita per year. We examined the relationship between BMI and medical care expenditure based on a sample of 423,682 Italian adults aged 18–95 in 2008–2010 (unpublished data).\n\nAt the organismic level, sufficient but not excessive energy intake is vital for promoting health and longevity2. At the extremes, both insufficient (i.e. starvation) and excessive (i.e. overweight and obesity) energy intake cause unfavorable alterations in body composition, metabolism, and organ function, eventually leading to premature death3. In contrast, data from a multitude of studies indicate that a moderate reduction in energy intake below usual ad libitum levels without malnutrition prevents or delays a wide range of chronic diseases, results in a dramatic increase in healthspan and lifespan, and preserves a number of measured metabolic and physiologic functions found in experimental animals in more youthful-like states1–3. The beneficial effects of dietary restriction (DR) in rodents can be achieved by reducing energy consumption, but also by reducing protein or methionine intake1–3,14–16. These data have recently been confirmed in nonhuman primates. In a 20-year study, adult rhesus monkeys subjected to a 30% reduction in dietary intake experienced no diabetes, a 50% decline in cancer and cardiovascular morbidity and mortality, and less sarcopenia and neurodegeneration17,18.\n\nFurthermore, data from recent clinical studies indicate that in humans, DR results in some of the same metabolic and physiologic adaptations related to healthy longevity found in DR rodents2,3. Individuals practicing moderate DR with adequate nutrition are powerfully protected against obesity, type 2 diabetes, high blood pressure, inflammation, and cardiovascular disease, and have lower cancer risk factors3,19,20. Moreover, it has been shown that reducing dietary protein intake lowers the circulating levels of a key growth factor (IGF-1) that plays an important role in the pathogenesis of prostate, breast and colon cancer, and in the aging process itself21,22. This finding is important because the recommended daily allowance (RDA) for protein intake is 0.83 g/kg of body weight/day, yet at least 50% of men and women in many developed countries chronically consume twice as much protein as the recommended intake23. Related to this problem is the fact that the majority of protein in the diet of North American and European citizens comes from foods of animal origin that promote weight gain and are rich in atherogenic saturated fatty acids24,25. Overconsumption of animal protein relative to other nutrients is not only a current epidemic among the affluent, but is a clear aspirational goal of millions of poor people in developing countries who are disproportionately increasing their consumption of meat and dairy as their wages rise above poverty level7. The pressure of this overconsumption of animal foods on water and land use is intense, with 70% of all land under tillage used to feed livestock, which can have as high as a 21:1 ratio of vegetable input to meat output26. By contrast, if global dietary patterns changed to reduce the consumption of animal source foods and led to the adoption of a diet rich in plant-based foods, only 30 to 40% of the crops cultivated currently would be needed, significantly reducing air, water and land pollution (from the intensive use of reactive nitrogen and phosphorus fertilizers, and pesticides, and the poor management of animal wastes in many regions)27–29, topsoil impoverishment, over-pumping of groundwater, agriculture-related fuel consumption, and greenhouse gas emissions30,31. Furthermore, if people ate fewer foods rich in empty calories, less meat- and dairy-derived foods and consumed more vegetables, fruits, beans, whole grains, seeds, and nuts, overweight and obesity rates could be reduced and many age-associated chronic diseases could be prevented, significantly reducing the gap between lifespan and healthspan, as well as health care costs3,12,13,19,24,25.\n\n\nSocial and environmental consequences of energy inefficiency\n\nAccording to the United Nations Population Division, the world’s population reached seven billion in 2012, and it is expected to reach nine billion by 205032. This unprecedented population expansion will require a massive increase in energy and food production to meet increasing demands. Although, we have the technology to further increase energy and food production (e.g. off-shore oil drilling, hydraulic fracturing for extracting natural gas from shale rock layers, biofuels, use of new pesticides, antibiotics, and more chemical fertilizers, genetically modified crops, etc.), this escalating demand will collide with the finite planet’s natural resources and the capacity to further absorb the increasing emissions produced by billions of people who live and work in energy-inefficient buildings; drive energy-inefficient, polluting motor vehicles; and desire to consume the same unhealthy, high-calorie diets rich in animal protein and fat that are typical of Western countries.\n\nToday, fossil fuels account for roughly 85% of total energy use worldwide for the heating/cooling of buildings, transportation, industrial activities, manufacturing, and other applications8,33. The use of this non-renewable energy mix is responsible for roughly 80% of the total anthropogenic greenhouse gas emissions, and for half of the short-lived greenhouse gases such as methane33. It has been estimated that intensive agriculture and animal farming alone already contribute almost 20% of worldwide annual greenhouse gas emissions31,33, and are responsible for increasing soil erosion and water resource pollution and depletion34. Soil erosion and water pollution associated with intensive farming in turn lead to even greater use of fossil fuels and more global warming, because more energy is needed to process polluted water and to produce more hydrocarbon-based fertilizers and pesticides in order to grow monoculture crops in a topsoil increasingly depleted of nutrients29,31,34.\n\nGlobal warming is now seen scientifically as a major threat to not only life but also livelihoods on our planet, and has serious environmental, social, and economic implications. Even the warming to which we are already committed in the coming decades (only a 1–2.5°C increase), is now predicted to have significant environmental consequences, including drought and land desertification, water shortages and reduced crop harvests, floods due to extreme weather and glacial retreat, inundation of coasts and small islands due to sea level rise, more frequent and devastating storms, extinction of plant and animal species, and diffusion of climate-sensitive diseases such as malaria9,10.\n\nWhile climate change is the hot-button issue in our global energy metabolism, other byproducts generated by the excessive use of pesticides, chemical fertilizers, and antibiotics in intensive agriculture, the combustion of fossil fuels, the mining of coal and other metals, drilling for oil, and nuclear accidents are also direct causal agents of serious morbidity and mortality events for humans (e.g. cancer, chronic respiratory disease, asthma, and heart disease) and for the environment (e.g. acid rain and eutrophication resulting in toxic algal blooms, hypoxia, increased incidences of fish kills, loss of biodiversity, topsoil erosion, and water pollution)34–36. Figure 2 shows a range of impacts for insufficient and excessive energy resource access.\n\nA schematic comparison of costs of energy poverty and excess energy consumption. The rough U-shape is characteristic of systems with excess impacts related to extremes of resource access and use. An associated aspect of the process of defining a regime of ‘wise use’ of resources is the role of efficiency relative to robustness of the system. A useful alternate representation is to place efficiency (“streamlining”) and diversity (“robustness”) as extremes on a single axis38.\n\nThe resource efficiency paradigm has clear and immediate impacts when applied to the global energy budget. It has been estimated that the development and wide-spread use of more energy-efficient residential and industrial buildings, ultra-efficient lighting technologies, energy-saving appliances, and light-weight ultra-low-drag hybrid-electric motor vehicles could save 70% or more of the energy that we consume every day and drastically reduce CO2 emissions and pollution8,33. These steps are important individually, but take on particular significance when these patterns of highly-efficient use are taken as more than one-off policies, but as guiding principles for the design of the entire energy and natural resource utilization systems. Further, fossil fuel use via simple-cycle turbines is inherently wasteful due to the thermodynamic requirement to reject, or emit to the environment all energy beyond the Carnot efficiency limit of η = (total work/heat transferred from the engine) = 1 – (Tcold/Thot), or roughly one third of the energy in the fuel for a standard power plant. This waste can be partially re-captured if overall system efficiency is taken as a design principle and a ‘combined heat and power’ system is utilized instead to capture the rejected heat for other uses such as warming homes or driving a second, lower energy engine cycle. Moreover, energy use of industrial equipment could be further reduced by changing their technical design, by using smart materials in conjunction with sensors and software that promote energy efficiency under all operating conditions. Finally, to improve energy resilience, it is necessary to combine energy efficiency with a steep increase in the development of renewable energy resources and the use of information and smart technology37. Using digital intelligence and smart technologies to improve the current grid systems could prevent outages and faults, restore outages faster, and help manage demands. For example, by assessing energy needs through use of meters, sensors, digital controls and analytic tools to monitor, control and automate the two-way flow of energy across operations, energy consumption could be substantially reduced. Smart grids can also integrate renewable energy sources (i.e. solar, wind and geothermal power), and interact locally with distributed power sources, or plug-in electric vehicles.\n\n\nConclusions\n\nSignificantly improving human and environmental health, societal wealth and well-being is possible, but requires a profound transformation in the way we live, and a new environment-centered industrial and economic system. Most of the needed knowledge and technology to enact a reshaping of our future and a new industrial revolution already exist today. In summary, we need to abandon the paradigm of producing more energy, food, and other products at lower cost in favor of a new paradigm that opts for less but high-quality energy, food and materials for a healthier life and environment. At the individual level, reducing the intake of calories by increasing the consumption of a variety of minimally processed plant foods and by significantly reducing the intake of animal foods will significantly increase health span and reduce health care costs, environmental pollution, soil erosion, water pollution and shortage, CO2 production and global warming, violent weather and associated planetary consequences. Similarly, making our houses more energy efficient and resilient (e.g. wall and roof insulation, energy-efficient windows and doors, ultra-efficient lighting technologies, energy-saving appliances, solar power to heat water and produce electricity, geothermal heat pumps, etc.), buying lightweight hybrid-electric motor vehicles, and reducing waste by choosing reusable products instead of disposables have huge effects in protecting human and environmental health as well. At the community level, we need more public/private investment and research in “green” chemistry, technologies and practices, including sustainable farming, breakthrough materials to improve building and vehicle efficiency, new technologies that better extract energy from renewable sources, hydrogen-fueled cars and buildings, and applications of modern information technology to maximize energy efficiency and resilience. The application of the energy efficiency and resource productivity paradigm offers a new ground for business invention, sustainable growth and economic development.\n\nWe also need to design and implement policies that enhance literacy about human and environmental health; improve the livability of our cities and towns by implementing, for example, projects for non-motorized transport, green spaces and parks; reward good behaviors, while enforcing the true costs of poor behavior (e.g. by lowering health insurance premiums for people with healthy lifestyles and metabolic profiles, taxing carbon and junk food, and ending subsidies for mining, oil, coal, corn, soy, and intensive factory animal farming).\n\nMost importantly, we need to understand that both individual and societal wealth, happiness, and well-being do not depend merely on the acquisition of material goods and on economic growth, but are powered by our physical and psychological health, the quality of life and the richness of our social relationships, and foremost by the health of the environment that supports all life on earth, our “natural capital” that must be preserved.", "appendix": "Author contributions\n\n\n\nLF, VA, DMK conceived and drafted the report. All authors contributed to critical revision of the report.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSupported by grants from the National Center for Research Resources (UL1 RR024992; a component of the National Institutes of Health and NIH Roadmap for Medical Research), the National Institute of Diabetes And Digestive And Kidney Diseases (P30DK056341), the Longer Life Foundation (an RGA/Washington University Partnership), AFAR, and a donation from the Bakewell Foundation and the Scott and Annie Appleby Charitable Trust. DMK gratefully acknowledges support from the Class of 1935 of the University of California, Berkeley, and the Karsten Family Foundation Endowment of the Renewable and Appropriate Energy Laboratory.\n\n\nAcknowledgments\n\nWe thank Edward L. “Ted” Bakewell III, Jim Dryden, Annie Gottlieb and Joanna Kopinska for their useful comments.\n\n\nReferences\n\nWeindruch R, Walford RL: The retardation of aging and disease by dietary restriction. Springfield, IL: Charles C Thomas Publisher, 1988. Reference Source\n\nFontana L, Partridge L, Longo VD: Extending healthy lifespan–from yeast to humans. Science. 2010; 328(5976): 321–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFontana L, Klein S: Aging, adiposity and calorie restriction. JAMA. 2007; 297(9): 986–994. 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Reference Source" }	[ { "id": "930", "date": "07 May 2013", "name": "Paul Terry", "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\nPeople can consume calories beyond their need and gain unhealthy weight. In ways that are somewhat analogous, societies can consume fossil fuel and other resources inefficiently, thereby transforming and polluting the environment in ways that are harmful. If this were the main point of the article, the reader might feel that the message is too obvious, the supporting facts too well known. Rather, the main thrust of the article is to delineate those analogous connections. Faithful to the article’s title, the authors assert: “A strong analogy exists between over/under consumption of energy at the level of the human body and the industrial metabolism of humanity.” They continue: “Similarly [with respect to the activity of energy-sensing cellular pathways in people], in a way that is not just analogical but organically and causally linked, energy and resource efficiency is vital at the level of society and the biosphere… We propose the concept that a deep parallel connection exists between over/under consumption of energy at the level of the human body and at the level of the biosphere, and that this connection has profound implications for human and environmental health.” Here, we were unsure as to the exact nature of their thesis, but it seemed the authors may be suggesting a remarkable synergy of very different effects on health, which may be measurable and ultimately help to define research priorities and improve study methods. However, the next two sections in the manuscript discuss the human health consequences of dietary energy over-consumption in individuals, and industrial over-consumption in societies, respectively, without showing many, if any, clear parallels. For example, it may be true that replacing meat consumption with that of vegetables may benefit both individuals and the environment, but clearly through different mechanisms and with different arrays of outcomes. This latter is a problem the authors don’t acknowledge. And, of course, under-consumption has very different implications to individuals, their cells, society and the environment, but except for the inclusion of the term in the sentences quoted above, under-consumption and its various implications are not discussed. In the previous examples and others, the “deep parallel connections” between cells and biosphere were neither self-evident nor made clear by the authors. Consequently, much of this opinion piece on energy consumption, particularly as expressed by the authors as an analogical link between human cells, society, and the biosphere, was not convincing. We were left with the obvious fact that over-consumption of personal or industrial fuel has consequences to human health. In summary, we found the main motif of the article, namely, the analogical linkage between personal and societal energy consumption, lacking in clarity, structure, and careful support. Data presented in tables lacked description of source and methods, making them difficult for us to interpret as well. Even so, we commend the authors for expressing poignant and relevant thoughts about how we as individuals, and as members of a society, may protect our future health, and how individual lifestyle may interact with the environment in that regard.", "responses": [ { "c_id": "455", "date": "13 May 2013", "name": "Luigi Fontana", "role": "Author Response F1000Research Advisory Board Member", "response": "This is an opinion article, and not a research or a systematic review article, in which a physician scientist expert of human nutrition and longevity, an engineer expert in energy efficiency, and an economist expert in health economics, joined in an interdisciplinary manner to highlight some of the main interconnected problems that our world is facing. We believe that the reviewers missed the aim of our “opinion” paper, and did not make any effort to read some of the reference articles that have been cited to support our thoughts. It is impossible, in twenty-eight hundred words, to present and discuss in detail all the topics that we have addressed in this paper. Our main aim was just to combine research data and concepts generated from our and other laboratories in order to stimulate the discussion and stress the importance of an interdisciplinary approach to address many of the problems that are negatively impacting human and environmental health around the developed and developing world. It is not true that we discussed the consequences of dietary energy over-consumption in individuals, but instead we discussed the health consequences of calorie and protein restriction without malnutrition on metabolic health. We highlighted the importance of a new set of data on the molecular mechanisms regulating healthy aging and longevity, and the importance of these data in redesigning the health and agriculture systems. We highlighted how new scientific data generated from metabolic and molecular studies on animal model of longevity and humans support the concept that less calories and proteins (especially animal proteins) are needed to promote human and environmental health and to reduce global warming. To our knowledge, nobody working in the field of sustainable agriculture and environmental health has ever attempted to link the molecular mechanisms regulating healthy longevity (i.e. down regulation of the cellular energy-sensing pathways by calorie and protein restriction) with environmental health. We also tried to highlight how ancestral cellular and molecular mechanisms have been designed to promote health when food is scarce, and energy is used efficiently not for growth but for cell repair and maintenance. Analogically, we stressed how energy efficiency, and not more production of food and energy, is essential to promote both environmental and human health, and sustainable economic development. In the medicine field, most of the research funding is devoted to study drugs that may block pathways that are promoting cancer, cardiovascular disease, type 2 diabetes, and other chronic disease. In contrast, a trivial amount of research money is spent to study and implement dietary interventions that promote healthy longevity. In the meantime, for example in the United States, an alarming 70% and 33% of the adults are overweight or obese, respectively. Moreover, only approximately 7% of the remaining normal weight (BMI 18.5-25 kg/m2) US men and women are lean because they are practicing a healthy lifestyle (i.e., eating a healthy moderate calorie restricted diet, exercising regularly, and not smoking). This misuse of public and private funding, that for example paradoxically promotes high protein diets as a tool for treating obesity and the chronic diseases associated with obesity, has serious detrimental consequences on the environment, because intensive animal farming contributes to almost 20% of worldwide annual greenhouse gas emissions, to soil erosion, and pollution." } ] }, { "id": "1207", "date": "05 Aug 2013", "name": "Riccardo Pietrabissa", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, an opinion article, points out a parallelism between systems (human body, environment and industry, earth) using energy consumption as a criteria for comparison. The paper does not introduce significant new knowledge on each of these systems and their relations with energy consumption, but expresses a global frame to rethink the use of energy. It may concern the goal of optimization in contrast with maximization or minimization, quality with respect to quantity. The consequence of these considerations are in wiser design of processes: prevention for health, efficiency for devices, integration for more complex systems as factories, buildings, crops, transportation. The considerations stated by authors address some political issues that require deeper analyses, certainly not possible in this short paper. Among these: is it possible to face the healthy aging and environment/industry sustainability jointly by public and private? Is it convenient to prevent diseases rather then treat them? Will it be possible, useful and profitable to continue to waste energy in the future as we have done in the past? It could be trivial to discuss all those questions and many others, each is well known. The parallelism introduced by this paper allows a greater vision on the problem of the quality of life in a better world not only for us in the remaining years of life, but for the future generations. The opinion of the authors is an interesting multidisciplinary viewpoint that can open a discussion on the role of science in suggesting integrated solutions for those open questions.", "responses": [] }, { "id": "1358", "date": "06 Aug 2013", "name": "Tony McMichael", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 important, big-picture, integration of the fundamental role of energy efficiency in the metabolism of organisms and of human societies - a role that provides guidance towards achieving balance and stability in complex systems, be they human bodies or energy-dependent economies. Energy flow and conservation is the currency of the world's processes of neg-entropy at all scales - the counter to the universal intrinsic tendency of all ordered systems to degrade and become disordered, as energy degrades to heat. The authors have been thorough in their review and consideration of the relevant published literature from different fields of inquiry, and offer a unifying perspective on the achievement of stability, balance and sustainability.", "responses": [] } ]	1	https://f1000research.com/articles/2-101
https://f1000research.com/articles/2-100/v1	28 Mar 13	{ "type": "Review", "title": "Nanotechnology-supported THz medical imaging", "authors": [ "Andreas Stylianou", "Michael A Talias" ], "abstract": "Over the last few decades, the achievements and progress in the field of medical imaging have dramatically enhanced the early detection and treatment of many pathological conditions. The development of new imaging modalities, especially non-ionising ones, which will improve prognosis, is of crucial importance. A number of novel imaging modalities have been developed but they are still in the initial stages of development and serious drawbacks obstruct them from offering their benefits to the medical field. In the 21st century, it is believed that nanotechnology will highly influence our everyday life and dramatically change the world of medicine, including medical imaging. Here we discuss how nanotechnology, which is still in its infancy, can improve Terahertz (THz) imaging, an emerging imaging modality, and how it may find its way into real clinical applications. THz imaging is characterised by the use of non-ionising radiation and although it has the potential to be used in many biomedical fields, it remains in the field of basic research. An extensive review of the recent available literature shows how the current state of this emerging imaging modality can be transformed by nanotechnology. Innovative scientific concepts that use nanotechnology-based techniques to overcome some of the limitations of the use of THz imaging are discussed. We review a number of drawbacks, such as a low contrast mechanism, poor source performance and bulky THz systems, which characterise present THz medical imaging and suggest how they can be overcome through nanotechnology. Better resolution and higher detection sensitivity can also be achieved using nanotechnology techniques.", "keywords": [ "Terahertz Medical Imaging", "Nanotechnology", "Nano-contrast Agents" ], "content": "Introduction\n\nNanotechnology is one of the newest fields of technology and science that has attracted the attention of the scientific community, since it is believed to possess the potential to entirely change our everyday life as we know it up to now. It is quite complicated to identify the origins of nanotechnology; however, no-one can deny the fact that the inspiring lecture of R. Feynman (29 December 1959) “There is plenty of room at the bottom” is the keystone to the field of nanotechnology1. This talk was so amazing for its time that many believe that it represented the birth of the new scientific field of nanotechnology.\n\nNot only are the origins of nanotechnology complicated, but also the definition of the term ‘nanotechnology’ is not as straightforward as it sounds since the field is very recent and there are many conflicting opinions. The first part of the words nanotechnology and nanoscience, the word nano, comes from the Greek word ‘nannos’, which means a very short man2 and indicates that we are referring to the technology and science that deal with the physical phenomena/technology in the nanoscale. Generally, we call nanotechnology the manipulation and study of the properties of objects that are, in at least one of their dimensions, smaller than 100 nm.\n\nThe importance of nanotechnology is the fact that on the nanometre scale, dimensions of materials are essential to characterise their properties3. At such dimensions, materials possess new physical properties or exhibit new physical phenomena. At such small dimensions, the properties of matter are completely different from what we have been taught and new uncommon properties are observed4. The new properties arise from the fact that at these dimensions the surface area per volume is increased and the material properties obey the rules of quantum mechanics and not the classical physics of the macroscopic scale5. Therefore, nanotechnology has not only to do with small dimensions, but also with new novel physical properties2.\n\nThe emerging applications of nanotechnology are so powerful that many scientists believe that it has the potential to radically change the world as we know it and some of them are even wondering whether nanotechnology can push forward the next 'nano-industrial revolution’6,7. One area that has been very promising is the application of nanotechnology to medicine5, the so-called nanomedicine. Through the developing field of nanomedicine, nanotechnology and medicine come together so that existing therapies and medical techniques can be improved8. Due to its significance for humans, nanomedicine has become one of the most crucial branches of nanoscience. It is considered to be the great challenge of medicine of the 21st century, mainly in three key areas: diagnosis, treatment and regenerative medicine9.\n\nA scientific and technological area with so many expectations will inevitably also positively affect the field of medical imaging and radiology. The field of medical imaging is very broad and since the discovery of X-rays, many non-invasive imaging modalities have been invented. Each modality presents its unique characteristics and its intrinsic limitations, and there are differences in their ionising or non-ionising nature, sensitivity, resolution, complexity, time of data acquisition, physical principles, performance conditions, provided information and of course the financial costs. Although the field of medical imaging has a quite long history, new innovative imaging modalities emerge in order to reduce the limitations and expand the capabilities of the existing modalities10. Unfortunately a ‘perfect and ideal’ imaging modality has not yet been developed and the existing modalities are characterised by different limitations. According to Boulaiz et al.8, an ideal imaging modality should have a non-invasive nature, high sensitivity and the ability to provide information on cell survival, function and localisation.\n\nAn area that attracts the interest of researchers is the use of non-invasive and non-ionising radiation for medical-imaging purposes. It has been stated that there is a revolution in non-invasive imaging modalities11 and imaging modalities that do not use ionising radiation minimise patient’s risks, enable imaging repeatability and in many cases are non-invasive and reduce patient’s suffering. According to Wallace et al.12 there is a gap between microscopy and medical imaging and consequently current efforts are focusing on developing non-ionising modalities that can fill this gap. One of the most recent and attractive modalities that satisfy these requirements is Terahertz (THz) imaging13–16.\n\nTHz radiation, also called ‘sub-millimetre radiation’ or ‘T-rays’, is generally defined as the frequency range from 100 GHz to 10 THz17 and is actually the gap between the infrared (IR) and microwaves13 (Figure 1). This region of the electromagnetic spectrum remained unexplored for many years since there were not appropriate sources (electronic or optical)18 available, although the characteristics of this radiation are unique (Table 1) and there are a number of potential applications. The development of ultra-short optical pulse lasers and the growth of semiconductor microfabrication techniques pushed for the expansion of THz radiation technology19.\n\nIt can be seen that THz radiation is the gap between the infrared and microwaves.\n\nThis paper aims to investigate whether nanotechnology can reform a specific imaging modality, THz imaging, and support it in order to overcome its limitations. Generally, it is believed that nanotechnology has the potential to change medicine, and modify it so as to enhance therapy and medical imaging techniques. Many of the proposed applications still remain far beyond what is now possible to achieve using nanotechnology techniques. The purpose of this work is to review the possible applications of not futuristic applications of nanotechnology-supported THz imaging modalities. THz imaging uses non-invasive radiation and although it is among the most attractive emerging imaging modalities, it has not yet become fully established. In order to achieve this, the limitations and drawbacks of the state-of-the-art THz imaging set-ups are first identified. Then nanotechnology-based techniques that were used to improve THz imaging are presented. How THz imaging can help nanotechnology and ethical/risk issues are briefly discussed. The focus is on both the improvement of the current imaging set-ups (detectors, emitting sources, etc.) and the use of nanotechnology-based contrast agents (nano-particles, nano-rods, etc.) to enhance their signals from specific targets.\n\n\nMethod\n\nIn the next section (Background), the current status of THz imaging is briefly presented and the limitations of these unique imaging modalities are presented. In the following section, the results of a systematic review on how nanotechnology can support THz imaging are presented. The searching of relevant material for the review was performed based on electronic resources including the online databases Scopus, Web of Science and PubMed. Google and Google Scholar were used for finding extra material. Furthermore, references found in the initial articles were also used. To capture the relevant studies the keywords and indexing terms that were used included: THz/THz imaging and nanotechnology/nanoscience, nano contrast agents, THz nano-imaging, terahertz nanoscopy. The searching procedure included the following limits: for the nanotechnology-supported THz imaging modalities, only articles published in English between January 2000 and January 2012 were included.\n\n\nBackground\n\nTHz radiation’s applications are expanding so quickly that they have an outstanding potential and social impact20. These applications can be expanded from medical, science and pharmaceutical applications to material non-destructive testing and security purposes. There is special interest in biomedical applications, such as the use of THz radiation as an imaging modality and for spectroscopy studies21, which have the potential for a serious clinical impact11. The first biomedical THz imaging was demonstrated in 199522 and since then a new non-invasive imaging modality has emerged.\n\nSince THz radiation has a long wavelength it can penetrate many materials23. Furthermore, polar molecules are sensitive to THz waves and consequently the detection of different hydration levels from tissues can be achieved13. The biomedical applications of THz waves are a consequence of the fact that THz radiation is sensitive to water and, what is more, biological molecules' characteristic energies lie in the THz region24. This is very important considering that water is one of the most important components of the tissue25. Actually, water molecules and all polar liquids absorb all the frequencies in the THz band. As a consequence, THz waves cannot penetrate moist tissue, a fact that enables both the development of imaging set-ups in transition (for in vitro studies) and reflection geometry (for in vitro and in vivo imaging)11. THz radiation is characterised by its ability to penetrate organic materials without ionisation, to distinguish different materials according to their water content and the fact that it can help the clarification of the unknown dynamics in the area of condensed matter physics (e.g. molecular recognition and protein folding)26.\n\nIn the case of THz medical imaging, the contrast mechanism arises from the fact that different absorption spectra and refractive indices characterise the different biological tissues when they interact with waves in the THz region17. Consequently, images and information from normal or pathological tissues (with abnormalities) can be obtained. Even from the very first demonstration of imaging from Hu and Nuss22, it was shown that the different water content of two different tissues (porcine muscle and fat) was the contrast mechanism12. Initial studies have demonstrated that biochemical and morphological features of the tissue provide contrast in images formed with THz pulses25.\n\nWhen THz radiation is used for spectroscopy, the key factor is the fact that the energy of the vibrational and rotation molecules (like proteins and DNA) corresponds to that of the THz photons25. THz spectroscopy can be used to investigate a variety of phenomena of great importance to scientists and engineers27. An interesting biomedical application is the demonstration of using THz spectroscopy to detect mutations and biomolecule conformational changes11. Also, the diagnosis and imaging of cancer is one very promising application of THz imaging technology28–32. This is a consequence of the fact that there is difference in the water content between healthy tissues and cancerous tumours and, what is more, there are differences due to cell alterations and abnormal protein density alterations that result in larger THz absorption and refractive index24,33. Although there are many expectations on this technique, the studies seem not to support any clinical application which can achieve high cancer detection rate24.\n\nAlthough the field of medical imaging in the last decades has seen great improvements and has significantly contributed towards a better medical practice both for diagnosis and therapy, THz is still developing in all of its components from technological concepts/set-ups to possible applications. THz-material/tissue interactions and the physical/biological mechanisms involved are still not well established and more research is still needed.\n\nThe lack of appropriate, low expense and compact-size emitters and detectors of T-rays has meant that the THz band has remained unexplored and unused for many years. Initial inappropriate set-ups were developed with expensive and bulky sources (e.g. free electron lasers, thermal sources), while the detectors demonstrated poor performance, like the liquid helium-cooled bolometers11. In the last decades, a number of novel techniques (like the ultrafast optical switch, the nonlinear method and quantum cascade lasers) innovated the field of THz optoelectronics19. The THz imaging systems can be separated into two main categories: Passive (also named Incoherent) and Active (also named Coherent) Pulsed or Continuous34,35. Here, we focus on the set-ups of the active categories, since these are the most extensively used for medical imaging purposes, while passive set-ups are most widely used for security purposes in airports and for weapon detection36,37. Pulsed and continuous THz imaging set-ups are both still in development, but pulsed systems are more widely used24.\n\nAt present there are many competitive techniques for generating THz waves (continuous or pulsed). The different THz sources can be separated into three basic categories: electronic sources, photonic sources and quantum cascade lasers. There are also some other smaller categories that are either emerging or not very popular, like p-germanium THz lasers38–40 and uni-travelling-carrier photodiode13,41, but they are not the focus here.\n\nThere are several ways of detecting THz radiation. There are a large number of published articles explaining the spectrum of the existing detectors and the new principles that are used for detecting T-rays are very broad42. They can be separated into direct detection (with Schottky-barried diodes and bolometers), heterodyne detection (like with super conducting hot electron bolometers) and heterodyne detection that uses photonical-generated THz local oscillators (electronic or photonic mixers)43. Photoconductors (PCs), electro-optic (EO) materials and photodiodes (PDs) are frequently used as photonic mixers. Direct and heterodyne detection are also referred to as incoherent and coherent detection, respectively44. Which method of detection will eventually be applied is mainly determined by the type of THz source that is used in the same set-up26 or which characteristics of the THz waves are intended to be detected.\n\nThe THz imaging systems, and THz technology in general, can be separated on whether they uses continuous waves (CWs) or pulses. CW THz is traditionally used for astronomy (like the study of Big Bang radiation), environmental monitoring and plasma diagnostics. Optically Pumped Terahertz Laser is a characteristic CW THz source26. For medical imaging, pulse THz sources are more attractive and have made THz imaging a challenging field for medical imaging.\n\nThe term “THz time-domain spectroscopy” (THz-TDS) refers to the technique where THz pulse methods are used for spectroscopy studies19. The same method system can be used for the formation of 2- and 3-D images. THz pulse-imaging is a quite simple methodology and a pump and probe beam are used. The pump beam interacts with the sample, and the detection of the coherent signal is obtained by combining the probe laser with the THz radiation25. The image of the subject can be built up due to the selective absorption of the THz radiation26. Consequently, the detector receives the signals with delay26 and by scanning the sample an image can be formed with each pixel representing the different time-series, which are the different adsorption characteristics25. The obtained data can then be processed with fast Fourier transform so as to move from the time to the frequency domain26.\n\nThe characteristics of THz radiation and already developed imaging set-ups also enable the use of this technique as an endoscope-based procedure45. The researchers involved in this study believed that if some limitations could be overcome (like the fact that water and the side-walls of the organs have a similar refractive index and power absorption), THz endoscopes would be a valuable tool for detecting tissue changes within the human body45.\n\nUnfortunately, THz imaging modalities are still characterised by a number of quite significant limitations. Particularly, THz radiation remained unexplored for many years due to the fact that detectors of THz waves were characterised by poor signal-to-noise ratio and slow processing. A second limitation was that the emitters were able to produce only incoherent and low-brightness THz radiation25 and some sources require cryogenic operating temperatures20. The development of both electronic and optical sources that emit at the THz spectrum is difficult to implement but very beneficial17. Some of the initial problems that THz technology has faced have found some solutions but there are also a number of significant drawbacks that still remain (Table 2)23,46.\n\nThe first drawback of THz imaging is a consequence of the nature of THz waves. Their long wavelength results in limitations in the imaging resolution compared with imaging modalities that use shorter wavelengths47. Due to the long wavelength, THz imaging can illustrate features in the range 1–3 mm, which is not enough for biomedical applications11. The limited penetration depth due to the high body-water component has until now limited possible applications and THz studies to surface tissues such as skin48,49, teeth50 and the cornea51. The use of the 0.5 THz frequency gives the highest contrast between normal and cancerous tissues, but this minimized the later resolution of THz imaging33. Furthermore, the contrast between healthy and pathological tissues is too low and there is a need for contrast agents33. Humphreys et al.11 also stressed the need for well informed and well organised databases that include the different responses of different tissues to THz radiation.\n\nAs was shown in the previous section, the use of femtosecond (fs) lasers is a common method for emitting THz radiation in the optical-to-THz conversion. The use of these sources make the commercialisation of THz imaging set-ups difficult due to both the high cost and the bulky dimensions of fs lasers17. This is why quantum cascade lasers are very attractive in the field, since they are expected to be cheaper and of appropriate size. Finally, it must be noted that although the potentials are great, the penetration depth of THz radiation is limited. Consequently, until now research has been concentrated on wound healing, burn diagnosis dermatology and dentistry, where a high depth is not required and the probed tissue is accessible without the need for waveguides11,25. Furthermore, the majority of the THz applications are still in the research phase, except for a few examples from the TeraView Company (Cambridge, UK), which has developed set-ups and techniques for detecting cancerous cells26. Currently, there are a number of companies that produce THz-related technology including Picometrix Inc. (Michigan, USA), Zomega Terahertz Corporation (New York, USA), Nikon Corporation (Tokyo, Japan), Toptica Photonics (Munich, Germany) and Hamamatsu Photonics (Tokyo, Japan), T-Ray Science Inc. (Vancouver, Canada).\n\n\nNanotechnology for THz imaging\n\nAs in all the emerging imaging modalities, THz presents some drawbacks that do not allow it to find its place in every day medical use. As was shown, these limitations cover a wide range from the low-performance of emitting sources to the low sensitivity or selectivity to pathological tissues. In order to overcome these drawbacks researchers are expanding their efforts in many different directions. Nanotechnology-based techniques seems to be a crucial key tool in their efforts to improve these imaging modalities. We believe that nanotechnology has the potential to improve the performance of imaging modalities. The next sections aim to discuss how current nanotechnology techniques can directly enhance THz medical imaging modalities. It will be demonstrated that nanotechnology can support THz imaging through several concepts, from using nanoparticles as contrast agents to the development of new THz sources and/or detectors (Figure 2).\n\nNanotechnology methods are used in all the components of THz imaging: contrast agents, sources and detectors (CNT: Carbon Nanotubes, QDs: Quantum Dots, NPs: Nanoparticles, NRs: nanorods, NGs: nanocages, WCNTs: multi-walled CNTs).\n\nOne area of THz imaging that nanotechnology could innovate is the use of contrast agents, also called contrast media or probes. Generally, contrast agents are used in order to increase image contrast from healthy and pathological tissue areas or molecules. Many contrast agents have been proposed and used for the existing imaging modalities (e.g. MRI contrast agents)52–54, but in the case of THz imaging, very few studies have been published. Although this area is in the very early stages, the results are very positive and open new horizons for the clinical application of THz medical imaging. By using contrast agents, it will be possible to enhance the sensitivity of cancer diagnosis by targeting tumours and by enabling the use of higher THz frequencies, which will allow better resolution33.\n\nNanotechnology can innovate THz imaging in this direction by the use of nanoparticles as contrast agents. With the term 'nanoparticles', a broad range of particles with dimensions in the nanoscale is implied, such as spherical particles, carbon nanotubes (CNT), fullerenes, quantum dots (QDs), cantilevers, nanorods (NRs), nanoshells, nanocages (NGs), nanowires and various metal and metal oxide nanoparticles. Nanoparticles that are characterised by their ability to produce surface plasmons, the so-called plasmon nanoparticles, are particularly interesting as they can be used for imaging and therapy purposes55. Gold (Au) is the most commonly used metal for fabricating nanoparticles for biomedical applications due to its biocompatibility, strong scattering around local surface plasmon (LSP) resonance wavelengths and the ability to accept the bio-conjunction process56.\n\nNanoparticle contrast agent techniques take advantage of the physical phenomenon known as the hyperthermia effect that occurs due to surface plasma polaritons (SPPs) when near-infrared laser beams irradiate nanoparticles. As a consequence of this phenomenon, the temperature of water in cancer cells (which are probed with nanoparticles) rises and since the THz signal is sensitive to water temperature alterations57, cancer cells can be probed and imaged (Figure 3).\n\na) First, the cancer cells are probed with nanoparticles (NPs) and are then irradiated with near-infrared (NIR) laser beams. b) After irradiation, surface plasma polaritons (SPPs) occur and as a result the temperature of water in the cancer cells is increased. Consequently, the cancer cells can be probed and imaged with THz radiation since the THz signal is sensitive to water temperature alterations.\n\nA significant work on this direction was published by Oh, Son and their colleagues in a series of four recent papers33,58–60. The new methodology was called nanoparticle-contrast-agent-enabled terahertz imaging58. Initially, hydroxyapatite gold nanocomposites and gold nanorods (GNRs) were studied and it was shown that contrast agents can enhance sensitivity in THz signals and can be bound in cancer cells and can consequently target cancerous tumours33. Their next in vitro experiments were performed in cancer cells with and without GNRs58. Their results showed that although there were not any significant differences in THz reflection images, the enhancement was high under IR irradiation and the differential mode enabled cancer diagnosis. They also demonstrated that tumours could be identified by monitoring the signal at a point without the need of imaging.\n\nOh et al. expanded their experiments in vivo by acquiring THz images in tumours of mice 24 hours after the injection of GNRs and the high sensitivity of the differential technique was shown59. Finally, in a recent publication THz molecular imaging (TMI) was demonstrated to be sufficiently sensitive to detect 15 mM of nanoprobes in vivo60. What is more, it was characterised in linear proportion to the nanoparticle concentration, which is a very useful quantification property. For their experiments, Oh et al. used a reflection-mode THz imaging set-up accompanied by a laser in the IR region for the surface plasma polariton induction59.\n\nOther research groups are also working in the field and Lee et al.24 studied gadolinium oxide (Gd2O3) nanoparticles as possible terahertz imaging contrast agents. Their results demonstrated that these kinds of particles are appropriate for terahertz medical imaging since their interaction with THz waves is very strong (the power absorption is ~3 orders higher than water)24. Moreover, as Gd2O3 nanoparticles are already used as MRI multi-functional contrast agents, their use for THz studies might enable the combination of the two imaging modalities (MRI-THz) and the combination and enhancement of the offered information.\n\nApart from the previously mentioned advantages of the use of nanoparticles as nanoprobes (nanocontrast agents), it is believed that they can offer further possibilities. The simultaneous use of the nanoparticles as hyperthermia therapeutic agents and THz imaging can achieve both diagnosis in early stages of cancer59 and therapy. Furthermore, THz imaging techniques can be applied for monitoring drug-delivery processes59,60 and finally, the use of infrared laser beams for imaging with THz set-ups opens the horizons for real practical THz endoscopy58.\n\nOne of the most suitable candidates for the development of compact THz sources is the quantum dot (QD) system, although the emission in the THz range has not yet been accomplished. QDs have dimensions between nanometres to a few microns and are characterised by the fact that they contain a tiny droplet of free electrons. Their size, shape and number of electrons can be precisely controlled depending on their possible applications. QDs are very attractive due to their intrinsic discreet energy level. After confirmation of the long carrier relaxation times and the ability to control these times, the way forward for QD-based THz optoelectronic devices was opened61. Takatori et al.62 demonstrated that InAc/GaAs (indium monoarsenide/Gallium arsenide) QDs have the potential to be intense terahertz emitters and recently the generation of THz radiation from InAc/GaAs quantum-based photoconductive antennae was achieved63. Moreover, a novel methodology for varying the QD growth parameters for manipulating the band gap in the THz emission range has been demonstrated64. In order to achieve 40 meV differences between intra-bands (E1 and E2), which is a necessary condition for intra-band THz emission, the effect of growth and monolayer coverage on the energy difference between the ground and excited states of two types of QD structures was investigated64. In a recent publication, a method for generating dual (or multiple) high-power THz difference frequency from a single QD laser diode was demonstrated65.\n\nOne other way that nanotechnology can innovate the THz sources, is the novel production of pulsed THz radiation by using nanostructured materials. It has been demonstrated by a group in Glasgow, UK, that appropriate nano-engineered surfaces can enhance THz radiation through surface plasmons (SPs) under a femtosecond laser simulation66. Initially, the emission of terahertz signal due to SPs has been confirmed from metal grating66, nanoparticle (ZnSe) surface67 and nanograin surface (ZnSe)68, while the studies have been expanded to different types of metal surfaces (mainly gold), such as nanoparticles, nanoparticle rings and pyramid-shaped particles69. When light interacts with metallic nanosurfaces, non-linear optical phenomena are enhanced due to the strong interactions and the high field strengths that are produced66. This concept enables the production of a terahertz pulse due to a new process of rectification. Gao and colleagues stated that this phenomenon is a consequence of the electrons' acceleration, which is a result of the surface plasmon excitation and believed that the mechanism is related to the multiphoton photoelectric effect69. In the case of surface nanoparticles, the mechanism of THz emission can be described in terms of dipole orientation67.\n\nApart from QDs and nanostructure materials, nanotubes, and especially CNTs, is another innovative nanotechnology area that is expected to highly influence THz emission and detection (see next section) of terahertz radiation. CNTs are molecular-scale tubes of graphitic carbon and were invented in 1991 by Iijima at Nec Fundamental Research Laboratories (Tsukuba Science City, Japan)70 and can be used in a variety of ways. Simulation studies have shown that CNT antenna properties can be improved by controlling the length, inter-tube distance and the number of nanotube elements so as to achieve better design of CNT-based sources/detectors for THz studies71. Recently, the traverse vibration of a novel composite NT was studied72. This NT was synthesized by coating CNT with piezoelectric zinc oxide (ZnO), which is a bio-safe and biocompatible material. The results showed than ZnO-CNT can be used for gigahertz/terahertz electromechanical nanoresonators. What is more, the tubular shape of CNT offers sharp tips that are appropriate for field emission, and consequently with the discovery of CNT a new class of field emitters was generated73. CNT bundle arrays have been developed as components of cathodes and have shown very promising results73. The CNTs were arranged as arrays and found to be appropriate for cold cathodes and able to operate at low voltages. It is believed that a high field enhancement, which produces efficient field emission, is caused by rearrangement of the free ends and outliers under an applied field, and is the reason of the bundle arrays' high emission73. Furthermore, in their work Manohara et al.73 showed that a highly compact field emission electron gun can be formed with monolithic integration of multiple electrodes. This technique enables electron-beam shaping and a novel miniature electron gun can be fabricated. In an older study, Manohara and colleagues presented the Nanoclystron, which is a novel micro-tube THz source74. In their circuit, the THz emission is achieved by CNTs, which are performing as electron emitters, and silicon-based reflex klystron-type cavities74. The use of highly ordered CNT arrays for THz emission has also recently demonstrated73.\n\nGamziha et al. (2011) are working towards miniaturised vacuum electronic devices that will be able to be used as high power THz sources75. One of the methods they have proposed is nanomaching, which offers many advantages such as rapid prototyping of any circuit. Their recent work on the development of a 0.22 THz circuit using nanocomputer numerical control (NCNC) presented very promising results. NCNC combined with UV lithography was also used by Shin et al.76 in order to develop a Travelling wave tube circuit for high power and broadband terahertz applications. The circuit was fabricated with ~50 nm surface roughness and a cascaded nanocomposite cathode was synthesized, a fact which opens the way for the future development of watt-level terahertz radiation sources.\n\nIn the previous section, it was shown that QDs are very attractive for the generation of new THz sources. QDs could also innovate the existing sensors for detecting and sensing terahertz radiation. QDs have been shown to be able to detect single THz photons with or without the assistance of a magnetic field77. Furthermore, carbon nanotube quantum dots can be used for developing highly sensitive detectors, which can also be frequency adjustable in the THz region78,79. Also, in a very recent publication, nanoscale carbon material was used for fabricating a tuneable quantum-dot sensing device80. Additionally, QD-type detectors can expand the performance of THz detectors at temperatures where state-of-the art THz detectors have limited sensitivity81. A novel sensor consisting of a QD (GaAS/AlGaAs QD), an electron reservoir and a superconducting single-electron transistor was presented with a cutting-edge performance82. The detector operating at temperatures below 1 K was able to perform single-THz photon counting by relying on photon-to-plasmon and plasmon-to-charge conversion, followed by charge measurement in a single-shot mode.\n\nAs in the case of QDs, nanotubes/CNTs can also be used for detection purposes with several ways as nanoantenna71,83, bolometers84 and even by using their mechanical properties85. Furthermore, CNTs can be coated with bio-safe and biocompatible materials, like piezoelectric zinc oxide (ZnO), and be safely used as terahertz electromechanical nanoresonators72. Their unique characteristics, like small junction areas, high electron mobilities and low estimated capacitances make them more attractive than solid-state components42. CNTs, especially multi-walled CNTs, can be used as antennas in THz detectors since it has been shown that they interact with light in the same manner as simple dipole radio antennas83. The polarisation and the length antenna effect are the key phenomena that can be used in optoelectronic devices. Furthermore, simulations have shown that CNT antenna arrays have better performance than single CNTs, and optimal design for receivers can be achieved. Very recently a novel resonant detector of THz radiation based on mechanically floating CNTs was presented85. The detector consists of two electrically coupled single-wall-CNTs, which lie parallel over an insulator and are characterised by a proportional response of the plasma-mechanical resonance to the mechanical oscillation quality factor. The detector output signal depends on an AC displacement current that occurs between the CNTs due to plasma-mechanical oscillations of CNTs. Nanotubes can also be used as bolometers. Of course the need for new, better performing, detectors has not been driven only from the field of THz medical imaging. Astrophysicists studying the universe radiation at THz radiation require improvement of the sensitivity of current bolometers86. To achieve this goal, the bolometers should be thermally isolated from the environment and have a very small capacity87. The development of nanobolometers would deliver the required characteristics and enables high sensitivity of even single THz photons88. The development of this kind of nanotechnology-based detectors pushes research to its current limits and possible future applications might be found in areas other than that of astronomy.\n\nFor some researchers, the development of novel electronic and semiconductor devices might improve and overcome many of the drawbacks that characterise current THz technology. In this direction, Balocco and his colleagues demonstrated novel planar nanodiodes, which are able both to emit and detect THz radiation at room temperature20. The diode concept relies on asymmetric device nanochannels and showed high efficiency and speed sensitivity. Additionally, nanostructure physics principles and an antennae approach were used for fabricating a compact THz detector performing at room temperature89. The inventors believed that these structures could contribute to the development of cost effective, compact and room-temperature operating THz emitters and detectors. One other significant limitation of current THz systems is that the detector and probe are not located close enough. As a consequence there is an affect on the sensitivity. New opportunities for high-resolution imaging can be achieved by integrating all the detection components on a semiconductor chip90. In the future nanotechnology could facilitate this by providing the tools for minimising the dimensions of all the components of THz imaging set-ups. Generally, there are many perspectives on nanotechnology concerning terahertz electronics and many novel electronic components are expected to be developed91.\n\nIn this section, it will be briefly discussed how nanotechnology can support THz imaging set-ups in areas that do not correspond directly to one of the previous discussed concepts (THz sources, detectors and contrast agents).\n\nAs already mentioned, terahertz radiation remained for many years unexplored and consequently a complete characterisation of its properties, physical characteristics and occurring phenomena have not yet been fully understood. An area that was affected by the absence of high-power THz sources is the study of the THz non-linear phenomena. In this direction, new sources that use nanotechnology will provide a useful tool for researchers in this field. Furthermore, other nanotechnology-based techniques could help the study of THz non-linear principles. For instance, nanostructures (nanoslits and nanogap split-ring resonators) were used in order to enhance the electric field and extend THz experiments into the non-linear regime92. The understanding of the THz-related non-linear phenomena might allow their application for medical imaging or other biomedical purposes as it happens with the non-linear optical phenomena.\n\nOne important limitation of THz imaging techniques is the limited imaging resolution47. One way that nanotechnology could help in minimising this drawback is the guide and focus of THz radiation by the use of SPPs on corrugated wires93. In addition, THz propagation on wires is the key area for the development of probes for biological investigations. It has been demonstrated that propagation on wires with a size around a nanometre can be achieved94. The small size of the wire opens the way for the development of MicroElectroMechanical Systems with sub-micrometre spatial resolution94. These systems can be applied for the THz spectroscopy of biomolecules in biological entities. Other nanotechnology techniques can also be applied in the same direction for the collection of THz spectroscopic signatures from individual biological molecules. For instance, a single-electron source and a THz radiation detection cell can compose a coupled three-quantum-dot structure that can be applied for single-molecule spectroscopy studies95. These novel THz spectroscopy techniques can be simultaneously used in the future with THz imaging methods.\n\nThe relationship between nanotechnology and THz is bidirectional, in the sense that the concurrent developments can contribute to both technologies. THz modalities have helped the expansion of nanotechnology. For instance, THz has made significant contributions in the study of semiconductor nanocrystals and quantum dots in recent years23. It is widely believed that terahertz nanoscopy will advance the development of new novel nanostructures since it overcomes the limited spatial resolution due to the diffraction limits of the traditional optical microscopy techniques96. Terahertz nanoscopy could be achieved by the development of novel probes for THz-scattering near-field optical microscopy (THZ-SNOM). The same concept has also been applied in the IR region with very promising results97. In the scattering-type of SNOM, optical amplitude and phase images with nanoscale resolution are formed. In this technique, Atomic Force Microscope tips are illuminated with laser and the elastically scattering light is recorded interferometrically98. Moreover, a resolution better than 40 nm was recently achieved by using IR and THz illumination96, since the resolution depends on the sharpness of the tip and not to the wavelength. Compared with other imaging modalities, it has the advantage of rich spectral contrast and consequently it can provide information concerning chemical composition, structural status and conductivity99. The possible applications of the technique are many and very promising. Recently it has been demonstrated that the method can be used for quantitative mapping of the local carrier concentration and mobility at the nanometre scale of different materials100. Finally, one very interesting field concerning nanotechnology is the development of techniques and methods that can be used for detecting and tracking nanoparticles especially in human bodies. A recent publication showed that nanoscale metal barriers embedded in nanoslot antennas can be used to detect a single nanoparticle101.\n\n\nSafety issues and ethical considerations\n\nAlthough THz imaging set-ups use non-ionising radiation, risk issues must be considered since hazards can arise from a variety of mechanisms other than ionisation102. Taking into account that emerging modalities are so new, it is obvious that a number of phenomena remain unexplored and their possible effects are unknown. These concepts are becoming even more complicated when nanotechnology-based techniques are used, since this innovative field is still in its infancy. The possible biological effects when electromagnetic (EM) radiation reacts with tissue include: thermal, acoustic, optical and photochemical mechanisms and their combinations25. The importance of the effects that EM radiation might have in humans is highlighted by the number of international and national bodies that are interested in the guidelines in relation to its effects102. A full and detailed understanding of the optical properties of tissues at THz radiation is necessary in order to achieve a safe in vivo image with THz imaging. Early studies have shown that the adsorption relies on the hydration of each tissue and tissues that are characterised by low water content possess a lower attenuation coefficient102,103. These results suggest that clinical imaging could be feasible only for certain applications and appropriate clinical protocols must be developed.\n\nInitial safety analysis, based on the available guidelines for skin exposure to radiation of 15–115 GHz, determined the maximum permissible exposure (MPE)25,103. The results showed that, according to the currently available guidelines, THz imaging set-ups are safe. But it must be noted that the majority of the published guidelines concerning THz radiation take into account only the heating effects and ignore other possibly damaging effects, e.g. thermo-mechanical damage. Furthermore, the guidelines were established for specific exposure durations and these are not always appropriate for THz imaging purposes102.\n\nResearch in the area is still in progress and as new applications are emerging, a full understanding of the THz-tissue interaction mechanism is imperative. For example, communication systems have started using frequencies of 300 GHz and above, which are not yet regulated104. In order for THz radiation to be used as a biomedical tool, researchers are trying to develop compact THz spectrometers that can be used for measuring optical properties of biological tissues105 and further investigations on the biological effects of THz radiation have been performed. Recently it has been shown that the 2.52 THz radiation generated primarily thermal effects in cells (fibroblasts) and thermal damage models can be used to predict the THz bioeffects106. A detailed review of the current state of the research on the biological effects of THz radiation can be found in a recent review paper, where projects, official regulations and publications are summarised107.\n\nOn the other hand, all medical nanotechnology-based techniques require special attention since possible risk, toxicity, ethical and social considerations must be taken into account2. This area cannot be ignored since nanotechnology is a new and still unknown field. Shape, size and morphology play a significant role in bio-toxicity, since at low dimensions, the surface increases and as a result has higher reactivity. The hazards due to nanoparticle toxicity are crucial108 and our current knowledge of the toxicity of the chemicals and materials is not sufficient when materials have dimensions in the nanoscale109. Moreover, due to their small size nanoparticles can penetrate humans through three paths: skin, breathing (mouth and/or nose) and digestive system (mouth)110. None of these three possible ways has been well studied at the moment. The risk of nanoparticles does not appear only during their application but also in every stage of their cycle from their production to transfer111,112, while possible environment pollution cannot be ignored113–115. There is evidence that nanoparticles are responsible for unusual diseases116 and what is more, the physical and biological mechanisms that are involved when nanoparticles are exposed to any kind of radiation within biological tissues remain unknown. Nonetheless, there is still a huge absence of clear regulatory guidelines, safety standards and MPE approvals for almost all the nanotechnology-related techniques117. Concerning regulation, the history of previous technologies must be taken into account so as to avoid repeating past mistakes118. Since nanotechnology is still in its infancy, new risks, ethical challenges and issues related with privacy and justice will arise while nanotechnology moves from research to clinical practice117. Finally, a crucial point has to do with how society will react and how willing it is to accept an innovative technology that has not yet proved to be either effective or safe119.\n\n\nConclusions\n\nThroughout this review, we have shown that nanotechnology can support emerging THz imaging modality in order to overcome some of its limitations. This can be achieved in many nanotechnology-based techniques and several drawbacks can be overcome, from the low performance of the emitting source to the miniaturisation of the whole set-up. These research results indicate that nanotechnology could help in the development of high-resolution, sensitive and portable detectors and new efficient sources for THz imaging purposes. What is more, the use of nanoparticles as contrast agents can enhance THz signals and detection, not only from healthy areas but also from specific pathological areas such as tumours. Although the techniques are in their infancy, it seems possible that nanotechnology may be applied to help THz imaging modality find its way into real everyday clinical use.", "appendix": "Author contributions\n\n\n\nAS had the original idea of the paper, conceived the study, carried out the research in the databases and prepared the first draft manuscript. MT supervised the research, contributed to the preparation of the manuscript and critically edited 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 were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nAS would like to gratefully and sincerely thank Dr. Marck McJury who introduced AS to this topic as well as for his guidance, understanding and support during his graduate studies at The Open University, UK.\n\n\nReferences\n\nFeynman RP: There's plenty of room at the bottom: An invitation to enter a new field of physics. Eng Sci. 1960; 23(5): 22–36. Reference Source\n\nAllhoff F: The coming era of nanomedicine. Am J Bioeth. 2009; 9(10): 3–11. PubMed Abstract \| Publisher Full Text\n\nDupas C, Houdy P, Lahmani M: Nanoscience: Nanotechnologies and nanophysics. 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Appl Phys Lett. 2004; 85(13): 2607–9. Publisher Full Text\n\nYngvesson KS, Fu K, Fu B, et al.: Experimental detection of terahertz radiation in bundles of single wall carbon nanotubes. 19th Int. Symp. Space THz Techn. 2008; 304–13. Reference Source\n\nStebunov Y, Leiman V, Arsenin A, et al.: Detection of modulated terahertz radiation using combined plasma and mechanical resonances in double-carbon-nanotube device. Appl Phys Express. 2011; 4(7): 075101. Publisher Full Text\n\nKarasik BS, Pereverzev SV, Wei J, et al.: Ultrasensitive hot-electron nanobolometers for terahertz astrophysics. 33rd international conference on infrared and millimeter waves and the 16th international conference on terahertz electronics, 2008, IRMMW-THz 2008; Nat Nanotechnol. 2008; 3(8): 496–500. PubMed Abstract \| Publisher Full Text\n\nWei J, Olaya D, Karasik BS, et al.: Ultrasensitive hot-electron nanobolometers for terahertz astrophysics. Nat Nanotechnol. 2008; 3(8): 496–500. PubMed Abstract \| Publisher Full Text\n\nKarasik BS, Sergeev AV, Prober DE: Nanobolometers for THz photon detection. IEEE Trans Terahertz Sci Technolog. 2011; 1(1): 97–111. Publisher Full Text\n\nSeliuta D, Kašalynas I, Tamošiunas V, et al.: Silicon lens-coupled bow-tie InGaAs-based broadband terahertz sensor operating at room temperature Electron Lett. 2006; 42(14): 825–7. Publisher Full Text\n\nKawano Y, Ishibashi K: An on-chip near-field terahertz probe and detector. Nat Photon. 2008; 2(10): 618–21. Publisher Full Text\n\nCha S, Choi JH, Baik CW, et al.: Perspectives on nanotechnology for RF and terahertz electronics. IEEE Trans Microwave Theory Tech. 2011; 59(10): 2709–2718. Publisher Full Text\n\nMerbol H, Bitze A, Feure T: Second harmonic generation based on strong field enhancement in nanostructured THz materials. Opt Express. 2011; 19(8): 7262–73. PubMed Abstract \| Publisher Full Text\n\nMaier SA, Andrews SR, Martín-Moreno L, et al.: Terahertz surface plasmon-polariton propagation and focusing on periodically corrugated metal wires. Phys Rev Lett. 2006; 97(17): 176805. PubMed Abstract \| Publisher Full Text\n\nTreizebré A, Bocquet B: Investigation on living cells with a THz BioMEMS. Joint 32nd international conference on infrared and millimetre waves, and 15th international conference on terahertz electronics, IRMMW-THz2007; 3–7 September 2007; Cardiff; 2007; p. 82–3. Reference Source\n\nWoolard DL, Zhao P: THz detection cell for sub-wavelength bio-molecular sensing. 2007 7th IEEE international conference on nanotechnology - IEEE-NANO 2007; 2–5 August 2007; Hong Kong; 2007; p. 320–5. Publisher Full Text\n\nHuber AJ, Keilmann F, Wittborn J, et al.: Terahertz near-field nanoscopy of mobile carriers in single semiconductor nanodevices. Nano Lett. 2008; 8(11): 3766–70. PubMed Abstract \| Publisher Full Text\n\nHuber AJ, Ziegler A, Köck T, et al.: Infrared nanoscopy of strained semiconductors. Nat Nanotechnol. 2009; 4(3): 153–7. PubMed Abstract \| Publisher Full Text\n\nHillenbrand R: Infrared and terahertz nanoscopy. 2010 IEEE photonics society summer topical meeting, PHOSST 2010; 19–21 July 2010; Playa del Carmen; 2010; p. 58–9. Publisher Full Text\n\nKeilmann F: Viewing the nanoworld in infrared/THz light. 34th international conference on infrared, millimeter, and terahertz waves, IRMMW-THz 2009; 21–25 September 2009; Busan; 2009. Publisher Full Text\n\nWittborn J, Weiland R, Huber AJ, et al.: Quantitative, nanoscale free-carrier concentration mapping using terahertz near-field nanoscopy. 49th international reliability physics symposium, IRPS 2011; 10–14 April 2011; Monterey, CA; 2011; p. 5C.1.1–5C.1.7. Publisher Full Text\n\nPark HR, Bahk YM, Ahn KJ, et al.: Controlling terahertz radiation with nanoscale metal barriers embedded in nano slot antennas. ACS Nano. 2011; 5(10): 8340–5. PubMed Abstract \| Publisher Full Text\n\nBerry E: Risk perception and safety issues. J Biol Phys. 2003; 29(2–3): 263–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFitzgerald AJ, Berry E, Zinov'ev NN, et al.: Catalogue of human tissue optical properties at terahertz frequencies. J Biol Phys. 2003; 29(2–3): 123–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKleine-Ostmann T, Münter K, Spitzer M, et al.: The electromagnetic environment above 100 GHz Electromagnetic compatibility, personal safety and regulation issues. IRMMW-THz 2006 - 31st international conference on infrared and millimeter waves and 14th international conference on terahertz electronics; 18–22 September 2006; Shanghai; 2006; p. 378. Publisher Full Text\n\nWilmink GJ, Ibey BL, Tongue T, et al.: Development of a compact terahertz time-domain spectrometer for the measurement of the optical properties of biological tissues. J Biomed Opt. 2011; 16(4): 047006. PubMed Abstract \| Publisher Full Text\n\nWilmink GJ, Rivest BD, Roth CC, et al.: In vitro investigation of the biological effects associated with human dermal fibroblasts exposed to 2.52 THz radiation. Lasers Surg Med. 2011; 43(2): 152–63. PubMed Abstract \| Publisher Full Text\n\nWilmink GJ, Grundt JE: Invited review article: Current state of research on biological effects of terahertz radiation. J Infrared Millim Terahertz Waves. 2011; 32(10): 1074–1122. Publisher Full Text\n\nOberdörster G, Stone V, Donaldson K: Toxicology of nanoparticles: A historical perspective. Nanotoxicology. 2007; 1(1): 2–25. Publisher Full Text\n\nde Jong WH, Roszek B, Geertsma RE: Nanotechnology in medical applications: Possible risks for human health. [Internet].: RIVM report 265001002; 2005. Reference Source\n\nBorm PJ, Robbins D, Haubold S, et al.: The potential risks of nanomaterials: A review carried out for ECETOC. Part Fibre Toxicol. 2006; 3: 11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuttagounder DS, Kalla DK, Zhang B, et al.: Sustainability in nanomanufacturing: Status and vision for the future. ASME 2011 international manufacturing science and engineering conference, MSEC 2011; 13–17 June 2011; Corvallis, OR; 2011; p. 585–90. Publisher Full Text\n\nSarahan PC, Bushong H: Managing your environmental, health & safety risk: A guide for nano companies and their insurers. Electronics, devices, fabrication, MEMS, fluidics and computational - 2011 NSTI nanotechnology conference and expo, NSTI-nanotech 2011; 13–16 June 2011; Boston, MA; Nanotech. 2011; 3: p. 561–4. Reference Source\n\nChen Z, Yadghar AM, Zhao L, et al.: A review of environmental effects and management of nanomaterials. Toxicol Environ Chem. 2011; 93(6): 1227–50. Publisher Full Text\n\nJiang G, Shen Z, Niu J, et al.: Nanotoxicity of engineered nanomaterials in the environment. Progr Chem. 2011; 23(8): 1769–81. Reference Source\n\nTurco RF, Bischoff M, Tong ZH, et al.: Environmental implications of nanomaterials: Are we studying the right thing? Curr Opin Biotechnol. 2011; 22(4): 527–32. PubMed Abstract \| Publisher Full Text\n\nSong Y, Tang S: Nanoexposure, unusual diseases, and new health and safety concerns. ScientificWorldJournal. 2011; 11: 1821–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBawa R: NanoBiotech 2008: Exploring global advances in nanomedicine. Nanomedicine. 2009; 5(1): 5–7. PubMed Abstract \| Publisher Full Text\n\nMarchant GE, Sylvester DJ, Abbott KW: What does the history of technology regulation teach us about nano oversight? J Law Med Ethics. 2009; 37(4): 724–31. PubMed Abstract \| Publisher Full Text\n\nKuiken T: Nanomedicine and ethics: Is there anything new or unique? Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2010; 3(2): 111–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "983", "date": "04 Jun 2013", "name": "Creidhe O’Sullivan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides not only a useful review of the ways in which nanotechnology can contribute to the field of THz medical imaging but also a review of the possibilities and shortcomings of the field in general. I believe that it will be of interest to both nanotechnology and THz researchers in general. A good list of references is provided. The authors concentrate on issues related to contrast agents, sources and detectors and mention many of the most widely-used and promising of these. Highlighting the potential impact of nanotechnology marks this review out from other THz review articles. The abstract and conclusion sections are appropriate and provide a useful summary for anyone briefly scanning the article. The publication search method used by the authors is clearly described.Minor commentsThe authors switch between THz and terahertz throughout the article.On page 4 (3rd paragraph). I find the sentence “The THz imaging systems can be separated into two main categories: Passive (also named incoherent) and Active (also named coherent) Pulsed or Continuous” confusing. Which are the 2 categories? Are all passive systems incoherent and pulsed? etc. Incoherent and coherent refer to the detection of the radiation, not whether the imaging set-up is active or passive. Should it be phrased “THz imaging systems can be separated according to whether they are active or passive, incoherent or coherent, continuous or pulsed”.Page 4 2nd column, 2nd paragraph “.. separated depending on whether …” or “separated on the basis of...” Would it be worth a line in the text (it is in table 2) that an advantage to THz is that it can propagate through materials such as bandages.Towards end of p.8 change \"capacity\" to \"heat capacity\".In the Conclusion, 1st line, “..emerging THz imaging modality...\" should be changed to “..emerging THz imaging modalities.”", "responses": [] }, { "id": "1066", "date": "16 Jul 2013", "name": "Safieddin Safavi-Naeini", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is a general review of nano-technology as applied to THz system for medical imaging. The review could have gone deeper than just a brief summary of other works, which is useful but not sufficient. The manuscript would be much more useful and informative if the authors would include a brief and simple explanation of some important THz imaging modalities by including a general block diagram of the system. The analytical content of the manuscript could be improved substantially by including a more rigorous description of various techniques and their assessment. The entire article only has 2 or 3 very general pictures which do not describe any specific technology or approach at any depth. More pictures, plots, and tables should be added to support authors’ arguments.The conclusions are very general and only based on summary of the other articles. No analytical method and independent data and/or results generated by the authors have been provided.", "responses": [] } ]	1	https://f1000research.com/articles/2-100
https://f1000research.com/articles/2-98/v1	28 Mar 13	{ "type": "Short Research Article", "title": "Over 30 million psychedelic users in the United States", "authors": [ "Teri S Krebs", "Pål-Ørjan Johansen", "Pål-Ørjan Johansen" ], "abstract": "We estimated lifetime prevalence of psychedelic use (lysergic acid diethylamide (LSD), psilocybin (magic mushrooms), mescaline, and peyote) by age category using data from a 2010 US population survey of 57,873 individuals aged 12 years and older. There were approximately 32 million lifetime psychedelic users in the US in 2010; including 17% of people aged 21 to 64 years (22% of males and 12% of females). Rate of lifetime psychedelic use was greatest among people aged 30 to 34 (total 20%, including 26% of males and 15% of females).", "keywords": [ "Psychedelic", "United States", "magic mushrooms", "psilocybin", "mescaline", "peyote", "LSD", "lifetime prevalence" ], "content": "Introduction\n\nThe classical serotonergic psychedelics, LSD (lysergic acid diethylamide), psilocybin (“magic mushrooms”), and mescaline (peyote and other cacti), have their main mechanism of action at the serotonin 2A receptor (5-HT2A), produce similar, often indistinguishable subjective effects, and elicit cross-tolerance1,2. The mechanisms of action, subjective effects, and risk profile of the classical serotonergic psychedelics distinguishes them from other drugs sometimes also labeled “hallucinogens”, such as entactogens, like methylenedioxymethamphetamine (MDMA; ecstasy) that act primarily at serotonin transporters, or dissociative anesthetics, like phencyclidine (PCP) or ketamine that act primarily at NMDA glutamate receptors1.\n\nPrevalence data on psychedelic use in the US is often reported for LSD alone (ignoring psilocybin and mescaline), or for psychedelics grouped together with PCP (popular in the 1970s), MDMA (popular since the 1990s), and/or other “hallucinogens” (some older estimates of hallucinogen use even included cannabis, amphetamine, and cocaine as hallucinogenic drugs), or for use among teenagers but not adults. Here, we present estimated lifetime prevalence of psychedelic use by age category using data from a large US population survey.\n\n\nMethods\n\nWe examined the estimated lifetime use of psychedelics by age based on 2010 data of the National Survey on Drug Use and Health (NSDUH). Results are presented for males and females separately. We counted participants as having any lifetime psychedelic use if they reported ever using LSD, psilocybin, mescaline, or peyote. The use of mescaline and peyote were combined into one category because mescaline is the active substance of the peyote cactus, but peyote use was also examined separately. Current age was only available as a categorical variable. This study was exempt from review by our Regional Committee for Medical Research Ethics because all data are available in the public domain without any identification of personal information.\n\nThe annual NSDUH survey provides estimates of substance use and mental health indicators from a randomly-selected sample representative of the US civilian non-institutionalized population aged 12 and older. The Substance Abuse and Mental Health Services Administration (SAMHSA) of the US Department of Health and Human Services is responsible for the NSDUH study design and methods of assessment. Trained interviewers met the randomly-selected participants in their homes, and participants listened to recorded questions via headphones and then entered their answers directly into a computer, providing a highly confidential and standardized setting. The response rate was approximately 78%. In addition, approximately 10% of participants were excluded from the public use data file, either because of excessive missing data on drug use or because they were excluded at random in order to increase anonymity. The total number of respondents in the public use file was 57,873. Detailed information on the sampling and data collection methods, including interview instructions and questionnaires, confidentiality and informed consent are available on the SAMHSA website (http://oas.samhsa.gov/nsduh.htm).\n\nEstimates were calculated using the online Survey Documentation Analysis from the Inter-university Consortium for Political and Social Research (http://dx.doi.org/10.3886/ICPSR32722.v3). Calculations of estimated population percentages and extrapolated total numbers of psychedelic users in the US took into account the weights provided with the NSDUH public use data file. Variance estimates took into account the complex sample design of the NSDUH survey using Taylor series linearization. Respondents with missing data on psychedelic use (less than 1% of the respondents) were assumed to have no use.\n\n\nResults\n\nAn estimated 32 million (95% confidence interval (CI): 30 to 33 million) US residents in 2010 reported lifetime use of LSD (23 million, 95% CI: 22 to 25 million), psilocybin (21 million, 95% CI: 20 to 22 million), mescaline (11 million, 95% CI: 10 to 12 million), or peyote (6 million, 95% CI: 5 to 7 million).\n\nFigure 1 shows the rate of lifetime psychedelic use in the US in 2010 by age category and gender. Lifetime rate of psychedelic use among people aged 50 to 64 years (the “baby boomer” generation) was similar to the rate among people aged 21 to 49 years. Among people aged 21 to 64 years, 17%, (95% CI: 15% to 18%) reported ever using LSD, psilocybin, or mescaline, including 22% (95% CI: 21% to 23%) of males and 12% (11% to 13%) of females. Prevalence of psychedelic use was low among people aged 65 and older (total 1.3%, 95% CI: 0.8% to 2.1%). Rate of lifetime psychedelic use was greatest among people aged 30 to 34 years (total 20%, 95% CI: 18% to 22%), with 26% (95% CI: 23% to 29%) of males and 15% (95% CI: 13% to 17%) of females.\n\nError bars show 95% confidence intervals. Any psychedelic includes LSD, psilocybin, mescaline, and/or peyote. Mescaline includes both mescaline and peyote.\n\n\nDiscussion\n\nPsychedelics continue to be widely used in the US. Common reasons given for using psychedelics include curiosity, mystical experiences, and introspection3. Rates of lifetime psychedelic use are greater in males than in females. Overall rates of lifetime psychedelic use are roughly the same among the “baby boomers” and younger adults. However, psilocybin was more common among younger adults, while LSD and mescaline or peyote were more common among older adults. Use of psilocybin mushrooms has increased since the 1970s in the US and worldwide, likely due to dissemination of simple home cultivation techniques, instructions on finding wild mushrooms, and information about effects and methods of psilocybin mushroom use4. This was a retrospective cross-sectional study. Self-reports of drug use behaviors could be influenced by memory errors and under-reporting; however, a 14-year longitudinal study reported good consistency over time in the reporting of lifetime LSD use5.", "appendix": "Author contributions\n\n\n\nTK conducted the data analysis. Both authors prepared and approved the manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nBoth authors were supported by the Research Council of Norway, grant number 185924.\n\n\nAcknowledgments\n\nThe Substance Abuse and Mental Health Data Archive provided the data files from the National Survey on Drug Use and Health, which was sponsored by the Office of Applied Studies of the Substance Abuse and Mental Health Services Administration.\n\n\nReferences\n\nNichols DE: Hallucinogens. Pharmacol Ther. 2004; 101(2): 131–181. PubMed Abstract \| Publisher Full Text\n\nGonzález-Maeso J, Weisstaub NV, Zhou M, et al.: Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior. Neuron. 2007; 53(3): 439–52. PubMed Abstract \| Publisher Full Text\n\nHallock RM, Dean A, Knecht ZA, et al.: A survey of hallucinogenic mushroom use, factors related to usage, and perceptions of use among college students. Drug Alcohol Depend. 2012; 130(1–3): 245–8. PubMed Abstract \| Publisher Full Text\n\nAndersson C, Kristinsson J, Gry J: Occurrence and use of hallucinogenic mushrooms containing psilocybin alkaloids. Nordic Council of Ministers. 2009. Reference Source\n\nJohnston LD, O’Malley PM: The recanting of earlier reported drug use by young adults. NIDA Res Monogr. 1997; 167: 59–80. PubMed Abstract" }	[ { "id": "874", "date": "03 Apr 2013", "name": "Wayne Hall", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is concise and well written and I have no major criticisms of the content of the article. The authors have appropriately analysed the best available data on the question that they address namely, what is the estimated life time use of various psychedelic drugs among US adults. They report analyses of a large representative US household survey data on lifetime experience with various psychedelic drugs. My issue is more with what the paper does not say.What’s missing from the paper is any indication of why the authors’ question is worth posing: 1. Why do lifetime rates of psychedelic drug use among US adults matter?2. Why did they only report lifetime use? Lifetime or ever use can overstate rates of use in the population. I would bet that most psychedelic use is very limited e.g. once or twice during late adolescence and early adulthood. This isn't clear from the lifetime data presented. Some data on frequency of use would make it clearer if most psychedelic use is in fact limited and experimental rather than more persistent as occurs with cannabis and MDMA. 3. Were any data collected on adverse events or experiences? If so, this could be briefly reported.4. It would be useful to provide a bit more data on the characteristic of psychedelic drug users other than their age and sex. How are they characterised by SES, education, geographic areas of residence and experience with other illicit drugs e.g. cannabis, MDMA and stimulants? 5. What if anything follows from the findings? Should we be concerned about this use or should we regard psychedelic drug use as being of much lower public health concern than the use of other illicit drugs such as cannabis, amphetamine type stimulants, cocaine and opioids?", "responses": [] }, { "id": "896", "date": "17 Apr 2013", "name": "Dave Nichols", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 is short and well written. It reports on lifetime incidence of use of various psychedelics. Without knowing the frequency of use, however, these data do not indicate whether the use of psychedelics is increasing.", "responses": [] } ]	1	https://f1000research.com/articles/2-98
https://f1000research.com/articles/2-97/v1	28 Mar 13	{ "type": "Research Article", "title": "Ouabain inhibition of Na/K-ATPase across the retina prevents signed refractive compensation to lens-induced defocus, but not default ocular growth in young chicks", "authors": [ "Melanie J Murphy", "Sheila Gillard Crewther", "Melanie J Murphy" ], "abstract": "Purpose: The relevance of retinal integrity and energy pathways to ocular growth and induction of refractive errors has seldom been investigated. Thus, we used ouabain to target the channels that are essential for the maintenance of membrane potentials in cells, sodium potassium ATPase (Na/K-ATPase), to examine refractive compensation and ocular growth in response to lens-induced defocus in the chick.Methods: A single intravitreal injection of 1 mM ouabain in dimethyl sulfoxide (DMSO) carrier or DMSO alone was followed by monocular defocus with positive or negative 10 D lens (or no lens) from post-hatching days 5-9 under 12/12 hr light/dark conditions. Biometry and dark-adapted flash and electroretinography (ERG) were conducted on day 9, followed by immunohistological analyses.Results: Ouabain inhibited differential ocular growth and refractive compensation to signed defocus compared to DMSO. By 4-days post-ouabain injection all components of the typical ERG responses to light had been eliminated, and widespread histological damage was apparent, though some ‘default state’ ocular growth was measurable. Immunohistochemistry demonstrated reduction in the specialized water channel Aquaporin 4 (AQP4) expression and increased evidence of caspase 3 expression (a cell death associated protein) in ouabain-treated eyes compared with DMSO alone.Conclusion: The current study demonstrates that blockade of photoreceptor and inner retinal responses to light onset and offset by ouabain inhibits differential refractive compensation to optical blur, but does not prevent ocular growth.", "keywords": [ "Na/K-ATPase", "occular growth", "chick", "oubain", "refractive compensation" ], "content": "Introduction\n\nRefractive compensation to optical blur in young animals has been attributed to environmentally-driven changes affecting the rate of ocular growth and axial dimensions of the eye1–5. To date only a few studies6–8 have investigated the impact of compromised retinal energetics and integrity on such growth, despite the retina being a highly metabolically active structure. Photoreceptor activity is highly dependent on aerobic glycolysis and the availability of adenosine triphosphate (ATP) energy resources9–12. In particular, an ATP source is needed to transport both the light induced changes in chloride, sodium and potassium ions out of the subretinal space (SRS) into the retinal pigment epithelium (RPE) and the obligatory accompanying fluid coming from the vitreous humour en route to the choroid. The sodium- [Na] potassium [K]-ATPase (Na/K-ATPase) channel is one of the most important channels involved in osmoregulation and fluid transport, particularly in the retina13.\n\nNa/K-ATPase activity is responsible for approximately 30 percent of total energy consumption in the body, and approximately 50 percent of the energy consumption in the retina14–16. Na/K-ATPase utilizes ATP hydrolysis to maintain electrolyte balance, and the associated electrochemical gradient across the cell membrane17,18 and cell volume by transporting Na+ out of cells and K+ in at a ratio of 3:2. Na/K-ATPase is critical to retinal function, regulating the Na+ and K+ gradients associated with the dark current in the photoreceptors, action potentials in the ganglion cells, Müller cell neurotransmitter uptake, light adaptation19 and synaptic activity. In the light, the Na/K-ATPase maintains the high extracellular Na+ concentration in the SRS, while also maintaining the potential difference across the apical membrane of the RPE and consequently driving the transport of ions and fluid from the retina across the RPE to the choroid20,21. Hence, changes in the pumping mechanism and/or expression of Na/K-ATPase by the selective inhibitor ouabain would be expected to alter the nature of phototransduction and affect outer and inner retinal neurotransmission.\n\nIndeed, ouabain has been shown to affect the resting potential of the optic nerve, disrupting retinal metabolism and inducing spreading depression in neural tissue22, and to affect cell density and the thickness of the inner plexiform layer (IPL) 2-days post-injection, with horizontal and amacrine cells being the primary sites of cell death. The extent of the morphological changes has been shown to affect the electrophysiological profile of the outer retinal response to a light stimulus in a dose-dependent manner15,23. At low concentrations, ouabain causes selective destruction of inner nuclear layer (INL) cells and the ganglion cell layer (GCL), while inducing reversible swelling in horizontal cells, preserving the Müller cells and photoreceptors24. At higher concentrations, ouabain completely destroys the retina25,26.\n\nTo date the functional consequences of ouabain injection on ocular growth and refractive compensation to optical defocus have not been investigated in great depth. A recent investigation examining the effect of a relatively low dose of 1 mM ouabain on refractive compensation in chicks wearing positive lenses reported that application of the drug prevented the development of hyperopia27. It was posited that this was due to the inhibition of Na/K-ATPase channels which then altered the nature of fluid transport across the RPE, leading to significant choroidal thinning and thus reduced refractive error. Unfortunately the effect of this dose of ouabain on photoreceptor function and general retinal morphology, choroidal dimensions, and on ocular growth and refractive compensation to negative lenses was not examined.\n\nThus, the current investigation aimed to examine the effect of a 1 mM dose of ouabain on refractive compensation and ocular elongation per se in response to negative as well as positive lenses or no optical adjustment. We hypothesize here that injection of ouabain, and subsequent inhibition of outer and inner retinal Na/K-ATPase activity, would not only prevent compensation to positive lenses as reported by Wong et al.27, but also prevent abnormal ocular elongation, refractive compensation and myopia to negative lenses due to the inability of any retinal elements to detect defocus and elicit the required driving force needed to alter ocular volume. It is further hypothesized that there would be no systematic change in choroid thickness across the experimental lens groups due to the inhibition of retinal responses to light rather than as a result of active ocular volume reduction following ‘ouabain-driven change’ in fluid transport. Although ouabain would be expected to thin the choroid due to increased retinal adhesion28, this effect is likely to be attenuated by the dilatory effect of dimethyl sulfoxide (DMSO) on this structure7.\n\nWe also examined whether changes in refractive compensation and ocular growth were associated with altered outer retinal function (i.e. altered photoreceptor, bipolar, Müller and RPE cell function), as assessed by electroretinography (ERG), and retinal organization via histological and immunocytochemical analysis. Changes in the expression of the specialized glial water channel aquaporin 4 (AQP4) across the retina were measured in view of the previously reported association between ouabain and retinal adhesiveness and cellular edema28. This work led us to expect altered intracellular and transretinal fluid production in negative lens versus no lens conditions. Apoptosis-related caspase 3 expression was also measured to assess the extent of cell death across the retina at the end of the chick rearing period.\n\n\nMethods\n\nAll procedures were conducted in accordance with La Trobe University Animal Ethics Committee guidelines and adhere to the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the ARVO Statement for the use of Animals in Ophthalmic and Vision Research. Well being of animals was monitored twice daily throughout the rearing period to ensure cleanliness and comfort. During surgical procedures, the level of anaesthesia was assessed continuously via toe squeeze, pupiliary reaction and respiration rate. Euthanasia for tissue collection was performed via decapitation while the animal was under heavy surgical anaesthesia.\n\nA total of 71 male hatchling chicks (Leghorn/New Hampshire) obtained from a local commercial hatchery were raised in a light (12/12 hour day/night cycle) and temperature controlled (30 ± 0.5°C) enclosure. On post-hatching day 5 (P5), animals were randomly assigned to one of 3 lens conditions (±10 dioptre or no lens) and 2 drug conditions (ouabain [Sigma-Aldrich, MO, USA] in DMSO [Sigma-Aldrich, MO, USA] carrier or DMSO alone) and reared until post-hatching day P9. Ambient luminance was maintained constantly at 183 lux during the 12 hour day cycle via a 20W halogen light globe in the roof of the enclosure. The group sizes for each experimental condition are shown in Table 1.\n\nOn day P5, chicks were anaesthetized (in the middle of the day cycle) with a intramuscular mixture of ketamine 45 mg/kg: xylazine 4.5 mg/kg and the right eye of each animal (experimental eye) was injected intravitreally with 5 μL of 1 mM ouabain in DMSO carrier solution (Sigma), to give an effective concentration of 2.5 μM in the eye assuming an ocular volume of 0.5 ml29. This is a low-medium dose of ouabain selected for consistency of comparison with Wong et al.27, and on the basis of previous reports of changes in retinal function and Na/K-ATPase activity in studies using effective concentrations of between 1.7 nM and 1 mM10,30–34. The left eye of each animal was injected with the DMSO carrier as a within subject control. Monocular defocusing goggles (±10 D) customised from modified human polymethyl methacrylate (PMMA) contact lenses (8.1 mm in diameter) were then attached to Velcro© and affixed to the periocular feathers of the chicks for 4 days. A no lens control group was also used. The general health of the chicks and the cleanliness of lenses were monitored twice daily.\n\nOn day P9, chicks were anaesthetized and both eyes were refracted by retinoscopy (Keeler, Vista Diagnostic Instruments, UK) and axial dimensions were obtained from the average of at least three A-Scan ultrasonography traces (A-Scan III, TSL: Teknar, Inc. St Louis, USA; 7 MHz probe).\n\nTo ascertain the functional effects of ouabain on outer retinal integrity 4 days post-injection (P9), 4 additional ‘no lens’ animals that had been injected with ouabain in DMSO carrier at day P5 and reared for the same experimental period as the animals included in the biometric analyses were prepared for ERG recording and compared to 4 ‘no lens’ chicks of the same age, that had also been injected with DMSO intravitreally, and 3 that had been injected with phosphate buffered saline (PBS). A square wave with a 500 ms light on duration and offset at 500 ms stimulation protocol (150 mm Ganzfeld stimulator with peak luminance 50 cd/m2, measured using a Tektronix J6523 narrow angle luminance probe [Tektronix, Inc, USA) was employed such that retinal ON and OFF responses could be separately observed. An intravitreal electrode (Ag/AgCl) was inserted under ketamine/xylazine anaesthesia (ketamine 45 mg/kg: xylazine 4.5 mg/kg i.m.) via a catheter placement unit, with scleral reference. Signals were recorded (under maintained anaesthesia) via a Powerlab amplifier (ADI, Australia) and band-pass filtered (0.3 – 1000 Hz). Twenty ERG recordings were obtained for each run, and 5 such runs were recorded consecutively for each animal. Grand mean averages with a 95% confidence interval for the mean recordings for each condition were determined using the IGOR common wave metrics software program (IGOR, Loswego, USA) for both drug conditions.\n\nTo assess the effect of ouabain on retinal morphology, patterns of cell death and the specialized AQP4 fluid transport mechanism, posterior eye cups of negative lens and ‘no lens’ animals were separated from the anterior portion of the eye at the midpoint of the ocular globe, and after the removal of the vitreous body, were then fixed in 4% paraformaldehyde (Sigma-Aldrich, MO, USA) for 30 minutes and washed in phosphate buffered saline (Sigma-Aldrich, MO, USA) three times before cryo-protection in 30% sucrose (Sigma-Aldrich, MO, USA) to inhibit crystal formation during freezing prior to embedding in Tissue-Tek OCT Compound (Sakura Finetek, USA) and cryosectioning in serial sections at 10 micron thickness. Samples were then stained with cresyl violet for general histological analysis or immunolabeled with AQP4 or caspase 3. Four slides, each with 3 sections, from 3 different animals per condition were examined under light microscopy using cresyl violet stain. The immunohistochemistry protocol and protein visualization was conducted as per the manufacturer’s instructions (Vector Laboratories Inc, CA, USA). This involved incubation of sections for 30 minutes in a 3% goat serum blocking solution (Vector Laboratories Inc, Burlingame, CA, USA) at room temperature, followed by primary antibody application overnight at 4°C with either rabbit anti-rat AQP4 antibody (1:20 dilution) or Caspase-3 (1:50; rabbit anti-rat; Millipore, CA, USA). Sections were then incubated for 30 minutes at room temperature with goat anti-rabbit biotinylated IgG (Vector Laboratories Inc, Burlingame, CA, USA) followed by incubation with ABC reagent (Vectastain ABC Kit with Rabbit IgG; Vector Laboratories Inc, CA, USA). Proteins were then visualised using a 3, 3'-diaminobenzidine (DAB) peroxidase substrate kit (DAB Peroxidase Substrate Kit, Vector Laboratories Inc, CA, USA). Slides were cover slipped with an anti-fade aqueous mounting medium (Gel Mount, Sigma-Aldrich, Australia). Ouabain- and DMSO-treated eyes were incubated together for each antibody localization protocol.\n\nProtein localization and variation (channel and receptor expression) was examined via a light transmission microscope (Leitz Dialux 22 Microscope). Images were captured using Spot Flex Cooled CCD digital camera and Spot software (Diagnostic Instruments, Inc, MI, USA). The staining intensity of captured images was measured with ImageJ (http://rsbweb.nih.gov/ij/; NIH, USA) analysis software.\n\nBiometric data in the preceding analyses utilised differences in values of the CE subtracted from the EE involved the following variables: refractive state, axial length, vitreous chamber depth and anterior chamber depth. The measurements for each animal were derived from an average of at least 3 scans, which were then averaged to obtain a mean difference for each dependant variable to control for within subject effects. Measurements were analysed via a series of 2 way Analysis of Variance (ANOVA) (3 lens × 2 drug) with an alpha = 0.05. Significant effects were further explored via Simple Main Effects Analysis, followed by either Student-Newman-Keuls (SNK) or Games-Howell (GH) Post Hoc testing when appropriate.\n\nHistological analysis involved the examination of sections for evidence of any anatomical abnormalities. Layer thickness of the ganglion cell layer, inner retina (including inner and outer plexiform layers) and outer retina, including approximate transverse number and spatial distribution of cells per layer of retina was assessed. Higher magnification inspection was made of retinal neurons and photoreceptor outer segments and individual RPE cells to check for evidence of hyper- or hypo-osmotic cell profiles, nuclei with irregular profiles, or apparent holes or vacuoles that are indicative of cellular dehydration or edema, as these are the classically accepted signs of generalised toxicity and cell damage (see Liang et al. 1996 and Liang et al. 200435,36 for examples of these signs). Changes in the immunoexpression of AQP4 and caspase 3 were identified by alterations in the intensity of DAB staining across the retinal layers.\n\nGrand mean average waves (with 95% confidence intervals) from the ERG recordings were obtained by averaging across the multiple recordings from the chicks in the DMSO and ouabain groups 4 days post injection. The main features of the ERGs analysed were the N50 (a-wave - photoreceptor onset peak), the P105 (b-wave bipolar amplitude peak), the response at light off (N500 - a surrogate measure of c-wave amplitude) and the P670 (d-wave peak, the photoreceptor + Off Bipolar response to light offset).\n\n\nResults\n\nBy day P9, 4 days after a single 5 μl injection of ouabain, it was clear that the treatment had notably altered the typical pattern of refractive compensation and ocular growth when compared with that seen in eyes with lens-induced defocus but injected with DMSO alone. Data presented in Table 1 and Figure 1a show that following ouabain injection, refractive compensation to both +10 D and -10 D defocus was severely inhibited, such that mean refractive state across all lens conditions varied by only 0.5 D while that of DMSO injected eyes varied by approximately 16 D.\n\nMean difference (± SE) measures of +/-10 D and ‘no lens’ eyes at 4 days for refractive state (a), axial length (mm) (b), vitreous chamber depth (mm) (c), and anterior chamber depth (mm) (d).\n\nThe lack of directional refractive change observed in the ouabain condition is reflected in measures of axial length (Figure 1b), with vitreous chamber depth and anterior chamber depth measures showing the same patterns of change (Figures 1c and 1d, respectively).\n\nOuabain treatment led to a general increase in vitreous chamber depth and shallower anterior chamber depth, irrespective of the lens condition. In comparison, rearing following DMSO injection resulted in the typical sign-dependent changes of increased axial growth and deeper vitreous and anterior chamber depths in the -10 D lens condition with the opposite being found in the +10 D lens condition. Little change in biometric measures was observed in ‘no lens’ eyes for either intravitreal injection of ouabain or DMSO alone conditions, except for an increase in the anterior chamber depth of ouabain-injected eyes.\n\nTwo way (Drug × Defocus) ANOVA revealed significant interaction effects between the Drug and Defocus condition for measures of refractive state, axial length and vitreous chamber depth (Table 2). The pattern of overall increased vitreous and overall decreased anterior chamber depths following single ouabain injection was supported statistically by a significant main effect for drug condition. Post hoc tests revealed significant differences between all lens conditions for refractive state, axial length and vitreous chamber depth in the DMSO condition, although the axial length and vitreous chamber depth for +10 D reared eyes were not significantly shorter than ‘no lens’ controls. Post hoc tests further confirmed that there was no significant difference between lens conditions for any measure for eyes injected with ouabain.\n\n*NS, not significant.\n\n\nHistology and immunohistochemistry\n\nAs shown in Figure 2, cresyl violet staining revealed widespread destruction of retinal elements in ouabain-injected eyes. Indeed, ouabain treatment induced fragility in tissue with all ouabain-injected eyes showing thinned choroids. DMSO alone did not induce any apparent histological damage and, as previously reported7, induced a thicker choroid with dilated blood vessels as shown by cresyl staining. Of note, in the ouabain-treated eyes was the observation of cone degeneration, while rods and Müller cells were still present 4 days post-injection when compared with left eyes from the same animal treated with the DMSO carrier alone. Müller cell bodies appeared prominent and swollen, indicative of retinal edema particularly in -10 D lens condition eyes, consistent with the previous observations of Marmor28 and Bringmann et al.37. Significant disruption of the nerve fibre layer (NFL) was also apparent in the cresyl violet-stained retinae.\n\nShows level of cell destruction on day 4 (P9) in chick wearing -10 D lens (a) and a control eye (b).\n\nThe expression of AQP4 in the NFL of ouabain-injected eyes was less than in the NFL of DMSO injected control eyes, with greater expression localised to the sublaminae A remnants of the IPL, particularly in the -10 D lens condition. The pattern of expression of caspase 3 staining was indicative of more widespread activation of apoptotic pathways across the retina following ouabain injection.\n\n\nElectrophysiology\n\nAs can be seen in Figure 3, intravitreal injection of ouabain in DMSO but not DMSO alone affected the typical ERG response, in comparison to PBS-injected eyes. The normal ERGs following PBS injection obtained under our conditions of dark-adapted short flash response demonstrate a clear photoreceptor (a-wave) and On bipolar (-wave) response occurring in conjunction with light onset, and an Off photoreceptor + OFF Bipolar response (referred to here as the -wave) being observed at light offset in ‘no lens’ eyes 4 days post-injection. As is obvious from Figure 3, and consistent with histological findings of photoreceptor and bipolar cell damage (Figure 2), recordings 4 days after injection of ouabain-treated eyes did not show either photoreceptor responses to light onset or offset a-, b-, or d wave response to light onset or offset, in ‘no lens’ eyes. However, some evidence of a residual RPE (c-wave) response was apparent in two of the four animals.\n\nOuabain-injected chicks (n = 4) are shown by the red line, DMSO-injected chicks (n = 3) by the black line and PBS-injected chicks (n = 3) by the dotted blue line. Each recording is the average of 20 potentials over 5 runs per animal recorded during 500 ms light on followed by light offset. The 95% confidence intervals of the waves are indicated by the shaded regions surrounding the mean waves.\n\n\nDiscussion\n\nThe electrophysiological results of the current study demonstrate that 1 mM of ouabain abolishes the photoreceptor ERG response to light onset and offset, and suppresses refractive compensation to both positive and negative lens-induced defocus but does not prevent ocular growth in ‘no lens’ conditions. In contrast, DMSO-injected eyes showed typical sign-dependent axial elongation or reduction in response to signed optical defocus (Figure 2b). Biometric results also revealed shallower anterior chamber depths in ouabain-injected eyes. This is consistent with previous findings that ouabain acts on the ciliary body as well as the retina to inhibit the volume regulatory mechanisms in the epithelia in the anterior portion of the eye38,39.\n\nThe atypical ERG recordings obtained for ouabain-injected ‘no lens’ eyes are indicative of total suppression of photoreceptor and outer retinal function. In agreement with previous studies19, our ERG recording data clearly demonstrated that the absence of a functional PR response following inhibition of the Na/K-ATPase transport system affects the regulation of electrical potential within the retina10,23,40–42 and leaves the eye unable to initiate any response to light onset or offset, and hence drive refractive compensation. The ERG recordings made in this study were also reminiscent of the effect of barium on the electrical response of the retina and RPE43–46. Barium, a potent K+ channel inhibitor, has also been shown to eliminate signed refractive compensation and ocular growth to lens-induced defocus46.\n\nThe biometric results suggest that the ouabain-induced loss of Na/K-ATPase activity in all cells in the retina and choroid eliminated the ability of the retina to detect blur and initiate sign-appropriate differential growth. Neither the biometric nor the immunohistochemical results indicate at what level in the retina this response was initiated. However, as indicated above, the ERG results show that functional activation of the retina from the level of the photoreceptors and beyond, has been eliminated. Indeed, the differential patterns of cell death across the retina are indicative of metabolic changes that would be expected following inhibition of Na/K-ATPase activity14,47. The swelling of the Müller cells observed in the current study is consistent with the changes observed in rat glioma cells undergoing osmotic challenge34,48. The widespread distribution of caspase 3 staining is further confirmation of the presence of apoptosis across the entire retinae of the ouabain-injected eyes.\n\nHistological analysis also showed that the eyes injected with the carrier DMSO (that showed a typical refractive compensation response) all showed the expected characteristic choroidal thickening7 irrespective of the lens group and resultant refraction. The choroidal response to DMSO confirmed our earlier observations7,49 of dissociation between the direction of ocular growth (and hence refractive compensation) and choroidal thickness. Light microscopy analysis revealed substantial disruption to retinal morphology and layer organization and no apparent systematic lens-induced change in choroidal thickness following ouabain injection.\n\nEarlier studies have suggested that survival of the outer retina is necessary for refractive compensation to blur, though sign appropriate growth can occur following the inhibition of the inner retina7,8,50. Indeed, the current findings are also consistent with reports of increased retinal adhesion due to swelling of photoreceptor outer segments and apical processes of the RPE28. Additionally, the observation of choroidal thinning across all lens groups following injection of ouabain in DMSO carrier further demonstrates that changes in choroidal thickness do not dictate refractive compensation, as previously suggested7,36,49.\n\nOuabain has been shown to destroy the ability of the retina to detect and respond to osmotic stress and adequately regulate cell volume28,51. This would be expected following ouabain-induced inhibition of Na/K-ATPase and the associated decrease in fluid flow out of retinal tissues and should be reflected by the expression of the retinal aquaporin AQP4 channel. Aquaporin channel expression has previously been shown to be involved in the mediation of fluid secretion and absorption in a variety of different functions within the eye52–54, including being associated with changes in ocular dimensions55,56. The growth changes seen in ouabain-treated animals in the current study are similar to the slower growth previously described in eyes compensating to positive lenses and the shift in the expression of AQP4 from primarily along the vitreal border of the NFL to the IPL layer in PBS- and DMSO-treated eyes to sublaminae A of the remaining IPL is also consistent with slowed vitreal volume change55. These results further support a role for the participation of AQP4 expression on Müller cells in the regulation of retinal cell volume changes following alterations of ionic fluid balance in the eye and also highlight the importance of active, directional fluid and ion exchange across the retina in refractive development5,57.\n\nFurther, consistent with the observations of widespread caspase 3 labeling across the retina at 4 days post injection in the current study, Li et al.58 demonstrated that the intensity of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) in rat retina increased over time from 12 hours post-ouabain injection, suggesting that ouabain initiated apoptotic processes within the retina. Micrographs from Li et al.58 showed widespread disorganization of the retinal layers at all levels, with significant findings of destruction of the IPL. These histological characteristics bear marked similarities to the retinae assessed in the current study that exhibited vacuoles, swelling, altered Müller cell processes and overall decreased retinal thickness (mostly due to destruction of the inner retina).\n\nTogether, the current results show that 4 days after a single injection of ouabain, a substance that was expected to severely impair the energetic systems of all cells, the transepithelial potential had been abolished and fluid movement had been altered, apparently preventing the retina from detecting and responding to signed defocus. Importantly, recent evidence links Na/K-ATPase function with the effects of hypoxia10,47 and with changes in immune function in the retina31,59–61, which could have important implications for conditions such as diabetic retinopathy62–64 and other metabolic conditions.\n\nIn conclusion, the results of the current investigation show that inhibition of Na/K-ATPase and photoreceptor responses to light onset and offset disrupt retinal organization and induce a non-sign-dependent choroidal thinning. This prevents signed ocular growth and refractive compensation to both positive and negative lens-induced defocus in the chick. Minimal non-sign-dependent growth of eyes still occurred irrespective of the functional state of the retina or the size of the choroid, also indicating that growth of the eye is a normal developmental condition even in the absence of a visual signal.", "appendix": "Author contributions\n\n\n\nSGC and MM conceptualized, designed and executed the study, MM performed data analysis and both authors drafted and revised the article for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAspects of the research were supported by Australia Research Council grant DP110103784 to SGC.\n\n\nReferences\n\nTroilo D, Wallman J: The regulation of eye growth and refractive state: an experimental study of emmetropization. Vision Res. 1991; 31(7–8): 1237–1250. PubMed Abstract \| Publisher Full Text\n\nWallman J, Turkel J, Trachtman J, et al.: Extreme myopia produced by modest change in early visual experience. Science. 1978; 201(4362): 1249–1251. 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PubMed Abstract\n\nVerkman AS: Role of aquaporin water channels in eye function. Exp Eye Res. 2003; 76(2): 137–143. PubMed Abstract \| Publisher Full Text\n\nGoodyear MJ, Crewther SG, Murphy MJ, et al.: Spatial and temporal dissociation of AQP4 and Kir4.1 expression during induction of refractive errors. Mol Vis. 2010; 16: 1610–1619. PubMed Abstract \| Free Full Text\n\nGoodyear MJ, Junghans BM, Giummarra L, et al.: A role for aquaporin-4 during induction of form deprivation myopia in chick. Mol Vis. 2008; 14: 298–307. PubMed Abstract \| Free Full Text\n\nCrewther SG, Liang H, Junghans BM, et al.: Ionic control of ocular growth and refractive change. Proc Natl Acad Sci U S A. 2006; 103(42): 15663–15668. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Zheng H, Liu PP, et al.: The new targets of ouabain in retinal interneurons of Sprague-Dawley rats. Brain Res Bull. 2010; 81(6): 617–624. PubMed Abstract \| Publisher Full Text\n\nCorrea Gde R, Cunha KC, Dos Santos AA, et al.: The trophic effect of ouabain on retinal ganglion cell is mediated by EGF receptor and PKC delta activation. Neurochem Res. 2010; 35(9): 1343–1352. PubMed Abstract \| Publisher Full Text\n\nSontheimer H: An unexpected role for ion channels in brain tumor metastasis. Exp Biol Med (Maywood). 2008; 233(7): 779–791. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAperia A: New roles for an old enzyme: Na,K-ATPase emerges as an interesting drug target. J Intern Med. 2007; 261(1): 44–52. PubMed Abstract \| Publisher Full Text\n\nDi Leo MA, Santini SA, Cercone S, et al.: Chronic taurine supplementation ameliorates oxidative stress and Na+ K+ ATPase impairment in the retina of diabetic rats. Amino Acids. 2002; 23(4): 401–406. PubMed Abstract \| Publisher Full Text\n\nKern TS, Kowluru RA, Engerman RL, et al.: Abnormalities of retinal metabolism in diabetes or galactosemia: ATPases and glutathione. Invest Ophthalmol Vis Sci. 1994; 35(7): 2962–2967. PubMed Abstract\n\nKrugel K, Wurm A, Pannicke T, et al.: Involvement of oxidative stress and mitochondrial dysfunction in the osmotic swelling of retinal glial cells from diabetic rats. Exp Eye Res. 2011; 92(1): 87–93. PubMed Abstract \| Publisher Full Text" }	[ { "id": "2232", "date": "25 Nov 2013", "name": "Debora Nickla", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the effects of a single injection of ouabain on ERGs, retinal histology, and the responses to positive and negative lenses in young chicks. Ouabain causes destruction of the retina as indicated by ERG amplitudes, caspase-3 expression and basic histology. It also prevents the compensation to both positive and negative lens-induced defocus. The writing is clear and concise, the results are convincing, and the science is sound. My only reservation is that it is unclear how much information about signal processing of defocus can be obtained by a drug that causes such massive damage to the retina, and so the results on the loss of the ability to compensate are not at all surprising. In figure 1 the apparent lack of difference between control and experimental groups in the “no lens” axial length (figure 1b) seem to be due to the reduction in anterior chamber depth in the ouabain eyes (1d), because the vitreous chamber grows more than the DMSO controls (1c). It appears to me that ouabain increases vitreous chamber growth in these normal eyes. If this is so, then whether or not the drug affects “default” growth is not clear, because what is the default growth for these eyes? If the “default” is growing faster than normal, then it should be stated and clarified. For the same reason, the use of the term “default” in the title is ambiguous. The authors state that \"ouabain treatment led to a general increase in vitreous chamber depth and shallower anterior chamber depth, irrespective of the lens condition\", but this is not so for the negative lenses when compared to DMSO controls (figs 1c and d). In the same paragraph, they say that ouabain increases anterior chamber depth in “no lens” eyes, but fig 1d shows a decrease in AC depth? This is confusing. Because the choroids thinned in the drug-injected eyes in all groups does not indicate that “changes in choriodal thickness do not dictate refractive compensation”, as none of these eyes were compensating to the refractive error. If it is true that the drug-eyes were not growing faster than normal (and I’m not convinced that they were not, given figure 1c), then a more correct statement would be that choroidal thinning is not associated with faster-growing eyes.", "responses": [] }, { "id": "6307", "date": "14 Oct 2014", "name": "Shanta Sarfare", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is well written and the results are properly analyzed and interpreted.An important control that has been performed but not described includes the PBS-injected eyes to demonstrate normal ocular growth at this age.There is no figure showing the AQP4 immunohistochemistry or caspase labelling, when it is discussed extensively in the discussion section.Figure 2 does not convincingly show increased swelling of Müller cells, and as such higher magnification images or specific antibody staining would be useful.The ERG a-, b-, c- and d-waves are not labelled in figure 3. It would be difficult for the uninitiated to understand the figure without the labels.The references should be thoroughly checked. For example, ref. 27 is incorrectly stated as published in IOVS, whereas it is actually only available as an ARVO abstract. It is indeed difficult to assess mechanism of oubain-induced refractive compensation due to the severe retinal toxicity of the chemical. Perhaps the authors ought to try lower doses with minimal toxic effects to tease out the true effect of ouabain?", "responses": [] } ]	1	https://f1000research.com/articles/2-97
https://f1000research.com/articles/2-95/v1	26 Mar 13	{ "type": "Research Article", "title": "I’m Like You, Just Not In That Way: Tag Networks to Improve Collaborative Filtering", "authors": [ "Jason Boorn", "Debra S Goldberg" ], "abstract": "Collaborative filtering aims to predict a person’s preferences by examining the preferences of similar people. Many collaborative filtering algorithms rely on a coarse notion of similarity, which assumes that if two people are sufficiently simiar in a few specific areas, each is likely to make good recommendations for the other in most areas. Our trust in the opinions of others, though, is rarely absolute; we often tend to trust recommendations from certain people in certain areas. In this paper we develop an algorithm which reflects this notion. Rather than capturing taste information at the user level, we capture taste at the topic level by making use of tags: arbitrary words or phrases which are often used to group online content. Previous attempts to improve collaborative filtering using tag information have attempted to determine tag meanings, and as a result have depended upon complex semantic analyses. Our algorithm avoids these complications by focusing instead on the clusters which tags establish. Using tags in this way provides a significant improvement in the accuracy of recommendations without a commensurate loss in coverage. These tag clusters also give rise to networks which can be exploited to further improve recommendation results.", "keywords": [ "Collaborative Filtering", "Graph Theory", "Information Retrieval", "Recommender Systems" ], "content": "1 Introduction\n\nAs digital repositories become larger and more numerous, discovering relevant information within them becomes more difficult. While there exist numerous tools (e.g. Google) to help us locate a piece of information in which we know we are interested, fewer options exist to guide us towards items of which we might be completely unaware.\n\nSoftware and algorithms which attempt to solve this problem are known as recommendation systems. Recommendation systems aim to find new items from a target repository which a user is likely to find valuable. One of the more successful and widespread methods for recommendation involves a process known as collaborative filtering. Collaborative filtering attempts to predict unknown user preferences based upon known past behavior. Most often, the process of predicting preferences for a target user proceeds in one of two ways: we either find those users whose item preference behavior most closely matches that of the target user (user-based), or we find those new items whose ratings are correlated with the target user's existing ratings across a group of users (item-based)1. Many current collaborative filtering algorithms can be applied to both user- and item-based prediction methods2,3. The decision on which method to apply is therefore often a computational one: because the problem is expressed in terms of nearest neighbors, it is preferable to limit the search space. That is, if the repository has many more users than items, item-based collaborative filtering is normally applied, and if the repository has more items than users, user-based collaborative filtering is normally applied.\n\nA crucial benefit of collaborative filtering is that it is domain free. That is, no knowledge of the underlying items is necessary, only a knowledge of user preferences. Because of this, collaborative filtering can be applied to a wide variety of repositories in a more or less generic form. This flexibility greatly improves its popularity, but also hampers its predictive power: when we ignore qualitative characteristics of the underlying items, we lose important information about why two users might share opinions on them.\n\nIn user-based collaborative filtering, this loss of information is especially relevant. Under this method, new item suggestions are drawn from the total number of items contained in a set of similar users. Under a user- based regime (where we have many more items than users), it is likely that this suggestion list contains a great many items which are completely unrelated to those which established the initial user similarity4. In a movie repository, for example, I might find a set of users whose taste in documentaries so closely matches my own that a collaborative filtering algorithm would deem them good recommenders for me. These same people, though, have presumably rated more than just documentaries, and if qualitative item information (such as genre) is absent, the suggestion algorithm is unable to separate out these unqualified results. In short, we tend to trust certain people in certain areas, but generic user-based collaborative filtering does not take this fact into account. In order to differentiate between items from different areas, some kind of item information is required. But in order to maintain domain independence, this item information must come from user behavior and not from the underlying item characteristics.\n\n\n2 Tag-based approaches\n\nUser-generated tagging systems have become popular ways to categorize online resources. in these systems, users are given the opportunity to attach a set of words or phrases to a resource which describe it in a way that makes sense to the user. in contrast to centralized hierarchical systems, these \"folksonomies\" are informal and unregulated. there is no defined schema for these descriptors; rather, users determine the words or phrases which best describe the associated item and attach these to the item when saving it.\n\nTags represent additional structure which may be used in a collaborative filtering algorithm. What is attractive about using tag information is that a tag can confer content information in a domain-independent manner. It can, in other words, give us a description of the underlying content without requiring a direct look at it. This comes at a cost, though, as the variety of tags that might be used to categorize a particular item seems endless. The flexibility which makes tagging so popular also limits its descriptive power.\n\nRecent studies have attempted to use tag information to improve collaborative filtering results5,6. In particular7, uses tag information to establish a \"context\" within which a user found the resource informative. This context becomes an additional layer through which suggestions can be filtered. Similarly8, uses tagging behavior to limit the number of possible items which can be suggested before applying a conventional user similarity approach. Here, tags are clustered by examining which items they are attached to across the entire dataset in an effort to characterize their meaning.\n\nStudies such as those above are indicative of a popular strategy when working with tag information. Because a tag is fundamentally a categorization term, it seems natural to first discover a tag's semantic meaning. This, however, can prove a difficult task, and efforts to employ tag information often get bogged down in larger questions of semantic analysis.\n\nIn fact, though, tags offer useful information apart from their semantic content which can be used to improve the results of user-based collaborative filtering. We propose a novel algorithm which analyzes tagging behavior to extend collaborative filtering in a natural manner, one that is both domain independent and free of semantic context. We find that suggestions generated from this graph outperform those generated from more conventional techniques, dramatically boosting accuracy without a commensurate decrease in coverage.\n\n\n3 Similarity by area\n\nWhen a user tags an item, he makes a statement about its content. Although this statement can be highly subjective, it is assumed to be consistent. That is, other items for this user which share this tag represent one of the user's interests. We refer to all the documents tagged by an individual user with a particular tag as a tag group. If our goal is to compare two people, we might use an approach which finds the documents they have in common. If our goal is to compare the interests of two people, we might alternatively find the documents which their tag groups have in common. We take a vector t to represent a given tag group. ti = 1 if the ith item in the data set is included in tag group t, ti = 0 otherwise. An expression for comparing different tag groups then uses cosine similarity over tag groups rather than over users:\n\n\n\nwhere t and g are vectors representing items in respective tag groups, < t, g > is the dot product of these vectors, and \|\|t\|\| is the magnitude (Euclidian norm) of the t vector.\n\nIt is important to note that this neighborhood does not take the semantic content of the tags into account. In this model they function in the same way that user names do in the conventional model: they serve only to delimit the group of items which are compared. In this way we sidestep the complicated issues of semantics which hamper many tag-based approaches, yet still manage to gain new information about content associations.\n\nThe most important consequence of comparing tag groups rather than users is that doing so limits the scope of the comparisons to a particular interest. This means that we have less information on which to base a comparison, but also that comparisons should be more focused. We see that these assumptions hold in a set of experiments performed using data from an online research paper repository.\n\n\n4 The CiteULike data set\n\nCiteULike is a social bookmarking website for researchers. It allows users to save and tag research articles. Because users may attach multiple tags to a document, it is possible (indeed highly likely) that a paper belongs to more than one of a user's tag groups, and also that it is contained in tag group(s) of other users (likely tagged with different words or phrases).\n\nEvery day, CiteULike publishes a data dump of its current repository. Here we use a dump from November 2007, because a number of other papers in the field have used the CiteULike data dump from this period8,9.\n\nBecause CiteULike publishes its data set on a daily basis, it has become an excellent resource for data in collaborative filtering. Unfortunately, it has also become an excellent target for spam. Unscrupulous marketers have seeded the data set with phony article descriptions designed to lead users to online offers. Many recent publications have used the data from CiteULike without acknowledging the amount of this material which is included in the daily data dumps. Before enacting experiments on the data set, we set out to scrub the underlying repository of extraneous spam data.\n\nCiteULike publishes two data files: one which includes an entry for each documentid/userid/tag combination10, and one which associates these documentids with external URLs (called a linkout file)11. CiteULike does not store full documents, only document descriptions. This URL information is therefore used to link a document description to its associated content. This content is usually the full text of a journal article, although any existing web page may be used.\n\nBecause setting up the target web page for an external URL requires some significant effort, we can be reasonably certain that any documentid which is associated with an external URL in the linkout file is not spam. As a first step to removing spam from the repository we simply removed all documents which did not have associated linkout entries. This made for a dramatic change in the dataset: based solely on the linkout test, roughly one third of the users, two thirds of the documents, and three quarters of the tags used in the corpus are associated with spam entries. We were somewhat surprised by this result, and so decided to verify the finding using an alternative method. A direct examination of those documents deemed questionable by the linkout test was undertaken. It is possible to query CiteULike by documentid directly. Legitimate documentid's produce their corresponding descriptions, even when these descriptions do not contain a linkout entry. Documents which correspond to spam entries, however, fail to produce a document description at all. By querying the site by documentid directly, we can determine whether an individual document is spam. Note that this procedure is different from the linkout test, where we were checking for the existence of an external URL associated with a given description. Here we check whether the description itself exists or not.\n\nOur questionable data set contains over half a million documents. Given that the time required to query each of these would be prohibitive, we adopt a more aggressive approach: we query a given document, and if it is found to be spam we remove it as well as all the users who have included it in their saved documents. We assume that few non-spammers will include spam entries in their saved articles, and that spammers have an incentive to include the same spam document in more than one of their \"false\" profiles. A manual examination of the resulting data sets (potentially good and still questionable) verified the approach: tags selected at random from the questionable set proved suspicious (e.g. \"free\", \"cash\", \"enlargement\"), while tags selected at random from the potentially good set matched tags from known good profiles.\n\nAfter performing this process on data determined to be questionable by the naive linkout file method, we found only 282 potentially good users, 2820 potentially good documents, and 10907 potentially good tag groups. These represented 1.0%, 0.3%, and 0.9% of the original data set, respectively. In other words, using just the linkout file to filter spam articles is a method which performs very well.\n\nFigures 1(a)–(c) show that spam removal tends to change the characteristics of the data set, which is to be expected. More importantly, removing spam from the data brings to light a crucial network property that can be used to dramatically improve the results of user-based collaborative filtering.\n\nWe plot the number of papers belonging to a given number of users, tags, and tag groups respectively, then plot a regression line through the data. All data are plotted in log/log format (base 10), and number of papers is plotted on the x-axis. The slope and mean squared error of the resulting regression line are displayed.\n\nWe alluded to a fundamental problem of accuracy: user-based collaborative filtering, because it does not take into account the fact that we trust certain people in certain areas, tends to return suggestion lists which are not very accurate. While these suggestion lists do contain relevant items, they contain a large number of irrelevant items. In Figure 1 we see that this is indeed the case. In each figure, the magnitude of the slope of the regression line indicates the skewness of the distribution: lower magnitudes indicate fatter tails. Shown in Figure 1(a), for plots of number of papers per user, this value is -1.26 and -1.51 for raw and filtered data, respectively. This indicates relatively fat tails for the number of papers by user. In other words, a large proportion of users in the data set have saved a large number of papers to their profiles. Yet the data set is sparse; the number of papers shared by users is small. Therefore a suggestion list based on user similarity will contain a relatively large number of papers, many of these not included on the basis of this similarity.\n\nWe can see a similar situation develop when we examine papers by tag. Tag in this case refers to the semantic token; any papers tagged with the term \"biology\" for example, would be grouped together. Here the slope of the regression line is -1.67 or -1.5, indicating fat tails. If we attempt to compare based on tag, then it seems we will also return a large number of extraneous documents.\n\nTurning to tag groups, however, we notice a change. A tag group in this case refers to just the set of papers which a user has grouped together using a tag; the semantic content of the tag itself is ignored. Here the slope of the regression line after filtering is -2.38, indicating much thinner tails. According to this, a comparison based on tag group should result in far fewer irrelevant documents. Moreover, we should get better results by ignoring the semantic content of the tags altogether, and just using them to define areas of trust.\n\nA further result emerges from the analysis of tag groups. Not only does the slope of the regression line decrease to a value of -2.38, so does the mean squared error of its fit to the data. In other words, the distribution of papers by number of tag groups follows a power law much more closely after filtering. This fact might be useful in estimating the prevalence of spam in an arbitrary data set which uses tag groups.\n\nIn order to apply the algorithms below to the data set, some additional preprocessing was required. In that singlet documents (documents saved by only one user) are impossible to analyze and represent only noise for purposes of investigation, these were removed. Additionally, because the investigation below depends upon tag group documents, we also removed tag groups which contained only one document. These two steps can depend on one another (i.e. removing tag groups with one document might create additional situations where a document is owned by only one user and vice- versa), so we ran both procedures iteratively until the number of documents converged to a representative set. Finally, we removed documents which were saved to the repository without any associated tags. Our final data set consisted of 4612 users, 32085 documents, and 40704 tag groups.\n\n\n5 Experimental results\n\nWe apply the collaborative filtering algorithms described above to the CiteULike data set after filtering with the linkout file and preprocessing as described above. We start with basic user collaborative filtering, which compares users to other users based upon which articles they share. We then apply the filtering algorithm described above which captures a more natural notion of trust in a given area. The results we obtain suggest a hybrid approach which combines the two; we apply this hybrid algorithm to the data set to achieve much improved suggestions. Results of this final experiment in turn suggest further areas of investigation which are discussed in the next section.\n\nIn order to evaluate the performance of our algorithm, we employ a set of metrics commonly used to validate leave-n-out suggestion algorithms. In these situations, we remove a target set of n items which we attempt to recover. Recall measures the percentage of items in the target set which appear in our suggestion list. Precision measures the percentage of the suggestion list which is made up of target set items. In a realistic suggestion environment, we care not only about how many items in the suggestion list match target list items, but also at what position in the list these matches occur4. We therefore measure accuracy, which represents the average position of a target item in the suggestion list. A common metric for evaluation which combines recall, precision, and accuracy is mean average precision, or MAP. Average precision is the sum of the precision at each relevant item in the suggestion list divided by the total number of relevant items in the collection. If we average this value over all suggestion lists, we get the MAP for the suggestion algorithm. A perfect MAP score, then, must recall all relevant documents and place them at the top of the suggestion list for a perfect score of 1.0. A final metric which we use in evaluation we call satisfaction, and is defined as the percentage of users in the set for whom at least one suggestion is found to match an item in the target set. Satisfaction is useful in isolating the percentage of cases which represent complete failures of the algorithm.\n\nTo see how user-based collaborative filtering performs on the CiteULike data set, and to establish a baseline for subsequent experiments, we apply a conventional form of user-based collaborative filtering. The procedure is as follows:\n\n1) Select a user at random who has saved at least 6 items; call this the target user.\n\n2) Remove half of the documents belonging to this user; call this the target set.\n\n3) After removing the target set documents from the target user document vector, find the k users in the repository whose document vectors are most similar to its remaining document vector using cosine similarity.\n\n4) The documents belonging to the document vectors of these k users, but not currently contained in the target user's document vector, represent the suggestion list. Score the suggestion list according to criteria described in section 5.1.\n\nThe above procedure depends upon a single parameter k: the size of the user neighborhood that is used to generate the suggestion documents. Previous studies on this data set suggest that generic user-based collaborative filtering algorithms perform best at values of k between 5 and 129. Table 2 lists results for values of k in (5,8,12). We see that satisfaction (percentage of users who received at least one good suggestion) is fairly high, but accuracy is dismal (recall lower accuracy scores are better). This agrees with our earlier assumptions about user-based filtering in the context of trust. The algorithm returns suggestions from all of a particular user's interests - not just those areas in which his interests align with the target user's. Because of this, a large number of the returned suggestions are irrelevant, and accuracy suffers.\n\nWe now apply the tag-based algorithm that implicitly specifies the interest area of a target user by comparing tag groups. We start with the natural approach detailed above; instead of comparing users to other users, we compare user interests to other user interests. The procedure is as follows:\n\n1) Select a tag group at random which contains at least 6 items; call this the target group.\n\n2) Remove half of the documents belonging to this group; call this the target set.\n\n3) After removing the target set documents from the target group document vector, find the k tag groups in the repository whose document vectors are most similar to its remaining document vector using cosine similarity.\n\n4) The documents belonging to the document vectors of these k tag groups, but not currently contained in the target group's document vector, represent the suggestion list. Score the suggestion list according to criteria described in section 5.1.\n\nAgain, we run the procedure for values of k in (5,8,12).\n\nResults are listed in Table 3. The assumptions relating to trust appear again to hold. Here we have paid a nominal price in satisfaction for a huge improvement in accuracy. The improvement in accuracy is sufficient to raise the overall MAP score between 25% and 65% depending on k.\n\n\n6 Analysis\n\nThe above results show that similarity comparisons performed with respect to particular subject areas outperform those performed on a simple user basis. They imply that, with respect to suggestions, we do tend to trust certain people in certain areas. Although we are less likely to recall as many of the target documents which represent good suggestions, those we do find appear much earlier in the suggestion list. In a practical setting, one could argue that the latter benefit greatly outweighs the former loss; few users will look further than 20 items deep in a suggestion list to ferret out an interesting recommendation4.\n\nLooking more closely at the results above, a fundamental tradeoff emerges. User-based collaborative filtering on this data set produces suggestions which are high in recall and satisfaction, but poor with respect to accuracy. Tag-based collaborative filtering on this data set produces suggestions which are lower in recall and satisfaction, but high with respect to accuracy. Tag-based filtering produces more accurate results because the denominator of the cosine similarity measure includes a term for the size of the tag group - effectively this penalizes larger tag groups. As a result, the suggestion list returned is smaller. This also explains the loss in recall we see above.\n\nIt is important to point out that the loss in recall we see with the tag-based algorithm lies mostly in a loss in satisfaction. Comparing the two algorithms, we see that using tags to drive similarity results in a loss of recall of 12%, 11%, and 11% for various k. We see a corresponding loss of satisfaction of 30%, 26%, and 21% respectively. In that satisfaction measures the number of groups for which any good recommendation was found, it appears that the price we pay in recall is primarily due to an inability to establish any good recommendations for a fairly large subset of the tag groups. That is, for roughly one-third of the tag groups tested, the algorithm is unable to find other tag groups which contain any of the hidden documents. The data set was constructed such that every document is guaranteed to exist in at least one other tag group. This seems, therefore, a rather large number of failures.\n\nOur network analysis of the data suggests why, and underscores the degree to which sparsity can be a problem in collaborative filtering. Recall that our algorithms hide half of the target data set; for user-based filtering we hide half of the user's documents and for tag-based filtering we hide half of the documents within that tag group. Because a user will be associated with at least as many documents as his largest tag group, removing half of his overall documents leaves us with a larger set on which to base recommendations. When we remove half of the documents which belong to a tag group, by contrast, this can leave us with far fewer (in some cases only 3) documents on which to base a similarity judgment. In an extremely sparse environment, this can have major consequences. It is quite probable, for example, that the documents removed from a tag group are part of a disconnected cluster: the other tag groups which contain these hidden documents do not also contain the non- hidden documents required to make them a part of the similarity neighborhood. This circumstance, moreover, is much more likely when dealing with smaller tag group sets than with larger user document sets.\n\nWe used a more realistic notion of trust to drive the tag-based algorithm. Taking a cue from13, the question we pose next is whether or not this notion also carries with it the prospect of transitivity. That is, can we use the transitive aspect of trust to establish \"trust networks\" and extend our model of similarly? If so, does such an extension present new alternatives which can achieve both high accuracy and high coverage?\n\n\n7 Network methods\n\nTo frame the basic similarity question in terms of a network, we first define the trust network. This structure is a graph, where tag groups are represented by nodes and similarities between tag groups by links. We use the cosine similarity measure described above to establish weights on these links. This structure can be represented as an adjacency matrix, where the (i,j) element of the matrix represents the cosine similarity between tag groups i and j. Our naive tag-based similarity algorithm can use this matrix to generate the k tag groups in a group's neighborhood by looking at the row corresponding to the target tag group. That is, if we are looking to generate the k tag groups which are most similar to a particular tag group t, we order the elements of the row corresponding to t and choose the first k of them. Our decision to use a network approach to boost satisfaction (and thus recall) derives from an analysis of the tag based algorithm's failures. The vast majority of tag groups for which no good suggestion can be found have a particular network structure, as illustrated in Figure 2. From our target group t we have hidden a set of documents. Documents of this hidden set, h, are also found in a set of tag groups G. The problem arises when an insufficient number of the documents in G are also contained in the non-hidden set of documents in t. Without this similarity, no links can be made between t and G using the algorithm above, and therefore no tag group of G is positioned to make recommendations. In that the set G is uniquely qualified to make recommendations on the hidden set of documents, the algorithm fails for t. It is this type of failure which most impacts the loss in recall seen above.\n\nWe know, however, that every document in the set G must also exist in some other tag group. Although it may be the case that all the hidden documents of t exist only between t and G, it is also the case that G contains many other documents, and these must link to the larger graph in some way. Moreover, even though a failure indicates that an insufficient number of the documents in G are also non-hidden documents of t, it is likely that some of the documents in G are also documents within k* (the neighborhood of similar tag groups initially constructed). If we suppose that strong links are likely to exist between k* and G, we can attempt to find these and use this information to generate suggestion lists of better recall.\n\nThe network methods we seek to apply here depend upon tag group comparisons over the entire tag group set. Attempting to analyze the entire network of tag groups would be either computationally intractable or at the very least extremely expensive. In order to produce a viable algorithm, we must find some way to limit the number of tag groups, and the resulting tag group network structure.\n\nOur hybrid approach can be broken down into two steps. In the first step, we calculate a neighborhood of users based upon overall document similarity (as in User-Collaborative Filtering, above). Once this neighborhood is established, we can filter the suggestion results by employing tag-based filtering. Here, instead of comparing over the entire data set, we limit our comparisons to tag groups included in the initial user neighborhood.\n\n1) Select a tag group at random which contains at least 6 items; call this the target group.\n\n2) Remove half of the documents belonging to this group; call this the target set.\n\n3) For the user associated with the target group, run the procedures detailed in section 5.2 to produce a neighborhood of k similar users.\n\n4) After removing the target set documents from the target group document vector, find the k tag groups in this user neighborhood whose document vectors are most similar to its remaining document vector using cosine similarity.\n\n5) The documents belonging to the document vectors of these k tag groups, but not currently contained in the target group's document vector, represent the suggestion list. Score the suggestion list according to criteria described in section 5.1.\n\nHere, we have limited the set of tag groups on which we compare to those in the initial user neighborhood. This approach is similar to8, except that the process is reversed. In8, a set of tags is employed to limit the neighborhood under which user similarity is calculated. Here, a set of users is employed to limit the neighborhood under which tag similarity is calculated. Additionally, our method does not make explicit use of tagging semantics, which are required in Zanardi's model in order to generate a tag neighborhood8.\n\nThe results of this modification are detailed in Table 4 at user neighborhood (k) values in (5,8,12).\n\nClearly, this method does not outperform the simple tag-based approach above. What is interesting, though, is that it does not suffer terribly. In other words, while limiting tag groups to a neighborhood of similar users does not improve the algorithm, it doesn't hamper it considerably, even when we consider relatively small user neighborhood sizes (e.g. 5). Under these conditions, the resulting tag group graph becomes a feasible target of network methods.\n\nWe also see that the naive user-based algorithm implemented above still offers considerably higher satisfaction and recall. In other words, the user neighborhood established under this naive approach contains a substantial number of documents that are not being found under the tag-based approach; there is still room for improvement.\n\nNow that we have established a neighborhood under which to apply network approaches feasibly, we return to the problem sketched above. Our goal is to find those tag groups in G that are not found using the basic tag- based algorithm. Because the user-based algorithm of section 5.2 delivers much higher levels of satisfaction and recall, we can be fairly confident that some of the tag groups in G lie within the neighborhood we've created.\n\nGenerally, this problem can be thought of in terms of clustering: what we are attempting to do is cluster k and G so that we can draw from documents in G even though G itself does not contain non-hidden documents from t. Many different attempts at graph clustering have been proposed for problems ranging from sociology to biology. Here, we first employ a straightforward graph metric which has been used to identify protein interactions in small-world networks12.\n\nThe clustering coefficient of a graph measures its cohesiveness. For an individual vertex, we count the number of links among its immediate neighbors and divide by the total number of possible links in this set. An average of this value for all vertices gives us the clustering coefficient. The mutual clustering coefficient extends this idea by measuring how many neighbors are shared between two vertices. For two vertices, we use a cumulative hypergeometric to generate a p value which corresponds to the likelihood of this or a more extreme shared configuration occuring by chance.\n\nFormally, for tag groups a and b, we take the neighborhood of each (N(a), N(b)), the number of neighbors in common (N(a) ∩ N(b)), and the total number of tag groups in the graph (Total) we calculate:\n\n\n\nThe summation represents the p value cited above. Taking the negative log allows us to compare different p values more intuitively.\n\nHigher mutual clustering coefficients signify a greater likelihood that a link should exist between two tag groups. This is useful in the situation we're addressing; a large number of shared neighbors among t and G should imply the existence of a similarity where the cosine algorithm has been unable to find one for reasons cited above.\n\nWe apply this measure to our graph, this time generating a neighborhood k by finding the tag groups with the highest mutual clustering coefficients to the target tag group t. Results for running this procedure for various k are listed in Table 5.\n\nThe algorithm delivers in one sense: we've gained in recall and satisfaction. However, we've paid a steep price in accuracy which serves to bring our overall MAP score down significantly. A closer look at our algorithm suggests a reason why: whereas the naive tag-based similarity network weighs document suggestions based directly on link weights, here we use link topology. In some sense, each individual similarity link here counts equally in the overall assessment of similarity. Once any type of similarity is encoded as a link, the weight of that link is ignored in the topological calculation above. Recall that the main strength of the naive tag-based algorithm was its ability to filter according to tag group size. This is precisely the information that is lost in the topological assessment above.\n\nIt is possible that the above algorithm could be modified to introduce a notion of threshold similarity and take link weight into account. That is, if we only include those links which are sufficiently powerful, we might recapture some of the lost information. Doing so would introduce another parameter to the model, though, one whose value appears on first glance to be somewhat subjective.\n\nIt is also possible that a version of mutual clustering coefficient which takes link weights into account directly might be applied. There have been efforts to establish a weighted version of the clustering coefficient, but to date no weighted version of the mutual clustering coefficient has been developed.\n\nIn our discussion of recent developments above, we noted a method which has been used in networks to leverage the putative transitivity of trust relationships. A notion of spreading activation is used in Massa to model trust in online networks13. Most models of spreading activation resemble Hopfield networks. In these, nodes are binary threshold units which can be activated if input to the node exceeds some threshold. Nodes are connected with weighted links, and the weight of a given link determines how much input it provides to its target once it has been activated.\n\nWhile a spreading activation model can be used to capture important qualities of a network based on transitive principles, its main benefit lies in its simplicity. Moreover, it is unclear why a binary mechanism better models a real-world notion of trust than would a conti- nous mechanism. Here, instead of a spreading activation model we employ a model which simulates a random walk on the similarity graph. This graph model allows for continuous similarity values in (0,1) and can be extended to an arbitrary number of steps.\n\nWe begin with the basic graph described above, which details the similarity of every tag group to every other within our user neighborhood. From this we generate n tag neighborhoods. Our first neighborhood is identical to the one we computed for the naive tag-based algorithm: it is generated by reading the top k similarities from the t row of the adjacency matrix. If we remove the (t,t) entry, and normalize the matrix to be row stochastic, this first set of tag groups can be seen as the most likely destinations in a one-step random walk from the t node in the similarity graph. Successive neighborhoods are simple matrix products: if we multiply the matrix generated in step n by the original (step 0), the t row gives us the probability of moving from t to each other tag group in the network in n + 1 steps. The probability of moving to a given tag group other than the target in n + 1 steps is added to the probability of moving to it in steps 1 through n and the tag groups with the highest probability scores overall are selected for the final neighborhood. Each document in a tag group receives that tag group's probability score (these are cumulative for each document across tag groups). The final suggestion list orders documents according to these scores.\n\nWith this algorithm, we have two parameters - one for the size of the initial user neightborhood, k, and one for the size of the final tag group neighborhood (with the highest probability scores) which we call z. We can also walk the graph for an arbitrary number of steps n. We try the algorithm for n = 2 and n = 3.\n\nWe first experiment with various values of (k,z) where n = 2 (Table 6). Here a random walk appears to perform better than mutual clustering. MAP scores for k = z are close to what they are for the hybrid model without network analysis, and accuracy scores are also roughly on a par. Unfortunately, we have not achieved a significant improvement in satisfaction or recall at k = z. When we move up to larger tag neighborhoods, we see that the accuracy diminishes to such an extent that the overall MAP score is decreased. While any practical algorithm for suggestion would not be based solely on this score, the benefit of MAP is that it puts a premium on accuracy - a metric which can be argued is more important than recall in a real system. As mentioned above, users are unlikely to peruse a list of suggestions beyond the first 20 or so items.\n\nOur next results detail a walk with various values of (k,z) where n = 3 (Table 7). Our results for a 3- step random walk appear slightly worse than those for the 2-step. Satisfaction and recall are roughly similar, but accuracy is slightly lower for the 3-step walk. This is probably due to the fact that our 3-step algorithm tends to weigh tag groups which are further out in the similarity graph a fraction higher than those closer to the target. As a result, those documents are slightly overweighted, and will appear higher in the suggestion list than is probably warranted.\n\nThe results above show that we can achieve good recall without leaving the initial user neighborhood. However, doing so often involves increasing the size of the tag group neighborhood significantly, and with such an increase comes a decrease in accuracy. While the accuracy of both random walk models is significantly better than that of the naive user-based algorithm, high levels of recall (e.g. over .4) are associated with low list position (e.g. over 30). Again, in a practical situation this is probably not a reasonable tradeoff.\n\nWe believe there exists a remedy, however. Overall, our strategy has been to look at the data set in sets of successively smaller neighborhoods: we first confine the results to a group of similar users, then confine this set according to tag groups. The former strategy does not appear to severely limit the number of good recommendations that can be made, and the latter strategy helps to remove items which do not relate to the target user's area of interest. If we continue this approach, and look to characterize the resulting tag group network in terms of its items, it is possible that we can improve the accuracy of recommendations and so improve the overall MAP score. If, for example, we look to find those items which are most important for maintaining cohesiveness of the tag graph, it would make sense that these items appear higher in the suggestion list. Approaches similar to this one are reserved for future study.\n\n\n8 Conclusions and future work\n\nWe have seen that a suggestion algorithm that takes into account a natural conception of trust outperforms one which does not. Moreover, recommendations from a user community should be sensitive to the fact that we trust certain people in certain areas. We can use the most widespread method of online categorization, tagging, to help delineate these areas of trust without resorting to semantic analysis of the tags themselves.\n\nIn a sparse environment, the network which emerges from a comparison based on tag groups is reasonably well-confined. That is, most of its structure is contained within a small set of similar users. But even in a sparse environment, suggestions made on the basis of similar users alone has the potential to be highly inaccurate. The act of categorization itself, however, provides a useful filter which does not depend on the content of the underlying items. Using tags, we can gain a huge improvement in accuracy with a modest decrease in recall. It is assumed that tags need not be used per se - that is, any system which allows users to group items into categories would aid in recommendation, even when that system does not derive from a shared ontology.\n\nA similarity model based on coarse user-user comparisons has very little cohesiveness: people outside a user's immediate neighborhood are unlikely to be similar enough to be useful as recommenders. However, because tag-based similarity is much more focused, it is possible to construct a similarity model which exhibits transitivity: if I trust person A in a particular area, and he trusts person B in the same area, the probability of B representing a good recommender is reasonably high. This property results in networks with more structure, and we believe this structure can be exploited to generate better suggestions.\n\nHere we relied upon a take-out-half approach, as we felt this method best reflected the circumstance of a typical user. In the future it would be helpful to apply a takeout-one approach, which is also common in this field, to compare the results. As mentioned above, we would also like to attempt a modification to mutual clustering which allows for weighted edges, as this would not require that we ignore this crucial information when making assessments of network topology.\n\nWe made attempts above to exploit the structure of the resulting tag group graph with mixed results. Mutual clustering posed accuracy problems because it did not take similarity measurements into account when arriving at link probabilities. A weighted version of this algorithm might fare better on this count.\n\nWe also suspect that a more thorough analysis of the networks which emerge would suggest additional methods to test. An understanding of how these graphs cluster, for instance, might shed light on when to look only at the initial neighborhood of tag groups and when to look deeper into the graph. A comparison of different networks across different data sets might also be helpful in understanding how the underlying network structure can help produce effective suggestions. An analysis of which items are most central to the tag graph structure might also prove valuable in determining their position in the recommendation list.", "appendix": "Author contributions\n\nJB conceived the project. JB and DSG designed the algorithms. JB wrote the code. DSG supervised the work. JB prepared the original manuscript. JB and DSG contributed to the final draft of the manuscript.\n\n\nCompeting interests\n\nNo relevant competing interests disclosed.\n\n\nGrant information\n\nThis work was funded by CU-Boulder and by National Science Foundation award MCB-0630250 to DSG.\n\n\nAcknowledgements\n\nWe thank Jem Corcoran and Juan Restrepo for their feedback.\n\n\nReferences\n\nHerlocker JL: Understanding and Improving Automated Collaborative Filtering Systems. Ph.D. Dissertation, University of Minnesota, 2000.\n\nAdomavicius G, Tuzhilin A: Towards the Next Generation of Recommender Systems: A Survey of the State-of-the-Art and Possible Extensions. IEEE Transactions on Knowledge and Data Engineering. 2005; 17: pp. 734–749.\n\nBell R, Koren Y, Volinsky C: Modeling Relationships at Multiple Scales to Improve Accuracy of Large Recommender Systems. ATT Research, Netflix Research Track Paper, 2007.\n\nMcNee S, Konstan JA: Meeting User Information Needs in Recommender Systems. Ph.D. Dissertation, University of Minnesota, 2006.\n\nLi X, Guo L, Zhao YE: Tag-based Social Interest Discovery. Proc. of the 17th International Conference on World Wide Web. 2008; pp. 675–684.\n\nTso-Sutter KH, Marinho LB, Schmidt-Thieme L: Tag-aware Recommender Systems by Fusion of Collaborative Filtering Algorithms. Proc. of the 2008 ACM symposium on Applied Computing. 2008; pp. 1995–1999.\n\nNakamoto R, Nakajima S, Miyazaki J, et al:Tag-Based Contextual Collaborative Filtering. 18th IEICE Data Engineering Workshop, 2007.\n\nZanardi V, Capra L: Social Ranking: Uncovering Relevant ContentUsing Tag-based Recommender Systems. Proc. of the 2008 ACM conference on Recommender systems. 2008; pp. 51–58.\n\nBogers T, van den Bosch A: Recommending Articles Using CiteULike. Proc. of the 2008 ACM Conference on Recommender systems. 2008; pp. 287–290.\n\nCiteULike, \"Who-posted-what data\" [Data File], http://static.citeulike.org/data/2007-11-02.bz2, Retreived November 8, 2008.\n\nCiteULike, \"Article linkout data\" [Data File], http://static.citeulike.org/data/linkouts-2008-02-02.bz2, Retreived November 8, 2008.\n\nGoldberg DS, Roth FP: Assessing experimentally derived interactions in a small world. Proc Natl Acad Sci U S A. 2003; 100: pp.4372–4376.\n\nMassa P, Avenasi P: Trust-aware Collaborative Filtering for Recommender Systems. International Conference on Cooperative Information Systems (CoopIS), 2004." }	[ { "id": "1348", "date": "19 Aug 2013", "name": "George Karypis", "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 paper explores an interesting use of tags within the context of recommender systems. My reservations regarding the paper have to do with: The baseline comparisonsThe baseline approach is a user-based content filtering of similar users. Though this is an acceptable baseline, it would also be nice to see how a pure collaborative-filtering approach works as well (i.e., no content, just article IDs). The interpretation of the resultsIt is not fair to compare the results coming from the first set of experiments, as they use a different approach when doing a train/test split. The results between the two methods can only be compared if the first step of both methods is the same. My suggestion is to use the \"Select a tag group at random which contains at least 6 items; call this the target group.\" approach for all experiments. The reason that the reported results are not comparable, is the the train/test scheme used for the tag-based results has created a very homogeneous test set relative to the training set, and as such, the prediction problem is easier. On the other hand, the train/test scheme used in the first set of experiments, when the user has rated many items, will be significantly more diverse, and as such it would be harder for the prediction method to identify the hidden articles, as they can cover only a subset of the user's interests.", "responses": [] }, { "id": "1578", "date": "27 Aug 2013", "name": "Zi-Ke Zhang", "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, entitled ‘I’m Like You, Just Not In That Way: Tag Networks to Improve Collaborative Filtering’, describes an algorithm to improve the performance of collaborative filtering, by introducing tag groups. In addition, in order to improve the performance of former naive tag-based approaches in metrics recall and satisfaction, the author also constructed trust tag networks and proposed several methods: hybrid approach, mutual clustering coefficient and random walk. It is interesting and innovative to introduce tag groups and tag networks which can avoid complex semantic analyses, and improve the performance of collaborative filtering in some aspects. The author also did well in the data pre-processing and methods discussion. However, the article needs to improve its clarity in the methods introduction. Also, the background of training steps is fuzzy. My technical comments are as follows: In most of the method introductions, the author may pay too much attention to semantic description of these approaches, but ignores their simplicity and clarity, which may seem somewhat confusing. For example, in the section of ‘a random walk’, much semantic description of random walk method in network may push readers to a confusing state. It would be clear and simple by introducing matrix multiplication and probability equations. The same situations happens in the section ‘a hybrid approach’, ‘mutual clustering coefficient’, and so on.My key concern is that all of approaches in this article have a step in the method where half of the documents belonging to target user or tag group are removed, which called the target set. In my mind, this step may mean separating training and testing sets for the recommendation task. However, it seems somewhat obscured. Based on prior experiences, all of training and testing samples are selected from a data set by specific or random ordering at one time. I do not sure whether this removing step is sound, without strong evidence cited to support this method. There are a few minor concerns in this article. For instance, values of metric precision in Table 4 are missing, without specific reasons. Above all, I suggest this paper is revised and submitted again.", "responses": [] } ]	1	https://f1000research.com/articles/2-95
https://f1000research.com/articles/2-94/v1	22 Mar 13	{ "type": "Review", "title": "Why do proteins aggregate? “Intrinsically insoluble proteins” and “dark mediators” revealed by studies on “insoluble proteins” solubilized in pure water", "authors": [ "Jianxing Song" ], "abstract": "In 2008, I reviewed and proposed a model for our discovery in 2005 that unrefoldable and insoluble proteins could in fact be solubilized in unsalted water. Since then, this discovery has offered us and other groups a powerful tool to characterize insoluble proteins, and we have further addressed several fundamental and disease-relevant issues associated with this discovery. Here I review these results, which are conceptualized into several novel scenarios. 1) Unlike 'misfolded proteins', which still retain the capacity to fold into well-defined structures but are misled to 'off-pathway' aggregation, unrefoldable and insoluble proteins completely lack this ability and will unavoidably aggregate in vivo with ~150 mM ions, thus designated as 'intrinsically insoluble proteins (IIPs)' here. IIPs may largely account for the 'wastefully synthesized' DRiPs identified in human cells. 2) The fact that IIPs including membrane proteins are all soluble in unsalted water, but get aggregated upon being exposed to ions, logically suggests that ions existing in the background play a central role in mediating protein aggregation, thus acting as 'dark mediators'. Our study with 14 salts confirms that IIPs lack the capacity to fold into any well-defined structures. We uncover that salts modulate protein dynamics and anions bind proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Accordingly, I modified my previous model. 3) Insoluble proteins interact with lipids to different degrees. Remarkably, an ALS-causing P56S mutation transforms the β-sandwich MSP domain into a helical integral membrane protein. Consequently, the number of membrane-interacting proteins might be much larger than currently recognized. To attack biological membranes may represent a common mechanism by which aggregated proteins initiate human diseases. 4) Our discovery also implies a solution to the 'chicken-and-egg paradox' for the origin of primitive membranes embedded with integral membrane proteins, if proteins originally emerged in unsalted prebiotic media.", "keywords": [ "Life represents extremely unique systems that supposedly emerged from inanimate nature", "but its origin remains a great mystery. As formulated by Schrödinger in 1944 in his book \"What is Life?\"1", "life is characteristic of two fundamental processes", "'order from order' and 'order from disorder'. Schrödinger predicted that the gene controlling the 'order from order' process in a species was an aperiodic crystal", "which was uncovered to be DNA a decade later by Watson and Crick. So far", "the mechanisms", "functions and structures of life associated with 'order from order' have been extensively unraveled. On the other hand", "another process for achieving 'order from disorder' that drove chemical evolution and led to the emergence of life from primitive physical systems has remained a subject of continuing debate and uncertainty1." ], "content": "Introduction\n\nLife represents extremely unique systems that supposedly emerged from inanimate nature, but its origin remains a great mystery. As formulated by Schrödinger in 1944 in his book \"What is Life?\"1, life is characteristic of two fundamental processes; 'order from order' and 'order from disorder'. Schrödinger predicted that the gene controlling the 'order from order' process in a species was an aperiodic crystal, which was uncovered to be DNA a decade later by Watson and Crick. So far, the mechanisms, functions and structures of life associated with 'order from order' have been extensively unraveled. On the other hand, another process for achieving 'order from disorder' that drove chemical evolution and led to the emergence of life from primitive physical systems has remained a subject of continuing debate and uncertainty1.\n\nAs self-assembly occurs at all levels of living systems, life appears to defy the second law of thermodynamics, which states that any spontaneous process is associated with the overall increase of entropy. In this regard, Schrödinger theorized that life, contrary to the general tendency dictated by the second law of thermodynamics, decreases or maintains its entropy by feeding on negative entropy. When Lovelock was asked in 1964 what he would do to look for life on Mars, he replied: \"I’d look for an entropy reduction, since this must be a general characteristic of life\"2. Later Prigogine proposed that living systems are dissipative structures existing far from equilibrium, into which importation and dissipation of energy could reverse the maximization of entropy rule imposed by the second law of thermodynamics3. Recently the self-organization in living systems has been proposed as a nature process resulting from the consumption of free energy4,5.\n\nProteins are the most important functional players that implement difficult, but essential tasks in living cells. They are linear heteropolymers composed of 20 common α-amino acids, which amazingly are all in L-mirror-image6,7. Intriguingly, a recent analysis indicated that the human genome has ~20,687 protein-coding genes8, which are remarkably smaller than those of many less complex organisms, such as the roundworm and the fruit fly. This may result from the extensive use of alternative splicing in humans, which provides the ability to build a very large number of modular proteins through the selective incorporation of exons.\n\nProteins also represent one of the best examples to illustrate the self-assembly of biomolecules. Remarkably, a portion of proteins will spontaneously self-organize into unique three-dimensional structures via protein folding processes9–17. It is widely recognized that the folding of cytosolic proteins is mainly resulting from solvophobic interactions of polar water molecules with the hydrophobic side chains of proteins. As a result, in the three-dimensional structure of a well-folded cytosolic protein, more than 80% of hydrophobic side-chains are buried in the internal core, thus being shielded from water, while most hydrophilic residues are exposed to polar water molecules. On the other hand, however, it has been now widely realized that many proteins are fully functional, but lack well-defined structures, and are thus called intrinsically unstructured/disordered proteins (IUPs). It has been estimated that ~50% of cellular proteomes encode proteins with long intrinsically unstructured domains/fragments18–23. Interestingly, a recent study indicates that there is a sharp increase in disorder associated with the transition from prokaryotic to eukaryotic cells23. Intrinsically unstructured proteins have been found to be significantly lacking in bulky hydrophobic (Ile, Leu, and Val) and aromatic (Trp, Tyr, and Phe) residues but dramatically enriched in polar (Arg, Gly, Gln, Ser, Pro, Glu, and Lys) and structure-breaking (Gly and Pro) residues18–23.\n\nModern life forms all begin with cells, which are separated from the external environment by a surface membrane, called the plasma membrane. Furthermore, eukaryotic cells have extensive internal membranes that further subdivide the cell into various compartments, which are all filled with watery media. Life on earth absolutely depends on water, which constitutes 70–80%, by weight, of most cells. Therefore, water is regarded as the ‘matrix of life’ which not only provides a passive scaffold, but also has many active roles in molecular biology24–30. The absolutely essential role of water for life is associated with its unique combination of many different properties, which are believed to be irreplaceable by another single molecule system. For example, water exhibits a solvophobic effect, which thus renders hydrophobic effects to play a dominant role in all self-organizing processes in biology12–17.\n\nOn the other hand, in addition to providing a barrier to prevent the free flow of molecules in and out of cells, biological membrane systems appear also to provide a hydrophobic environment in cells. All biological membranes are composed of lipid molecules, consisting of a hydrophilic headgroup, and one or more hydrophobic fatty acid hydrocarbon tails. Due to their amphiphilic nature, lipid molecules spontaneously self-organize into bilayers in cells, consisting of two sheets of lipids with their hydrophobic tails facing each other to form the hydrophobic core, which is shielded from the aqueous surroundings by a polar interface consisting of the hydrophilic headgroups. There is no sharp border between the hydrophobic core and the surrounding water, as this interface region has gradually changing hydrophobicity31. In 1992, Wiener and White determined the structure of a bilayer, in which the hydrophobic core is about 30 Å thick, while the interface region extended about 15 Å on either side32. As a consequence, it is anticipated that biological membranes also provide a non-polar phase to accommodate hydrophobic proteins. Indeed, it has been estimated that more than 30% of proteins are located in membrane environments, which are highly hydrophobic33–35. Nevertheless, it appears to be much more complex for the folding of membrane proteins as lipid bilayers show large variations in density and polarity on a nanometer scale. While the center is highly hydrophobic and significantly disordered, the surfaces consist of diverse mixture of polar functional groups including the carbonyl and glycerol groups, which extensively interact with water molecules. As such, all attempts to use organic solvent to mimic membranes failed33–42.\n\nOne intriguing phenomenon associated with proteins is their insolubility in aqueous buffers. Protein aggregation/insolubility is not only problematic for in vitro protein research and industry applications, but is commonly associated with a large spectrum of human disease, in particular neurodegenerative/aging diseases including Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), spinocerebellar ataxias (SCA), amyotrophic lateral sclerosis (ALS), aging and many others. These diseases have the common characteristic of the aggregation of disease-specific proteins and are thus called 'conformational diseases'43–54. Recently, protein aggregation has been increasingly revealed to be involved in other human diseases55,56, and also has important implications in fields other than biology, including nanotechnology and material science57.\n\nInterestingly, both genetic mutations (familiar) or environmental insults (sporadic) can trigger protein aggregation diseases, implying that a common mechanism may exist to link these clinically distinct diseases. It has been extensively thought that the aggregation of these proteins is due to misfolding. In other words, these proteins still have the intrinsic ability to fold into well-defined structures, but have been misled to aggregation through 'off-pathway' misfolding triggered by abnormal cellular and environmental factors. Indeed, it has been shown that all folded proteins could be induced to form amyloid aggregates in vitro by manipulating solution conditions45,46. Nevertheless, recent studies increasingly reveal that protein aggregation is not a rare event under abnormal conditions. Most unexpectedly, a recent study on human cells reveald that approximately 30% of cellular proteins are 'wastefully synthesized'58. Immediately after their synthesis, these proteins get aggregated and are thus rapidly degraded by proteasomes58. This strongly implies that even under normal cellular environments, aggregation is an evitable destination for a large portion of proteins. As accumulation of aggregated protein can dramatically perturb protein homeostasis and can lead to extensive cell and tissue damage through a variety of mechanisms, cells have developed various quality control systems in evolution to remove misfolded/aggregated proteins58–65.\n\nSo why do proteins become insoluble/aggregated? A complete answer to this question not only sheds light on the fundamental properties associated with proteins, but is also crucial to deciphering the mechanisms underlying a variety of human diseases as well as to further developing therapeutic strategies and agents. Certainly, some proteins become aggregated from well-folded soluble forms through 'off-pathway' misfolding, which can be significantly suppressed by cellular chaperone systems. However, there exist a portion of proteins which appear to be not refoldable and completely insoluble in various buffer conditions, as particularly demonstrated by the results from structural genomics projects66. Previously, biophysical studies have been exhaustively conducted on misfolded proteins to understand the factors mediating their aggregation. In contrast, high-resolution studies on unrefoldable and insoluble proteins have not been possible due to the absence of a general method to solubilize these insoluble proteins without adding denaturants and detergents. Nevertheless, many disease-causing protein mutants appear to belong to this category, as exemplified by the ALS-causing P56S mutant of the VAPB-MSP domain67. Because of this, a fundamental question remains unanswered as to by which degree these mutations alter the folding properties of these mutant proteins.\n\nMarvelously, in 2005 we discovered that all 11 unrefoldable and insoluble proteins we had could be solubilized in unsalted water for high-resolution nuclear magnetic resonance (NMR) investigations68,69. The results led to the classification of these 'insoluble proteins' into three groups: group 1, with no secondary and tertiary structures; group 2, with only secondary structure but no tertiary packing; and group 3, with secondary structure and loose tertiary packing, like molten globule states. Remarkably, we found that all these insoluble proteins lack tight tertiary packing. Therefore, I have proposed that unrefoldable and insoluble proteins may lack the intrinsic ability to fold into well-defined structures, thus with many hydrophobic side chains exposed to the polar water molecules. As such, a very low ionic strength is sufficient to non-specifically screen out intrinsic repulsive interactions and, thus, trigger hydrophobic clustering/aggregation68. In 2008, I further proposed a model to rationalize our discovery and explain why our results appear to be inconsistent with the well-established dogma about the effect of salt on protein solubility26.\n\nOur discovery led to the establishment of a general and powerful tool to characterize unrefoldable and insoluble proteins. Indeed, with this tool other groups have studied insoluble proteins, including those involved in biomineralization70–76. On the other hand, this also allows us to address several fundamental and disease-relevant issues associated with the discovery. Here I review the results of the studies based on our discovery. Analysis of these results in the relevant contexts leads to the conceptualization of several novel scenarios.\n\nFirstly, we characterized solution conformations and dynamics of several unrefoldable and insoluble proteins including those with low complexity sequences, as well as resulting from the splicing variation and insertion/mutation on well-folded domains such as SH3 and MSP. The results uncover that unlike 'misfolded proteins', which still have the capacity to fold into uniquely well-defined structures, unrefoldable and insoluble proteins all lack this ability due to either low complexity sequences, splicing variation, or insertion/mutation in the well-folded domains. As a consequence, they will unavoidably aggregate in vivo with ~150 mM ion concentration, and are thus here designated as 'intrinsically insoluble proteins (IIPs)'.\n\nSecondly we assessed the upper limit of our discovery with the 25-residue integral membrane peptide of the influzene M2 channel, one of the most hydrophobic protein sequences in nature. To our great surprise, it could also be solubilized in unsalted water without lipid molecules to form a highly helical conformation.\n\nThirdly, the fact that insoluble proteins including integral membrane proteins could all be solubilized in unsalted water, but become aggregated upon being exposed to salt ions logically suggests that salt ions play central roles in mediating protein aggregation, and are thus designated as 'dark mediators' here. To delineate the underlying mechanisms, we systematically assessed the effects of 14 salts with 8 anions on the conformation of unrefoldable and insoluble proteins dissolved in unsalted water. This led to the discovery that anions have asymmetric binding to both unstructured and well-folded proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Very recently, we discovered that different salts have very diverse effects on protein dynamics.\n\nFourthly, we have also tested whether insoluble proteins are generally capable of interacting with membranes. This leads to our discovery that the ALS-causing P56S mutation and the truncation in the VAPB-3 splicing variant transform the seven-strand immunoglobulin-like β-sandwich adopted by the native MSP domain into a helical structure with the majority of residues buried in membranes.\n\nFifthly, in light of our discovery, I hypothesize a solution to the 'chicken-and-egg' paradox for the origin of primitive cells embedded with integral membrane proteins if all proteins originally emerged in unsalted prebiotic oceans/media.\n\n\nIntrinsically insoluble proteins\n\nDue to low-complexity sequences, lacking of hydrophobic and aromatic and enrichment of polar residues, intrinsically unstructured proteins have been generally thought to be highly soluble in buffers. However, by solubilizing it in unsalted water, we have characterized a transcriptional activator, ApLLP, to be both insoluble and intrinsically unstructured22.\n\nThe 120-residue ApLLP (Aplysia LAPS18-like protein) is a transcription factor which is required for long-term synaptic facilitation in Aplysia neurons. As seen in Figure 1A, ApLLP is characteristic of a protein with low hydrophobicity, containing only 25.83% hydrophobic residues. Further assessment by DISPROT in Figure 1B also revealed that it has a disorder probability larger than 0.8 over the whole sequence, strongly suggesting that ApLLP is intrinsically unstructured.\n\n(A) The sequence of ApLLP, with two Nuclear Localization Signals (NLS) boxed. (B) Analysis results of the globularity and disordered regions of ApLLP by VSL2B.\n\nIntriguingly, despite its low hydrophobicity, ApLLP was highly insoluble in a variety of buffers tested, but could be solubilized in unsalted water at high concentrations (>200 µM). CD and NMR characterization clearly indicated that it was predominantly disordered without any stable secondary and tertiary structures (Figure 2). Unexpectedly, if one or two nuclear localization signals located at N- and C-termini were removed, the truncated ApLLP suddenly became soluble in buffers such as 20 mM phosphate buffer. However, the two nuclear localization signal sequences have no detectable difference of hydrophobicity from the rest of the protein.\n\n(A)1H–15N NMR HSQC spectrum of the 15N-labeled full-length ApLLP solubilized in unsalted water. (B–C) Superimposition of the 1H–15N NMR HSQC spectra of ApLLP-87 (B) and ApLLP-55 (C) in the absence and in the presence of the DNA fragment at a molar ratio of 1/2 (protein/DNA). Shifted HSQC peaks arising from the Gly backbone and Trp side-chain (inlet) only presented in ApLLP-87 are indicated by green arrows.\n\nAs demonstrated by NMR titrations in Figure 2A–2B, both truncated ApLLP forms were able to bind the CRE DNA element. However, even upon forming the ApLLP-DNA complex, ApLLP still appeared to be highly disordered, as evident from NMR and CD results (Figure 2). As such, we had a very difficult time to get the results, eventually published ~2.5 years after the first submission, because some reviewers could not be convinced by two observations, 1) ApLLP is highly insoluble in buffer despite owning a highly degenerative sequence and with low hydrophobicity. 2) Even upon complexing with DNA, the intrinsically unstructured ApLLP failed to form a well-folded complex structure. Now, this phenomenon is starting to be recognized to exist in a large variety of protein-protein and protein-DNA complexes involved in intrinsically unstructured proteins, and thus designated as the 'fuzzy complex'73,77–80.\n\nSimilar features have since been observed by another group on the basic helix-loop-helix motif (bHLH) region of a transcription factor NGN173. It was found that \"bHLHN was soluble in pure water at any concentration\". On the other hand, upon binding to two DNA E-boxes, the protein also appeared to be highly disordered.\n\nRemarkably, a group of proteins involved in biomineralization that are highly insoluble in buffers has also been demonstrated to be soluble in unsalted water at high protein concentrations that are suitable for detailed biophysical characterizations70–72,75,76. Biomineralization, which generates the hard tissues of living organisms, is a process under tight regulation by hormones, enzymes and various regulatory proteins. However, many of them have resisted structural characterization because of their high insolubility75,76,81–83. For example, the 172-residue porcine amelogenin, found in mammalian tooth enamel, is one of the most highly mineralized materials of vertebrates. This protein is essential for normal enamel development and undergoes self-association to form supramolecular assemblies under defined conditions in the laboratory70. However, its aggregation could be suppressed in unsalted water and exhaustive biophysical studies including detailed assignments by three-dimensional NMR experiments revealed that it is intrinsically disordered with an extended configuration in the monomeric form70.\n\nRecently, a systematic bioinformatics study unraveled that all proteins in SwissProt that have been annotated for biomineralization show an extremely high level of predicted disorder (with a mean of 53%), which represents the most disordered class of the protein world. Furthermore, the same feature was associated with evolutionarily more distant proteins involved in the formation of the silica wall of marine diatoms and the shell of oysters and other mollusks. The general and very strong correlation between biomineralization and structural disorder has been proposed to indicate that controlled growth of the mineral phase in biology may only be achieved with the assistance of highly disordered proteins82. In this regard, both structural disorder and high insolubility seem essential for their functions in coordinating biomineralization.\n\nOur previous studies also uncovered the fact that many insoluble proteins with non-degenerative sequences are also highly unstructured. These sequences may escape detection by bioinformatics tools for identifying IUPs as their sequences show no detectable difference from those that adopt well-folded structures. For example, VAPB-3, a splicing variant of VAPB, is both insoluble and unstructured84,85.\n\nThe human VAPB protein is composed of three conserved domains (Figure 3A), namely an N-terminal 125-residue domain homologous to the major sperm protein (MSP), a central coiled-coil domain, and a C-terminal transmembrane fragment67,79. Previously we have determined the crystal structure of the human VAPB-MSP domain, which assumes a classic MSP fold with a seven-strand immunoglobulin-like β sandwich (Figure 3B). Several VAPB splicing variants lacking the transmembrane segment such VAPC and VAPB-3 have been identified to contain truncated MSP domains79,80,85. For example, VAPC possesses N-terminal 70 residues that are completely identical to the 125-residue MSP domain of VAPB, and the C-terminal 29 residues unique in VAPC. Interestingly, despite being intrinsically unstructured, VAPC is buffer-soluble and serves as an endogenous inhibitor of HCV infection80.\n\n(A) 243-residue human VAPB protein is composed of the 125-residue major sperm protein (MSP), coiled coil (CC) and transmembrane (TM) domains. (B) Crystal structure of the 125-residue MSP domain of the human VAPB protein. Two residues, Pro56 and Thr46 are displayed in spheres, whose mutations to Ser and Ile respectively lead to familiar amyotrophic lateral sclerosis. The N-terminal 105 residues (in blue) are identical in both the MSP domain and VAPB-3, a splicing variant of VAPB, while the last 20 residues (in green) of the MSP domain are absent in VAPB-3.\n\nIn contrast, VAPB-3 is another splicing variant of VAPB, composed of N-terminal 105 residues identical to the MSP domain, plus a short unique C-tail (Figure 3B). Recently we demonstrated that it was both unrefoldable and insoluble, but again could be solubilized in unsalted water84. CD and NMR characterization indicates that VAPB-3 is predominantly disordered (Figure 4A–4B). Furthermore, we achieved the sequential assignment of most residues with triple resonance experiments HNCACB and CBCA(CO)NH. Subsequently, we obtained chemical shifts and NOE connectivities by analyzing HSQC-TOCSY and HSQC-NOESY spectra. Very small (ΔCα-ΔCβ) values over the majority of VAPB-3 residues (Figure 4C) clearly indicate that it is highly unstructured in unsalted water, completely consistent with the CD results. The lacking of stable secondary structures in VAPB-3 is strongly demonstrated by its NOE patterns (Figure 4D). For most residues, only sequential NOEs could be identified. dαN (i, i + 2), but not dαN (i, i + 3) NOEs were found over several short segments, indicating that the helical conformation weakly populated over these regions is very dynamic. No long-range NOE was detected, as expected for such a predominantly disordered protein without any tight tertiary structure. Based on our CD and NMR analyses, it can be concluded that the truncation in VAPB-3 completely eliminates the intrinsic capacity to fold into the MSP fold, and consequently VAPB-3 no longer has the ability to fold into the MSP fold or any other well-defined structure, thus becoming predominantly disordered in unsalted water. Upon being exposed to salt ions, the exposed hydrophobic side chains will be clustered to result in aggregation through a mechanism I previously proposed26.\n\n(A) Far-UV CD spectrum. (B) 1H–15N NMR HSQC spectrum acquired at 25ºC. (C) Residue-specific (ΔCα–ΔCβ) values of VAPB-3 and P56S-MSP. For VAPB-3, blue bars are used to indicate (ΔCα–ΔCβ) values for the first 105 residues identical to those of MSP, while green bars for unique residues. For P56S-MSP, red bars are used to indicate (ΔCα–ΔCβ) values for the first 105 residues, while brown bars for the C-terminal 20 residues. (D) NOE connectivity pattern of VAPB-3 defining secondary structures.\n\nIn this section, I have reviewed the studies on the unrefoldable and insoluble proteins with full-length native sequences. The results reveal that surprisingly a portion of native protein sequences with both low- and high-complexity completely lacks the intrinsic ability to fold into any uniquely defined structures and is also completely insoluble in buffers. While high insolubility of such proteins involved in biomineralization has been recognized to be essential for their functions76,81–83, how living systems utilize the insolubility of the proteins represented by ApLLP and VAPB-3 is an important topic to be further explored. Remarkably, the genomes of eukaryotic organisms are anticipated to be abundant in splicing variants, some of which may be similar to VAPB-3, and also have truncation on well-folded domains. As this kind of splicing variants show non-degenerative sequences and high hydrophobicity, they are usually not spotted by the bioinformatics tools for detecting IUPs. Therefore, markedly different from the classic IUPs, VAPB-3 represents a previously undetectable subgroup of IUPs which is characteristic of non-degenerative sequences, with high insolubility and hydrophobicity comparable to that of well-folded proteins. As a consequence, the number of IUPs in eukaryotic genomes may be much larger than currently detected.\n\nOur results also unveil that the mechanism for the misfolding-triggered aggregation shows a fundamental difference from that underlying the aggregation of the proteins represented by ApLLP and VAPB-3. For the 'misfolded proteins', they still retain the intrinsic capacity to fold into well-defined three-dimensional structures, but are misled into aggregation via 'off-pathway' misfolding, triggered by unfavorable conditions such as their over-expression. As such, their aggregation can be significantly recovered with the assistance of cellular chaperone systems. In contrast, the proteins represented by ApLLP and VAPB-3 have no ability to fold into any well-defined structures, due to the low complexity sequences, or truncation of well-folded domains. Therefore, the exposure of hydrophobic side chains in these proteins will unavoidably lead to aggregation upon being exposed to salt ions. Since this category of proteins is naturally occurring, and even in the presence of chaperone systems the aggregation is their evitable destination in vivo with ~150 mM ion concentration, here I designate them as 'intrinsically insoluble proteins' (IIPs).\n\nThe results also highlight an extreme complexity underlying protein insolubility. Despite having a low hydrophobicity and being unstructured, the full-length ApLLP protein is completely buffer insoluble. In particular, although the nuclear location signaling sequences have no detectable difference in hydrophobicity from the rest of the protein, their deletion renders the truncated ApLLP forms to remain similarly unstructured, but to suddenly become buffer-soluble. This observation imposes a great challenge to precisely predict protein solubility from amino acid sequences by current bioinformatics methods. So why is it so difficult to achieve the prediction? One possibility is that like protein folding, protein aggregation is also highly sequence-dependent. Another scenario has been recently proposed that protein folding/aggregation is a dissipative process of the non-Euclidian manifold in which structural hierarchy builds up by diminishing energy density gradients in the quest for a stationary state determined by surrounding density-in-energy4,5,86. The non-Euclidian landscape for protein folding/aggregation is not predetermined. Instead, it is forming and deforming during the folding/aggregation. Hence, the prediction of folding/aggregation is not just technically challenging, but is non-deterministic polynomial time (NP) complete87.\n\n\nSolubilizing integral membrane peptide in unsalted water\n\nEven during the preparation of our first two manuscripts on solubilizing insoluble proteins in unsalted water21,68, I asked myself the question ‘what kind of proteins would be insoluble in unsalted water?’ To assess the upper limit of our discovery, we have attempted, but failed to chemically synthesize and Escherichia coli -express peptides containing only bulky hydrophobic residues. Therefore, I decided instead to use the 25-residue M2 transmembrane peptide of the influenza A proton channel, which is one of the most hydrophobic sequences in nature, with a sequence of SSDPL VVAAS IIGIL HLILW ILDRL. Previously, the three-dimensional structures of this peptide have been determined in the presence of lipid molecules by crystallography88, liquid and solid NMR spectroscopy, respectively89,90. In all these structures, the M2 fragment is a well-formed helix, which is further assembled into a tetramer (Figure 5A).\n\n(A) Tetrameric structure in a membrane environment with the sequence of SSDPL VVAAS IIGIL HLILW ILDRL. (B) Far-UV CD spectra of the chemically synthetic peptide solubilized in unsalted water (pH 4.0) at a concentration of 100 µM without any lipid molecules, at 20ºC (blue), 95ºC (red) and 20ºC after the thermal unfolding (green).\n\nTo our great surprise, the M2 peptide could again be solubilized in unsalted water to reach at least ~100 μM concentration without any lipid molecules91. Furthermore, in unsalted water, the peptide also forms a high helical conformation which remains almost unchanged during thermal unfolding up to 95ºC, as monitored by CD spectroscopy (Figure 5B). However, in the absence of lipid molecules, the peptide appeared to be specifically self-organized into a supramolecule complex consisting of a large amount of individual helical peptides as the NMR resonance peaks were too broad to be detected. However, its far-UV CD spectra typical of a helical conformation could not result from non-specific aggregation, as aggregated proteins give rise to far-UV CD spectra similar to that for a β-sheet protein. On the other hand, as expected, the peptide would precipitate immediately upon adding NaCl up to 1 mM.\n\nNow, results by us and other groups have demonstrated that proteins previously thought to be insoluble could indeed be solubilized in unsalted water. In particular, the most hydrophobic integral membrane peptide is also soluble in unsalted water without lipid molecules, but gets aggregated upon the introduction of salt ions. These results together constitute a fact that logically suggests that despite existing in the background, salt ions play at least an equal important role in mediating protein aggregation. Therefore, here I designate salt ions as 'dark mediators' for protein aggregation, analogous to 'dark matter', which controls the global structure of the Universe in the background, but whose nature still remains almost unknown92.\n\nTherefore, in aqueous environments, factors mediating protein aggregation are composed of two key categories, one associated with proteins and another with the salt ions existing in solution. The interplay between them governs protein aggregation and can be symbolized by the Taiji diagram (Figure 6). While the 'Yang' part from proteins has been clearly recognized and exhaustively assessed, the 'Yin' part from salt ions stays largely elusive in the background, and certainly needs to be explored in the future. In the next sections, I will review our results with both 'Yang' and 'Yin' factors modulating protein aggregation.\n\nFactors modulating protein aggregation are symbolized by Taji dagram, which are constituted by those from proteins ('yang') and salt ions existing in the background ('yin'). The 'yang' part has been exhaustively characterized, which includes misfolding and modifications of proteins such as mutation, deletion and post-translational modifications, etc. By contrast, the 'yin' part is only beginning to be recognized, and is involved in the ability of salt ions in imposing non-specific electrostatic screening, specific anion-binding, altering protein dynamics and water structure, etc.\n\n\n'Yang' of protein aggregation\n\nDue to the marginal stability and dynamic nature, even wild-type proteins which have the intrinsic capacity to fold into well-defined three-dimensional structures have been extensively demonstrated to 'off-pathway' misfold into aggregates, or amyloid fibers in vitro by manipulating solution conditions45,46. On the other hand, it has been well known that some modifications, especially mutations of certain specific proteins, render them to become unrefoldable and insoluble both in vivo and in vitro, thus leading to a variety of human disease, in particular neurodegenerative diseases. As a consequence, these modified proteins have been exhaustively characterized by various biophysical methods. However, due to the previous absence of a general method to solubilize these insoluble proteins in the aqueous solution without adding denaturants and detergents, the solution conformations of these unrefoldable and insoluble proteins remained completely unknown. The lack of such knowledge leads to the inability to answer a fundamental question: whether these mutants still possess the intrinsic capacity to fold into well-defined structures, and their aggregation is only due to 'off-pathway' misfolding, or whether they have lost this ability; and consequently aggregate following the mechanism underlying 'intrinsical insoluble proteins'.\n\nWith our discovery as a powerful tool, in the past several years we have addressed this question by high-resolution NMR characterizations of several model systems. Below I review two of them. One is an unrefoldable and insoluble SH3 mutant triggered by a single-residue insertion, and another is an unrefoldable and insoluble MSP mutant triggered by one residue P56S mutation, which causes a familiar ALS.\n\nSH3 modular domains, containing ~60 residues, play a critical role in transmitting, as well as integrating, cellular signals. Structurally, all SH3 domains share a common β-barrel fold comprising five β-stands (Figure 7A). So far, more than 4,000 SH3 domains have been identified in a variety of organisms93–95. Very interestingly, we found that the first SH3 domain of the human Nck2 protein, GenBank sequence AAC04831, originally cloned from a tumor tissue, was completely insoluble and not refoldable in all aqueous buffers we tested94. Later on, we found that this sequence had an extra Val insertion at position 22, the tip of the diverging turn linking the RT-loop and the second β-strand (Figure 7A), thus designated as V22-SH393.\n\n(A) NMR structure of the first SH3 domain of the human Nck2 protein (2B86) with the secondary structures and position of the inserted Val residue indicated. (B) Far-UV CD spectra of the wild-type SH3 and its four insertion mutants which were collected at protein concentrations of 20 µM at 25ºC. The wild-type SH3 was dissolved in 5 mM phosphate buffer (pH 6.2) while the insertion mutants were solubilized in unsalted water (pH 4.0).\n\nNevertheless, with our discovery, we succeeded in characterizing its conformation and backbone dynamics by CD and NMR spectroscopy93. As shown in Figure 7B, far-UV CD spectra clearly indicate that V22-SH3 is highly disordered as compared with the native protein (Figure 7B). To assess whether the insolubility is a result of the introduction of the bulky Val hydrophobic side chains, we replaced Val with Ala, Asp and Lys residues, respectively. However, the results demonstrated that any of the four insertions at this position would lead to the complete buffer insolubility as well as elimination of the native SH3 fold (Figure 7B). This notion was further supported by the 1H-15N HSQC spectra of these insertion mutants, which have very narrow spectral dispersions at both 1H and 15N dimensions (Figure 8). The result thus indicates that the insolubility of V22-SH3 is not due to the introduction of the large Val hydrophobic side chain. It is also worthwhile to point out that no significant difference was identified for the HSQC spectral dispersions of V22-SH3 at pH 4 and 6.2 (Figure 8F), suggesting that being unstructured is not due to the low pH value.\n\n1H–15N NMR HSQC spectra of the wild-type SH3 and its four insertion mutants at protein concentrations of 100 µM acquired. The wild-type SH3 was dissolved in 5 mM phosphate buffer (pH 6.2) while four insertion mutants were solubilized in unsalted water (pH 4.0). (F). Superimposition of HSQC spectra of V22-SH3 in unsalted water at pH 4.0 (blue) and at pH 6.2 (red).\n\nWe further conducted extensive NMR characterization on conformations and dynamics of the V22-SH3 domain solubilized in salt-free water. As seen in Figure 9A, its Cα chemical shift deviations are already similar to those of the wild-type in the presence of 8 M urea, thus indicating that V22-SH3 becomes significantly disordered93. More specifically, in V22-SH3, the N- (residues 1–6) and C- (residue 47–57) termini are largely unstructured, without any significant secondary structure populated. However, it seems that in V22-SH3, the non-native helical conformation is populated over the secondary region but not the first region, as we have observed on the wild-type SH3 domain at pH 2.0 or 4Ala mutant at pH 6.595.\n\n(A) Bar plot of Cα chemical-shift deviations from their random-coil values for V22-SH3 (red), and wild-type in the presence of 8 M urea (blue). (B) Characteristic NOEs defining secondary structures identified for V22-SH3. (C) {1H}-15N steady-state NOE intensities for V22-SH3 (red bars) and wild-type (cyan bars).\n\nIndeed, in V22-SH3 there exist many non-native αH(i)-NH(i + 2) and αH(i)-NH(i + 3) NOEs over the sequence, in particular over residues 28–42 (Figure 9B). These NOEs are totally incompatible with the well-formed and rigid β-barrel structure of the SH3 domain in the native condition. As such, the existence of these non-native medium-range NOEs, together with the chemical shift deviations, suggesting that in V22-SH3, the non-native helical conformation is indeed populated over the second region, but not the first region as observed in the wild-type SH3 domain at pH 2.0 and 4Ala mutant at pH 6.595. On the other hand, in V22-SH3, there still exist many native-like long-range NOEs93, implying that despite severe disrupted tight packing and populated non-native helical conformations, V22-SH3 may still have a rudiment tertiary topology similar to its native SH3 fold.\n\nWe have assessed the backbone dynamics of V22-SH3. As shown in Figure 9C, V22-SH3 has significant reduced hNOE values over the whole sequence if compared with the wild-type SH3 domain at pH 6.5, in particular over the N- and C-termini which are characterized to be highly unstructured by chemical shift deviations. Nevertheless, except for the C-terminal two residues, all V22-SH3 residues still have positive hNOE values, with many >0.4. In particular, hNOE values >0.6 are found for two residues, Trp35 and Trp36, which are located at the central positions of the region that is characterized to own a highly populated helical conformation.\n\nFurthermore, we calculated reduced spectral densities at three frequencies from the relaxation data of different forms of this SH3 domain, which reflect relaxation contributions from the motions on different timescales. As seen in Figure 10C, if compared to the wild-type SH3 domain at pH 6.5, V22-SH3, like wild-type SH3 at pH 2.0 and 4Ala mutant, have significantly increased J(0.87ωH) over the whole sequence, indicating that a dramatic increase in the fast motions on the ps-ns timescale. Interestingly, out of three large unfolded SH3 forms, V22-SH3 uniformly has the highest J(0.87ωH) values, suggesting that V22-SH3 has the largest increase of the fast motions. It is particularly interesting to point out that the region with the largest J(0) values are over Glu24-Arg25-Leu26-Trp27-Leu-28-Leu29 (Figure 10A). This observation implies that this region undergoes slow motions on the µm-ms time scale and/or dynamic aggregation. Strikingly, this region was previously revealed to play a critical role in coordinating the transformation from the non-native helical conformation to native all-β SH3 fold during the folding of this SH3 domain. It is thus likely that in the SH3 domain, there exists an overlap between the interactions responsible for the insolubility and critical for integrating the formation of the native β-barrel fold, as the 4Ala mutant with this region mutated suddenly became soluble in buffer95.\n\nSpectral densities of V22-SH3 solubilized in unsalted water at pH 4.0 (red); wild-type at pH 6.5 (cyan); wild-type at pH 2.0 (blue) and 4Ala mutant at pH 6.5 (green), calculated from the 15N backbone relaxation data measured at 800 MHz. (A) J(0), (B) J(ϖN), and (C) J(0.87ϖH).\n\nTo understand how salt ions affect the conformation and solubility of V22-SH3, we have conducted extensive titrations of NaCl into various V22-SH3 samples solubilized in salt-free water. The results revealed that V22-SH3 has no significantly different conformation in the absence of and in the presence of NaCl, indicating that the failure to form any well-defined structure is an intrinsic feature of V22-SH3. On the other hand, both V22-SH3 and NaCl concentrations are important for its aggregation. If the V22-SH3 concentration is high (>300 µM), addition of NaCl even to 5 mM would result in rapid visible aggregation. As such, we lowered the V22-SH3 concentration down to 50 µM and subsequently collected a series of 1D and 2D HSQC spectra by gradually increasing the NaCl concentrations. As shown in Figure 11, addition of NaCl caused almost no change of the spectral dispersion of V22-SH3, convincingly demonstrating that no fundamental difference exists for its conformations in the absence and presence of salt. However, although no visible aggregate was observed during the experiments, addition of salt even to 2 mM induces the NMR line broadening which leads to the disappearance of some HSQC peaks. This implies that addition of salt, even at a very low concentration, induces dynamic aggregation and/or conformational exchanges on the µs-ms time scale. At a NaCl concentration of 40 mM, most HSQC peaks disappear except for those of several C-terminal residues. When the NaCl concentration reaches 100 mM, all peaks become too broad to be detected. Moreover, after more than 5 hours, the visible aggregates formed in the V22-SH3 sample in the presence of only 5 mM NaCl.\n\nSuperimposition of the 1H–15N NMR HSQC spectra of V22-SH3 at a protein concentration of 50 µM, solubilized in unsalted water (pH 4.0) (blue), and with gradual introduction of NaCl (red) to different concentrations. The HSQC spectra were acquired on an 800 MHz NMR spectrometer at 25ºC. The blue font is used for labeling the residue with its HSQC peak intensity significantly reduced or disappeared, while the red is for the residue with its HSQC peak still observed.\n\nWe also characterized an in vitro and in vivo insoluble P56S mutant of the VAPB-MSP domain which is a causative mutation for the neurodegenerative disease ALS. This mutant causes a familial ALS, and could not be studied previously due to its high insolubility which is even resistant to solubilization by Triton X-10067,84. We first determined the crystal structure of the native VAPB-MSP domain which adopts a seven-strand immunoglobulin-like β-sandwich in which Pro56 and Pro12 adopt the unusual cis-peptide bond conformation that appears to be critical in maintaining two characteristic S-shaped loops (Figure 12A).\n\n(A) Crystal structure of the wild-type VAPB MSP domain, with Pro12 and Pro56 displayed in spheres. The two Pro residues appear to play a key role in maintaining two characteristic S-shaped loops in the MSP domain. (B) Far-UV CD spectra of the wild-type MSP (black) and P56S mutant at pH 3.5 (red), 4.5 (brown), 5.5 (green), and 6.5 (blue). (C) Far-UV CD spectra of the P56S mutant at different pH values and salt concentrations. (D) Superimposition of the HSQC spectra of the wild-type MSP domain (blue) and P56S mutant at pH 3.5 (red).\n\nDespite showing severe aggregation both in vivo and in vitro, the P56S mutant protein could again be solubilized at high protein concentrations in unsalted water. Preliminary CD characterization reveals that the P56S mutation eliminates its β-sandwich structure of the wild-type MSP domain, and renders the mutant to be predominantly disordered in water at different pH values (Figure 12B), and in the presence of NaCl at different concentrations (Figure 12C). This conclusion is further supported by its narrow HSQC spectral dispersion (Figure 12D).\n\nWe have performed detailed NMR characterization of both wild-type and P56S MSP domains by acquiring a large set of three-dimensional heteronuclear NMR spectra at a 500 µM protein concentration. As shown in Figure 13A, the wild-type MSP domain has very large Cα chemical shift deviations typical of a fully folded protein. By contrast, the P56S mutant has dramatically reduced deviations characteristic of an unfolded protein. Very surprisingly, it appears that in Pro56Ser, the native β-sheet secondary structure is totally eliminated. Instead, based on the negative Hα chemical shift deviations (Figure 13B), it appears that the non-native helical conformation is weakly populated over the sequence, in particular over residues Thr97-Glu108. In the wild-type MSP structure, a helix is also formed, but it is much shorter, only over Ala104-Glu108. Further analysis of the NOE connectivity pattern (Figure 13C) indicates that except for the missing residues, sequential dNN(i, i + 1) and medium-range dRN(i, i + 2) NOEs could be observed over the majority of the sequence, suggesting that the non-helical conformation is indeed populated to some degree. However, dNN(i, i + 2) NOEs could be found only over two segments (Gly33-Thr46 and Thr97-Glu108), while only two dαN(i, i + 3) NOEs could be identified between Ala104 and Lys107, and between Val105 and Glu108. As such, the non-native helical conformation is only dynamically populated in the P56S mutant.\n\n(A) Bar plot of chemical shift deviations (Δδ = δobs - δcoil) of Cα atoms from their random-coil values for the wild-type (blue) and P56S (red). (B) Chemical shift deviations of Hα atoms for P56S. (C) Characteristic NOEs defining secondary structure identified for the P56S mutant.\n\nInterestingly, as seen in Figure 12A, two characteristic S-shaped loops constrained by P12 and P56 respectively, are present in the MSP domain. Thus we have explored whether Pro12 also plays a similar role to P56 in maintaining the MSP fold. The obtained results showed that indeed the P12S mutant also became unrefoldable and insoluble, but again could be dissolved in unsalted water. CD studies indicate that P12S is highly unstructured at different pH values (Figure 14A). This conclusion is further supported by its HSQC spectra at different pH values. In particular, most HSQC peaks of the P12S mutant can be superimposed with those of the P56S mutant (Figure 14B), strongly suggesting that the P12S and P56S mutants share similar conformational properties over the majority of the molecules.\n\n(A) Far-UV CD spectra of the wild-type MSP (black) and P12S mutant in unsalted water at pH 3.5 (red), 4.5 (brown), 5.5 (green), and 6.5 (blue). (B) Superimposition of HSQC spectra of P56S (blue) and P12S mutants in unsalted water at pH 3.5 (red).\n\nWe have characterized conformations of unrefoldable and insoluble proteins resulting from one-residue insertion/mutation on the well-structured folds such as SH3 and MSP shared by a large number of sequences. The results uncover a surprising fact that such one-residue variations, which also occur naturally, are sufficient to completely eliminate the intrinsic capacity of the wild-type sequences to fold into the native folds, thus rendering the mutants to become 'intrinsically insoluble proteins'. Therefore, in addition to the low complex sequences and truncation of well-folded domains as exemplified by ApLLP and VAPB-3, the mutation and insertion of well-folded domains represents another mechanism underlying the emergence of 'intrinsically insoluble proteins' in genomes. As eukaryotic genomes contain a large number of random mutations, it is thus reasonable to expect that part of these mutations may lead to 'intrinsically insoluble proteins'.\n\nThe results also strongly suggest that there indeed exists a small set of critical residues which make a dominant contribution to the formation/maintenance of the native structures17. Intriguingly, as shown in the SH3 domain, the interactions critical for coordinating the formation of the SH3 fold have an overlap with those responsible for its aggregation. This might represent a general phenomenon which implies that even for proteins adopting well-folded structures, nature may not or may be unable to optimize their sequences to completely avoid aggregation.\n\n\n'Yin' of protein aggregation\n\nSo far, my lab has encountered ~60 unrefoldable and insoluble proteins; and we found that they were all soluble in unsalted water but lacking in well-defined structures. The same results have also been reported by other groups on insoluble proteins including those involved in biomineralization. On the other hand, when I presented speeches on our discovery in conferences, one always-asked question is whether being unstructured for such insoluble proteins solubilized in unsalted water is due to the absence of salt ions. Although previously we have addressed this question by titrating unrefoldable and insoluble proteins solubilized in unsalted water with NaCl, and demonstrated that the addition of NaCl triggered no formation of well-folded structures, NaCl, however, is generally considered to be neutral in the Hofmeister series and therefore it is of fundamental interest to evaluate the effects of other salts.\n\nWe thus extended our previous investigations by titrating the 83-residue cytoplasmic domain of ephrin-B2 with 14 salts whose 8 anions are located in the middle, on the left and right sides of the Hofmeister series96. Previously the entire domain was found to be insoluble and consequently its structure remains unstudied97. However, by a truncation approach, the last 33 residue functional subdomain was found to be soluble even in 50 mM sodium phosphate buffer and consequently its NMR structure was determined to assume a well-packed hairpin followed by largely unstructured C-terminal 11 residues (Figure 15A). Interestingly, the phosphorylation of three Tyr residues on the β-hairpin would disrupt the structure and lead to the binding to the downstream Nck2-SH2 domain for signal transduction98. Therefore, the last 33 residues can serve as an internal reference to report its conformations in buffer and unsalted water.\n\n(A) Three-dimensional structure of the last 33 residues designated as ephrinB-33 that we previously determined by NMR spectroscopy. (B) Far-UV CD spectrum of the entire ephrin-B2 cytoplasmic domain at a protein concentration of 20 µM (pH 4.0) at 25ºC. (C) 1H–15N NMR HSQC spectrum at a protein concentration of 300 µM (pH 4.0). (D) Hα conformational shifts of the entire ephrin-B2 cytoplasmic domain with a 16-residue His-tag (blue bars) and of the isolated last 33 residues ephrinB-33 (red bars). The first 7 residues of the His-tag could not be assigned due to missing side-chain resonances. Lys17 is the starting residue of the entire cytoplasmic domain while Cys67 is the starting residue of ephrinB-33.\n\nThe full-length domain was indeed buffer-insoluble but again could be solubilized in unsalted water. Preliminary characterizations by far-UV CD (Figure 15B) and NMR HSQC spectroscopy (Figure 15C) indicate that the entire domain is largely unstructured without any tight tertiary packing. On the other hand, we have achieved the sequential assignment of the whole domain; and the Hα conformational shifts support the conclusion that the entire domain is largely disordered (Figure 15D). Most strikingly, the last 33 residues of the entire domain in unsalted water have Hα chemical shifts almost identical to the isolated 33-residue subdomain in 50 mM sodium phosphate buffer. This clearly indicates that the last 33 residues adopt almost the same conformation in unsalted and buffer water.\n\nWe subsequently monitored conformational changes by HSQC spectroscopy upon titrating the domain with 14 salts: namely Na2SO4, NaF, NaSCN, Na2HPO4, NaCl, NaBr, NaNO3, NaI, MgCl2, KCl, CaCl2, guanidinium chloride (GdmCl), LiF, and KCl. As exemplified by the HSQC spectra with Na2SO4 located on the left, NaCl in the middle, and NaSCN on right sides of the Hofmeister series (Figure 16), gradual introduction of three salts up to 100 mM results in no significant change of the HSQC spectral dispersions, but only slight shifts of HSQC peaks. The results clearly demonstrate that no significant conformational change occurs upon adding 14 salts up to 100 mM. Therefore, being unstructured for unrefoldable and insoluble proteins solubilized in unsalted water is not a result of the absence of salt ions, but reflects their absence of the intrinsic capacity to form any well-folded structures.\n\nSuperimposition of two-dimensional 1H–15N NMR HSQC spectra of the entire cytoplasmic domain of ephrin-B2 at a protein concentration of 300 μM (pH 4.0) at 25ºC, in the absence and in the presence of Na2SO4, NaCl and NaSCN at varying salt concentrations.\n\nTo our surprise, however, the salt effects on shifting of HSQC peaks are not uniform over the whole protein, as represented by Figure 16. By superimposing the HSQC spectra at different salt concentrations, we mapped out the residue-specific changes of 1H and 15N chemical shifts upon salt titrations, as represented by the results with three salts (Figure 17). Although upon addition of salts, most of the residues with HSQC peaks shifts are located over the N-terminal half of the protein, the patterns of the chemical shift changes are very diverse for salts whose anions are located on the left side of the Hofmeister series, while they are largely uniform for those anions that are on the right side of the series including NaCl and NaSCN. To determine the relative contribution of cations and anions to the HSQC peak shifts induced by salts, we monitored the shifts by titrating chloride salts with different cations, including MgCl2, KCl, CaCl2, and GdmCl. The different chloride salts caused very similar patterns of HSQC peak shifts, suggesting that the observed effects are mostly due to the chloride anion96. These results suggest that the HSQC peak shifts observed here are mostly triggered by the asymmetric binding of different anions to the protein residues.\n\nChemical shift difference (CSD) of the amide proton (1H) and nitrogen (15N) for residues of the entire ephrin-B2 cytoplasmic domain plus His-tag induced by the addition of Na2SO4, NaCl and NaSCN at three different concentrations.\n\nWe then plotted the chemical shift changes induced by ions as a function of salt concentration for the residues that demonstrate significant peak shifts, and very unexpectedly, all curves appear to be saturable (Figure 18). This implies that these anions all have specific binding to the protein. Using the one binding site model, we separately fitted these titrations based on 1H and 15N chemical shifts. To our surprise again, the majority of the apparent Kd values are found to be less than 50 mM, thus indicating that all eight anions bind to the ephrin-B2 cytoplasmic domain with high affinity. Of particular significance, Na2SO4 is found to be the strongest binder to most residues, with an average apparent Kd of only 1 mM96.\n\nThe fitting curves for 1H and 15N chemical shift changes using the one binding site model for significant shifted residues of the entire ephrin-B2 cytoplasmic domain, which are induced by gradually adding Na2SO4, NaCl and NaSCN. The blue and red lines are used to indicate the curves associated with the highest and lowest Kd values, respectively.\n\nOur study on the unstructured ephrin-B2 cytoplasmic domain96 suggests that the protein-anion interaction is mostly modulated by anion type, protein conformation and electrostatic property. Therefore, a high-resolution study of the effects of different salts on a well-folded protein is crucial. We thus investigated interactions of three physiologically relevant salts with the well-folded WW4 domain99. Previously we have determined its NMR structure and binding with a Nogo-A peptide100. By use of the same NMR HSQC titrations, we monitored its binding to three salts (Na2SO4, NaCl and NaSCN) with salt concentrations up to 200 mM (800 protein molar equivalents), under three solution conditions: 1) in unsalted water at pH 6.4; 2) in 20 mM phosphate buffer at pH 6.4; and 3) in water at pH 4.0.\n\nAs seen in Figure 19A, WW4 has a far-UV CD spectrum characteristic of a β-turn rich protein, and no significant difference is found for its spectra in water at pH 6.4 and 4.0, demonstrating that WW4 has very similar structures at two pH values. Furthermore, we determined the exposure degree of the WW4 amide protons to solvent by NMR H/D exchange experiments (Figure 19B–D), as well as calculated its electrostatic potential surfaces at pH 6.4 (Figure 19E) and 4.0 (Figure 19F). Under three solution conditions, we acquired a series of HSQC spectra of WW4 with progressive addition of Na2SO4, NaCl and NaSCN. The results showed that addition of three salts up to 200 mM leads to no dramatic change of HSQC spectral dispersions, suggesting no significant alternation of tertiary packing. Nevertheless, addition of three salts does induce shifts of distinctive sets of HSQC peaks. Specifically, in water at pH 6.4, NaCl induces significant shifts (>0.03 ppm) for only two residues (Arg35 and Asn36) (Figure 20D); Na2SO4 for three (Phe31, Lys32 and Asn36) (Figure 20A) and NaSCN for nine (Trp9, Glu10, Glu16, Gly17, Asp23, Arg27, Lys32, Arg35 and Asn36) (Figure 20G). Markedly, the overall shift patterns induced by each salt are highly similar in unsalted water at pH 6.4 and 4.0.\n\n(A) Far UV CD spectra of the WW4 domain (25 μM) in unsalted water at pH 6.4 (blue) and 4.0 (red). (B)–(C) Experimental (dots) and fitted (lines) values are shown for HSQC peak intensities in NMR H/D exchange experiments for WW4 residues whose peaks are significantly (B) and not significantly (B) perturbed by salts. (D) WW4 structure colored with H/D exchange rates (Kex): blue for residues with HSQC peaks disappeared in 2 min after the lyophilized sample was dissolved in D2O; green for residues with Kex >5 h-1 and red for residues with Kex <5 h-1. (E)–(F) The electrostatic potential of WW4 at pH 6.4 and 4.0 respectively, which is calculated with APBS and visualized at the level of the accessible surface of the protein, with blue and red corresponding to positive and negative potential values respectively.\n\nResidue-specific chemical shift differences (CSD) of amide protons of the WW4 domain triggered by titration of Na2SO4, NaCl and NaSCN. (A)–(C) Na2SO4; (D)–(F) NaCl; and (G)–(I) NaSCN titrations of WW4 in unsalted water at pH 6.4; in 20 mM phosphate buffer at pH 6.4, and in water at pH 4.0 respectively. Residues with significant 1H chemical shift changes (>0.03 ppm) are labeled: red for residues with amide proton H/D exchange rate (Kex) <5 h-1 and blue for residues with significant changes only in the presence of 20 mM sodium phosphate buffer (pH 6.4).\n\nWe fitted all titration tracings with 1H chemical shift differences >0.03 ppm to obtain the apparent dissociation constants (Kd) based the one binding site model (Table 1). Intriguingly, although Na2SO4 perturbs far fewer numbers of residues than NaSCN, it has the strongest binding affinity, with average Kd values of 32.0, 15.7 and 86.3 mM respectively for backbone amide protons under three conditions. NaCl and NaSCN have similar affinity but lower than Na2SO4, with average Kd values of ~100 mM even in water (Table 1). On the other hand, shifts of the HSQC peaks of side-chain amide protons of WW4 could also be monitored upon titrating and subsequently their shift tracings were fitted to obtain apparent Kd values (Table 1). Interestingly, only Na2SO4 appears to extensively interact to side-chain amide protons, in particular at pH 4.0. It is worthwhile to note that the Kd values for binding to the side-chain amide protons are approximately 3- to 4-fold larger than those of the backbone ones at the same condition, indicating that anions interact with backbone and side-chain amide protons separately.\n\na“Buffered” refers to “in 20 mM sodium phosphate (pH 6.4)”.\n\nbKd values are in mM;\n\ncIn calculating the average values, Kd values >250 mM are not included.\n\nStrikingly, the pre-existence of 20 mM sodium phosphate buffer significantly reduces the binding affinity of all three salts, as exemplified by titration curves and Kd values of several representative residues (Figure 21). For Na2SO4, although the presence of the buffer leads to an approximately 3-fold affinity reduction for backbone amide protons, titration curves still show saturation to some degree (Figure 21A and Table 1). However, for NaSCN, the presence of the buffer renders the titration curves to appear to be almost linear which thus could not be fitted with good confidence (Figure 21C and Table 1). For the side chain amide protons, the presence of the buffer leads the titration curves by both Na2SO4, and NaSCN to become completely linear (Figure 21B and 21D).\n\n(A)–(B) Residue-specific apparent dissociation constants (Kd) for backbone amide proton of Phe31 and side-chain amide proton of Asn36 titrated by Na2SO4 under different conditions. (C)–(D) Residue-specific apparent dissociation constants (Kd) for backbone amide proton of Glu16 and side-chain amide proton of Asn25 titrated by NaSCN under different conditions. Experimental (dots) and fitted (lines) values are shown for the 1H chemical shift changes induced by gradual addition of two salts (Na2SO4 and NaSCN). Red is for the data in water (pH 6.4), green for those in water (pH 4.0), and blue for those in 20 mM sodium phosphate buffer (pH 6.4).\n\nStrikingly, the pre-existence of 20 mM sodium phosphate also considerably changes the shift patterns by all three salts. In the buffer, some residues, which are not perturbed either by that salt or sodium phosphate separately (Figure 22A), suddenly appeared to be significantly perturbed (Figure 20). For example, in the phosphate buffer, Na2SO4 is suddenly able to significantly perturb Trp9, Asp23 and Asn25 (Figure 20B), which are not largely perturbed by Na2SO4 alone in water either at pH 6.4 or 4.0 (Figure 20A–C).\n\n(A) Residue-specific chemical shift differences of amide protons (1H) of the WW4 domain upon addition of Na2HPO4 at 150 mM (blue bars) and 200 mM (red circles). (B) Residue-specific apparent dissociation constants (Kd) for Glu16 and Lys32. Experimental (dots) and fitted (lines) values are shown for the 1H chemical shift changes induced by gradual addition of Na2HPO4. (C) Residue-specific chemical shift differences of amide protons (1H) upon addition of Na2SO4 at 200 mM to the WW4 sample in unsalted water at pH 6.4 (red bars) and in unsalted water with the pre-existence of 150 mM NaCl at pH 6.4 (blue bars). (D) Residue-specific apparent dissociation constants (Kd) for Phe31 and Lys32. Experimental (dots) and fitted (lines) values are shown for the 1H chemical shift changes induced by gradual addition of Na2SO4.\n\nTo understand the effect of the phosphate buffer, we titrated the WW4 domain with Na2HPO4 and the result showed that it only weakly binds to WW4, with only two amide protons having significant shifts of HSQC peaks (Figure 22A) and subsequent fitting give rise to their apparent Kd value similar to those by NaSCN (Figure 22B and Table 1). To assess whether the observed change of shift patterns is completely due to the non-specific electrostatic screening imposed by the presence of 20 mM sodium phosphate, we also titrated Na2SO4 to a WW4 sample in the pre-existence of 150 mM NaCl, which is considered to be the physiological concentration in blood and has an ionic strength larger than that of 20 mM sodium phosphate. Very unexpectedly, as shown in Figure 22C, the presence of 150 mM NaCl appears to largely attenuate the shift amplitudes but lead to no significant change in shift patterns. Noticeably, the presence of 150 mM NaCl does lead to ~3-fold reduction of the binding affinity of Na2SO4, to Phe31 and Lys32 (Figure 22D).\n\nNa2SO4, NaCl and Na2HPO4 appear to bind only a subset of well-exposed amide protons which are located on loops/turns or a short 310-helix over Pro34-Arg35-Asn36 (Figure 19D). By a sharp contrast, NaSCN is even able to bind well-protected amide protons such as from Trp9, Glu10, Val22 and Asp23, which are on two central β-strands. In particular, Glu10 is one of two residues with the most protected amide protons (another is Ile11) with a Kex of 0.97 h-1. Further analysis of electrostatic potential surfaces reveals a surprising picture: the amide protons interacting with Na2SO4, NaCl and Na2HPO4 are almost all located on the electrostatically positive regions while NaSCN is also able to bind to the amide protons of Trp9, Glu10, Val22 and Asp23 located on electrostatically negative regions (Figure 19E-F). This implies that Na2SO4 binding is highly electrostatically driven while NaSCN is not. Indeed, as shown in Table 1, the binding affinities of Na2SO4 at pH 4.0 have a ~2-fold increase as compared to those at pH 6.4 while no significant difference is found for NaSCN at two pH values. Remarkably, the presence of 20 mM sodium phosphate renders Na2SO4 to behave like NaSCN, capable of significantly interacting Trp9 and Asp23 on two core β-strands with well protected amide protons (Figure 19E and Figure 20B), while those two residues are not significantly perturbed by sodium phosphate alone even at a concentration up to 200 mM (Figure 22A).\n\nBy use of the last 33-residue subdomain as a reporter, we again demonstrate that protein conformations have no fundamental difference in unsalted water and buffer. Remarkably, our systematic study with 14 salts with 8 anions located differently in the Hofmeister series clearly indicates that the absence of the unique folded tertiary structures in unrefoldable and insoluble proteins solubilized in unsalted water is the manifestation of their intrinsic properties, not due to the absence of salt ions.\n\nSurprisingly, we reveal that if we remove the interference of the pre-existing ions, 8 anions are all able to bind distinctive sets of ephrin-B2 residues with very high affinity. This selective and high-affinity binding by anions was further confirmed on a well-folded WW domain, despite having a ~10-fold reduction of the affinity. Markedly, sulfate anion is the tightest binder to both unstructured and well-folded proteins. The asymmetric anion binding appears to be modulated by three major factors: anion type, protein conformation and surface electrostatic potential. Highly hydrated sulfate, phosphate and chloride anions appear to only bind well-exposed amide protons, mostly driven by electrostatic interactions. The highest charge-density of sulfate anion appears to be the key factor responsible for the tightest interactions with amide protons. By contrast, thiocyanate is able to bind the largest set of amide protons including some well-protected amide protons located on electrostatically negative patches. This observation implies a fundamental difference between thiocyanate and other three anions. Indeed, thiocyanate was characterized to be weakly solvated with low charge density and consequently it seems that van der Waals interactions play a key role.\n\nThe scenario we uncovered is completely inconsistent with the current belief that protein-ion interactions are predominantly non-specific, electrostatic interactions at physiologically relevant ion concentrations (<100 mM)101. It seems that the previous failure to observe selective anion binding to proteins with high affinity is most likely due to the pre-existence of buffer ions. Indeed, we have shown that the pre-existence of 20 mM sodium phosphate exactly as previously used101 not only reduces the binding affinities of the anion-binding, but also alters the binding patterns, which is not observed in the pre-existence of 150 mM NaCl. Now we have obtained NMR relaxation evidence that different salts have very diverse effects on protein dynamics, particularly on µs-ms time scale and this effect is dependent of both cations and anions.\n\nOur results also reveal that a well-folded protein is significantly shielded from anion binding. This result also implies that salt ions also play a key role in provoking misfolding for some protein mutants such as the T46I-MSP domain, which still possesses the capacity to fold into the native MSP structure, and only has cooperative unfolding lost and protein dynamics increased102. The mutation-causing increase of protein dynamics may allow salt ions to more easily access the core region of the T46I mutant, which results in tighter anion-binding, and/or further changes of protein dynamics. All alterations together result in an enhanced misfolding of the mutant in the macromolecular crowded cells102,103.\n\n\nTransformation from non-membrane to membrane proteins, and emergence of primitive membranes embedded with integral membrane proteins\n\nThe basic building block of all organisms is the cell, which is generally regarded as a space with a watery interior separated from the external environment by two layers of phospholipid molecules called the plasma membrane. Eukaryotic cells also have extensive internal membranes that further subdivide the cellular space into various compartments. However, the middle of membranes is constituted by fatty acid hydrocarbon tails, which are highly hydrophobic. Therefore, cellular membrane systems appear not only to build up a barrier for preventing the free flow of molecules in and out of cells, as well as among different cellular compartments, but also provide a hydrophobic phase, in particular in eukaryotic cells. As such, two phases with opposite polarity properties already coexist in cells, one is watery polar while another is membrane-based hydrophobic phases. The membrane-based hydrophobic phase may play many unrecognized but extremely important roles as the transition from prokaryotic to eukaryotic cells is associated with a dramatic emergence of large internal membrane systems.\n\nAs our studies on various insoluble proteins suggest that protein insolubility/aggregation mostly results from the unfavorable interaction between polar water molecules and hydrophobic side chains26, it is thus tempting to speculate that insoluble proteins may have a stronger capacity to interact with membranes containing a hydrophobic phase in the middle. With our discovery as a powerful tool, we have tested this possibility by solubilizing insoluble proteins in unsalted water followed by gradual adding the lipid mimetic dodecylphosphocholine (DPC). The results show that the insoluble proteins tested are indeed all able to interact with lipids to different degrees.\n\nIn particular, we found that the ALS-causing P56S-MSP mutant and VAPB-3 are able to transform into well-formed helical structures with most residues buried in a membrane environment84. The wild-type VAPB-MSP adopts a well-folded β-sandwich structure which is highly soluble in buffer (Figure 12A) and shows no detectable interaction with DPC84. By contrast, both P56S mutant (Figure 23A) and VAPB-3 variant (Figure 23B) are completely buffer insoluble, and are predominantly unstructured in unsalted water. However, both of them are able to interact with DPC and gradually transform into a highly helical structure (Figure 23). As monitored by HSQC spectroscopy, the transformation is involved in almost all residues of P56S and VAPB-3. Once fully formed, the helical conformations account for 68% for both P56S and VAPB-3 and most residues appear to be buried in the DPC micelle. This suggests that for P56S-MSP and VAPB-3, it is thermodynamically more favorable to insert into membranes to form high helical state than to stay in unsalted water as a predominant disordered state.\n\n(A)–(B) Far-UV CD spectra of P56S-MSP (A) and VAPB-3 (B) in the absence of (blue) and in the presence of DPC at different ratios (protein:DPC): 1:6 (cyan), 1:10 (bright green), 1:20 (purple), 1:50 (pink), 1:100 (brown), 1:150 (green) and 1:200 (red). (C) Ellipticity values at 222 nm of P56S-MSP (red) and VAPB-3 (blue) vs. the ratios (protein:DPC). (D)–(E) 1H–15N NMR HSQC spectra of P56S-MSP (D) and VAPB-3 (E) in the absence of (blue) and presence of DPC at two ratios (red) as indicated (P56S:DPC). (E) Cartoon model of the wild-type VAPB protein anchored onto ER membrane, with the well-folded MSP domain in the cytosol. (F) Cartoon model proposed here to rationalize a recent report that the P56S VAPB protein triggers the formation of a novel form of organized smooth endoplasmic reticulum with stacked cisternae. Briefly, the P56S-MSP domain becomes insoluble in the cytosol and thus will have a strong preference to insert into ER cisternae. If the C-terminal transmembrane fragment and P56S-MSP insert into different ER cisternae, a novel form of organized smooth ER will form with stacked cisternae.\n\nOur results provide a mechanism to rationalize a recent report that the P56S mutant is able to trigger the formation of a novel form of organized smooth endoplasmic reticulum (ER) with stacked cisternae104,105, despite failing to detect significant formation of aggregated inclusions in motor neurons derived from induced pluripotent stem cells of patients carrying the P56S mutation106. In light of our discovery, the formation of ER with stacked cisternae can be nicely explained. Briefly, as shown in Figure 23E, the wild-type VAPB protein is anchored onto the ER by the C-terminal transmembrane fragment, and its well-folded MSP domain is in the cytosol. However, on the advent of the P56S mutation, the mutated MSP domain becomes completely insoluble in the cytosol and will spontaneously insert into ER membranes: some into the same cisternae, some into the different cisternae (Figure 23F). The insertion into different cisternae will lead to the formation of an ER with stacked cisternae, which has been observed in cells104,105. Furthermore, if the P56S mutant protein is over-expressed in cells, the membrane systems may not be able to accommodate all of it, and so it gets accumulated as visible inclusions/aggregates.\n\nOur results thus demonstrate that many insoluble proteins are potentially able to interact with membranes, and non-membrane proteins can easily transform into membrane-interacting proteins by slight mutations/truncations/modifications. However such membrane-interacting proteins cannot be detected by current bioinformatics tools and consequently the number of membrane-interacting proteins may be much larger in cells than currently recognized. Also attacking membrane systems before the accumulation of visible aggregates/inclusion may represent a general mechanism by which insoluble proteins trigger human diseases such as neurodegenerative diseases. Our results also imply a potential mechanism to connect sporadic and familiar human diseases. For example, the fact that VAPB-3 shares similar properties with P56S-MSP as regards membrane interactions, means that even without carrying any ALS-causative mutation, a person might develop ALS through a mechanism underlying ALS8, if VAPB slicing variants like VAPB-3 get accumulated in cells to a certain degree, due to proteasomal inhibition triggered by pathological and/or environmental conditions. Indeed, the VAPB-3 protein was found to become detectable in cells only upon proteasomal inhibition, a condition commonly found in all neurodegenerative diseases85.\n\nIn a TIBS article107, Mulkidjanian and colleagues found it enigmatic how the integral membrane proteins could reach the primeval membranes, since these proteins, having extremely high hydrophobicity, are water-insoluble and consequently \"even if occasionally synthesized, would remain stuck in the ribosome\"107. As a consequence, emergence of protein-embedded primeval membranes is called a 'chicken and egg paradox'. However, in light of our discovery, this paradox can be solved as some evidence suggests that proteins and primitive membranes with integral membrane proteins might emerge in unsalted oceans with a slightly acidic pH107,108. This prebiotic condition is amazingly very similar to what my lab commonly used to solubilize insoluble/membrane proteins. Consequently, as supported from our results, even the most hydrophobic integral membrane peptide would not be stuck in the ribosome in unsalted oceans. Instead, they would be certainly soluble in such a prebiotic unsalted medium. In particular, the concentrations for most, if not all, proteins might also be very diluted in the primitive oceans and consequently they would be able to freely diffuse around to reach the primeval membranes to achieve spontaneous assembly, as shown for P56S-MSP and VAPB-3.\n\nOur discovery might also shed light on another mysterious issue associated with the diversification of proteins. It was proposed that the space of realized protein folds appears to just account for one-tenth of the space of possible folds109. This implies that a large portion of the sequence space remains unexplored in the life forms on Earth. Here I hypothesize that this might be associated with ocean salinity. The machinery generating proteins is believed to have existed before the emergence of the membrane-enveloped primitive cells109,110. In unsalted oceans and without membranes, proteins created with their sequences highly randomized were all soluble and thus could diffuse freely to encounter other proteins, other biomacromolecules, organic and inorganic small molecules for self-assembly into various complexes/machineries. The slight increase in salt concentrations in oceans might facilitate the assembly of some protein-based complexes or machineries. Indeed, we frequently observed that in unsalted water, protein-protein interactions are dramatically suppressed due to the repulsive electrostatic interactions between individual protein molecules. The presence of salt ions is expected to reduce repulsive electrostatic interactions by a non-specific screening effect and/or specific anion-binding, thus allowing protein-protein, and other, interactions.\n\nOn the other hand, once the membrane-contained cells were formed and the oceans became highly salted, proteins with high hydrophobicity would become aggregated and/or start to attack membranes. This might trigger the emergence of mechanisms to halt the high randomized sampling of the sequence spaces. In this regard, all modern proteins regardless of being well-folded; intrinsical unstructured and membrane-associated might be all diverged from the constrained numbers of the primordial proteins which were randomly created in unsalted oceans. This scenario perfectly rationalizes my previous proposal that \"[modern] proteins appear so designed that in pure water their intrinsic repulsive interactions are sufficient to suppress the attractive forces, thus preventing them from severe precipitation/aggregation\"26,68. It might also be possible that the current 20 natural α-amino acids in the L-image were selected because their polymerized products, proteins, were all soluble in the primitive unsalted oceans.\n\n\nConclusion remarks and further directions\n\nIn the past several years, our discovery has provided a powerful tool for us and other groups to characterize a variety of unrefoldable and insoluble proteins. So far, my lab has encountered ~60 such insoluble proteins but found that all of them could be solubilized in unsalted water for high-resolution biophysical studies, some of which have been published22,67–69,84,91,93,96,111–113. Most strikingly, we demonstrated that the 25-residue M2 transmembrane peptide of influenza A proton channel, one of the most hydrophobic sequences in nature, could also be solubilized in unsalted water without lipid molecules to form a highly helical conformation. This implies that only in unsalted water are all proteins are able to manifest their intrinsic conformations, regardless of their hydrophobicity and whether they are well-folded, partial folded or predominantly unstructured (Figure 24). So why are all proteins soluble in unsalted water? One scenario that rationalizes this phenomenon is that the prebiotic aqueous medium where proteins originally emerged was largely unsalted. The current 20 α-amino acids in the L-image conformation were selected because their products, proteins, are all soluble in such a medium. Indeed, there exists evidence indicating that when primitive proteins were made, the ocean was highly unsalted and slightly acidic107,108. Moreover, it has been shown that the presence of ions even at concentrations much lower than those of contemporary oceans would impose adverse effects on membrane self-assembly and RNA polymerization, and consequently the prebiotic medium could not have been salted114.\n\nIn the unsalted water, which may mimic the prebiotic medium where proteins originally emerged, all proteins, regardless of their hydrophobicity, being well-folded or unstructured, are all soluble and being able to diffuse freely. However, upon becoming salted, only well-folded proteins and a portion of intrinsically unstructured proteins (IUPs) remain soluble in salted aqueous solution. The salted conditions may facilitate the formation of various protein-based complexes such as among well-folded proteins, between well-folded protein and unstructured peptides/proteins; and among unstructured peptides/proteins. On the other hand, upon encountering lipid molecules, some proteins such as those with high hydrophobicity may transform into membrane proteins. Nevertheless, there might be a portion of proteins designated as 'intrinsically insoluble proteins (IIPs)', which completely lack the intrinsic capacity to fold into any well-defined structures and also have their hydrophobic patches improperly exposed to aqueous bulky solution. For certain reasons, these proteins fail to reach and/or cannot be completely accommodated by biological membrane systems. Aggregation is consequently their evitable destination in vivo with ~150 mM ion concentration. As the aggregation of IIPs cannot be recovered by cellular chaperone systems, to minimize their harmful effects eukaryotic cells have developed many complex degradation machineries to degrade IIPs and 'misfolded proteins', which include ubiquitin–proteasome pathway (UPP) and autophagosome–lysosome pathway (ALP).\n\nAmazingly, only this scenario can provide a solution to the 'chicken and egg paradox' for the origin of the primeval membranes embedded with integral membrane proteins. As the most hydrophobic integral membrane peptide is soluble in unsalted water, all primitive membrane proteins regardless of their hydrophobicity are anticipated to be soluble in unsalted prebiotic oceans and to diffuse around freely. Upon encountering primitive membranes, these proteins would insert into the hydrophobic phase of the membranes and transform into membrane proteins (Figure 24). This transformation process appears to be thermodynamically favorable as implied from our result that the unstructured VAPB-3 and P56S-MSP domain can spontaneously insert into membranes in unsalted water and transform into high helical structures. The transformation into membrane proteins might also serve to protect proteins with high hydrophobicity from becoming aggregated upon the later increase in salt concentrations. Consequently protein aggregation might not be a severe problem in primitive cells even with high salt concentrations. It appears that even in prokaryotic cells, under normal physiological conditions, protein aggregation is mainly involved in 'off-pathway' misfolding of proteins which can assume well-folded structures and are soluble in the salted cytosol, rather than 'intrinsically insoluble proteins'. Indeed, the presence of molecular chaperone systems plus minor degradation activity appears to be sufficient to minimize the accumulation of aggregated proteins. Consequently, complex protein degradation machineries have been underdeveloped in prokaryotic cells.\n\nIn contrast, eukaryotic cells appear to face a serious problem of protein aggregation even at unstressed conditions. Very surprisingly, based on results with human cell lines, it has been estimated that ~30% of newly synthesized proteins get aggregated and rapidly degraded by proteasomes58,115,116. These proteins were called as defective ribosomal products (DRiPs), \"that never attain native structure owing to errors in translation or post-translational processes necessary for proper protein folding\"58, but their exact nature remains to be defined. Here I propose that the 'intrinsically insoluble proteins (IIPs)' we establish here may account for a large portion of DRiPs. As implied from the results with 'intrinsically insoluble proteins' in vitro, the aggregation might be also an inevitable destination for them in vivo due to their lack of the intrinsic capacity to fold into unique tertiary structures and high ion concentrations in vivo. In particular, unlike 'misfolded proteins', the aggregation of 'intrinsically insoluble proteins' cannot be overcome by chaperone systems. Therefore, despite being an immense waste, the only option for cells to minimize their harmful effects is to remove them by degradation immediately after their synthesis58,115,116.\n\nThe emergence of a large amount of 'intrinsically insoluble proteins' in eukaryotic cells appears to be associated with at least three well-known characteristics of eukaryotic genomes: 1) increase of intrinsically unstructured proteins with low-complexity sequences; 2) emergence of the splicing variation; and 3) accumulation of random mutations. The underlying mechanisms for generating 'intrinsically insoluble proteins' by these three processes can be exemplified by our results with three naturally occurring insoluble proteins: ApLLP, a full-length wild-type protein with a low complexity sequence; VAPB-3, a splicing variant with truncation on the well-folded MSP domain; and P56S-MSP, a ALS-causing mutant with only a single-residue mutation on the MSP domain. On the other hand, as we frequently observed, the interactions responsible for protein aggregation are also important for the folding and interactions. Therefore, nature may or may not be able to optimize protein sequences to completely avoid aggregation. As a result, eukaryotic genomes are abundant in 'intrinsically insoluble proteins' and, to cope with them, eukaryotic cells have developed many complex machineries to remove them, which include the ubiquitin–proteasome pathway (UPP) and the autophagosome–lysosome pathway (ALP) (Figure 24). Indeed, proteins involved in proteasomes have been found to account for 1% of total cellular protein58. Degradation of various aggregated proteins might represent a central mission for eukaryotic cells to survive and the failure of the degradation systems might have catastrophic consequences for the organisms, which include various aggregation-causing diseases and aging.\n\nWonderfully, a cell is already composed of two opposite, but complementary phases in terms of polarity. While cellular compartments contain the polar watery phase, cellular membrane systems provide the hydrophobic hydrocarbon phase. As we have shown, insoluble proteins have a preference to interact with membranes to different degrees. This implies that insoluble proteins may have potential to transform into membrane-interacting proteins. Indeed, as we recently uncovered, upon encountering lipids the insoluble VAPB-3 and P56S-MSP mutant transform into integral membrane proteins with the majority of residues buried in the membrane environment. Therefore, it is tempting to hypothesize that biological membranes emerge in evolution not only to separate the interior space of a cell from environments, and to achieve compartmentalization within eukaryotic cells, but also serve to provide a hydrophobic phase to host proteins with high hydrophobicity. It is possible that the emergence of large internal membrane systems in eukaryotic cells might also represent part of cellular response to the dramatic increase of 'intrinsically insoluble proteins' in eukaryotic genomes. In other words, the emergence of internal membrane systems may help accommodate many insoluble proteins by facilitating their transformation into membrane proteins, and, mutually, these newly formed membrane proteins might also contribute to the formation and maintenance of novel structures of the internal membrane systems. Indeed, it has been extensively found that over-expression of membrane proteins would trigger expansion and structural changes of eukaryotic internal membranes and, in particular, it even resulted in the formation of internal systems in E. coli cells117–121. Moreover, ER membrane structures and dynamics have been demonstrated to be critically regulated by the presence of various membrane-interacting proteins104,105,118–121.\n\nTheoretically, the existence of both watery and membrane phases in cells would be sufficient to accommodate proteins with various hydrophobicity. Nevertheless, in modern cells with ~150 mM ion concentration, without the specific assistance by complex machineries such as translocon31, most 'intrinsically insoluble proteins' would be stuck in ribosomes before they are able to reach membranes. Consequently they will be either degraded immediately after they are synthesized, or accumulated upon over-expression and/or inhibition of the degradation machineries triggered by pathological conditions. The presence of ~150 mM ions may also represent a cellular mechanism to prevent the insoluble proteins to access membranes. Nevertheless, a portion of such proteins, in particular those that are abnormally over-expressed, might still be able to reach membranes. Therefore, to attack biological membranes may represent a general mechanism by which over-expression/accumulation of insoluble proteins initiates various aggregation-causing diseases.\n\nAll results together by us and other groups reveal a fact that unrefoldable and insoluble proteins, including the most hydrophobic integral membrane peptide, are all soluble in unsalted water, but become aggregated upon being exposed to salt ions. This logically suggests that salt ions play a role at least as important as proteins in mediating protein aggregation. As salt ions exist in the background and such key roles have been largely unrecognized, I thus designate them as 'dark mediators' as analogous to 'dark matter'92. The factors from proteins and salt ions are equally important in modulating protein aggregation, which can be nicely symbolized by the Taji diagram (Figure 6).\n\nThe most frequently asked question, when I presented speeches on our discovery in conferences, is whether the lack of the well-defined structures observed on unrefoldable and insoluble proteins solubilized in unsalted water is due to the absence of salt ions. To systematically address this question, we have selected the insoluble cytoplasmic domain of ephrin-B2 as a model system and titrated it with 14 salts with 8 anions located in the middle, on the left and right sides of Hofmeister series. This set of results, together with those we previously obtained by titrating unrefoldable and insoluble proteins with NaCl, clearly demonstrate that the solution conformations have no fundamental difference in unsalted and salted water. Therefore, the lack of tight tertiary packing has been confirmed to represent an intrinsic feature associated with unrefoldable and insoluble proteins, as I have previously proposed26,68.\n\nUnexpectedly, however, our studies unveil that in contrast to the common belief, anions are able to asymmetrically bind both unstructured and well folded proteins with high selectivity and affinity at physiological relevant concentration (<200 mM). The anion binding has been characterized to be mediated by anion type, protein conformation and surface electrostatic potential. Remarkably, the well-folded protein is significantly shielded from anion binding in terms of the number of binding sites and affinity. Surprisingly, the selective anion binding with high-affinity can be dramatically masked by the pre-existence of salt ions. Intriguingly, the pre-existence of 20 mM sodium phosphate not only masks the selective anion binding but also alters the binding patterns. Now we have obtained NMR relaxation data revealing that this is mostly due to very diverse effects by different salts on protein dynamics, particularly on µs-ms time scale.\n\nIn 2008, I proposed a model to rationalize why unrefoldable and insoluble proteins can be soluble in unsalted water, but become aggregated upon introduction of salt ions even at very low concentration. However, in the previous model only the non-specific electrostatic screening effect is considered26. Briefly, due to the lacking of the tight tertiary packing, these proteins have a substantial amount of hydrophobic side chains exposed to the bulk water. If a protein of this family is dissolved in unsalted water with pH deviated from its pI, the individual molecules will bear a significant amount of net charges (Figure 25A). In this regard, the repulsive electrostatic interaction and/or large protein hydration shell will constitute an energy barrier unfavorable for inter-molecular interactions. Consequently, in unsalted water, protein aggregation is significantly suppressed. However, even if a small amount of salt ions is introduced, the repulsive electrostatic interaction will be screen out and/or the protein hydration shell may be disrupted to some extent. As a sum, the hydrophobic interaction will become dominant, thus leading to immediate aggregation (Figure 25C).\n\n(A) An unrefoldable and insoluble protein in unsalted water with the solution pH several unites away from its pI. Small cyan spheres stand for water molecules and green ellipsoids for protein molecules with a large amount of hydrophobic side chains exposed. (B) Protein molecules in the presence of a small amount of salt ions (larger red spheres). In addition to imposing non-specific electrostatic screening as I previously proposed, the presence of salts also provides specific anion binding to protein residues, thus altering the surface electrostatic potential. Furthermore, salts may also changes water clustering structure as well as protein dynamics as represented by the broken lines of green ellipsoids. (C) The complex interplay of these salt effects may result in aggregation of the protein.\n\nHere, based on our new results, I modify the model to include both specific anion binding and salts’ effect on protein dynamics (Figure 25B). The presence of the two specific effects appears to suddenly complicate the relationship between ion type/concentration and protein solubility. Upon introducing salt ions, the reduction of repulsive electrostatic interactions by non-specific screening may still represent a major consequence. On the other hand, the specific anion binding can either result in neutralizing the net charges, or leads to the introduction of extra charges. For example, a protein with a neutral pI will have a positive net charge in acidic solutions. The binding by mono-valent anions such as chloride only to the positively charged regions of the protein is expected to neutralize the positive charges, thus leading to further reduction of repulsive electrostatic interactions. Nevertheless, the sign of the net charge may be reversed if this protein has a large number of positively charged patches which are tightly bound by multi-valent anions such as sulfate; or both hydrophobic and hydrophilic patches of this protein are extensively bound by anions such as thiocyanate. For such special situations, the relationship between ion concentration and protein solubility could be extremely complex. Here it is tempting to hypothesize that in human fluids, chloride has been selected as the dominant anion in evolution, at least partly due to its minimal ability in binding to proteins and in altering protein dynamics.\n\nElectrostatic interactions have been recently proposed to play key roles in modulating protein structures, interactions and assemblies in cellular crowding environments122. The specific but extremely complex effects we found for salt ions also on well-folded proteins imply that in addition to their central roles in mediating protein aggregation, salt ions may also act to modulate various biological functions of proteins. The variation of salt types and concentrations in cells may have significant impacts on proteins, including aggregation, which are ultimately associated with human diseases. Therefore, it would be critical to decipher their molecular mechanisms by experimental and computational approaches for establishing therapeutic strategies. On the other hand, the existence of specific anion-binding also implies a marked challenge to simulate protein-protein, protein-ligand interactions due to the current difficulty in computationally assessing specific interactions between proteins and anions.\n\nOver the past few years, our discovery has allowed extensive characterizations by us and other groups on unrefoldable and insoluble proteins, which not has elucidated previously unknown regimes associated with proteins; but has also provided critical insights into aggregation-causing diseases. On the other hand, fundamental questions have arisen which can be grouped into the following four categories.\n\nFirst of all, protein aggregation in watery media is most likely to result from the unfavorable interactions between polar water molecules and hydrophobic side chains of proteins. However, the relationship between hydrophobicity of a protein sequence and its solubility seems extremely complex. This is even true for the intrinsically unstructured ApLLP. ApLLP has a low complexity sequence and low hydrophobicity (~30%). However, it is completely insoluble in buffer. More intriguingly, removal of the N- or C-terminal fragment to give a slightly lower hydrophobicity could result in buffer soluble forms22. Also, while the P56S mutant and splicing variant of the MSP domain are completely buffer-insoluble and predominantly disordered in unsalted water, another splicing variant VAPC is buffer-soluble with a disordered conformation in buffer similar to those of P56S-MSP and VAPB-3 in unsalted water80. Therefore, it is of fundamental interest to delineate whether this complexity is just due to the context-dependent nature for protein aggregation, or as recently proposed, is due to its non-Euclidian landscape. If so, the prediction of protein aggregation from sequences is not just technically challenging, but is non-deterministic polynomial time (NP) complete87.\n\nSecondly, previously it has been well-established that many well-folded proteins can misfold into aggregated/amyloid forms to trigger human diseases, due to their dynamic nature and co-existence of many partial folded intermediates induced by slight changes of solution conditions45,46,102,123–125. Here, we establish the existence of 'intrinsically insoluble proteins (IIPs)', some of which also cause neurodegenerative diseases such as ALS. The concept of 'IIPs' is in a nice agreement with the recent observation that ~30% of cellular proteins are DRiPs. Therefore, in the future, it is of particular essence to experimentally characterize the conformations of DRiPs by solubilizing them in unsalted water. This study will offer an estimation of the percentages of 'misfolded proteins' and 'intrinsically insoluble proteins' in DRiPs. This knowledge is crucial for future development of therapeutic strategies for treating aggregation-causing human diseases. To treat misfolding-triggered diseases, the effort may be devoted to preventing 'off-pathway' misfolding and/or to recover misfolded proteins, such as by enhancing the chaperone function. By contrast, to deal with 'IIPs'-causing diseases, the major focus might be to warrant the normal function of degradation machineries.\n\nThirdly, now it has been increasingly recognized that the chameleon transformation of protein conformations is not that uncommon. For example, the β-barrel structure of the hNck2 SH3 domain could transform into a similar helical conformation triggered by either acid-induced unfolding or mutation to disrupt tertiary interactions93,95,126. However, it is still surprising to discover the transformation of the unstructured P56S-MSP domain in unsalted water into a well-folded helical structure in a membrane environment, as its hydrophobicity is much lower than that expected for a membrane protein. This implies that becoming a membrane is not highly dependent on the sequence hydrophobicity. In this regard, the P56S-MSP domain represents a unique model for further deciphering the general principles which direct folding, stability and evolution of membrane proteins.\n\nFinally, our in vitro studies and the in vivo identification of DRiPs accounting for 30% cellular proteins58 implies that protein sequences have not been successfully optimized to avoid aggregations. Therefore, a fundamental question arises whether this failure is due to the overlap of interactions responsible for aggregation with those requested for folding and interaction, or whether in nature there might be no so-called optimization restrained by 'functions'. Living systems might only be a natural manifestation of principles of self-organization in terms of structures and dynamics of all biomolecules and surrounding components. 'Function' is nothing but just an interpretation of these structures and dynamics in the context of relevant interaction networks.", "appendix": "Competing interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nOur studies reviewed here are supported by Ministry of Education of Singapore (MOE) Tier 2 Grant R-154-000-388-112, MOE 2011-T2-1-096 and National Medical Research Council (NMRC) grant R154-000-454-213 to Jianxing Song.\n\n\nAcknowledgement\n\nI would like to thank all the lab members for their critical contribution.\n\n\nReferences\n\nSchrödinger E: What is Life? Cambridge University Press, 1944. Reference Source\n\nLovelock J: GAIA – A New Look at Life on Earth; Oxford University Press, 1979. Reference Source\n\nPrigogine I, Nicolis G, Babloyants A: Thermodynamics of Evolution. Phys Today. 1972; 25(11): 23–28. Publisher Full Text\n\nAnnila A, Annila E: Why did life emerge? Int J Astrobiol. 2008; 7(3–4): 293–300. 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Biophys Chem. 2013; 175–176: 39–46PubMed Abstract \| Publisher Full Text" }	[ { "id": "869", "date": "27 Mar 2013", "name": "H Jane Dyson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 lengthy review on the contributions of dissolved ions to the behavior (aggregation, membrane interaction) of proteins. It is rather long and rambling (and seems somewhat repetitive) and may be construed to some extent as “propaganda” for the author’s opinion in this case. There also does seem to be quite a lot of primary data, ie, data appearing in this review that have not received peer review in other journals. However, on balance, the review is interesting, and should spark dialogue if not controversy in the scientific community.", "responses": [] }, { "id": "919", "date": "02 May 2013", "name": "Monika Fuxreiter", "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 abstract is fine but I think the title is somehow misleading. The review does not give an answer as to why proteins aggregate and how pathological aggregations can be reversed or prohibited. The title should be more specific and scientific (avoid ‘dark mediators’). Article content: The self-assembly of proteins is a very important problem not only from a folding point of view, but also from evolutionary or pathological aspects. The proposal that pure water can prohibit or reverse aggregation is interesting. I do not feel the presented results are convincing enough to provide a deeper insight into the mechanism of the aggregation phenomenon. Conclusions are made based on a few examples and miss robust statistical tests on larger databases. For example, low-complexity sequences that are unstructured and insoluble, or proteins with high-complexity sequences that are unstructured and insoluble. Protein disorder is associated with a particular composition of amino acids, and has many other characteristics that are easy to detect. Intrinsically disordered (ID) proteins however are soluble, in many cases ID segments help to solubilize aggregation-prone regions. The relationship between ID and aggregation is controversial. Aggregation-prone regions also have a particular composition (for example see R. Pomès et al., 2006 for differences between elastic and aggregating sequences). This review does not reflect the complexity of the theme and often runs into simplified conclusions in this respect.I would like to see the mechanism for how ions mediate aggregation and what the impact of their removal is. The relationship between ions and protein structure has been analyzed by various authors, and these works would be worth a mention (e.g. B. M. Pettitt et al., PMID: 19548651, PMID: 20151732, PMID: 20306490)The author argues for ‘specific binding’ of ions. It is obvious from various other works that ions bind at particular locations, but specificity has not been demonstrated clearly in this paper. Would ions make a difference between a WT protein and a single mutant?I think that the conclusions go beyond the scope and validity of the results, in particular ‘birth, transformation and death of proteins’. This process is so complex and presented in an over-simplified manner here. How ID, which is identified here as one of the main factors, is related to degradation for example? How can pure water reverse the effect of aggregation promoting ions? Just removing the ions does not guarantee that the protein will find its correct folding pathway. What about the hydrophobic patches in general? Will they still dislike water and try to avoid?I would strongly argue with the expression ‘dark mediators’. Ions are not dark. By neutron scattering and some modern SAXS data we can have detailed structural information on them.What would be the real practical application of the work?I feel the evolutionary arguments are rather speculative. There are some studies on ID evolution, maybe a good point to start with.Overall, I feel the review – although many experimental data are presented – to be speculative and conclusions are not supported with robust analysis. The proposal is interesting but should be corroborated more. I also miss a general mechanistic picture, with thermodynamic and structural arguments given.", "responses": [] }, { "id": "1022", "date": "24 Jun 2013", "name": "Jonathan W Yewdell", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/2-94
https://f1000research.com/articles/2-32/v1	06 Feb 13	{ "type": "Short Research Article", "title": "Mutation detection in cholestatic patients using microarray resequencing of ATP8B1 and ABCB11", "authors": [ "Kirsten E McKay", "Christopher K Bruce", "Jane L Hartley", "A S Knisely", "Ulrich Baumann", "Sonja-Stephanie Bockisch", "Ekkehard Sturm", "Christian J Hendriksz", "Deidre A Kelly", "Fiona Macdonald", "Paul Gissen", "Christopher K Bruce", "Jane L Hartley", "A S Knisely", "Ulrich Baumann", "Sonja-Stephanie Bockisch", "Ekkehard Sturm", "Christian J Hendriksz", "Deidre A Kelly", "Fiona Macdonald", "Paul Gissen" ], "abstract": "Background: Neonatal cholestasis is a common presentation of childhood liver diseases and can be a feature of various conditions including disorders of bile acid biogenesis and transport, various inborn errors of metabolism and perinatal infections. Some inherited metabolic diseases can be easily screened using biochemical assays, however many can only be accurately diagnosed by DNA sequencing. Fluorescent capillary Sanger sequencing (FS) is the gold standard method used by clinical laboratories for genetic diagnosis of many inherited conditions; however, it does have limitations. Recently microarray resequencing (MR) has been introduced into research and clinical practice as an alternative method for genetic diagnosis of heterogeneous conditions. In this report we compared the accuracy of mutation detection for MR with FS in a group of patients with ‘low-normal’ gamma glutamyl transpeptidase (gGT) cholestasis without known molecular diagnoses.Methods: 29 patient DNA samples were tested for mutations in the ATP8B1 and ABCB11 genes using both FS and MR. Other known causes of “low gGT cholestasis” such as ARC syndrome and bile acid biosynthesis disorders were excluded.Results: Mutations were identified in 13/29 samples. In 3/29 samples FS and MR gave discordant results: MR had a false positive rate of 3.4% and a false negative rate of 7%.Conclusions: The major advantage of MR over FS is that multiple genes can be screened in one experiment, allowing rapid and cost-effective diagnoses. However, we have demonstrated that MR technology is limited in sensitivity. We therefore recommend that MR be used as an initial evaluation, with FS deployed when genetic and clinical or histopathological findings are discordant.", "keywords": [ "Microarray resequencing", "Neonatal cholestasis", "ATP8B1", "ABCB11" ], "content": "Introduction\n\nNeonatal cholestasis is characterised by persistent hyperbilirubinaemia and has an incidence of around 1 in 2500 live births1. It can occur as a result of impaired bile acid biosynthesis, defective bile secretion by hepatocytes (due to membrane transporter defects), or extrahepatic obstruction to biliary flow. Patients present with jaundice, dark urine, pale acholic stools and hepatomegaly. Older infants may have pruritus, develop fat-soluble vitamin deficiencies and fail to thrive1. The aetiology of cholestasis is varied and includes multiple inherited causes, such as progressive familial intrahepatic cholestasis (PFIC), Niemann-Pick disease type C and arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome1. Furthermore cholestasis can be subdivided into ‘low-normal’ and high gamma glutamyl transpeptidase (gGT) categories depending on the level of serum gGT activity.\n\nAccurate diagnosis is essential in order to inform decisions on treatment and management. As an example, familial intrahepatic cholestasis 1 (FIC1) protein deficiency caused by mutations in ATP8B1 (PFIC type 1) and bile salt export protein (BSEP) deficiency caused by mutations in ABCB11 (PFIC type 2) have similar presentations, however liver transplantation is used more frequently in PFIC type 2 then type 16. Genetic testing allows accurate assessment of the genetic risk of cholestasis in families, in addition to providing definitive diagnoses. Clinical laboratories tend to use fluorescent capillary sequencing (FS; Sanger method), because it lends itself to automation and is highly sensitive. However, FS is limited by machine capacity and relatively high consumables costs.\n\nMicroarray resequencing (MR) is an alternative sequencing method previously used to detect mutations in patients with intrahepatic cholestasis2. We have performed initial testing of a 300kb custom-designed microarray (Birmingham ReseqUencing Microarray, BRUM1) in patients with known mutations4. BRUM1 includes probes to sequence two genes (ATP8B1 and ABCB11) involved in PFIC. This short research article describes further evaluation of the BRUM1 microarray using samples simultaneously tested by FS and MR in patients without previously known mutations and assesses the advantages and disadvantages of MR in clinical laboratory use.\n\n\nMaterials and methods\n\nDNA samples from 29 children (15 females) who presented with intrahepatic cholestasis in the first year of life, low-normal range gGT activity without ARC syndrome or defects in bile acid biosynthesis were used. ARC syndrome was excluded by clinical examination3 and bile acid biosynthesis disorders were excluded by determination of urinary bile acid profile by electrospray ionisation mass spectrometry. Thus there was a high likelihood of detecting mutations in ATP8B1 and ABCB11 in these samples. The study was approved by South Birmingham Research Ethics committee (reference number CA/5175).\n\nPolymerase chain reaction was used to amplify the coding exons of ATP8B1 and ABCB11 plus the intron-exon boundaries prior to both FS and MR. The PCR reaction used 50ng of DNA, 0.5μM each primer (Sigma), 0.5mM dNTPs (Bioline) and 2 units of Taq DNA polymerase (Biomix Red, Bioline UK) in 1x PCR buffer.\n\n5μl of PCR product was treated with ExoSap IT (GE Healthcare) for 30 mins at 37°C before being denatured at 85°C for 15 mins. Sequencing primers (at a final concentration of 0.5μM) was added to 1μl of BigDye 3.1 and 1x BigDye buffer (Applied Biosystems) in a final volume of 10μl. The sequencing programme was thermal cycling for 34 cycles of denaturing at 95°C for 20 seconds, annealing 50°C for 20 seconds and extension at 60°C for 4 minutes. Resulting products were precipitated using EDTA/Na acetate and ethanol and centrifuged at 4000 rpm for 30 minutes. Products were washed in 70% ethanol before being air dryed, resuspended in 10μl of HiDi formamide (Applied Biosystems) and denatured. Resuspended products were analysed using a DNA analyzer 3730xl (Applied Biosystems) and the resulting sequence traces were analysed using Sequence Analysis 5.2.2 (Applied Biosystems).\n\nPCR product quantitation, pooling, fragmentation, labelling and hybridisation were performed according to the GeneChip Custom Resequencing Array Protocol V2.1 (Affymetrix). Arrays were washed and stained using a FS450 fluidics station before being scanned with a GCS3000 7G scanner (Affymetrix). Intensity files were generated using AGCC (Command Console V1.0, Affymetrix) and processed in GSeq 4.1 (Affymetrix). Base calling assumed the diploid model and a quality score threshold of 2 (default settings were used for all other parameters).\n\n\nResults\n\nATP8B1 and ABCB11 were analysed using FS and MR. The sequence variants detected in 13 of the samples (44.5% detection rate) are listed in Table 1. Eight variants had previously been described in association with PFIC and ten variants were novel. Four of the variants were nonsense mutations (22%), two were splice site changes (11%) and the rest were missense (67%). No insertions or deletions were detected using the specifically designed probes. No mutations were detected by either technique in the remainder of the patients and therefore the cause of ‘low-normal gGT’ cholestasis remains undetermined in these cases. The negative results in these cases may be explained by whole exon deletions or duplications, intronic mutations affecting splicing, or promoter region mutations in ATP8B1 and ABCB11, as such mutations would not be detected by either strategy. In addition, it is possible that further genes are involved in the phenotype of neonatal cholestasis with low gGT.\n\na - Variant was not confirmed by FS and is therefore a false positive finding.\n\nb - DNA changes were experimentally determined by sequencing.\n\nc - Protein changes are predicted rather than experimentally determined.\n\nd - Novel missense changes are of unclear pathogenicity.\n\nIn sample 17, MR detected a variant not confirmed by FS, constituting a false positive result. Two samples (6 and 22) had compound heterozygous changes in ATP8B1 detected by FS but not by MR. Both samples had two variants in close proximity; therefore we speculate that these are in cis and that each sequence variant has impaired the hybridisation of surrounding probes. Our experience of testing patients for mutations in these genes suggests that false negative results arising in this manner are likely to be uncommon. However, in this cohort they occurred in 7% of samples.\n\n\nDiscussion\n\nMR is an attractive alternative to traditional FS for genetic testing in neonatal cholestasis. The BRUM1 microarray used in this study has a much larger capacity than was utilised in this experiment, as it contains probes for 92 genes associated with inherited disorders. Analysis of the ability of this microarray to detect known gene mutations has shown a 97% mutation detection rate for base substitutions4. The major advantage of MR is increased capacity, allowing sequencing of multiple genes in one experiment as quickly as one gene using FS. The main bottleneck is the requirement to quantify and to pool individual PCR products before hybridisation. This step is time-consuming and prone to error (due to pipetting volume variations and potential sample mix-ups). Therefore, automation to minimise such errors would be useful, if not essential, for adoption of this method into clinical laboratories. Alternatively, use of long range PCR as the preparatory step for the MR protocol could reduce the number of reactions to be pooled, although it is more sensitive to poor DNA quality than standard PCR. Finally, microfluidics-based PCR systems, such as the 48.48 Access Array (Fluidigm Corporation, San Francisco, CA), might be combined with MR to avoid the quantification and pooling step altogether. This system allows the simultaneous but separate amplification of up to 48 PCR products for up to 48 samples, in nanolitre-sized reactions, using a semi-automated process. The end product of the process is a pool of PCR products for each sample, and it is commonly used for target enrichment for benchtop next-generation sequencers.\n\nOf the mutations recorded in the Human Gene Mutation Database5 in ATP8B1 and ABCB11, 24% and 16% respectively were small insertions or deletions (indels), although their combined frequency in PFIC cases is unknown as most are private mutations. The major disadvantage of MR is it’s insensitivity for the detection of indels4. Whilst known indels can be detected with proper microarray design, novel indels will be missed. Larger insertions and deletions involving whole exons, of the type routinely detected by multiplex ligation-dependent probe amplification (MLPA; MRC-Holland, Netherlands), are not detected by either MR or FS. Another disadvantage, underscored by the results of this study, is that mis-called base substitutions (both false negative and false positive calls), are frequent and were found in approximately 10% of samples in this study.\n\nIn conclusion, MR allows rapid and cost-effective genetic screening in neonatal cholestasis and yields results relevant for patient management. Many clinical laboratories are experienced in oligoarray comparative genome hybridization (CGH) and thus have access to equipment required for MR, suggesting that MR could be implemented for a relatively small monetary investment. In principle, MR could be applied to many clinical scenarios involving heterogeneous conditions, especially if the mutations tend to be base substitutions. We recommend that MR methods be used for initial evaluation, with FS deployed when genetic and clinical or histopathologic findings are discordant.\n\n\nConsent\n\nConsent for research into the genetic cause of the patients’ cholestasis was obtained from the patients’ legal guardian at the time of obtaining the sample.", "appendix": "Author contributions\n\n\n\nKEM performed the experiments, analysed the data and prepared the first draft of the manuscript. CKB performed the experiments and analysed the data. SSB recruited patients and performed experiments. UB, ES, DAK, CJK and JLH recruited patients and analysed the data. ASK and FMcD analysed the data. PG conceived the study and analysed the data. All authors were involved in the revision of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nReferences\n\nSuchy FJ, Sokol RJ, Balistreri WF: Liver disease in children. Cambridge University Press 2007. Reference Source\n\nLiu C, Aronow BJ, Jegga AG, et al.: Novel resequencing chip customized to diagnose mutations in patients with inherited syndromes of intrahepatic cholestasis. Gastroenterology. 2007; 132(1): 119–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith H, Galmes R, Gogolina E, et al.: Association among genotype, clinical phenotype and intracellular localization of trafficking proteins in ARC Syndrome. Hum Mutat. 2012; 33(12): 1656–1664. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBruce CK, Smith M, Rahman F, et al.: Design and validation of a metabolic disorder resequencing microarray (BRUM1). Hum Mutat. 2010; 31(7): 858–865. PubMed Abstract \| Publisher Full Text\n\nHuman Gene Mutation Database. Accessed July 2012. Reference Source\n\nPawlikowska L, Strautnieks S, Jankowska I, et al.: Differences in presentation and progression between severe FIC1 and BSEP deficiencies. J Hepatol. 2010; 53(1): 170–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nByrne JA, Strautnieks SS, Ihrke G, et al.: Missense Mutations and Single Nucleotide Polymorphisms in ABCB11 Impair Bile Salt Export Pump Processing and Function or Disrupt Pre-Messenger RNA Splicing. Hepatology. 2009; 49(2): 553–567. PubMed Abstract \| Publisher Full Text\n\nvan Mil SW, van der Woerd WL, van der Brugge G, et al.: Benign Recurrent Intrahepatic Cholestasis Type 2 Is Caused by Mutations in ABCB11. Gastroenterology. 2004; 127(2): 379–384. PubMed Abstract \| Publisher Full Text\n\nKlomp LW, Vargas JC, van Mil SW, et al.: Characterization of Mutations in ATP8B1 Associated With Hereditary Cholestasis. Hepatology. 2004; 40(1): 27–38. PubMed Abstract \| Publisher Full Text\n\nStrautnieks SS, Byrne JA, Pawlikowska L, et al.: Severe Bile Salt Export Pump Deficiency: 82 Different ABCB11 Mutations in 109 Families. Gastroenterology. 2008; 134(4): 1203–1214. PubMed Abstract \| Publisher Full Text\n\nKlomp LW, Bull LN, Knisely AS, et al.: A Missense Mutation in FIC1 Is Associated With Greenland Familial Cholestasis. Hepatology. 2000; 32(6): 1337–1341. PubMed Abstract \| Publisher Full Text\n\nLiu LY, Wang ZL, Wang XH, et al.: ABCB11 gene mutations in Chinese children with progressive intrahepatic cholestasis and low gamma glutamyltransferase. Liver Int. 2010; 30(6): 809–15. PubMed Abstract \| Publisher Full Text" }	[ { "id": "769", "date": "14 Feb 2013", "name": "Struan Grant", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "770", "date": "14 Feb 2013", "name": "Andrea Ballabio", "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 compare, in a very thorough and detailed manner, the performance of Microarray resequencing (MS) vs. Sanger sequencing (FS) for mutation detection in the ATP8B1 and ABCB11 genes in cholestatic patients. They recommend the cautious use of microarray resequencing as an initial evaluation tool and propose to limit the use of Sanger sequencing to more problematic and discordant cases.However, given the non-negligible discordance observed by the authors between the two approaches (MR had a false positive rate of 3.4% and a false negative rate of 7%), the definition of the cases that should be analyzed by FS is not entirely clear. Moreover, a number of other resequencing procedures that can be applied to the simultaneous analysis of multiple genes are now available. Some of them are very competitive in terms of efficacy and cost with MS and it is quite likely that in the near future they will be more widely used with respect to MS. Ideally, the authors should have discussed about this issue.", "responses": [] } ]	1	https://f1000research.com/articles/2-32
https://f1000research.com/articles/2-91/v1	15 Mar 13	{ "type": "Research Article", "title": "Intervention- versus physiology-based risk assessment scores for predicting cardiac arrest: a pilot study", "authors": [ "Christopher G Choukalas", "Suzanne Kellman", "Michelle L Keese", "Michelle Loor", "Marzanna Vasington", "Michael F O’Connor", "Dana P Edelson", "Christopher G Choukalas", "Michelle L Keese", "Michelle Loor", "Marzanna Vasington", "Michael F O’Connor", "Dana P Edelson" ], "abstract": "There is increasing interest in predicting and avoiding cardiac arrest in hospitalized patients. Multiple studies have used vital signs or scores based upon them, such as the Modified Early Warning Score (MEWS). Scoring systems that measure supportive care, such as the Sequential Organ Failure Assessment (SOFA) and the 28-item Therapeutic Interventions Scoring System (TISS-28) might be superior to systems used in previous studies. This study was performed to determine if a system using SOFA and/or TISS would be superior in detecting clinical deterioration prior to cardiac arrest.Using a retrospective chart review, MEWS, SOFA and TISS-28 scores were calculated for twenty patients at baseline and then in the 24 hours prior to their cardiac arrest. Supportive interventions and nursing care (SOFA and TISS-28) changed more than measures of physiology (MEWS) in the period prior to cardiac arrest, likely due to the fact that vital sign deterioration can be delayed by supportive measures. These results support the idea that a SOFA and/or TISS-28 scoring system might be superior to the MEWS, which could be used to make hospital rapid response teams more effective.", "keywords": [ "TISS-28", "SOFA", "MEWS", "cardiac arrest", "risk assessment" ], "content": "Introduction\n\nCardiac arrest in hospitalized patients outside the intensive care unit (ICU) carries a high mortality. Many studies have shown that these cardiac arrests are rarely sudden, as demonstrated by abnormal vital signs in the hours leading up to these events1–7. The challenge lies in developing robust algorithms to predict cardiac arrests in order to better target interventions. In fact, one potential explanation for the failure of rapid response systems (RRS) to better impact patient outcomes is an inability to identify which patients actually require RRS intervention8.\n\nThe Modified Early Warning Score (MEWS) is one attempt at a risk prediction algorithm. This scale, which assigns point values for abnormal vital signs or mental status assessments, has been used to predict requirement for hospital admission in emergency department patients9 and to predict hospital mortality10. However, although a vital sign-based tool is appealing for general ward patients, it is limited by the quality of the data, specifically with respect to respiratory rate and mental status, which are poorly assessed and documented outside the intensive care unit. In addition, physiology-based tools do not take into account what has been done to the patient to maintain that physiology, such as the use of supplemental oxygen to maintain oxygen saturation or vasopressor agents to maintain blood pressure.\n\nIn the ICU setting, scales such as the Sequential Organ Failure Assessment (SOFA)11 have been developed to account for both physiology and supportive measures, but have not been validated for use outside the ICU. Yet another ICU scale with the potential to fill this critical role for floor patients is the 28-item Therapeutic Interventions Scoring System (TISS-28)12. This system was designed to quantify nursing workload in the intensive care setting, and as such does not include any physiology-based scales. Rather, the scale assesses the intensity of caregiver interventions such as the frequency of vital signs, use of invasive catheters and drains, and the frequency and intensity of certain treatments.\n\nWe hypothesized that a scoring system that includes supportive measures and interventions (such as the SOFA and/or TISS-28) would be superior to a system relying only on physiology (such as the MEWS) in detecting clinical deterioration preceding cardiac arrest on the floors.\n\n\nMethod\n\nWe conducted a retrospective chart review of patients between 2006 and 2008 at the University of Chicago Medicine, which is a tertiary-care, university teaching hospital with approximately 600 beds, without a RRS at the time of the study. The study protocol was approved by the University of Chicago Institutional Review Board.\n\nA convenience sample of patients suffering cardiac arrest in the hospital were included in the analysis. Paper charts were reviewed and data sufficient to calculate the SOFA, TISS-28, and MEWS for each patient was extracted. This included vital signs, laboratory results for creatinine, bilirubin, arterial blood gases, vasoactive drug doses, and nursing notes related to mental status and care duties. When arterial gas data were not available, we estimated the PaO2 necessary for the SOFA using a validated conversion algorithm13.\n\nThe TISS-28 is comprised of 28 items with point values from one to eight, with higher scores equating to a higher nursing workload. The items are subdivided into component physiologic systems (Basic Monitoring, Cardiovascular, Respiratory, Interventions, Renal, Neurologic and Metabolic subscales) to isolate the dynamic aspects of the scale in the time preceding arrest; each subscale consists of one to several items. For example, the Neurologic subscale consists solely of the presence or absence of intracranial pressure monitoring (worth four points), whereas the Cardiovascular subscale consists of the presence of single or multiple vasoactive medications, the presence of a central line, and the presence of an arterial line (worth three, four, two, and five points, respectively). The Basic Monitoring scale consists of items related to the care of drains and wounds, and the frequency of vital sign checks and lab draws. The Intervention subscale encompasses recent procedures and studies the patient might undergo, such as endotracheal intubation, echocardiography, and trips to radiology or the operating room. The Respiratory and Renal subscales consist of items pertaining to the support of those particular organ systems (ventilatory management, diuresis and hemodialysis, respectively). Finally, the Metabolic subscale consists of items related to treatment of acid-base abnormalities and malnutrition.\n\nMEWS, SOFA and TISS-28 scores were calculated for the calendar day of the arrest (pre-arrest) and the day prior to the arrest (baseline). MEWS scores were based on vital signs such as heart rate, respiratory rate, temperature, systolic blood pressure and patient responsiveness. Data obtained during or after the arrest was excluded. In accordance with studies using the SOFA score14,15, we used the highest score for each organ system to calculate the total score, and then used the highest score from each time point. For the sake of consistency, we used this approach to calculate the TISS-28 and MEWS scores as well. Data were stored in a spreadsheet (Microsoft Excel, Redmond, WA) and analyzed using SPSS (Chicago, IL). Baseline and pre-arrest scores were compared using two-sided paired sample t-tests. Percentages of change in the various scales from baseline to pre-arrest were compared using chi-squared statistics. An alpha of 0.05 was used for all analyses.\n\n\nResults\n\nA total of twenty patients were included in the study. Demographic data are presented in Table 1. The mean age of subjects was 68±15 years. Eighty five percent of patients were black and sixty percent were female. Admission diagnoses for the 20 patients were highly variable and included dyspnea, subdural hematoma, and congestive heart failure, amongst others. For three subjects, cardiac arrest took place on the day of admission, and hence no baseline data were available. In the remaining 17 patients, the SOFA score increased from 1.29±0.40 at baseline to 1.76±0.45 (p=0.03) on the calendar day before the event, representing an increase of 36%, while the TISS-28 increased from 9.9 to 15.0 (p=0.04), representing a 52% increase. There was no significant change in the MEWS (see Table 2).\n\nSE denotes standard error. CVA; cardiovascular accident.\n\nPaired samples t-test.\n\n+Out of the 17 subject with baseline data.\n\nAnalysis of the TISS-28 subscales revealed that the primary driver of the increase in TISS-28 was due to an increase in the Interventions score, which more than tripled (0.59±0.40 at baseline vs. 2.00±0.73 the day before arrest; p=0.03). There was no significant difference in the Basic Monitoring, Cardiovascular, and Respiratory subscales, despite a trend in the same direction (see Table 3).\n\nPaired samples t-test.\n\n+Out of the 17 subject with baseline data.\n\n\nDiscussion\n\nIn this study of patients suffering cardiac arrest, we found that the measure of nursing care (TISS-28) changed to a greater degree than the physiology-based measures (MEWS) in the period preceding the arrest. While some patients experience sudden arrest, many are believed to decompensate over a period of hours or even days prior to their ultimate cardiac or respiratory arrest5. Schein and colleagues identified respiratory disorders as the most common antecedent to arrest, and noted that most patients experiencing arrest had \"documented observations of clinical deterioration or new complaints within eight hours of arrest\"7. In a retrospective, case-controlled study Hodgetts et al16 observed that abnormal breathing, heart rate, or systolic blood pressure were powerful predictors of arrest. In contrast, Kause4 identified hypotension and altered mental status as the primary predictors of arrest in their study examining patients from the United Kingdom and New Zealand.\n\nThe detection of patient deterioration by vital signs-based systems can be delayed by supportive measures, suggestive of low sensitivity. In many decompensating patients, vital signs may be altered by a variety of supportive measures instituted by caregivers. These supportive measures will delay the detection of deterioration by physiologic based scoring systems such as the MEWS, but would be detected by a scoring system of supportive measures such as the TISS. Intervention and laboratory based scoring systems may be more sensitive or earlier predictors of patient deterioration than any scoring system limited to only one of these dimensions.\n\nStudies of RRS have varied in their ability to demonstrate an effect on cardiac arrest and mortality17–19. The systems in most of these studies rely on nursing or medical staff to notice the sometimes subtle changes in patients’ well-being in order to activate the RRS. The attraction of simple severity of illness scales like those studied here is that, in conjunction with an electronic medical record, they can be calculated automatically. Such a system could prompt caregivers to consider RRS consultation when scale scores (i.e., caregiver duties) increase or pass some threshold, and this might better direct RRS resources to the sickest patients.\n\nOur study demonstrates that the intensity of caregiver intervention, particularly those interventions related to procedures and cardiovascular and respiratory systems, is superior to MEWS in detecting patient decompensation. In our patients, the liver and coagulation components of the SOFA score changed less than the other parts of the SOFA score, though it is possible that the indicators of liver and coagulation function (i.e. bilirubin and platelet count) would be more useful in a larger sample of patients in which these parameters changed significantly. Similarly, the CNS component of the TISS-28, the measurement of intracranial pressure, was not performed on any patient in our study at any time point. Using only the presence or absence of intracranial pressure monitoring as a metric for CNS deterioration would likely miss a patient’s deteriorating mental status as a sign of clinical worsening.\n\nOur study has several limitations. First, our sample size is small. It is possible that the MEWS would prove to be a significant predictor in a larger study. However, as an exploratory study of the utility for intervention-based assessment, either in addition to or in place of physiology-based predictors, these data show promise for the TISS-28 as a measure of clinical deterioration. Second, our study is a retrospective case series. These findings will need to be validated in a larger prospective, controlled trial. Third, although the TISS-28 describes caregiver interventions across subscales and organ systems, some subscales are more comprehensive than others (e.g., as mentioned above, the Neurologic subscale consists of one item, whereas other subscales cover a broader range of potential interventions). While changes in mental status are widely believed to foreshadow decompensation, variable and generally incomplete documentation of mental status frustrates abstracting SOFA and MEWS from charts.\n\nAssessment of the central nervous system is probably sub-par in all three clinical scales. Whereas the TISS-28 focuses exclusively on ICP monitoring, the SOFA CNS subscale consists solely of the Glasgow Coma Scale (GCS) and the MEWS CNS variable captures patients’ responsiveness to stimulation. Each of these measures probably misses the subtle mental status alterations that precede clinical decompensation. Furthermore, the various CNS measures in this study were the most likely variables to have missing data. For the GCS, when data were lacking, we inferred a value of 15 (normal), and did the same for the CNS measure of the MEWS. This approach may lower the total scale scores, but we believe this is the most conservative approach to handling these missing data.\n\nIn conclusion, our data show that an intervention-based scoring system changes more than physiology-based scoring systems in the period prior to cardiac arrest. This may be due to support used to maintain normal vital signs in the early period of decompensation, which is not captured by vital sign-based scoring systems. This finding needs to be validated prospectively.", "appendix": "Author contributions\n\nChristopher G Choukalas, conceived study and analyzed data. Suzanne Kellman, analyzed data and prepared manuscript. Michelle L Keese, collected/extracted and analyzed data. Michelle Loor, collected/extracted data. Marzanna Vasington, collected/extracted data, developed data collection tool. Michael F O’Connor, conceived study and analyzed data. Dana P Edelson, analyzed data. All authors reviewed and approved the manuscript for publication.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nReferences\n\nSmith AF, Wood J: Can some in-hospital cardio-respiratory arrests be prevented? A prospective survey. Resuscitation. (1998); 37: 133–7.\n\nBerlot G, Pangher A, Petrucci L, et al:Anticipating events of in-hospital cardiac arrest. Eur J Emerg Med. (2004); 11(1): 24–8.\n\nBuist M, Bernard S, Nguyen TV, et al:Association between clinically abnormal observations and subsequent in-hospital mortality: A prospective study. Resuscitation. (2004); 62: 137–41.\n\nKause J, Smith G, Prytherch D, et al:A comparison of antecedents to cardiac arrests, deaths and emergency intensive care admissions in Australia and New Zealand, and the United Kingdom-The ACADEMIA study. Resuscitation. (2004); 62: 275–82.\n\nFranklin C, Mathew J: Developing strategies to prevent in-hospital cardiac arrests: analysing responses of physicians and nurses in the hours before the event. Crit Care Med. (1994); 22: 244–7.\n\nHillman KM, Bristow PJ, Chey T, et al:Antecedents to hospital deaths. Inter Med J. (2001); 31: 343–8.\n\nSchein RM, Hazday N, Pena M, et al:Clinical antecedents to in-hospital cardiac arrest. Chest. (1990); 98: 1388–92.\n\nEdelson DP: A weak link in the rapid response system. Arch Intern Med. (2010); 170(1): 12–3.\n\nSubbe CP, Kruger M, Rutherford P, et al:Validation of a modified Early Warning Score in medical admissions. QJM. (2001); 94: 521–6.\n\nGoldhill DR, McNarry AF, Mandersloot G, et al:A physiologically-based early warning score for ward patients: The association between score and outcome. Anaesthesia. (2005); 60: 547–53.\n\nVincent JL, Moreno R, Takala J, et al:The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. On behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Med. (1996); 22: 707–710.\n\nMoreno R, Morais P: Validation of the simplified therapeutic intervention scoring system on an independent database. Intensive Care Med. (1997); 23(6): 640–4.\n\nPandharipande PP, Shintani AK, Hagerman HE, et al:Derivation and validation of SpO2/FiO2 ratio to impute for PaO2/FiO2 ratio in the respiratory component of the Sequential Organ Failure Assessment score. Crit Care Med. (2009); 37(4): 1317–21.\n\nMoreno R, Vincent JL, Matos R, et al:The use of maximum SOFA score to quantify organ dysfunction/failure in intensive care. Results of a prospective, multicentre study. Working Group on Sepsis related Problems of the ESICM. Intensive Care Med. (1999); 25(7): 686–96.\n\nFerreira FL, Bota DP, Bross A, et al:Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA. (2001); 286(14): 1754–8.\n\nHodgetts TJ, Kenward G, Vlachonikilis IG, et al:The identification of risk factors for cardia arrest and formulation of activation criteria to alert a medical emergency team. Resuscitation. (2002); 54: 125–31.\n\nBuist MD, Moore GE, Bernard SA, et al:Effects of a medical emergency team on reduction of incidence of and mortality from unexpected cardiac arrests in hospital: preliminary study. BMJ. (2002); 324(7334): 387–90.\n\nTee A, Calzavacca P, Licari E, et al:Bench-to-bedside review: The MET syndrome--the challenges of researching and adopting medical emergency teams. Crit Care. (2008); 12(1): 205.\n\nHillman KM, Chen J, Cretikos M, et al:Introduction of the medical emergency team (MET) system: A cluster-randomised controlled trial. Lancet. (2005); 365: 2091–7." }	[ { "id": "1000", "date": "14 Jun 2013", "name": "Anders Aneman", "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 conclusion is not justified based on the presented data because of the following concerns:Small convenience sample (n=17) studied retrospectively. Article does not specify DNR status or comment on limitations of care preceding cardiac arrest. Only 48 hrs of hospitalization studied. No detail provided on level of care, level of monitoring, or level of nursing.TISS-28 and SOFA has not been validated outside ICU and present sample size precludes any meaningful correlations between scoring systems.TISS-28 and SOFA excludes the intuitive criterion (concern for patient) that is included in RRS trigger track systems and found to be both common and predictive of outcome. For example, normal physiology maintained with significant support would not meet numerical triggers but definitely be very concerning for clinical staff (cf. discussion MEWS vs. TISS-28/SOFA). This limits both the novelty and the validity of the study. Insufficient detail for SOFA provided in article to delineate cardiorespiratory pathology (only aggregate SOFA) and notably TISS-28 did not change significantly in any domain apart for “Interventions” (again, concern for patient).", "responses": [] }, { "id": "1309", "date": "13 Aug 2013", "name": "Una Kyriacos", "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\nStudies on patient monitoring systems such as this are important. The study has limitations and the authors acknowledge this: it is an exploratory study of limited scope. This limits the conclusions to tentative assumptions at best.The study could be strengthened by acknowledging that the two scoring systems (TISS-28 and SOFA) are not intended for general ward use, whereas this is the overt intention with the MEWS, with which the former systems are compared. Morrice and Simpson (2006) did this successfully in a cross-sectional survey using the EWS, TISS-28 and APACHE II. Neither the abstract, nor the main paper, forefront the difference in purpose of the MEWS, TISS-28 AND SOFA. The results are therefore not surprising: both the TISS-28 and SOFA systems, used within high dependency areas, require staff-intensive input and supportive interventions. The TISS-28 point equals 10.6 minutes of a nurses' shift (a total of 46.55 points per shift delivered by one nurse) (Miranda, de Rijk & Schaufeli, 1996). The MEWS, on the other hand, although also designed for early recognition of physiological deterioration in ward patients, but by non-specialist nurses, is used in a context characterised by sicker, more dependent patients than previously described in the literature.Inclusion and exclusion criteria for patient records have been omitted making replication difficult. The results do not indicate if the MEWS was used as a single parameter or aggregate weighted track-and-trigger system for the record review.", "responses": [] }, { "id": "3361", "date": "03 Feb 2014", "name": "José Machado", "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 presents a study that compares three forecasting models used in healthcare.The models are based on scores for predicting cardiac arrests. The idea is useful and the theme is very important due to the huge number of people that are affected by cardiac arrests worldwide. However, there are some gaps that are crucial to validate the success of the study. The small sample is indeed the main flaw of this study and consequently, it compromises the results interpretation. It is very dangerous to take solid conclusions with just a few examples. In other words, the reliability of this study is not enough to evaluate and to compare these three forecasting models. A larger sample, as well as a more heterogeneity is demanded. The description of the models could be more detailed, in order to a better comprehension. The bibliography is adequate.", "responses": [] } ]	1	https://f1000research.com/articles/2-91
https://f1000research.com/articles/2-89/v1	14 Mar 13	{ "type": "Research Article", "title": "Campylobacter jejuni genomes exhibit notable GC variation within housekeeping genes", "authors": [ "Vathsala Mohan", "Mark Stevenson", "Mark Stevenson" ], "abstract": "Campylobacter jejuni (C. jejuni) is a rapidly evolving bacterial species with massive genetic recombination potential to generate niche specific genotypes. Generally the housekeeping gene lineage has been evidenced to undergo lateral gene transfer and recombination quite frequently compared to the information processing gene lineage. During such exchanges, genetic amelioration takes place over time acquiring the host genomes’ molecular characteristics. In this study, fifty genes that comprised twenty five metabolic housekeeping lineage genes and twenty five information processing lineage genes from nineteen C. jejuni genomes were studied. These nineteen genomes included seven C. jejuni isolates that belonged to the same genotype or multilocus sequence type ST-474 that were sequences in New Zealand. The genes from both lineages were tested for recombination and the guanine-cytosine (GC) variation. There was a positive correlation between the GC variance and the number of recombination events amongst the metabolic housekeeping genes. Genes that showed wider GC variance had a relatively high number of recombination events. In contrast, although recombination was evident in all of the informational genes, there was no correlation between the GC variance and recombination. The observation of a positive correlation between the GC variance and the recombination events in the metabolic housekeeping genes may reflect the recent events of exchange of DNA and the regions that are constantly dynamic to undergo recombination under certain circumstances. While in the case of informational genes, the demand of stringent homology between genes may be a limiting factor for the absence of such correlation, however, the sites that involved in recombination may also represent the hotspots of recombination in those genes.", "keywords": [ "Molecular events such as mutation", "deletion", "recombination and gene transfer play paramount roles in shaping the evolution of prokaryotes (reviewed by1", "2). As a consequence the genomes are more prone for nucleotide base compositional fluctuations. Particularly", "the evolutionary forces pose a major impact on the guanine-cytosine (GC) content of bacteria at the level of genes and genomes3. Amongst all the important evolutionary forces", "the impact of recombination (homologous", "non-homologous or illegitimate) on the evolution of bacteria has been evidenced as the major driving force or factor of microevolution4–8. However", "the rate of recombination may differ greatly amongst different bacterial species", "while some species recombine more frequently to have multiple recombination events than mutations that render them weakly clonal", "whereas in other species it appears to be a rare incident leading to distinct clonal lineages5", "6", "8", "9. Studies of genetic diversity in the bacterial kingdom have shown that bacteria form clusters of genetically related strains and that extensive recombination among related clusters have been regarded as normal rather than exceptional events10. However", "not every single gene is involved in recombination or horizontal gene transfer11", "12." ], "content": "Introduction\n\nMolecular events such as mutation, deletion, recombination and gene transfer play paramount roles in shaping the evolution of prokaryotes (reviewed by1,2). As a consequence the genomes are more prone for nucleotide base compositional fluctuations. Particularly, the evolutionary forces pose a major impact on the guanine-cytosine (GC) content of bacteria at the level of genes and genomes3. Amongst all the important evolutionary forces, the impact of recombination (homologous, non-homologous or illegitimate) on the evolution of bacteria has been evidenced as the major driving force or factor of microevolution4–8. However, the rate of recombination may differ greatly amongst different bacterial species; while some species recombine more frequently to have multiple recombination events than mutations that render them weakly clonal, whereas in other species it appears to be a rare incident leading to distinct clonal lineages5,6,8,9. Studies of genetic diversity in the bacterial kingdom have shown that bacteria form clusters of genetically related strains and that extensive recombination among related clusters have been regarded as normal rather than exceptional events10. However, not every single gene is involved in recombination or horizontal gene transfer11,12.\n\nThe lineages of the genes were broadly classified into informational and operational or metabolic housekeeping genes. The informational genes include genes of translation (T), transcription (S), and replication (R) and also the ATPases, GTPases (G) and tRNA synthetases whereas the operational genes are those involved in cell operations such as amino acid synthesis (A), biosynthesis of co-factors (B), cell envelope proteins (C), energy metabolism (E), intermediary metabolism (I), fatty acid and phospholipid biosynthesis (L), nucleotide biosynthesis (N), and regulatory genes (Z)12. The operational genes are the most modular genes in the cells that are inclined to be horizontally transferred or recombined most often11,13. As a result of this behaviour of the housekeeping genes it is therefore prudent to speculate that housekeeping genes may exhibit notable molecular differences compared to the informational genes.\n\nCampylobacter jejuni (C. jejuni), is a zoonotic pathogen that colonises the gut of a wide variety of birds and mammals and has been attributed to the majority of bacterial gastroenteritis cases in developed countries14. Most predominantly the disease is caused by C. jejuni and often the disease is self limiting, however on rare occasions there can be serious sequelae such as Guillain-Barré syndrome and reactive arthritis15. The natural competency and the plasticity of C. jejuni were not investigated until Dingle et al. (2001)16 designed an MLST scheme for C. jejuni which has subsequently been exploited to structure and investigate the association of C. jejuni populations with different hosts and the environment from which human clinical infection originated17–21. Further, Wilson et al.22 used the population genetics-phylogenetics approach to demonstrate the massive evolutionary potential of C. jejuni inferring that recombination plays a major role in the generation of diversity at twice the rate of mutation per se.\n\nAs C. jejuni is an actively recombining bacterial species, an attempt to investigate the GC variations in a subset of operational genes and informational genes was made in an effort to understand the difference between these two lineages of genes within C. jejuni genomes. The assumptions to conduct this analysis were: (1) amelioration or coalescence of GC content or the nucleotide base composition takes relatively long time23 which means that an event of recombination in a given group of genes with lack of time for coalescence will exhibit notable GC variation compared to other genes in the population; (2) housekeeping genes are relatively more prone for recombination where interspecies recombination has been demonstrated between donor and recipient DNA molecules that differ by up to 25–30% of their nucleotide sites24–26. Given the fact that C. jejuni is a competent bacterial species where generation of host adapted variants has been documented using MLST datasets by several studies27–31, it may be hypothesised that the housekeeping genes may exhibit notable differences in their GC content24; (3) Informational genes require highly stringent homology for an event of recombination to occur and recombination takes place as a part of DNA repair32 and hence these genes may not exhibit such notable GC variation even in the presence of recombination events. In order to test these hypotheses, nineteen C. jejuni genomes were analysed in this study.\n\n\nMethods\n\nHousekeeping genes were located on the C. jejuni NCTC 11168 genome (GenBank accession number NC002163) and a total of 50 genes were selected for the analyses33–35. The genes were further classified into operational genes (metabolic housekeeping genes) and informational genes based on their function by referring to the KEGG pathway and the gene function websites. The positions of the categorised selected subset of genes (metabolic housekeeping and informational genes) are marked on the reference C. jejuni NCTC 11168 (GenBank accession number NC 002163) circular genome and are shown in Figures 1A to Figures 1D, respectively. Here after in this paper the genes are referred as housekeeping genes (metabolic housekeeping genes) and informational genes for the purpose of plain comparison and interpretation. Twelve fully sequenced C. jejuni genomes (Table 1) were used to compare 50 selected genes. The gene sequences were downloaded from the GenBank database. Seven C. jejuni MLST ST-474 isolates were sequenced at the Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand. The overall GC and GC3 contents of individual genes were compared using DnaSP v536. The frequency distribution graphs of GC contents from all the 19 genomes compared were generated in R programming language37. Inferences on recombination within each gene under investigation were drawn using Dual-Brothers within Geneious v.5.3.438 and the number of recombination events, referred to as Rm was estimated using DnaSP v5. The relationship between the GC variance and the recombination events was analysed using linear models by having Rm as a dependent variable, and the log GC variance and the length as independent variables. Detailed descriptions of methods used in the article are available from a related data article39.\n\nST: Multilocus sequence sequence type; GBS: Guillain-Barré syndrome; ORFs: Open reading frames; Mb: Mega bases; GC: Guanine: Cytosine; ns: not stated; *:Date of start of project.\n\n\nResults and Discussion\n\nGuanine-Cytosine (GC) content ranged between 31.9% and 36.4% across the metabolic housekeeping genes in general, where both the high and low ranges of the GC contents was evident in the C. jejuni subsp. doylei (CJJD269.97269.97) genome. The GC content of the housekeeping alleles varied among genomes with the MLST scheme alleles the tkt and gltA alleles showing a relatively wider variation followed by glyA, glmM, aspA, glnA and uncA alleles. GC contents were identical amongst all genes across all the ST-474 genomes except for two genes, namely fumC and trpC however, the GC3 content of the pycA gene varied between the ST-474 genomes. Analysis of recombination events showed that the infB gene possessed the highest number of 27 sites (Rm [the number of recombination events] = 27) (Table 2a) while the trpB and uncA possessed the least number of sites (n = 1) to be involved in recombination. The linear model showed that the number of recombination events was positively correlated with the GC variance, where the genes that showed a wider GC variance showed a high number of recombination events. However, the infB and gltB genes showed relatively high numbers of recombination events (n = 27 and n = 26, respectively) and they did not reveal convincing GC variation between genomes as it was observed in other genes. While the GC variance and the number of recombination events was found to be positively correlated (p value = 0.009), the length was not found to influence the number of recombination events significantly (p value = 0.7). Figures 1A and Figures 1B illustrate the relationship between the GC variance and the recombination events, and the relationship between the length and the recombination events, respectively. The frequency distribution graphs of the GC contents for all the 25 genes are provided in Mohan et al.37 in figures 2–5.\n\nRm: Number of recombination sites.\n\nThe overall guanine-cytosine (GC) content varied greatly between informational genes in general, where genes such as mfd, ogt, polA, recJ, recN and xseA showed lower ranges of GC contents (from 0.265 to 0.298) whereas the remainder showed a relatively higher GC content with rplB showing the highest GC content of 0.392. The analysis of informational genes for recombination events showed that ogt gene and ssb possessed a single recombination site, while mutS was found to possess the highest number of recombination events (n = 38) (Table 2b describes the recombination events that occurred in the informational genes). The linear model showed that Rm was dependent on the length of the genes (p value = 0.05) rather than on the GC variance (p value = 0.9). Figures 1C and Figures 1D illustrate the relationship between the GC variance and the number of recombination events, and the relationship between the length and recombination events, respectively. (Frequency distribution graphs of GC contents for the informational genes are provided in Mohan et al.37 in figures 6–9).\n\nRm: Number of recombination sites.\n\nDifferences in the nucleotide base composition of a gene and/or genome is a fundamental element shaping the genomic evolution which in turn, directly influences the GC contents of genes and/or genomes40. GC variation has been thought to be driven both by neutral mutational effects and adaptive selection pressures41,42. In this study, GC contents of the housekeeping and informational genes were measured across 19 C. jejuni genomes. Variation in the GC contents amongst the housekeeping genes investigated in this study within the 19 genomes was clearly evident. However, it should be noted that the GC contents of the housekeeping genes within the seven ST- 474 genomes were identical except for two genes, the fumC and trpC, with a variation in the GC3 content of the pycA gene (but showed an identical overall GC content across all the ST-474s). There was an association between the GC variance and the number of recombination sites that occurred within different housekeeping genes, where the majority of the genes (investigated in this study) that possessed a wider GC variance showed a higher number of recombination sites.\n\nThe variation in base composition is a consequence of differences in the patterns of evolutionary events43 where variation in the GC content is dependent on the mutational patterns and/or the evolutionary events that had occurred in a given nucleotide sequence43,44. GC content has also been shown to be correlated with various biological factors45 and is a potential research area where there has been a significant level of research carried out3,41,46–52 as well as is ongoing. Previous reports that investigated the causes for the differences in the GC content have evidenced that the GC content at the third codon position (GC3) and the conversion of GC to AT and AT to GC at this position to occur in favour of a selection for the GC content of any given genome47,53. Further, this selection pressure possesses a great impact on evolution47,53. Furthermore, the major changes in the GC content has been shown to predict the future direction of the evolutionary changes of the genomic GC content54. The tRNA abundances in a genome has been shown to be yet another important selective pressure that determines the synonymous codon usage (change in the GC3 position) which in turn reflects the differential evolution in organisms50,55. Hence, in the light of previous research reports it will be prudent to hypothesise that since the housekeeping genes are involved in metabolic processes in a genome this may be a reflection of the different metabolic evolutionary pressures that acted upon these genes as a measure of adaptation to the prevailing environmental conditions. Also, the base composition may have changed as a result of recombination between the same species and /or between bacteria with a similar base composition and tRNA pools. It is very intriguing to determine the driving forces for the change in the base composition which may help to better understand the biological reasons behind the frequent nucleotide changes within housekeeping genes in C. jejuni.\n\nFurther, there may be few additional explanations for the variation in the GC contents that might have arisen as a result of recombination such as (1) they may not have ameliorated or coalesced after an event of recombination; or (2) may not have had ample time for amelioration after recombination; or (3) those sites may be the hotspots or active spots on the genes that are dynamic and continuously engaging in recombination.\n\nReports using MLST datasets have revealed mosaic alleles within the seven housekeeping alleles and have also suggested and/or raised the possibilities of both host adaptation and convergence of C. jejuni and Campylobacter coli (C. coli) species27,30,31. There may be an additional influence which may be relatively biological that triggers such recombination between C. jejuni isolates present in a host and/or convergence of C. jejuni with C. coli that may in turn enable better survival of the bacteria in certain hosts. Hybrid alleles of tkt and gltA have been frequently documented in previous reports30,31 where in our study, these two genes showed wider GC variance within genomes. In contrast, atpD and trpB showed the least number of recombination sites where, the GC content did not vary as evidenced in the tkt and the gltA alleles across the investigated genomes. However, there was one exception, the infB gene that showed a high number of recombination sites showed relatively small GC variance.\n\nIn the case of informational genes, the overall GC content varied greatly amongst genes investigated within the genomes. Although there was variation in the GC contents within genes, it was not positively correlated with the number of recombination sites, whereas it was correlated with the length of genes which is not surprising. It may be speculated that since an event of recombination in informational genes demand a relatively high degree of homologies56, recombination in this subset of genes may have occurred between sequences with high homologies. Moreover, according to the complexity theory11,12 the repair and ribosomal genes belong to the lineages of higher complex, which may not allow them to compromise or tolerate GC variation during recombination. Further, apart from the differences between individual genomes, the overall GC variation amongst the informational genes investigated in this study, (lower GC content in mfd, ogt, polA, recJ, recN and xseA genes and higher GC contents in the remainder) reflect the differences in the functional conservation and complexity. For example, rplB showed the highest GC content of 39.2% which is relatively complex and conserved as it is a 50S ribosomal protein – L2, involved in several discrete steps of polypeptide synthesis such as peptidyl transferase activity, binding of aminoacyl-tRNA to A and P-sites57.\n\n\nFuture research\n\nEven though C. jejuni has been shown over the past decade through a significant amount of research to be a promiscuous and a competent bacterial species, the biological triggers behind recombination which leads to host adaptation and emergence of new variants are very unclear. Further, most of the studies are based on the seven housekeeping alleles that cover the internal fragments (approximately 400–500 base pairs) of those genes used in the MLST scheme for typing the isolates16,58. The nucleotide differences outside the typing region always get neglected which is important when a functional gene is to be evaluated for evolution. Housekeeping genes involved in various metabolic functions and cellular processes play a critical role in the overall integrity and survival of micro-organisms in general. The correlation between GC variance and recombination indicates the vulnerability of housekeeping genes to evolutionary forces and further it also shows how dynamic the regions on these genes are to continuously respond to such stimuli. Although base composition varies with mutation and recombination events (which is an expected biological plausibility) it is intriguing to speculate (1) how frequently the nucleotide base composition change in C. jejuni; (2) how much nucleotide changes can a C. jejuni gene and/or genome tolerate; (3) what are the biological triggers that generate new C. jejuni variants; (4) in the event of recombination how long does the exchanged gene portion take to coalesce; and (5) what are the functional genes mostly affected by evolutionary forces?\n\nEven though our study has made an effort to differentiate between two lineages of genes in C. jejuni and to substantiate and speculate the possible triggers for GC variation and its relationship with recombination, the data we used is very small to make concrete statements. However, larger genomic datasets will be able to provide distinctive resolution on the hypotheses formulated in our study and will also provide answers to the questions that we are raising in this paper.", "appendix": "Author Contributions\n\nVM did the data collection, analyses and wrote the paper in partial fulfillment of her PhD thesis. MS was VM’S supervisor and worked alongside her to get this manuscript out.\n\n\nGrant Information\n\nThis work was part of Vathsala Mohan’s PhD work and this project was supported by:\n\n1. Building Research Capability in Strategically Relevant Areas (BRCSRA) fund;\n\n2. Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North (RM12767); and\n\n3. The Royal Society of New Zealand Marsden Fund (MAU0802).\n\nThis was an original research work and the funders had no role in any of the processes or in the preparation of the manuscript.\n\n\nCompeting Interests\n\nNo competing interests were disclosed.\n\n\nAcknowledgements\n\nI thank Prof. Nigel French and Dr. Patrick Biggs, mEpiLab, IVABS, Massey University, for giving their comments on this piece of work. I thank Dr. Patrick Biggs for kindly providing me with the gene predictions for my PhD work. I thank Massey University Doctoral Scholarship and New Zealand International Doctoral Scholarship for funding me. This work was carried out in mEpiLab, IVABS, Massey University, New Zealand.\n\n\nReferences\n\nRocha EP: Evolutionary patterns in prokaryotic genomes. Curr Opin Microbiol. 2008; 11: p. 454–460.\n\nFeil EJ: Small change: keeping pace with microevolution. Nat Rev Microbiol. 2004; 2: p. 483–495.\n\nGenereux DP: Evolution of genomic GC variation. 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Ann Rev Genet. 2006; 60: p. 561–588." }	[ { "id": "922", "date": "02 May 2013", "name": "David Ussery", "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 article examines variation in GC content and recombination in two different sets of genes, across nineteen Campylobacter jejuni genomes. After reading the abstract several times, I'm still not sure exactly what hypothesis is being tested here. I am confused by the first sentence in the abstract, which states that C. jejuni is 'rapidly evolving' and has 'massive genetic recombination potential'. Compared to what? Certainly compared to a virus, C. jejuni is quite slowly evolving. Further, it seems from the larger picture of whole genome comparison, there is not THAT much difference within the C. jejuni genomes, compared to say for example E. coli, which can be three to four times the size of C. jejuni, and has a very large pan-genome - about ten times the size of any individual E. coli genome. Several years ago, based on a smaller set of genomes, we found that the C. jejuni genome was much less 'open' than the E. coli genome. (see PMID 19691844). I had to read and re-read the first paragraph of the Introduction section many times. Going through just that first paragraph:\"Molecular events such as mutation, deletion, recombination and gene transfer play paramount roles in shaping the evolution of prokaryotes (reviewed by1,2).\"I'm curious as to what exactly a 'molecular event' is? I would think that most molecular biologists are 'atomists' that is, they think in terms of biochemistry. Is there an alternative, perhaps vitalism or some supernatural event that is an alternative to a 'molecular event'. What is the difference between 'mutation' and 'deletion'? Would a 'deletion' not be considered a subset of 'mutation'? And what is meant by 'recombination'? Is this different to 'mutation'?\"As a consequence the genomes are more prone for nucleotide base compositional fluctuations.\"The [prokaryotes] are MORE prone than what? Viruses? Eukaryotes? Not sure what is being referred to here. Are the authors saying that mutations, deletions, recombination, gene transfer happen more often in bacteria than in eukaryotes? Or PERHAPS the fact that, because bacterial genomes tend to be more coding-rich (80% or more of the genome codes for proteins), then variations in the third codon position might allow genomes to become more AT rich or GC rich??\"Particularly, the evolutionary forces pose a major impact on the guanine-cytosine (GC) content of bacteria at the level of genes and genomes3.Amongst all the important evolutionary forces, the impact of recombination (homologous, non-homologous or illegitimate) on the evolution of bacteria has been evidenced as the major driving force or factor of microevolution4–8.\"So recombination is driving the changes in GC content? How does that work exactly? Or perhaps the authors here are talking about something other than differences in GC content? If so, this could be elaborated.\"However, the rate of recombination may differ greatly amongst different bacterial species; while some species recombine more frequently to have multiple recombination events than mutations that render them weakly clonal, whereas in other species it appears to be a rare incident leading to distinct clonal lineages5,6,8,9.\"And where does Campylobacter fit on this scale? Is there more variation, more recombination, or less? And, once again, perhaps it would be nice for the authors to mention that Campylobacter is very AT rich.\"Studies of genetic diversity in the bacterial kingdom have shown that bacteria form clusters of genetically related strains and that extensive recombination among related clusters have been regarded as normal rather than exceptional events10. However, not every single gene is involved in recombination or horizontal gene transfer11,12.\"This is comforting perhaps, but an indication of the fraction of genes undergoing recombination in Campylobacter would be good…In the Methods section, the NCBI refseq number is given, rather than a true 'GenBank accession number', which for the C. jejuni genome is AL111168. The methods section states that the genomes were downloaded from GenBank - but it appears that perhaps NCBI RefSeq was used instead. RefSeq is a somewhat curated database, with different gene annotations (and hence there could be different gene lengths which might give different %GC contents) - so it is very important to clearly state WHICH database was actually used here. The INSDC accession numbers are shared between GenBank, EMBL, and the DNA DataBase of Japan (DDBJ). To be honest, I really do not see that large of a difference between the variation in %GC in the housekeeping genes vs. information genes. I think a simple box and whiskers plot, showing the genome distribution, compared to the distribution for the housekeeping vs. information genes is the best way to visualise and compare the three distributions. I suspect that there's not that much of a difference here. (Which by the way, I looked through the manuscript several times - nowhere could I find a mention or even brief discussion that C. jejuni is quite AT rich, compared to other bacterial genomes).The conclusion, that the information genes have less recombination than the housekeeping genes is perhaps not surprising, but I'm not sure how 'recombination' is measured here - the authors refer to a computer program which was used to measure recombination in HIV sequences, with high rates of changes. However, applying this model to something like Campylobacter, where the variation is extremely low (compared to HIV certainly). So if there is a single nucleotide difference, is this called a 'recombination event'? Are SNPs recombination events? Maybe sometimes?In summary, I find this short paper would reflect a nice student project, done as part of a course. It is a good exercise to write up what has been done, but I'm still missing the hypothesis that is being tested, the anticipated results, and what sorts of results would falsify the hypothesis. Why not come up with a very clear hypothesis that can be tested, and run it across the roughly 80 C. jejuni genomes available from NCBI? http://www.ncbi.nlm.nih.gov/genome/149", "responses": [] }, { "id": "932", "date": "07 May 2013", "name": "Trudy M. Wassenaar", "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 compared 50 genes, obtained from 12 Campylobacter genomes, to test two hypotheses. I rephrase them for clarity here as: (1) Does GC content provide evidence of recombination events, and can this shed light on the history of recombinational events in the Campylobacter genome? (2) Are housekeeping genes more prone for recombination than informational genes in Campylobacter? From the 'Results and discussion' section it is not clear whether the authors have confirmed or rejected these hypotheses.I have difficulties with both of these hypotheses. My concerns with the first hypothesis are the following: The authors consider only recombination between donor DNA with a higher GC-content into an AT-rich Campylobacter genome. They ignore that the vast majority of recombination cab occur within a Campylobacter species, or, to a lesser extent, between closely related species (C. jejuni and C. coli), in which case there is no base composition difference between donor and acceptor and you wouldn't see amelioration of GC content. The 'recombination' that the authors have identified and use in their analysis (they even mention recombination sites although it is unclear how these were identified) have more likely taken place between alleles of different Campylobacter clones than between genes of different species.My concern with the second hypothesis is that there is no fundamental genetic or physiological difference between 'housekeeping genes' and 'informational genes'. It is a man-made division only, while the cell just maintains its physiology by means of all these proteins. The only relevant distinction here is whether a gene product is active as a sole contributor to a process (an enzyme, say, that works on a single substrate-to-product conversion without interaction with other factors) or whether a gene product acts in close contact with many other gene products (a ribosomal protein, say). The latter are constrained in mutation, but only for non-synonymous mutations. The authors ignore codon usage effects, expression levels (highly expressed genes employ different codons), and compositional constraints of proteins that are reflected by their codons and thus affect the gene's GC content.A recent paper studied evolution in the complete core genome of 27 Campylobacter genomes. It covered more than 1100 genes and employed various statistical methods. That paper reported that there was NO division in evolutionary signature between informational versus housekeeping genes (Snipen et al., (2012) Analysis of evolutionary patterns of genes in Campylobacter jejuni and C. coli. Microb. Inform. Expt. 2:8).", "responses": [] } ]	1	https://f1000research.com/articles/2-89
https://f1000research.com/articles/2-87/v1	14 Mar 13	{ "type": "Data Article", "title": "Molecular data analysis of selected housekeeping and informational genes from nineteen Campylobacter jejuni genomes", "authors": [ "Vathsala Mohan", "Mark Stevenson", "Mark Stevenson" ], "abstract": "Campylobacter jejuni (C. jejuni) is a rapidly evolving bacterial species with massive genetic recombination potential to generate niche specific genotypes. Generally the housekeeping gene lineage has been evidenced to undergo lateral gene transfer and recombination fairly frequently compared to the information processing gene lineage. During such exchanges, gene amelioration takes place over time acquiring the host genomes’ molecular characteristics. In this study fifty genes that comprised twenty five housekeeping lineage genes and twenty five information processing lineage genes from nineteen C. jejuni genomes were studied. These nineteen genomes included seven C. jejuni isolates that belonged to the same genotype or multilocus sequence type ST-474. The genes from both lineages were tested for recombination and the guanine-cytosine (GC) variation. This paper details about the data collected and the analyses performed in the corresponding research article entitled ”Campylobacter jejuni genomes exhibit notable GC variation within housekeeping genes”. Further, this paper provides details on the results that are not included in the research paper to provide completeness to the study conducted. The gene sequences from the seven C. jejuni ST-474 isolates were submitted to the GenBank and the corresponding gene IDs are provided for referencing purposes.", "keywords": [ "Mulitlocus sequence typing (MLST) is a technique devised to sub-divide bacterial populations1. MLST exploits the internal fragments that are approximately 470 to 500 base pairs of the housekeeping genes and constructs allelic profiles based on variations in the nucleotide sequences of (usually) seven housekeeping genes. Bacterial isolates are grouped into different clusters2. C. jejuni is a zoonotic pathogen that causes gastroenteritis in humans worldwide and the natural competency and plasticity of C. jejuni have not been investigated outside the MLST housekeeping genes3–8. This technique has subsequently been exploited to structure and investigate the association of C. jejuni populations where", "previous researches inferred that a mutation at any site can occur somewhere in the population within the space of a week7–9. Hybrid alleles were shown to be more common in some of the MLST loci (tkt", "aspA", "gltA and glyA) and in the MOMP gene of C. jejuni (interchangeably designated as porA) as a result of recombination and", "has been interpreted as a consequential measure of convergence of C. jejuni and C. coli7", "8", "10." ], "content": "Background\n\nMulitlocus sequence typing (MLST) is a technique devised to sub-divide bacterial populations1. MLST exploits the internal fragments that are approximately 470 to 500 base pairs of the housekeeping genes and constructs allelic profiles based on variations in the nucleotide sequences of (usually) seven housekeeping genes. Bacterial isolates are grouped into different clusters2. C. jejuni is a zoonotic pathogen that causes gastroenteritis in humans worldwide and the natural competency and plasticity of C. jejuni have not been investigated outside the MLST housekeeping genes3–8. This technique has subsequently been exploited to structure and investigate the association of C. jejuni populations where, previous researches inferred that a mutation at any site can occur somewhere in the population within the space of a week7–9. Hybrid alleles were shown to be more common in some of the MLST loci (tkt, aspA, gltA and glyA) and in the MOMP gene of C. jejuni (interchangeably designated as porA) as a result of recombination and, has been interpreted as a consequential measure of convergence of C. jejuni and C. coli7,8,10.\n\nHousekeeping alleles of C. jejuni strains (genotypes) share 86% nucleotide sequence identity with each other however, the housekeeping alleles cover lesser than < 0.2% of the entire genome11. The molecular differences at the genome level and/or at the full gene level may not be detected even when MLST is combined with other antigenic genes such as porA. Moreover, the housekeeping alleles or genes are involved in specific metabolic function which may not reflect the evolutionary events in other genes involved in a different function in a genome. Further, there is no evidence to suggest that MLST alleles are identical at their full length level and it is not known if a detailed knowledge of MLST alleles can capture the true evolutionary history of the other genes in a genome.\n\n\nObjectives\n\nBased on these background information and knowledge gap, the nucleotide sequence data from two different lineages of genes, the metabolic housekeeping and the informational genes were collected from 19 C. jejuni genomes. The main objectives were\n\n(1) To select genes those are categorised as housekeeping genes.\n\n(2) To ensure the presence of these genes in all of the C. jejuni genomes compared without segmentation.\n\n(3) To categorise them according to the functions they are involved in.\n\n(4) To create two different nucleotide datasets for comparison and,\n\n(5) To test different genetic hypotheses.\n\n\nMaterials and methods\n\nAlthough genes have been classified as housekeeping and informational genes, the list of genes that can be categorised as housekeeping genes have not be defined very clearly. Hence in this study we collected the set of genes that have been used in routine multilocus sequence typing (MLST) schemes for different bacterial species from the PubMLST database and from previous phylogenetic studies that used housekeeping genes for analyses12–14. The gene names were matched with the C. jejuni NCTC 11168 genome (GenBank accession number NC002163) that is fully annotated and has been used as reference in many other genome comparative studies15–20. Following the identification of genes, a total of 50 genes were selected and these were further examined for their presence in other C. jejuni reference genomes to be analysed in this study. Full length nucleotide sequences of the selected set of housekeeping genes were retrieved in FASTA format from the GenBank from all of the selected C. jejuni genomes. Further the genes were classified into operational genes (metabolic housekeeping genes) and informational genes based on their function by referring to the KEGG pathway and the gene function websites. The positions of the categorised selected subset of genes (metabolic housekeeping and informational genes) are marked on the reference C. jejuni NCTC 11168 (GenBank accession number NC002163) circular genome and are shown in Figure 1A and Figure 1B, respectively. Here after in this paper the genes are referred to as housekeeping genes (metabolic housekeeping genes) and informational genes for the purpose of plain comparison and interpretation.\n\nDetails of the reference C. jejuni genomes, housekeeping and informational genes, their names and their functions are provided in Table 1, Table 2 and Table 3, respectively. Twelve fully sequenced C. jejuni genomes were used to compare 50 selected genes. The gene sequences were downloaded from the GenBank database. Seven C. jejuni MLST ST-474 isolates were sequenced at the Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand. The gene predictions were kindly provided by Dr. Biggs Patrick from Massey Genome Service, Massey University, Palmerston North for the seven C. jejuni MLST ST-474 isolates. These genomes were studied in partial fulfilment of the PhD project by the first author of this paper. The nucleotide sequences for the selected genes (n = 50) were retrieved from the gene predictions from the seven C. jejuni ST-474 genomes in FASTA format and gene sequences for five of the four ST-474 isolates P179a, H704a, H73020, P569a and P694a were submitted to the GenBank and their sequence ids are given in Table 4 and Table 5 for metabolic and informational or repair genes, respectively. In addition this data is accessible below:\n\nST: Multilocus sequence sequence type; GBS: Guillain-Barré syndrome; ORFs: Open reading frames; Mb: Mega bases; GC: Guanine: Cytosine; ns: not stated; *:Date of start of project.\n\nThe length of the house keeping and the informational genes and sites of recombination can be found below:\n\nThe results of the guanine-cytosine (GC) analysis with the GC range for all of the genes from 19 C. jejuni genomes can be found below:\n\nThe overall GC and GC3 contents of individual genes were compared using DnaSP v521. The frequency distribution graphs of GC contents from all the 19 genomes compared were generated in R programming language22. Inferences on recombination within each gene under investigation were drawn using Dual-Brothers within Geneious v.5.3.423. This function uses a double change point model that detects spatial variation in the phylogenetic tree topology and spatial variation of the nucleotide substitution process23. The gene sequences were aligned using Geneious v5.3.4 and the recombination detection functionality was applied on each gene alignment to infer changes in the topologies and substitution processes. In addition, the aligned sequences were examined using DnaSP v5 to identify the sites involved in recombination. The number of recombination events, referred to as Rm was estimated using DnaSP v5. The GC variance for the genes were estimated using the variance function in Microsoft Office Excel and the relationship between the GC variance and the recombination events was analysed using linear models by having Rm as a dependent variable, and the log GC variance and the length as independent variables.\n\nFigure 2–Figure 9 describe the guanine-cytosine (GC) variation in the housekeeping and the informational genes. The plots describe the frequency distribution of GC content across 19 C. jejuni genomes and the number inside the parenthesis denotes the number of recombination sites for the respective genes.\n\nThe plots describe the distribution of GC across 19 C. jejuni genomes and the number inside the parenthesis denotes the number of recombination sites.\n\nThe plots describe the distribution of GC across 19 C. jejuni genomes and the number inside the parenthesis denotes the number of recombination sites.\n\nThe plots describe the distribution of GC across 19 C. jejuni genomes and the number inside the parenthesis denotes the number of recombination sites.\n\nThe plots describe the distribution of GC across 19 C. jejuni genomes and the number inside the parenthesis denotes the number of recombination sites.\n\nThis plot describes the GC distribution across all 19 C. jejuni genomes. The number of recombination sites is provided in parentheses.\n\nThis plot describes the GC distribution across all 19 C. jejuni genomes. The number of recombination sites is provided in parentheses.\n\nThis plot describes the GC distribution across all 19 C. jejuni genomes. The number of recombination sites is provided in parentheses.\n\nThis plot describes the GC distribution across all 19 C. jejuni genomes. The number of recombination sites is provided in parentheses.", "appendix": "Author Contributions\n\nVM did the data collection, analyses and wrote the paper in partial fulfillment of her PhD thesis. MS was VM’S supervisor and worked alongside her to get this manuscript out.\n\n\nGrant Information\n\nThis work was part of Vathsala Mohan’s PhD work and this project was supported by:\n\n1. Building Research Capability in Strategically Relevant Areas (BRCSRA) fund;\n\n2. Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North (RM12767); and\n\n3. The Royal Society of New Zealand Marsden Fund (MAU0802).\n\nThis was an original research work and the funders had no role in any of the processes or in the preparation of the manuscript.\n\n\nCompeting Interests\n\nNo competing interests were disclosed.\n\n\nAcknowledgements\n\nI thank Prof. Nigel French and Dr. Patrick Biggs, mEpiLab, IVABS, Massey University, for giving their comments on this piece of work. I thank Dr. Patrick Biggs for kindly providing me with the gene predictions for my PhD work. I thank Massey University Doctoral Scholarship and New Zealand International Doctoral Scholarship for funding me. This work was carried out in mEpiLab, IVABS, Massey University, New Zealand.\n\n\nReferences\n\nMaiden MCJ: Multilocus Sequence Typing of Bacteria. Annu Rev Microbiol. 2006; 60: p. 561–588.\n\nMaiden MCJ, Bygraves JA, Feil E, et al:Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A. 1998; 95: p. 3140–3145.\n\nColles FM, Dingle KE, Cody AJ, et al:Comparison of Campylobacter populations in wild geese with those in starlings and free-range poultry on the same farm. Appl Environ Microbiol. 2008; 74: p. 3583–3590.\n\nDingle KE, Colles FM, Wareing DR, et al:Multilocus sequence typing system for Campylobacter jejuni. J Clin Microbiol. 2001; 39: p. 14–23.\n\nMcCarthy ND, Colles FM, Dingle KE, et al:Host-associated genetic import in Campylobacter jejuni. Emerg Infect Dis. 2007; 13: p. 267–272.\n\nSheppard SK, Colles F, Richardson J, et al:Host association of Campylobacter genotypes transcends geographic variation. Appl Environ Microbiol. 2010; 76: p. 5269–5277.\n\nSheppard SK, McCarthy ND, Falush D, et al:Convergence of Campylobacter Species: implications for Bacterial Evolution. Science. 2008; 320: p. 237–239.\n\nSheppard SK, McCarthy ND, Jolley KA, et al:Introgression in the genus Campylobacter: Generation and spread of mosaic alleles. Microbiology. 2011; 157: p. 1066–1074.\n\nWilson DJ, Gabriel E, Leatherbarrow AJ, et al:Rapid Evolution and the Importance of Recombination to the Gastroenteric Pathogen Campylobacter jejuni. Mol Biol Evol. 2009; 26: p. 385–397.\n\nClark CG, Beeston A, Bryden L, et al:Phylogenetic relationships of Campylobacter jejuni based on porA sequences. Can J Microbiol. 2007; 53: p. 27–38.\n\nDingle KE, Maiden MC: In Campylobacter: Molecular and Cellular Biology. ed. M.E.K.E.p. Ketley JM. Ketley JM, Konkel ME, Eds Horizon Bioscience, Wymondham, Norfolk, UK, 2005.\n\nWertz JE, Goldstone C, Gordon DM, et al:A molecular phylogeny of enteric bacteria and implications for a bacterial species concept. J Evol Biol. 2003; 16: p. 1236–1248.\n\nChristensen H, Kuhnert P, Olsen JE, et al:Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae. Int J Syst Evol Microbiol. 2004; 54: p. 1601–1609.\n\nMargos G, Gatewood AG, Aanensen DM, et al:MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc Natl Acad Sci U S A. 2008; 24: p. 8730–8735.\n\nFouts DE, Mongodin EF, Mandrell RE, et al:Major structural differences and novel potential virulence mechanisms from the genomes of multiple Campylobacter species. PLoS Biol. 2005; 3: e15.\n\nGundogdu O, Bentley SD, Holden MT, et al:Re-annotation and re-analysis of the Campylobacter jejuni NCTC11168 genome sequence. BMC Genomics. 2007; 8: p. 162.\n\nHofreuter D, Tsai J, Watson RO, et al:Unique features of a highly pathogenic Campylobacter jejuni strain. Infect Immun. 2006; 74: p. 4694-4707.\n\nLefebure T, Stanhope MJ: Pervasive, genome-wide positive selection leading to functional divergence in the bacterial genus Campylobacter. Genome Res. 2009; 19: p. 1224–1232.\n\nParker CT, Quiñones B, Miller WG, et al:Comparative genomic analysis of Campylobacter jejuni strains reveals diversity due to genomic elements similar to those present in C. jejuni strain RM1221. J Clin Microbiol. 2006; 44: p. 4125–4135.\n\nParkhill J, Wren BW, Mungall K, et al:The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature. 2000; 403: p. 665–668.\n\nLibrado P, Rozas J: DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009; 25: p. 1451–1452.\n\nZhou BB, Elledge SJ: The DNA damage response: putting checkpoints in perspective. Nature. 2000; 408: p. 433–439.\n\nMinin VN, Dorman KS, Fang F, et al:Dual multiple change-point model leads to more accurate recombination detection. Bioinformatics. 2005; 21: p. 3034–3042." }	[ { "id": "921", "date": "02 May 2013", "name": "David Ussery", "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 article describes a comparison of 'housekeeping genes' with 'information processing genes', across 19 Campylobacter jejuni genomes. I cannot fully evaluate this paper as the data is not available. I understand that this is a new journal, and as such perhaps F1000Research has not agreed that all sequences should be deposited to GenBank or EMBL before publication, but I find this frustrating that the results are published, but the data is missing.Since currently there are only 11 C. jejuni genomes listed as 'complete', I was curious as to what additional genomes were used in this study. See:http://www.ncbi.nlm.nih.gov/genome/browse/Unfortunately, it seems that only about half of the finished genomes were used (that is, 6 out of 11), and an additional set of apparently randomly chosen 6 unfinished, draft genome sequences were used, in addition to the apparently draft sequences of another 7 genomes of sequence type ST-474. Although it is stated that seven of the genomes have been deposited to GenBank, they are still not present in the database of complete genomes, as of 1 May, 2013 (the article was published about 6 weeks ago, on 14 March, 2013). In Table 1, I can see accession numbers for DRAFT sequences for two of the seven ST-474 genomes, and the remaining five do not appear to be listed in the NCBI genomes project pages, which includes even planned projects with no data yet. But the two ST-474 genomes listed in Table 1 do not appear to have the sequence type (ST-474) in the GenBank file; instead, all that is listed is \"rare MLST strain\". This missing meta-data makes it difficult to know if the other five genomes are perhaps there, but I cannot find them, even if I search for the strain name (e.g., P179a) provided for in Table 1.Figure 1 shows the location of the housekeeping genes in the NCTC 11168 genome (which I recognise because I know that the GenBank accession number is for that strain - but wouldn't it be much more clear to just label this chromosome as 'C. jejuni', and then put the strain name (NCTC 11168)? I am curious as to whether there's a significant AT content difference in the genes in the top half (e.g., closer to the replication origin), compared to the lower half (replication terminus). How does this difference compare to that found between the two gene sets?From my perspective, Figures 2 - 9 are not very informative at all. These could be collapsed perhaps into a SINGLE box and whisker's plot, where the distributions could easily be compared with each other.", "responses": [] }, { "id": "3557", "date": "07 Feb 2014", "name": "Daniel Falush", "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\nI do not understand either from the abstract or reading the earlier paper why this data was collected. Neither the strain collection nor the quantities calculated have any obvious special features. The information presented is of a similar character to statistics that are automatically generated for example by the pubMLST database. There is no reason to clog up the scientific literature with such reports.", "responses": [] } ]	1	https://f1000research.com/articles/2-87
https://f1000research.com/articles/1-25/v1	09 Oct 12	{ "type": "Short Research Article", "title": "Longhorned beetle (Coleoptera: Cerambycidae) diversity in a fragmented temperate forest landscape", "authors": [ "Daniel M Pavuk", "Andrea M Wadsworth", "Andrea M Wadsworth" ], "abstract": "Longhorned beetles (Coleoptera: Cerambycidae) are an important component of temperate forest ecosystems. We trapped longhorned beetles in forests in northwest Ohio during 2008 to test the hypothesis that larger forests have greater species diversity than smaller forests. Large forests had a significantly greater cerambycid species richness than small forests (t = 3.16. P = 0.02), and there was a significant relationship between forest size and cerambycid species richness.", "keywords": [ "Longhorned beetles", "or cerambycids", "are important species in temperate forest ecosystems", "due to their feeding impacts on trees. Many cerambycids feed on dead wood and therefore assist in the decomposition of dead trees in forest ecosystems. Saproxylic cerambycids (dead wood dependent) and other saproxylic beetles are thought to be useful indicators of forest biodiversity1. We were interested in testing the hypothesis that larger forests have greater cerambycid species diversity than smaller forests in NW Ohio", "a highly fragmented landscape in terms of forest ecosystems." ], "content": "Introduction\n\nLonghorned beetles, or cerambycids, are important species in temperate forest ecosystems, due to their feeding impacts on trees. Many cerambycids feed on dead wood and therefore assist in the decomposition of dead trees in forest ecosystems. Saproxylic cerambycids (dead wood dependent) and other saproxylic beetles are thought to be useful indicators of forest biodiversity1. We were interested in testing the hypothesis that larger forests have greater cerambycid species diversity than smaller forests in NW Ohio, a highly fragmented landscape in terms of forest ecosystems.\n\n\nMethodology\n\nThree types of traps (Lindgren funnel trap, Intercept Panel trap, and Window trap) were set up in each of 8 forests in northwestern Ohio. 95% ethanol was used to attract beetles (Figure 1–Figure 3).\n\nWe started collecting beetles in early June, and we continued to collect them until early October (Figure 4).\n\nWe put the traps into 8 different forest areas. Four forests were large (>100 hectares) and four forests were classified as small (<20 hectares).\n\nStrophiona nitens (top left), Gaurotes cyanipennis (top right) Urographus fasciatus (bottom left) and Microgoes oculatus (bottom right).\n\n\nResults and discussion\n\nThe number of individual species that were collected at the 8 different forest sites can be viewed in Table 1–Table 8.\n\nLarge forests had greater cerambycid species richness than small forests (Figure 5–Figure 6).\n\nFuture research should focus on the landscape matrix and degree of isolation of forests, especially isolation of smaller forests.\n\nMany other beetle species from other families were also captured (e.g., Elateridae, Curculionidae), so these data should also be examined.\n\nTotal Number of individuals of each species caught during the entire trapping period (early June to early October). Traps were positioned in the approximate center of each forest and all checked each week.\n\nTotal Number of individuals of each species caught during the entire trapping period (early June to early October). Traps were positioned in the approximate center of each forest and all checked each week.\n\nTotal Number of individuals of each species caught during the entire trapping period (early June to early October). Traps were positioned in the approximate center of each forest and all checked each week.\n\nTotal Number of individuals of each species caught during the entire trapping period (early June to early October). Traps were positioned in the approximate center of each forest and all checked each week.\n\nTotal Number of individuals of each species caught during the entire trapping period (early June to early October). Traps were positioned in the approximate center of each forest and all checked each week.\n\nTotal Number of individuals of each species caught during the entire trapping period (early June to early October). Traps were positioned in the approximate center of each forest and all checked each week.\n\nTotal Number of individuals of each species caught during the entire trapping period (early June to early October). Traps were positioned in the approximate center of each forest and all checked each week.\n\nTotal Number of individuals of each species caught during the entire trapping period (early June to early October). Traps were positioned in the approximate center of each forest and all checked each week.\n\nThe four large forests were Oak Opening, Secor, Pearson, and Wildwood, and the four small forests were Bradner Preserve, Fuller Preserve, Carter Woods, and Environmental Studies Woods. The t-test was significant (t = 3.16, df = 6, P = 0.02).\n\nOak Openings, Secor, Wildwood, and Pearson were the Large Forests, and Bradner Preserve, Carter Woods, Fuller Preserve, and ENVS Woods were the small forests.", "appendix": "Author contributions\n\n\n\nDaniel M. Pavuk set the traps, collected the beetles, identified the beetles to species, and performed the statistical analysis. Andrea M. Wadsworth assisted with the collection of the beetles and sorting the beetles.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by funds provided by The Department of Biological Sciences, Bowling Green State University, Bowling Green Ohio.\n\n\nAcknowledgements\n\nWe thank Mr. John Jaeger, Director of Natural Resources, Toledo, Metroparks, and Mr. Chris Smalley, Stewardship Director, Wood County, OH, Parks, for permission to do research in their parks and preserves. Thanks also to Dr. Jeff Holland, Purdue Univ., for his advice and suggestions on this research project.\n\nThis work was previously presented as a poster (PS 90-42) at the Ecological Society of America Annual meeting 2012.\n\n\nReferences\n\nHolland JD: Sensitivity of Cerambycid indicators to definition of high diversity. Biodivers Conserv. 2007; 16(9): 2599–2609. Publisher Full Text" }	[ { "id": "329", "date": "15 Oct 2012", "name": "Patrick Tobin", "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 overall merits of the study, general aspects of the experimental design, and hypothesis tested are interesting and conceptually sound.The incredible amount of work involved in such a study is also recognized. A primary concern is that only one year of data was collected, which limits the interpretation of the findings. Measuring populations in the field over only one year, from which species richness was estimated, can be subject to annual variation that may lead to over- or underestimates of individuals in any given year. Moreover, some species of Cerambycidae may take more than one year to undergo a generation and thus these species could be undercounted. Regardless, the finding of increased richness with increased forest size confirms prior observations in related diversity studies. Also, the methods applied in this study are sound and thus provide a protocol in future endeavors.One modification I would like to see in a revised manuscript is that instead of examining differences in richness between two categories (large versus small forests), I would prefer to see richness examined along a continuous measurement (i.e., the size of each forest), assuming that there is variation in sizes among the forests. In looking at Figure 6, I wonder if the respective area of each forest could explain the difference in species number. It also could be insightful to consider estimating diversity using established indices (e.g., Shannon, Simpson), which could help relate your work to other similar studies.", "responses": [] }, { "id": "330", "date": "17 Oct 2012", "name": "Peter Silk", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/1-25
https://f1000research.com/articles/2-44/v1	13 Feb 13	{ "type": "Research Article", "title": "Reactive oxygen production induced by near-infrared radiation in three strains of the Chl d-containing cyanobacterium Acaryochloris marina", "authors": [ "Lars Behrendt", "Marc Staal", "Simona M Cristescu", "Frans JM Harren", "Martin Schliep", "Anthony WD Larkum", "Michael Kühl", "Lars Behrendt", "Marc Staal", "Simona M Cristescu", "Frans JM Harren", "Martin Schliep", "Anthony WD Larkum" ], "abstract": "Cyanobacteria in the genus Acaryochloris have largely exchanged Chl a with Chl d, enabling them to harvest near-infrared radiation (NIR) for oxygenic photosynthesis, a biochemical pathway prone to generate reactive oxygen species (ROS). In this study, ROS production under different light conditions was quantified in three Acaryochloris strains (MBIC11017, HICR111A and the novel strain CRS) using a real-time ethylene detector in conjunction with addition of 2-keto-4-thiomethylbutyric acid, a substrate that is converted to ethylene when reacting with certain types of ROS. In all strains, NIR was found to generate less ROS than visible light (VIS). More ROS was generated if strains MBIC11017 and HICR111A were adapted to NIR and then exposed to VIS, while strain CRS demonstrated the opposite behavior. To our knowledge, this is the first study of ROS generation associated with NIR-driven oxygenic photosynthesis and it suggests that Acaryochloris can avoid a considerable amount of light-induced stress by using NIR instead of VIS for its photosynthesis, adding further evolutionary arguments to their widespread appearance.", "keywords": [ "Most oxyphototrophs use visible light (VIS", "400–700 nm) for chlorophyll (Chl) a-driven photosynthesis", "while cyanobacteria in the genus Acaryochloris largely employ Chl d", "thereby enabling them to use near-infrared radiation (NIR", ">700 nm) for oxygenic photosynthesis1", "2. Two of the strains are well described in their growth and photopigment composition: the type strain Acaryochloris marina MBIC11017 was isolated from the didemnid ascidian Lissoclinum patella from coral reef habitats in Palau2", "3 and was later genome sequenced4. Since its first discovery", "other Acaryochloris strains have been obtained from Japanese macroalgae (Strain Awaji5)", "from surfaces in a hypertrophic lake in the US (strain CCMEE5410)6", "from swipes of coral substrate collected on Heron Island", "Australia (strain HICR111A)7", "and most recently from an Australian mangrove (strain MPGRS1)8 and stromatolites in Shark Bay", "Western Australia (strain ssball1)9." ], "content": "Introduction\n\nMost oxyphototrophs use visible light (VIS, 400–700 nm) for chlorophyll (Chl) a-driven photosynthesis, while cyanobacteria in the genus Acaryochloris largely employ Chl d, thereby enabling them to use near-infrared radiation (NIR, >700 nm) for oxygenic photosynthesis1,2. Two of the strains are well described in their growth and photopigment composition: the type strain Acaryochloris marina MBIC11017 was isolated from the didemnid ascidian Lissoclinum patella from coral reef habitats in Palau2,3 and was later genome sequenced4. Since its first discovery, other Acaryochloris strains have been obtained from Japanese macroalgae (Strain Awaji5), from surfaces in a hypertrophic lake in the US (strain CCMEE5410)6, from swipes of coral substrate collected on Heron Island, Australia (strain HICR111A)7, and most recently from an Australian mangrove (strain MPGRS1)8 and stromatolites in Shark Bay, Western Australia (strain ssball1)9.\n\nThe light microenvironment in natural habitats occupied by Acaryochloris spp. has a high contribution of NIR relative to visible light10–12 and such habitats appear to create similar niche differentiation with bacteria carrying specialized photopigments such as Chl d/f or bacteriochlorophylls10,13. The notion of a global distribution of Chl d and cyanobacteria in the genus Acaryochloris11,14 further reinforces the need to obtain information on the photobiology of Chl d-containing oxyphototrophs. Understanding the adaptive mechanisms in oxyphototrophs capable of using wavelengths beyond VIS is of interest as it provides information concerning the usability, stress levels and limitations associated with NIR-driven oxygenic photosynthesis.\n\nOf all biological pathways, photosynthetic electron transport is particularly prone to produce reactive oxygen species (ROS) due to the very high (positive) redox potential of the primary donor of photosystem II needed to oxidize water, and the low redox potential of the primary electron acceptor of photosystem I needed to reduce ferredoxin; here, singlet oxygen (1O2) is produced by PSII and superoxide anions (-O2)/hydrogen peroxide (H2O2) in the Mehler-ascorbate peroxidase (MAP) pathway of PSI15–17. ROS encompasses the production of singlet oxygen, superoxide anions, hydrogen peroxide and hydroxyl radicals (OH), all of which are derived through the local energization of O2. If not properly quenched by protective mechanisms, ROS can damage proteins, DNA and other cellular macromolecules, and this damage can ultimately lead to cell death. Known quenching mechanisms encompass enzymes such as superoxide dismutase and catalase or non-enzymatic antioxidants like glutathione, carotenoids and α-tocopherol (vitamin E)18. In plants, ROS and in particular 1O2 production has been shown to occur at photosystem II upon illumination with visible light19,20. In cyanobacteria, shorter wavelengths such as ultraviolet radiation (UVR, <400 nm) are known to induce ROS, causing DNA damage, lipid-peroxidation and overall decreased photosynthetic efficiency21,22. To our knowledge, no study has investigated the effect of NIR on ROS production in cyanobacteria.\n\nRelative levels of ROS can be estimated through measurements of for example gene expression23, ROS-sensitive fluorescence probes24 and enzyme activity25. These methods provide integrated values of ROS production over incubation time intervals ranging from minutes to hours. In this study we used a fast and sensitive laser photo-acoustic gas detector26 that can measure the ethylene produced from the reaction of certain types of ROS with the substrate 2-keto-4-thiomethylbutyric acid (KMBA), previously added to the samples. Such near real-time ROS detection is valuable in determining the immediate effect of treatments on the physiological state and stress level within living organisms. KMBA is thought to diffuse into intact cells27 and, when supplied at saturating concentrations, outcompetes other radical scavenging mechanisms. In the KMBA assay, the butyric acid moiety reacts with ROS like peroxynitrite, hydroxyl radicals and peroxyl radicals28, resulting in the formation of ethylene, which can then be quantified. In other studies, KMBA has been used to test the antioxidant capacity of radical scavengers via their ability to inhibit ethylene formation relative to a control reaction (total oxyradical scavenging capacity, TOSC)28,29.\n\nIn this study, we report the effect of light intensity and spectral composition on ROS generation, as measured in real-time using a laser-photoacoustic gas detector in three different strains of NIR utilizing cyanobacteria belonging to the genus Acaryochloris, including a new strain (named Acaryochloris CRS), isolated from phototrophic biofilms growing on dead coral branches collected on Heron Island, Australia.\n\n\nResults and discussion\n\nWe aimed to determine the stress levels associated with Chl d-driven oxygenic photosynthesis and tested NIR, VIS and narrower wavebands for their capacity to induce ROS in three strains of Acaryochloris: i) The Acaryochloris marina type strain MBIC110172,3, ii) strain HICR111A7, and iii) a novel strain, named CRS, that we isolated from dead coral branches which, based on 16S rRNA gene sequencing, grouped within the genus Acaryochloris (Figure 1). Acaryochloris strains MBIC11017 and HICR111A are both well described in terms of their photopigmentation, genomic content, ultrastructure and their capability to perform photoacclimation4,7,30. To test whether photoacclimation, i.e., light-dependent change in pigment levels, was associated with increased resistance or sensitivity towards ROS, we acclimated the strains to NIR or VIS prior to experiments. By taking advantage of the real-time ROS detection method, we could for the first time demonstrate the immediate effects of NIR, VIS and other wavelengths on the ROS pools within living cyanobacteria Specifically, we found that:\n\n(i) Depending on strain and previous adaptation, ROS levels were lower in cells exposed to NIR than in those exposed to VIS (Figure 2A);\n\n(ii) Exposure to shorter wavelengths such as blue and cyan, generated the most ROS in strain MBIC11017 and HICR111A, while less ROS was produced upon exposure to longer wavelengths (green, amber and red) (Figure 2C).\n\n(iii) Strain CRS generated less ROS upon exposure to VIS when previously acclimated to NIR, while strains MBIC11017 and HICR111A appear more sensitive to VIS when adapted to NIR (Figure 2A).\n\nSequences from other cyanobacteria (35 in total) were obtained from the SILVA database while CRS-specific sequences were obtained through PCR amplification and subsequent sequencing. Phylogeny was calculated using Neighbor-joining methods and Jukes-Cantor substitution models as implemented in MEGA5. Tree stability was assessed using bootstrapping at 10000 replications. Only bootstrap values >50% are displayed within the tree. The scale represents 0.02 substitutions per nucleotide position. The green-sulphur bacterium Chlorobium tepidum TLS was chosen as an outgroup.\n\nAll values were normalized to Chl d concentrations as determined by spectrophotometry. All cultures were grown under either near-infrared radiation (NIR, 720 nm) or visible light (VIS, 400–700 nm) before subsequent light exposure. (A) Cleveland dot-plot of ROS production (in nl Ethylene h-1 µg-1 Chl d) measured during exposure of VIS or NIR adapted Acaryochloris cells to either VIS (blue dots, 340–480 µmol photons m-2 s-1, as denoted on the graph) or NIR (red squares, 400 µmol photons m-2 s-1). (B) Growth forms of the different Acaryochloris strains MBIC11017, HICR111A and CRS. All strains displayed were grown under NIR and are approximately one week old. Please note the natural formation of biofilms in Acaryochloris strain HICR111A (arrow ) and CRS. (C) Action spectrum of ROS-induced ethylene production (nl Ethylene h-1 µg-1 Chl d) in VIS or NIR adapted strains MBIC11017 and HICR111A. Due to lack of sufficient culture material, action spectra were not determined for Acaryochloris strain CRS. Peak emissions of the monochromatic LEDs used for illumination were: red (645 nm), amber (595 nm), green (535 nm), cyan (495 nm) and blue (470 nm). The irradiance in this experiment was adjusted to 300 µmol photons m-2 s-1.\n\n\n\nIn Acaryochloris, VIS irradiance is primarily absorbed by the photopigments Chl d (with maximum absorption occurring at 440–470/710 nm), Chl a (440–470/675 nm), carotenoids (440–520 nm) and if present, phycobiliproteins (560–650 nm). NIR provides a more targeted stimulation of photosynthesis and is almost exclusively absorbed by Chl d. At comparable photon irradiances (VIS = 340–480 µmol m-2 s-1 versus NIR = 400 µmol m-2 s-1), we found that, depending on strain and previous adaptation, ROS levels were lower in cells exposed to NIR than in those exposed to VIS (Figure 2A). Based on pulse-amplitude modulated (PAM) fluorometry measurements, the light intensities used in our experiments are known to saturate relative-electron transport rates in the type strain MBIC1101712. Photoinhibition was not observed even at higher photon irradiance, but we hypothesize that prolonged exposure to relatively high irradiances (10–20 fold more irradiance than during culturing for 15–20 min) could result in the over-reduction of the primary acceptors on the PSI and PSII side, resulting in the production of ROS15,23. Alternatively, ROS could be the result of photosensitized light-harvesting pigments31; however, in intact light-harvesting complexes the efficiency of electron transfer towards the reaction centers is usually outcompeting the formation of long-lived (ROS-forming) chlorophyll triplet states32. This appears particularly true for the unique phycobiliprotein antenna rods in A. marina MBIC11017, in which excitation electron transfers to PSII were found to be significantly faster than in Chl a-containing cyanobacteria33. Additionally, it is known that within light-harvesting complexes carotenoids are outcompeting O2 in the de-excitation of triplet chlorophyll states34. All three strains used in the current study were found to contain the carotenoid zeaxanthin (Table 1), which can play a crucial role in the quenching of singlet oxygen and general non-photochemical quenching of excited Chl states32,35.\n\nThe strains were adapted to either visible light (VIS) or far-red light (NIR). Photopigments were identified manually from HPLC chromatograms and ratios calculated based on the derived peak areas. Average values and standard error from the mean from two independent growth experiments are displayed.\n\nExposure to shorter wavelengths, such as blue (470 nm) and cyan (495 nm) light generated the most ROS in strains MBIC11017 and HICR111A, while less ROS was produced upon exposure to longer wavelengths (green, amber and red) (Figure 2C). Blue and cyan light-induced ROS production in MBIC11017 and HICR111A is probably due to spectral overlap with the Soret-band absorption of Chl a/d (440–470 nm) (Figure 2B) and the above-mentioned mechanisms in ROS generation. Red (645 nm), amber (595 nm) and green (535 nm) light overlaps with the absorption spectra of phycobiliproteins which, if present, aid in light harvesting and excitation energy transfer towards the photosystems33. Strain MBIC11017 is known to express the phycobiliproteins phycocyanin and allophycocyanin36, while strain HICR111A reportedly lacks phycobiliproteins7.\n\nComparable ROS levels were observed in strains HICR111A and MBIC11017 under yellow and green light, suggesting the presence of pigments absorbing these wavelengths or the possibility of other light-induced ROS production mechanisms. Spectrophotometric analysis of the strains showed weak absorption in the phycobiliprotein-specific region within all three strains (Figure 3). This would corroborate the excitation energy transfer to PSI and II in strain HICR111A and could explain the observed ROS production under yellow and green light. However, this would also refute previous reports on the absence of phycobiliproteins in this strain7. Given that phycobiliproteins were not purposely extracted and further analyzed in the present study, we can at this point only speculate about their presence and relative expression under different growth conditions.\n\nAll strains were adapted to either visible light (VIS) or near infrared radiation (NIR) prior to measurements. All spectra were normalized to the maximal absorbance of Chl d at 710 nm.\n\nThere is a long history of associating pigment compositions within phototrophs with the spectral composition of ambient light and exposure history: for recent work see Stomp et al. 200737 and Apel and Hirt 200323, respectively. These two factors are likely to determine the sensitivity of phototrophs to irradiance and their capability to cope with ROS levels generated upon irradiation. Interestingly, we found that strain CRS generated very little ROS upon exposure to VIS when previously acclimated to NIR, while strains MBIC11017 and HICR111A appear to be more sensitive to VIS when previously adapted to NIR (Figure 2A). HPLC analysis revealed a much higher ratio of zeaxanthin/α-carotene in NIR-acclimated CRS cells than in the other two strains (Table 1). In A. marina MBIC11017, α-carotene was found to be an integral part of both photosystem reaction center cores38,39 and zeaxanthin is predominantly found in the periphery of light-harvesting complexes35.\n\nBased on the higher zeaxanthin/α-carotene ratios in NIR-adapted cells of all three strains, we hypothesize that there are slightly more antenna complexes (zeaxanthin) per reaction center cores (α-carotene) in NIR-adapted cells than in those pre-adapted to VIS. Given that the antenna complexes are predominantly composed of Chl d and thus absorb in the NIR part of the light spectrum, this chromatic photoacclimation is expected and further corroborated by higher Chl d/zeaxanthin ratios in NIR-adapted strains in this study. Besides their light harvesting capability, the carotenoids, zeaxanthin and α-carotene, are also known for their antioxidative capabilities15,34. Particularly zeaxanthin was shown to play a crucial role in non-photochemical quenching and energy dissipation from sensitized chlorophyll molecules or singlet oxygen15. We hypothesize that the higher contribution of zeaxanthin in the NIR-adapted strain CRS could aid in effectively capturing NIR but also in quenching almost all ROS produced during illumination with either VIS or NIR.\n\nIn addition, it is possible that strain-specific differences in ROS production are associated with dissimilarities in growth forms: Strain HICR111A forms cell aggregates7, and so does strain CRS (Figure 2B), whereas strain MBIC11017 usually grows as dispersed cells7 but can be immobilized into biofilms40. The formation of aggregates in strain HICR111A and CRS might provide photoprotection through self-shading, a behavior reportedly less pronounced in strain MBIC110177. Both HICR111A and CRS originate from concretionary substrata in shallow reef flats, a high irradiance habitat. In contrast, strain MBIC11017 was isolated from a didemnid ascidian2, a light environment depleted of VIS but with sufficient NIR10–12.\n\nBased on these first measurements, we suggest that through utilization of NIR, Acaryochloris can avoid a considerable amount of light stress, while harvesting a portion of the electromagnetic radiation spectrum not used by other oxyphototrophs. Additionally, aggregation of certain strains could protect against excess amounts of ROS generated during high irradiance exposure. Overall, this would add a further argument as to why Acaryochloris is a successful and apparently globally widespread oxyphototroph11.\n\nTo the best of our knowledge, we are the first to provide data on ROS levels associated with NIR-driven oxygenic photosynthesis. Unfortunately, due to measuring time constraints at the trace gas facility, we were unable to provide replicated measurements of ROS levels under various light regimes. This emphasizes the need to perform additional measurements of NIR induced ROS production in A. marina and other far-red utilizing oxyphototrophs.\n\n\nMaterials and methods\n\nDead coral branches with patches of faint yellow-greenish pigmentation were collected during low tide from coral patches on the inner reef flat off Heron Island, Queensland, Australia (see more details on the sampling site in Behrendt et al. 201210). The samples were transported back to the laboratory in a container with seawater and immediately placed into outdoor aquaria that were continuously flushed with aerated ambient seawater pumped in from the reef flat. Bacterial cells found on the dead coral branch were removed using a sterile scalpel and immediately placed into KESM media and kept under continuous dim visible light for three days. After transportation to Sydney, the cells were kept at ~26–28°C in KESM media3 under NIR LEDs (centered at 720 nm, Cat. No. L720-04AU, Epitex Inc., Japan). NIR irradiance was set to ~5 µmol photons m-2 s-1 using a SKP200 light meter equipped with a SKP216ER irradiance sensor with a 550 to 750 nm light sensitivity range (Skye Instruments, United Kingdom). After three weeks, the growing cells were diluted into aliquots of fresh KESM medium. After additional incubation, the pigmentation of the cells was inspected by measuring their absorption characteristics using a spectrophotometer (UV-2550, Shimadzu, Japan). The cells were hereafter maintained in KESM media under NIR.\n\nAcaryochloris strains MBIC110771, HICR111A (obtained from the Roscoff Culture Collection, Station Biologique de Roscoff, France); and the newly isolated Acaryochloris strain CRS were grown in 200 ml cell culture flasks in KESM media (salinity of 30) in a shaking incubator at 28°C as previously reported1. All cultures were shaken at 100 rpm under a 12/12 h light-dark shift. Near infrared radiation (NIR) was provided by narrow band LEDs (L720-04AU, 700–740 nm, centered at 720 nm, Epitex Inc., Japan) at an irradiance of 20–40 µmol photons m-2 s-1. Another set of cultures was grown under the same irradiance but using visible light delivered by a halogen lamp equipped with a heat filter (HQ Power, Brinck Elektronik, Denmark). Absolute irradiance measurements of NIR and visible light were done with a calibrated spectroradiometer (Jaz ULM-200, Ocean Optics, Dunedin, FL, USA).\n\nSix ml of dense cell culture was spun down and then extracted using the FastDNA for Soil kit (MP Biomedicals, France) using the manufacturers standard protocol. The resulting DNA was quantified using the Qubit system (Invitrogen, Life Technologies Europe, USA) and diluted 1:10 using molecular grade water. The 16S rRNA gene was amplified using the primers 16SCYfw (5´-GGCTCAGGATGAACGCTGGCGG-3´) and 16SCYrv (5´-ACCTTGTTACGACTTCACCCCAGTC-3´) using the PCR Master (Roche, Switzerland) with 30 amplification cycles. The resulting PCR product was purified on an agarose gel and the band excised using a sterile scalpel. DNA was extracted from the excised gel using the QiaexII gel extraction kit (Qiagen Nordic, Sweden) and then cloned into the pCR4-TOPO cloning vector (Invitrogen, Life Technologies Europe, USA) and transformed into One-Shot TOP-10 chemically competent cells (Invitrogen, Life Technologies Europe, USA). Clones were subsequently grown in LB-medium, plasmids were extracted using the Qiaprep kit (Qiagen Nordic, Sweden), and checked for correct sized inserts using gel electrophoresis. Three clones were sent off for subsequent sequencing by a commercial provider (Macrogen, Seoul, Korea).\n\nCyanobacterial 16S rRNA gene sequences were retrieved from the SILVA database and aligned together with sequences retrieved from clones using MUSCLE as implemented in the Molecular Genetic Analysis (MEGA) software package version 5.0. Neighbor-joining (NJ) was used to infer phylogenetic relationships among sequences; support values with Jukes-Cantor distances and 10,000 bootstrap replicates are displayed next to branches displayed in the phylogenetic tree (Figure 1).\n\nTwo ml of each culture were pelleted by centrifugation at 8000 × g. The supernatant was removed, while the resulting pellet was re-suspended in 96% ethanol and incubated at 4°C for 60 min in darkness. During the ethanol extraction, the samples were vortexed at maximal speed every 15 minutes. After one hour, the cells were pelleted by centrifugation at 8000 × g and the supernatant was used to determine Chl d concentrations via spectrophotometry (UV-2101PC, Shimadzu, Japan) according to Ritchie41. The same spectrophotometer was used to measure the in vivo absorbance spectra of the different cultures. Acaryochloris strains HICR111A and CRS proved very difficult to keep in suspension and were therefore sonicated (Misonix sonicator 4000, Qsonica LLC., Newtown, CT, USA) for one minute at maximum speed prior to spectrophotometric measurements. To prevent bleaching of the photopigments, all handling was done as quickly as possible and under low-light conditions.\n\nFor HPLC analysis, 2 ml of Acaryochloris cultures were spun down at maximum speed (~13,000 rpm) in a bench centrifuge, the supernatant was removed and the remaining pellet resuspended in cold acetone-methanol (7:2 by vol) and the cells sonicated for 20s using a Misonix sonicator 4000 (Qsonica LLC., Newtown, CT, USA) according to Frigaard et al. 199642. The cells were incubated for 2 min on ice in complete darkness, centrifuged again and the extract filtered through a Minisart 0.2-µm pore-size filter (Sartorius, Germany). Ammonium acetate (15 µl; 1.0 M) was added to the extracts (150 µl) to further improve pigment resolution before subsequent injection. Pigment separation was performed on an Agilent 1260 infinity HPLC machine (Agilent Technologies, Santa Clara, CA, USA) and a Nova-pak C18 column (dimensions: 3.9 × 300 mm). A 1260 Infinity Multiple Wavelength Detector (Agilent Technologies, Santa Clara, CA, USA) was used for the detection of compound specific absorption wavelengths. Acaryochloris culture extracts were run with solvent A (methanol:acetonitrile:water, 42:33:25 by vol) and solvent B (methanol:acetonitrile:ethyl acetate, 39:31:30 by vol) in a gradient comprised of 40% solvent B at time of injection, a linear increase to 100% B at 60 min and back to 40% B in 3 minutes. Flow rate was kept constant at 1 ml min-1 and the column at a temperature of 30°C. Photopigments were identified manually from HPLC chromatograms and ratios calculated based on the derived peak areas. Average values and standard errors from the mean from two independent growth experiments were calculated and are displayed in Table 1.\n\nReal-time ethylene production was measured using a laser-based photo-acoustic ethylene detector (ETD-300, Sensor Sense, The Netherlands) combined with an in-line gas-flow through system (Valve Controller VC 6, Sensor Sense, The Netherlands). The system was described in Cristescu et al. 200826. Custom-made gas-tight incubation chambers were connected via the valve controller to the ETD, which could sequentially sample ethylene fluxes from the different incubators. The incubator was made of anodized aluminum (51ST quality) and contained a cooling/heating channel to control temperature and a glass window to supply light to the samples (see details in Staal et al. 200143). The incubator could hold 2 ml aliquots of Acaryochloris culture. Culture samples were augmented with α-keto-γ-(methylthio)butyric acid sodium salt (KMBA) (>97% purity, K6000, Sigma-Aldrich) made to a final concentration of 2.8 mM in KESM media; earlier experiments on cyanobacterial cultures demonstrated saturating levels of KMBA when supplied at this concentration (M Staal, SM Cristescu, L Stal & FJM Harren, unpublished observations). After addition of KMBA, the cultures were mixed using a pipette to obtain a uniform distribution of the chemical. All measured ethylene concentrations were normalized to the Chl d concentration in the samples.\n\nTo ensure steady state ethylene fluxes at the moment a sample was connected to the ETD, we supplied a continuous flow of moisturized air (2l h-1) over every individual incubator during the experiments. The air was moisturized by flushing it through gas tight vials filled with de-ionized water; this was necessary to prevent evaporation of media in the incubator. The system was continuously controlled for gas leaks, by automated comparison of the incoming and outgoing gas flow. The outlet of the incubator was connected to a CO2 trap (KOH pellets) and water scrubber (CaCl2) placed before the ethylene detector. The valve controller allowed each measuring chamber to be alternately connected for 20 minutes to the ethylene detector. Steady state ETD readings from the cultures were obtained within ~4 minutes after connection to the ETD. The ETD-300 has a sample frequency of ~12 samples min-1 and the concentrations of the last two minutes per treatment were averaged. Typical standard deviations were 0.15 ppbv for ethylene measurements under steady state conditions. The averaged concentrations were normalized to the amount of Chl d present in the cultures to correct for differences in biomass between samples.\n\nLight-dependent ROS production was measured using both VIS (400–700 nm) and NIR. For visible wavelengths, we used an incubator setup (Mini-Incubator, Sensor Sense, Nijmegen, The Netherlands), fitted with an array of 11 1W cool white LEDs (Luxeon Star, 1W, Lumileds, USA) connected to a PC-driven controller. Irradiance levels were set between 340 and 480 µmol photons m-2 s-1 for visible light. Different irradiance levels were adjusted by varying the electrical current of the LED array via a special software routine (Sensor Sense, Nijmegen, The Netherlands) and measuring the downwelling irradiance with a calibrated light meter (LI250, LiCOR Biosciences, Lincoln, USA).\n\nFor NIR exposure, the actinic light was provided by four collimated NIR LEDs (M3L1-720–30, 700–740 nm, centered at 720 nm, Roithner Lasertechnik, Vienna, Austria) at an intensity of 400 µmol photons m-2 s-1. The absolute NIR irradiance level was measured with an irradiance sensor attached to a calibrated spectroradiometer (Jaz ULM-200, Ocean Optics, USA). Subsequently, the measured irradiance spectrum was integrated over a spectral range of 650–800 nm. All samples were incubated at 28°C using a cooler/heater bath (HD-25, Julabo, Germany).\n\nAction spectra of ROS production were measured using three high-power LEDs (Luxeon star, 1W, Lumileds, USA) mounted onto an aluminum plate for efficient cooling. The following colors and centered wavelengths were used in the action spectra: Blue (470 nm, 25 nm spectral half-width, LXHL-MB1D), Cyan (495 nm, 30 nm spectral half-width, LXHL-ME1D), Green (535 nm, 35 nm spectral half-width, LXHL-MM1D), Amber (595 nm, 14 nm spectral half-width, LXHL-ML1D), and Red (645 nm, 20 nm spectral half-width, LXHL-MD1D). The LEDs were powered by LED power supplies (LED31, Velleman, Belgium). For each LED color, the incident irradiance was adjusted to 300 µmol photons m-2 s-1 by adjusting the distance from the LEDs to the incubation chamber window. Photon irradiances were measured with a calibrated light meter (LI250, LiCOR Biosciences, Lincoln USA) before each measurement.", "appendix": "Author contributions\n\n\n\nL Behrendt, M Kühl and AWD Larkum conceived the study. M Kühl, L Behrendt and M Staal designed the experiments. L Behrendt, M Kühl and SM Cristescu carried out the research. FJM Harren and SM Cristescu contributed to the design of experiments and provided access to the trace-gas facility. AWD Larkum and M Schliep isolated the novel A. marina strain CRS. L Behrendt, AWD Larkum and M Kühl 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 were disclosed.\n\n\nGrant information\n\nThe work was supported by the Danish Council for Independent Research \| Natural Sciences (FNU), project 11-108257, and the EU-FP6-Infrastructures-5 program, project FP6-026183 ‘Life Science Trace Gas Facility’.\n\n\nAcknowledgments\n\nThe work was conducted under a Marine Parks Permit (G06/178151.1) from the Great Barrier Reef Authority (Australia). We thank Niels-Ulrik Frigaard for help with the HPLC based pigment analysis and Søren J Sørensen for help with the 16S rRNA gene sequence analysis. We thank the staff at Heron Island Research Station for excellent technical assistance during the field work.\n\n\nReferences\n\nMiyashita H, Adachi K, Kurano N, et al.: Pigment composition of a novel oxygenic photosynthetic prokaryote containing chlorophyll d as the major chlorophyll. Plant Cell Physiol. 1997; 38(3): 274–281. Reference Source\n\nMiyashita H, Hisato I, Norihide K, et al.: Chlorophyll d as a major pigment. Nature. 1996; 383: 402. Publisher Full Text\n\nMiyashita H, Ikemoto H, Kurano N, et al.: Acaryochloris marina gen. et sp. nov.(cyanobacteria) an oxygenic photosynthetic prokaryote containing Chl d as a major pigment. J Phycol. 2003; 39(6): 1247–1253. Publisher Full Text\n\nSwingley WD, Chen M, Cheung PC, et al.: Niche adaptation and genome expansion in the chlorophyll d-producing cyanobacterium Acaryochloris marina. Proc Natl Acad of Sci U S A. 2008; 105(6): 2005–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurakami A, Miyashita H, Iseki M, et al.: Chlorophyll d in an epiphytic cyanobacterium of red algae. Science. 2004; 303(5664): 1633. 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PubMed Abstract \| Publisher Full Text\n\nHe YY, Klisch M, Häder DP: Adaptation of cyanobacteria to UV-B stress correlated with oxidative stress and oxidative damage. Photochem Photobiol. 2002; 76(2): 188–96. PubMed Abstract \| Publisher Full Text\n\nHe YY, Häder D: Reactive oxygen species and UV-B: effect on cyanobacteria. Photochem Photobiol Sci. 2002; 1(10): 729–736. PubMed Abstract \| Publisher Full Text\n\nApel K, Hirt H: Reactive oxygen species: metabolism oxidative stress and signal transduction. Annu Rev Plant Biol. 2004; 55: 373–99. PubMed Abstract \| Publisher Full Text\n\nSoh N: Recent advances in fluorescent probes for the detection of reactive oxygen species. Anal Bioanal Chem. 2006; 386(3): 532–43. PubMed Abstract \| Publisher Full Text\n\nKohen R, Nyska A: Invited review: Oxidation of biological systems: oxidative stress phenomena antioxidants redox reactions and methods for their quantification. Toxicol Pathol. 2002; 30(6): 620–650. PubMed Abstract \| Publisher Full Text\n\nCristescu SM, Persijn ST, te Lintel Hekkert S, et al.: Laser-based systems for trace gas detection in life sciences. Appl Phys B. 2008; 92(3): 343–349. Publisher Full Text\n\nMansouri S, Bunch AW: Bacterial ethylene synthesis from 2-oxo-4thiobutyric acid and from methionine. J Gen Microbiol. 1989; 135(11): 2819–27. PubMed Abstract \| Publisher Full Text\n\nRegoli F, Winston GW: Quantification of total oxidant scavenging capacity of antioxidants for peroxynitrite peroxyl radicals and hydroxyl radicals. Toxicol Appl Pharmacol. 1999; 156(2): 96–105. PubMed Abstract \| Publisher Full Text\n\nRegoli F, Winston GW: Applications of a new method for measuring the total oxyradical scavenging capacity in marine invertebrates. Mar Environ Res. 1998; 46(1–5): 439–442. Publisher Full Text\n\nDuxbury Z, Schliep M, Ritchie RJ, et al.: Chromatic photoacclimation extends utilisable photosynthetically active radiation in the chlorophyll d-containing cyanobacterium Acaryochloris marina. Photosynth Res. 2009; 101(1): 69–75. PubMed Abstract \| Publisher Full Text\n\nRinalducci S, Pedersen JZ, Zolla L: Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II. Biochim Biophys Acta. 2004; 1608(1): 63–73. PubMed Abstract \| Publisher Full Text\n\nSundstrøm V: Photosynthetic light harvesting charge separation and photoprotection: the primary steps. Photobiology the science of life and light. 2008; 289–319. Publisher Full Text\n\nTheiss C, Schmitt FJ, Pieper J, et al.: Excitation energy transfer in intact cells and in the phycobiliprotein antennae of the chlorophyll d containing cyanobacterium Acaryochloris marina. J Plant Physiol. 2011; 168(12): 1473–87. PubMed Abstract \| Publisher Full Text\n\nYoung AJ, Frank HA: Energy transfer reactions involving carotenoids: quenching of chlorophyll fluorescence. J Photochem Photobiol B. 1996; 36(1): 3–15. PubMed Abstract \| Publisher Full Text\n\nBlankenship R: Molecular mechanisms of photosynthesis. 1–321 (Blackwell Publishing: 2002). Publisher Full Text\n\nChan YW, Nenninger A, Clokie SJ, et al.: Pigment composition and adaptation in free-living and symbiotic strains of Acaryochloris marina. FEMS Microbiol Ecol. 2007; 61(1): 65–73. PubMed Abstract \| Publisher Full Text\n\nStomp M, Huisman J, Stal LJ, et al.: Colorful niches of phototrophic microorganisms shaped by vibrations of the water molecule. ISME J. 2007; 1(4): 271–82. PubMed Abstract \| Publisher Full Text\n\nTomo T, Kato Y, Suzuki T, et al.: Characterization of highly purified photosystem I complexes from the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC11017. J Biol Chem. 2008; 283(26): 18198–209. PubMed Abstract \| Publisher Full Text\n\nTomo T, Okubo T, Akimoto S, et al.: Identification of the special pair of photosystem II in a chlorophyll d-dominated cyanobacterium. Proc Natl Acad Sci U S A. 2007; 104(17): 7283–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBehrendt L, Schrameyer V, Qvortrup K, et al.: Biofilm growth and near-infrared radiation-driven photosynthesis of the chlorophyll d-containing cyanobacterium Acaryochloris marina. Appl Environ Microbiol. 2012; 78(11): 3896–3904. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRitchie RJ: Consistent sets of spectrophotometric chlorophyll equations for acetone methanol and ethanol solvents. Photosynth Res. 2006; 89(1): 27–41. PubMed Abstract \| Publisher Full Text\n\nFrigaard N, Larsen KL, Cox RP: Spectrochromatography of photosynthetic pigments as a fingerprinting technique for microbial phototrophs. FEMS Micriobiology Ecology. 1996; 20(2): 69–77. Publisher Full Text\n\nStaal M, Lintel-Hekkert ST, Harren F, et al.: Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on-line monitoring. Environ Microbiol. 2001; 3(5): 343–51. PubMed Abstract \| Publisher Full Text" }	[ { "id": "776", "date": "18 Feb 2013", "name": "Helen Yap", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 evaluation: This is a well-written paper describing potentially ground-breaking research which has universal application, considering the ubiquitous occurrence of the cyanobacteria studied, and the crucial role they play in primary production of shallow-water habitats worldwide.Limitation of the study: The authors acknowledge that the experiment suffers from lack of replication of measurements of ROS levels under the different light regimes. They also do not include statistical analyses that would have confirmed significant differences among the different strains, and between treatments (NIR- and VIS-adapted).Nevertheless, the results are interesting and merit further investigation. In general, the experiment appears to have been conducted following appropriate scientific standards and protocols, and using valid methods. Results and Discussion:3rd from last paragraph: “concretionary” is not a common term to define coral reef substrata", "responses": [] }, { "id": "778", "date": "18 Feb 2013", "name": "Susanna López Legentil", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 evaluation: In this manuscript, Behrendt and coworkers present some novel results about the generation of reactive oxygen species (ROS) by a cyanobacterium (Acaryochloris marina) that relies on chlorophyll d for photosynthesis. Although I am not an expert on the biochemical pathways involved in photosynthesis, the methodology chosen appears appropriate to address the question at hand and the experiments appear to have been well conducted. Additional replicates and tests are still necessary to confirm the pattern found, but I believe the subject matter addressed in this manuscript and the results obtained are of enough interest to warrant immediate publication.Limitation of the study: As the authors point out, additional measurements of ROS levels under various light regimes are necessary. Data should also be subjected to appropriate statistical analyses to derive stronger conclusions. In addition, it will be interesting to test another strain that does not aggregate to reinforce some of the conclusions presented in the article. Introduction: In the first paragraph you mentioned that 2 strains of A. marina have been well described, but then proceed to describe only one (MBIC11017). Please, add some information about the second well-described strain.", "responses": [] }, { "id": "795", "date": "22 Feb 2013", "name": "Alison Telfer", "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 shows experiments on several cyanobacterial strains of the Acaryochloris genus, in which chlorophyll a is replaced by Chl d which absorbs in the near infra red (NIR) - 25-30 nm to the red of Chl a. It shows that under stress levels of NIR excitation (and also adaptation to growth under NIR) less reactive oxygen species (ROS) are formed. The authors conclude that this is a strategy of this genus (more species of which are being discovered widely around the planet) to protect against photodamage during high irradiance exposure - a more and more likely occurrence these days.The authors stress the point (page 7, left, para 3) that this is a preliminary study in which fully replicated measurements of ROS were not possible. I understand this constraint and accept that reporting measurements of ROS, using the novel technique of real time ethylene detection, from Acaryochloris is very interesting but I feel the paper goes too far in its claims. It also is unclear about the different types of ROS detected and the mechanism by which protection is provided by carotenoids.3, right para 2.The ROS detection method (real time ethylene detection RTED) described in ref 28 (Regoli and Winston) appears to only directly detect very strong oxidants hydroxyl and peroxyl radicals and peroxynitrite. It does not directly detect superoxide or singlet oxygen (1O2). This should be made clearer in the text and there should be discussion about production of hydroxyl radicals. Relating to point 1.: The correlation between light-induced increase in relative amounts of the zeaxanthin containing antenna complexes and the ROS levels detected by the RTED system should be explained in more detail. Carotenoids in photosynthetic complexes mainly operate by quenching chlorophyll triplet states before they can form 1O2 or they directly quench any 1O2 that is formed. They do also quench oxygen radicals but these are more likely to be produced in the aqueous phase (e.g. from reduced ferredoxin in PSI) where they are usually quenched by antioxidant enzymes such as ascorbate peroxidase etc. Though it is possible alpha-Car in the PSII reaction center could quench radicals produced from reduced quinone. When the ROS levels, detected by RTED, increase it is likely that other ROS (e.g. superoxide) and perhaps 1O2 also increase and so carotenoids would be helpful. However, the text makes it sound as if in CRS the zeaxanthin level increases relative to chlorophyll when it is simply more antenna (Zea plus Chl d) being produced and so there is more potential for 1O2 production.Essentially I feel the text, though cautious, claims too much. The errors on the pigment levels especially for CRS (they are huge) suggest the very different values for NIR and VIS adapted cells could be a fluke. Also the single point for CRS in Fig. 2A NIR adapted under VIS exposure could be a fluke.Minor Pointsp. 4 end para beginning ‘In Acaryochloris....:\n\nChange last sentence round so it says: zeaxanthin will bring about rapid quenching of excited chlorophyll states and if necessary can also quench singlet oxygen - or something similar to this.p.6 end para 3:\n\nRephrase: ...and zeaxanthin is restricted to the peripheral light-harvesting complexes (PCB proteins).p.6 Second sentence from bottom: I do not like the sentence emphasising that zeaxanthin quenches singlet oxygen. The a-carotene in the reaction centre is at least as likely to be quenching singlet oxygen as it is well known it cannot directly quench the radical pair triplet state as it is bound to far away from the highly oxidising primary electron donor. The reference to energy dissipation by zeaxanthin is irrelevant here. Fig. 2C\n\nFor clarity use different symbols (but same colour) for NIR adapted and VIS adapted points.", "responses": [] } ]	1	https://f1000research.com/articles/2-44
https://f1000research.com/articles/2-84/v1	12 Mar 13	{ "type": "Research Article", "title": "Positive or negative allosteric modulation of metabotropic glutamate receptor 5 (mGluR5) does not alter expression of behavioral sensitization to methamphetamine", "authors": [ "Peter R Kufahl", "Natali E Nemirovsky", "Lucas R Watterson", "Nicholas Zautra", "M Foster Olive", "Natali E Nemirovsky", "Lucas R Watterson", "Nicholas Zautra", "M Foster Olive" ], "abstract": "We investigated the role of metabotropic glutamate receptor type 5 (mGluR5) in methamphetamine-induced behavioral sensitization. The mGluR5 positive allosteric modulator (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) and negative allosteric modulator fenobam were tested in separate experiments. Sprague-Dawley rats were repeatedly injected with 1 mg/kg methamphetamine or saline, and then given a locomotor challenge test using a dose of 0.5 mg/kg methamphetamine. Prior to the challenge test session, rats were injected with CDPPB, fenobam, or a vehicle. Doses from previous studies showed reduced drug-conditioned behavior; however in this study neither CDPPB nor fenobam pretreatment resulted in an altered expression of behavioral sensitization, indicating a lack of mGluR5 involvement in sensitized methamphetamine-induced locomotion. Additionally, the high dose (30 mg/kg) of fenobam resulted in decreased methamphetamine-induced locomotion in rats regardless of drug exposure history, which suggests evidence of nonspecific behavioral inhibition.", "keywords": [ "methamphetamine", "sensitization", "mGluR5", "allosteric modulator" ], "content": "Introduction\n\nCompulsive drug use and associated maladaptive behaviors are cardinal features of methamphetamine (METH) addiction, and have been strongly associated with the neurochemical consequences of repeated METH abuse1–3. Among the various neurotransmitter systems affected by METH exposure is the glutamate system, where long-lasting drug-induced changes are suspected factors underlying craving and persistent vulnerability to relapse4. Due to their dual roles in mediating glutamatergic synaptic plasticity and control of synaptic glutamate release, the metabotropic glutamate receptors (mGluRs) have emerged as therapeutic targets of interest in the study of drug addiction5. Antagonizing the excitatory postsynaptic metabotropic glutamate receptor 5 (mGluR5) has been recently shown to attenuate the reinforcing effects of METH on a progressive ratio schedule, as well as attenuating drug-seeking behavior in rats previously trained to self-administer METH6. Selective stimulation of mGluR5 has been found to improve the rate of extinction learning in rats previously conditioned to the reinforcing effects of cocaine. This study investigated the role of mGluR5 in the behavioral changes induced by repeated exposure to METH, using positive and negative allosteric modulators of mGluR5 function in separate experiments.\n\nThe consequences of chronic METH abuse are often studied in the rat model of behavioral sensitization, where chronic METH injections reliably induce an elevated locomotor response to a subsequent METH challenge, relative to rats with no prior history of METH exposure8–11. Through their interactions with the dopaminergic projections of the medial forebrain, mGluRs have been found to have roles in both the development and expression of psychostimulant sensitization12. mGluR5 has been associated with the locomotor response and reinforcement attributes of psychostimulants since mice lacking this receptor were found not to respond to or self-administer cocaine as wild-type mice13. While antagonism of group I mGluRs, which includes mGluR5, in subsequent experiments has generally failed to convincingly affect locomotor sensitization to cocaine14, the effects of positive allosteric modulation on psychostimulant sensitization have so far remained untested. We evaluated the effect of the mGluR5 positive allosteric modulator (PAM) 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) and the mGluR5 negative allosteric modulator (NAM) fenobam on the expression of behavioral sensitization to METH. We utilized doses of CDPPB that have been shown to improve extinction learning after METH [30 mg/kg15], and cocaine [60 mg/kg7], self-administration training, and doses of fenobam (10–30 mg/kg) that have effectively reduced drug-seeking in METH-trained rats in our laboratory16.\n\n\nMethods and materials\n\nEighty-eight male Sprague-Dawley rats (Harlan Laboratories, Livermore, CA), weighing 250–275 g, were pair-housed on arrival in a humidity-controlled colony room and maintained in a reversed light/dark cycle with free access to food and water throughout the experiment. All experimentation was conducted during the dark phase of the light/dark cycle. All procedures were conducted with the approval of the Institutional Care and Use Committee at Arizona State University and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals (National Research Council)17.\n\n3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, custom synthesized by Chemir Analytical Services, Maryland Heights, MO) was suspended in 10% v/v Tween 80 via sonication to form a 60 mg/ml concentration for intraperitoneal (i.p.) administration. Fenobam (1-(3-chlorophenyl)-3-3-methyl-5-oxo-4H-imidazol-2-yl) urea (custom synthesized by Chemir Analytical Services) was suspended in 0.3% v/v Tween 80 vehicle to form a 30 mg/ml concentration for i.p. administration. (+)Methamphetamine hydrochloride (Sigma Aldrich, St Louis, MO) was dissolved in sterile saline for i.p. administration.\n\nLocomotor activity was assessed in a Rotorat System apparatus (Med Associates, Mt. St Albans, VT) that measured rotational ambulation, quantified as quarter turns in both directions, within a bowl-shaped arena (Figure 1A). The rats (N=43 in the CDPPB experiment, N=45 in the fenobam experiment) were divided into groups where half of the rats received five injections of 1 mg/kg METH dissolved in saline (1 ml/kg, i.p.), separated by 48 hours, and the other half received injections of saline of matching volume (Figure 1B). Each injection was immediately followed by a 90 min locomotor test session. After a 6-day waiting period in the colony room, all rats were given a saline injection (1 ml/kg, i.p.) and subjected to a locomotor test session. The next day, rats were injected with 0 (N=7), 30 (N=8) or 60 mg/kg (N=6–7) CDPPB in one experiment; or 0 (N=8), 10 (N=8) or 30 mg/kg (N=6–7) fenobam in the other experiment, and 30 min later given a challenge dose of 0.5 mg/kg METH and subjected to a 90 min locomotor test session.\n\nThe locomotor apparatus (A) consists of a rotating actuator anchored to a U-shaped bracket over a steel bowl-shaped arena (Med Associates; 18 in top diameter, 6 in bottom diameter, 6 in depth) containing a layer of Sani-chip bedding. The rat is attached to the actuator via 45 cm spring leash terminated with an alligator clip, which is hooked onto a cable tie around the neck for the duration of the test session. The apparatus registers rotational movements as the rat causes the actuator to pivot, accumulated by computer as quarter turns. The experimental procedure (B) consisted of three days of acclimation sessions in the locomotor arenas, followed by five injections of METH (1.0 mg/kg, i.p.) or saline separated by 48 hr (Days 1, 3, 5, 7 and 9). After each injection, rats were placed into the locomotor arenas for 90 min and their rotational data were recorded as quarter turns. Rats underwent locomotor testing following a saline injection on Day 15, and these data were balanced between groups assigned to mGluR5 treatment or vehicle treatment. On Day 16, the rats were given an injection of the mGluR5 ligand (CDPPB or fenobam) or vehicle, and tested 30 min later following a probe injection of METH (0.5 mg/kg, i.p.).\n\nAdditional experiments were conducted to examine the effects of mGluR5 modulation on baseline locomotion. Rats were acclimated to the apparatus in 90 min sessions for two consecutive days, and on the next day given a 90 min locomotor test session 30 min after treatment with 0, 30 or 60 mg/kg CDPPB in one experiment (N=5); or 0, 10 or 30 mg/kg fenobam in another experiment (N=5).\n\nData analysis procedures were performed using Prism 5 (GraphPad, La Jolla, CA). For the sensitization experiments, quarter turn data (in either direction, totaled over 90 min) taken during the five chronic treatment sessions were analyzed using 2-way ANOVA with METH history (naïve, METH-treated) as a between-subjects factor and day (1, 3, 5, 7 or 9) as a within-subjects factor. Locomotor behavior exhibited during the challenge sessions were quantified as quarter turns and analyzed using 2-way ANOVA with METH history and treatment (0, 30 or 60 mg/kg for the CDPPB experiment, and 0, 15 or 30 mg/kg for the fenobam experiment) as between-subjects factors. Significant interaction effects were followed by pairwise comparisons (Fisher’s LSD tests).\n\nIn the baseline locomotion experiments, quarter turn data were analyzed using one-way ANOVA with CDPPB or fenobam treatment as the main factor.\n\n\nResults\n\nIn the CDPPB experiment, rats treated with repeated METH injections exhibited progressively increasing amounts of quarter turns, as confirmed by a significant main effect of METH history (F1,164 = 51.8, p < 0.0001) and a day × METH history interaction (F4,164 = 3.4, p < 0.05). In these rats, locomotion was significantly elevated from Day 1 levels (2110 ± 284) on Day 5 (3117 ± 401, p < 0.05, Fisher’s LSD test) and Day 7 (3432 ± 433, p < 0.01), but not Day 9 (Figure 2A and Table S1–Table S2). Similarly, in the fenobam experiment, repeated injections of METH but not saline resulted in elevated quarter turns, as confirmed by significant main effects of day (F4,172 = 4.1, p < 0.005) and METH history (F1,172 = 60.9, p < 0.0001) and a day × METH history interaction (F4,172 = 6.0, p < 0.0005). In these rats, locomotion was significantly elevated from Day 1 levels (2175 ± 320) on Day 5 (3136 ± 297, p < 0.05, Fisher’s LSD test), Day 7 (3548 ± 388, p < 0.01) and Day 9 (3469 ± 438, p < 0.05, Figure 2B and Table S3–Table S4).\n\nIn locomotor sessions prior to mGluR5-targeted treatment (A-B), rats were chronically given 1 mg/kg METH (filled circles) or saline (open circles). In both the CDPPB (A) and fenobam (B) experiments, the reported quarter turns progressively increased above first-day levels in the METH-exposed groups. P < 0.05 different from Day 1 levels. In the subsequent test using 0.5 mg/kg METH in all groups (C), rats with a history of chronic METH exposure exhibited elevated locomotor behavior, but CDPPB pretreatment had no effect. In the fenobam experiment (D), rats with a history of chronic METH exposure also exhibited elevated locomotor activity, and this behavioral sensitization was not affected by 10 mg/kg fenobam pretreatment. After 30 mg/kg fenobam treatment, the METH-sensitized locomotor response was reduced from the vehicle level. P < 0.05 difference between METH history groups, regardless of mGluR5 ligand treatment. +P < 0.05 different from vehicle treated group with matching history of METH exposure. PAM stands for positive allosteric modulation, and NAM stands for negative allosteric modulation.\n\nIn the CDPPB experiment, rats with a history of repeated METH treatments exhibited a greater number of quarter turns following a probe injection of 0.5 mg/kg METH, evidence of locomotor sensitization (Figure 2C and Table S5–Table S6). This elevated response to METH was not attenuated by CDPPB pretreatment, as shown by the existence of a main effect of METH history (F1,37 = 10.7, p < 0.005) but no other main effects or interactions.\n\nIn the fenobam experiment, rats with a history of repeated METH treatments also exhibited elevated quarter turns following the 0.5 mg/kg METH probe (Figure 2D and Table S7–Table S8). Pretreatment with fenobam attenuated the locomotor response to METH, regardless of METH exposure history, as revealed by the presence of main effects of METH history (F1,39 = 20.1, p < 0.001) and treatment (F2,39 = 6.7, p < 0.005), but no METH history × treatment interaction. However, pretreatment with the large dose of fenobam (30 mg/kg) resulted in significantly reduced METH-induced locomotion in rats with a history of chronic 1 mg/kg METH injections (0 mg/kg fenobam: 1192 ± 105 quarter turns vs. 30 mg/kg fenobam: 597 ± 150 quarter turns, p < 0.01, two-sample t-test), and produced a trend toward a significant reduction in rats with a history of saline injections (0 mg/kg fenobam: 622 ± 493 quarter turns vs. 30 mg/kg fenobam: 405 ± 106 quarter turns, P = 0.08).\n\nAll of the tested doses of CDPPB and fenobam had negligible effects on baseline locomotion, measured 30 min after time of injection. Both the 60 mg/kg dose of CDPPB (300 ± 92 quarter turns, vs. 345 ± 43 for the vehicle) and the 30 mg/kg dose of fenobam (389 ± 59 quarter turns, vs. 407 ± 74 for the vehicle) produced slightly attenuated locomotor responses, but no significant effects were revealed by ANOVA in either experiment (Figure 3 and Table S9–Table S10).\n\nCDPPB (A) or fenobam (B) was injected 30 min prior to locomotor testing. No significant effects were reported from the quarter turns collected over 90 min sessions.\n\n\nDiscussion\n\nAs expected, rats repeatedly injected with 1 mg/kg METH exhibited greater locomotor activity than the saline-treated rats, and demonstrated more activity during the latter sessions than the initial session. Treatment with CDPPB did not significantly alter METH-induced rotational locomotion, and treatment with fenobam only significantly reduced rotational locomotion at its highest dose (30 mg/kg). Neither CDPPB nor fenobam significantly attenuated the baseline locomotor activity of drug-naïve animals, although the small effect found for 30 mg/kg fenobam in that experiment (Figure 3B) could explain the moderate reduction of quarter turns exhibited by METH-challenged rats (Figure 2D) as a non-specific phenomenon. Thus, locomotor effects of mGluR5 modulation were largely absent at the dose ranges that have been shown in earlier studies to reduce operant behavior motivated by METH or cocaine training7,15,16,18,19.\n\nThese largely negative findings indicate that the maintenance of behavioral sensitization is likely mediated by neurobiological substrates other than mGluR5. These data are also in agreement with previous observations that mGluR5 function does not appear critical for the expression of locomotor sensitization to cocaine14,20, and extends them to include METH sensitization. Furthermore, the contribution of mGluR5 to initial locomotor responses to injected psychostimulants13 appears to be replaced by other neurochemical substrates with chronic drug exposure.\n\nWhile mGluR5 is an important therapeutic target in researching treatments for addiction to psychostimulants as well as other abused substances, there is building evidence that the role of this receptor in drug-related behaviors changes with increasing exposure. A recent study of rats chronically exposed to METH sufficient to induce measurable conditioned place preference found a reduction of surface expression of mGluR5 in the medial prefrontal cortex21, an area known to contribute to the expression of behavioral sensitization4. The current findings using the behavioral sensitization model therefore suggest that the changes in the degree to which mGluR5 mediates drug-stimulated and drug-conditioned behavior previously shown to occur with chronic cocaine exposure might also take place in rats with a history of chronic METH exposure. The possibility of the changing roles among the various mGluR subfamilies as a result of drug exposure merits further studies utilizing animal models of METH-induced activity and motivated behavior.", "appendix": "Author contributions\n\n\n\nPRK and MFO conceived of the study and designed the experiments. PRK, NEN, LRW and NZ carried out the research. PRK and MFO prepared the initial draft of the manuscript and all further revisions. All authors approved of the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was completed with the support of NIH/NIDA grants DA025606 and DA024355 to MFO.\n\n\nAcknowledgements\n\nThe authors wish to acknowledge Angel Villa, Kaveish Sewalia, Elisabeth Moore, Casey Halstengard, Piroska Barabas and Lauren Hood of Arizona State University for providing valuable technical assistance.\n\n\nSupplementary tables\n\nIn locomotor sessions prior to mGluR5-targeted treatment, rats were chronically given 1 mg/kg METH i.p. In this experiment, the reported quarter turns progressively increased above first-day levels.\n\nIn locomotor sessions prior to mGluR5-targeted treatment, rats were chronically given 1 ml/kg saline i.p. The reported quarter turns did not significantly change from first-day levels.\n\nIn locomotor sessions prior to mGluR5-targeted treatment, rats were chronically given 1 mg/kg METH i.p. In this experiment, the reported quarter turns progressively increased above first-day levels.\n\nIn locomotor sessions prior to mGluR5-targeted treatment, rats were chronically given 1 ml/kg saline i.p. The reported quarter turns did not significantly change from first-day levels.\n\nIn the Day 16 tests using 0.5 mg/kg METH in all groups, rats with a history of chronic saline injections exhibited elevated locomotor behavior, but CDPPB pretreatment had no effect.\n\nIn the Day 16 tests using 0.5 mg/kg METH in all groups, rats with a history of chronic METH exposure exhibited elevated locomotor behavior, but CDPPB pretreatment had no effect.\n\nIn the Day 16 tests using 0.5 mg/kg METH in all groups, rats with a history of chronic saline injections exhibited elevated locomotor behavior, but fenobam pretreatment had no effect.\n\nIn the Day 16 tests using 0.5 mg/kg METH in all groups, rats with a history of chronic METH exposure exhibited elevated locomotor behavior, and 30 mg/kg but not 10 mg/kg fenobam resulted in reduced quarter turns relative to vehicle-pretreated animals.\n\n\nReferences\n\nMcLellan AT, Lewis DC, O'Brien CP, et al.: Drug dependence, a chronic medical illness: implications for treatment, insurance, and outcomes evaluation. JAMA. 2000; 284(13): 1689–95. PubMed Abstract \| Publisher Full Text\n\nWHO, Amphetamine-type stimulants : a report from the WHO meeting on amphetamines, MDMA and other psychostimulants. W. Substance Abuse Dept., Editor, World Health Organization (WHO): Geneva 1996. Reference Source\n\nBarr AM, Panenka WJ, MacEwan GW, et al.: The need for speed: an update on methamphetamine addiction. J Psychiatry Neurosci. 2006; 31(5): 301–13. PubMed Abstract \| Free Full Text\n\nTzschentke TM, Schmidt WJ: Glutamatergic mechanisms in addiction. Mol Psychiatry. 2003; 8(4): 373–82. PubMed Abstract \| Publisher Full Text\n\nGass JT, Olive MF: Glutamatergic substrates of drug addiction and alcoholism. Biochem Pharmacol. 2008; 75(1): 218–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGass JT, Osborne MP, Watson NL, et al.: mGluR5 antagonism attenuates methamphetamine reinforcement and prevents reinstatement of methamphetamine-seeking behavior in rats. Neuropsychopharmacology. 2009; 34(4): 820–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCleva RM, Hicks MP, Gass JT, et al.: mGluR5 positive allosteric modulation enhances extinction learning following cocaine self-administration. Behav Neurosci. 2011; 125(1): 10–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFujiwara Y, Kazahaya Y, Nakashima M, et al.: Behavioral sensitization to methamphetamine in the rat: an ontogenic study. Psychopharmacology (Berl). 1987; 91(3): 316–9. PubMed Abstract \| Publisher Full Text\n\nFujiyama K, Kajii Y, Hiraoka S, et al.: Differential regulation by stimulants of neocortical expression of mrt1, arc, and homer1a mRNA in the rats treated with repeated methamphetamine. Synapse. 2003; 49(3): 143–9. PubMed Abstract \| Publisher Full Text\n\nOhmori T, Abekawa T, Koyama T: Environment modifies the expression of behavioral sensitization produced by methamphetamine: behavioral and neurochemical studies. Behav Pharmacol. 1995; 6(2): 133–142. PubMed Abstract \| Publisher Full Text\n\nOhmori T, Abekawa T, Koyama T: Scopolamine prevents the development of sensitization to methamphetamine. Life Sci. 1995; 56(14): 1223–9. PubMed Abstract \| Publisher Full Text\n\nVezina P, Kim JH: Metabotropic glutamate receptors and the generation of locomotor activity: interactions with midbrain dopamine. Neurosci Biobehav Rev. 1999; 23(4): 577–89. PubMed Abstract \| Publisher Full Text\n\nChiamulera C, Epping-Jordan MP, Zocchi A, et al.: Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice. Nat Neurosci. 2001; 4(9): 873–4. PubMed Abstract \| Publisher Full Text\n\nDravolina OA, Danysz W, Bespalov AY: Effects of group I metabotropic glutamate receptor antagonists on the behavioral sensitization to motor effects of cocaine in rats. Psychopharmacology (Berl). 2006; 187(4): 397–404. PubMed Abstract \| Publisher Full Text\n\nKufahl PR, Hood LE, Nemirovsky NE, et al.: Positive Allosteric Modulation of mGluR5 Accelerates Extinction Learning but Not Relearning Following Methamphetamine Self-Administration. Front Pharmacol. 2012; 3: 194. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatterson LR, Kufahl PR, Nemirovsky NE, et al.: Attenuation of reinstatement of methamphetamine-, sucrose-, and food-seeking behavior in rats by fenobam, a metabotropic glutamate receptor 5 negative allosteric modulator. Psychopharmacology (Berl). 2013; 225(1): 151–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCouncil NR Guide for the care and use of laboratory animals. 8 ed, Washington, DC: National Acadamies Press 2011. PubMed Abstract\n\nGass JT, Olive MF: Positive allosteric modulation of mGluR5 receptors facilitates extinction of a cocaine contextual memory. Biol Psychiatry. 2009; 65(8): 717–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWidholm JJ, Gass JT, Cleva RM, et al.: The mGluR5 Positive Allosteric Modulator CDPPB Does Not Alter Extinction or Contextual Reinstatement of Methamphetamine-Seeking Behavior in Rats. J Addict Res Ther. 2011; S1(4). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerzig V, Schmidt WJ: Effects of MPEP on locomotion, sensitization and conditioned reward induced by cocaine or morphine. Neuropharmacology. 2004; 47(7): 973–84. PubMed Abstract \| Publisher Full Text\n\nHerrold AA, Voigt RM, Napier TC: Brain region-selective cellular redistribution of mGlu5 but not GABA(B) receptors following methamphetamine-induced associative learning. Synapse. 2011; 65(12): 1333–43. PubMed Abstract \| Publisher Full Text" }	[ { "id": "841", "date": "18 Mar 2013", "name": "David Triggle", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 this is a report of primarily negative findings it is not without value and should be published. The premise of the research is reasonable, the methods appropriate and the conclusions appropriate and not overreaching. Essentially, the workers have demonstrated through behavioural studies in rats that allosteric modulation – either positive or negative – of the metabotropic glutamate receptor 5 does not modify methamphetamine-induced behavioural sensitization. This adds to our knowledge of the effects of methamphetamine in its abuse.", "responses": [] }, { "id": "952", "date": "14 May 2013", "name": "Bianca Jupp", "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 publication by Kufahl and colleagues presents an investigation into the effect of positive and negative allosteric modulators of mGluR5 on the expression of locomotor sensitization to the psychostimulant methamphetamine, the results of which apparently support previous data regarding a lack of involvement of this receptor in the expression of sensitized locomotion. While the study is well designed, a critical component of the results was omitted making the interpretation of the current data impossible, and severely undermines the author’s conclusions. Specifically, while the authors methodologically included a saline challenge when assessing the expression of sensitization, they failed to report these results. Without this it is not possible to determine if indeed the increase in locomotor activity observed in the METH pre-treatment group is due to expression of conditioned hyperactivity or locomotor sensitization. I suspect it may be the former due to the apparently reduced locomotor activity (approx 1200) observed during this challenge session even when compared to acute METH (approx 2000). Usually expression of locomotor sensitization is much greater than the final conditioning session. It is therefore unreasonable for the authors to conclude that PAM or NAM of mGluR5 has no effect on expression of sensitization as it is not even clear if the animals are expressing sensitized behaviour. Inclusion of the saline challenge data will clarify this point. Have the authors considered using a longer ‘waiting’ period between development and testing expression? A recent study by Timmer and Steketee, 2012 found that intra-prefrontal cortex injections of the mGluR5 PAM MTEP reduced the expression of locomotor sensitization to cocaine following 21 days but not 7 days. The authors should include this in the discussion of their results.", "responses": [] }, { "id": "987", "date": "06 Jun 2013", "name": "Sharon Rosenzweig-Lipson", "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 present studies investigated the effects of positive and negative allosteric modulation of mGluR5 receptors on methamphetamine sensitization. The authors conclude that “Positive or negative allosteric modulation of metabotropic glutamate receptor 5 (mGluR5) does not alter expression of behavioral sensitization to methamphetamine”. While the data, in part, support those conclusions; the presence of an effect of 30 mg/kg fenobam on methamphetamine sensitization suggests at least some role of mGlur5 NAM activity. Evaluation of an additional NAM or a higher dose of fenobam would allow for a firmer conclusion on this point.", "responses": [] } ]	1	https://f1000research.com/articles/2-84
https://f1000research.com/articles/2-83/v1	11 Mar 13	{ "type": "Research Article", "title": "Nephron filtration rate and proximal tubular fluid reabsorption in the Akita mouse model of type I diabetes mellitus", "authors": [ "Jurgen Schnermann", "Mona Oppermann", "Yuning Huang", "Mona Oppermann", "Yuning Huang" ], "abstract": "An increase of glomerular filtration rate (hyperfiltration) is an early functional change associated with type I or type II diabetes mellitus in patients and animal models. The causes underlying glomerular hyperfiltration are not entirely clear. There is evidence from studies in the streptozotocin model of diabetes in rats that an increase of proximal tubular reabsorption results in the withdrawal of a vasoconstrictor input exerted by the tubuloglomerular feedback (TGF) mechanism. In the present study, we have used micropuncture to assess single nephron function in wild type (WT) mice and in two strains of type I diabetic Ins2+/- mice in either a C57Bl/6 (Akita) or an A1AR-/- background (Akita/A1AR-/-) in which TGF is non-functional. Kidney glomerular filtration rate (GFR) of anesthetized mice was increased by 25% in Akita mice and by 52% in Akita/A1AR-/-, but did not differ between genotypes when corrected for kidney weight. Single nephron GFR (SNGFR) measured by end-proximal fluid collections averaged 11.8 ± 1 nl/min (n=17), 13.05 ± 1.1 nl/min (n=23; p=0.27), and 15.4 ± 0.84 nl/min (n=26; p=0.009 compared to WT; p=0.09 compared to Akita) in WT, Akita, and Akita/A1AR-/- mice respectively. Proximal tubular fluid reabsorption was not different between WT and diabetic mice and correlated with SNGFR in all genotypes. We conclude that glomerular hyperfiltration is a primary event in the Akita model of type I diabetes, perhaps driven by an increased filtering surface area, and that it is ameliorated by TGF to the extent that this regulatory system is functional.", "keywords": [ "The development of both type I and type II diabetes mellitus (DM) is often associated with an increase of glomerular filtration rate (GFR) (usually referred to as glomerular hyperfiltration)", "and the same phenomenon has been observed in various experimental models of DM1", "2. The issue of diabetic hyperfiltration has attracted substantial interest because of the evidence that the occurrence of hyperfiltration may have some value in predicting the development of diabetic nephropathy3. This concept seemed plausible because of the evidence that hyperfiltration may be caused by increased glomerular capillary pressure and that intraglomerular hypertension represents a general risk factor for glomerular disease4. Despite the continuing debate about the reality behind the link between diabetic hyperfiltration and diabetic nephropathy", "the issue of the causation of glomerular hyperfiltration has been intensely pursued in rodent models of diabetes. Among the proposed mechanisms responsible for diabetic hyperfiltration", "relaxation of afferent arterioles in response to reduced input from tubuloglomerular feedback (TGF) has played a prominent role." ], "content": "Introduction\n\nThe development of both type I and type II diabetes mellitus (DM) is often associated with an increase of glomerular filtration rate (GFR) (usually referred to as glomerular hyperfiltration), and the same phenomenon has been observed in various experimental models of DM1,2. The issue of diabetic hyperfiltration has attracted substantial interest because of the evidence that the occurrence of hyperfiltration may have some value in predicting the development of diabetic nephropathy3. This concept seemed plausible because of the evidence that hyperfiltration may be caused by increased glomerular capillary pressure and that intraglomerular hypertension represents a general risk factor for glomerular disease4. Despite the continuing debate about the reality behind the link between diabetic hyperfiltration and diabetic nephropathy, the issue of the causation of glomerular hyperfiltration has been intensely pursued in rodent models of diabetes. Among the proposed mechanisms responsible for diabetic hyperfiltration, relaxation of afferent arterioles in response to reduced input from tubuloglomerular feedback (TGF) has played a prominent role.\n\nTGF is an intrarenal regulatory system that operates at the level of the juxtaglomerular apparatus, and that translates changes in NaCl concentration at a distal tubular site, probably the macula densa, into inverse changes of glomerular capillary pressure and nephron filtration rate5. Two different concepts have been advanced as to how TGF may be involved in the dysregulation of GFR in DM. One hypothesis argues that the primary process is the growth of the proximal tubule leading to enhanced water and solute reabsorption with the consequence that NaCl delivery to the macula densa decreases, the TGF-imposed vasomotor tone relaxes, and glomerular capillary pressure and GFR increase6. This “tubulo-centric” concept has been supported by substantial experimental evidence coming for the most part from experiments in rats with streptozotocin-induced type I DM6. Alternatively, it has been suggested that diabetic hyperfiltration is primary, driven by structural changes and/or by largely unknown derangements in the spectrum of vasoactive mediators, and that TGF serves as a mechanism that prevents the full extent of the effects of these abnormalities on GFR7. These two concepts are not easily reconcilable because vasorelaxation is caused by a normally functioning TGF in the first, whereas, in the second, vasorelaxation is TGF-independent and is in fact counteracted by it to the extent TGF is functional. This “glomerulo-centric” theory has found support in the finding that type I diabetic mice of the Akita strain without a functional TGF system, achieved by breeding the Ins2 mutation of the Akita mice into the TGF-less A1 adenosine receptor (A1AR) null background8, display exaggerated hyperfiltration compared to Akita mice with a presumably intact TGF9.\n\nIn a recent extensive review of the complex issues surrounding renal function in diabetic models, it has been argued that the failure to detect a clear TGF relaxation in the Akita mouse model of diabetes might be due to excessively high plasma glucose levels and the inability of proximal tubules to enhance reabsorption sufficiently10. This may prevent distal NaCl levels from falling, thereby maintaining some TGF activation and preventing hyperfiltration. Even though this argument does not explain the exaggerated hyperfiltration in the Akita mice with the A1AR-/- background in which the absence of TGF makes variations of distal NaCl irrelevant, we have taken this argument as an incentive to directly assess proximal fluid reabsorption by micropuncture in Akita diabetic mice with both native and A1AR-/- backgrounds. While confirming the presence of hyperfiltration in the TGF-less diabetic mice, our data show that there are no measurable reductions in the rates of proximal fractional fluid reabsorption between WT mice and diabetic animals with or without TGF. We therefore maintain the view that, at least in this particular model of type I diabetes, TGF serves as a mechanism protecting against the development of uncontrolled hyperfiltration.\n\n\nMethods\n\nMale Akita mice heterozygous for the Ins2 mutation (Ins2+/–; C57Bl/6 background) from Jackson Laboratories (Bar Harbor, ME, USA) were crossed with female C57Bl/6 WT mice in the NIH animal facility. To generate Ins2+/–/A1AR-/- double mutants, female A1AR-/- mice (C57Bl/6 background) were crossed with male F2 mice heterozygous for both the Ins2 (Akita) and A1AR mutations. All micropuncture experiments were performed in male animals Successful experiments were performed in 16 animals (WT=5, Akita=5, Akita/A1AR-/-=6). Mice were housed in the NIH animal facility at a room temperature of 22°C and a 12 hour dark/12 hour light cycle. Animal experimentation was approved and carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Genotyping for A1AR was done on tail DNA using PCR as described previously8. Genotyping of Ins2 was done by standard PCR (primers: sense TGCTGATGCCCTGGCC TGCT, antisense TGGTCCCACATATGCACATG) using AmpliTaq DNA Polymerase (Applied Biosystems, Foster City, CA, USA). The PCR product was then digested for 2 h with Fnu4HI restriction enzyme (Cat# R01785; New England Biolabs, Ipswich, MA, USA) and separated on agarose/ethidium bromide (3% [w/v]) gel to yield bands of 140 bp for WT, and 280 bp for the Ins2 mutation.\n\nFor micropuncture experiments, mice were anesthetized with 100 mg/kg thiobutabarbital (inactin) intraperitoneally and 100 mg/kg ketamine subcutaneously. Body temperature was maintained at 37.5°C by placing the animals on an operating table with a servo-controlled heating plate. The trachea was cannulated, and a stream of 100% oxygen was blown towards the tracheal tube throughout the experiment. The left carotid artery was catheterized with hand-drawn polyethylene tubing for continuous measurement of arterial blood pressure and blood withdrawal. A hand-drawn polyethylene catheter connected to an infusion pump was inserted into the right jugular vein for an intravenous maintenance infusion of saline at 400 µl/hr. The bladder was cannulated from a suprapubic midline incision for urine collections. Following a flank incision, the kidney was carefully dissected free of surrounding fat and placed in a lucite holder. The opening of the cup at the hilum was obstructed with fat and the kidney was covered with mineral oil.\n\nTo measure rates of proximal fluid reabsorption, an infusion of 125I-iothalamate (Glofil-125, Iso-Tex Diagnostics, Friendswood, TX, USA; ~40 µCi/hr) was started 20–25 minutes before micropuncture. Nephron filtration and absorption rates were determined by free-flow micropuncture as previously described11. Following tubular identification by dye injection, proximal collections were done in the last surface segment (collection duration 2.5 min in most cases ) using oil-filled pipettes. Fluid volume was determined from column length in a 0.5 µl Drummond microcap. Samples were transferred into a counting vial and radioactivity was determined in a gamma counter (Riastar, Packard Instrument Company, U.S.A.). Blood samples were collected in heparinized 5 µl microcaps at the beginning and at the end of the experiment. Temporal spacing between the two blood samples was between 38 and 55 minutes. Plasma reference values for each tubular sample were obtained by linear interpolation. 125I-iothalamate radioactivity was measured in duplicates using 0.5 µl samples of plasma and urine using Drummond 0.5 µl microcaps for sample transfer.\n\nAll reported statistical comparisons were made by one way ANOVA using the Bonferroni post hoc test at p<0.05 as showing significance (GraphPad Prism, GraphPad Software Inc., San Diego U.S.A.).\n\n\nResults\n\nA summary of measurements of GFR and a number of functional variables in WT (n=5), Akita (n=5), and Akita/A1AR-/- mice (n=6) during anesthesia is shown in Table 1. As observed previously in conscious mice, GFR was lower in WT than in diabetic mice reaching significance in the Akita diabetic mice with the A1AR deletion. Because body weights (BW) were not different between genotypes, the same increase was observed when GFR was normalized for 100 g of body weight. Interestingly, however, significant differences disappeared when GFR was expressed per kidney weight (KW), reflecting the fact that kidney weights were significantly higher in both groups of diabetic mice compared to WT. As indicated by the increased KW/BW ratio, the increase of kidney weight occurred without similar changes in body weight. It is safe to assume that glucosuria was the cause of the significantly higher urine flows in both strains of Akita mice as shown previously12. There were no significant differences between WT and diabetic mice in mean arterial blood pressure, body weight, or age. An estimate of the number of nephrons filtering at the level of measured SNGFRs (Kidney GFR/SNGFR) suggests that nephron numbers were not significantly different between the genotypes used in this study.\n\nGFR, glomerular filtration rate; MAP, mean arterial blood pressure; UV, urine flow rate; BW, body weight; KW, kidney weight; GFR/SNGFR, mean GFR divided by mean single nephron GFR (number of functional nephrons for both kidneys).\n\np<0.05, *p<0.01 (ANOVA with Bonferroni post hoc test; statistics given for comparison with wild type).\n\nMeasurements of SNGFR and fluid reabsorption along the proximal tubule by micropuncture confirmed the presence of hyperfiltration at the single nephron level (Figure 1A). Mean SNGFR was 11.8 ± 1 nl/min in WT (n=17), 13.05 ± 1.1 nl/min in Akita (n=23; p=0.27), and 15.4 ± 0.84 nl/min in Akita/A1AR-/- mice (n=26; p=0.009 compared to WT; p=0.09 compared to Akita). The 10.6% and 23% rise of SNGFR in Akita and double mutant mice respectively was less than the 24.8% and 56% increments of whole kidney GFR. Fractional fluid absorptions expressed as the ratio of iothalamate concentration in tubular fluid (TF) over that in plasma (P) (TF/Piothalamate) (Figure 1B) or converted to fractional fluid reabsorption in percent of GFR were not significantly different between WT and diabetic animals, averaging 1.84 ± 0.07 or 44.3 ± 2.3% in WT, 1.72 ± 0.05 or 40.7 ± 1.8% in Akita (p=0.27) and 2.1 ± 0.1 or 49.6 ± 2.3% in Akita/A1AR double mutant mice (p=0.06 compared to WT). Fluid reabsorption in absolute terms (Figure 2A) was not significantly different between WT and diabetic mice, but a tendency for slightly higher SNGFR and TF/Piothalamate values in the Akita/A1AR-/- mice added up to a significantly higher reabsorption rate compared to Akita mice (p<0.05 by ANOVA). Glomerulotubular balance, the relationship between GFR and reabsorption, was not disrupted and was not markedly different in the three strains of mice (Figure 2B).\n\nA: SNGFR in individual tubules; solid lines indicate mean values and broken lines are 95% confidence intervals. B: TF/Piothalamate ratios in individual tubules; solid lines are mean values and broken lines are 95% confidence intervals; numbers in brackets are numbers of mice/numbers of tubules.\n\nA: Fluid absorption rates in individual nephrons; solid lines are means, and broken lines are 95% confidence intervals. B: Relationship between SNGFR and reabsorption rate; lines indicate linear regressions.\n\n\n\n\n\n\n\n\n\n\nDiscussion\n\nPrevious measurements of GFR in conscious young animals have shown that type I diabetic Akita mice tend to show hyperfiltration that became highly significant in the A1AR-null genetic background12. Similarly, the induction of diabetes by alloxan was associated with hyperfiltration in both WT and A1AR-/- mice13. The present results confirm these observations in anesthetized animals in which the GFR of Akita mice increased by 25% in the C57Bl/6 background (nonsignificant) and by 56% (p<0.05) in the A1AR-/- background. As we have argued previously, the augmented hyperfiltration cannot be mediated by TGF since the A1AR-deficiency in both mixed WT and Akita diabetic genetic backgrounds renders TGF non-functional9,12,14. We cannot exclude the possibility that A1AR-deficiency directly enhanced GFR through some unknown mechanism. However, A1AR-deficient mice have been shown previously to have normal filtration rates9,15 so that the GFR-raising effect would have to be linked to A1AR deficiency under diabetic conditions. The present micropuncture results corroborate the irrelevance of TGF for hyperfiltration in diabetic Akita mice in another way. Hyperfiltration at the single nephron level was seen during withdrawal of fluid at late proximal tubular sites, thereby preventing fluid from gaining access to sites beyond the proximal tubule and effectively normalizing distal fluid delivery to zero. Thus, by eliminating TGF influences, this opening of the feedback loop reveals TGF-independent effects on GFR. To the extent that TGF is functional in a given animal or condition, SNGFR measured by proximal fluid collections represents a non-steady-state that overestimates SNGFR by acute removal of the GFR-suppressing action of TGF (Figure 3). In the present experiments, one may assume that SNGFR is closest to steady-state values in the Akita/A1AR-/- mice in which TGF is non-functional under all circumstances, and that proximal SNGFRs in WT and Akita mice overestimate true filtration rates to probably different degrees. We suggest that this overestimation is the reason why the relative changes of SNGFR in diabetic mice compared to WT are less than those of kidney GFR (10.6% and 23% vs. 25% and 56% in Akita and Akita/A1AR-/-, respectively). Our data are consistent with the notion that the rise of SNGFR in Akita/A1AR-/- mice is primary, and that it is made possible by absence of a TGF-mediated vasoconstrictor input. The tendency for GFR and SNGFR to increase in the Akita mice on a C57Bl/6 background may reflect the reduced TGF efficiency previously observed in both type I and type II diabetes9,16,17. Our study was not designed to identify the factors responsible for the increased filtration rate. Nevertheless, it is noteworthy that kidney GFR was not significantly different between control and diabetic animals when GFR was related to kidney weight. As also documented in the present studies, renal hypertrophy out of proportion to body weight is a well known early symptom of diabetes mellitus in patients, and this growth includes an increase in glomerular capillary surface area and thus presumably in the filtration coefficient18.\n\nSNGFR values (black dots) on the y axis represent SNGFR values from the present experiments; SNGFR values at LP flow of 30 nl/min come from our earlier study in which the effect of raising LP flow on early proximal flow rate was determined9. The negative lines connecting SNGFRs represent the TGF relationship that, for reasons of simplicity, is drawn as a linear relation, although it is known to be sigmoidal19,20. The slope of the positive line reflects proximal reabsorption, and it was calculated assuming a TF/Piothalamate ratio of 2. The intersects between the TGF and reabsorption relationships indicated by the open circles represent steady-state values for reabsorption and SNGFR19.\n\nThe issue of whether proximal fluid reabsorption is overwhelmed in Akita mice cannot be decided authoritatively due to the non-steady-state conditions and limited statistical power. Nevertheless, the present experiments did not show a significant reduction in either fractional or absolute proximal fluid reabsorption in Akita mice compared to WT mice (40.7 vs. 44.3% and 5.4 vs. 5.2 nl/min). The relative increase in plasma glucose levels of Akita mice at a young age is about threefold based on previous measurements9, from about 200 to 600 mg/dl, and therefore comparable to what has been reported in the streptozotocin model of diabetes in rats, except that baseline glucose in C57Bl/6 mice was higher7,17. The increase of proximal absorption in Akita/A1AR double mutant mice is for the most part due to the increase of SNGFR, reflecting the maintenance of load-dependent fluid reabsorption (Figure 2B). The inability to detect clear GFR-independent changes in proximal fluid reabsorption in our study is consistent with previous evidence that wide variations of glucose reabsorption rates have little effect on net fluid retrieval. For example, severe reductions of proximal glucose transport in SGLT2-deficient mice were not associated with significantly reduced proximal fluid fluxes21,22. Conversely, raising plasma glucose by infusion has little effect on proximal fluid reabsorption consistent with mathematical modeling, showing small and biphasic effects of glucose on water flux over a three- to fourfold variation of glucose around normal values23,24. While the links between Na and glucose uptake and between Na and water transport demand that fluid and glucose absorption rates should co-vary, the magnitude of the estimated effects is too small to be detectable by micropuncture.\n\nIn summary, our results confirm at the single nephron level that GFR increases in diabetic Akita mice as a function of TGF non-functionality, consistent with the notion that TGF prevents hyperfiltration in this model of type I diabetes. Rates of absolute and fractional fluid reabsorption were found to be comparable between control and diabetic animals. While hyperfiltration is seen in both streptozotocin-induced diabetes and in the genetic diabetes of the Akita mice, the mechanisms underlying this functional abnormality may be different in the two models.", "appendix": "Author contributions\n\n\n\nJS performed experiments, designed the study, and wrote the manuscript; MO and YH performed experiments, and approved of the manuscript.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the intramural research program of the National Institute of Diabetes and Digestive and Kidney Diseases, NIH (DK043408-12 KDB).\n\n\nReferences\n\nHelal I, Fick-Brosnahan GM, Reed-Gitomer B, et al.: Glomerular hyperfiltration: definitions, mechanisms and clinical implications. Nat Rev Nephrol. 2012; 8(5): 293–300. PubMed Abstract \| Publisher Full Text\n\nLevine DZ: Can rodent models of diabetic kidney disease clarify the significance of early hyperfiltration?: recognizing clinical and experimental uncertainties. Clin Sci (Lond). 2008; 114(2): 109–118. PubMed Abstract \| Publisher Full Text\n\nMagee GM, Bilous RW, Cardwell CR, et al.: Is hyperfiltration associated with the future risk of developing diabetic nephropathy? A meta-analysis. Diabetologia. 2009; 52(4): 691–697. PubMed Abstract \| Publisher Full Text\n\nHostetter TH, Rennke HG, Brenner BM: The case for intrarenal hypertension in the initiation and progression of diabetic and other glomerulopathies. Am J Med. 1982; 72(3): 375–380. PubMed Abstract \| Publisher Full Text\n\nSchnermann J, Briggs JP: Function of the juxtaglomerular apparatus: control of glomerular hemodynamics and renin secretion. In: The Kidney, Physiology and Pathophysiology, edited by Alpern RJ and Hebert SC. Burlington-San Diego-London: Elsevier Academic Press, 2008; p. 589–626.\n\nVallon V, Blantz RC, Thomson S: Glomerular hyperfiltration and the salt paradox in early [corrected] type 1 diabetes mellitus: a tubulo-centric view. J Am Soc Nephrol. 2003; 14(2): 530–537. PubMed Abstract \| Publisher Full Text\n\nPollock CA, Lawrence JR, Field MJ: Tubular sodium handling and tubuloglomerular feedback in experimental diabetes mellitus. Am J Physiol. 1991; 260(6 Pt 2): F946–952. PubMed Abstract\n\nSun D, Samuelson LC, Yang T, et al.: Mediation of tubuloglomerular feedback by adenosine: Evidence from mice lacking adenosine 1 receptors. Proc Natl Acad Sci U S A. 2001; 98(17): 9983–9988. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFaulhaber-Walter R, Chen L, Oppermann M, et al.: Lack of A1 adenosine receptors augments diabetic hyperfiltration and glomerular injury. J Am Soc Nephrol. 2008; 19(4): 722–730. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVallon V, Thomson SC: Renal function in diabetic disease models: the tubular system in the pathophysiology of the diabetic kidney. Annu Rev Physiol. 2012; 74: 351–375. PubMed Abstract \| Publisher Full Text\n\nHashimoto S, Adams JW, Bernstein KE, et al.: Micropuncture determination of nephron function in mice without tissue angiotensin-converting enzyme. Am J Physiol Renal Physiol. 2005; 288(3): F445–F452. PubMed Abstract \| Publisher Full Text\n\nFaulhaber-Walter R, Huang YG, Jou W, et al.: Adenosine A1 receptor deficiency in C57Bl/6 mice is associated with abnormal glucose tolerance, reduced insulin sensitivity, increased body weight and body fat fraction. Mid Atlantic Diabetes Research Meeting (abstract), 2006.\n\nSallstrom J, Carlsson PO, Fredholm BB, et al.: Diabetes-induced hyperfiltration in adenosine A(1)-receptor deficient mice lacking the tubuloglomerular feedback mechanism. Acta Physiol (Oxf). 2007; 190(3): 253–259. PubMed Abstract \| Publisher Full Text\n\nBrown R, Ollerstam A, Johansson B, et al.: Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor-deficient mice. Am J Physiol Regul Integr Comp Physiol. 2001; 281(5): R1362–1367. PubMed Abstract \| Publisher Full Text\n\nVallon V, Richter K, Huang DY, et al.: Functional consequences at the single-nephron level of the lack of adenosine A1 receptors and tubuloglomerular feedback in mice. Pflugers Arch. 2004; 448(2): 214–221. PubMed Abstract \| Publisher Full Text\n\nHashimoto S, Yamada K, Kawata T, et al.: Abnormal autoregulation and tubuloglomerular feedback in prediabetic and diabetic OLETF rats. Am J Physiol Renal Physiol. 2009; 296(3): F598–604. PubMed Abstract \| Publisher Full Text\n\nVallon V, Blantz RC, Thomson S: Homeostatic efficiency of tubuloglomerular feedback is reduced in established diabetes mellitus in rats. Am J Physiol. 1995; 269(6 Pt 2): F876–883. PubMed Abstract\n\nSchwieger J, Fine LG: Renal hypertrophy, growth factors, and nephropathy in diabetes mellitus. Semin Nephrol. 1990; 10(3): 242–253. PubMed Abstract\n\nBriggs J: A simple steady-state model for feedback control of glomerular filtration rate. Kidney Int Suppl. 1982; 12(Suppl. 12): S143–S150. PubMed Abstract\n\nBriggs JP, Schubert G, Schnermann J: Quantitative characterization of the tubuloglomerular feedback response: effect of growth. Am J Physiol. 1984; 247(5 Pt 2): F808–F815. PubMed Abstract\n\nLy JP, Onay T, Sison K, et al.: The Sweet Pee model for Sglt2 mutation. J Am Soc Nephrol. 2011; 22(1): 113–123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVallon V, Platt KA, Cunard R, et al.: SGLT2 mediates glucose reabsorption in the early proximal tubule. J Am Soc Nephrol. 2011; 22(1): 104–112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBishop JH, Green R, Thomas S: Free-flow reabsorption of glucose, sodium, osmoles and water in rat proximal convoluted tubule. J Physiol. 1979; 288: 331–351. PubMed Abstract \| Free Full Text\n\nWeinstein AM: Osmotic diuresis in a mathematical model of the rat proximal tubule. Am J Physiol. 1986; 250(5 Pt 2): F874–884. PubMed Abstract" }	[ { "id": "835", "date": "13 Mar 2013", "name": "Helle Praetorius", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 'Nephron filtration rate and proximal tubular fluid reabsorption in Akita mouse model of type I diabetes mellitus' is a follow up to the same groups previous publication on hyperfiltration in diabetic mice model. The manuscript is very clear and easy to read despite the relatively complicated content. It addresses essential points for the nature of DM- induced hyperfiltration and the conclusion of TGF as a relative protector against DM-induced hyperfiltation is well founded. In this paper the authors look at younger mice, which is commendable as the hyperfiltration is seen as an early event in diabetes that precedes significant DM-induced renal failure. The experiments are of a high quality and the figures are generally well prepared.Minor concerns:The current manuscript builds on previous results that firmly substantiate the used diabetic models. In this study the age of the mice are not completely comparable to the previous study. It would, however, be helpful if it was clear from the text that the relevant data is already available - that the animals in fact have high blood glucose and are seemingly slightly dehydrated. It is unclear where the data comes from in Figure 3 (30 nl/min). The legend states that the values come from reference 9 - but the mean values for EPFR given in that publication is: (5.0 nl/min - WT; 6.9 nl/min - Ins+/-; 11.5 nl/min Ins+/-A1AR-/-) – so this must apparently be new data for the 30 nl/min? – or is there some conversion of the numbers that is not perfectly clear. This point is relatively important as a fall from 15.2 nl/min (current value, 0 nl/min) to 11.5 nl/min (value from old paper, 30 nl/min) in the Ak/A1AR-/- is quite substantial and does witness about some TGF in this mouse. Moreover, what is the age of the animals (30 nl/min) that are compared here? Is it reasonable to make a line between the two points if they are indeed measured in separate experiments.It is not explicit that the n in the measurements of SNGFR is number of experiments and not animals. The test used to test for normal distribution of the data, which is a prerequisite for using the ANOVA test, is not stated under statistics. The values in the text are not given with the same number of decimals. The method for measuring GFR in general is not included in the method section.", "responses": [ { "c_id": "391", "date": "14 Mar 2013", "name": "Jurgen Schnermann", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Dr. Praetorius, We appreciate your comments, and provide the following in response to the concerns that you have raised: The youngest cohort of mice in our previous study (Faulhaber-Walter et al.) had an age of 14 weeks. While the age-matching between strains in the present experiments was not perfect, the mean of the ages is about 14 weeks. According to our previous data diabetic mice at this age have plasma glucose levels of between 600 and 700 mg/dl. This is an agreement with the original description of the Akita mice, in which diabetes had been shown to be of early onset with an approximate threefold increase of plasma glucose by 7 weeks of age (Yoshioka et al.). A manifestation of diabetes in the current experiments is the increased urine flow, which is a consequence of non-absorbed glucose as qualitatively established by dipstick, which affirms that hyperglycemia and the increase of filtered glucose must have exceeded the maximal glucose absorptive capacity. Figure 3 was meant to facilitate the understanding of the non-steady-state problem in SNGFR measurements in the proximal tubule rather than being a presentation of hard data. Nevertheless, the endpoint values at 30 nl/min were derived from reductions of the current SNGFR values by the relative EPFR decrements seen in the different strains previously which were 48% in wild type, 29% in Akita, and 0% in the double mutants. One might also point out that even if TGF didn’t exist at all and the SNGFRs were in fact steady-state values, the main argument could still be made in that measured GFR’s increased in the diabetic mice (at least in those without A1AR). The ages of the animals used at that time were between 9.8 and 17 weeks. It is hard to affirm Gaussian distribution if the sample size is relatively small, but all groups passed the KS test for normality. Nevertheless, subjecting the SNGFR data to a nonparametric test (Kruskal-Wallis) showed that the probability of the null hypothesis to be correct, 0.026 suggesting that the medians between the groups are in all likelihood different." } ] }, { "id": "840", "date": "18 Mar 2013", "name": "Bellamkonda Kishore", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Schnermann and associates entitled, “Nephron filtration rate and proximal tubular fluid reabsorption in the Akita mouse model of type I diabetes mellitus” addresses a critical question in the development of diabetic nephropathy. To address the critical question of the role of TGF in the glomerular hyperfiltration in diabetes nephropathy, the authors used transgenic mouse models. Diabetic nephropathy in human patients is a complex disease and no single transgenic mouse model of diabetic nephropathy can perfectly match the human disease. Despite these limitations, Schnermann and associates defined well the problem they addressed, and used appropriate mouse models of type I diabetes mellitus and skilled techniques. In this context, their approach is well controlled and provided meaningful data vis-à-vis the problem they attempted to address. Thus, overall it is convincing to this reviewer that their conclusions are valid and acceptable to the scientific community. I do not have any major issues, and only present a few minor concerns.Although, the manuscript is well written, at some critical places the use of long sentences is not advisable to convey the meaning smoothly, especially to the not-so-experienced readers and junior scientists.It is not clear to me whether +/- data presented in the Table 1 represent SD or SEM. It should be mentioned in the Table footnote as well as under the Statistics description.Although the scattered data points show the trends, however, looking at the Akita group in Fig 1A, and Ak/A1AR-/- group in Fig 1B and 2A, it is clear that the distribution is not normal. The Ak/A1AR-/- group has distinctive distributions as compared to the other two groups in these figure panels. Hence, the data presented in Figures 1 & 2 will be better interpretable by including parallel box plots. The box plots also provide more insights by showing the median and quartiles. The authors should consider this suggestion.", "responses": [ { "c_id": "430", "date": "02 Apr 2013", "name": "Jurgen Schnermann", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Dr. Kishore,Thanks for your comments!You talk about model limitations in your introduction, and I wanted to confirm that we agree and are fully aware of this. One of the most intriguing aspects of the human disease is its variability in that some diabetics have hyperfiltration and some don’t, and some of those that do may get renal disease and some don’t. I am not aware that any of the experimental models mimic this critical phenotype, in part perhaps because the statistical database isn't large enough to show it. In any case, the mechanism of hyperfiltration, something that one may get out of animal studies, is much less important than its predictive value for developing renal disease, and this one can get only out of human studies. Thus, hyperfiltration in mouse models (and the mechanisms causing it) may be more of academic than clinical interest. Certainly the question of whether an increase of plasma glucose into the diabetic range increases or decreases proximal tubular fluid reabsorption is a defined question that should be answerable, and the impression that even this limited question appears to be model-dependent is somewhat disappointing.The deviations presented in Table 1 are SEMs. In regard to the figures, we thought that by showing every single measurement we leave the issue of data distribution open. Box plots don’t add much other than 25% and 75% quartiles and minimum and maximal values, and I simply doubt that there is much information in this. As to distribution, we agree that some of the data look not normally distributed, but to conclude that this is an inherent property of one strain vs. another is most likely wrong. It is much more likely that data from the 7th, or 8th, or 9th or nth mouse would fill the void and make the data more and more normally distributed. In regard to the long sentences, we know we are no Shakespeares, but the writing does not seem too bad – comparatively." } ] } ]	1	https://f1000research.com/articles/2-83
https://f1000research.com/articles/1-64/v1	13 Dec 12	{ "type": "Case Report", "title": "Capecitabine-induced radiation recall phenomenon: a case report", "authors": [ "José Aguilar", "Elena García", "Elisa García-Garre", "Elena García", "Elisa García-Garre" ], "abstract": "Radiation recall dermatitis is defined as an inflammatory reaction of the skin at the site of previous irradiation. Different drugs have been associated with triggering this phenomenon, and it can also affect other areas and organs where previous radiotherapy has been administered. The time gap between the inflammatory reaction and previous radiation can range from days to several years.We report what we believe to be the first case of Capecitabine-induced Radiation Therapy Oncology Group (RTOG) Grade 4 recall skin toxicity (ulcerating dermatitis), which occurred three years after skin irradiation. Clinicians should be aware of this phenomenon, even when considering patients for whom it has been a long time since previous radiation therapy. This unusual and late drug side effect should be borne in mind in the differential diagnosis and management of advanced-disease patients as it may be confused with local relapse or infectious complication of previously operated areas.", "keywords": [ "We report a case of a radiation recall phenomenon after the administration of Capecitabine", "consisting of pain", "hyperpigmentation", "and ulceration in the field of postmastectomy irradiation (which the patient received 3 years previously)." ], "content": "Introduction\n\nWe report a case of a radiation recall phenomenon after the administration of Capecitabine, consisting of pain, hyperpigmentation, and ulceration in the field of postmastectomy irradiation (which the patient received 3 years previously).\n\n\nCase report\n\nA 78-year old woman allergic to salicylics was diagnosed with a T4dN3M0 (American Joint Committee on Cancer) infiltrating ductal left breast carcinoma (inflammatory breast cancer) in March 2006. Owing to her general condition and advanced local disease, she was initially treated with primary hormonotherapy consisting of Letrozole 2.5 mg/d over a period of 6 months with a good local response as measured by ultrasound scanning. In October 2006, she was operated on and a modified radical mastectomy was performed. Pathology reported a 6 cm in diameter infiltrating ductal carcinoma pT4dN2a positive for both estrogen and progesterone receptors, and Her2-neu negative. After surgery she started on chemotherapy (Taxol 80 mg/m2 on a weekly schedule for 4 weeks) and adjuvant radiotherapy (50 Gy over left hemithorax and supraclavicular nodes in February 2007). Immediately after initial radiotherapy, in 2007, she developed skin toxicity Radiation Therapy Oncology Group (RTOG) grade 2, which was successfully managed with topical medication (Radiocrem® Rotthafarm SL. 3 times a day). She started Letrozole 2.5 mg/d again in January 2007. In September 2009 she developed a neoplasic left pleural involvement and began hormonotherapy with Fulvestrant 500 mg/monthly for 5 months, followed by Exemestane 25 mg/d due to clinical and radiological progression. In May 2010, she developed new pleural progression, which was treated with Capecitabine at a dose of 1000 mg/m2/12h (3 cycles). Three months later in July 2010, she was noted to have developed a series of ulcers on the previous mastectomy scar, which had changed in colour (hyper and dispigmentation) and elasticity (stiffness and extreme fragility) over the skin of the previously irradiated area in the left hemithorax (Figure 1). A punch-biopsy was performed and carcinoma in the involved skin was ruled out. The diagnosis of a recall radiodermitis (ulcerating dermatitis, grade 4 RTOG) was thus established in July 2010 and so Capecitabine was withdrawn and palliative 20 mg Tamoxifen started. The skin began to improve after 3–4 weeks following withdrawal of Capecitabine and treatment with topical steroids (Menaderm® Menarini; Beclomeatsone 0.025%). She remains on Tamoxifen treatment, and the disease is now stable.\n\n\nDiscussion\n\nRadiation-recall dermatitis is an inflammatory reaction of the skin at the site of previous irradiation. Many chemotherapy drugs have been presumed to cause this phenomenon and a database to collect these rare reaction cases has even been proposed1. It can also affect other anatomical areas such as the digestive system (when abdominal radiotherapy has been used)2. Although a closer time gap is more usual, the time gap between the inflammatory reaction and previous radiation can range from days to several years3.\n\nThere are few reported cases of Capecitabine-induced radiation recall phenomenon, the first one being authored by Ortmann et al. in 20024. Their hypothesis relied on the pro-drug entity of Capecitabine being capable of being activated in previously irradiated tissue.\n\nMore recently, Ghosal and Misra have reported a case5 with thoracic hyperpigmentation instead of inflammation being the main clinical finding. Other rare though well known Capecitabine side effects are hyperpigmentation associated with palmar-plantar erythrodisesthesia6 or even Stevens-Johnson syndrome7.\n\n\nConclusions\n\nTo our knowledge, this is the first reported case of Capecitabine-induced RTOG grade 4 (ulcerating dermatitis) recall skin toxicity of previously irradiated skin. We suggest it is relevant for differential diagnosis with other entities such as local cancer relapse or even surgical site infection. In case of any doubt such as in our case, punch biopsy can help. Clinicians should be aware of this phenomenon, even if a long period of time has lapsed since the previous radiation therapy.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details and clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nEG, EGG and JA were the clinicians who treated the woman reported in the case. JA prepared the first draft of the manuscript which was revised by EG and EGG. JA prepared the text. All authors have been involved in the revision of the 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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAzria D, Magne N, Zouhair A, et al.: Radiation recall: a well recognized but neglected phenomenon. Cancer Treat Rev. 2005; 31(7): 555–70. PubMed Abstract \| Publisher Full Text\n\nSaif MW, Black G, Johnson M, et al.: Radiation recall phenomenon secondary to capecitabine: possible role of thymidine phosphorylase. Cancer Chemother Pharmacol. 2006; 58(6): 771–5. PubMed Abstract \| Publisher Full Text\n\nBurris HA 3rd, Hurtig J: Radiation recall with anticancer agents. Oncologist. 2010; 15(11): 1227–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrtmann E, Hohenberg G: Treatment side effects. Case 1. Radiation recall phenomenon after administration of capecitabine. J Clin Oncol. 2002: 20(13): 3029–30. PubMed Abstract\n\nGhosal N, Misra V: A case of capecitabine-induced hyperpigmentation and radiation recall phenomenon. Clin Oncol (R Coll Radiol). 2009; 21(8): 632. PubMed Abstract \| Publisher Full Text\n\nPui JC, Meehan S, Moskovits T: Capecitabine induced cutaneous hyperpigmentation: report of a case. J Drug Dermatol. 2002; 1(2): 202–205. PubMed Abstract\n\nSendur M, Kilickap S: Stevens-Johnson síndrome after treatment with capecitabine. Clin Oncol (R Coll Radiol). 2008; 20(2): 202–203. PubMed Abstract \| Publisher Full Text" }	[ { "id": "397", "date": "17 Dec 2012", "name": "Jay Thomas", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "398", "date": "19 Dec 2012", "name": "Rita De Sanctis", "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 give this article an ‘approved with reservations’. The reason for this is that I found that it lacks a few fundamental aspects:What was the response after 3 cycles of capecitabine? Please remember (and eventually cite) some reports of skin toxicity as predictor of response.It would be interesting to provide a description of the inflammatory characteristics of the punch-biopsy (even with iconografic report, if available).What was the presentation of the skin toxicity? Sudden or progressive? Were some topical medications used before the appearance of Figure 1 aspects?“Palliative” is all the setting of the present clinical case and not only when the authors decided to withdrawn capecitabine. What is the reason for the choice of tamoxifen instead of another chemotherapy,i.e. vinorelbine?Radiocrem (R): please provide the active components and avoid the commercial brand.What was the duration of stable disease (“the disease is now stable”).Palmar-plantar erythrodisesthesia is not so rare with capecitabine (my group and I have reported a 8% of G3/G4 skin toxicity with capecitabine). Moreover, according to guidelines of side effects management, do the authors believe that a rechallenge with capecitabine after dose reduction could be performed?", "responses": [ { "c_id": "371", "date": "08 Feb 2013", "name": "Jose Aguilar", "role": "Author Response", "response": "Dear Dr De Sanctis,Thank you for your detailed and relevant comments. We have sent an amended text to the editor that includes the following answers to your questions (and 2 figures from the skin biopsy): 1) After 3 cycles of capecitabine, the disease remained stable. Some authors have suggested that skin toxicity might be a predictor of response, though it has been better related to anti-epidermal growth factor receptor monoclonal antibodies (Petrelli F, et al. The predictive role of skin rash with cetuximab and panitumumab in colorectal cancer patients: a systematic review and meta-analysis of published trials. Target Oncol. 2013 Jan 16. [Epub ahead of print] ) http://www.ncbi.nlm.nih.gov/pubmed/233217772) In the biopsy, ulcers can be easily seen over granulation tissue. The area with preserved epidermis shows acanthosis and parakeratosis with loss of epidermal ridges. Vascular ectasia, hyalinized collagen and loss of skin adnexa is seen in the dermis. Infiltration by neoplasm was ruled out. We have enclosed 2 new figures with these findings to the original text. You can view these in Version 2 of our article, Figure 2.3) Skin toxicity appeared in a rapidly progressive pattern, no more than 2 weeks after the patient felt the first symptoms (skin stiffness and a burning local sensation) . No topical medication was used before the skin rash appeared.4) The term “palliative” has been used throughout the text to describe chemotherapy in the context of a patient with metastatic disease, with the objective of relieving symptoms and to delay the expected evolution. The possibility of the ulcers being the origin of an opportunistic infection was the reason why we chose the option of tamoxifen treatment instead of further chemotherapy.5) Radiocrem ® was used to avoid description of the different active components of the cream that include: tocopheryl acetate, disodium EDTA, Silybum marianum, Vitis vinifera, etc.6) Tamoxifen treatment allowed the patient to have 4 months with stable disease and serologic response. Afterwards, pleural progression was diagnosed and vinorebiline treatment was started with a good response; after 8 cycles, the patient suffered a new episode of skin toxicity that was managed with vinorebiline withdrawal and Letrozole treatment, which allowed for a 9 month stability period. On March 2012, progression was seen (liver metastasis and greater pleural effusion with clinical deterioration) and cyclophosphamide treatment was started. All active medication was stopped in May and palliative care lasted until the patient died a few months afterwards.7) We agree with your statement. “Rare” might not be the best word for this side effect frequency. Anyway, though palmar-plantar erythrodisesthesia is a well known dose-dependent effect, recall radiodermatitis does not seem to have that characteristic. That is why we tried to find another treatment and did not use capecitabine again in this patient." } ] } ]	1	https://f1000research.com/articles/1-64
https://f1000research.com/articles/2-80/v1	06 Mar 13	{ "type": "Short Research Article", "title": "Hygienic habits are a risk factor for adult-onset asthma", "authors": [ "Anna G Polunina" ], "abstract": "Multiple etiologies have been shown to contribute to asthma development, with excessive hygiene and microbial deprivation being one of the strongest risk factors for asthma onset in pediatric populations. The present study evaluated the contribution of hygienic habits in the development of adult-onset asthma. Twenty three adult-onset asthma patients (age of onset ranged from 21 to 71 years old) and 36 controls were asked to respond to a questionnaire concerning their frequency of shower taking and hand washing. Nine of the 23 (39.1%) asthmatic patients reported taking showers twice per day, compared to 2 controls (5.6%; χ2 = 15.4, p=0.017). In addition, sixteen (69.6%) of the asthmatic patients reported very frequent hand washing (≥ 7 times per day), whereas only 6 (16.7%) controls reported less frequent (2 – 6 times per day) hand washing habits. These data confirm that excessive hygienic habits are associated with the development of adult-onset asthma.", "keywords": [ "bronchial asthma", "hand washing", "hygienic hypothesis", "shower taking" ], "content": "Introduction\n\nAsthma is an important cause of quality of life impairment1,2, complications in long-term corticosteroid therapy3,4, increased health care utilization5 and mortality1. For instance, a recent study by Jia and colleagues1 demonstrated that the loss of the quality-adjusted life expectancy was 7.0 years for people with asthma compared to those without asthma. The prevalence of asthma has considerably increased during the twentieth century in both Western and Eastern Europe. For instance, in the UK, the rate of children consulting for asthma showed an eightfold increase from 1955/6 to 1991/2, and the rate of adults showed a three to fourfold increase6. Although from the mid-1990s the incidence of asthma stabilized or even somewhat decreased in Western societies6, this has not been true in Russia’s case. The Ministry of Healthcare of the Russian Federation reported an increase of asthma prevalence from 0.62% to 0.82% during the period from 2000 to 20087.\n\nMultiple factors have been shown to be associated with an increased risk of asthma. Allergy in parents and genetic factors8–10, bronchitis or pneumonia in infancy8, viral infections11, air pollution12, insufficient aerobic exercise13, obesity14, and special dietary patterns15 have all been associated with an increased asthma risk. At the same time, growing up or living in rural settings has been consistently shown to be associated with a reduced risk of asthma and allergies9,16,17. A study of 13,889 Belarusian children confirmed earlier findings in Western populations of the protective effects of rural settings, pet ownership and the presence of siblings on the risk of asthma development18. The latter findings underlie the hygiene hypothesis, which postulates that infections and unhygienic contact may confer protection against the development of allergic illnesses16,17.\n\nAdult-onset asthma and allergies appear to be highly prevalent in contemporary Russia. The official epidemiologic data do not show the real prevalence and incidence of asthma due to poor diagnostics at ambulatory centers and imperfect statistical service. However, even the official statistics showed an increase of adult-onset asthma incidence from 0.048% to 0.060% in 2008 compared to 20027. In a recent Swedish study, the incidence rate of adult-onset asthma (defined as “physician-diagnosed” asthma with onset at or after 16 years of age) was 2.3%19. In the Russian Federation, the prevalence of adult-onset allergy and asthma appeared to increase after the boundaries of the country were opened in the 1990s, and many Russian citizens visited European countries and adopted European life-styles, including daily showers and frequent hand washing.\n\nNo previous studies on the effects of hygienic habits on the risk of adult-onset asthma could be identified. Here the results of a pilot investigation of hygienic habits in asthmatic patients are presented. The findings presented here were discovered as part of a larger investigation on the evaluation of neurological complications of chronic asthma medications with a special attention to steroid myopathy20. Therefore, only two short questions concerning hygienic habits were added to the study protocol. Nevertheless, the findings of excessive hand washing and shower taking in asthmatic patients were significant in comparison with controls, and, therefore suggest excessive hygiene as a risk factor in asthma development in adults.\n\n\nMethods\n\nThe design of the present study was reviewed and approved by the administration of the Municipal Ambulatory Medical Service N124 of Moscow Health Care Department.\n\nPatients with bronchial asthma attending the Municipal Ambulatory Medical Service N124 of the Moscow Health Care Department were invited to participate in the study. The inclusion criteria for patients with asthma were: 1) recent consultation in the Pulmonology department, which confirmed that the patient suffered bronchial asthma; 2) asthma onset over the age of 20. The exclusion criteria were: 1) age younger than 20 and older than 85 years; 2) serious concomitant diseases. The first control group included patients attending the medical service due to non-allergic diseases (i.e., arterial hypertension, diabetes mellitus, etc.) or subjects who visited the medical center for prophylactic examination. In addition, we included patients with autoimmune diseases (sarcoidosis, pulmonary fibrosis, etc.) who received steroids, as the primary aim of our main study was the evaluation of symptoms of steroid myopathy. Patients with autoimmune diseases constituted the second control group. An age of 75 years or over, emergency cases and severe conditions were exclusion criteria for patients of control groups.\n\nIt was explained to all patients that the examination did not concern the medical management of their disease and was conducted as a part of scientific research. The methods of the examination were explained to all patients, and 23 asthmatic patients, 24 controls and 12 patients with autoimmune diseases gave a verbal informed consent to participate in the study as is customary in Russia.\n\nWe asked patients and healthy subjects to complete a two item questionnaire by choosing one of four possible responses. The first item contained the following question: ‘How often do you take shower or bath?’ The optional responses were: 1) twice per week or less; 2) 3 – 4 times per week; 3) once per day; 4) twice per day. The second item was: ‘How often do you wash hands?’ The optional responses were: 1) once per day or less; 2) 2 – 3 times per day; 3) 4 – 6 times per day; 4) 7 times and more per day. Responses demonstrating infrequent hygienic behaviors were evaluated as 1 point, and excessive hygienic behaviors (e.g., taking showers twice per day and hand washing over 7 times per day) were evaluated as 4 points, whilst intermediate habits ranged from 2 to 3 points.\n\nAll analyses were performed using SPSS software for windows (SPSS 17.0, Chicago, IL, USA).\n\nGroup characteristics were compared by ANOVA with a post hoc Bonferroni multiple comparisons correction. The distribution of hand washing and shower taking frequency in the three groups were compared by Pearson χ2-tests. Mann-Whitney tests were used for comparing mean scores on the hygienic habits questionnaire.\n\n\nResults\n\nPatient characteristics and statistics are presented in Table 1. The mean age of asthma onset was 43.4±12.8 years old in our patient group (range 21 – 71 years old). Patients of the first control group visited the medical service due to diabetes mellitus, arterial hypertension, myelopathy, gastritis, pancreatitis, dorsopathy and coronary disease. The autoimmune disease control group included patients with sarcoidosis, pulmonary fibrosis, chronic inflammatory demyelinating polyneuropathy and rheumatoid arthritis.\n\n*Means and standard deviations are presented where appropriate.\n\nAbbreviations: ADG, autoimmune disease group; AH, arterial hypertension; CD, coronary disease; CIDP, chronic inflammatory demyelinating disease; DM, diabetes mellitus; Do, dorsopathy; Ga, gastritis; My, myelopathy; NS, not significant; Pa, pancreatitis; Pro, prophylactical examination; PulmFibr, pulmonary fibrosis; RA, rheumatoid arthritis.\n\nThe asthma and both control groups did not show significant differences in age, education, height or weight. The asthma patients (mean age = 56 ± 13 years) were older in comparison with the autoimmune disease group (44 ± 15 years). In addition, the asthma patients were significantly shorter (mean height = 168 ± 10 cm) in comparison with the patients with autoimmune disease (177 ± 7 cm). Although, there was a larger proportion of females in the asthma group, this was not significant.\n\nAsthma patients significantly differed in hygienic habits from both control groups (Table 2). At the same time no differences in hygienic habits between the two control groups were found.\n\nNine of the 23 asthmatic patients reported taking showers twice per day, whereas only two patients with the autoimmune disease, and none in the non-autoimmune controls reported taking showers twice per day. Although 4 patients with asthma reported rare shower taking (twice per week or less), the same four patients reported excessive hand washing (7 or more times per day). When both control groups were combined together, asthma patients showed significantly higher shower taking scores (2.87 ± 1.14) in comparison with controls (2.11 ± 0.95; z=-2.59, p=0.01).\n\nSixteen (69.6%) asthmatic patients reported very frequent hand washing (7 or more times per day), whereas only 2 of the 24 (8.3%) patients without allergic or immune diseases and 2 of 12 (16.7%) patients with autoimmune diseases reported very frequent hand washing habits. When the two control groups were combined together, asthmatic patients showed significantly higher hand washing scores (3.61 ± 0.72) in comparison with controls (2.69 ± 0.75; z=-4.64, p < 0.001).\n\nWhen only males or only females were included in the analysis, the distribution of shower taking frequency did not reach significance. However, the distribution of hand washing frequency was still significantly higher in asthmatic patients in comparison with the combined control group in males [χ2 = 13.0, p=0.005] and females [χ2 = 8.7, p=0.013].\n\n\nDiscussion\n\nThese results support the hypothesis that excessive hygiene may underlie the high prevalence of allergic diseases in the contemporary world. Here it was found that excessive hygiene is significantly associated with adult-onset bronchial asthma. Interestingly, it was found that excessively frequent hand washing was more strongly related to bronchial asthma in comparison with shower taking. In contrast, non-allergic autoimmune diseases did not show associations with hygienic habits in this pilot study.\n\nInvestigations of the effects of infectious diseases on the risk of allergy development show an inverse and frequency dependent relationship: the more infections subjects have encountered as assessed by positive serology, the lower the observed prevalence of atopy, allergic rhinitis and asthma17. Many epidemiologic studies suggest that ‘microbial deprivation’ considerably contributes to the etiology of allergy and asthma21.\n\nA range of recent pediatric studies demonstrated that intestinal microbiota imbalances in infants are associated with the development of asthma and allergies in children over the age of three22,23. Higher counts of Lactobacilli, Bifidobacteria and Enterococci in stool samples were characteristic for non-allergic children, whereas an increased number of Clostridia were typical for children who developed allergies22. Collectively, these studies demonstrated the importance of gut microbial community as a correlate of the development of atopy23. Many studies have demonstrated the effectiveness of probiotic treatment for reducing atopic eczema in children, though studies of probiotic effects on asthma symptoms are limited24. In addition, a regular intake of probiotics was consistently shown to reduce respiratory symptoms of the common cold in both schoolchildren25 and adult26 cohorts.\n\nOverall, the negative effects of excessive hygiene in this patient cohort may be explained by an insufficient microbial load (brought about through excessive hygienic habits) and, therefore, abnormal functioning of the immune system in asthmatic patients. Insufficient immune system stimulation in infants has consistently been recognized as an important contributor to allergy development in previous studies22–24. These results provide evidence that excessive hygiene, perhaps mediated through microbial deprivation, is associated with adult-onset asthma as well.", "appendix": "Competing interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author declares that no grants were involved in supporting this work.\n\n\nReferences\n\nJia H, Zack MM, Thompson WW: The effects of diabetes, hypertension, asthma, heart disease, and stroke on quality-adjusted life expectancy. Value Health. 2013; 16(1): 140–7.\n\nSyk J, Alving K, Undén AL: Association between self-rated health and asthma: a population-based study. Clin Respir J. 2012; 6(3): 150–8.\n\nDahl R: Systemic side effects of inhaled corticosteroids in patients with asthma. Respir Med. 2006; 100(8): 1307–17.\n\nAngeli A, Guglielmi G, Dovio A, et al:High prevalence of asymptomatic vertebral fractures in post-menopausal women receiving chronic glucocorticoid therapy: a cross-sectional outpatient study. Bone. 2006; 39(2): 253–9.\n\nChipps BE, Zeiger RS, Borish L, et al:TENOR Study Group. Key findings and clinical implications from The Epidemiology and Natural History of Asthma: Outcomes and Treatment Regimens (TENOR) study. J Allergy Clin Immunol. 2012; 130(2): 332–42.\n\nAnderson HR, Gupta R, Strachan DP, et al:50 years of asthma: UK trends from 1955 to 2004. Thorax. 2007; 62(1): 85–90.\n\nOvsyannikov NV, Lyapin VA: Bronchial asthma in a large industrial center of the Western Siberia. Omsk: Izdatelstvo, 2010.\n\nGlushkova AV, Grjibovski AM: Prevalence and correlates of asthma among children in central St. Petersburg, Russia: cross-sectional study. Croat Med J. 2008; 49(6): 741–50.\n\nWennergren G, Ekerljung L, Alm B, et al:Asthma in late adolescence--farm childhood is protective and the prevalence increase has levelled off. Pediatr Allergy Immunol. 2010; 21(5): 806–13.\n\nFreĭdlin MB, Bragina Elu, Fedorova OS, et al:Genome-wide association study of allergic diseases in Russians of Western Siberia. Mol Biol (Mosk). 2011; 45(3): 464–72.\n\nHanania NA, King MJ, Braman SS, et al:Asthma in the elderly: Current understanding and future research needs--a report of a National Institute on Aging (NIA) workshop. J Allergy Clin Immunol. 2011; 128(3 Suppl): S4–24.\n\nJacquemin B, Kauffmann F, Pin I, et al:Air pollution and asthma control in the Epidemiological study on the Genetics and Environment of Asthma. J Epidemiol Community Health. 2012; 66(9): 796–802.\n\nAlexander CJ: Asthma: a disuse contracture. Med Hypotheses. 2005; 64(6): 1102–1104.\n\nKilic H, Oguzulgen IK, Bakir F, et al:Asthma in obese women: outcomes and factors involved. J Investig Allergol Clin Immunol. 2011; 21(4): 290–6.\n\nKim JH, Ellwood PE, Asher MI: Diet and asthma: looking back, moving forward. Respir Res. 2009; 10: 49.\n\nStrachan DP: Family size, infection and atopy: the first decade of the \"hygiene hypothesis\". Thorax. 2000; 55(Suppl. 1): S2–10.\n\nvon Mutius E: 99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: farm lifestyles and the hygiene hypothesis. Clin Exp Immunol. 2010; 160(1): 130–5.\n\nKramer MS, Matush L, Bogdanovich N, et al:The low prevalence of allergic disease in Eastern Europe: are risk factors consistent with the hygiene hypothesis? Clin Exp Allergy. 2009; 39(5): 708–16.\n\nTorén K, Ekerljung L, Kim JL, et al:Adult-onset asthma in west Sweden - Incidence, sex differences and impact of occupational exposures. Respir Med. 2011; 105(11): 1622–8.\n\nPolunina AG, Isaev FV, Demianova MA: Steroid-induced myopathy. Zh Nevrol Psikhiatr Im S S Korsakova. 2012; 112(10–2): 60–64.\n\nBjörkstén B: The hygiene hypothesis: do we still believe in it? Nestle Nutr Workshop Ser Pediatr Program. 2009; 64: 11–8.\n\nVael C, Vanheirstraeten L, Desager KN, et al:Denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma. BMC Microbiol. 2011; 11: 68.\n\nYoo J, Tcheurekdjian H, Lynch SV, et al:Microbial manipulation of immune function for asthma prevention. Proc Am Thorac Soc. 2007; 4: 277–282.\n\nToh ZQ, Anzela A, Tanq ML, et al:Probiotic therapy as a novel approach for allergic disease. Front Pharmacol. 2012; 3: 171.\n\nRerksuppaphol S, Rerksuppaphol L: Randomized controlled trial of probiotics to reduce common cold in schoolchildren. Pediatr Int. 2012; 54(5): 682–7.\n\nWest NP, Pyne DB, Cripps AW, et al:Lactobacillus fermentum (PCC®) supplementation and gastrointestinal and respiratory-tract illness symptoms: a randomised control trial in athletes. Nutr J. 2011; 10: 30." }	[ { "id": "858", "date": "22 Mar 2013", "name": "Dina Czeresnia", "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 article aims to evaluate a causal association between hygiene habits (washing hands and taking showers) and the onset of asthma in adults. In the introduction, the author justifies this option by using the hygienic hypothesis theory, which according to her 'postulates that infections and unhygienic contact may confer protection against the development of allergic diseases'. In my view this is a superficial proposal because the hygienic hypothesis theory, although so named, has a much wider scope than just considering hygiene habits as a risk factor to asthma. The hygienic hypothesis theory is concerned with the maturation of the immune system after birth driven by exposure to commensal or pathogenic microbes. The origin of asthma and other allergies could be related to a failed or underdeveloped maturation of the immune system occurring mainly during the early infancy. This is not necessarily restricted to a relation with washing hands or taking showers frequently. What is the theory underlining the hypothesis on the relationship between hygiene habits and the onset of asthma in adults? Furthermore, the research design is flawed. For instance, the questions about hygiene habits are investigated at the present time, and this disagrees with Hill's criteria about the temporal relationship between causes and effects.", "responses": [ { "c_id": "422", "date": "23 Mar 2013", "name": "Anna Polunina", "role": "Author Response", "response": "1) The hygienic hypothesis of allergies is yet the hypothesis but not the theory or completely clear scientific conception. Although, the association between the demographic characteristics of the populations and the prevalence of allergies, including asthma, is well established and reproduced in tens of contemporary studies, the immunological mechanisms underlying this association remain unclear. Dina Czeresnia states that immunological system develops only in infancy, and therefore all effects of microbiological environment may be of importance only in that early period of life. I don't think that the human brain develops all through the life untill the old age, whereas immunity develops only in infancy. Clearly, that many healthy and unhealthy immonological processes continue all through the life of the people. Therefore, changing of hygienic habits may influence immunity all through the life. For instance, my own allergy started when I was 21 years old, and I changed greatly my hygienic habits at that period due to my study in medical clinics and phobias of the infections in those clinic. I had no problems with allergy in my childhood, never. 2) I realize that the design of the study is far from the ideal, and the reported findings could be considered only as preliminary and pilot. However, if I could find in the available contemporary publications studies of the effects of the hygienic habits on allergy prevalence I would never spend my energy and time on making this study and article. My interest to this problem relates to my observations as a practicing physician of tens of asthmatic patients with steroid myopathy, and many of them could not walk (I visited them at home). Steroid inhalers are considered to be safe, but they are not safe. Therefore, I consider asthma prevention as a very important and highly perspective field of the research. Asthma prevention is possible, and control of hygienic habits is a very important issue. I think that the mechanism of negative effects of excessive shower taking and hand washing relates to excessive elimination of dust and microbes from the skin. OK, we can eliminate the dust and microbes from the skin, but we can't eliminate the latter from our bronches or mucosa of the respiratory tract. Therefore, the whole power of our immunity has nothing to do only to attack our bronches and other parts of respiratory tracts, and perhaps intestinum. Contemporary people do not have helminths, but we still have 'antihelminth' immunity, i.e. eosinophiles and IgE. This immunity attacks dust on the skin and bronches, and mucosa of the respiratory tract. And when the dust is completely removed from the skin, bronchs is the only place of immunological atacks. Clearly, the future evidence-based studies are needed in this field." } ] }, { "id": "870", "date": "27 Mar 2013", "name": "Paul Licciardi", "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 manuscript by Polunina assesses the negative association of hand washing and shower taking with the development of asthma in adults. The premise for this work is based on the hygiene hypothesis where reduced microbial exposures in early life can lead to the development of allergic diseases.While the study is interesting and shows a decreased frequency of hand washing and shower taking in the control group(s) compared to the asthmatic group, there are still some major issues with the design of the study and interpretation of the data. Most significantly, the data described in this paper was collected at a single time point and so the author should be careful not to over-interpret the findings in relation to cause and effect since it cannot be established that the hygienic habits resulted in the asthma symptoms. It is also likely that individuals with asthma may have an increased likelihood of undertaking additional hygienic habits. As the author notes, the hygiene hypothesis is complex and there are many different variables that can influence the development of allergic disease. Factors such as household size, vaccinations and diet can also have important contributions to this effect and should be taken into account in this cohort.", "responses": [] } ]	1	https://f1000research.com/articles/2-80
https://f1000research.com/articles/2-78/v1	06 Mar 13	{ "type": "Research Article", "title": "Anacardic acid, a histone acetyltransferase inhibitor, modulates LPS-induced IL-8 expression in a human alveolar epithelial cell line A549", "authors": [ "Tetsuo Yasutake", "Hiroo Wada", "Manabu Higaki", "Masuo Nakamura", "Kojiro Honda", "Masato Watanabe", "Haruyuki Ishii", "Shigeru Kamiya", "Hajime Takizawa", "Hajime Goto", "Tetsuo Yasutake", "Hiroo Wada", "Manabu Higaki", "Masuo Nakamura", "Kojiro Honda", "Masato Watanabe", "Haruyuki Ishii", "Shigeru Kamiya", "Hajime Goto" ], "abstract": "Objective and design: The histone acetylation processes, which are believed to play a critical role in the regulation of many inflammatory genes, are reversible and regulated by histone acetyltransferases (HATs), which promote acetylation, and histone deacetylases (HDACs), which promote deacetylation. We studied the effects of lipopolysaccharide (LPS) on histone acetylation and its role in the regulation of interleukin (IL)-8 expression. Material: A human alveolar epithelial cell line A549 was used in vitro.Methods: Histone H4 acetylation at the IL-8 promoter region was assessed by a chromatin immunoprecipitation (ChIP) assay. The expression and production of IL-8 were evaluated by quantitative polymerase chain reaction and specific immunoassay. Effects of a HDAC inhibitor, trichostatin A (TSA), and a HAT inhibitor, anacardic acid, were assessed. Results: Escherichia coli-derived LPS showed a dose- and time-dependent stimulatory effect on IL-8 protein production and mRNA expression in A549 cells in vitro. LPS showed a significant stimulatory effect on histone H4 acetylation at the IL-8 promoter region by ChIP assay. Pretreatment with TSA showed a dose-dependent stimulatory effect on IL-8 release from A549 cells as compared to LPS alone. Conversely, pretreatment with anacardic acid inhibited IL-8 production and expression in A549 cells. Conclusion: These data suggest that LPS-mediated proinflammatory responses in the lungs might be modulated via changing chromatin remodeling by HAT inhibition.", "keywords": [ "Pneumonia is an important socio-medical problem and one of the leading causes of death in the world1. Gram-negative rods", "crucial pathogens in hospital- as well as community-acquired pneumonia", "produce and release endotoxins which constitute lipopolysaccharides (LPS). Once inhaled via respiratory routes", "LPS stimulate alveolar structural as well resident cells to release many kinds of bioactive agents such as proinflammatory cytokines into local microenvironments2." ], "content": "Introduction\n\nPneumonia is an important socio-medical problem and one of the leading causes of death in the world1. Gram-negative rods, crucial pathogens in hospital- as well as community-acquired pneumonia, produce and release endotoxins which constitute lipopolysaccharides (LPS). Once inhaled via respiratory routes, LPS stimulate alveolar structural as well resident cells to release many kinds of bioactive agents such as proinflammatory cytokines into local microenvironments2.\n\nRecent studies have emphasized a crucial role for the lung epithelium as an important sentinel and effector system of innate immunity3–5. Upon infectious agents and their products being inhaled into the respiratory systems, activated lung epithelium may contribute to the regulation of the immune response as the first-line defense mechanism6. Among those defense responses, it seems important that alveolar epithelial cells express and release a variety of pro-inflammatory cytokines and chemokines into alveolar microenvironments6,7. A CXC chemokine, interleukin (IL)-8, plays an important role in the acute recruitment of immune/inflammatory cells, especially neutrophils, to the site of infection in the lung8,9. LPS bind to Toll-like receptors (TLR) and thereby activate downstream signal transduction pathways which ultimately phosphorylate cytosolic I-κB kinase10,11. Then, I-κB is phosphorylated to induce free-form of NF-κB, which translocates into the nucleus. The NF-κB binds to its specific binding sites on the promoter regions and enhances the expression of IL-8 gene12,13.\n\nIncreasing evidence has indicated that the expression of many inflammatory genes involves the remodeling of the chromatin structure provided by histone proteins14,15. Remodeling of chromatin within the nucleus is controlled by the degree of acetylation/deacetylation of histone residues on the histone core around which DNA is coiled. Histone acetylation results in the unwinding of the chromatin structure, which enhances the binding of transcription factors to their specific promoter sites on the DNA16. Nuclear histone acetylation is a reversible process and is regulated by a group of histone acetyltransferases (HATs) which promote acetylation, and histone deacetylases (HDACs) which promote deacetylation17,18. The loosening of DNA-histone interactions and the subsequent unmasking of transcription factor binding sites is controlled by specific covalent modifications of accessible N-terminal histone tails19. Among the four core histone proteins that comprise the central chromatin core (H2A, H2B, H3, and H4), acetylation processes on H3 and H4 seem particularly important in gene regulation. For example, Gilmour and associates20 found that acetylation on H4 played an important role in environment particle-induced IL-8 production in A549 cells. Viable Listeria monocytogenes-stimulated endothelial cells showed increased expression of IL-8, and that process depended on modifications of H3 and H421. Although the host response in pneumonia is characterized by massive cytokine production, and altered histone modifications have been observed in diseased lungs22, it is not fully elucidated how histone modifications contribute to innate immune regulation in the lung.\n\nIn this study, we tried to determine whether Escherichia coli-derived LPS, one of the mainstream stimuli upon bacterial respiratory infection, altered histone acetylation/deacetylation balance, and to see whether the modulation of HDACs or HATs by their specific inhibitors (i.e. trichostatin A [TSA] for HDACs and anacardic acid for HATs) affected IL-8 gene expression and protein production in an alveolar epithelial cell line A549 in vitro.\n\n\nMaterials and methods\n\nHuman alveolar epithelial cell line A549 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) via DS Pharma Biomedical Co., Ltd (Tokyo, Japan). A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, NY, USA) and 1% penicillin-streptomycin (Sigma-Aldrich, St, Louis, MO, USA), and incubated at 37°C in 5% CO2 atmosphere. Cells were cultured to 80% confluency as judged under inverted microscopy before the medium was replaced with serum-free Dulbecco’s Modified Eagle’s Medium and incubated for a further 15 h. The cells were stimulated with different concentrations of E.coli-derived LPS (026:B6, Sigma-Aldrich, St, Louis, MO, USA) for further experiments. To evaluate the effects of HDAC and HAT inhibitors, cells were pre-treated with TSA or anacardic acid (Sigma-Aldrich, St, Louis, MO, USA; dissolved in dimethyl sulfoxide [DMSO] and further diluted for use) at the concentrations indicated, 1h prior to stimulation with LPS (10 μg/ml).\n\nTotal RNA from A549 cells was isolated with the RNeasy Mini kit (Qiagen, Hamburg, Germany), and cDNA was prepared using the Im Prom II reverse transcription system (Promega, Madison, WI, USA) for reverse transcription, and all procedures were conducted according to the manufacturers’ instructions. Gene transcript levels of IL-8, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified by real-time PCR using a reaction mixture with SYBR Premix Ex Taq (Takara, Tokyo, Japan) on a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primer sets for IL-8 were: (forward) 5’-CTGATTTCTGCAGCTCTGTG-3’; (reverse) 5’-TTCACTGGCATCTTCACTG-3’ and for GAPDH were: (forward) 5’-TGAACGGGAAGCTCACTGG-3’ (reverse) 5’-TCCACCACCCTGTTGCTGTA-3’ The relative amount of gene transcript was estimated after normalization by dividing the calculated value for the gene of interest by the GAPDH value.\n\nChromatin immunoprecipitation was performed using Millipore’s ChIP kit (Billerica, Massachusetts, USA) with an acetyl-histone H4 antibody. Cells were cultured to 80% confluency in 100mm culture plates. Cells were cross-linked by adding 1% formaldehyde for 10 minutes at room temperature in shaking. Then, 1ml of 10×glycine was added to 10ml of growth media to each dish to quench unreacted formaldehyde at room temperature for 5 minutes. Cells were washed twice with cold 10ml phosphate buffered saline (PBS). One ml PBS containing 1×protease inhibitor cocktail II prepared in the ChIP kit was added to each dish. Cells were scraped from each dish into a conical tube. The tubes were centrifuged at 700×g at 4°C for 5 minutes to pellet cells. Each cell pellet was resuspended in 1ml of SDS lysis buffer containing 1×protease inhibitor cocktail II prepared in the ChIP kit. Chromatin was sonicated to an average DNA length of 200–1000 bp using an Ultrasonic Disruptor UD-200 sonicator (TOMY, Tokyo, Japan). Sonicated samples were centrifuged and the supernatant was collected. Samples (100μl) of the extracted chromatin were diluted with 900μl ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl), precleared (1 hour) by incubation with 60μl Protein G Agarose containing 1×protease inhibitor cocktail II, and subjected to immunoprecipitation with a specific antibody with rotation overnight at 4°C. The antibody used for ChIP assays was anti-acetyl histone H4 antibody (Millipore, Billerica, Massachusetts, USA). Immunocomplexes were collected by adsorption onto 60μl Protein G Agarose and the beads were washed five times sequentially with Low Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), High Salt Immune Complex Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl) and LiCl Immune Complex Wash Buffer (0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1), which were prepared in the ChIP kit. Precipitates were washed twice with TE Buffer (10mM Tris-HCl, pH 8.0, 1mM EDTA), and antibody-chromatin fragments were eluted from the beads with 1% sodium dodecyl sulphate in 0.1 M NaHCO3. Cross-links were reverted by adding 200mM NaCl and heating at 65°C for 5 hours. In total, 10mg/ml RNase A were added and samples were then incubated for 30 minutes at 37°C. 10mg/ml proteinase K, 10mM EDTA and 40mM Tris-HCl were added and samples were then incubated for 2 hours at 45°C.\n\nA total of 1ml of Bind Reagent A was added to each sample, and mixed well by pipetting. The sample was transferred up to a spin filter placed in a collection tube, and was centrifuged for 30 seconds at 12000×g. Then, 500μl of Wash Reagent B was added to each spin filter placed in a collection tube, and was centrifuged for 30 seconds at 12000×g. Then, 50μl of Elution Buffer C was added to each spin filter placed in a clean collection tube, and was centrifuged for 30 seconds at 12000×g to recover purified DNA. The IL-8 enhancer regions were quantified by real-time PCR using a reaction mixture with SYBR Premix Ex Taq (Takara, Tokyo, Japan) on a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primer sets for IL-8 were: (forward) 5’- CAGAGACAGCAGAGCACAC-3’; (reverse) 5’- ACGGCCAGCTTGGAAGTC-3’. All PCR signals from immunoprecipitated DNA were normalized to PCR signals from non-immunoprecipitated input DNA.\n\nThe concentration of IL-8 in the media was determined by sandwich ELISA kit (R&D Systems, San Diego, CA) according to the manufacturer’s instructions.\n\nData were analyzed with the Statistical Package for Social Science (SPSS) version 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Values are expressed as mean ± standard error (SE). Mann-Whitney U test was performed for comparisons between groups. A P-value < 0.05 was considered significant. All P-values were two-sided.\n\n\nResults\n\nWe analyzed IL-8 production in LPS stimulated A549 cells. A549 cells were stimulated by different concentrations of LPS (10 ~ 200μg/ml) for indicated time periods. As shown in Figure 1, LPS showed a time- and dose-dependent stimulatory effect on IL-8 release.\n\nA549 cells were cultured until 80% confluence and stimulated by different concentrations of lipopolysaccharide (LPS) (10 ~ 200μg/ml). LPS showed a time- and dose-dependent stimulatory effect on IL-8 release at each concentration. Data were expressed in mean±SEM. P<0.05, compared to LPS unstimulated cells at each concentration and time point (Mann-Whitney U test), n=4.\n\nA549 cells were stimulated by 10μg/ml LPS, and the time course in levels of IL-8 mRNA was analyzed by qRT-PCR. IL-8 mRNA levels showed a gradual increase in response to LPS, reaching a maximum level 2 h after initial stimulation with 10μg/ml, which then decreased after that point (Figure 2).\n\nA549 cells were cultured until subconfluence and stimulated by LPS at 10μg/ml. After 0 ~ 8 hrs, IL-8 mRNA expression was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The IL-8 mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Data were expressed in mean±SEM. P<0.05: compared to LPS 0hr stimulated cells (Mann-Whitney U test), n=4.\n\nBased on the previous reports showing that modifications of histones, in particular acetylation of H4, seem to contribute to the regulation of inflammatory genes such as IL-820, we assessed acetylation of H4 after stimulation of LPS in A549 cells. We analyzed histone modifications at the IL-8 gene promoter by ChIP assay. A549 cells were stimulated with LPS (10μg/ml) for 0 min to 3 h. LPS induced a time-dependent increase of acetylation of H4 at the IL-8 promoter, and this increase peaked after 1 h (P<0.05, Mann-Whitney U test), and then decreased after 3 h of LPS stimulation (Figure 3).\n\nA549 cells were stimulated 0 ~ 3 hr with LPS (10μg/ml). The results presented are from ChIP analyses using anti-acetyl H4 antibodies. All PCR signals from immunoprecipitated DNA were normalized to PCR signals from non-immunoprecipitated input DNA. Results are expressed as percentage of the input. Data were expressed in mean±SEM. P<0.05: compared to LPS 0hr stimulated cells (Mann-Whitney U test), n=4.\n\nNext, we wondered whether inhibition of HDACs by TSA or blocking of HATs by anacardic acid impacts on LPS-induced IL-8 expression. We increased global histone acetylation by incubation of A549 cells with TSA (10nM, 24 h) which did not induce IL-8 secretion per se (Figure 4). Preincubation for 1 h with TSA (10nM) before subsequent treatment with LPS significantly increased IL-8 release as compared to LPS alone (Figure 4). Pretreatment with TSA (10nM) showed a tendency to increase IL-8 mRNA levels as assessed by qPCR analysis, but did not reach statistical significance (Figure 5).\n\nTSA at 1 ~ 1000nM treated 1 hr before LPS stimulation (10μg/ml) showed a significant stimulatory effect on LPS-induced IL-8 release. Data were expressed as mean±SEM. P<0.05: compared to LPS-stimulated cells (Mann-Whitney U test), n=4.\n\nTSA at 10nM treated 1 h before LPS stimulation (10μg/ml) tended to show a stimulatory effect on LPS-induced IL-8 gene activation. After 0 ~ 8 h, the levels of IL-8 mRNA were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The IL-8 mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels, n=4.\n\nSuppression of histone acetylation by blocking of HATs via anacardic acid showed a dose-dependent inhibitory effect on LPS-stimulated IL-8 production (Figure 6), indicating that histone deacetylation regulates IL-8 expression in LPS-treated epithelial cells. The effects of anacardic acid on IL-8 mRNA expression were analyzed by qRT-PCR. Anacardic acid at 100μM administered 1 h before LPS (10μg/ml) stimulation showed a significant inhibitory effect on LPS-induced IL-8 mRNA levels (Figure 7). These observations indicated that modulation of histone acetylation by anacardic acid regulated LPS-stimulated IL-8 gene expression in A549 cells.\n\nAnacardic acid at 10 ~ 100μM treated 1 hr before LPS stimulation showed a significant inhibitory effect on LPS-induced IL-8 release. Data were expressed as mean±SEM. DMSO: dimethyl sulfoxide used for solvent. P<0.05: compared to LPS (10μg/ml) stimulated cells (Mann-Whitney U test), n=4.\n\nAnacardic acid at 100μM treated 1 hr before LPS (10μg/ml) stimulation showed a significant inhibitory effect on LPS-induced IL-8 gene activation. After 0 ~ 8hrs, the levels of IL-8 mRNA were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The IL-8 mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Data were expressed as mean±SEM. P<0.05 (Mann-Whitney U test), n=4.\n\n\nDiscussion\n\nIn the present study, we demonstrated that E. coli-derived LPS stimulate human alveolar epithelial A549 cells to express and release IL-8, an important chemokine for the local recruitment of neutrophils as an initial defense mechanism8,9. The specific histone acetylation processes were evaluated by ChIP assay, which clearly showed that LPS induced histone H4 acetylation in A549 cells. Next, we studied the effects of TSA, a HDAC inhibitor on LPS-induced IL-8 expression. TSA showed a significant stimulatory effect on IL-8 production, whereas this agent tended to increase, although not significantly, IL-8 mRNA levels in LPS-stimulated A549 cells. Finally, a HAT inhibitor, anacardic acid, significantly decreased IL-8 mRNA levels as well as protein release in a dose-dependent fashion. These results suggested that LPS-induced IL-8 gene expression is, at least in part, regulated by histone H4 acetylation/deacetylation balance at the IL-8 promoter region.\n\nIt has been reported that alveolar epithelial cells respond to bacterial products such as LPS to produce a variety of inflammatory cytokines and mediators including IL-82. We23,24 and others9,25,26 have previously shown that airway and alveolar epithelial cells, including A549 cells, are activated by a variety of endogenous agents such as cytokines as well as exogenous stimuli including LPS and fine particles, and express biologically active compounds including cytokines and chemokines.\n\nThe transcription of many genes is known to correlate with levels of acetylated nuclear histone proteins14–16. The expression of many inflammatory genes involves the remodeling of the chromatin structure provided by histone proteins. Histone acetylation causes the unwinding of the chromatin structure, thereby enabling transcription factors to bind to their specific promoter sites on the DNA. Such acetylation processes are reversible and regulated by HATs, which promote acetylation, and HDACs, which promote deacetylation18,19.\n\nProinflammatory gene transcription regulation is a multifaceted process that requires integrated sequential molecular events for maximal gene transcription to occur. Activation of specific gene expression needs to be associated with co-operated chromatin remodeling by histone acetylation and binding of transcription factors to their specific binding sites on DNA. In the present experiments, the levels of histone H4 acetylation peaked 1 h after LPS stimulation followed by the maximal levels of IL-8 mRNA at 2 h; such a time course was consistent with the above scenarios.\n\nOur study supports a hypothesis that histone H4 acetylation could play a key role in inflammatory gene transcription such as IL-8 in A549 cells caused by LPS; however, the role of acetylation of the other histones in this model is yet to be established. It has been reported that a variety of histone acetylations are involved in cytokine transcriptional processes. Miyata et al.27 have shown that H3 and H4 are acetylated in murine epithelial cells in response to granulocyte-colony stimulating factor (G-CSF) at the myeloperoxidase gene promoter site, a response dependent on MAPK activation.\n\nAnacardic acid is a bioactive phytochemical found in the nutshell of Anacardium occidentale28. The promising effect of anacardic acid on IL-8 gene regulation in the current study has already been reported by Schmeck and associates29 in Legionella pneumophila-derived flagellin-induced IL-8 expression. Although the available records are promising, more detailed investigation into the therapeutic properties of anacardic acid, particularly the anti-cancer and anti-inflammatory activities, are needed30.\n\nLPS-mediated IL-8 production is generally expected to act as a defense mechanism which recruits neutrophils in the local environment and facilitates bacterial elimination. However, it is now also known that excessive accumulation of activated neutrophils in the lung is likely to cause excessive inflammatory changes that damage lung tissues8. In this context, appropriate control of local inflammation would be an important strategy for the management of severe pneumonia22. Recently, it has been attracting attention that macrolide antibiotics added to guideline-based therapy has shown a better outcome than respiratory quinolones among intensive care unit (ICU) hospitalized patients with severe pneumonia and/or sepsis31. It has been speculated that certain anti-inflammatory actions of macrolides were involved in such clinically beneficial effects32. Therefore, the suppressive effects of anacardic acid on IL-8 gene expression and production might become a novel strategy for controlling excessive inflammation in order to lessen the risks of acute lung injury and, ultimately, respiratory failure to death.\n\nIn conclusion, we have shown that E. coli-derived LPS-induced expression and release of IL-8 are associated with increased acetylation of histone H4 expression. The HDAC inhibitor TSA increased IL-8 release. In contrast, a HAT inhibitor, anacardic acid, significantly decreased IL-8 mRNA levels as well as protein release in a dose-dependent fashion. These results suggested that regulation of HDAC and HAT activity by low molecular weight agents might become a novel strategy for the appropriate control of inflammation by modulating inflammatory mediators such as IL-8.", "appendix": "Author contributions\n\n\n\nTY, HW, HT and HG conceived the study. TY and MH designed the experiments. TY, MN, KH, MW and HI carried out the research. SK contributed to the design of experiments and provided expertise in ChIP experiments. TY, HW and HT prepared the first draft of the manuscript. MW and HI contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported in part by the scientific grants from the Japanese Ministry of Education, Science and Culture (Hajime Goto, M.D., Ph.D.: grant number 21591298, and Hiroo Wada, M.D., Ph.D.: grant number 23591481).\n\n\nAcknowledgements\n\nWe thank Ms Makiko Sakamoto for technical assistance during this project. We also thank Ms Yumiko Date for secretarial assistance.\n\n\nReferences\n\nMizgerd JP: Acute lower respiratory tract infection. N Engl J Med. 2008; 358(7): 716–727. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThorley AJ, Grandolfo D, Lim E, et al.: Innate immune responses to bacterial ligands in the peripheral human lung--role of alveolar epithelial TLR expression and signalling. PLoS One. 2011; 6(7): e21827. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGilmour PS, Rahman I, Hayashi S, et al.: Adenoviral E1A primes alveolar epithelial cells to PM(10)-induced transcription of interleukin-8. Am J Physiol Lung Cell Mol Physiol. 2001; 281(3): L598–L606. PubMed Abstract\n\nHashimoto S, Gon Y, Takeshita I, et al.: Diesel exhaust particles activate p38 MAP kinase to produce interleukin 8 and RANTES by human bronchial epithelial cells and N-acetylcysteine attenuates p38 MAP kinase activation. Am J Respir Crit Care Med. 2000; 161(1): 280–5. PubMed Abstract \| Publisher Full Text\n\nNakamura H, Yoshimura K, Jaffe HA, et al.: Interleukin-8 gene expression in human bronchial epithelial cells. J Biol Chem. 1991; 266(29): 19611–7. PubMed Abstract\n\nChuquimia OD, Petursdottir DH, Rahman MJ, et al.: The Role of Alveolar Epithelial Cells in Initiating and Shaping Pulmonary Immune Responses: Communication between Innate and Adaptive Immune Systems. PLoS One. 2012; 7(2): e32125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanders CJ, Doherty PC, Thomas PG: Respiratory epithelial cells in innate immunity to influenza virus infection. Cell Tissue Res. 2011; 343(1): 13–21. PubMed Abstract \| Publisher Full Text\n\nChollet-Martin S, Montravers P, Gibert C, et al.: High levels of interleukin-8 in the blood and alveolar spaces of patients with pneumonia and adult respiratory distress syndrome. Infect Immun. 1993; 61(11): 4553–9. PubMed Abstract \| Free Full Text\n\nLi B, Dong C, Wang G, et al.: Pulmonary epithelial CCR3 promotes LPS-induced lung inflammation by mediating release of IL-8. J Cell Physiol. 2011; 226(9): 2398–405. PubMed Abstract \| Publisher Full Text\n\nAkira S: Toll-like receptor signaling. J Biol Chem. 2003; 278(40): 38105–38108. PubMed Abstract \| Publisher Full Text\n\nMitchell JA, Paul-Clark MJ, Clarke GW, et al.: Critical role of toll-like receptors and nucleotide oligomerisation domain in the regulation of health and disease. J Endocrinol. 2007; 193(3): 323–30. PubMed Abstract \| Publisher Full Text\n\nHenkel T, Machleidt T, Alkalay I, et al.: Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B. Nature. 1993; 365(6442): 182–5. PubMed Abstract \| Publisher Full Text\n\nPark GY, Christman JW: Nuclear factor kappa B is a promising therapeutic target in inflammatory lung disease. Curr Drug Targets. 2006; 7(6): 661–8. PubMed Abstract \| Publisher Full Text\n\nEgger G, Liang G, Aparicio A, et al.: Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004; 429(6990): 457–63. PubMed Abstract \| Publisher Full Text\n\nBarnes PJ, Adcock IM, Ito K: Histone acetylation and deacetylation: importance in inflammatory lung diseases. Eur Respir J. 2005; 25(3): 552–63. PubMed Abstract \| Publisher Full Text\n\nKuo MH, Allis CD: Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays. 1998; 20(8): 615–626. PubMed Abstract \| Publisher Full Text\n\nTang YA, Wen WL, Chang JW, et al.: A novel histone deacetylase inhibitor exhibits antitumor activity via apoptosis induction, F-actin disruption and gene acetylation in lung cancer. PLoS One. 2010; 5(9): e12417. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClaus R, Fliegauf M, Stock M, et al.: Inhibitors of DNA methylation and histone deacetylation independently relieve AML1/ETO-mediated lysozyme repression. J Leukoc Biol. 2006; 80(6): 1462–72. PubMed Abstract \| Publisher Full Text\n\nGrunstein M: Histone acetylation in chromatin structure and transcription. Nature. 1997; 389(6649): 349–52. PubMed Abstract \| Publisher Full Text\n\nGilmour PS, Rahman I, Donaldson K, et al.: Histone acetylation regulates epithelial IL-8 release mediated by oxidative stress from environmental particles. Am J Physiol Lung Cell Mol Physiol. 2003; 284(3): L533–40. PubMed Abstract \| Publisher Full Text\n\nSchmeck B, Beermann W, van Laak V, et al.: Intracellular bacteria differentially regulated endothelial cytokine release by MAPK-dependent histone modification. J Immunol. 2005; 175(5): 2843–50. PubMed Abstract\n\nGoodman RB, Strieter RM, Martin DP, et al.: Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am J Respir Crit Care Med. 1996; 154(3 Pt 1): 602–11. PubMed Abstract \| Publisher Full Text\n\nTakizawa H, Ohtoshi T, Kawasaki S, et al.: Diesel exhaust particles induce NF-kappa B activation in human bronchial epithelial cells in vitro: importance in cytokine transcription. J Immunol. 1999; 162(8): 4705–11. PubMed Abstract\n\nYamauchi Y, Okazaki H, Desaki M, et al.: Methotrexate induces interleukin-8 production by human bronchial and alveolar epithelial cells. Clin Sci (Lond). 2004; 106(6): 619–25. PubMed Abstract \| Publisher Full Text\n\nArae K, Hirata M, Kurata S, et al.: Mycoplasma pneumoniae induces interleukin-8 production via the epidermal growth factor receptor pathway. Microbiol Immunol. 2011; 55(10): 748–50. PubMed Abstract \| Publisher Full Text\n\nSchleimer RP, Kato A, Kern R, et al.: Epithelium: at the interface of innate and adaptive immune responses. J Allergy Clin Immunol. 2007; 120(6): 1279–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiyata Y, Towatari M, Maeda T, et al.: Histone acetylation induced by granulocyte colony-stimulating factor in a map kinase-dependent manner. Biochem Biophys Res Commun. 2001; 283(3): 655–60. PubMed Abstract \| Publisher Full Text\n\nSullivan JT, Richards CS, Lloyd HA, et al.: Anacardic acid: molluscicide in cashew nut shell liquid. Planta Med. 1982; 44(3): 175–7. PubMed Abstract \| Publisher Full Text\n\nSchmeck B, Lorenz J, N'quessan PD, et al.: Histone acetylation and flagellin are essential for Legionella pneumophila-induced cytokine expression. J Immunol. 2008; 181(2): 940–7. PubMed Abstract\n\nHemshekhar M, Sebastin Santhosh M, Kemparaju K, et al.: Emerging Roles of Anacardic Acid and Its Derivatives: A Pharmacological Overview. Basic Clin Pharmacol Toxicol. 2011. PubMed Abstract \| Publisher Full Text\n\nAsadi L, Eurich DT, Gamble JM, et al.: Guideline adherence and macrolides reduced mortality in outpatients with pneumonia. Respir Med. 2012; 106(3): 451–8. PubMed Abstract \| Publisher Full Text\n\nMouktaroudi M, Giamarellos-Bourboulis EJ: Macrolides for the therapy of nosocomial infections. Curr Opin Infect Dis. 2012; 25(2): 205–10. PubMed Abstract \| Publisher Full Text" }	[ { "id": "817", "date": "07 Mar 2013", "name": "Bruce Rubin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 extremely well conducted study from a top research group. Takizawa and colleagues clearly demonstrate the role of histone acetylation in airway epithelial cell signal transduction after exposure to bacterial endotoxin. These findings are novel and may help in identifying novel targets for modulating chronic airway inflammation.", "responses": [] }, { "id": "830", "date": "11 Mar 2013", "name": "Shu Hashimoto", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well written and conducted. The data and conclusions are clear and worthwhile for the readers.", "responses": [] } ]	1	https://f1000research.com/articles/2-78
https://f1000research.com/articles/2-76/v1	05 Mar 13	{ "type": "Research Article", "title": "Drosophila immune priming against Pseudomonas aeruginosa is short-lasting and depends on cellular and humoral immunity", "authors": [ "Theodoulakis Christofi", "Yiorgos Apidianakis", "Theodoulakis Christofi" ], "abstract": "Immune responses are traditionally divided into the innate and the adaptive arm, both of which are present in vertebrates, while only the innate arm is found in invertebrates. Immune priming experiments in Drosophila melanogaster and other invertebrates during the last decade have challenged this dogma, questioning the boundaries between innate and adaptive immunity. Studies on repeated inoculation of Drosophila with microbes reveal a long-lasting cellular immunity adaptation against particular microorganisms. Here we study the lasting effect of immune priming against infection with Pseudomonas aeruginosa, an opportunistic human pathogen that is lethal to the common fruit fly. Drosophila priming with heat-killed or low in virulence P. aeruginosa extends fly survival during a secondary lethal infection with a virulent strain of the same species. The protective immune response can last for more than 10 days after exposure to a persistent low-in-virulence live infection, but it is eliminated 7 days after the host is primed with heat-killed bacteria. Moreover, not only the cellular, but also the systemic NF-κB-mediated immune responses contribute to immune priming. Thus each microbe might elicit different mechanisms of immune priming that may or may not last for long.", "keywords": [ "Pseudomonas aeruginosa", "immune priming", "adaptive immune response", "cellular immunity", "humoral immunity" ], "content": "Introduction\n\nOrganisms are targets of various infectious microbes that attack a host by penetrating its body in order to feed and reproduce. To cope with infection, each host has developed physical barriers that inhibit microbial entry and tissue homeostasis factors and immune responses that may increase tolerance or resistance to infection. Immune responses can directly target microbes and are observed in most species, from bacteria to mammals through a variety of versatile mechanisms that may be of a broad or of a very microbe-specific nature. In terms of immediacy and specificity, the immune responses have been traditionally divided into innate and adaptive1.\n\nDefence barriers, such as the physical barrier of the skin or insect cuticle, intestinal mucus or insect peritrophic membrane and the low or high acidity in the gastrointestinal tract are the first lines of defence against invading microorganisms1. In addition, innate immunity can be elicited as a fast and broad response against pathogens. Specialised immune cells such as macrophages and neutrophils can internalise and digest microbes initiating an inflammatory response at the site of infection or systemically to produce a hostile environment for the intruder. The complement group of proteins can also be activated to fight invading microorganisms2–5.\n\nComponents of the immune system can exhibit further specificity and acquire memory of past infections. This evolutionary step, termed ‘adaptive immunity’, can only be seen in vertebrates and displays antigenic specificity, diversity, immunologic memory and self/non-self recognition. Adaptive immunity depends on innate immune responses such as phagocytosis and inflammation that trigger the utilisation of specific immune response on the invader6. Adaptive immunity can produce a variety of immune responses specific to antigenic challenges through a variety of effectors. Cooperation between lymphocytes and antigen presenting cells is the main mechanism of action, according to which naive B lymphocytes expressing a membrane-bound antibody molecule are activated when they bind to their specific antigen and divide quickly into memory B cells and effector B cells that induce humoral immunity7. T lymphocytes recognise cell antigens only from major histocompatibility complex molecules and proliferate into memory and effector T cells. T lymphocytes can be subdivided into T helper (TH) and T cytotoxic (TC) cells that are responsible for the tight regulation of the immune response and cytotoxic T lymphocyte activity (CTL)8. During a primary immune response, naive T and B lymphocytes become antigenically committed and expand rapidly in a process called clonal selection9. Immunologic memory can be attributed to these memory cells, which have long life spans and exhibit a heightened response during secondary exposure.\n\nDrosophila is the main model organism for studying innate immunity among invertebrate species. Drosophila immune defences include physical barriers10, homeostatic factors11 and local and systemic immune responses. Three systemic responses have been described in the fly: the humoral response, melanization and the cellular response12. Similarly to other arthropods, Drosophila contains a circulating hemolymph with blood cells called hemocytes. These can be sub-divided into three cell types with different functions: plasmatocytes, lamellocytes and crystal cells12. Plasmatocytes, which comprise the majority of mature hemocytes, can clear unwanted cells and pathogens through phagocytosis12. Lamellocytes can only be observed in larvae where they encapsulate and neutralise larger objects, and crystal cells are involved in the melanization process12. The synthesis and deposition of melanin in the affected area is thought to play an important role in wound healing, captivation and encapsulation of invading microbes and production of toxic substances for subsequent microbial destruction12. Coagulation occurs to prevent hemolymph loss but can also trap microorganisms and facilitate their destruction13.\n\nThe Drosophila fat body is analogous to the mammalian liver where humoral response molecules are produced14. Bacteria and fungi activate the Toll pathway indirectly via production of a “danger” signal15. In addition, bacteria and fungi induce the Toll and Imd pathways directly by the recognition of bacterial peptidoglycan and fungal beta-glucan via peptidoglycan recognition proteins and Gram-negative binding protein 3 respectively16. Upon systemic immune response Toll and Imd pathways induce the NF-κB factors Dif and Rel respectively, which in turn induce the expression of several antimicrobial peptides (AMP)17. Besides AMPs, plasmatocytes locate and phagocytose bacteria through the help of scavenger receptors Eater and Dscam18,19. The epithelial barrier also exhibits local immunity where production of reactive oxygen species (ROS) and AMPs provides a defence mechanism in the gut20. In addition to the plasmatocyte-expressed cytokine unpaired 3 (Upd3) induces the JAK/STAT pathway mediating robust responses to bacterial and fungal infection12; while the same pathway can be induced upon tissue damage or viral infection21,22.\n\nThe aforementioned innate immune responses have not been proven to exhibit adaptive properties such as memory or specificity. However, the classic division between innate and adaptive immunity has recently been brought into question by a number of studies in invertebrate organisms, which challenge the currently defined boundaries of immunological memory23.\n\nRecent evidence suggests that arthropods can display selected 'specificity' towards particular microorganisms. Pham and colleagues demonstrated that the fruit fly exhibits a specific primed immune response dependent on plasmatocytes24. They tested various pathogens including bacteria and fungi and found that flies mount a prolonged protective response against Streptococcus pneumoniae after being primed with a sub-lethal or heat-killed dose of the bacterium. S. pneumoniae bacteria are killed by the host within 1 day of infection only in primed flies whereas unprimed flies still contained bacteria indicating that survival depends on the elimination rate of S. pneumoniae24. They also found a similar adaptation with the natural fungal pathogen Beauveria bassiana.\n\nProtection against other bacteria was not observed by priming with S. pneumonia. Conversely, other heat-killed bacteria – known to be strong immune activators - did not exert a protective response against S. pneumoniae. Immune pathway mutants demonstrated that immune priming is due to the activation of the Toll pathway but not due to the expression of AMPs. These findings illustrate the selective adaptability of the immune system through the activation of Toll pathway and plasmatocytes. However it is important to note that not all pathogens respond in the same way. In this report, we use the example of Pseudomonas aeruginosa, a gram-negative bacterium that induces the Imd and the Toll pathways, as well as the cellular immune response.\n\nPrevious studies show that live P. aeruginosa infection with the low-in-virulence CF5 strain primes the immune system and helps to protect Drosophila from subsequent lethal infection with the virulent PA14 strain (UCBPP-PA14)25. This protection is evident 6, 12 and 24 hours post immune priming and involves the activation of both the Imd and the Toll pathway25. Here we assess the duration of this protective response and the involvement of humoral and cellular immune responses. We find that immune priming with heat-killed P. aeruginosa CF5 confers protection for less than 7 days and that the Imd and the Toll pathways, as well as phagocytosis, contribute to host protection at 2 and 5 days respectively post-immune priming.\n\n\nMethods\n\nWild type Oregon R and Eater mutant flies were a gift from Christine Kocks18. Canton S (CS) was obtained from Bloomington stock Center. Imd1, RelE20 and Dif1 mutant flies were a gift from Bruno Lemaitre.\n\nP. aeruginosa strains PA14 and CF5 are previously described human isolates25. For inoculation with live CF5 cells flies were pricked with a needle previously dipped into a solution of 3 x 108 CF5 cells/ml or in PBS as a control as previously described25,26. For CF5 colony forming units (CFUs) enumeration, 3 flies per time point, in triplicates, were ground and plated every two days. Using the injection method 9.2 nl of a bacterial solution was introduced into the fly thorax to prime or infect the flies and host survival was measured every hour as previously described25,26. 9.2 nl of a solution containing 3 x 108 heat-killed CF5 cells/ml or the equivalent volume of PBS was injected to prime flies with heat-killed P. aeruginosa. Primed flies were subsequently injected with 9.2 nl of a live bacteria solution containing 3 x 107 PA14 cells/ml.\n\nFly survival kinetics were analyzed using the MedCalc software (www.medcalc.org/). Survival curve analyses were performed using the Logrank test of the Kaplan-Meier survival analysis27. The supplementary data tables (Table S1–Table S7) accompanying this work provides the actual number of flies per experiment and individual results used for analysis.\n\n\nResults\n\nTo test if long-term protection could be achieved by immune priming, we initially infected wild type male Oregon R flies with 130 colony forming units (CFUs) of the low-in-virulence P. aeruginosa strain CF5. Infection was persistent for at least 10 days when >100 CFUs/fly were still present in the flies (Figure 1). Injections with the virulent PA14 strain were performed on the 11th day of priming with CF5 and in control non-primed flies. In primed flies, the 50% survival time was over two hours longer and over 10% of primed flies had survived at 30 hours post-infection (Figure 2). It should be noted that P. aeruginosa infection with the PA14 strain is reproducibly 100% lethal under these conditions and short time differences between survival curves are biologically and statistically significant using the Kaplan-Meier survival kinetic analysis11,26. Here we observe a protective role against a virulent bacterium when the host is primed with live bacteria of the same species (P<0.0001) (Figure 2).\n\nWild type Oregon R male flies were infected with 130 colony forming units (CFUs) of the low virulent P. aeruginosa CF5 strain. Infection is persistent for at least 10 days with more than 100 CFUs per fly.\n\nP. aeruginosa PA14 injection in primed (dashed line) and non primed (continuous line) Oregon R flies with live CF5 bacteria showed an extension in the 50% survival time for over of two hours in contrast to controls and more that 10% survived at 30 hours post infection (P<0.0001). n=40–49 flies per condition.\n\nTo assess the duration of immune priming when bacteria are not able to replicate and persist in the host, we primed flies with bacteria that were heat-killed for 10 minutes at 60°C. ~3000 heat-killed CF5 cells were injected per fly 2, 5 or 7 days prior to infection with the lethal strain PA14. Under these conditions, no CFUs could be recovered from flies prior to PA14 infection.\n\nInitially wild type Oregon R (OR), Rel mutant (Imd pathway) and Dif mutant (Toll pathway) mutant flies were primed with heat-killed (H.K.) CF5 cells and infected with the PA14 strain 2 days later. OR flies showed a prominent extension in survival (P<0.0001) with more than 30% of primed flies surviving the infection at 30 hours post-infection (Figure 3). However the Rel mutant primed and non-primed flies died at similar rates (P=0.2199), suggesting that protective priming responses against P. aeruginosa depends on the rel gene. Dif mutant flies could elicit a protective response (P=0.0010), which was nevertheless less prominent compared to that of OR flies. Thus rel and to a lesser extent dif, the 2 main NF-κB immune factors of Drosophila, appear to contribute to the protective immune response that lasts for at least 2 days.\n\nWild type Oregon R (OR) and mutant flies of the Imd (Rel) and Toll (Dif) pathways were primed with heat-killed (H.K.) CF5 cells (doted lines) 2 days prior to PA14 challenge. Primed OR flies showed extended survival times and 30% survivors (P<0.0001). Rel mutant control and primed flies died at similar rates indicating a protective role of the rel gene (Rel P=0.2199). Dif mutant flies exhibited a low but significant protective effect (P=0.001). n=19–29 flies per condition.\n\nTo examine if phagocytosis is important for immune priming against P. aeruginosa we primed Eater deficient flies for 2 days. Mutant Eater non-primed flies were more susceptible to infection than OR non-primed flies (P<0.0001), and contrary to wild type flies, priming of mutant flies did not lead to any survivors at 30 hours (Figure 4). Nevertheless, primed Eater deficient flies survived longer when primed (P<0.0001), suggesting that additional immune responses contribute to host protection at 2 days post priming. The role of cellular responses in immune priming was nevertheless clear when the PA14 strain was injected 5 days after priming, when primed Eater mutant flies are equally susceptible to non-primed flies (P=0.5616), while wild type flies were still significantly protected by priming (P=0.0037) (Figure 5). This suggests that immune priming at 5 days depends heavily on phagocytosis.\n\nEater mutant and wild type Oregon R flies (OR) were primed two days prior to PA14 injection to investigate the role of phagocytosis in immune priming. Eater deficient flies were more susceptible to infection than wild type flies (P<0.0001) in the absence of priming. Nevertheless Eater mutants survived longer when primed indicating additional immune responses of the host at 2 days post priming. n=18–20 flies per condition.\n\nWild type Oregon R (OR) and Eater mutant flies where challenged with PA14 5 days post injecting with heat-killed CF5. Eater mutants were equally susceptible to non primed flies (P=0.5616) in contrast to control OR flies (P=0.0037). This apparent cellular response suggests that phagocytosis efficacy depends on the priming period prior to challenge. n=19–20 flies per condition.\n\nTo assess if the priming effect can last for longer, we primed wild type but also Imd and Rel mutant flies 7 days prior to PA14 infection, and we noticed that priming had no significant effect in the survival rates of any of the genotypes tested: Canton S (P=0.0726), Rel (P=0.9163), Imd (P=0.0663) (Figure 6). To assess if 7 day primed flies are incapable of mounting a protective immune response or if priming diminishes after 7 days, we double primed wild type flies 5 and 7 days prior to PA14 infection. We noticed that double priming extends the survival of flies (P=0.0007) (Figure 7), thus priming with dead P. aeruginosa has a short-lasting protective effect of less than a week.\n\nWild type Canton S (CS), Imd and Rel mutant primed and non primed flies were tested. All fly genotypes exhibited no significant effect, CS (P=0.0726), Rel (P=0.9163), Imd (P=0.0663), indicating that priming is transient. n=18–24 flies per condition.\n\nWild type Canton S flies (CS) were primed with heat-killed CF5 cells 5 and 7 days before P. aeruginosa PA14 injection. A protective response was observed (P=0.0007) in the primed flies. n=29–35 flies per condition.\n\n\nDiscussion\n\nCollectively our data indicate that low-in-virulence P. aeruginosa can prime the Drosophila humoral and cellular immune responses against a subsequent lethal infection with a more virulent strain. Nevertheless, unlike priming with S. pneumoniae or B. Bassiana, this is not a long-lasting effect. It is thus pivotal that future studies assess in detail the differences in the immune responses among many different microbes and in time points that last for many days rather than hours as is customary. Long-term responses to single or repeated challenges of the immune system might pinpoint novel aspects of immunological memory. One aspect of immune responses that might be related to immunologic memory in invertebrates is specificity. A recent breakthrough in the specificity of immune responses in insects came with the discovery of the multi-variable gene Dscam28. Dong and his team found that different immune elicitors in the mosquito direct the production of pathogen-specific splice variants of the Down Syndrome Cell Adhesion Molecule receptor necessary for the protection of the host from infection with Plasmodium. Though no experiments were done to test the duration of this specific response, this work illustrates the adaptability of the insect immune system. There are additional examples of specific immune responses in invertebrates such as the snail Biomphalaria glabrata, in which fibrinogen related proteins (FREPS) exhibit a high rate of diversification at a genomic level, and the expression profiles of the scavenger receptor cysteine-rich proteins in the sea urchin, although how these proteins respond to re-challenge is not known29,30. Protection against a secondary infection is also seen in the mealworm beetle, Tenebrio molitor31. Prolonged protection was observed when initial exposure to lipopolysaccharides (LPS) before infection with spores of the entomopathogenic fungus Metarhizium anisopliae occurred. This was attributed to a long-lasting antimicrobial response of the LPS-challenged larva, which provided a survival advantage when it was exposed to fungal infection. Thus invertebrate hosts can be further studied to understand the parameters of long-lasting immune responses and their relation to immune specificity and memory.\n\nIn conclusion, the area of immunological memory remains elusive in the invertebrate world and only recently have small steps been made to investigate this aspect of the immune system. Specific responses can occur against particular pathogens. Generalisations on the defence mechanisms do not represent the true complexity of the immune system. Therefore, the immune system of invertebrates is still a field that can advance our understanding of how organisms defend themselves from intruders.", "appendix": "Author contributions\n\n\n\nYA designed the research and performed experiments; TC analyzed the data; YA and TC wrote the paper. 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\nMarie Curie PCIG-GA-2011-303586 grant to YA.\n\n\nSupplementary tables\n\nInfected flies were ground and plated, 3 individuals per replicate, at days 0, 2, 4, 6, 8 and 10 post infection where the colony forming units (CFU) were measured and calculated per fly. The experiment was performed in triplicates where the average CFU numbers were plotted with the corresponding standard deviation.\n\nOregon R (OR) uninfected flies (n=49) and OR infected with live CF5 bacteria (n=40) for 11 days were injected with the virulent PA14 strain. Top row numbers indicate the time in hours post injection whereas lower rows the corresponding number of surviving flies in naive and primed flies respectively. Sample size is indicated at 20h.\n\nWild type Oregon R (OR) and mutant Dif and Rel flies were primed with heat-killed (H. K.) CF5 cells. Naive and primed flies were injected with the virulent PA14 strain after 2 days. Top row numbers indicate the time in hours post challenge whereas lower rows indicate the corresponding number of surviving flies in naive and primed flies respectively for each genotype. Sample size is indicated at 0h.\n\nOregon R (OR) (n=18) and Eater (n=20) mutant uninfected flies, and primed with heat-killed (H. K.) CF5 bacteria were inoculated at 2 days with the virulent PA14 strain. Top row numbers indicate the time in hours post challenge whereas lower rows indicate the corresponding number of surviving flies in naive and primed flies respectively for each genotype. Sample size is indicated at 18h.\n\nWild type Oregon R (OR) and Eater mutant uninfected flies (n=19, n=20) and primed with heat-killed (H. K.) CF5 bacteria (n=19, n=20) were inoculated at day 5 with PA14. Top row numbers indicate the time in hours post challenge whereas lower rows indicate the corresponding number of surviving flies in naive and primed flies respectively for each genotype. Sample size is indicated at 18h.\n\nWild type Canton S (CS) and mutant Rel and Imd flies were primed with heat-killed (H. K.) CF5 cells. Flies were injected with the virulent PA14 strain 7 days post priming. Top row numbers indicate the time in hours post injection whereas lower rows indicate the corresponding number of surviving flies in naive and primed flies respectively for each genotype. Sample size is indicated at 18h.\n\nCanton S (CS) wild type flies were primed twice with heat-killed (H. K.) CF5 cells at days 5 and 7 prior to injection with PA14. Naive (n=35) and primed (n=29) flies were inoculated with the virulent PA14 strain 7 days post priming. Top row numbers indicate the time in hours post challenge whereas lower rows indicate the corresponding number of surviving flies in naive and primed flies respectively. Sample size is indicated at 0h.\n\n\nReferences\n\nKindt TJ, Goldsby RA, Osborne BA, et al.: Kuby Immunology. W. H. Freeman; 2007. Reference Source\n\nBeutler B: Innate immunity: An overview. Mol Immunol. 2004; 40(12): 845–59. PubMed Abstract \| Publisher Full Text\n\nMorgan BP, Marchbank KJ, Longhi MP, et al.: Complement: Central to innate immunity and bridging to adaptive responses. Immunol Lett. 2005; 97(2): 171–9. PubMed Abstract \| Publisher Full Text\n\nCarroll MC: The complement system in regulation of adaptive immunity. Nat Immunol. 2004; 5(10): 981–6. PubMed Abstract \| Publisher Full Text\n\nAkira S, Takeda K, Kaisho T: Toll-like receptors: Critical proteins linking innate and acquired immunity. Nat Immunol. 2001; 2(8): 675–80. 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Publisher Full Text\n\nDong Y, Cirimotich CM, Pike A, et al.: Anopheles NF-kappaB-regulated splicing factors direct pathogen-specific repertoires of the hypervariable pattern recognition receptor AgDscam. Cell Host Microbe. 2012; 12(4): 521–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPancer Z: Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin. Proc Natl Acad Sci U S A. 2000; 97(24): 13156–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang SM, Adema CM, Kepler TB, et al.: Diversification of ig superfamily genes in an invertebrate. Science. 2004; 305(5681): 251–4. PubMed Abstract \| Publisher Full Text\n\nMoret Y, Siva-Jothy MT: Adaptive innate immunity? responsive-mode prophylaxis in the mealworm beetle, tenebrio molitor. Proc Biol Sci. 2003; 270(1532): 2475–80. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "832", "date": "12 Mar 2013", "name": "Petros Ligoxygakis", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors study a controversial issue in insect immunity: the existence of memory or in other words whether sub-lethal doses of a pathogen may “prime” the insect to respond more efficiently (and survive) a subsequent infection with an otherwise lethal dose of the same pathogen.Memory in insect immunity has been reported since the field began in the classic work of Metalnikow [Metalnikow S (1929) Immunité d’adaptation et immunité de defense SR. Soc. Biol. 101, 34–67] and more recently in a paper by David Schneider’s lab [Pham LN, Dionne MS, Shirasu-Hiza M, Schneider D (2007) A specific primed response in Drosophila is dependent on hemocytes. PLoS Pathog. 3, e26] as well as in a paper published by Siva-Jothy’s lab in Sheffield [Moret Y & Siva-Jothy MT (2003). Adaptive innate immunity? Responsive-mode prophylaxis in the mealworm beetle, Tenebrio molitor. Proceedings of the Royal Society of London B, 270: 2475-2480].Therefore, the possible existence of memory (or “priming”) is an interesting subject and warrants further investigation to explore the limitations of such a response and its characteristics. The types of experiments that one would use to do this (and are indeed used in this work) are mainly survival experiments following infection. Any differences must be very well documented with appropriate statistical tests. Regarding statistical analysis one comment that I would like to make (prompted by Figure 6; curves for other figures seem OK) is that crossing survival curves between different treatments/genotypes indicate non-proportional hazards (so one needs to check for crossing hazards to be sure). Such a scenario increases the probability of a Type II error when using the log-rank test (and weighted log-rank tests) i.e. concluding there is no statistical difference when there actually is one. There are alternative tests to analyse data with crossing hazards (e.g. Renyi-type), and applying one of these to the data set of Figure 6 in addition to their current analysis is a must.Regardless of statistics, their wild-type-background result (and the basis of the research) is a shift in survival of 2 – 4 hours if flies are pre-primed: what is the significance of that? For Figures 3 and 4 the OR flies survive at approx. 30% by 30 hours (although I am not clear of how many times these experiments were repeated? See below comment) – what would be of more interest is whether these flies have recovered from the PA14 infection i.e. do they continue to survive after 30 hours (and for how long)? Have the flies cleared / controlled the PA14 bacteria. The authors therefore need to look at survival and CFUs beyond 30 hours. Further, the biggest shift at 50%_survival - from all the experiments - between primed and non-primed is 4 hours (Fig. 3, OR): is this a real difference or is it within the boundaries of variation? Generally, from what I can tell, experiments have only been performed once, and mostly this is with 19 – 35 flies: if more flies were used, along with biological repeats, would the same trends consistently be observed? Finally, I think the authors really need to show the fly survival with CF5 and heat-killed CF5; but more importantly, inject live CF5 into the pre-seeded flies as they did with PA14 i.e. if then, the pre-seeded CF5-live flies survive slightly better as they did with PA14, it points to a general mechanism rather than one relating to the virulence of the bacteria. And vice versa, where if a strain is more virulent, then “priming” may be advantageous. Minor: Generally, data is better presented as Kaplan-Meier curves; since you are dead or not dead, having slopes between time-of-death is misleading (unless the data set is modelled).In figure legends: if error bars are shown, it should be indicated what they are describing. And the test used to derive the P-value should be given with this value.", "responses": [] }, { "id": "847", "date": "18 Mar 2013", "name": "Bernard Mathey-Prevot", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Christofi and Apidianakis present an extension of their previous work on immune priming with Pseudomonas aeruginosa. They focus on the duration of the protective response elicited against the virulent PA14 strain by pre-treating adult Drosophila flies with live or heat-killed low-in virulence CF5 bacteria. There are few new insights into the mechanisms by which the protective effect is mediated (involvement of the Toll, Rel and Imd pathways as well as phagocytosis), but the report makes the important point that the duration and extent of priming will vary greatly with the bacteria or fungi used in the experiments, and strongly advocates that future studies into the adaptive characteristics of insect immunity should be carried out over more than a day rather than a few hours as it is commonly done. Careful and extended time-courses will uncover important differences in how priming can lead to significantly diverse responses to infection with one strain of bacteria or another.General: I do have some issues and would like the authors to comment on the following:The authors present reasonably performed experiments, but I do have some reservation about the conclusions drawn from their experiments. In particular, I was struck that OR and CS strains show some differences in response to priming (Fig. 3). While the end point is similar at 30h for both strains, they show very distinct survival rates between 25 and 29h. This apparent difference might suggest that different genetic background will play an important role in the priming response. In that regard, I wonder why the OR or CS were chosen as controls, rather than strains that are of the same genetic background than the mutants used in this study. What is also surprising is that the authors chose to ignore the apparent discrepancy between the two strains, and furthermore go on to perform additional experiments where they either chose OR (Fig. 4, 5) or CS (Fig. 6, 7) without really justifying or giving a rationale why they included a particular control strain rather than the other. For consistency sake, it would have been better to select one or the other, or include both stains in each experiment.I am probably missing something but I don’t get the logic in Fig 7. The double priming at 5 and 7 days prior to infection is said to extend the protective effect over that of 7 days, but in reality they are looking at flies which had been primed last at 5 days prior the challenge. To me, the correct comparison should have been doubly primed CS flies vs. 5 day primed CS flies, rather than untreated CS. Untreated flies should have been included, but only to serve as a reference.One potential explanation for the modest involvement of plasmatocytes and phagocytosis in mediating protection against a challenge with PA14 might be that the initial priming leads to a transient increase in plasmatocytes. It would be nice to have a sense of whether the number of circulating plasmatocytes is increased after priming. I realize that the protection observed in priming is supposed to be strain specific (no cross protection against other bacteria); however, the protective effect related in this report is rather modest. As such, one wonders whether there are two types of responses: 1) The increase in plasmatocytes, which alone would confer a broad protection but be too weak to have a significant effect on other bacteria, and 2) The humoral and recognition pathways that confer specificity against a particular strain.Minor: The sentence on page 2, starting with “In addition to the plasmatocyte-expressed…” to the end of the paragraph is awkward and needs to be edited.", "responses": [] }, { "id": "854", "date": "20 Mar 2013", "name": "Dimitrios Kontoyiannis", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting report on a complex and somewhat controversial area (insect immune memory responses) by an investigator who has done nice work in the field. The experimental methods are sufficient, although some methodological issues might create some difficulties in data interpretation;The results would be more convincing if isogenic strains of Pseudomonas and Drosophila were used, as differences are rather small (and possibly biologically non-significant), raising the question of genetic-background influences. In addition, backing up the mortality experiments with bacterial burden data (CFU/gr) or even histopathology in selected differences would make the claims much stronger.It would be of interest to have, in addition to heat-killed bacteria, septic injury in the same day as the control. Perhaps priming could occur even by septic injury, in a bacteria-independent factorFuture experimental direction could be to investigate the effect of different inocula and whether there is a critical minimal threshold of exposure that results in priming.Another way to investigate the role, if any, of phagocytosis would be to feed wild flies corticosteroids (see Chamilos G et al. PNAS) and evaluate if wild type flies have sluggish priming compared to corticosteroid-unexposed flies.I would also analyze survival data of figs 3 and 6 by alternative statistical method in view of the close overlap.", "responses": [] }, { "id": "859", "date": "22 Mar 2013", "name": "Bruno Lemaitre", "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 short paper on the influence of priming on the Drosophila immune response.The results are worthy enough to be published but require major improvements.1) The paper, especially the introduction, is poorly written. I just mention few problems*All the parts on the vertebrate adaptive immune should be double checked by an immunologist. Many sentences make no sense.Ex.: 'Cooperation between lymphocytes and antigen presenting cells is the main mechanism of action, according to which naive B lymphocytes expressing a membrane-bound antibody molecule are activated when they bind to their specific antigen and divide quickly into memory B cells and effector B cells that induce humoral immunity'This is a short cut. B cells are activated upon direct recognition of antigen and a signal coming from T cell that has been previously activated by a DC This is not a mechanism of action... • Intro part on Drosophila- (ref 11) is a paper unrelated to what it is linked ('homeostatic factors').- Can melanization be considered as a part of the humoral response?- What does 'captivation' mean?- The role of dScam in phagocytosis is poorly established compared to NimC1. I am not aware that Eater has been shown to 'locate' bacteria. - 'also exhibit local immunity': 'Also' seems weird when the part above discusses systemic immunity.• Intro part on specificity- Specificity has been shown. Flies activate adapted immune response to aggressors (ex. Toll/Imd, encapsulation only against parasites). This is not new. What is possibly new is the high degree of specificity.- What is the meaning of 'selected specificity' and 'selective adaptability'?- 'They also found similar adaptation': the term adaptation is unclear.• Boman, in his famous 1972 Nature paper, has already shown that priming with E. clocae could protect fly from an infection by P. aeruginosa. This could be mentioned.Figure 4: Eater is not a cellular deficient mutant but a phagocytic mutant. Idem for 'humoral response mutant' in figure 6. This is not precise enough.2) All the experiments in figures 3, 4, 5, 6, 7 should be repeated at least a second time in a way to have an independent repeat and a higher number of flies.3) The observed effect is not striking: improvement of only 2h. From my point of view, this actually suggests that priming is not very efficient and could be simply explained by the long-lasting effect of AMPs or other immune factors. This hypothesis should be discussed first before evoking dScam, other complex mechanisms or the concept of memory. The memory hypothesis of dSCAM is clearly not needed to explain the observed results. 4) Mutation should be better described and written in italic. Is there an eater mutant or are the authors using a set of deficiencies to generate a mutant?5) Figure 3: this author has already reported that Toll pathway contributes to host defense against P. aeruginosa. In this case, can they explain why dif mutant survives better than the the wild-type?", "responses": [] } ]	1	https://f1000research.com/articles/2-76
https://f1000research.com/articles/2-74/v1	05 Mar 13	{ "type": "Research Article", "title": "Dissecting the role of TRPV1 in detecting multiple trigeminal irritants in three behavioral assays for sensory irritation", "authors": [ "CJ Saunders", "Winston Y Li", "Tulsi D Patel", "Jeffrey A Muday", "Wayne L Silver", "Winston Y Li", "Tulsi D Patel", "Jeffrey A Muday", "Wayne L Silver" ], "abstract": "Polymodal neurons of the trigeminal nerve innervate the nasal cavity, nasopharynx, oral cavity and cornea. Trigeminal nociceptive fibers express a diverse collection of receptors and are stimulated by a wide variety of chemicals. However, the mechanism of stimulation is known only for relatively few of these compounds. Capsaicin, for example, activates transient receptor potential vanilloid 1 (TRPV1) channels. In the present study, wildtype (C57Bl/6J) and TRPV1 knockout mice were tested in three behavioral assays for irritation to determine if TRPV1 is necessary to detect trigeminal irritants in addition to capsaicin. In one assay mice were presented with a chemical via a cotton swab and their response scored on a 5 level scale. In another assay, a modified two bottle preference test, which avoids the confound of mixing irritants with the animal’s drinking water, was used to assess aversion. In the final assay, an air dilution olfactometer was used to administer volatile compounds to mice restrained in a double-chambered plethysmograph where respiratory reflexes were monitored. TRPV1 knockouts showed deficiencies in the detection of benzaldehyde, cyclohexanone and eugenol in at least one assay. However, cyclohexanone was the only substance tested that appears to act solely through TRPV1.", "keywords": [ "Trigeminal", "TRPV1", "Airway Irritation", "Behavioral Aversion", "Respiratory Reflex", "Apnea" ], "content": "Introduction\n\nChemesthesis, the ability to detect chemical stimuli by the somatosensory system, enables the avoidance of potentially dangerous substances and is of considerable importance to an organism’s survival. Nociceptive fibers originating in both the dorsal root ganglia (DRG) and trigeminal ganglia (TG) respond to a variety of chemesthetic stimuli1. In most mammals, nociceptive DRG fibers are protected from chemesthetic compounds by a keratinized epithelium2,3. Therefore, chemesthetic sensations are primarily mediated by trigeminal fibers that innervate the mucus membranes of the nasal cavity, nasopharynx, oral cavity and cornea. Additionally, chemesthetic agents can also stimulate vagal nerve fibers innervating the gut and respiratory tract. A wide variety of chemical irritants stimulate the polymodal nociceptors of the trigeminal nerve4. While our knowledge of the receptors found on these nociceptive neurons is expanding, the mechanism of stimulation is known for only a few of these trigeminal chemical stimuli. For example, capsaicin, a compound found in chili peppers, activates transient receptor potential vanilloid 1 (TRPV1) channels5.\n\nTRPV1 is a member of the transient receptor potential (TRP) family of nonspecific cation channels6, which were first identified in the Drosophila visual system7. TRP channels are found in a multitude of mammalian tissues and are of particular importance in sensory systems8. TRPV1 is activated by stimuli as diverse as plant metabolites, such as capsaicin5, eugenol9 and resiniferatoxin10; low pH5; temperatures above 43°C5; endocannabinoids, such as anandamide11; lipid-derived secondary messengers such as phosphoinositides12; and metal ions, such as Ni2+13. The channel is highly expressed by Calcitonin gene-related peptide- and substance P-positive nociceptive fibers originating in the DRG and TG14. In addition to TRPV1 and TRPV family members 2–4, the nociceptive Aδ and C fibers of the trigeminal nerve also express TRP ankyrin 1 (TRPA1), TRP melastatin 8 (TRPM8), acid sensing ion channels (ASIC) and nicotinic acetylcholine receptors (nAChR)2, which mediate chemical sensitivity to allyl isothiocyanate15, menthol16, acids17,18 and nicotine19.\n\nFibers from the ophthalmic branch of the trigeminal nerve, cranial nerve V1, innervate the most rostral portion of the airway. Stimulation of these fibers results in protective airway reflexes, including coughing, vasodilation and decreased respiration rate1,20,21. Chemesthetic compounds activate the trigeminal nerve either through direct stimulation of free nerve endings or by stimulation of solitary chemosensory cells. However, TRPV1 is found only on the free nerve ends and not on solitary chemosensory cells3,21,22.\n\nThese facts support the concept that the trigeminal system is a warning system tuned to be responsive to diverse classes of potentially dangerous substances23. As would be expected in such a warning system, the trigeminal system exhibits many aspects of redundancy, such as the presence of three different receptors that are sensitive to low pH: TRPV1, TRPA1 and ASIC18 and responsiveness to very different chemical stimuli, indicated by the presence of multiple broadly tuned receptor proteins, such as TRPV1 and TRPA1. Much of the difficulty in determining the mechanism of stimulation of trigeminal irritants can be attributed to the redundancy and promiscuity of the trigeminal system.\n\nTo determine whether TRPV1 is necessary for the detection of trigeminal irritants other than capsaicin, we tested wildtype and TRPV1 knockout mice (Trpv1–/–) in three different behavioral assays for trigeminal irritation. In one assay, mice were presented with a chemical via a cotton swab and their responses scored on a 5-level scale. In another assay, a modified two-bottle preference test, which avoids mixing irritants with the animal’s drinking water to prevent post-ingestive effects, was used to assess aversion. In the final assay, a custom-built air-dilution olfactometer was used to administer volatile compounds to mice restrained in a double-chambered plethysmograph where respiratory reflexes were monitored, this representing a modified “Alarie Test”24,25. Our data suggest that, although many compounds may activate TRPV1, only a few, such as cyclohexanone, are exclusively detected by the channel, a finding that underlies the biological redundancy typified by the trigeminal system.\n\n\nMaterials and methods\n\nAdult, female, wildtype (C57Bl/6J) and TRPV1 knockout mice (B6.129X1-Trpv1tm1Jul/J) were purchased from Jackson Laboratories in Bar Harbor, ME, USA, for the present study. Animals were given at least one week to acclimate after delivery to the Winston Hall animal facility. The mice were housed in conventional shoebox-type polycarbonate caging, changed once every 7 days. The bedding material was 1/8” Bed-o-cob, and they were provided with ad-lib water and rodent chow (Purinalab 5P00). Nesting material was provided in the form of Enrich-Nest. All animals were housed in groups of 4, provided with food and water ad libitum and were maintained on a 12 hour light cycle. 16 wildtype and 16 Trpv1-/- mice were used in this study; each mouse was tested in each experimental paradigm. At the end of the experiment, mice were tail biopsied and PCR was used to confirm the presence or absence of TRPV1. TRPV1 forward primer (5´-CGA GGA TGG GAA GAA TAA CTC ACT G-3´) and reverse primer (5´-GGA TGA TGA AGA CAG CCT TGA AGT C-3´) were purchased from Operon Biotechnologies (Huntsville, AL, USA). All experimental procedures were approved by Wake Forest University’s Animal Care and Use Committee (ACUC protocol # A07-104).\n\nCompounds tested included acetic acid, amyl acetate, benzaldehyde, capsaicin, cyclohexanone, eugenol (4-allyl-2-methoxyphenol), (-)-nicotine and toluene. All chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA) and had been certified ≥99% pure.\n\nThe cotton swab test has been used previously to test the aversion of TRPA1 knockout mice to a number of chemicals25. For liquid stimuli, cotton applicators (Puritan Medical Products, Guilford, ME, USA) were saturated with the pure compound before presentation to the animal. For powdered stimuli, the applicator was saturated with distilled water then placed in the compound so that it would adhere to the cotton.\n\nTo prevent the animals from becoming conditioned to associate the cotton swabs with aversive stimuli, several non-aversive substances were also presented to the mice. The favorable substances included chocolate powder (Nesquick, Glendale, CA, USA), water and wheat flour (King Arthur, Norwich, VT, USA); the noxious compounds tested were acetic acid, amyl acetate, benzaldehyde, capsaicin, cyclohexanone, eugenol, nicotine and toluene. Presentation was alternated between aversive and rewarding stimuli and an individual’s first three responses to the noxious substances were recorded.\n\nEach animal was presented with each compound three times and its reaction to the stimulus was scored as follows:\n\n-2: Rapid, reflexive withdrawal, removing the head from the stimulus source\n\n-1: Rejection of stimulus marked by a slow head turn movement\n\n0: No response or lack of interest in stimulus presentation\n\n+1: Investigation of stimulus source, marked by an increase in inspiratory sniffing\n\n+2: Attempted manipulation of stimulus source or attempted feeding behavior.\n\nWe have previously used this assay to assess the response of TRPA1 knockouts25 and capsaicin-desensitized rats26 to trigeminal irritants. For an individual mouse, a mean behavioral score was calculated for each substance and the scores from each animal were then averaged to ensure normally distributed data. To detect differences between the responses of wildtype and TRPV1-/- mice, a two-way ANOVA followed by Bonferroni post hoc tests was conducted with GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego, CA, USA).\n\nFelt washers (Duro-Felt Products, Little Rock, AR, USA) were placed over the sipper tube of two water bottles and protected with a wire screen. The wire screen prevented the mouse from manipulating the washers or coming into direct contact with the compounds on the washers. One washer was soaked in undiluted irritant. The washer on the opposite tube was saturated with distilled water in place of irritant but otherwise prepared identically. No chemicals were mixed with the drinking water in either bottle. To approach and drink from a water bottle the mouse had to inhale the vapors created as the volatile compounds evaporated from the felt washer. Previous studies have used a traditional two-bottle preference paradigm where the capsaicin was mixed with the drinking water27. While the traditional approach has the advantage of being able to test non-volatile compounds, such as the prototypical TRPV1 agonist capsaicin, it also allows the chemical to stimulate the taste system and possibly stimulate sensory fibers in the gut. Our modified two-bottle test avoids these confounding issues.\n\nIndividual mice were housed with two of the drinking tubes and water consumption was measured in each over 24 hrs. After an initial recording period of 24 hrs, the washers were renewed and the locations were switched to control for side bias. Water consumption was then measured over another 24 hrs. The values from the irritant-treated tube on the two days were summed and normalized to the total amount of water consumed (water consumed from irritant treated tube / (water consumed from irritant treated tube + water consumed from untreated tube). Tubes were of identical construction and all individual drinking tubes were tested over night before the experiment to ensure that they did not lose water through dripping. Data underwent an arcsine transformation to meet the assumptions of a two-way ANOVA. A two-way ANOVA followed by Bonferroni post hoc tests was conducted with GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego, CA, USA) to detect differences between wildtype and TRPV1-/- mice.\n\nA custom built, relatively inexpensive computer-controlled air-dilution olfactometer was used to administer volatile compounds to unanesthetized mice restrained in four double-chambered plethysmographs (Kent Scientific Corp, Torrington, CT, USA; Figure 1). The plethysmographs monitored the respiratory cycle via pressure transducers. Polytetrafluoroethylene tubing, 3/16” in diameter (Cole-Parmer, Vernon Hills, IL, USA), was used to connect all the individual components of the olfactometer and to construct the four-channel manifold.\n\nEMFC, electronic mass flow controller.\n\nThe olfactometer was constructed so that air from a compressor was fed through desiccant and into a charcoal scrubber. This clean dry air flowed to two different lines, the clean airline and the irritant line. In the clean air line, air flowed to a glass rotometer (Cole-Parmer, Vernon Hills, IL, USA) set for a flow rate of 2 l/min. The air was humidified by passage through a saturator tube filled with deionized water. Humidification prevented dry air from desiccating the nasal mucosa of the mice. This line finally fed into a ganged three-way solenoid valve (Nacom Industries, Tustin, CA, USA) and provided clean air to mice at all times except for the stimulation period.\n\nThe irritant line was divided into two subdivisions, each of which was connected to an electronic mass flow controller (EMFC) (Sierra Instruments Inc, Monterey, CA, USA). One of these subdivisions fed into a saturator tube filled with distilled water, while the other fed into a saturator tube filled with undiluted liquid irritant. On both ends of the irritant saturator tube there was a two-way isolatch solenoid valve (General Valve Corporation, Pine Brook, NJ, USA) that controlled the forward flow and prevented backflow of the irritant. The saturator tube containing distilled water only required one valve to control forward flow because backward contamination was not a concern in this case. The two subdivisions of the irritant air line joined and led into a second ganged three-way solenoid valve. By using the two EMFCs to control the ratio of irritant saturated air to clean air, it was possible to control the concentration of irritant to which the animals were exposed. To maintain the air at a constant temperature, all saturator tubes were placed in a 21°C water bath. The irritant-laden air produced by the EMFCs flowed at a rate of 2 l/min.\n\nSince the trigeminal nerve is polymodal and responds to mechanical as well as chemical stimuli1, it is important to have a smooth transition between the clean air and the irritant-laden air. This was accomplished by smoothly switching the ganged three-way valves so that the clean air line flowed out via the exhaust and the irritant air line air flowed towards the mice. Air was sent through the EMFCs to produce the irritant 1 min before it was delivered to the mice in order to prevent excessive loss of the chemical in the saturator tube. Manipulation of the hardware was accomplished by writing a computer program in Visual Basic™ (program available below) which controlled a USB multi-Function data acquisition module from B & B Electronics (Model UD 128A8D; Ottawa, IL, USA) that was wired to the hardware.\n\nThe output of both the three-way solenoid valves was connected to a four-channel manifold that equally divided the air flow among the four plethysmograph chambers. The manifold was constructed from the same Teflon tubing used to connect the individual components of the olfactometer. By maintaining constant flow rates in the chambers, the pressure difference caused by inhalation by the test mouse provided sufficient pressure change to be used as an indicator of respiration. Each chamber was divided by a plastic ring, around which a latex dam was fitted, which formed a seal around each animal’s neck. The chambers were then connected to a pressure transducer (Kent Scientific, Torrington, CT, USA) which monitored the pressure change due to respiration. The outputs of the transducers were recorded using Acqknowledge 3.73 software (BIOPAC Systems, Goleta, CA, USA).\n\nRespiration rate depression for female wildtype (C57Bl/6J) mice was calculated with AcqKnowledge and compared with that of female TRPV1 knockout mice for a variety of compounds in an attempt to determine whether TRPV1 was responsible for the detection of these irritants. Baseline respiration was recorded for 5 min. Respiration was then recorded for 5 min during irritant exposure. Wildtype and TRPV1-/- mice were sampled once per chemical at one concentration. Four mice were tested at the same time, each isolated in a separate plethysmograph chamber unable to interact with the other mice. The concentrations of amyl acetate tested were 2400 ppm, 2800 ppm, 3200 ppm and 4000 ppm. The concentrations of cyclohexanone tested were 1200 ppm, 1600 ppm, 2000 ppm and 3000 ppm. The concentrations of eugenol tested were 10 ppm, 15 ppm, 20 ppm and 25 ppm. The concentrations of nicotine tested were 10 ppm, 20 ppm, 30 ppm and 40 ppm. Concentrations were determined by using the Clausius-Clapeyron equation as previously described by Bryant and Silver1. A two-way ANOVA followed by Bonferroni post hoc tests was conducted for each chemical with GraphPad Prism 5.03 for Windows (GraphPad Software, San Diego, CA, USA) to detect differences between wildtype and TRPV1-/- mice.\n\n\nResults\n\nCotton swabs saturated with an irritant were presented to wildtype (n=7) and TRPV1 (n=7) knockout mice to determine whether TRPV1 is necessary for detection of an acute presentation of an irritating compound. The response to these compounds was scored on a five-point scale. The scale ranged from -2 for a reflexive trigeminal response, to 0 for no response to the cotton swab, to +2 for feeding behavior (summarized in Figure 2A). Two-factor ANOVA revealed a significant difference between irritants (P<0.001), genotype (P<0.01) and a significant interaction between irritant and genotype (P<0.05).\n\n(A) A five-point scale ranging from -2 for a trigeminal reflex to +2 for a highly favorable response was used to assign behavioral scores for each substance. (B) Mean ± SEM behavioral score for wildtype mice (n=7), filled bars, and TRPV1-/- knockout mice (n=7), unfilled bars. Two-way ANOVA revealed a significant difference among irritants (P<0.001), genotype (P<0.01) and a significant interaction between irritant and genotype (P<0.05). Knockout mice had a significantly more favorable response to capsaicin (P<0.01) and cyclohexanone (P<0.05) than wildtype mice. P<0.05, P<0.01, *P<0.001.\n\nWildtype mice showed aversive responses to all noxious substances (Figure 2B). Acetic acid produced the most pronounced aversive response of all the substances tested, typically a distinct trigeminal reflex followed by the animal retreating from the cotton swab. Amyl acetate, benzaldehyde, cyclohexanone, nicotine and toluene all produced aversive behaviors in the wildtype mice, although the responses were not as extreme as the response to acetic acid. Capsaicin and eugenol were both slightly aversive but did not produce responses as extreme as those induced by the other noxious compounds.\n\nThe response of TRPV1 knockout mice to most substances tested was similar to the response of wild type mice (Figure 2B). The only substances to which TRPV1 knockout mice showed significantly less aversion to than the wildtype mice were capsaicin (p<0.01) and cyclohexanone (p<0.05). The response of the TRPV1 knockouts to capsaicin was in the non-aversive range. However, the response of the TRPV1 knockout mice to cyclohexanone was still in the aversive range. There appeared to be some divergence in the response of TRPV1 knockouts to eugenol but statistical analysis revealed these disparities were not significant.\n\nIn this assay, mice were able to control their exposure to vapors of the chemical irritant being tested, unlike the cotton swab and respiratory assays, where mice were exposed to a chemical irritant and their behavior monitored. In previous two-bottle preference experiments with trigeminal irritants, the chemicals were mixed with the drinking water27. A disadvantage of this approach is that it might result in stimulation of gustatory or gut chemosensors in addition to the trigeminal system28. We avoided mixing chemicals with the drinking water by treating felt washers with the chemical irritants and placing these washers over the sipper tube of the water bottles. However, a disadvantage of our approach is that the non-volatility of capsaicin prevented it from being tested.\n\nIf TRPV1 alone is required to detect a noxious chemical then TRPV1 knockout mice should not avoid water bottles surrounded by vapors of that chemical. A control experiment conducted without irritant applied to the washers on either drinking tube (both washers treated with distilled water) indicated no significant side bias and no differences between the amount of water consumed by wildtype and TRPV1 knockout mice (data not shown).\n\nTo compare water consumption between wildtype and TRPV1 knockout mice, the mean and SEM water consumption values from the irritant-treated tube were normalized to the total amount of water consumed from both bottles. TRPV1 knockout mice drank significantly more water from bottles treated with benzaldehyde (p<0.05), cyclohexanone (p<0.05), eugenol (p<0.01) and toluene (p<0.05) than wildtype mice (Figure 3). There was no significant difference in the amount of water consumed from bottles treated with acetic acid, amyl acetate and nicotine between wildtype and TRPV1 knockout mice. Two-way ANOVA also revealed a significant difference between irritants (P<0.001), genotype (P<0.001) and interaction between the two factors (genotype and irritant, p<0.01).\n\nResults are from the two-bottle preference test. Mean ± SEM percentage of water consumed from the irritant-treated tube by wildtype and TRPV1-/- mice. Two-way ANOVA revealed a significant difference among irritants (P<0.001), genotype (P<0.001) and interaction between the two factors (P<0.01). Knockout mice drank significantly more than wildtype mice from bottles treated with benzaldehyde (P<0.05), cyclohexanone (P<0.05), eugenol (P<0.01) and toluene (P<0.05). The number of mice in each experimental group is indicated at the foot of each bar. P<0.05, P<0.01, P<0.001.\n\nSince many trigeminal irritants are also detectable by the olfactory system, the aversion to chemicals observed in the cotton swab and two-bottle preference tests could possibly be due to detection by that system. To overcome this uncertainty, an assay of sensory irritation was needed that was specific to trigeminal irritation. Trigeminal stimulation causes stereotyped changes to the normal exhalation pattern. Specifically, trigeminal stimulation results in a reflexive increase in airway resistance and is characterized by an increase in the period of initial expiration (Figure 4A). This “braking” during the first stage of expiration results in a longer period between breaths and thus effectively decreases respiratory frequency and has been used extensively as an assay of trigeminal irritation21,22,27,29–32. A typical trace of baseline respiration and the response to 3000 ppm cyclohexanone from a wildtype and TRPV1 knockout mouse are shown in Figure 4A. To compare respiration rate depression between wildtype and TRPV1 knockout mice, the respiration rate during the 5 min irritant exposure was normalized to the 5 min baseline recording.\n\n(A) Sample traces from a wildtype and TRPV1 knockout mouse in a double-chambered plethysmograph showing the typical respiratory response to 3000 ppm cyclohexanone (marked by horizontal bar). Tick marks on the x-axis indicate 6 sec. (B–E) Percent respiratory depression of four animals expressed as mean ± SEM (n=4). (B) The effect of amyl acetate exposure was not significantly different between wildtype and knockout mice. A two-way ANOVA detected a significant difference between concentrations (P<0.01) but no significance between genotype or interaction between the two. (C) The effect of cyclohexanone exposure was significantly different between wildtype and knockout mice at concentrations of 1000 ppm (P<0.05), 2000 ppm (P<0.001) and 3000 ppm (P<0.001). A two-way ANOVA detected a significant difference between genotype (P<0.001) and a significant interaction (P<0.01) but no significant difference between concentration. (D) The effect of eugenol exposure was not significantly different between wildtype and knockout mice. A two-way ANOVA detected a significant difference between concentrations (P<0.001) but no significance between genotype or interaction between the two. Finally, (E) the effect of nicotine exposure was not significantly different between wildtype and knockout mice. A two-way ANOVA detected a significant difference between concentrations (P<0.001) but no significance between genotype or interaction between the two. P<0.05, P<0.01, *P<0.001.\n\nAmyl acetate (Figure 4B), eugenol (Figure 4D) and nicotine (Figure 4E) caused respiratory rate depression in wildtype and TRPV1-/- mice in a dosage-dependent manner. There were no significant differences in the respiratory rate depression seen in wildtype and TRPV1-/- mice at any concentration of amyl acetate, eugenol or nicotine. There was a significant difference in the respiratory depression seen between different concentrations of amyl acetate (P<0.01), eugenol (P<0.001) and nicotine (P<0.001) but no significant differences were detected between genotype or from interaction between genotype and concentration for these compounds.\n\nA significant interaction was detected between genotype and cyclohexanone concentration (P<0.01) by two-way ANOVA. There was also a significant difference between genotype (P<0.001) but not concentration. There was no significant difference between the respiration rate of wildtype and TRPV1 knockout mice at 1200 ppm cyclohexanone, the lowest concentration tested. At higher concentrations, the respiration rate of wildtype mice was significantly lower than TRPV1-/- mice at 1600 ppm (P<0.05), 2000 ppm (P<0.001) and 3000 ppm (P<0.001) cyclohexanone (Figure 4C). Cyclohexanone depressed the respiration rate of wildtype mice in a dosage-dependent manner. However, cyclohexanone did not alter the respiration rate of TRPV1 knockout mice from its baseline at any concentration.\n\n\nDiscussion\n\nPolymodal nociceptive trigeminal fibers are sensitive to a wide range of chemical stimuli4. This sensitivity is due to the expression of a diverse population of receptor proteins, many of which are expressed by the same fiber2. For example, TRPV1 and TRPA1 are coexpressed by the same trigeminal fibers33. This arrangement of receptors is largely responsible for the broad responsiveness of the trigeminal nerve and also provides redundant detection mechanisms for many chemicals. Understanding these features of the trigeminal system is of considerable importance in understanding its role as a broadly tuned warning system of potentially dangerous compounds23. Additionally, the presence of multiple broadly responsive receptors with overlapping sensitivity has made it difficult to determine the exact mechanisms by which a chemical stimulates trigeminal fibers and represents a profound experimental challenge. In the present study, we have taken advantage of the availability of TRPV1 knockout mice to elucidate the role of TRPV1 in detecting several known trigeminal irritants.\n\nCapsaicin, the active component of chili peppers, was used to clone the TRPV1 channel5 and has been used as a topical TRPV1 agonist in a multitude of studies34–36. While capsaicin is the prototypic exogenous TRPV1 agonist, the non-volatility of capsaicin restricts its use in experimental paradigms where the stimulus is administered as a vapor. Our previous experiments established capsaicin as a potent trigeminal irritant, but administration to the upper respiratory tract required surgical manipulation4. The current study examines the involvement of TRPV1 in the detection of trigeminal irritants in unanesthetized and minimally restrained animals. This approach has the benefit of more accurately simulating how terrestrial vertebrates, including humans, naturally encounter upper respiratory tract irritants but also highlights the complexity of using capsaicin in studies of upper respiratory tract irritation.\n\nCapsaicin was tested in the cotton swab assay where TRPV1 knockout animals found it less aversive than wild type mice. This result is entirely consistent with the multitude of experiments indicating that TRPV1 is responsible for detecting capsaicin34–36. Wildtype mice found capsaicin to be the least aversive of all the irritants tested in this assay. This result supports the concept that despite the potency of capsaicin as a painful stimulus when applied topically, the poor volatility of the chemical prevents it from reaching the pain fibers of the upper respiratory tract under normal conditions.\n\nNicotine occurs naturally in a number of plant species where it is thought to act as a repellent to potential predators. Humans have cultivated many of these plants to allow for the recreational use of nicotine37. Previous work has established nicotine as a potent trigeminal irritant that is primarily detected via nicotinic acetylcholine receptors (nAChRs)19 or TRPA138. Since nicotine is known to have a mechanism of stimulation independent of TRPV119,38, it was included in this study to ensure that TRPV1 knockout mice respond normally to trigeminal irritants. In every case, the response of TRPV1 knockout mice to nicotine did not differ from wildtype mice, indicating that these mice were capable of detecting and responded to trigeminal irritants normally.\n\nAcetic acetate is produced by a variety of bacteria during fermentation. Dilute acetic acid is commonly used as a flavor enhancer while concentrated acetic acid is used in a variety of industrial processes39. Due to its low pH, acetic acid is a potent chemesthetic stimulus that is capable of activating both ASIC, TRPA118 and TRPV140 proteins on the trigeminal nerve2,4. TRPV1 knockout mice showed no deficiencies in their ability to detect concentrated acetic acid in either the cotton swab or the two-bottle preference tests. Previous experiments established that TRPV1 knockout mice show normal expiratory pause reflex in response to acetic acid20. Presumably, trigeminal ASIC and TRPA1 receptors respond to acetic acid even in the absence of TRPV1, allowing the TRPV1 knockout mice to detect and avoid acids through this redundancy. Additionally, the olfactory system could be mediating or contributing to the avoidance of acetic acid in the cotton swab or the two-bottle preference tests.\n\nAmyl acetate occurs naturally in fruits such as bananas and pears. It is also produced synthetically for use as an artificial flavoring because of its distinctly fruity odor39. In addition to stimulating the olfactory system, at high concentrations amyl acetate also stimulates the trigeminal system4,25. Previous work has demonstrated that while amyl acetate activates TRPA1 it is not the only mechanism by which amyl acetate is detected25.\n\nIn the present study, TRPV1 knockout mice showed no deficiencies in their ability to detect and avoid amyl acetate in the cotton swab or the two-bottle preference tests. Additionally, amyl acetate induced the expiratory pause reflex to the same degree in TRPV1 knockout mice and wildtype mice. These findings are consistent with previous work that demonstrated that TRPV1 does not respond to amyl acetate in vitro4. These results establish that TRPV1 is not one of the redundant mechanisms by which amyl acetate is detected.\n\nBenzaldehyde occurs naturally in almonds and is produced synthetically to be used as a reactant and solvent in industrial chemistry39. The sap of the tolu balsam tree contains toluene and has been used as an ingredient in cough syrups41. Both benzaldehyde and toluene stimulate the trigeminal nerve4,24, fail to activate TRPV1 in vitro4, activate TRPA1 in vivo and fail to induce the expiratory pause reflex in TRPA1 knockouts25. However, TRPA1 knockouts find benzaldehyde less aversive in the cotton swab paradigm but respond to toluene normally25. In the current study, TRPV1 knockouts respond normally to an acute presentation of benzaldehyde and toluene in the cotton swab test but drink significantly more from drinking tubes treated with either chemical than wildtype mice. These contradictory results may indicate that both TRPV1 and TRPA1 are required for the normal detection of these chemicals. If both these chemicals are irritating the respiratory epithelium via trigeminal TRPA1 channels then the subsequent tissue inflammation will result in increased levels of prostaglandins and bradykinin6. Prostaglandins and bradykinin are known to sensitize TRPV1 channels42 and it is possible that, when sensitized by these molecules, TRPV1 channels could be activated by benzaldehyde or toluene. Additionally, most TRPA1-expressing fibers also express TRPV133. Stimulation of TRPA1 by benzaldehyde or toluene could lead to increased production of lipid-derived secondary messengers which can stimulate TRPV1 in the same fiber12. Alternatively, interactions between the olfactory and trigeminal systems that involve both TRPV1 and TRPA1 may be responsible for the detection of these compounds25,43–45. Future studies should consider the use of TRPV1/TRPA1 double knockout mice to explore this possibility that both channels are involved in the detection of benzaldehyde and toluene.\n\nEugenol is a major component of clove oil. Eugenol has a number of uses in fragrance and flavor industries and is commonly used as a dental analgesic46. Previous work has established that eugenol is capable of activating TRPV19, TRPA1 and TRPM847 in vitro. Eugenol induced a normal expiratory pause reflex in TRPV1 knockout mice. To our knowledge, this study is the first to demonstrate a eugenol-induced expiratory pause reflex. TRPV1 knockout mice showed no deficiencies in their ability to detect an acute presentation of eugenol in the cotton swab assay. These results are consistent with the presence of TRPA1 and TRPM8 being sufficient to mediate protective airway reflexes that are mediated solely though the trigeminal stimulation. However, TRPV1 knockout mice drank significantly more from drinking tubes treated with eugenol than wildtype mice. It is possible that eugenol stimulation activates TRPA1 and results in the sensitization of TRPV1 as described above.\n\nCyclohexanone is synthesized in large quantities for its application in the production of polymers48 and has no known natural source49. Exposure to residual cyclohexanone present in medical devices composed of polymers is thought to cause dangerous physiological effects49. Cyclohexanone is a potent trigeminal irritant19 and activates TRPV1 channels, but not TRPA1, in vitro4,50. TRPV1 knockout mice show a significant reduction in sensitivity to cyclohexanone in all three assays used in this study. Strikingly, TRPV1 knockout mice fail to alter their respiration rate when exposed to concentrations of cyclohexanone sufficient to depress respiration in wildtype mice by over 30% (Figure 4C). While TRPV1 knockout mice find cyclohexanone significantly less aversive than wildtype mice in the cotton swab and two-bottle drinking assays, they still avoid cyclohexanone. This result is logically consistent with the concept that some chemicals stimulate both the olfactory and trigeminal systems. In the case of the cotton swab and two-bottle drinking assays, cyclohexanone can still be detected by olfactory receptors, allowing the animal to perceive and avoid the chemical. However, only the trigeminal system is involved in inducing the expiratory pause reflex21,30. TRPV1 knockout mice show no expiratory pause reflex when exposed to cyclohexanone, and therefore TRPV1 is necessary for cyclohexanone-induced sensory irritation.\n\n\nConclusions\n\nAcetic acid, amyl acetate, benzaldehyde, capsaicin, cyclohexanone, eugenol, nicotine and toluene are all potent trigeminal irritants. All of these compounds, except cyclohexanone, have botanical or bacterial sources that could easily be encountered by mammals. Cyclohexanone is commonly used in the synthesis of polymers, and exposure to residual cyclohexanone can have negative effects on human health. Of the eight compounds tested, five (acetic acid, amyl acetate, benzaldehyde, eugenol, and toluene) are thought to have redundant mechanisms of action. TRPV1 knockout mice showed deficiencies in their ability to detect each of these compounds in only one of the three assays of trigeminal irritation. Cyclohexanone was the only compound tested that was significantly less aversive to TRPV1 knockouts in all three assays. Furthermore, TRPV1 is required for the detection of cyclohexanone by the trigeminal system because TRPV1 knockout mice lacked respiratory reflexes in response to this compound. It is notable that redundant mechanisms used by the trigeminal system to detect the naturally occurring chemicals tested are also sufficient to detect at least one potentially dangerous compound that has no natural source, cyclohexanone.", "appendix": "Author contributions\n\n\n\nCJS and WLS conceived and designed the study. CJS and JAM constructed the olfactometer and wrote the controlling program. CJS carried out the cotton swab and respiratory assays. WYL and TDP carried out the two-bottle preference testing. CJS and WLS prepared the manuscript. All authors approved the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a Wake Forest University Science Research Fund award to WLS.\n\n\nAcknowledgments\n\nWe would like to thank Helen Murphy (Duke University, Biology Department) for technical assistance with PCR. We are also grateful for Thomas Finger (University of Colorado School of Medicine) for commenting on earlier drafts of this manuscript.\n\n\nReferences\n\nBryant BP, Silver W: Chemesthesis: The common chemical sense. In: Finger TE, Silver W, Restrepo D, editors. Neurobiology of Taste and Smell. New York: Wiley-Liss, Inc; 2000; 73–100. Reference Source\n\nSilver W, Richards P, Saunders CJ: Functional Neuroanatomy of the Upper Airway in Experimental Animals. In: Morris JB, Shusterman D, editors. Toxicology of the Nose and Upper Airways. New York: Informa Healthcare. 2010; 45–64. Reference Source\n\nSilver WL, Finger TE: The anatomical and electrophysiological basis of peripheral nasal trigeminal chemoreception. Ann N Y Acad Sci. 2009; 1170: 202–205. PubMed Abstract \| Publisher Full Text\n\nSilver WL, Clapp TR, Stone LM, et al.: TRPV1 receptors and nasal trigeminal chemesthesis. Chem Senses. 2006; 31(9): 807–812. PubMed Abstract \| Publisher Full Text\n\nCaterina MJ, Schumacher MA, Tominaga M, et al.: The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. 1997; 389(6653): 816–824. PubMed Abstract \| Publisher Full Text\n\nSzallasi A, Cortright DN, Blum CA, et al.: The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept. Nat Rev Drug Discov. 2007; 6(5): 357–372. PubMed Abstract \| Publisher Full Text\n\nAbramowitz J, Yildirim E, Birnbaumer L: The TRPC Family of Ion Channels: Relation to the TRP Superfamily and Role in Receptor- and Store-Operated Calcium Entry. In: Liedtke W, editors. TRP Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades. Boca Raton, Fl: CRC Press. 2007; 1–30. PubMed Abstract\n\nDamann N, Voets T, Nilius B: TRPs in Our Senses. Curr Biol. 2008; 18(18): R880–R889. PubMed Abstract \| Publisher Full Text\n\nYang BH, Piao ZG, Kim YB, et al.: Activation of vanilloid receptor 1 (VR1) by eugenol. J Dent Res. 2003; 82(10): 781–785. PubMed Abstract \| Publisher Full Text\n\nCalixto JB, Kassuya CA, Andre E, et al.: Contribution of natural products to the discovery of the transient receptor potential (TRP) channels family and their functions. Pharmacol Ther. 2005; 106(2): 179–208. PubMed Abstract \| Publisher Full Text\n\nZygmunt PM, Petersson J, Andersson DA, et al.: Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature. 1999; 400(6743): 452–457. PubMed Abstract \| Publisher Full Text\n\nLukacs V, Thyagarajan B, Varnai P, et al.: Dual regulation of TRPV1 by phosphoinositides. 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PubMed Abstract \| Publisher Full Text\n\nRichards PM, Johnson EC, Silver WL: Four Irritating Odorants Target the Trigeminal Chemoreceptor TRPA1. Chemosensory Perception. 2010; 3(3–4): 190–199. Publisher Full Text\n\nAlimohammadi H, Silver WL: Patterns of variation in the behavioral responses of rats to irritants after neonatal capsaicin treatment. Presented at the Twenty Sixth Annual Meeting of the Association for Chemoreception Sciences. Sarasota, FL. 2004. Reference Source\n\nSimons CT, Dessirier JM, Jinks SL, et al.: An animal model to assess aversion to intra-oral capsaicin: increased threshold in mice lacking substance P. Chem Senses. 2001; 26(5): 491–497. PubMed Abstract \| Publisher Full Text\n\nSclafani A, Ackroff K: Role of gut nutrient sensing in stimulating appetite and conditioning food preferences. Am J Physiol Regul Integr Comp Physiol. 2012; 302(10): R1119–33. 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PubMed Abstract \| Publisher Full Text\n\nCaterina MJ, Leffler A, Malmberg AB, et al.: Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 2000; 288(5464): 306–313. PubMed Abstract \| Publisher Full Text\n\nPark TJ, Lu Y, Juttner R, et al.: Selective inflammatory pain insensitivity in the African naked mole-rat (Heterocephalus glaber). PLoS Biol. 2008; 6(1): e13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTzabazis AZ, Niv SH, Manering NA, et al.: Trigeminal antihyperalgesic effect of intranasal carbon dioxide. Life Sci. 2010; 87(1–2): 36–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTso TC: Tobacco. In: Elvers B, Hawkins S,Schulz G, editors. Ullmann's Encyclopedia of Industrial Chemistry. fifth ed. Weinheim, Germany:Wiley-VCH, 2000; A27. : 123–146. Publisher Full Text\n\nTalavera K, Gees M, Karashima Y, et al.: Nicotine activates the chemosensory cation channel TRPA1. Nat Neurosci. 2009; 12(10): 1293–1299. PubMed Abstract \| Publisher Full Text\n\nAsh M, Ash I: Handbook of Flavors and Fragrances. New York: Synapse Information Resources; 2008. Reference Source\n\nArai T, Ohkuri T, Yasumatsu K, et al.: The role of transient receptor potential vanilloid-1 on neural responses to acids by the chorda tympani, glossopharyngeal and superior laryngeal nerves in mice. Neuroscience. 2010; 165(4): 1476–1489. PubMed Abstract \| Publisher Full Text\n\nFabri J, Graeser U, Simo TA: Toluene. In: Elvers B, Hawkins S,Schulz G (eds.), Ullmann's Encyclopedia of Industrial Chemistry. fifth ed. Weinheim, Germany:Wiley-VCH 2000; A27. : 147–157.Publisher Full Text\n\nMoriyama T, Higashi T, Togashi K, et al.: Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain. 2005; 1: 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchaefer ML, Bottger B, Silver WL, et al.: Trigeminal collaterals in the nasal epithelium and olfactory bulb: a potential route for direct modulation of olfactory information by trigeminal stimuli. J Comp Neurol. 2002; 444(3): 221–226. PubMed Abstract \| Publisher Full Text\n\nDaiber P, Genovese F, Schriever VA, et al.: Neuropeptide receptors provide a signalling pathway for trigeminal modulation of olfactory transduction. Eur J Neurosci. 2013; 37(4): 572–582. PubMed Abstract \| Publisher Full Text\n\nDesesa CR, Vaughan RP, Lanosa MJ, et al.: Sulfur-containing malodorant vapors enhance responsiveness to the sensory irritant capsaicin. Toxicol Sci. 2008; 104(1): 198–209. PubMed Abstract \| Publisher Full Text\n\nPrashar A, Locke IC, Evans CS: Cytotoxicity of clove (Syzygium aromaticum) oil and its major components to human skin cells. Cell Prolif. 2006; 39(4): 241–248. PubMed Abstract \| Publisher Full Text\n\nBandell M, Story GM, Hwang SW, et al.: Noxious Cold Ion Channel TRPA1 Is Activated by Pungent Compounds and Bradykinin. Neuron. 2004; 41(6): 849–857. PubMed Abstract \| Publisher Full Text\n\nMusser MT: Cyclohexanol and Cyclohexanone. In: Elvers B, Hawkins S, Schulz G, editors. Ullmann's Encyclopedia of Industrial Chemistry. fifth ed. Weinheim, Germany:Wiley-VCH; 2000; A8. : 217–226. Publisher Full Text\n\nHoward PH: Cyclohexanone. Handbook of Environmental Fate and Exposure Data For Organic Chemicals.Boca Raton. Fl: CRC Press 1997; 528. Reference Source\n\nWillis DN, Liu B, Ha MA, et al.: Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants. FASEB J. 2011; 25(12): 4434–4444. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "822", "date": "08 Mar 2013", "name": "Bruce Bryant", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a nicely designed study, well executed. Since there were 3 assays used I was expecting more comparison of the 3 assays. Also, in the discussion is capsaicin a typical or topical TRPV1 agonist?", "responses": [] }, { "id": "833", "date": "12 Mar 2013", "name": "Gunnar D Nielsen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 hypothesis is that trigeminal induced (sensory) irritation of chemicals is due to multiple receptors, which is summarized in the Introduction section. Thus an irritant response may be due to activation of several receptors providing redundant detection of an irritant effect of a chemical. This study investigates the role of the TRPV1 (“capsaicin receptor”) in activation of the trigeminal nerve by seven substances (toluene, nicotine eugenol, cyclohexanone, capsaicin, benzaldehyde, amyl acetate and acetic acid).Mice with and without the TRPV1 receptor (wild type and knockout mice) were used. Each substance was studied in both types of mice by three methods: 1) Withdrawal from a cotton swab soaked in the compound;2) A two bottle drinking water preferred choice test, where the atmosphere around one of the bottles was clean air and the other was with the air containing the test compound, and; 3) A reflex induced decreased in respiratory rate due to activation of the trigeminal nerve, a relatively specific method.It was convincingly demonstrated that cyclohexanone was less irritating in all three test methods in the knockout mice which indicates that the irritation response at least partly was mediated by the PRPV1 receptor. In the specific respiratory rate test, the TRPV1 receptor was essential for its detection. The manuscript is consistent and well written. The last author (Wayne L Silver) is a pioneer within sensory irritation research.", "responses": [] } ]	1	https://f1000research.com/articles/2-74
https://f1000research.com/articles/2-73/v1	04 Mar 13	{ "type": "Review", "title": "Assets of the non-pathogenic microorganism Dictyostelium discoideum as a model for the study of eukaryotic extracellular vesicles", "authors": [ "Irène Tatischeff" ], "abstract": "Dictyostelium discoideum microvesicles have recently been presented as a valuable model for eukaryotic extracellular vesicles. Here, the advantages of D. discoideum for unraveling important biological functions of extracellular vesicles in general are detailed. D. discoideum, a non-pathogenic eukaryotic microorganism, belongs to a billion-year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split. During growth and early starvation-induced development, it presents analogies with lymphocytes and macrophages with regard to motility and phagocytosis capability, respectively. Its 6-chromosome genome codes for about 12,500 genes, some showing analogies with human genes. The presence of extracellular vesicles during cell growth has been evidenced as a detoxification mechanism of various structurally unrelated drugs. Controls led to the discovery of constitutive extracellular vesicle secretion in this microorganism, which was an important point. It means that the secretion of extracellular vesicles occurs, in the absence of any drug, during both cell growth and early development. This constitutive secretion of D. discoideum cells is very likely to play a role in intercellular communication. The detoxifying secreted vesicles, which can transport drugs outside the cells, can also act as \"Trojan horses\", capable of transferring these drugs not only into naïve D. discoideum cells, but into human cells as well. Therefore, these extracellular vesicles were proposed as a new biological drug delivery tool. Moreover, Dictyostelium, chosen by the NIH (USA) as a new model organism for biomedical research, has already been used for studying some human diseases. These cells, which are much easier to manipulate than human cells, can be easily designed in simple conditioned medium experiments. Owing to the increasing consensus that extracellular vesicles are probably important mediators of intercellular communication, D. discoideum is here suggested to constitute a convenient model for tracking as yet unknown biological functions of eukaryotic extracellular vesicles.", "keywords": [ "Four decades ago", "the extracellular medium was considered to be no more than a waste reservoir of cell life", "exclusively occurring inside the eukaryotic cell delimited by the plasma membrane." ], "content": "Introduction\n\nFour decades ago, the extracellular medium was considered to be no more than a waste reservoir of cell life, exclusively occurring inside the eukaryotic cell delimited by the plasma membrane.\n\nHere are a few landmarks of the story of extracellular vesicles (EVs): \"Exosomes\" were first mentioned by R. Johnstone in 1970 and further, thoroughly studied during maturation of reticulocytes1,2. \"Shed\" vesicles were observed in 19813. Exosomes in blood cells and their function in the transfer of immunity were thoroughly studied in the early 2000s4,5. Clinical observations of exosomes, as normal and pathogenic biomarkers in urine, were first mentioned in 20046. The presence of nucleic acids, mRNAs and microRNAs in exosomes, was pointed out in 20077, as a first strong indication of vesicle-mediated intercellular communication.\n\nThe increasing importance of EVs can be evaluated by international meetings devoted first to exosomes in Montreal, Canada in 20058, then in 2011, in the International Workshop on Exosomes in Paris, France9, and more recently, in 2012, to EVs in general, at the first meeting of the International Society of Extracellular Vesicles (ISEV) in Gothenburg, Sweden10. An increasing number of workshops are currently devoted to this exploding field of research.\n\nIn the absence of a consensus on the nomenclature for various kinds of extracellular vesicles, the term \"extracellular vesicles\" is used throughout this paper to designate secreted vesicles in general, with no qualification based on defined criteria. For well known subtypes, like exosomes, the more precise qualification is mentioned, when appropriate.\n\nAfter a widespread observation of many different kinds of extracellular vesicles, emerging from quite different fields of biology and medicine, the modern puzzling questions deal about the life functions of these vesicles.\n\nWhy do so many cells expand their action field beyond the cell membrane? Why do cells release vesicles?11. What are the physiological functions of all these EVs? The current state of the art about the role of extracellular vesicles has recently been presented12, whereas exosomes seem to be appealing candidates for mediating intercellular communication13.\n\nHuman complexity is indeed much too high to suggest one particular human cell type as a model for understanding the respective roles of the \"inside/outside\" parts of the cell. To address such a fundamental question requires a convenient well-known eukaryotic model, easy to handle and to follow in a variety of extracellular environments.\n\n\nA model for the study of eukaryotic extracellular vesicles: Dictyostelium discoideum\n\nDictyostelium discoideum came to scientific life in 1935, when it was discovered by Raper14 in the USA Virginia forest, but belongs to a billion year-old Amoeboza lineage, which diverged from the animal-fungal lineage after the plant animal-split15. To survive for such a long time as a unicellular protist, it had to develop a well-conserved life strategy. One of its original biological tricks was to fight against starvation through the first known eukaryotic tentative step towards multicellularity, that is, the aggregation of undifferentiated cells. This aggregation is followed by differentiation into two main cell species: dying stalk cells and \"everlasting\" spores. Thus, D. discoideum experiences specific time-separated physiological states of growth and differentiation during its life cycle. During growth, as unicellular non-pathogenic amoebae, D. discoideum cells, about 10 µm in diameter, are very similar to some human blood cells; they are often compared to lymphocytes with regard to motility, and to macrophages concerning their capability for phagocytosis. After about 24h of starvation-induced development, through cell aggregation (of about 105 cells), followed by differentiation and morphogenesis, it ends up as a \"mushroom-like\" organism of 1–2 mm height, forming a fruiting body, composed of a stalk (about 20% of the cells) and a spore bag containing viable spores (about 80% of the cells)16.\n\nThus, whereas differentiation pulls the microorganism towards a plant-like structure, growing and aggregating cells remain as animal-like cells. Therefore, D. discoideum can offer a simple model of individual eukaryotic cells during growth and also mimic an undifferentiated \"tissue\" during starvation-induced aggregation. The above-defined \"animal\" state of D. discoideum during growth and aggregation, completely separated from differentiation, is the first asset of D. discoideum as a model for eukaryotic cells. Some molecular approaches to cell biology using D. discoideum are detailed in Methods in Cell Biology17.\n\nAnother advantage of D. discoideum is the good knowledge of its genetic material18. Its 6-chromosome genome has been completely sequenced15. The relatively small genomic DNA (3.4 × 107 bp) is efficiently transcribed (90%) into about 12,500 genes, some of which show analogies with human genes. Its 55.5 kb mitochondrial genome has also been sequenced19. A multicopy 90 kb extrachromosomal element that carries the ribosomal RNA genes, and some plasmids complete the DNA components. The RNA amount is about 10 times the DNA amount and due to this RNA excess, the cell cycle does not require a G1 RNA-synthesis step before the DNA-synthesis S phase. Moreover, Dictyostelium cells can be easily transformed by restriction enzyme-mediated integration (REMI)20.\n\nA third asset of D. discoideum lies in its simple growth conditions. In nature, D. discoideum cells grow by bacterial phagocytosis, with a 3h generation time. A widely used laboratory double axenic mutant, Ax-2, can grow in semi-synthetic HL5-medium21, either as adhering cells or in agitated suspension, with a 8 - 11h generation time. A completely defined medium has also been elaborated22 and is now commercialised, as are many other Dictyostelium growth media (Foremedium, UK). Moreover, D. discoideum cells can reach a density of 2–3 × 107 cells ml-1 in the stationary phase and can also be grown in bioreactors23, when huge amounts of cell materials are needed.\n\nIt is worth noting that, owing to all these advantages, D. discoideum was recognised, in 1999, by the National Institutes of Health (NIH, USA) as a new interesting model for biomedical research. Indeed, Dictyostelium had already helped in the understanding of the function of nucleoside diphosphate kinase (NDP) involved in tumour metastasis24,25, and in elucidating the structure of vaults26,27 linked to the drug resistance-related protein LRP28. More recently, R. Escalante gathered various studies that have used D. discoideum as a model for human diseases29. They include pathogen infections, sensitivity to cisplatin and other anticancer drugs, pathobiology of cell motility, lissencephaly, bipolar disorder treatments, lysosomal trafficking and mitochondrial diseases.\n\nDrug detoxification in D. discoideum cells is mediated through release of extracellular vesicles containing the drug. In 1998, it was shown that Dictyostelium cells, known to be highly resistant to xenobiotics, get rid of Hoechst 33342 (HO342), a vital DNA stain for most cells, but not for D. discoideum cells, by means of secretion of extracellular vesicles embedding the dye30. This capability for D. discoideum extracellular vesicle-mediated detoxification has also been checked for hydrophobic drugs, like the photosensitizer hypericin, used in some cancer diagnoses and aimed to photodynamic therapy31.\n\nD. discoideum extracellular vesicles can transfer their vesicular cargos to other cells. Previous studies have found that D. discoideum drug-containing extracellular vesicles are able to transfer HO342 not only to the nuclei of naïve HO342-resistant Dictyostelium cells, but also to the nuclei of human leukaemic multidrug-resistant K562r cells32. In the same way, it it has been shown that hypericin can be transferred from hypericin-containing Dictyostelium extracellular vesicles to the Golgi of tumoral HeLa cells31. Taking into account these experiments, Dictyostelium extracellular vesicles have been proposed as a new biological therapeutic drug delivery tool33,34.\n\nD. discoideum extracellular vesicles are not only a cell detoxifying tool. These vesicles are constitutively released during both growth30 and starvation-induced aggregation35, and are thus candidates for the mediation of intercellular communication.\n\nQuite recently, C. A. Parent found and characterised exosomes during Dictyostelium aggregate formation, suggesting that these organelles are conserved in both Dictyostelium and mammals36,37 (personal communication).\n\nIn order to use D. discoideum EVs as a model for the functional study of eukaryotic EVs, their characterisation is needed. Recently, these vesicles were used to propose a fast characterisation of EVs by means of three joined techniques: nanoparticle tracking analysis, cryo-electron microscopy and Raman tweezer microspectroscopy38.\n\nWith regard to the precise characterisation of Dictyostelium EVs, much remains to be done.\n\nFirst chacterisations of EVs lipids and nucleic acids30 and of EVs proteins31 have been performed. A more precise protocol for the purification of these extracellular vesicles should be devised, followed by a thorough characterisation of the components of all the different subclasses of D. discoideum extracellular vesicles.\n\nHowever, before performing a specialised study of each subclass of D. discoideum extracellular vesicles, it is worth questioning the whole distribution panel of EVs accompanying each important physiological state of D. discoideum cell life. R. Gomer was a pioneer in stressing the importance of the conditioned extracellular medium of D. discoideum cells as a source of secreted autocrine factors during both growth and early starvation39,40. He showed that the aggregate sizes were controlled by autocrine protein factors in the extracellular medium41 and showed the presence of autocrine cell growth inhibition42.\n\nExperiments are currently undertaken to test the plausible hypothesis that cells are not programmed to grow, differentiate or die by an apoptotic \"clean\" death, by their only genomic content and that cells would need intercellular communications to define their individual fate. The necessary signals would be conditioned by all the surrounding cells, according to their own history, and the most convenient way to communicate should indeed be searched for in the cell extracellular medium.\n\nPreviously, we found that an apoptotic-like death could be induced with full efficiency by starving a naïve D. discoideum cell population in a conditioned medium. This conditioned medium (t22) was prepared by starving a first D. discoideum cell population in agitated suspension in a phosphate buffer (pH 6.8) for 22 h. When starved in this cell-depleted t22 conditioned medium, the second D. discoideum cell population completely lost its aggregation competence and was directed to an apoptosis-like death35,43. Preliminary (unpublished) experiments indicated that the extracellular vesicles present in the t22 conditioned medium might be responsible for changing the fate of D. discoideum cells from \"social\" aggregation to mitochondrial-mediated apoptotic death.\n\nIn order to understand the important biological communication signals potentially carried by D. discoideum EVs, it is interesting to take advantage of the relative simplicity of this eukaryotic model. D. discoideum is indeed a wonderful tool to question the respective functions of and interplay between extracellular vesicles and soluble secreted autocrine factors in conditioned medium experiments. This should help in understanding the main, still mostly unknown, biological functions of the secreted vesicles.\n\n\nConclusion\n\nIn 2012, Dictyostelium discoideum was recognized as an interesting model for the study of eukaryotic extracellular vesicles10,44. Due to the important knowledge accumulated on this eukaryotic microorganism since 1935, and to the wide panel of well-defined mutants available in the Stock Center (see dictyBase), a lot of simple experiments can be designed to question the physiological functions of EVs during cell growth, aggregation or apoptotic-like cell death. Moreover, the findings obtained with D. discoideum are likely to be extrapolated to higher eukaryotic cells, in the same way as the extracellular vesicle-mediated detoxification mechanism of D. discoideum cells was a clue towards the introduction of a new multidrug resistance mechanism against antitumoral chemotherapy45.", "appendix": "Competing interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\n\n\n\nAcknowledgments\n\nPierre-Yves Turpin is greatly acknowledged for his helpful assistance in writing the manuscript.\n\n\nReferences\n\nJohnstone RM, Adam M, Hammond JR, et al.: Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem. 1987; 262(19): 9412–20. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValadi H, Ekstrom K, Bossios A, et al.: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007; 9(6): 654–9. PubMed Abstract \| Publisher Full Text\n\nCouzin J: Cell biology: The ins and outs of exosomes. Science. 2005; 308(5730): 1862–3. PubMed Abstract \| Publisher Full Text\n\nIWE. Final Program IWE 2011. International Workshop on Exosomes (IWE) 2011. January 19–22, Paris, France. Reference Source\n\nISEV.Abstracts from the First International Meeting of the International Society for Extracellular Vesicles 2012. Gothenburg, Sweden. Journal of Extracellular Vesicles 2012; 1. Publisher Full Text\n\nNieuwland R, Sturk A: Why do cells release vesicles? Thromb Res. 2010; 125(Suppl 1): S49–51. PubMed Abstract \| Publisher Full Text\n\nGyorgy B, Szabo TG, Pasztoi M, et al.: Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci. 2011; 68(16): 2667–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThéry C: Exosomes: secreted vesicles and intercellular communications. F1000 Biol Rep. 2011; 3(15). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaper KB: A new species of slime mold from decaying forest leaves. J Agr Research. 1935; 50: 135–147.\n\nEichinger L, Pachebat JA, Glockner G, et al.: The genome of the social amoeba Dictyostelium discoideum. Nature. 2005; 435(7038): 43–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoomis WF: Dictyostelium discoideum: A developmental System. New York: Academic Press, Inc. 1975; 214p. Reference Source\n\nSpudich JA: Dictyostelium discoideum: Molecular Approaches to Cell Biology. London: Academic Press, Inc. Methods Cell Biol. 1987; 28: 1–516PubMed Abstract\n\nLoomis WF, Kuspa A: The genome of Dictyostelium discoideum, in Dictyostelium: A model System for Cell and Developmental Biology. Proceedings of the Yamada Conference XLVI on International Dictyostelium Conference, October 14–18, Sendai, Japan, Maeda Y, Inouye K, Takeuchi I, Editors. Universal Academy Press, Inc: Tokyo, Japan 1996; p. 15–30.\n\nOgawa S, Yoshino R, Angata K, et al.: The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization. Mol Gen Genet. 2000; 263(3): 514–9. PubMed Abstract \| Publisher Full Text\n\nKuspa A, Loomis WF: Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc Natl Acad Sci U S A. 1992; 89(18): 8803–7. PubMed Abstract \| Publisher Full Text\n\nWatts DJ, Ashworth JM: Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem J. 1970; 119(2): 171–4. PubMed Abstract \| Free Full Text\n\nFranke J, Kessin R: A defined minimal medium for axenic strains of Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1977; 74(5): 2157–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu Y, Knol JC, Linskens MH, et al.: Production of the soluble human Fas ligand by Dictyostelium discoideum cultivated on a synthetic medium. J Biotechnol. 2004; 108(3): 243–51. PubMed Abstract \| Publisher Full Text\n\nLiotta LA, Steeg PS: Clues to the function of Nm23 and Awd proteins in development, signal transduction, and tumor metastasis provided by studies of Dictyostelium discoideum. J Natl Cancer Inst. 1990; 82(14): 1170–2. PubMed Abstract \| Publisher Full Text\n\nWallet V, Mutzel R, Troll H, et al.: Dictyostelium nucleoside diphosphate kinase highly homologous to Nm23 and Awd proteins involved in mammalian tumor metastasis and Drosophila development. J Natl Cancer Inst. 1990; 82(14): 1199–202. PubMed Abstract \| Publisher Full Text\n\nKedersha NL, Miquel MC, Bittner D, et al.: Vaults. II. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes. J Cell Biol. 1990; 110(4): 895–901. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVasu SK, Rome LH: Dictyostelium vaults: disruption of the major proteins reveals growth and morphological defects and uncovers a new associated protein. J Biol Chem. 1995; 270(28): 16588–94. PubMed Abstract \| Publisher Full Text\n\nScheffer GL, Wijngaard PL, Flens MJ, et al.: The drug resistance-related protein LRP is the human major vault protein. Nat Med. 1995; 1(6): 578–82. PubMed Abstract \| Publisher Full Text\n\nEscalante R: Dictyostelium as a model for human disease. Semin Cell Dev Biol. 2011; 22(1): 69–130. PubMed Abstract \| Publisher Full Text\n\nTatischeff I, Bomsel M, de Paillerets C, et al.: Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342. Cell Mol Life Sci. 1998; 54(5): 476–87. PubMed Abstract \| Publisher Full Text\n\nLavialle F, Deshayes S, Gonnet F, et al.: Nanovesicles released by Dictyostelium cells: a potential carrier for drug delivery. Int J Pharm. 2009; 380(1–2): 206–15. PubMed Abstract \| Publisher Full Text\n\nTatischeff I, Lavialle F, Pigaglio-Deshayes S, et al.: Dictyostelium extracellular vesicles containing Hoechst 33342 transfer the dye into the nuclei of living cells: a fluorescence study. J Fluoresc. 2008; 18(2): 319–28. PubMed Abstract \| Publisher Full Text\n\nTatischeff I, Alfsen A: A New Biological Strategy for Drug Delivery: Eucaryotic Cell-Derived Nanovesicles. J Biomater Nanobiotechnol. 2011; 2(5): 494–9. Publisher Full Text\n\nTatischeff I, Alfsen A, Lavialle F: Extracellular vesicles from non-pathogenic amoeba useful as vehicle for transferring a molecule of interest to an eukaryotic cell. (DRITT-UPMC) 2003–2012: European Priority No. 03 291 752. European Patent (Danemark, Deutschland, France, Great Britain, Italy, Netherland, Spain) USA and Canada Patents.\n\nTatischeff I, Petit PX, Grodet A, et al.: Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis. Eur J Cell Biol. 2001; 80(6): 428–41. PubMed Abstract \| Publisher Full Text\n\nEM. Exosomes and Microvesicles 2012 Conference. Orlando, Florida, USA September 29th October 2nd 2012. Reference Source\n\nKriebel PW, Jenkins L, Zhang L, et al.: Isolation and proteomic analysis of Dictyostelium exosomes in Exosome and Microvesicles Conference. Orlando, Florida USA, 2012.\n\nTatischeff I, Larquet E, Falcon-Pérez JM, et al.: Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy. J Extracellular Vesicles. 2012; 1: 19179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClarke M, Dominguez N, Yuen IS, et al.: Growing and starving Dictyostelium cells produce distinct density-sensing factors. Dev Biol. 1992; 152(2): 403–6. PubMed Abstract \| Publisher Full Text\n\nClarke M, Gomer RH: PSF and CMF, autocrine factors that regulate gene expression during growth and early development of Dictyostelium. Experientia. 1995; 51(12): 1124–34. PubMed Abstract \| Publisher Full Text\n\nBrock DA, van Egmond WN, Shamoo Y, et al.: A 60-kilodalton protein component of the counting factor complex regulates group size in Dictyostelium discoideum. Eukaryot Cell. 2006; 5(9): 1532–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoe JM, Bakthavatsalam D, Phillips JE, et al.: Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation. BMC Biochem. 2009; 10: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArnoult D, Tatischeff I, Estaquier J, et al.: On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death. Mol Biol Cell. 2001; 12(10): 3016–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTatischeff I, Kruglik S, Larquet E, et al.: Microvesicles of Dictyostelium discoideum as a model of eukaryotic extracellular vesicles. ISEV: F1000 Posters2012; 3. : 427. Reference Source\n\nTatischeff I: Cell-derived microvesicles and antitumoral multidrug resistance. C R Biol. 2012; 335(2): 103–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "813", "date": "06 Mar 2013", "name": "Richard Gomer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 brief review of the work on Dictyostelium exosomes.", "responses": [] }, { "id": "878", "date": "04 Apr 2013", "name": "Lawrence Rajendran", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 review, Dr. Tatischeff presents the advantages of using D. discoideum as a model system to study extracellular vesicle release. It is a very well written review and is up to date. Dr. Tatischeff has performed pioneering work in this field and has now, in this recent review, provided a comprehensive overview on the history of D. discoideum extracellular vesicle release research.", "responses": [] } ]	1	https://f1000research.com/articles/2-73
https://f1000research.com/articles/2-72/v1	04 Mar 13	{ "type": "Research Article", "title": "A p53-like transcription factor similar to Ndt80 controls the response to nutrient stress in the filamentous fungus, Aspergillus nidulans ", "authors": [ "Margaret E Katz", "Kathryn Braunberger", "Gauncai Yi", "Sarah Cooper", "Heather M Nonhebel", "Cedric Gondro", "Kathryn Braunberger", "Gauncai Yi", "Sarah Cooper", "Heather M Nonhebel", "Cedric Gondro" ], "abstract": "The Aspergillus nidulans xprG gene encodes a putative transcriptional activator that is a member of the Ndt80 family in the p53-like superfamily of proteins. Previous studies have shown that XprG controls the production of extracellular proteases in response to starvation. We undertook transcriptional profiling to investigate whether XprG has a wider role as a global regulator of the carbon nutrient stress response. Our microarray data showed that the expression of a large number of genes, including genes involved in secondary metabolism, development, high-affinity glucose uptake and autolysis, were altered in an xprGΔ null mutant. Many of these genes are known to be regulated in response to carbon starvation. We confirmed that sterigmatocystin and penicillin production is reduced in xprG- mutants. The loss of fungal mass and secretion of pigments that accompanies fungal autolysis in response to nutrient depletion was accelerated in an xprG1 gain-of-function mutant and decreased or absent in an xprG- mutant. The results support the hypothesis that XprG plays a major role in the response to carbon limitation and that nutrient sensing may represent one of the ancestral roles for the p53-like superfamily. Disruption of the AN6015 gene, which encodes a second Ndt80-like protein, showed that it is required for sexual reproduction in A. nidulans.", "keywords": [ "xprG", "Ndt80", "Aspergillus nidulans", "nutrient stress" ], "content": "Introduction\n\nXprG and two non-catalytic hexokinase-like proteins (HxkC and HxkD) were first identified as regulators of extracellular protease production in Aspergillus nidulans through genetic analysis1–3. In A. nidulans, extracellular proteases are produced in response to carbon, nitrogen or sulfur starvation4. Genetic evidence indicates that XprG activates expression of extracellular protease genes in response to nutrient stress and that HxkC and HxkD are negative regulators of XprG1–3,5,6. The hxkCΔ1 and hxkDΔ3 null mutations and the xprG1 gain-of-function mutation increase production of extracellular proteases1–3,5. In contrast, loss-of-function mutations in xprG abolish carbon-starvation-induced production of extracellular proteases and are epistatic to the hxkCΔ1 and hxkDΔ3 null mutations3,6,7. The production of an acid phosphatase in response to phosphate limitation and of extracellular proteases in response to nitrogen- and sulfur-starvation is also reduced in xprG- mutants7. Thus, there is evidence that XprG could be involved in a general response to starvation.\n\nXprG is similar to VIB-1 of Neurospora crassa, and both are members of the Ndt80 family of p53-like, Ig-fold transcriptional activators (Pfam PF05224)7. VIB-1 is required for expression of genes involved in heterokaryon incompatibility, a type of programmed cell death (PCD)8. XprG is also similar to the Saccharomyces cerevisiae meiosis-specific transcriptional activator, Ndt809. Ndt80 activates the transcription of more than 150 genes during the middle phase of meiosis and is required for progression through meiosis10. It has recently been shown that Ndt80 is also involved in resetting lifespan during meiosis and that transient expression of NDT80 extends the lifespan of aging yeast cells11.\n\nHxkC and HxkD are similar in sequence to catalytic hexokinases but lack some of the conserved residues found in the sugar-binding and ATP-binding domains1. In addition, both possess an extra stretch of amino acids within the adenosine-binding domain. Several plant hexokinase-like proteins that lack catalytic activity also possess an insertion in this same position12,13. The hxkC- and hxkD- mutants have similar phenotypic effects on extracellular protease production but the proteins encoded by these genes are located in different subcellular compartments1. HxkD is a nuclear protein and HxkC is the first fungal hexokinase shown to be associated with mitochondria. Binding of hexokinase to mitochondria blocks apoptosis in human cells and PCD in plants14–16.\n\nAs meiosis in S. cerevisiae requires nutrient deprivation and genes expressed during heterokaryon incompatibility are also expressed in response to starvation, we have suggested that nutrient sensing may be a feature of all Ndt80 family members7. Previous studies have shown that XprG regulates production of extracellular proteases and an acid phosphatase in response to starvation2,3,5–7. In this report, we show that XprG has a wider role as a global regulator of the carbon nutrient stress response and is involved in triggering autolysis, a form of fungal programmed cell death induced by starvation.\n\n\nMaterials and methods\n\nA. nidulans was cultured at 37°C in Aspergillus complete or minimal medium17 except that glucose was omitted from media that contained other carbon sources. For media that contained 1% skim milk as a carbon source, sodium deoxycholate (0.08%) was used to induce compact colony formation. For RNA extraction, mycelia were grown for 24 h in minimal medium containing glucose and then transferred to minimal medium containing glucose or no carbon source for 16 h. To monitor autolysis, six flasks containing 50 mL of minimal medium, 10 mM ammonium tartrate and vitamin supplements were each inoculated with 3×108 conidia and placed on an orbital shaker. Flasks were removed at 24 or 48 h intervals, the submerged mycelia harvested using Miracloth (Calbiochem/Merck) and samples of filtered culture medium collected. To observe conidiophore development on solid medium, strains were inoculated into 1 cm2 blocks of complete medium on microscope slides as described by Larone18. The techniques used for genetic analysis of A. nidulans have been described19. The Aspergillus strains used in this study are listed in Table 1.\n\naThe gene symbols are described in the Aspergillus Genome Database.\n\nTotal RNA was prepared using a procedure developed by Reinert et al.20. mRNA was prepared from total RNA using the PolyATtract® mRNA Isolation System IV as described by the manufacturer (Promega Corp.). DNA was removed from total RNA or polyA+ RNA with the Ambion Turbo DNA-free Kit™ (Applied Biosystems) prior to quantification with a NanoDrop® spectrophotometer. The primers (Supplementary Table 1) used in qRT-PCR experiments were designed using the Primer3 program (http://frodo.wi.mit.edu/primer3/). Each primer pair was first tested with serial dilutions of MH2 RNA to determine the linear range of the qRT-PCR assays using SuperScript III Platinum SYBR Green One-Step qRT-PCR Kits (Invitrogen). The experiments were performed using a Corbett CAS1200 liquid handling robot and Corbett Rotor-Gene 3000 real-time thermal cycler (QIAGEN). In the assays to determine relative transcript levels, 1 ng of total RNA was added to each reaction. Each reaction was performed in duplicate or triplicate and the actA control reactions were included in each run.\n\ncDNAs labeled with Alexa Fluor® 555 and Alexa Fluor® 647 were prepared from mRNA using the SuperScript™ Plus Indirect cDNA Labeling System according to the instructions of the manufacturer (Invitrogen). A. nidulans DNA microarrays, supplied by the Pathogen Functional Genomics Resource Center (PFGRC) at The Institute for Genomic Research (TIGR) were hybridized with the labeled cDNAs using the TIGR protocol21. The A. nidulans microarrays consisted of 11,481 unique 70-mer oligonucleotides spotted in duplicate on the array plus an additional 1,000 control probes from Arabidopsis thaliana and 1,430 empty features (negative controls). The hybridized slides were scanned immediately in an Axon 4200AL scanner (Molecular Devices). The intensity values for the two channels for each spot were acquired by automatic photomultiplier tube gains to obtain the highest intensity with 0.05% saturated pixels. The resulting images were analyzed by measuring the fluorescence of all features on the slides using GenePix Pro 6.1 software (Molecular Devices). The median fluorescence intensity of these pixels within each feature was taken as the intensity value for the feature.\n\nThe NCBI Gene Expression Omnibus (GEO) accession number for the microarray data reported in this paper is GSE36235 and the data are available at http://www.ncbi.nlm.nih.gov/geo/. Also available for download from this GEO accession is a Supplementary Analysis File containing all pre-processing analyses, annotated lists of differentially expressed genes with links to NCBI as well as gene ontology, pathway analyses and other relevant images and diagrams (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE36235&submit.x=15&submit.y=14).\n\nQuality control measures, pre-processing and analyses were performed using the statistical computing language R22 and Bioconductor23. All microarray images and quality control measurements were within recommended limits24. The quality of the arrays was assessed through standard quality control measures: pseudo-images of the arrays (to detect spatial effects), MA (M is the intensity ratio and A is the average intensity) scatter plots of the arrays versus a pseudo-median reference chip, and other summary statistics including histogram and boxplots of raw log intensities, signal-to-noise ratios on both channels, boxplots of plates and print tips, boxplots of normalized log ratios, among others. Transcription intensities in adjusted log2 were estimated after normalization within arrays using maximum likelihood25 followed by between array variance stabilization26. Briefly, the data were adjusted by an affine transformation and then all slides were log2 transformed to stabilize the variance. Prior to testing for differential expression, the data were filtered to remove control (n=1,000 from Arabidopsis thaliana) and empty spots (n=1,430) and spots flagged as bad in over 90% of the slides (n=4,754), thus leaving 9,104 unique features to be tested.\n\nDifferential expression was tested on a gene by gene basis using a moderated t-test with intensities adjusted using an Empirical Bayes approach27. A covariance structure to account for the duplicate probes and within array variability was also fitted to the model. Features were considered significantly differentially expressed for a false discovery rate adjusted p-value of 0.05 using the Benjamini-Hochberg correction28.\n\nThe annotation of the array features was derived from the AspGD – Aspergillus Genome Database29 and identifiers were annotated to gene ontology terms and pathway information for testing gene set enrichment in GO and KEGG (Kyoto Encyclopedia of Gene and Genomes). In subsequent text the term probe is replaced by gene. The differentially expressed genes were analyzed in the context of their Gene Ontology (GO)30 and involvement in KEGG biological pathways31,32.\n\nFunctional profiles for the differentially expressed genes were derived for each of the GO categories: cellular component, molecular function and biological process. Differentially expressed genes were mapped from their Entrez identifier to their most specific GO term and these were used to span the tree structure and test for gene enriched terms. Profiles for each category were also constructed for the differentially expressed genes for different tree depths (Supplementary Analysis File). To avoid over-inflated p-values, the background for both GO and KEGG pathway analyses consisted exclusively of the array probes used in the analyses after the removal of control probes, unexpressed probes and unannotated probes. Gene ontologies and KEGG pathways reported in this manuscript include those with a significance value of p < 0.05.\n\nFor sterigmatocystin assays, flasks containing 50 mL of Aspergillus minimal medium were inoculated with 3 x 108 conidia scraped from cultures grown on complete medium containing 2.2% agar. After 24 h, the growth medium was collected and the mycelia were transferred to carbon-free medium for 24 h. Sterigmatocystin was extracted from 10 mL aliquots of filtered growth medium using the method described by Keller et al.33 with the following modifications. An equal volume of chloroform was added to each sample, mixed vigorously and agitated on a shaking platform for 15 min. After centrifugation at 1600 x g for 5 min, the aqueous phase was transferred to a fresh tube and the chloroform extraction was repeated. The chloroform from the first and second extractions was pooled, dried in a rotary evaporator and the residue resuspended in 50 µL chloroform. A 5 µL sample of each extract was applied to aluminum-backed, silica thin layer chromatography sheets (Merck) and separated using a mixture of benzene and glacial acetic acid (95:5). After drying, the plate was sprayed with 15% AlCl3 dissolved in 95% ethanol, baked at 65°C for 15 min and photographed under 365 nm UV illumination. Sterigmatocystin (Sigma) was used as a standard.\n\nSterigmatocystin was also extracted from three 16 mm plugs taken from conidiating colonies grown on solid minimal medium using the method described by Keller et al.33 with the following modifications. Chloroform (1 mL) was added to the agar plugs and mixed vigorously. After centrifugation at 1000 x g for 5 min, the chloroform containing the extracted sterigmatocystin was transferred to a fresh tube, washed twice with 0.5 mL Milli-Q water (QPAK 2 purification pack, Millipore) and then evaporated. The residue was resuspended in 0.1 mL chloroform.\n\nPenicillin levels in filtered penicillin production broth containing 3% lactose or 3% glucose were assayed as described by Espeso and Peñalva34. 5 mL aliquots of filter-sterilized culture medium were lyophilised and resuspended in 300 µL of 10 mM sodium phosphate buffer pH 6.8. The volume (35–50 µL) corresponding to the penicillin produced by 9.3 mg mycelium (dry weight) was applied to 6 mm wells in Luria Broth plates seeded with Micrococcus luteus (UNE014). Penicillin G (Sigma) dissolved in 10 mM sodium phosphate buffer pH 6.8 was applied as a control. The filtrates were left to diffuse for 18 h at 4°C and then incubated at 30°C for 32 h. For samples treated with penicillinase (Sigma Aldrich), 1 µL containing 1 U of enzyme in 100 mM Tris-HCl pH7 with 0.1% BSA was added and the samples were incubated at 25°C for 15 min before they were applied to the plates. The samples that were not treated with penicillinase were treated in an identical manner except that the 1 µL of 100 mM Tris-HCl pH7 0.1% BSA did not contain any enzyme.\n\nThe uptake of D-[U-14C] glucose (10.6 GBq/mmol, Amersham) was measured in germinating conidia as described previously35. Conidia were germinated in minimal medium containing 1% glucose, 0.1% yeast extract, 10 mM ammonium tartrate and vitamins and then washed five times with carbon-free minimal medium containing 10 mM NH4Cl and vitamins. Glucose uptake was measured in aliquots of 2.5 x 107 germinating conidia 5, 30, 60 and 90 s after transfer to media containing 0.025, 0.125, 0.5 or 2 mM glucose.\n\nThe AN6015 gene (ndtA) was disrupted in an nkuAΔ strain (MH11036) so as to increase the frequency of gene targeting events36. The entire predicted coding region of AN6015 (nucleotides 21661–23381, contig 103; Aspergillus Comparative Database) was replaced with the Aspergillus fumigatuspyroA gene using a similar strategy to the one described in Nayak et al.36. Gene disruption was confirmed by PCR and Southern blot analysis. Double mutants with lesions in AN6015 (ndtA) and hxkC, hxkD or xprG were generated in crosses and the presence of ndtA::A. fumigatus pyroA was confirmed by PCR using primers MK261 (5´-AACGGTTACCTCCCAATTGC-3´) complementary to sequences upstream of the A. nidulans ndtA coding region and MK323 (5´-GATGGTCTCGAACTGACCTT-3´) complementary to the A. fumigatus pyroA gene.\n\n\nResults\n\nA. nidulans microarrays provided by the Pathogen Functional Genomics Resource Center (PFGRC) were used to compare transcript levels in an xprG+ strain and an xprGΔ null strain after transfer to medium containing glucose as a carbon source or medium lacking a carbon source (carbon starvation) for 16 h. These four experiments (Figure 1) were designed to detect differences in transcript levels between the two strains (Experiments 2 and 4) and changes in transcript levels in each strain due to the different nutrient conditions (Experiments 1 and 3). The NCBI Gene Expression Omnibus (GEO) accession number for the microarray data reported in this paper is GSE36235 and is available at http://www.ncbi.nlm.nih.gov/geo/. A total of 516 probes that hybridized to differentially expressed transcripts were detected in Experiment 1, which examined the effect of carbon starvation in an xprG+ strain. One hundred and ninety seven were up-regulated and 319 were down-regulated during carbon starvation (Figure 2). The top five biological processes identified in the Gene Ontology analysis of Experiment 1 were sterigmatocystin biosynthesis, ergosterol biosynthesis, conidial spore wall assembly, the purine salvage pathway and autolysis. In the xprGΔ1 mutant, the number of transcripts that showed a significant change in response to carbon starvation was lower (Figure 2). All of the 73 up-regulated and 222 down-regulated transcripts in Experiment 3 showed similar responses (in direction) to carbon starvation in Experiment 1.\n\nThe arrowheads point to the samples labeled with Alexa Fluor® 555. Each experiment consisted of three biological replicates, indicated by arrows, and included a dye swap. The full genotypes of the xprG+ (MH2) and xprGΔ (MK422) strains are given in Table 1.\n\nIn Experiments 1 and 3, the number of transcripts up-regulated during carbon starvation is shown in blue and the number down-regulated is shown in black. In Experiment 4, the number of transcripts that are down-regulated in the xprGΔ1 mutant is shown in blue and number up-regulated is shown in black.\n\nIn Experiment 4, which examined the effect of the xprGΔ1 mutation on A. nidulans’ response to carbon starvation, 133 probes hybridized to transcripts that were either up- or down-regulated (Figure 2). Ninety four probes hybridized to transcripts that were down-regulated in the xprGΔ1 mutant and 39 genes were up-regulated. Fifteen of the down-regulated transcripts, including four of the top five, belonged to the sterigmatocystin gene cluster (Table 2). The pathway for the synthesis of sterigmatocystin, a carcinogen closely related to aflatoxin, is encoded by a cluster of 25 co-regulated genes37. Transcripts from an additional four genes from the cluster (aflR, stcA, stcO, and stcS) had lower levels in the xprGΔ1 mutant with p-values of less than 0.05 prior to applying the Benjamini-Hochberg correction28. The tdiB gene, which is down-regulated in the xprGΔ1 mutant, belongs to another secondary metabolism gene cluster, tdiA-E, that controls the biosynthesis of the anti-tumor compound terrequinone A38,39. A second gene in the cluster, tdiA, was down-regulated in the xprGΔ1 mutant with a p-value of 0.002 prior to adjustment and 0.073 after application of the Benjamini-Hochberg correction. It is interesting that disruption of the laeA gene, which encodes another regulator of the tdi gene cluster, produced similar effects on the members of the cluster; the reduction in tdiB transcript levels was greater than that of tdiA and the levels of the tdiC, D and E transcripts were affected to an even lesser extent in the laeAΔ mutant39.\n\naThe genes are described in the Aspergillus Genome Database. Only named genes (and genes with a similar function to the named genes) are listed. The fold change (log2 scale) is given in parentheses, with a negative value indicating that the gene is down-regulated in the xprGΔ1 mutant during carbon starvation. The full data set for differentially expressed genes is available through NCBI Gene Expression Omnibus (GEO) accession number GSE 36235, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE36235).\n\nbThe effect of the xprGΔ1 mutation on transcript levels during carbon starvation was determined in microarray experiments.\n\nOther genes with documented functions that showed differential expression in response to carbon starvation in the xprGΔ1 mutant include two genes encoding extracellular proteases (prtA and pepJ) which are known to be expressed during starvation5,40,41. The expression of prtA in response to carbon or nitrogen starvation has been shown to be XprG-dependent6. HxkC is involved in the regulation of extracellular protease production. Disruption of the hxkC gene, which is down-regulated in the xprGΔ1 mutant, increases extracellular protease production1.\n\nThe microarray data indicated that a key regulator of conidiophore development brlA42 was down-regulated in the xprGΔ1 mutant, while the veA gene, which activates sexual development43 was up-regulated. Genes encoding a putative sex pheromone (ppgA) and pheromone receptor (preA) were also expressed at higher levels in the xprGΔ1 mutant. Carbon starvation is known to induce transcription of the brlA gene44.\n\nAutolysis is a process of hyphal fragmentation and digestion that occurs in stationary cultures of A. nidulans after carbon source depletion45. Though autolysis and apoptotic cell death occur concurrently during carbon starvation, genetic evidence indicates that the two processes are regulated independently46. The chitinase encoded by the chiB gene plays an important role in autolysis47 while nagA is involved in apoptotic cell death48. Both chiB and nagA, which were up-regulated in response to carbon starvation in the xprG+ strain in Experiment 1, are down-regulated in the xprGΔ1 mutant.\n\nIn contrast to Experiment 4, only two probes on the array showed significantly different intensities when hybridized with cDNA prepared from xprG+ and xprGΔ1 strains grown in medium containing glucose in Experiment 2. This confirms that the role of XprG is mainly confined to the starvation response. Only one of the two probes identified in Experiment 2 is annotated as a gene, hpdA, which encodes a putative 4-hydroxyphenylpyruvate dioxygenase with a predicted role in pyomelanin production. In Aspergillus fumigatus, disruption of the hpdA homolog (hppD) abolished pyomelanin pigment production and no pigment was detected in mycelia or culture medium of the mutant when it was grown in liquid medium49.\n\nThree genes that were down-regulated (brlA, chiB, tdiB) and two that were up-regulated (ppgA, veA) in the xprGΔ1 mutant (Experiment 4) were analyzed in qRT-PCR experiments using new preparations of RNA (Table 3), and by agarose gel electrophoresis of qRT-PCR products (Supplementary Figure 1). The housekeeping gene encoding actin (actA) was used as a control. The level of the actin transcript was lower in carbon-free medium than in glucose in both strains. In previous studies we have observed, using Northern blot analysis, that the level of the actA transcript is reduced (relative to rRNAs) during carbon starvation5. The transcript levels in the three down-regulated genes were all higher in the xprG+ strain than in xprGΔ1 mutant during carbon starvation and were higher during carbon starvation than in nutrient-sufficient conditions in a xprG+ strain as predicted by the microarray results. The qRT-PCR data for the up-regulated ppgA gene showed much higher expression in the xprGΔ1 mutant than the wild-type strain during carbon starvation and higher levels in carbon-free medium than glucose for the xprGΔ1 mutant, consistent with the results in microarray Experiments 4 and 3, respectively. However, no significant difference in ppgA expression was detected in microarray Experiment 1, whereas the qRT-PCR data suggest that ppgA transcript levels are higher during carbon starvation in the xprG+ strain. For the veA gene, no differences between the wild-type and mutant strains were detected.\n\naThe average cycle threshold (Ct) values for threshold of 0.03 normalized fluorescence units and standard errors are shown. A lower Ct value indicates higher transcript levels. Relative expression levels (REL), based on the Takeoff point and reaction efficiency, were calculated using the Corbett Rotor-Gene Comparative Quantitation program, using the xprG+/glucose reactions for each gene as the calibrator. The relative expression levels do not take into consideration the differences in the actA transcript levels.\n\nThe results of the microarray experiments suggested that expression of genes in the sterigmatocystin gene cluster was reduced in the xprGΔ1 mutant. To confirm that sterigmatocystin levels were altered, sterigmatocystin was extracted from the growth medium of strains carrying two different xprG- mutations (xprG2 and xprGΔ1) and a strain carrying the xprG1 gain-of-function mutation. The xprG2 loss-of-function mutation is due to the insertion of two base pairs which causes a frameshift mutation in the ninth codon of the xprG gene7. The xprGΔ1 mutation, which lacks codons 248–344, was constructed by gene disruption and has a phenotype that is identical to the xprG2 mutant7. The xprG1 mutation is a missense mutation in the putative DNA-binding domain of XprG7. Sterigmatocystin levels were reduced in both the gain- and loss-of-function mutants (Figure 3A). In the wild-type strain very low levels of sterigmatocystin were detected after 24 h growth in medium containing glucose and much higher levels after transfer, for 24 h, to medium lacking a carbon source. No sterigmatocystin was detected in the xprG2 or xprGΔ1 mutants in either growth condition and the level of sterigmatocystin in the xprG1 gain-of-function mutant was much lower than in the wild-type strain. Production of a blue-green pigment which co-migrates with sterigmatocystin33 was reduced in the xprG- mutants but not in the gain-of-function mutant. Sterigmatocystin production was also reduced in the xprG1, xprG2 and xprGΔ1 cultures grown on solid medium (Supplementary Figure 2).\n\nA. Sterigmatocystin, extracted from the filtered growth medium of an xprG+ strain (MH2), an xprG1 strain (MK85) and two xprG- strains, MK198 (xprG2) and MK422 (xprGΔ1), was analyzed using thin layer chromatography. Sterigmatocystin fluoresces yellow after treatment with AlCl3. Sterigmatocystin (ST) (Sigma) was applied as a standard. The cultures used in the assays were generated by inoculating growth medium with 3 x 108 conidia. After transfer to carbon-free medium for 24 h, the dry mycelial weights were 100 mg (xprG+), 71 mg (xprG1), 129 (xprG2) and 152 mg (xprGΔ1). B. Penicillin bioassay based on inhibition of bacterial growth. Samples of filtered, concentrated growth medium from strains MH2 (xprG+), MK85 (xprG1), and MK198 (xprG2) was applied to wells in medium seeded with the Micrococcus luteus. 400 ng of penicillin G (penG) and 10 mM sodium orthophosphate buffer pH 6.8 (buffer) were used as controls. The Aspergillus growth medium contained either 3% glucose or 3% lactose. In the right-hand plate the samples were treated with 1 U of penicillinase (Sigma Aldrich) before they were applied to the wells. The full genotypes of the strains are given in Table 1.\n\nPenicillin is also a product of secondary metabolism in A. nidulans. Although no significant changes in the expression of penicillin biosynthetic genes were detected in the microarray experiments, this may have been due to the fact that the growth medium was not optimal for penicillin production. Bioassays were used to detect penicillin levels in broth cultures optimised for penicillin production34. The results showed that penicillin levels, as measured by bacterial growth inhibition, were greatly reduced in an xprG2 loss-of-function mutant and increased in an xprG1 gain-of-function mutant (Figure 3B). When glucose was included in the growth medium, no penicillin was detected in the culture medium of any strains (Figure 3B).\n\nBrlA is a DNA-binding protein that is required for conidiophore development42,50. The microarray and qRT-PCR data showed that expression of brlA is induced during carbon-starvation but is at lower levels in the xprGΔ1 mutant. The RNA used in the microarray and qRT-PCR experiments was extracted from mycelia grown in submerged cultures. While conidiation does not normally occur under these conditions, transfer to medium lacking a carbon source does induce conidiation in submerged cultures44. All xprG- mutants produce conidia though they are abnormally pale in color7 (Figure 4A). The conidophore structure of xprG mutants was examined and appeared to be normal (Figure 4A, Table 4). The conidiophore stalk length was highly variable in all strains but the difference between the xprG+ and xprG2 is marginally significant (p = 0.05). Asexual spore production was also highly variable in the gain- and loss-of-function mutants (Table 4). Both xprG1 and xprG- mutants were slightly slower to initiate conidiophore development.\n\nA. Conidiophores of strains MH2, MK198, and MK85 were photographed after 2 days growth at 37°C on solid complete medium on microscope slides followed by treatment with diluted Lactophenol Cotton Blue stain. For the lower set of pictures, conidia were scraped from MH2, MK422 and MK85 colonies on complete medium. Scale bars: 50 µm (upper row), 20 µm (lower row). B. Conidiophores of strains MH2, MK422 and MK85 after transfer to carbon-free liquid medium for 24 h. Scale bars: 10 µm. The full genotypes of the xprG+ (MH2), xprG2 (MK198), xprGΔ1 (MK422) and xprG1 (MK85) strains are given in Table 1.\n\naThe full genotypes are given in Table 1. Strains MH2 (xprG+) and MK85 (xprG1) were used for all analyses. Strain MK422 was used for all xprG- analyses except for mean conidiophore stalk length, which used MK198 (xprG-). Conidiophore morphology in surface cultures was examined in both MK198 and MK422.\n\nbConidiophores were photographed at 400 x magnification after growth at 37°C on microscope slides. Measurements were carried out using the ImageJ program (http://rsbweb.nih.gov/ij/). The mean length (± SD) for over 100 conidiophores are given. The difference between the xprG- and xprG+ strains was marginally significant (unpaired t-test, p=0.05)\n\ncThe number of asexual spores (conidia) per mm2 was determined by removing three plugs from colonies on complete medium containing 2.2% agar. The conidia from each plug were suspended in a solution of 0.01% TWEEN80 and counted in a haemocytometer. The number per mm2 (± SD) is the mean from four experiments which used different batches of media. No significant differences were found using an unpaired t-test.\n\ndConidiophore development was monitored after transfer to carbon-free medium.\n\nExpression of the ivoC gene was lower in the xprGΔ1 mutant. IvoC encodes a putative cytochrome P450 that is required for conidiophore pigmentation (A.J. Clutterbuck, personal communication). The ivoB gene also showed lower expression in the xprGΔ1 mutant with an unadjusted p-value of 0.002. Mutants lacking a functional copy of ivoA, B or C have ivory-coloured conidiophores42. Microscopic examination showed that the conidiophore stalks of xprG2 mutants display normal pigmentation (Figure 4A).\n\nInitiation of conidiophore development occurs irrespective of nutrient limitation in A. nidulans cultures exposed to air51 and can be induced in submerged cultures by carbon starvation44. We found that conidophore development occurred in carbon-starved submerged cultures of both the xprGΔ1 loss- and xprG1 gain-of-function mutants, though the number of metulae appeared to be reduced (Figure 4B). Thus, XprG is not essential for triggering conidiophore development in response to carbon starvation.\n\nWe investigated the genetic interactions between the xprG mutations and mutations in genes encoding key regulators of conidiophore development. VeA is a component of the light sensor which regulates the switch from sexual to asexual development. Laboratory strains of A. nidulans produce abundant asexual spores (conidia) in the absence of light because of a point mutation in the veA gene43. To investigate the interaction between the xprG and veA genes, strains carrying the xprG1 and xprG2 mutations were crossed to a ve+strain, which requires light to trigger asexual spore formation. When xprG2 ve+ segregants were grown in complete darkness, the colonies produced even fewer conidia than xprG+veA+ strains, whereas the xprG1 gain-of-function mutation partially suppressed VeA-mediated repression of conidiophore development (Figure 5). Programmed initiation of conidiation in surface cultures depends on FluG, but fluG- mutants can be induced to undergo conidiophore development by nutrient stress52. We found that the xprG1 mutation partially suppresses the conidiophore development defect in the fluG701 mutants (Figure 5). In contrast, the xprG1 mutation did not suppress the brlA1 defect in conidiation.\n\nA. Conidiation is suppressed by VeA in the dark but XprG1 partially restores conidiation in a veA+ strain. The plate was photographed after 3 days of growth on complete medium at 37°C. Light was excluded by wrapping the plate in aluminum foil. The full genotypes of the xprG+ (MH2), xprG2 (MK198), xprG1 (MK85), xprG+ veA+ (WIM-126), xprG2 veA+ (MK565), and xprG1 veA+ (MK563) strains are given in Table 1. B. The fluG gene is involved in producing an extracellular signal for the induction of conidiophore development67. The fluG701 mutation is partially suppressed by the xprG1 gain-of-function mutation. The full genotypes of the strains (top left MK593, top right MK592, bottom left MK595, bottom right MK594) are given in Table 1.\n\nThe Aspergillus niger mstA gene encodes a high-affinity sugar transporter that is highly expressed during carbon starvation and repressed by glucose53. The A. nidulans homologue of mstA was among the top five genes that were up-regulated in response to carbon starvation in an xprG+ strain in Experiment 1, and was down-regulated in the xprGΔ1 mutant. The effect of xprG loss- and gain-of-function mutations on glucose transport was examined (Figure 6). In the xprGΔ1 mutant, glucose uptake was significantly reduced when low levels of glucose were present but was unaltered when the concentration of glucose was high, indicating that only high-affinity glucose uptake was decreased. Both high- and low-affinity uptake of glucose was reduced in the xprG1 gain-of-function mutant.\n\nThe results are the average for four (xprGΔ1, xprG1) and five (xprG+) experiments and standard errors are shown. The rate of glucose uptake was compared with the uptake of the xprG+ strain at each concentration of glucose using an unpaired t-test. Values which differed significantly from the value for the xprG+ strain are indicated with asterisks (p < 0.5, p < 0.1) The full genotypes of the xprG+ (MH2), xprGΔ (MK422) and xprG1 (MK85) strains are given in Table 1.\n\nThe chiB gene, which plays an important role in autolysis, was among the top five genes that were up-regulated in response to carbon starvation in the xprG+ strain in Experiment 1, and was down-regulated in the xprGΔ1 mutant. Production of extracellular proteases also increases during autolysis54. The genes encoding two extracellular proteases, PrtA and PepJ, were down-regulated in the xprGΔ1 mutant. Cultures of the xprG1 and xprG2 mutants were observed over a period of eight days to determine whether XprG plays a role in autolysis, which occurs in stationary, submerged cultures of A. nidulans after carbon source depletion45. The disintegration of mycelial pellets, decline in mycelial mass, increase in culture medium turbidity due to hyphal fragmentation and accumulation of brown pigment which accompany autolysis occurred more rapidly in the xprG1 gain-of-function mutant. In contrast, mycelial pellets were still present in the cultures of the xprG2 and xprGΔ1 mutants (the two xprG-genotypes) after 8 days and there was no evidence of hyphal fragmentation or pigment accumulation (Figure 7). These results indicate that XprG is required for autolysis in response to carbon starvation. Thus, XprG, like Vib-1 of N. crassa has a role in regulating programmed cell death.\n\nLoss of mycelial mass (A) and changes in the appearance of cultures (B) were monitored for 8 days in submerged cultures inoculated with the same number of conidia. The results in A are the average for the three experiments and standard errors are shown. The mycelial mass at each time point was compared with the mass of the xprG+ strain using an unpaired t-test. Values which differed significantly from the value for the xprG+ strain are indicated with asterisks (p < 0.5, p < 0.1, *p < 0.001) The full genotypes of the xprG+ (MH2), xprG1 (MK85), xprG2 (MK198), xprGΔ1 (MK422) and xprG2 ndtAΔ (MK505) strains are given in Table 1.\n\nThe microarray experiments showed that expression of the hpdA gene was reduced in the xprGΔ1 mutant. The A. fumigatus hppD gene is the ortholog of the A. nidulans hpdA gene and has been shown to be essential for the production of pyomelanin49. A ΔhppD mutant has colourless mycelia and does not release pyomelanin in liquid mediuam. Thus, it is likely that the pale mycelia and absence of released pigment in the xprG-mutants during autolysis is due to reduced hpdA expression.\n\nNdt80 is a transcriptional activator required for progression through meiosis in S. cerevisiae9,10 whereas A. nidulans mutants lacking a functional copy of the xprG gene are able to complete meiosis. S. cerevisiae is unusual among ascomycete fungi in that it possesses only one transcription factor in this class (Table 5). In A. nidulans, a second putative member of this class (AN6015) shows greater similarity to Ndt80 (17.1% identity overall and 23.5% in the DNA-binding domain) than does XprG (12.4% identity overall and 13.8% identity in the DNA-binding domain). To investigate the role of AN6015, the gene was disrupted. Strains carrying a disrupted copy of AN6015 could be crossed to wild-type strains but no cleistothecia (fruiting bodies) were observed when AN6015Δ mutants were crossed. These results suggest that AN6015 is required for sexual reproduction in A. nidulans and, as in S. cerevisiae, mutations in AN6015 are recessive. We suggest that AN6015 be named NdtA.\n\naGenome sequences were obtained from the Fungal Genome Initiative of the Broad Institute with the exception of the P. chrysosporium, P. placenta and P. blakeleeanus sequences which were from the DOE Joint Genome Institute.\n\nUnlike xprG loss-of-function mutations, ndtAΔ does not affect conidial pigmentation (Fig 8A), prevent extracellular protease production or suppress mutations in hxkC and hxkD (Figure 8B and Figure 8C). If no ammonium is present, wild type strains produce a halo, due to extracellular protease activity, on medium containing milk as a nitrogen source. The ndtAΔ mutant also displays a halo but the xprG2 mutant, which is protease-deficient, does not when grown on medium containing milk as a nitrogen source (Figure 8B). Extracellular protease activity is low on medium containing milk as a carbon source, as carbon starvation is required to stimulate extracellular protease production when ammonium is present3. The hxkCΔ and hxkDΔ mutants have elevated levels of extracellular protease and produce large halos on this medium1,2. The xprG2 mutation suppresses this phenotype but the ndtAΔ mutation does not (Figure 8C). xprG2 ndtAΔ double mutants had the same pale conidia as xprG2 strains. Like the xprG2 single mutant, the xprG2 ndtAΔ double mutant produced no halo on medium containing milk as a carbon or nitrogen source and did not undergo autolysis in response to nutrient stress (Figure 7).\n\nColony morphology and extracellular protease production of wild-type and mutant strains on (A) minimal medium (B) medium containing milk as a nitrogen source and (C) medium containing milk as a carbon source. The clear halo surrounding colonies on medium containing milk is due to extracellular protease activity. The full genotypes of strains MH97 (WT), MK198 (xprG2), MK481 (6015Δ), MK320 (hxkDΔ3), MK186 (hxkD1xprG2), MK532 (hxkDΔ3 6015Δ), MK388 (hxkCΔ1), MK408 (hxkCΔ1 xprG2), and MK531 (hxkCΔ 6015Δ) are given in Table 1.\n\n\nDiscussion\n\nThe transcriptional profiling data reported here reveal that XprG plays a major role in the activation of gene expression in response to carbon starvation. More than 37% of the 197 probes that hybridized to transcripts that were significantly up-regulated during carbon starvation, were down-regulated in the xprGΔ1 mutant. This proportion is higher if less stringent criteria are used to identify differentially regulated transcripts; 60% of the transcripts up-regulated during carbon starvation show more than a two-fold decrease in transcript levels in the xprGΔ1 mutant and 91% show at least some decrease. In contrast, less than 5% of the 319 probes that hybridized to transcripts that were down-regulated during carbon starvation were up-regulated in the xprGΔ1 mutant and none were down-regulated. As XprG is a putative transcriptional activator, it is not surprising that it does not appear to be involved in repression of gene expression during carbon starvation. Secondary effects (e.g. down-regulation of repressors) may be responsible for the few transcripts14 that are down-regulated during carbon starvation and up-regulated in the xprGΔ1 mutant. XprG also does not appear to play a role in regulating gene expression during growth in medium containing glucose as a carbon source.\n\nHxkC and HxkD are hexokinase-like proteins which are negative regulators of extracellular protease production and may modulate the activity of XprG1,3. It has previously been reported that contrary to expectations, hxkD transcript levels increase during carbon starvation1. The microarray data reported here showed that the hxkC gene, is also up-regulated during carbon starvation, and that increased expression of hxkC is dependent on XprG. It was not expected that hxkC and hxkD transcript levels would increase during carbon starvation, because HxkC and HxkD are negative regulators and production of extracellular proteases increases during carbon starvation. As noted previously, these results could indicate that HxkC and D have other functions during carbon starvation1.\n\nWe have shown here that XprG regulates the expression of brlA, a key regulator of conidiophore development, in submerged cultures during carbon starvation. However, conidiophore development is essentially normal in xprG- mutants grown on solid media and can be induced by carbon starvation in submerged cultures. Thus, the reduction of brlA expression observed in the xprGΔ1 mutant is not sufficient to block conidiophore development. Nevertheless, the genetic evidence suggests that XprG plays some role in triggering asexual development as the xprG1 mutation stimulates conidiophore development in a veA+strain incubated in the dark and in a fluG701 mutant.\n\nSecondary metabolism and asexual/sexual development are linked in filamentous fungi. XprG appears to be a member of a group of regulatory proteins that control both secondary metabolism and development (reviewed in Bayram et al.55). This group includes the light regulator VeA, which is required for sexual development43 and has been shown to regulate sterigmatocystin production56, LaeA, the global regulator of secondary metabolism57 which is also required for asexual development58, and components of a heterotrimeric G protein signaling pathway which is required for both asexual development and sterigmatocystin production59. All of the proteins in this group act upstream of BrlA, the transcription factor that activates genes required for conidiophore development60, but is not required for sterigmatocystin production61. The A. nidulans homologue of S. cerevisiae Ime2 protein kinase is also a member of this group. An imeBΔ null mutant does not produce sterigmatocystin and overproduces sexual fruiting bodies in light in a veA+ strain62. In S. cerevisiae Ime2 activates transcription of Ndt80 and also controls Ndt80 activity through phosphorylation63. XprG, as an Ndt80-like protein, could be a target of ImeB in A. nidulans.\n\nIn addition to the link between asexual development and secondary metabolism in A. nidulans, there is a link between asexual development and autolysis46,54,64. Thus, XprG may play a direct role in regulating autolysis through regulation of chitinase (ChiB), extracellular proteases (PrtA, PepJ) and other hydrolytic enzymes or XprG could act indirectly through BrlA, which is involved in the induction of autolysis54.\n\nThe xprG1 gain-of-function mutant had previously been shown to have the reverse phenotype to xprG- mutants with respect to extracellular protease and pigment production7. Here we show that the xprG1 mutation leads to accelerated autolysis and increased penicillin production, whereas autolysis and penicillin production is reduced or absent in an xprG- mutant. Likewise, conidiation is increased in an xprG1 veA+ strain but decreased in an xprG-veA+ strain. In contrast, glucose uptake and sterigmatocystin levels were reduced in both the xprG1 and xprG- mutants. The reason for this difference in phenotypic effect is not known. The xprG1 allele contains a missense mutation (R186W) in the putative DNA-binding domain of XprG7. It may be that this amino acid substitution increases the affinity of the XprG1 for some binding sites but decreases the affinity for others. Missense mutations with this type of gene specificity effect have been documented in the DNA-binding domain of AreA, the A. nidulans regulator of genes involved in nitrogen metabolism65.\n\nWe have shown that the two genes encoding Ndt80-like proteins in A. nidulans perform different functions. Among fungi, there is considerable variation in the number of genes in the NDT80 family (Table 5). Most basidiomycetes and the unicellular ascomycete Schizosaccharomyces pombe do not possess any genes encoding Ndt80/PhoG-like proteins. In contrast, the zygomycetes have large numbers of these genes. The number of NDT80-like genes varies within genera (e.g. Aspergillus) and even within the same species (e.g. Candida albicans). As most ascomycetes have a gene similar to NDT80 and one or more genes similar to xprG (data sourced from the Fungal Genome Initiative), it seems likely that the unicellular S. cerevisiae has lost the xprG-like gene.\n\nThe p53-like transcription factor superfamily (http://supfam.org/) is comprised of seven families containing the following DNA-binding domains: p53, Rel/Dorsal, T-box, STAT, Runt, Ndt80, and the LAG-1/CSL. Many of the proteins in this superfamily, including MRF (myelin gene regulatory factor), a mammalian member of the Ndt80 family, are involved in development. The Ndt80 and LAG-1 families include both animal and fungal proteins and the Ndt80 family is also found in the slime molds Dictyostelium discoideum and Dictyostelium purpureum. The Ndt80 family is present in all ascomycete fungi, with the exception of the Schizosaccharomyces species, but is absent from most of the basidiomycete fungi that have been sequenced to date. In contrast, LAG-1 family members are found in all basidiomycetes but are lacking in all ascomycetes except Schizosaccharomyces species.\n\nWe have previously proposed that the common feature of fungal p53-like proteins is a role in nutrient sensing, and this may be the original role for this group of transcriptional activators7. It has recently been shown that Ndt80 is involved in resetting lifespan during meiosis and transient expression of NDT80 extends the lifespan of aging yeast cells11. Pathways responsible for the response to nutrient status appear to play an important role in controlling lifespan66. We speculate that the ability of Ndt80 to sense nutrient status could be crucial in determining lifespan.", "appendix": "Author contributions\n\n\n\nMK conceived the study, MK, KB, and HN designed the experiments, MK, KB, GY, and SC carried out the experiments, CG analysed the microarray data, MK, HN and CG contributed to the preparation of the manuscript. All authors, except GY, were involved in the revision of the draft manuscript and have agreed to the final content. In spite of repeated attempts, MK has not been able to contact GY in China, but does not wish to omit him from the manuscript as he carried out important experimental work when he was visiting MK’s laboratory.\n\n\nCompeting interests\n\n\n\nNo competing interest have been disclosed.\n\n\nGrant information\n\nK Braunberger was supported by an Australian Postgraduate Award scholarship.\n\n\nAcknowledgments\n\nWe gratefully acknowledge the NIAID-sponsored Pathogen Functional Genomics Resource Center (PFGRC) for provision of A. nidulans microarrays, the Fungal Genetics Stock Center (Kansas City, Missouri USA) and M J Hynes for provision of A. nidulans strains, and the following individuals for their help: N Keller and W B Yin (sterigmatocystin assays), A MacCabe (glucose uptake assays), K Quinn (microarrays), J Clay (photography), S. Walkden-Brown and S Burgess (qRT-PCR).\n\n\nSupplementary figures\n\nThe template was total RNA extracted from an xprG+ (+) and xprGΔ1 (-) strains transferred to glucose (+) or carbon-free medium (-) for 16 h. A 100 bp ladder (Axygen) was used as a standard in the first and last lanes of the 2% agarose gel. The full genotypes of the xprG+ strain (MH2) and the xprGΔ1 strain (MK422) are given in Table 1.\n\nSamples of two xprG+ strains (lane 1 MH2, Lane 2 MH97), two xprG1 strains (lane 3 MK85, lane 4 MK86) and four xprG- strains (lane 5 MK198, lane 6 MK413, lane 7 MK414, lane 8 MK422) was analyzed using thin layer chromatography with a benzene: glacial acetic acid (95:5 vol/vol) as described in the experimental procedures. A sterigmatocystin (ST) standard (Sigma) was applied as standard).\n\n\nReferences\n\nBernardo SM, Gray KA, Todd RB, et al.: Characterization of regulatory non-catalytic hexokinases in Aspergillus nidulans. Mol Genet Genomics. 2007; 277(5): 519–532. 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PubMed Abstract \| Publisher Full Text\n\nEmri T, Molnar Z, Pocsi I: The appearances of autolytic and apoptotic markers are concomitant but differently regulated in carbon-starving Aspergillus nidulans cultures. FEMS Microbiol Lett. 2005; 251(2): 297–303. PubMed Abstract \| Publisher Full Text\n\nYamazaki H, Yamazaki D, Takaya N, et al.: A chitinase gene, chiB, involved in the autolytic process of Aspergillus nidulans. Curr Genet. 2007; 51(2): 89–98. PubMed Abstract \| Publisher Full Text\n\nShin KS, Kwon NJ, Kim YH, et al.: Differential roles of the ChiB chitinase in autolysis and cell death of Aspergillus nidulans. Eukaryot Cell. 2009; 8(5): 738–746. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmaler-Ripcke J, Sugareva V, Gebhardt P, et al.: Production of pyomelanin, a second type of melanin, via the tyrosine degradation pathway in Aspergillus fumigatus. Appl Environ Microbiol. 2009; 75(2): 493–503. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEmri T, Molnar Z, Pusztahelyi T, et al.: The fluG-BrlA pathway contributes to the initialisation of autolysis in submerged Aspergillus nidulans cultures. Mycol Res. 2005; 109(Pt 7): 757–763. PubMed Abstract \| Publisher Full Text\n\nBayram O, Braus GH: Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins. FEMS Microbiol Rev. 2012; 36(1): 1–24. PubMed Abstract \| Publisher Full Text\n\nKato N, Brooks W, Calvo AM: The expression of sterigmatocystin and penicillin genes in Aspergillus nidulans is controlled by veA, a gene required for sexual development. Eukaryot Cell. 2003; 2(6): 1178–1186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBok JW, Keller NP: LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryot Cell. 2004; 3(2): 527–535. 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PubMed Abstract \| Publisher Full Text\n\nBayram O, Sari F, Braus GH, et al.: The protein kinase ImeB is required for light-mediated inhibition of sexual development and for mycotoxin production in Aspergillus nidulans. Mol Microbiol. 2009; 71(5): 1278–1295. PubMed Abstract \| Publisher Full Text\n\nSopko R, Raithatha S, Stuart D: Phosphorylation and maximal activity of Saccharomyces cerevisiae meiosis-specific transcription factor Ndt80 is dependent on Ime2. Mol Cell Biol. 2002; 22(20): 7024–7040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPocsi I, Leiter E, Kwon NJ, et al.: Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production. J Appl Microbiol. 2009; 107(2): 514–523. PubMed Abstract \| Publisher Full Text\n\nKudla B, Caddick MX, Langdon T, et al.: The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J. 1990; 9(5): 1355–1364. PubMed Abstract \| Free Full Text\n\nTóth ML, Sigmond T, Borsos E, et al.: Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans. Autophagy. 2008; 4(3): 330–338. PubMed Abstract\n\nLee BN, Adams TH: The Aspergillus nidulans fluG gene is required for production of an extracellular developmental signal and is related to prokaryotic glutamine synthetase I. Genes Dev. 1994; 8(6): 641–651. PubMed Abstract \| Publisher Full Text\n\nButnick NZ, Yager LN, Hermann TE, et al.: Mutants of Aspergillus nidulans blocked at an early stage of sporulation secrete an unusual metabolite. J Bacteriol. 1984; 160(2): 533–540. PubMed Abstract \| Free Full Text" }	[ { "id": "860", "date": "25 Mar 2013", "name": "Amir Sharon", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paper. The work presented in this paper is well planned, experiments are well designed and executed, and the analyses are comprehensive and provide clear answers to the main questions. Particularly, the interpretation of the micro array data, analysis of differentially expressed genes, and reference to most significantly changed gene/gene clusters between the wild type and mutant are excellent. The conclusions are all well supported by the data and interpreted in a conserved manner. The results are novel and interesting. All experiments are detailed and clear.", "responses": [] }, { "id": "881", "date": "08 Apr 2013", "name": "Michael Hynes", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCarbon starvation is likely to be a common stress that fungi encounter in the environment. This group has previously identified the xprG gene, which contains a p53 like Ndt80 DNA binding domain, as being involved in the response to starvation. Here they have studied by microarrays the effects of xprG on the response to 16 hours of carbon starvation. They have verified some of the responses by qRT-PCR as well as physiological studies. Effects on glucose uptake, conidial and hyphal pigmentation, secondary metabolite production and autolysis were verified and are consistent with previous studies. The effects of an xprG gain of function mutation support the results. This work therefore provides strong support for XprG playing an important role in the response to starvation – a novel and significant result which adds to the large body of data relating to genes involved in development and secondary metabolite production in A. nidulans.A further significant result is the finding that a second Ndt80 domain containing gene, AN6015- designated ndtA, when deleted results in loss of sexual development. This may be related to the known role of Ndt80 in meiosis in Saccharomyces cerevisiae. Interestingly an Ndt80 homolog has been found to be involved in biofilm formation in Candida albicans (Cell 148, 126–138).", "responses": [] } ]	1	https://f1000research.com/articles/2-72
https://f1000research.com/articles/2-69/v1	04 Mar 13	{ "type": "Research Article", "title": "Factors associated with the use of probiotics in patients with inflammatory bowel disease", "authors": [ "Claire Louise Agathou", "Ian LP Beales", "Claire Louise Agathou" ], "abstract": "Background: Probiotic preparations are heavily promoted in the United Kingdom and are widely available to purchase. Probiotics have multiple effects on gastrointestinal functions and may have beneficial or even harmful effects in inflammatory bowel disease (IBD). Various complementary and alternative medicines are commonly used by IBD patients but there is much less data specifically on the use of probiotics. Aim: To examine the current use of probiotics by IBD patients and determine the factors associated with probiotic use.Methods: Subjects with IBD undergoing routine care at a UK teaching hospital underwent a standardized structured questionnaire-interview. Current use of probiotics was explored and patient- and disease-related factors examined. IBD-related quality of life was assessed with the short inflammatory bowel disease questionnaire (S-IBDQ). Logistical regression was used to explore factors associated with probiotic use.Results: Forty subjects were interviewed. Probiotic use was common, 40% of subjects being regular users. Probiotic use was significantly associated with a shorter duration of IBD since diagnosis, a diagnosis of Crohn’s disease, formal post-18 education and lower quality of life as assessed by the S-IBDQ. A preference for the taste of the preparation was as common a reason for using probiotics as were potential disease modifying effects. Non-users reported that the costs of the preparations and doubts about efficacy were the primary reasons for non-use.Conclusions: In this study probiotic use was common in IBD patients. Several patient- and disease- related factors, including a lower perceived quality of life, were associated with the use of probiotics.", "keywords": [ "The use of probiotics within the field of Gastroenterology is an area of significant current interest. The gut is home to millions of microorganisms and collectively this is often referred to as the “gut microbiome”1. This refers to coexistence of beneficial and pathological microorganisms within the gut flora", "which under usual physiological “healthy” states are considered normal. Clostridium difficile (C. difficile) demonstrates this balance – many normal individuals carry this organism within their large bowel and yet exhibit no associated symptoms", "however", "when the more beneficial organisms are reduced", "for example by use of antibiotics", "C. difficile dominates the GI tract resulting in an acute diarrhoeal-type disease2. The prospect that we may be able to improve the natural history of bowel diseases through manipulation of the normal gut homeostasis between various health-promoting and health-endangering microorganisms is the basis of potential for the role of probiotics in health and disease3–5. Probiotics are defined as live microbial supplements which exert a beneficial effect on health and are non-pathogenic or toxic6." ], "content": "Introduction\n\nThe use of probiotics within the field of Gastroenterology is an area of significant current interest. The gut is home to millions of microorganisms and collectively this is often referred to as the “gut microbiome”1. This refers to coexistence of beneficial and pathological microorganisms within the gut flora, which under usual physiological “healthy” states are considered normal. Clostridium difficile (C. difficile) demonstrates this balance – many normal individuals carry this organism within their large bowel and yet exhibit no associated symptoms; however, when the more beneficial organisms are reduced, for example by use of antibiotics, C. difficile dominates the GI tract resulting in an acute diarrhoeal-type disease2. The prospect that we may be able to improve the natural history of bowel diseases through manipulation of the normal gut homeostasis between various health-promoting and health-endangering microorganisms is the basis of potential for the role of probiotics in health and disease3–5. Probiotics are defined as live microbial supplements which exert a beneficial effect on health and are non-pathogenic or toxic6.\n\nRecent evidence suggests that the presence of different bacterial species in the colon can have a significant impact on the immune functioning of the gastrointestinal tract and that manipulation of normal gut microbiome homeostasis can alter local immunity within the luminal gut as well as systemically3,4,7,8. Idiopathic chronic inflammatory bowel diseases (IBD) such as Crohn’s disease and ulcerative colitis are characterised by persistent or episodic inflammation of the gastrointestinal mucosa and it appears that these patients are mounting overwhelming immune responses to non-pathogenic gut bacteria which would otherwise be ignored in the gut of a healthy host9. The gut microbiome differs significantly between healthy controls and IBD patients, particularly within the inflammatory colonic lesions, which are found to contain greater numbers of unfavourable bacteria7,9. Luminal colonic flora and the immunological response of the gut play a major role in initiation and perpetuation of chronic IBD7,8,10, although whether these alterations in gut microbiome are primary causes of the diseases or secondary phenomena resulting from the disease currently remains undecided.\n\nProbiotics are non-pathogenic, live microbial supplements which when taken on a regular basis, claim to offer an immune advantage by increasing the balance of health-promoting bacteria such as lactobacilli. Prolonged exposure of supplemental lactobacilli induces their translocation and adherence to human intestinal epithelial cells which are capable of activating macrophages9. Enhancing the immunomodulatory effect of intestinal flora to inflammation by such means is thus of great current interest for patients with IBDs yet studies supporting this theory are limited.\n\nThe most commonly used bacterial micro-organisms are bifidobacterium and lactobacillus and marketing strategies promote these heavily on the basis that they will improve health and be of benefit to the gastrointestinal tract. For the past decade, probiotics have been marketed as food supplements, most commonly in the form of drinks or tablets, widely available without prescription from supermarkets or from internet sites. Thus, there is uncertainty about the prevalence of use within the general population and in particular amongst patients who suffer with inflammatory bowel conditions. We have hypothesized that these preparations would be especially attractive to patients with chronic IBDs: these conditions may require continued therapy with powerful immunosuppressive drugs and the concept of restoring the balance of bacteria in the gut with a food supplement is likely to be attractive to many people. However we do not know how many people with IBD are using these preparations. Although widely regarded as completely safe and natural, it is even possible that probiotics could be harmful: the powerful immunosuppressive drugs taken by many patients with IBD or the underlying disease could alter the response to these otherwise harmless bacteria, and rare cases of invasive systemic disease have been reported with probiotics6. It is now clear that many foodstuffs and dietary supplements can interact with prescription drugs and either increase or decrease the effect of these drugs10,11; other theoretical problems with probiotics in IBD include the possible transmission of bacterial antibiotic resistance from non-pathogenic to pathogenic bacteria and the generation of as yet unreported negative effects upon the gastrointestinal immune system6. Also probiotics have been shown to accelerate gastrointestinal transit and could induce diarrhoea or a change in stool frequency in an IBD patient that might otherwise be assumed to be a flare of active disease10,12.\n\nDespite the potential benefits or adverse effects of probiotics in IBD as outlined above, we do not currently know how prevalent the use of these supplements are. Neither do we know why certain patients may be taking them. Therefore it is important to determine how commonly these supplements are used in IBD and how interested IBD patients are in taking them. Furthermore, determining which are the main drivers of probiotic usage may shed light on issues with the current delivery of care. Probiotic uses may reflect a dissatisfaction with current therapies or demonstrate a positive interest in more natural therapies; thus we may be able to identify areas of need within our service and hence provide additional resources and support for patients. Despite the widespread availability of probiotics, there are relatively few studies examining the use of these preparations in the IBD population13–17, and in particular there is a paucity of information related to patients in the United Kingdom.\n\nWe wherefore aimed to determine the prevalence of probiotic use amongst patients with IBD and assess which disease-related and demographic factors are influencing their consumption. As probiotics are relatively new “therapies”, often classified as complementary alternative medication (CAM), it is important to determine variables that may influence their usage as these may create bias in later studies seeking to quantify their overall effects.\n\n\nMethods and materials\n\nStudy subjects were selected from IBD outpatient clinics and inpatients under the care of the Adult Gastroenterology Department of the Norfolk and Norwich University Hospital. Consecutive adult (> 18 years old) patients with a confirmed diagnosis of Crohn’s disease or ulcerative colitis, from sessions when the student researcher was available, were eligible for recruitment.\n\nPatients requiring enteral nutrition were excluded as oral intake was dictated by their prescription and not personal choice. Patients with indeterminate colitis were excluded to aid classification of the results. Subjects unable to complete the interview questionnaire in English were excluded. All participants gave informed consent and the study was approved by the Norfolk and Norwich University Hospital research governance committee and Cambridge 3 Research Ethics committee.\n\nAll subjects underwent a structured interview and completion of a structured questionnaire, (see Appendix 1) administered by the same trained student-researcher (CLA). Questions were separated into sociodemographic and disease-related variables including an assessment of the patient’s health-related quality of life by incorporating the S-IBDQ (Short Inflammatory Bowel Disease Questionnaire), a validated screening tool for patients with IBD13.\n\nThe study was designed as an exploratory study aiming to test the feasibility and acceptability of the questionnaire and methodology in the IBD population and provide estimates on the prevalence of probiotic use in the IBD population, which could be possibly used to inform the design of a subsequent larger study. For this study probiotic use was defined as regularly using at least one single probiotic preparation per week for at least one month. For the purposes of analysis, age was split into 2 groups, younger (18–55) and older (56+). SPSS 19.0 was used for statistical analysis: Fisher’s Exact Test was used to examine categorical data on age, duration of diagnosis and education level and Mann-Whitney U-test was used to examine the results of the S-IBDQ. Unconditional logistic regression was used to calculate odds ratios (OR) with 95% confidence intervals (CI) for the use of probiotics adjusted for age, gender, IBD type, duration of diagnosis and educational status.\n\n\nResults\n\n40 patients were recruited between October 2010 and February 2011, 8 inpatients and 32 outpatients. Subjects were evenly split between Crohn’s Disease (20) and ulcerative colitis (20), and the overall age range was 18–78 (median age range 51–55, mode age range 61–65). Thirteen subjects were male and 27 female. Sixteen patients (40%) were considered to be regular users of probiotics (that is, using probiotics at least once per week). Crude data are appended in Appendix 2.\n\nProbiotic use was much commoner in subjects with Crohn’s disease (13/20) than in those with ulcerative colitis (3/20) (odds ratio (OR) 9.8, 95% confidence interval 2.20 – 54.9) (p < 0.01).\n\nThe results describing probiotic use with different sociodemographic variables are shown in Table 1. Men used probiotics less commonly than women, but this was not statistically significant. Similarly, probiotic use was non-significantly commoner in the younger age group. However, probiotic use was significantly more common in those subjects who had continued in formal education after the age of 18 (OR 8.15, 95% CI 1.8 – 44.9) (p < 0.01).\n\nDisease-related factors are shown in Table 2. Within our sample there appeared to be a different distribution of disease duration (time since the diagnosis of IBD) between probiotic users and non-users (Figure 1). There was a greater variability in disease duration since diagnosis in users than in non-users. When probiotic use was examined by duration of disease, probiotic use was commoner in those with a shorter duration of disease (< 36 months) (OR 9.83, 95% CI 1.19 – 258.3) (p < 0.05).\n\nLogistic regression was used to adjust odds ratios for age, sex, duration of disease and educational attainment. The results are shown in Table 3. After adjustment, probiotic use was found to be significantly associated with Crohn’s disease compared to ulcerative colitis, shorter duration of disease and higher educational attainment.\n\nData from the S-IBDQ were used to analyze the relationship between IBD symptoms and probiotic use. As shown in Table 4, probiotic use was significantly associated with overall poorer perceived health-related quality of life. However on comparison of each individual domain, there appears to be no significant difference in S-IBDQ scores in both systemic and bowel-specific outcomes. Non-users scored more favourably in both emotional and social domains and these were both found to show statistical significance. Within our population, scores from the S-IBDQ did not correlate with other sociodemographic or disease related factors.\n\n(The lower the score, the poorer the health related QOL of the patient)\n\nWe also explored the reasons subjects gave for using or non-using probiotics, and the results are shown in Table 5. Although the numbers in each group were small, there were some potentially different reasons that seemed to drive probiotic use. In the non-users, the relatively high costs and a perception that the probiotics would not significantly improve symptoms were common reasons given; interestingly a substantial minority group (8 subjects) said they avoided probiotics because of perceived lactose intolerance and the possibility that milk-containing products would make the symptoms worse. Half of probiotic users gave the taste of probiotic preparations as their primary reason for using them; the other half suggested that they used probiotics primarily to improve their IBD symptoms in some way.\n\n\nDiscussion\n\nAlthough our study is relatively small, we have found that probiotic use in the IBD population is associated with Crohn’s disease rather than ulcerative colitis, shorter disease duration since diagnosis, higher educational attainment and a poorer perceived quality of life. All probiotics were purchased, none were obtained on prescription. The majority of probiotic use was in the form of yoghurts or drinks, although one patient with ulcerative colitis regularly used the specific probiotic preparation VSL #3 (a concentrated probiotic food supplement preparation, Ferring, West Drayton, UK), which he purchased via the internet. Interestingly probiotic use was common in the IBD population, despite the lack of evidence supporting the use of these drinks and yoghurts; this probably reflects popular interest in healthy lifestyles and non-drug therapies. Proprietary probiotic drinks have had significant marketing through television advertisements and many users said they were inclined to purchase brands which were being actively promoted. Interestingly, over one third of non-users stated that their primary reason for not using probiotics in their diet was on account of cost, thus indicating an important reason which may deter patients from purchasing them, particularly as their effects are considered beneficial only with long-term, continuous use.\n\nWithin our total population, level of education appeared to have a strongly significant association with probiotic use: over 80% of the participants who claimed to use probiotics had also continued to higher education. This again may be related to the cost of probiotics which is deflecting participants on lower salaries (higher education being one determinant of earning power). Increased probiotic use in those continuing with post-18 education may also reflect individual awareness or social awareness about probiotics.\n\nThe commonest reason provided by users regarding their primary choice to consume probiotics was that they liked the taste. This was perhaps slightly surprising, as we may have expected disease-related issues to be the primary drivers. In light of this it is interesting that none of the non-users gave taste as a primary reason for not using probiotics. Within both groups, reasons governing the decision to either use or not use probiotics were taste preference (users) or high cost (non-users), respectively – this reflects that probiotics may be considered more as a luxury/food item, rather than a medical therapy. In the current study we did not explore experiences with previous use of probiotics.\n\nSubjects with Crohn’s disease were more commonly users of probiotics than those with ulcerative colitis and this did not seem to be related to any specific factors and was not obviously related to perceived quality of life. At present, the numbers in the study are too small to provide any further data on previous surgeries or drug exposures as potential drivers to the use of probiotics. Within the free text of the questionnaire, of the non-users, 7 ulcerative colitis patients commented that they avoided the use of probiotics as they considered them to be similar to milk and, as they avoid all dairy produce for symptom prevention, this ruled out any desire or option to use them. Only one patient with Crohn’s disease stated that she avoided probiotics for similar reasons. If probiotic supplementation does prove in future to offer a health benefit, the promotion and availability of a preparation acceptable for those who avoid dairy-like products should be considered.\n\nProbiotic use was commoner in subjects relatively early in the course of their disease, but this was independent of the age of the patient. It will be interesting to explore if this association is present in larger cohorts: one explanation may be that newly diagnosed patients are keen to explore many avenues to help their disease, yet those with established disease may have adjusted to their disease and have no desire for, nor awareness of, other potential therapies.\n\nPerhaps not surprisingly, probiotic use was associated with a perceived lower quality of life, suggesting that those most disaffected with current disease management are more likely to look elsewhere for alternatives. Interestingly probiotic use was associated with lower scores on the domains measuring the emotional and social domains assessing quality of life relative to the psychosocial impact of the disease but not those measuring the physical effects of the disease. Therefore, this may indicate that those who are more affected by the disease in an emotional or social respect are more likely to seek further means to control or improve their disease. Thus specifically asking about probiotic use may be a useful surrogate marker for detecting those IBD patients with the most dissatisfaction or difficulty with their current situation and may allow more tailored individual interventions.\n\nDespite the ready availability and possible advantages of using probiotics in IBD, there is relatively little data concerning the use of probiotics in this patient group. Several studies have included an examination of probiotic use within the wider sphere of “complementary and alternative therapies”, which obviously has a much wider reach than just probiotics, and certainly it can be hypothesized that probiotics may have a more specific and targeted effect on gastrointestinal health compared with more general well-being than may be seen with other CAMs. Given these possible more direct benefits of probiotics compared to other CAMs, the specific factors associated with probiotic use remain to be determined. One study in IBD outpatients from a Canadian teaching centre showed that overall 56% of patients were using some form of CAM but only about half of this use was probiotics13. Similar to our study, higher educational achievement was associated with CAM use13. Again, problems with, and dissatisfaction with, current medical therapies seemed to be an important driver in CAM use: although in that study probiotic use was higher in those apparently with more active disease (assessed by the Harvey-Bradshaw index), there was no difference in S-IBDQ scores between CAM users and non-users13. A postal study of Canadian IBD patients14 again confirmed that CAM use was common (47% had ever used, with 23% current users). Probiotics, in the form of Acidophilus species, were commonly used (19% of patients being current users) but overall herbal and plant therapies were more common (41%) and massage therapies (18%) were almost as common14. Interestingly between the Canadian provinces there were regional variations, with probiotic use being more common in all other provinces (20–25% of patients) compared with Quebec (8%). A further internet-based cross sectional survey of IBD patients (mainly from North America) demonstrated that 34% of patients were current users of at least one type of CAM with vitamins and herbal products the most popular15. The only factors that seemed associated with CAM use in this study were not having had previous IBD-related surgery or not having received steroids16; data for probiotics were not reported separately. Further studies from Germany16 and Hungary17 have again confirmed that CAM use is common in the IBD population but also illustrate that different products predominate in different areas. Probiotics were less commonly used in these populations, despite probiotics being prescribable for IBD in Germany. In Germany homeopathy was most common16, whilst in Hungary herbal tea and homeopathy were predominant, with minimal use of probiotics17. These studies suggest that many other factors are important in determining the choice of any specific CAM and that the factors which relate specifically to probiotic use in different geographical and sociodemographic groups use remain to be determined.\n\nIn conclusion, despite the relatively small size of our sample, we have shown that the use of probiotics is common in a UK IBD cohort. Use of probiotics was associated with Crohn’s disease more than ulcerative colitis, relatively short duration of disease since orginal onset, lower perceived quality of life and higher educational attainment. The taste of the probiotic supplements was equally as popular as potential disease-modifying effects as a reason for using probiotics. Non-users were influenced by the costs involved, perceived lack of benefit and a concern that diary-based products might make symptoms worse. Further studies are warranted to determine the different patient- and disease-related factors that influence the use of probiotics and also to determine any positive or negative effects of probiotic use on the behaviour of IBD, complications, concordance with prescribed medication and patient well-being.", "appendix": "Author contributions\n\n\n\nCLA and ILBP jointly conceived and designed the study. CLA performed the interviews and data collection. CLA and ILPB analysed the data. CLA wrote the initial draft of the paper. ILPB wrote the final draft and is the guarantor of the paper. Both authors have seen the raw data and agree with publication.\n\n\nCompeting interests\n\n\n\nCLA has no competing interests. ILPB has received has received funding for travel, conferences and educational activities from Ferring Pharmaceuticals, who hold the licence for the distribution of VSL#3 in Europe.\n\n\nGrant information\n\nFinancial support for the study was provided by the Norfolk and Norwich University Hospital Inflammatory bowel disease research fund (NNUH Fund E33).\n\n\nAcknowledgments\n\nWe would like to thank Dr Sabeena Pheerunggee and all of the Gastroenterology team at NNUH and for the excellent co-operation and organisation from all the nurses and auxiliary staff working at the weekly Inflammatory Bowel Disease outpatient clinics on Wednesdays and Thursdays, and Professor Lee Shepstone for statistical support. The study formed part of CLA’s MBBS degree.\n\n\nReferences\n\nChild M, Macfarlane G: The Human Ecosystem. Student BMJ. 2008; 16: 450–451. Publisher Full Text\n\nLeffler DA, Lamont JT: Treatment of Clostridium difficile associated disease. Gastroenterology. 2009; 136(6): 1899–912. PubMed Abstract \| Publisher Full Text\n\nKim HJ, Vazquez Roque MI, Camilleri M, et al.: A randomized controlled trial of a probiotic combination VSL#3 and placebo in irritable bowel syndrome with bloating. Neurogastroenterol Motil. 2005; 17(5): 687–96. PubMed Abstract \| Publisher Full Text\n\nTung JM, Dolovitch LR, Lee CH: Prevention of Clostridium difficile diarrhoea infection with Saccharomyces bouladii: a systematic review. Can J Gasttoenterol. 2009; 23(12): 817–21. PubMed Abstract \| Free Full Text\n\nSummers RW, Elliot DE, Urban JF Jr, et al.: Trichuris suis therapy for active ulcerative colitis: a randomized controlled trial. Gastroenterology. 2005; 128(4): 825–32. PubMed Abstract \| Publisher Full Text\n\nSnydman DR: The safety of probiotics. Clin Infect Dis. 2008; 46(Suppl 2): S104–11. PubMed Abstract \| Publisher Full Text\n\nTargan SR, Shannahan F, Karp LC: Inflammatory Bowel Disease: From Bench to Bedside. Mechanisms of systemic inflammation associated with intestinal injury. Springer US 2nd edition, 2005; 305–335. Publisher Full Text\n\nGionchetti P, Rizzello F, Tambasco R, et al.: Which therapies are advisable in pouchitis? Inflamm Bowel Dis. 2008; 14(Suppl 2): S241–2. PubMed Abstract \| Publisher Full Text\n\nIweala OI, Nagler CR: Immune privilege in the gut: the establishment and maintenance of non-responsiveness to dietary antigens and commensal flora. Immunol Rev. 2006; 213(1): 82–100. PubMed Abstract \| Publisher Full Text\n\nKoretz RL, Rotblatt M: Complementary and alternative medicine in gastroenterology: the good, the bad, and the ugly. Clin Gastroenterol Hepatol. 2004; 2(11): 957–67. PubMed Abstract \| Publisher Full Text\n\nFujita K: Food-drug interactions via human cytochrome P450 3A (CYP3A). Drug Metabol Drug Interact. 2004; 20(4): 195–217. PubMed Abstract \| Publisher Full Text\n\nKatan MB: The probiotic yoghurt Activia shortens intestinal transit, but has not been shown to promote defecation. Ned Tijdschr Geneeskd. 2008; 152(13): 727–30. PubMed Abstract\n\nWeizman AV, Ahn E, Thanabalan R, et al.: Characteristics of complementary and alternative medicine use and its impact on medication adherence in inflammatory bowel disease. Aliment Pharmacol Ther. 2012; 35(3): 342–9. PubMed Abstract \| Publisher Full Text\n\nHilsden RJ, Verhoef MJ, Best A, et al.: Complementary and alternative medicine use by Canadian patients with inflammatory bowel disease: results from a national survey. Am J Gastroenterol. 2003; 98(7): 1563–8. PubMed Abstract \| Publisher Full Text\n\nHilsden RJ, Meddings JB, Verhoef MJ: Complementary and alternative medicine use by patients with inflammatory bowel disease: An internet survey. Can J Gastroenterol. 1999; 13(4): 327–32. PubMed Abstract\n\nJoos S, Rosemann T, Szecsenyi J, et al.: Use of complementary and alternative medicine in Germany-a survey of patients with inflammatory bowel disease. BMC Complement Altern Med. 2006; 6: 19–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLakatos PL, Czegledi Z, David G, et al.: Association of adherence to therapy and complementary and alternative medicine use with demographic factors and disease phenotype inpatients with inflammatory bowel disease. J Crohns Colitis. 2010; 4(3): 283–90. PubMed Abstract \| Publisher Full Text\n\nJowett SL, Seal CJ, Barton JR, et al.: The short inflammatory bowel disease questionnaire is reliable and responsive to clinically important change in ulcerative colitis. Am J Gastroenterol. 2001; 96(10): 2921–8. PubMed Abstract \| Publisher Full Text\n\n\n\n\n" }	[ { "id": "865", "date": "26 Mar 2013", "name": "Richard Pollok", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 small study but well designed and well written. It might have been interesting to learn what type of probiotics were being used, this could be submitted as an appendix. The conclusion is reasonable, it is an interesting observation that these products seem to be popular early in diagnosis but interest then drops off.", "responses": [] }, { "id": "887", "date": "09 May 2013", "name": "Sara McCartney", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is excellently written, clear and interesting. It addresses some of the issues of widely used but often undocumented and poorly evaluated therapies readily available to patients with IBD. The design and methods are clearly laid out and the discussion was relevant and appropriate.My reservations relate to: Although this is an observational study some comparator data in an age-matched group without IBD would be helpful. The numbers are small and the age distribution of the study group is older than one might expect with IBD (mode age range 61-65). This may limit generalisation to the wider IBD population. Further information as to which probiotic preparations were taken would be helpful.Overall, however, I feel this should be approved, particularly as a pilot to a larger study when the issues raised above could be fully addressed.", "responses": [] }, { "id": "886", "date": "09 May 2013", "name": "Stephen Lewis", "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 enjoyed reading this small study but it is clearly underpowered and has no control group making it difficult to draw meaningful conclusions.AbstractBackground: The second sentence implies that probiotics have an effect on gastrointestinal function, which contradicts some of the later statements saying that their effects are unsubstantiated. I would tend to favour focusing on the study and not entering into any discussion on the effectiveness of probiotics.Introduction:This is far too long and could easily be cut in half. Particularly the first paragraph is repeated elsewhere in the introduction and is largely irrelevant to the study. The grammar generally could be more precise. Materials and methods:I have significant reservations on the over interpretation of statistics in such a small sample size. In particular where you are drawing conclusions using Fisher exact tests on a sample of three patients some groups, this leaves the results open to speculation even if statistically significant. Results:When stating a result is statistically significant it is appropriate just to say that the findings are significant, 'statistically' becomes a redundant word. If something is not statistically significant then it is not significant. So to state 'men use probiotics less commonly than women but this was not statistically significant' is incorrect, statistically there is no difference. This is repeated throughout the paper and should be corrected.What would be really interesting is if there was a control group of patients who did not have IBD to see whether their probiotic consumption was similar to those with IBD and whether the same confounding factors occurred. At least some comment should be made to this. Given that taste preference was one of the main reasons for using probiotics, this factor should be explored in more detail. In particular some examples of costing would be appropriate. How much more expensive are probiotic yoghurts than normal yoghurts? Would patients have taken the probiotics even if they did not have IBD?I would also like to see some data on length of probiotic use. Presumably if it were used for a while with no benefit then patients would stop taking it. The difference between ulcerative colitis and Crohn’s is interesting and I just wonder if this represents differences in quality of life.Was there any perception that probiotics actually reduced or improved patient’s quality of life or reduce the symptoms?Discussion:Given that taste preference was one of the major findings this perhaps should be more dominant in a discussion. I guess it also would be linked to marketing side, which is quite powerful. Overall there is a bit of repetition and the text is a bit verbose, it could be edited down a little.", "responses": [] } ]	1	https://f1000research.com/articles/2-69
https://f1000research.com/articles/2-68/v1	04 Mar 13	{ "type": "Software Tool Article", "title": "The Focal Adhesion Analysis Server: a web tool for analyzing focal adhesion dynamics", "authors": [ "Matthew E Berginski", "Shawn M Gomez", "Matthew E Berginski" ], "abstract": "The Focal Adhesion Analysis Server (FAAS) is a web-based implementation of a set of computer vision algorithms designed to quantify the behavior of focal adhesions in cells imaged in 2D cultures. The input consists of one or more images of a labeled focal adhesion protein. The outputs of the system include a range of static and dynamic measurements for the adhesions present in each image as well as how these properties change over time. The user is able to adjust several parameters important for proper focal adhesion identification. This system provides a straightforward tool for the global, unbiased assessment of focal adhesion behavior common in optical microscopy studies. The webserver is available at: http://faas.bme.unc.edu/.", "keywords": [ "Focal Adhesion Analysis Server", "web too", "focal adhesion", "dynamics" ], "content": "Introduction\n\nThe quantitative analysis of focal adhesion (FA) structures in motile cells commonly relies on the use of fluorescently tagged protein components and time-lapse fluorescence microscopy. Traditionally, the resulting images are analyzed using NIH ImageJ1 or related tools, but we have recently developed a set of computer-vision algorithms designed to automate many of these analysis steps. These core methods have been documented in a prior publication2 and made available as an open source download; however, they require substantial expertise with the command line interface for their use. With the focal adhesion analysis server (FAAS), we have created a web application that allows users to submit time-lapse fluorescence image sets of FA proteins and have these images automatically analyzed.\n\nThe methods implemented by the analysis system have been previously used in several studies to investigate the quantitative properties of FAs in cells under various conditions. For example, adhesion static and dynamic properties were quantified with fluorescently labeled FAK, vinculin and paxillin3–5. Global, whole-cell changes to adhesion and cytoskeletal architecture when the Arp 2/3 complex is disabled have also been characterized4. By integrating these image analysis methods into a straightforward web application, we hope to make them more broadly accessible to the cell-imaging community.\n\n\nFeatures\n\nThe primary interface is a set of webpages that allow a user to upload a stacked tiff set of images for processing. After the images are uploaded to the server, the processing pipeline is run, and the results are returned as a downloadable zip file. This results file contains all the intermediate processing steps as well as a set of visualizations. These visualizations show which regions of the cell were identified as adhesions and how the tracking algorithm followed single adhesions through time (Figure 1).\n\nThe results section shows examples from the visualizations produced by the pipeline. In the top example, the entire cell is shown, with an individual adhesion outlined and tracked through time. The bottom three examples show single adhesions, outlined in green, with other nearby adhesions outlined in blue.\n\nThe analysis pipeline extracts and quantifies a wide range of properties. FA properties characterized in each individual image include adhesion area, marker protein intensity and the lengths of the major and minor axes. In addition to these static properties, the system also collects dynamic adhesion properties, which are quantified by recording the changes in individual adhesions between frames in the image stack. Dynamic properties currently include the FA assembly and disassembly rates6 and the focal adhesion alignment index4. All of these results are saved in CSV format, which is suitable for import into statistical or graphing software. For users only interested in static results derived from individual images, as in an analysis of a set of fixed-cell images, all the other dynamic properties can be safely ignored.\n\nThe user is also provided with two types of visualizations that show either the entire field of view or single adhesions over time. The visualization of the entire field of view is produced for every image in the submitted image set and outlines each adhesion with a unique color (Figure 1). This visualization can be used to verify that the adhesions were correctly detected, segmented and tracked. The second visualization type shows a single adhesion segmented and tracked through time (Figure 1).\n\nProvided that adhesions are present for at least 10 sequential images, this visualization allows the user to compare an individual FA’s properties with the appearance of the adhesion in the original image data. This suite of automatically extracted properties and visualizations enables the user to minimize the amount of laborious manual analysis normally required to quantify FA image sets.\n\nSeveral of the parameters used to analyze the images can be specified when an image set is submitted for analysis. The most important of these is the threshold used to identify the regions of the image that qualify as FAs versus background. The appropriate threshold will vary depending on the type of cell imaged and the imaging conditions. To make setting this parameter easier, we have added a feature where a single image can be submitted, segmented using various thresholds and then immediately returned to the user for visual inspection of the results obtained when the threshold is varied. The user also has the option to turn off the default watershed-based segmentation that is used to split adjacent FAs and modify the minimum or maximum FA size accepted by the system. Finally, the time between images can also be specified to ensure that the calculation of the rates of assembly or disassembly are made in the correct units.\n\nUsers have the option of providing an email address when an image set is submitted. If an email address is provided, notification of the completion of the image processing pipeline, along with a link to download the results, is sent. The system can also be used without an email address, but the user must return to the web interface to check on the status of the processing. The processing time is dependent on the number of images in the set and how many adhesions are detected during processing. Using typical input data, we tested the system throughput and found that the average processing and analysis time per image, under full load, is 13 seconds. Because the system can handle four image sets at once, we expect experimental throughput to be acceptable for everyday usage.\n\n\nConclusion\n\nThe Focal Adhesion Analysis Server provides an automated image processing pipeline in an easy-to-use web-based application. A wide range of FA properties are automatically collected from the image sets submitted, and the results are returned in CSV formatted files. Users have the option to adjust the parameters used to process their image sets to suit their specific imaging conditions and cell types of interest.", "appendix": "Author contributions\n\n\n\nMEB and SMG conceived the study, MEB coded the web application, MEB and SMG wrote the manuscript. All authors read and approved the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding was provided to SMG by the University Cancer Research Fund http://unclineberger.org/ucrf\n\n\nAcknowledgements\n\nWe would like to thank the initial users of the beta version of the FAAS website, who provided helpful feedback concerning the website interface and results.\n\n\nReferences\n\nSchneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7): 671–675. PubMed Abstract \| Publisher Full Text\n\nBerginski ME, Vitriol EA, Hahn KM, et al.: High- resolution quantification of focal adhesion spatiotemporal dynamics in living cells. PLoS One. 2011; 6(7): e22025. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen K, Tolbert CE, Guilluy C, et al.: The vinculin c-terminal hairpin mediates f-actin bundle formation, focal adhesion, and cell mechanical properties. J Biol Chem. 2011; 286(52): 45103–45115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu C, Asokan SB, Berginski ME, et al.: Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis. Cell. 2012; 148(5): 973–987. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Z, Lessey E, Berginski ME, et al.: Gleevec, an abl family inhibitor, produces a profound change in cell shape and migration. PLoS One. 2013; 8(1): e52233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWebb DJ, Donais K, Whitmore LA, et al.: Fak-src signalling through paxillin, erk and mlck regulates adhesion disassembly. Nat Cell Biol. 2004; 6(2): 154–161. PubMed Abstract \| Publisher Full Text" }	[ { "id": "810", "date": "05 Mar 2013", "name": "Jonathan Jones", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 generated a valuable tool for analysing adhesion site dynamics. The article is clear, logical and focused. This should be a useful resource for the field.", "responses": [] }, { "id": "814", "date": "07 Mar 2013", "name": "Kenneth Yamada", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 automated web-based analysis system provides a helpful system for considerably simplifying analyses of dynamic focal adhesions of cells in 2D cell culture. Although two examples of the analysis of single adhesions are clear, the example at the bottom left of Figure 1 seems to show appearance of a rather large, complex, multi-pronged cell adhesion that may be a composite of several smaller focal adhesions – it may be useful to discuss this conceptual issue briefly. After further validation and use by other colleagues, this analysis server may become a valuable shared resource for the field.", "responses": [] } ]	1	https://f1000research.com/articles/2-68
https://f1000research.com/articles/2-67/v1	01 Mar 13	{ "type": "Research Article", "title": "Streptococcal taxonomy based on genome sequence analyses", "authors": [ "Cristiane C Thompson", "Vanessa E Emmel", "Erica L Fonseca", "Michel A Marin", "Ana Carolina P Vicente", "Vanessa E Emmel", "Erica L Fonseca", "Michel A Marin", "Ana Carolina P Vicente" ], "abstract": "The identification of the clinically relevant viridans streptococci group, at species level, is still problematic. The aim of this study was to extract taxonomic information from the complete genome sequences of 67 streptococci, comprising 19 species, by means of genomic analyses, multilocus sequence analysis (MLSA), average amino acid identity (AAI), genomic signatures, genome-to-genome distances (GGD) and codon usage bias. We then attempted to determine the usefulness of these genomic tools for species identification in streptococci. Our results showed that MLSA, AAI and GGD analyses are robust markers to identify streptococci at the species level, for instance, S. pneumoniae, S. mitis, and S. oralis. A Streptococcus species can be defined as a group of strains that share ≥ 95% DNA similarity in MLSA and AAI, and > 70% DNA identity in GGD. This approach allows an advanced understanding of bacterial diversity.", "keywords": [ "Comparative Genomic", "Genomic Taxonomy", "Streptococci species" ], "content": "Introduction\n\nBacteria are subjected to numerous forces driving their diversification. As a consequence, different strains of a single bacterial species sometimes have the ability to explore distinct niches, to be pathogenic or non-pathogenic and to present different metabolic pathways1,2. In such a scenario, the identification of bacteria isolates to the species level is a hard task1,2.\n\nCurrently, the genus Streptococcus comprises 99 recognized species, many of which are associated with disease in humans and animals (http://www.bacterio.net/s/streptococcus.html). The viridans group streptococci (VGS) encompass four phylogenetic clusters: Mitis, Mutans, Salivarius and Anginosus, which are part of the human microbiota, being isolated mainly from the oral cavity, gastrointestinal and genitourinary tracts3. The Mitis group currently includes the important pathogen S. pneumoniae and 12 other recognized species, S. australis, S. cristatus (formerly S. crista), S. gordonii, S. infantis, S.mitis, S. oligofermentans, S. oralis, S. parasanguinis (formerly S. parasanguis), S. peroris, S. pseudopneumoniae, S. sanguinis (formerly S. sanguis) and S. sinensis. The Anginosus group includes three recognized species, S. anginosus, S. constellatus (including two subspecies S. constellatus subsp. constellatus and S. constellatus pharyngis) and S. intermedius, and the Salivarius group includes S. salivarius, S. vestibularis, and S. thermophilus.\n\nCurrently, bacterial species are considered to be a group of strains (including the type strain) that are characterized by a certain degree of phenotypic consistency, showing > 70% DNA-DNA hybridization values and over 97% 16S rRNA sequence similarity4,5. Identification of streptococci is based on the current taxonomic standards using a combination of 16S rRNA gene sequence analyses, DNA-DNA hybridization, serologic and phenotypic data; however, they have been strikingly resistant to satisfactory classification, reflected in frequently changing nomenclature6,7. For instance, the 16S rRNA gene sequences of S. mitis and S. oralis are almost identical (> 99%) to S. pneumoniae, making the use of this information alone insufficient to distinguish these species8.\n\nRecent studies have used whole genome analysis to determine the taxonomic relationships among bacterial species9–14. In order to determine the robustness of genomic markers in streptococci species delineation, we analyzed a collection of 67 complete genomes. The availability of whole genome sequences of several closely related species, for instance, S. mitis - S. oralis - S. pneumoniae, and S. salivarius - S. thermophilus - S. vestibularis, formed an ideal test case for the establishment of the genomic taxonomy of streptococci.\n\n\nMaterial and methods\n\nThe genomic sequences of 67 streptococci that were publicly available for download by June 2nd, 2011 at the National Center for Biotechnology Information (NCBI) under the project accession number indicated in Table 1 were used in this study. The following analyses were performed according to Thompson et al. (2009)13 and are briefly described below.\n\nG+C content (%): guanine + cytosine content (%). No. of CDs: number of coding DNA sequence. Nc: effective number of codons.\n\nThe 16S rRNA gene sequences and the gene sequences used for MLSA were obtained from GenBank (http://www.ncbi.nlm.nih.gov). The MLSA approach was based on the concatenated sequences of five house-keeping genes (aroE, ddl, gki, pheS and recA)15,16. The concatenated sequences were aligned with ClustalX program17. The phylogenetic inference was based on the neighbour-joining genetic distance method (NJ)18 using MEGA519. Distance estimations were obtained according to the Kimura-2-parameter20 for 16S rRNA gene and MLSA. The reliability of each tree topology was checked by 2000 bootstrap replications21.\n\nThe AAI of all conserved protein-coding genes was calculated as described previously22. Conserved protein-coding genes between a pair of genomes were determined by whole-genome pairwise sequence comparisons using the BLASTp algorithm23. For these comparisons, all protein-coding sequences (CDSs) from one genome were searched against the genomic sequence of the other genome. The genetic relatedness between a pair of genomes was measured by the AAI of all conserved genes between the two genomes as computed by the BLAST algorithm. By this approach, a value of < 95% AAI of protein-coding genes indicates separate species.\n\nCodon usage bias was calculated for each genome. The effective number of codons used in a sequence (Nc)24 was calculated using CHIPS (http://emboss.bioinformatics.nl/cgi-bin/emboss/chips) with the default parameters.\n\nMononucleotide and dinucleotide frequencies were calculated using COMPSEQ (http://emboss.bioinformatics.nl/cgi-bin/emboss/compseq) with default parameters. Dinucleotide relative abundances (ρXY) were calculated using the equation ρXY = fXY/fXfY where fXY denotes the frequency of dinucleotide XY, and fX and fY denote the frequencies of X and Y, respectively. The difference in genome signature between two sequences is expressed by the genomic dissimilarity (δ), which is the average absolute dinucleotide of relative abundance difference between two sequences, and were calculated using the equation: δ(f,g) = 1/16Σ\|ρXY (f) - ρXY (g)\| (multiplied by 1000 for convenience), where the sum extends over all dinucleotides25.\n\nThe genome distance was calculated using genome-to-genome distance calculator (GGDC)26. Distances between a pair of genomes were determined by whole-genome pairwise sequence comparisons using BLAST23. For these comparisons, algorithms were used to determine high-scoring segment pairs (HSPs) for inferring intergenomic distances for species delimitation. The corresponding distance threshold can be used for species delimitation26.\n\n\nResults and discussion\n\nIn this work we compared complete genomes for 67 streptococci comprising 19 species to address their taxonomic position. A previous study with a small set of streptococci genomes (eight) and species (four), using a combination of several genomic analyses, showed the applicability of this approach in streptococci taxonomy9. Overall our analysis, using a large data set, showed that genomic taxonomy is an accurate approach to clearly define the streptococci species. The taxonomic resolution of the 16S rRNA, AAI, MLSA, GGD and codon usage analysis for streptococci species definition is summarized in Table 2.\n\nMLSA: multilocus sequence analysis. AAI: amino acid identity. GGD: genome to genome distance. Nc: effective number of codons.\n\n\n\n\n\n\n\nThe complete genome of the streptococci comprised a single chromosome. The estimated size of the genomes ranged from 1.7 Mb (S. infantis) to 2.3 Mb (S. sanguinis). The number of CDS varied from 1,700 (S. pyogenes) to 2,352 (S. pneumoniae) (Table 1). The average G+C content of streptococci genomes ranged from 35% to 43%. These species presented a variable interspecies genome size and G+C content, indicating heterogeneity within the genus Streptococcus. One of the reasons for this variability could be associated with the frequent occurrence of horizontal gene transfer events27–29.\n\nMLSA and 16S rRNA phylogenetic trees showed similar topologies (Figure 1). The MLSA was performed using five instead of the seven genes applied in the pneumococcus multilocus sequence typing (MLST) scheme (http://spneumoniae.mlst.net/)15,16. Three genes, aroE, ddl and gki, are from the MLST scheme, and pheS and recA were included in this work. The concatenation of these genes (7741 bp) allowed an accurate delineation of the streptococci species considered here. The nucleotide sequence similarities were much lower for MLSA than 16S rRNA gene. A pairwise comparison of MLSA among the species revealed sequence similarity between 67% and 100%, while the 16S rRNA gene sequence similarities varied from 92% to 100%. At the intraspecies level, the similarity values ranged from 95% to 100% for MLSA, and 99% to 100% for the 16S rRNA gene sequences. The closest species within the Mitis (S. pneumoniae - S. oralis - S. mitis) and Salivarius groups (S. vestibulares - S. salivarius - S. thermophilus) were clearly placed apart from each other by MLSA, while these species had almost identical 16S rRNA gene sequences (≥ 99% sequence similarity). A previously study showed that recA analysis is a valuable tool for proper identification of pneumococci in routine diagnostics, but limitations on discrimination of other members of the Mitis group were observed30. S. sanguinis ATCC 49296 showed a much closer relationship with S. oralis ATCC 35037T (95% similarity) than to other S. sanguinis strains (77% similarity), suggesting it belongs to the species S. oralis. In addition, S. bovis ATCC 700338 was placed in the S. gallolyticus cluster with 98% MLSA sequence similarity. This work showed that MLSA, using this new combination of five concatenated genes (aroE, ddl, gki, pheS and recA), distinct from the Streptococcus MLST scheme, allowed a proper identification of most streptococci species, even within the VGS group.\n\nThe numbers at the nodes indicate the values of bootstrap statistics after 2000 replications, and values below 50% are not shown. Bars, 0.005% and 0.02% estimated sequence divergence.\n\nThe percentage of average amino acid identity (AAI) among streptococci species ranges from 68% to 94%, while within species it varies from 95% to 100%. The VGS species S. pneumoniae, S. mitis and S. oralis shared 89–93% AAI. The species S. salivarius, S. thermophilus and S. vestibularis showed a maximum AAI of 93%. S. sanguinis ATCC 49296 and S. oralis ATCC 35037 showed 96% identity and S. bovis ATCC 700338 and S. gallolyticus strains had 98% identity. These findings suggest that strains ATCC 49296 and ATCC 700338 belong to the species S. oralis and S. gallolyticus, respectively. According to our analyses the AAI and MLSA are the most useful genomic features for the elucidation of streptococci taxonomy.\n\nThe genomic dissimilarity values among streptococci were between 3 and 127, while the intraspecies values were between 0 and 17. Streptococci within the VGS group, for instance, S. salivarius, S. thermophilus and S. vestibularis species, showed dissimilarity values between 5 and 12 and S. pneumoniae, S. mitis and S. oralis species had dissimilarity values between 3 and 14. Thus, there was not a clear differentiation of these closely related species within the VGS group on the basis of the genomic dissimilarity values. This could be due to the extensive recombination and horizontal gene transfer events which occur between closely related streptococci species that share ecological niches12,30.\n\nOn the other hand, species within the Pyogenic group had a distinct genomic signature, with values ranging from 13 to 85. However, genome signatures alone have significant limitations when used as phylogenetic markers for differentiating members of the VGS. The exact mechanisms that generate and maintain the genome signatures are complex, but possibly involve differences in species-specific compositional bias, i.e., G+C content, G+C and A+T skews, codon bias, and mutation bias32,33.\n\nNc values provide a meaningful measure of the extent of codon preference in a genome, values range between 20 (extremely biased genome where one codon is used per amino acid) and 61 (all synonymous codons are used). Within the set of 67 complete streptococci genomes examined in this study, the Nc ranged from 44.0 to 54.5 (Table 1). For instance, S. pneumoniae - S. oralis - S. mitis species had Nc values of 50, 51 and 50, respectively. The Salivarius group (S. vestibulares - S. salivarius - S. thermophilus), and S. bovis ATCC 700338- S. gallolyticus showed Nc values of 47 and 44.5, respectively. Overall, codon usage bias was very similar among the streptococci species investigated. However, S. sanguinis ATCC 49296 showed a much closer Nc value with the S. oralis ATCC 35037 (51.7 and 51.4, respectively) than other S. sanguinis strains (54.5), which was in agreement with the other analyses used in this study.\n\nThe GGD was calculated only for closely related species that were not differentiated by 16S rRNA gene sequence analysis (Figure 1). Based on GGD analysis the species within the Mitis and Salivarius groups were identified as separate species, showing GGD values analogous to the < 70% discriminatory value used for DNA-DNA hybridization. Conversely, S. bovis ATCC 700338 and S. gallolyticus were identified as belonging to the same species by GGD.\n\nS. bovis ATCC 700338 (biotype II) and S. gallolyticus as well as S. sanguinis ATCC 49296 and S. oralis ATCC 35037T were not separated and, therefore, according to this analysis would be classified as the same species, respectively. It was shown that S. bovis biotype I and II/2 isolates were, in fact, S. gallolyticus34, and S. sanguinis ATCC 49296 was placed into S. oralis species by GGD analysis. A misidentification of S. sanguinis ATCC 49296 has already been shown by means of biochemical and serological properties by Narikawa and colleagues35.\n\nAnother interesting result is that the S. parasanguinis ATCC 15912 and F0405 strains were found to be at the upper limits for definition as members of the same species based on different genomic analyses. For instance, they shared 95% AAI, 94% identity by MLSA, a value of 17 on the basis of genomic signature and < 70% similarity in GGD. Therefore, based on these genomic markers, these S. parasanguinis strains could, in fact, be separate species. This data reflects the complexity of bacterial species delineation, since these organisms are all under a constant evolutionary process.\n\n\nConclusion\n\nThe delineation of closely related streptococci species was evident in this genomic study. Different methods produced different levels of taxonomic resolution. The methods with the higher resolution for species identification were MLSA and AAI, while closely related species had similar Nc values and genomic signatures. Based on the genomic analyses, a Streptococcus species can be defined as a group of strains that shares ≥ 95% identity in MLSA and AAI, and > 70% identity in GGD. This definition may be useful to advance the taxonomy of Streptococcus. This approach allows an advanced understanding of bacterial diversity and identification.", "appendix": "Author contributions\n\n\n\nCCT and VEE carried out the computational and genomic analyses and analyzed the results. All authors (ACPV, CCT, ELF, MAM and VEE) participated in discussing and writing the manuscript. All authors have agreed to the final contents of the article.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nVEE had a PRODOC-CAPES fellowship. CCT has a PNPD-CAPES fellowship, ELF has a PNPD-FAPERJ fellowship and MAM has a CAPES fellowship.\n\n\nReferences\n\nGevers D, Cohan FM, Lawrence JG, et al.: Opinion: Re-evaluating prokaryotic species. Nat Rev Microbiol. 2005; 3(9): 733–9. PubMed Abstract \| Publisher Full Text\n\nCohan FM, Koeppel AF: The origins of ecological diversity in prokaryotes. Curr Biol. 2008; 18(21): R1024–34. PubMed Abstract \| Publisher Full Text\n\nAlam S, Brailsford SR, Whiley RA, et al.: PCR-Based methods for genotyping viridans group streptococci. J Clin Microbiol. 1999; 37(9): 2772–6. PubMed Abstract \| Free Full Text\n\nStackebrandt E, Goebel BM: Taxonomic Note: A place for DNA-DNA reassociation and 16S ribosomal-RNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol. 1994; 44(4): 846–849. Publisher Full Text\n\nWayne LG, Brenner DJ, Colwell RR, et al.: Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol. 1987; 37(4): 463–464. Publisher Full Text\n\nHoshino T, Fujiwara T, Kilian M: Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients. J Clin Microbiol. 2005; 43(12): 6073–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKawamura Y, Hou XG, Sultana F, et al.: Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol. 1995; 45(2): 406–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuzuki N, Seki M, Nakano Y, et al.: Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization. J Clin Microbiol. 2005; 43(9): 4528–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoenye T, Vandamme P: Extracting phylogenetic information from whole-genome sequencing projects: the lactic acid bacteria as a test case. Microbiology. 2003; 149(pt 12): 3507–17. PubMed Abstract \| Publisher Full Text\n\nCoenye T, Gevers D, Van de Peer Y, et al.: Towards a prokaryotic genomic taxonomy. FEMS Microbiol Rev. 2005; 29(2): 147–67. PubMed Abstract \| Publisher Full Text\n\nRichter SS, Heilmann KP, Dohrn CL, et al.: Accuracy of phenotypic methods for identification of Streptococcus pneumoniae isolates included in surveillance programs. J Clin Microbiol. 2008; 46(7): 2184–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCroucher NJ, Harris SR, Fraser C, et al.: Rapid pneumococcal evolution in response to clinical interventions. Science. 2011; 331(6016): 430–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson CC, Vicente ACP, Souza RC, et al.: Genomic taxonomy of Vibrios. BMC Evol Biol. 2009; 9: 258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson CC, Vieira NM, Vicente AC, et al.: Towards a genome based taxonomy of Mycoplasmas. Infect Genet Evol. 2011; 11(7): 1798–804. PubMed Abstract \| Publisher Full Text\n\nEnright MC, Spratt BG: A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology. 1998; 144(Pt 11): 3049–60. PubMed Abstract \| Publisher Full Text\n\nHanage WP, Fraser C, Spratt BG: Sequences, sequence clusters and bacterial species. Philos Trans R Soc Lond B Biol Sci. 2006; 361(1475): 1917–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson JD, Gibson TJ, Plewniak F, et al.: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997; 25(24): 4876–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4(4): 406–25. PubMed Abstract\n\nTamura K, Peterson D, Peterson N, et al.: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28(10): 2731–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol. 1980; 16(2): 111–120. PubMed Abstract \| Publisher Full Text\n\nFelsenstein J: Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution. 1985; 39(4): 783–791. Publisher Full Text\n\nKonstantinidis KT, Tiedje JM: Towards a genome-based taxonomy for prokaryotes. J Bacteriol. 2005; 187(18): 6258–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltschul SF, Madden TL, Schäffer AA, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWright F: The 'effective number of codons' used in a gene. Gene. 1990; 87(1): 23–9. PubMed Abstract \| Publisher Full Text\n\nKarlin S, Mrázek J, Campbell AM: Compositional biases of bacterial genomes and evolutionary implications. J Bacteriol. 1997; 179(12): 3899–913. PubMed Abstract \| Free Full Text\n\nAuch AF, Klenk HP, Göker M: Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci. 2010; 2(1): 142–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang A, Yang M, Hu P, et al.: Comparative genomic analysis of Streptococcus suis reveals significant genomic diversity among different serotypes. BMC genomics. 2011; 12: 523. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellanger X, Roberts AP, Morel C, et al.: Conjugative transfer of the integrative conjugative elements ICESt1 and ICESt3 from Streptococcus thermophilus. J Bacteriol. 2009; 191(8): 2764–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarvey RM, Stroeher UH, Ogunniyi AD, et al.: A variable region within the genome of Streptococcus pneumoniae contributes to strain-strain variation in virulence. PloS One. 2011; 6(5): e19650. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZbinden A, Köhler N, Bloemberg GV: recA-based PCR assay for accurate differentiation of Streptococcus pneumoniae from other viridans streptococci. J Clin Microbiol. 2011; 49(2): 523–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDonati C, Hiller NL, Tettelin H, et al.: Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species. Genome Biol. 2010; 11(10): R107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarlin S: Global dinucleotide signatures and analysis of genomic heterogeneity. Curr Opin Microbiol. 1998; 1(15): 598–610. PubMed Abstract \| Publisher Full Text\n\nFoerstner KU, von Mering C, Hooper SD, et al.: Environments shape the nucleotide composition of genomes. EMBO Rep. 2005; 6(12): 1208–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDevriese LA, Vandamme P, Pot B, et al.: Differentiation between Streptococcus gallolyticus strains of human clinical and veterinary origins and Streptococcus bovis strains from the intestinal tracts of ruminants. J Clin Microbiol. 1998; 36(12): 3520–3. PubMed Abstract \| Free Full Text\n\nNarikawa S, Suzuki Y, Takahashi M, et al.: Streptococcus oralis previously identified as uncommon \"Streptococcus sanguis\" in Behçet’s disease. Arch Oral Biol. 1995; 40(8): 685–90. PubMed Abstract \| Publisher Full Text" }	[ { "id": "818", "date": "03 Apr 2013", "name": "Tomoo Sawabe", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 are good enough to attract readers in the scientific community.Genome based multi-gene sequence comparison is one of the promising tools to analyse bacterial populations. To achieve the analysis for Streptococcus, the authors carefully designed massive data genome analysis. The results are strong enough and supported by the results of the analysis.The conclusion is clear that authors proposed a threshold value on the basis of genome-based MLSA in Streptococcus bacteria.", "responses": [] }, { "id": "872", "date": "03 Apr 2013", "name": "Bruno Gomez-Gil", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well written with an appropriate title and abstract. The methods are adequate for the aims of the study, but I would suggest that including the Average Nucleotide Identity (ANI) analysis as suggested by Rosello-Mora et al. 2006, would certainly improve the manuscript. The online analysis can be found here http://www.imedea.uib.es/jspecies/index.html.The conclusions are adequate and the data sufficient to replicate all the analyses.The data are openly accessible at GenBank.", "responses": [] } ]	1	https://f1000research.com/articles/2-67
https://f1000research.com/articles/2-65/v1	28 Feb 13	{ "type": "Research Article", "title": "Reactive microglia after taste nerve injury: comparison to nerve injury models of chronic pain", "authors": [ "Dianna L Bartel", "Thomas E Finger", "Thomas E Finger" ], "abstract": "The chorda tympani (CT), which innervates taste buds on the anterior portion of the tongue, is susceptible to damage during inner ear surgeries. Injury to the CT causes a disappearance of taste buds, which is concurrent with significant microglial responses at central nerve terminals in the nucleus of the solitary tract (nTS). The resulting taste disturbances that can occur may persist for months or years, long after the nerve and taste buds have regenerated. These persistent changes in taste sensation suggest alterations in central functioning and may be related to the microglial responses. This is reminiscent of nerve injuries that result in chronic pain, where microglial reactivity is essential in maintaining the altered sensation (i.e., pain). In these models, methods that diminish microglial responses also diminish the corresponding pain behavior. Although the CT nerve does not contain nociceptive pain fibers, the microglial reactivity after CT damage is similar to that described in pain models. Therefore, methods that decrease microglial responses in pain models were used here to test if they could also affect microglial reactivity after CT injury. Treatment with minocycline, an antibiotic that dampens pain responsive microglia, was largely ineffective in diminishing microglial responses after CT injury. In addition, signaling through the toll-like 4 receptor (TLR4) does not seem to be required after CT injury as blocking or deleting TLR4 had no effect on microglial reactivity. These results suggest that microglial responses following CT injury rely on different signaling mechanisms than those described in nerve injuries resulting in chronic pain.", "keywords": [ "chorda tympani", "nucleus of the solitary tract", "chronic pain", "microglia", "minocycline", "TLR4" ], "content": "Introduction\n\nThe chorda tympani (CT) nerve is the sensory branch of the seventh cranial nerve that innervates taste buds on the anterior tongue. The CT runs through the middle ear where is particularly susceptible to injury during ear surgeries1. CT transection (CTx) is accompanied by a disappearance of taste buds on the denervated side of the tongue2,3. At the same time, significant microglial responses also occur in the first central gustatory relay – the nucleus of the solitary tract (nTS)4. This nerve damage can cause a loss or distortion of taste, or dysguesia, that can persist for months or years long after the CT nerve and taste buds have regenerated5. Such long-lasting dysguesias suggest alterations in central nervous function.\n\nThe central glial reactivity that occurs after damage to other sensory nerves actively contributes to abnormal sensations that arise after nerve damage. For example, increasing evidence from animal models suggests that microglial reactivity in particular is essential to initiate and maintain chronic pain (reviewed in6–9. This in large part explains why traditional pain drugs that directly target neuronal cells do not completely quiet persistent pain messages – because the neurons’ heightened sensitivity is also driven by microglia. A similar phenomenon might explain the lasting dysguesias after injury to the CT. Hence, understanding the mechanisms involved in microglial responses has important implications for treating abnormal sensations caused by nerve injury.\n\nEven though the CT nerve does not contain any nociceptive C-fibers10, which are themselves a source of microglial responses11,12, the general profile of the microglial responses are similar with CT injury as that seen in pain models. Specifically, damage to the CT causes significant microglial responses in terms of morphological reactivity and an increased density of microglial cells. Within a day after CT injury, microglia in the vicinity of afferent CT terminals changed their morphology from the characteristic ramified morphology to a hypertrophied reactive morphology, characterized by shorter thicker processes and an amoeboid shape. The increased microglial population primarily resulted from microglial proliferation, which was supplemented by microglial migration within sub-divisions of the nTS4.\n\nWhile the details of how nerve injury activates microglia are not entirely known, in nerve injuries that result in neuropathic pain pharmaceutical treatments can diminish pain behavior as well as the corresponding microglial responses. Therefore, various treatments and models that decrease microglial responses in experimental pain models were used here to test if they could dampen the microglial responses in the nTS after CT injury.\n\nMicroglia express multiple purinergic receptors. Upon sensing changes in the environment, this purinergic signaling results in several functional phenotypes such as process extension, migration, proliferation and phagocytosis (reviewed in13). Here, we used mice that lack the P2X2 and P2X3 receptors to test whether these subunits are required for microglial responses following CT injury. Further, because the gustatory nerves of the P2X-dlbKO mice are not responsive to taste stimulation14, this model also tested whether the disruption of normal gustatory signaling might alter microglial reactivity.\n\nThis antibiotic is commonly used to treat chronic pain in animal models. Minocycline is a tetracycline derivative with anti-inflammatory and neuroprotective effects that are unrelated to its anti-microbial action (see reviews15,16). This compound is highly hydrophobic and readily crosses the blood-brain barrier. Multiple effects can account for the alleviation of pain behavior with minocycline, such as inhibition of matrix metalloproteinase as well as other anti-inflammatory, antioxidant and anti-apoptotic properties17–19. Minocycline treatment also results in reduced expression of the microglial marker Iba120. Although this treatment has many modes of action, minocycline can only prevent but not reverse neuropathic pain21. Therefore, to test if minocycline could diminish microglial reactivity in the nTS, the minocycline was administered to the animals before they received CTx and the animals continued to receive treatment for the duration of their survival.\n\nRecent studies have shown that TLR4 signaling is involved in nerve injury-induced microglial reactivity and related pain behavior22,23. Deletion of TLR4 can prevent pain and microglial reactivity from developing after spinal nerve injury22. Hence, we tested whether TLR4 signaling is necessary for microglial reactivity after CT damage by performing CTx on the C3H mice that lack functional TLR4.\n\nTLR4 signaling can also be inhibited with naloxone. Recent studies demonstrated that both the opioid antagonist (-)-naloxone and the non-opioid (+)-naloxone inhibit toll-like receptor 4 (TLR4) signaling and reverse neuropathic pain following spinal nerve injuries23,24. Hence, we performed CTx on the C57BL6/J animals that were then treated with naloxone as has been done in neuropathic pain models24.\n\nThe current study used these methods and models and examined their effects on microglial reactivity in the nTS following CT injury. Specifically, we examined microglial morphology, counted the number of microglia and measured the fluorescent intensity of microglial markers.\n\n\nMaterials and methods\n\nThe experiments were conducted on male and female mice aged three to nine months (strains listed below and in Table 1). All animals were housed on a 14-hour light cycle with access to standard chow ad-libitum. The following protocols were approved by the Institutional Animal Care and Use Committees at the University of Colorado Anschutz Medical Campus (Aurora, CO).\n\n\nMouse strains\n\nC57BL6/J – These mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and were bred in-house. A total of 32 C57BL6/J animals were used in these experiments.\n\nP2X2/P2X3-/- – These double-knockout mice (P2X-dblKO) and their wild-type controls (P2X-WT) were originally obtained from Roche Palo Alto (Palo Alto, CA;25) and were subsequently bred at Charles River Transgenics Laboratory or in-house at the University of Colorado. The gustatory nerves in these mice are unresponsive to taste stimuli14. A total of four P2X-dblKO animals and four P2X-WT animals were used in these experiments.\n\nC3H/HeJ (Tlr4Lps-d) – The C3H mice carry a point mutation in the cystolic domain of the Toll-like receptor 4 protein (TLR4), resulting in the exchange of amino acid 712 from proline to histidine that disrupts the functionality of the receptor26. These mice and their wild-type controls (C3HeB/FeJ) were obtained from The Jackson Laboratory. A total of three C3H animals and three C3HeB animals were used in these experiments.\n\nThe chorda tympani transection and perfusion procedures were performed according to methods detailed in a previous publication4 and are described briefly here.\n\nCT transection (CTx): The animals of the different treatment groups (a total of 41 mice) were anesthetized with intramuscular injections of dexmedetomadine hydrochloride (0.4mg/kg; Pfizer, Finland) and ketamine hydrochloride (40mg/kg; Bioniche Pharma; Lake Forest, IL). After placing the animal in a non-traumatic head holder, we visualized and transected the CT using the transaural approach on the right side. The left CT remained intact to serve as an internal control. The anesthesia was then reversed with antipamezole (2mg/kg: Pfizer, Finland), the antidote to dexmedetomadine. The animals survived for 3 days.\n\nPerfusion and tissue fixation: The animals were deeply anesthetized with 50mg/kg sodium pentobarbital (Ovation Pharmaceuticals Inc; Deerfield, IL) and perfused transcardially with 15 mL of 0.9% sodium chloride followed by 25 mL of 4% paraformaldehyde (PFA in 0.1M phosphate buffer). The brainstem was dissected from the skull, which was then postfixed in PFA for 4 hours at room temperature and cryoprotected with 20% sucrose in 0.1M phosphate buffer overnight at 4°C. The next day, the tissue was mounted in OCT compound (Fisher Scientific; Pittsburgh, PA) and cut on a cryostat. Free-floating 40 µm sections were collected into PBS (0.1M phosphate buffered saline).\n\nAfter three ten-minute washes in PBS, tissue sections were incubated for one hour in blocking solution [1% bovine serum albumin (Sigma, St. Louis, MO), 0.3% triton (American Bioanalytical, Natick, MA) and 2% normal goat serum (Jackson Immunoresearch, West Grove, PA) in PBS]. Incubation with rabbit anti-Iba1 (Wako; Richmond, VA; polyclonal; #19-19741, dilution 1:500) or rat anti-cd11b (Serotec; Raleigh, NC; monoclonal; #MCA74GA, dilution 1:50) antibody diluted in blocking solution was carried out overnight at 4°C. The next day, three PBS washes were followed by 2 hours of incubation with the appropriate secondary antibody, Alexa488 goat anti-rabbit or Alexa488 goat anti-rat (Invitrogen, Carlsbad, CA; dilution 1:400 in blocking solution). For the final 25 minutes, green fluorescent Nissl stain (NeuroTrace; Molecular Probes, Eugene, OR) was added to the incubation solution at 1:100. After the sections were washed several times in buffer, they were mounted onto slides and coverslips were applied with Fluormount G (Southern Biotechnology Associate, Inc.; Birmingham, AL).\n\nThe densest projections of chorda tympani fibers occur in rostral levels of the nTS, designated R1, R2 and R34,27. Using the fluorescent Nissl stain, we could delineate the rostral central (RC) and ventral (V) subdivisions of the nTS according to previous studies (28,29 and see Figure 1). The borders of the nTS were outlined on grayscale fluorescent Nissl images in Adobe Illustrator 10 and superimposed onto the color images. All representative images shown in figures are taken at levels R2 or R3.\n\nA. Borders of the nTS and surrounding nuclear regions were identified with fluorescent Nissl staining (green). The P2X2 staining in magenta delineates the area of incoming CT fibers. B. The majority of CT projections terminate within the rostral central subnucleus (RC) though some finer fibers extend into the ventral subnucleus (V). Images in all figures are displayed in the orientation shown above (medial - left, dorsal - top). Cun - external cuneate, nTS - nucleus of solitary tract, 10N - dorsal motor nucleus of vagus, SpVe - spinal vestibular nucleus, RF - reticular formation, IV - fourth ventricle.\n\nConfocal images were acquired with an Olympus Fluoview Laser Scanning Confocal Microscope using 20× oil-immersion objective (N.A. 0.80) or 60× oil-immersion objective (N.A. 1.4). The green and red channels were obtained sequentially and merged together to prevent sideband excitation of the fluorophores.\n\nImages that were used for counting microglial cells were obtained with the same acquisition parameters (i.e., laser intensity, gain and offset) on the lesioned and unlesioned sides to permit quantitative comparisons. Cell counts were done as previously described4. Briefly, using the optical dissector method a pair of parallel images spaced 5 µm apart was used to count Iba1+ microglia. A microglial cell body was counted in the ‘reference’ image if the same cell body was not present in the partner ‘look-up’ image.\n\nConventional epifluorescent images were obtained using an Olympus BX41 upright microscope and a 20× objective (N.A. 0.50). These images were used to measure fluorescence levels of Iba1 and cd11b. Using the ImageJ version 1.62 software (National Institutes of Health; Bethesda, MD), fluorescence levels were measured within a defined circular region of interest (ROI=1,596 40 µm2). For each animal, four ROI measurements were taken on intact and transected sides in the following regions: rostral central nTS (RC), ventral nTS (V) and spinal vestibular nucleus (SpVe).\n\nThe cell counts and intensity measurements for each experiment were analyzed using SPSS software (Chicago, IL) with appropriate ANOVAs and Tukey’s post-hoc analysis. Significance was defined as p<0.05.\n\nTo test if P2X2 and P2X3 receptors are necessary for microglial responses, CTx was performed on four P2X-dblKO mice and four P2X-WT mice. Microglia were stained with Iba1, imaged and then counted.\n\nTo test if minocycline could decrease microglial responses after CTx, three C57BL6/J mice received minocycline before and after CTx. Minocycline hydrochloride (Sigma M9511) was administered via intraperitoneal injection at 50mg/kg every 12 hours based on previous studies17,21,30. Treatment began one day prior to CTx surgery and continued until the animal was sacrificed three days later. Microglia were stained with Iba1 and cd11b and imaged to quantify cell numbers and levels of fluorescence.\n\nTLR4 mutants (C3H mice): These experiments tested whether a functional TLR4 was required for the nerve damaged-induced microglial response. CTx was performed on three C3H mice, which lack a functional TLR4, as well as three of their wild-type siblings (C3HeB) to compare to the C57BL6/J animals. The animals survived for three days. Microglia were stained with Iba1, imaged and then counted.\n\nNaloxone: These experiments sought to block TLR4 with naloxone treatment at the onset of the CT injury. A total of eight C57BL/6 animals received CTx and were then treated with naloxone hydrochloride (Sigma, #N7758, racemic mixture; administered subcutaneously), seven C57BL/6 animals were treated with naloxone but did not received CTx. Naloxone has a short half-life of ~60 minutes in the serum31. To examine whether naloxone could prevent the microglial response days after CTx, it was necessary to administer the naloxone continuously, which was achieved by means of osmotic minipumps. The total doses of naloxone, 10mg, in these experiments is comparable to previous studies on rats using chronic intrathecal and acute subcutaneous administration23 and other studies on mice using chronic subcutaneous administration32.\n\nThe ALZET® pump model 1003D (delivers 1 μl/hr, up to 3 days) was used in these experiments. The pumps were filled according to company instructions. So as not to delay the delivery of drug, prior to implantation, the pump was allowed to reach its steady-state pumping rate by incubating in sterile saline at 37°C for 6–12 hours.\n\nAccording to the manufacturer, the solubility of naloxone is 50mg/mL. Since the total volume of the pumps is 100 μl, the total amount of naloxone that could be administered by one pump was 5mg. This dosage of naloxone was as follows:\n\n5mg/0.02kg/3dy = 1.67mg/0.02kg/1dy = 69.58μg/0.02kg/1hour\n\n69.58μg/0.02kg = 0.06958mg/0.02kg = 3.48mg/kg/1hour\n\nTo achieve the highest dosage of naloxone (10mg), the animals received two of these pumps for three days. Hence, the dose of naloxone administered was 6.96mg/kg/1hour.\n\nAfter the animal was sedated, the animal was given a starter injection of the naloxone solution at the same concentration/day determined to be delivered by the pump (i.e., in the example above 1.67mg/0.02kg). This ‘booster’ shot was given via intraperitoneal (I.P.) injection in order to get naloxone to an effective level before nerve injury. To insert the pump, the area on the back of the neck between the scapulae was shaved and surgically prepped. After making a ~1-cm incision in the skin, a hemostat was inserted under the skin, then gently opened and closed to create a pocket for the pump. The pump was inserted into the pocket, exit port first, and the incision was closed with several stitches with Decon suture. While the animal was still sedated, the CT was transected.\n\n\nBehavior testing with naloxone treatment\n\nWe first wanted to test whether naloxone was gaining access to the CNS. Previous studies have shown that naloxone causes a decreased intake of preferred foods such as sweet-tasting chow or solutions33–37. Therefore, we tested the animals’ sweet preference as an indication of the effectiveness of the naloxone treatment. Sweet preference was measured using a two-bottle taste preference test in which the animals are given a choice between water and sucrose. The preference score was calculated [sucrose (mL)/total fluid (mL)] such that the higher the score the greater the sweet preference with 0.5 representing no preference. The general protocol involved training the animals for several days with the two bottles before they received naloxone pumps. At the end of each training and testing period, fluid intake was recorded. Several different testing parameters were used in these experiments and are described as follows.\n\n\nBehavior testing with naloxone and CTx\n\nThe following describe experiments that used naloxone treatment (10mg) and CTx.\n\nA. Mice were given continual access to two-bottles: one with water and one with 300mM (10%) sucrose where the bottles were switched sides every 24 hours at which time the fluid intake was recorded. Mice were trained for 4 days before receiving pumps with 10mg Naloxone or saline and CTx. The mice recovered for 6 hours with full access to water and were then again given the two bottles with water and sucrose for 2 days and were then sacrificed.\n\nB. Mice were placed on a 22.5 hour water deprivation schedule and were trained for 3 days during which they had access to fluids for a total of 1.5 hours per day: during the first 30 minutes both bottles contained water, then mice were given one bottle of water and one bottle of 300mM (10%) sucrose for 60 minutes during which bottles were switched sides after 30 minutes. On day 4, pumps filled with 10mg naloxone or saline were implanted followed by CTx. The mice recovered for 6 hours with full access to water and were then tested with water and sucrose for 3 days and were then sacrificed.\n\n\nBehavior testing with naloxone only\n\nTo ensure that CTx was not affecting the taste preference behavior, animals that treated with 10mg of naloxone but did not undergo CTx were also tested. The concentration of sucrose was also lessened in these experiments to ensure that more subtle effects of NLX could be detected.\n\nA. Mice were given continual access to two-bottles: one with water and one with 200mM (7%) sucrose where the bottles were switched sides every 24 hours at which time the fluid intake was recorded. Mice were trained for 2 days before receiving pumps with 10mg naloxone. The mice recovered for 6 hours with full access to water, were tested with water and sucrose for 2 days and then sacrificed.\n\nB. Mice were water restricted for 21.5 hours and were trained for 2 days during which they had access to fluids for a total of 3.5 hours per day: during the first 90 minutes both bottles contained water, then mice were given one bottle of water and one bottle of 200mM (7%) sucrose for 2 hours during which bottles were switched sides after 60 minutes. On day 3, pumps filled with 10mg naloxone were implanted. The mice recovered for 6 hours with full access to water, were tested with water and sucrose for 2 days and then sacrificed.\n\n\nResults\n\nStaining for P2X2 and P2X3 receptors in the rostral medulla delineates the incoming CT terminations within the nTS4. As shown in Figure 1, P2X2 staining in magenta identifies terminal CT fibers, which are primarily contained within the rostral central (RC) subnucleus of the nTS. The RC subnucleus is also where reactive microglia appear following CT injury4 and (see Figure 2).\n\nA. As revealed with Iba1 staining, microglia on the intact side (Int) of the nTS occupy distinct spatial areas throughout the nTS and surrounding areas. B. At 3 days post CTx on the transected side (Tx), a dense cluster of Iba1 immunoreactivity appears in the RC subnucleus of the nTS and microglia display reactive morphologies. C. The number of Iba1 labeled microglia in the RC subnucleus on the Tx side in the P2X-dblKO animals (light blue squares) does not appear to differ from wild-type animals (dark grey squares) at 3 days post CTx. Each square represents the mean from one case. D. At 10 days post CTx, the cluster of reactive microglia is still present in the RC subnucleus of C57BL6/J mice. E. Similarly, reactive microglia remain at 10 days post CTx in the P2X-dblKO animals.\n\nTo test whether P2X2 and P2X3 receptors are necessary for microglial reactivity in the nTS, we performed CTx in mice that lacked these functional receptors (P2X-dlbKO). In both the P2X-WT and the P2X-dblKO cases, Iba1+ microglia on the unlesioned intact side (Int) of the nTS were evenly distributed throughout the tissue (Figure 2A shows the Int side of P2X-dblKO). By the third day after injury, a dense cluster of Iba1 immunoreactivity appeared in the nTS on the transected side (Tx). Specifically, this aggregate of Iba1+ microglia was primarily confined to the RC subnucleus, which contains the densest projections of CT terminal fibers (Figure 2B shows Tx side of P2X-dlbKO). These microglial cells displayed reactive morphologies with thickened processes and enlarged somata. This result parallels the course of microglial responses following CTx in C57BL6/J animals4. Further, the number of microglial cells in the RC subnucleus in P2X-dblKO did not differ from the number of microglia in their wild-type siblings (Figure 2C). That is, the density of microglia increased in the RC subnucleus in the P2X-dblKO, just as seen in the wild-type siblings as well as C57BL6/J animals. At ten days post CTx, the cluster of reactive microglia remains in the RC subnucleus in C57BL6/J animals (Figure 2D). Similarly, reactive microglia are also present at 10 days after CTx in the P2X-dblKO animals (Figure 2E). Hence, even in the absence of P2X2 and P2X3 transection of the CT still resulted in reactive microglia.\n\nIn minocycline treated cases, microglial cells still reacted throughout the CT terminal field. At three days after injury, the number of microglia in the RC subnucleus still increased on the transected sides compared to the intact sides whether the animals received minocycline, saline or no treatment (Figure 3A; n=3 each). Microglia were also morphologically reactive in the RC subnucleus, as seen when labeled with Iba1 or cd11b (Figure 3B,C).\n\nA. The number of Iba1-labeled microglia in the RC subnucleus increases on the transected sides (Tx) compared to the intact sides (Int) with ‘No treatment’ and saline treatment. The number of microglia also increases on the transected side with minocycline treatment (Mino). An asterisk () denotes a significant difference on the transected side compared to the corresponding intact side with p<0.05, SEM error bars, n=3 for each group. B,C. After minocycline treatment, staining for Iba1 and cd11b reveals the typical cluster of reactive microglia in the RC subnucleus on the transected sides.\n\nSince the number of microglial cells was similar after injury with or without minocycline treatment, we compared the levels of expression of two microglial markers: Iba1, a cytoplasmic calcium binding protein, and cd11b, a membrane-bound integrin receptor involved in phagocytosis. At the R2 and R3 rostral levels of the nTS27, we measured the intensity of Iba1 and cd11b staining on the intact and transected sides. The fluorescent levels on the transected sides were normalized by subtracting the fluorescent levels from the corresponding intact sides. These normalized measurments were compared between no treatment (white bars) and minocycline treatment (dark bars; Figure 4). At level R3, minocycline did not affect the levels of Iba1 staining [F(1,4)=1.1, p=0.3] or cd11b staining [F(1,4)=10, p>0.05] in the rostral central (RC) or ventral (V) subnuclei of the nTS nor the immediately adjacent spinal vestibular region (SpVe; Figure 4A). However, at level R2 minocycline treatment did result in significantly decreased levels of cd11b but only in the RC [F(2,8)=23.9, p<0.05] (see Figure 4B). The fact that only the levels of cd11b, an intergrin receptor involved in phagocytic activity, were decreased with minocycline treatment might suggest that reactive microglia in the nTS are involved in phagocytosis.\n\nFluorescent levels were measured on both intact and transected sides. In order to normalize the levels on the transected side (shown in the figure), the levels from the intact sides were subtracted from the transected sides. A. At the rostral level R3, minocycline treatment (dark bars) did not affect the intensity of Iba1 or cd11b staining compared to no treatment (white bars). The fluorescent intensity in the rostral central (RC) and ventral (V) subregions of the nTS as well as the neighboring spinal vestibular nucleus (SpVe) is not affected by minocycline treatment. B. At the rostral level R2, minocycline treatment did not affect Iba1 levels but resulted in significantly lower fluorescence levels of cd11b in the RC subnucleus, though the levels in V and SpVe were not affected. The asterisk () denotes a significant difference on the transected side compared to the intact side with p<0.05, SEM error bars, n=3 for each group.\n\nTLR4 mutants (C3H): To test the possibility that TLR4 signaling is essential for microglial activation, CTx was performed on the TLR4 mutant animals (C3H) as well as their wild-type siblings (C3HeB). The typical cluster of Iba1-labeled microglial cells appeared in the RC subnucleus on the transected side in the TLR4 mutant animals just as seen in wild type animals (Figure 5A,B). Microglia on the intact sides did not change (not shown). Further, the density of microglial cells also increased on the transected side compared to intact sides in the TLR4 mutant animals (Figure 5C) just as in wild type controls. Thus, these data suggest that TLR4 signaling is not required for microglial activity after CT injury.\n\nA. The number of Iba1+ microglia in the rostral central (RC) subnucleus of the nTS increases on the transected (Tx) side compared to the intact (Int) side in the C3H (TLR4 mutant) animals, as seen in C3HeB and C57BL6/J wild-type animals. An asterisk () denotes a significant difference on the transected side compared to the corresponding intact side with p<0.05, SEM error bars, n=3 for each group. B,C. Further, as seen in C57BL6/J wild-type animals the typical cluster of reactive Iba1+ microglia also appears in the RC subnucleus on the transected side in C3H (TLR4 mutant) animals.\n\nNaloxone: These experiments were performed in C57BL6/J animals and sought to block TLR4 signaling with naloxone treatment at the onset of the CT injury.\n\n\nBehavior testing with naloxone and CTx\n\nWe first sought to test whether naloxone was affecting functionality in the CNS. In other studies on rats, the administration of naloxone causes a decreased intake of preferred foods such as sweet-tasting chow or sugar solutions33–37. Here, we determined the animals’ sweet preference score as an index of naloxone effectiveness.\n\nDuring the training period, animals preferred the sucrose solution whether they were water deprived or not (Figure 6). The preference score was calculated [sucrose (mL)/total fluid (mL)] so that the higher the score the greater the sweet preference. Hence, a score > 0.5 is indicative of sucrose preference. After placement of minipumps with 10mg naloxone or saline followed by CT injury, the overall sucrose preference in naloxone-treated animals did not differ from saline control animals. Although two naloxone treated animals showed a decreased intake of sucrose the first day after treatment (Figure 6A, red and green lines), this behavior was not maintained. On the whole, the preference for sucrose did not consistently decrease in naloxone-treated animals that also underwent CTx.\n\nA. When mice had continual access to water and sucrose, the animals preferred the sucrose solution to water during the three-day training period. The preference score was calculated [sucrose (mL)/total fluid (mL)] such that the higher the score indicates the greater the sweet preference with 0.5 representing no preference (each line represents an individual animal). After surgery to implant naloxone or saline pumps followed by CTx, the mice that received 10mg naloxone still preferred sucrose on the whole. B. When mice were water deprived for 22.5 hours with access to water and sucrose the remaining 1.5 hours, all mice preferred the sucrose solution before and after surgery (each line represents an individual animal).\n\n\nBehavior testing with naloxone only\n\nTo test if CTx might be interfering with the animals’ sweet preference behavior, the animals were tested with naloxone treatment only. When animals were not water deprived, there was a significant decrease in the intake of sucrose on the second day of training (Figure 7A). As the mice had not yet received treatment, this suggests a disturbance or distraction during the training session. The next training day, mice resumed their intake of sucrose. However, after treatment with 10mg of naloxone the mice continued to prefer sucrose (i.e., scores near 1). Similarly, when animals were water deprived and only had access to the sucrose and water bottles for a few hours a day, treatment with 10mg naloxone did not cause a significant and consistent decrease in intake of sucrose solution (Figure 7B).\n\nA. When mice had continual access to water and sucrose, the animals showed less preference for sucrose on the second day of training (each line represents an individual animal). Because the animals had not yet received naloxone, this was likely a disturbance during the experiment. However, after naloxone treatment the animals still prefer sucrose solution to water. The preference score was calculated [sucrose (mL)/total fluid (mL)] such that the higher the score indicates the greater the sweet preference with 0.5 representing no preference (each line represents an individual animal). B. Similarly, when mice were water deprived for 21.5 hours with access to water and sucrose the remaining 2.5 hours, they maintained their preference for sucrose after treatment with 10mg naloxone (each line represents an individual animal).\n\nIn summary, 10mg of naloxone administered subcutaneously by minipumps did not cause a reduced intake in sucrose. This behavior had not been affected by CT injury, since the naloxone-treated animals with and without CTx showed similar taste preference behavior. This suggests that naloxone was not accessing the brain at a sufficient level to affect this neuronally mediated taste behavior.\n\n\nMicroglial studies with naloxone treatment\n\nIn naloxone-treated animals that received CTx, microglia still reacted throughout the CT terminal field at 3 days post injury. The typical cluster of Iba1 labeled microglial cells appeared in the RC subnucleus (Figure 8B–D). Further, the number of microglial cells on the transected side after nerve injury remains increased in the RC subnucleus with naloxone (naloxone; blue bars) treatment compared to no treatment (grey bars; Figure 8A). Thus, naloxone treatment did not affect microglial reactivity in the nTS in terms of morphology or increased density. This suggests that naloxone-sensitive TLR4 signaling is not required for microglial activity in the nTS.\n\nA. The number of microglia in the rostral central (RC) subnucleus increases on the transected side (Tx) compared to the intact side (Int) when treated with naloxone. An asterisk () denotes a significant difference on the transected side compared to the corresponding intact side with p<0.05, SEM error bars, n=3 for each group. B. At 3 days after CTx and ‘No treatment’ Iba1 staining reveals reactive microglia in the RC subnucleus on the transected side. C,D. Similarly, reactive microglia are present in the RC subnucleus in mice treated with 5mg or 10mg NLX.\n\n\nDiscussion\n\nThe current study sought to elucidate the mechanisms involved in microglial responses following chorda tympani transection (CTx). These experiments were designed to address different possibilities of microglial activation in the nTS.\n\nUsing the P2X-dlbKO mice, we tested whether the P2X2 and P2X3 receptors were necessary for microglial reactivity with CTx. Microglial cells in the nTS increased in number and were morphologically reactive in the P2X-dblKO animals just as seen in wild type animals. These experiments suggest that these purinergic receptors are not required for microglial reactivity in the nTS.\n\nInterestingly, the gustatory nerves of the P2X-dlbKO mice are not responsive to taste stimulation14. Hence, the fact that microglial reactivity still occurs following CT injury also suggests that microglia are reacting to more than just an interruption in normal neuronal signaling. One possible explanation is that microglia only respond to acute interruptions in normal neuronal activity. P2X-dblKO animals, however, represent a permanent loss of ‘normal’ nerve activity. Future experiments to acutely block activity of the CT nerve and subsequently examine microglial responses will be of interest.\n\nAlthough minocycline treatment reduces microglial reactivity in pain models, this treatment did not affect the microglial responses after CT injury. Rather, microglia in the nTS displayed reactive morphologies and increased in number in the RC subnucleus. Even though the number of microglia increased with minocycline, we measured the intensity levels of two microglial markers. While the level of Iba1 was not affected by minocycline treatment, the level of cd11b with minocycline treatment was significantly decreased compared to the ‘No treatment’ group in the RC but only in the R2 rostral level of the nTS. That minocycline did not have a greater effect on microglial responses after CT damage may be an issue of dosage. While the half-life of minocycline is about 15 hours in humans38, its half-life in rodents is reported to be ~3 hours39,40. Hence, to achieve steady state concentrations more frequent administration of larger doses of minocycline is likely necessary. Nevertheless, the level of cd11b did not significantly decrease in neighboring regions suggesting that the decreased level in the RC subnucleus is a specific effect. The fact that only the levels of cd11b, an intergrin receptor involved in phagocytic activity, were decreased with minocycline treatment is also suggestive that reactive microglia in the nTS are involved in phagocytosis.\n\nIn the central nervous system TLR4 is expressed by microglia and perhaps by astrocytes and endothelial cells but not by neurons41. Although best known as the receptor that detects cell wall components of gram-negative bacteria (i.e., LPS), TLR4 can also respond to “endogenous danger signals” or “alarmins”42. These are signals of cellular stress or disruption including the release of DNA, heat shock proteins and similar components that are normally concealed from immune surveillance. Many such substances are released after peripheral nerve injury, which in turn activate microglia to release a variety of neuroexcitatory and pain-enhancing substances43.\n\nIndeed, recent studies have shown that TLR4 signaling is involved in microglial signaling and related pain behaviors following spinal nerve injuries. Not only does deletion of TLR4 prevent pain from developing after spinal nerve injury22, but inhibiting TLR4 with naloxone can also reverse established pain states23,24.\n\nTLR4 mutants (C3H): The role of TLR4 signaling was tested by performing CT injury in TLR4 mutant animals. Following CT damage, microglial cells in the nTS increased in number and were morphologically reactive throughout the CT terminal field in the mutant animals, just as seen in wild type animals. These data suggest that TLR4 signaling is not necessary for microglial reactivity following CT injury. This might suggest that microglia in the nTS may rely on different signaling mechanisms than those described in models of chronic pain.\n\nNaloxone: This treatment has been demonstrated to diminish microglial reactivity in pain models. Interestingly, in addition to binding the classic opioid receptors, naloxone also binds to the Toll-like receptor 4 (TLR4) on microglia44. In the current experiments, naloxone was used to test whether microglial responses in the nTS were dependent on TLR4 signaling. First, to gage whether naloxone was indeed accessing the CNS, the animals’ sweet preference was measured. This is based on a wealth of literature demonstrating that opioid antagonists such as naloxone cause a reduced intake of sweet-tasting substances. The appetite suppressant effect of naloxone is centrally mediated, resulting from neuronal interactions in the reward pathway45. Based on other studies in rats, naloxone caused a decreased intake in sweet foods34,35. While this hypophagic effect is a result of naloxone binding to neuronal opiate receptors, the behavior is centrally mediated46 and hence indicates that naloxone is accessing the CNS. In the current experiments, animals preferred the sucrose solution during the training period as expected (i.e., preference score > 0.5). After treatment with naloxone, however, the overall sucrose preference did not differ and the animals continue to prefer sucrose. On the whole, the preference for sucrose did not consistently decrease in naloxone treated animals.\n\nWhile the anorectic effect of systemic naloxone is detectable in a variety of species, including humans47, rodent studies have relied entirely on the use of rats. Herein lies the primary difference between previous studies in rats and the current study in mice. Earlier reports from rats repeatedly show that subcutaneous doses of naloxone from 0.5–10mg/kg/hour caused a significantly reduced intake of sweet tasting food or drink for about 2 hours after treatment33–37. However, a continuous dosage of 6.96mg/kg/hour did not consistently reduce the sucrose intake in mice in the current study. This may also reflect the difference in the means of administration, i.e., experiments in rats routinely used acute subcutaneous injections of a higher concentration of drug rather than a chronic subcutaneous delivery of a lower dose. Regardless, the current experiments do not provide evidence that naloxone was accessing the CNS at a level sufficient to affect the neuronally mediated sweet preference behavior.\n\nNaloxone treatment also did not affect the microglial responses after CT injury. Rather, microglia in the nTS displayed reactive morphologies and increased in numbers. Naloxone has been shown to affect microglia via an acute subcutaneous administration to rats at 100mg/kg23. This caused full reversal of neuropathic pain behavior as well as decreased expression of the microglial marker, cd11b. Because the half-life of naloxone is about an hour, this acute administration can be roughly compared to the hourly dose received with chronic subcutaneous delivery. However, the highest dose that could be achieved in the current study was 6.96mg/kg/hour, less than ten times that of the acute dose. This is one possibility why microglial responses in the nTS were not affected by naloxone treatment.\n\nHowever, an alternate interpretation of these data is that naloxone did indeed access the CNS but did not affect the microglial responses. This could be explored by performing a sciatic nerve lesion in mice with the subcutaneous minipumps to deliver naloxone. The chronic naloxone treatment used here can be compared to the effects of acute naloxone, which effectively dampened the microglial response triggered by sciatic nerve injury23. If the subcutaneous minipumps used in the current studies block the microglial responses after sciatic injury, this would suggest that naloxone is accessing the CNS at an effective dose. Further, such a result would argue that signaling mechanisms involved in pain-related microglia reactivity are different than non-pain microglial reactivity, i.e., in the nTS in response to CT injury.\n\nAdditionally, naloxone could be administered directly into the brainstem with an intracisternal injection. Subcutaneous administration was chosen in the current study so as not to compromise the blood-brain barrier, a disturbance that can activate microglia. Yet a study in rats experiencing chronic pain used minipumps to deliver naloxone by injection into the intrathecal space (fluid-filled area of spinal cord located between the pia mater and the arachnoid mater). This treatment method successfully reversed pain behavior as well as decreased the expression of microglial markers23. This suggests that naloxone effectively dampened microglial responses that might have arisen from the intrathecal method (namely, making a small hole through the skull and disrupting the blood brain barrier). Using a similar intracisternal injection route of naloxone with CT injury would ensure that naloxone is reaching the brainstem, a question that could not be answered here either in terms of microglial responses or neuronal changes (i.e., taste behavior).\n\n\nSummary\n\nPeripheral injury to the CT results in central microglial responses. Even though the CT lacks pain signaling, the general profile of this microglial activation in terms of morphological reactivity and increased density is similar to that seen in nerve injury models of chronic pain4. The current experiments examined several possible mechanisms that could be involved in microglial reactivity after CT injury. Treatment with minocycline, a compound that dampens microglial responses in pain models, was largely ineffective in diminishing microglial responses after CT injury. Also, TLR4 signaling, which is central to microglial reactivity following spinal nerve injury, does not seem to be required for microglial responses following CT injury. Finally, signaling through P2X2 and P2X3 is also not necessary for microglial reactivity following CT damage, which might also suggest that microglia are responding to more than an interruption of nerve signaling. Taken together, these data suggest that reactive microglia in the nTS rely on different signaling mechanisms than those described in models of chronic pain. Nevertheless, alternative treatment protocols with minocycline and naloxone warrant further investigation.", "appendix": "Author contributions\n\n\n\nDLB conceived the concept of the study, carried out the research and prepared the first draft of the manuscript. TEF contributed to the experimental design and analysis of data. Both authors were involved in the revision of the draft manuscript and have agreed to the final content and submission of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by NIH grants DC009762-01 F31 (D.L.B.), NIDCD RO1 DC0000147 (T.E.F.), R56 DC0000147 (T.E.F.), HD41697-05 T32, P30 DC04657 (D. Restrepo) and Dept Public Health Service, Training Grant #HD41697-05 (Type 5, T32).\n\n\nReferences\n\nClark MP, O'Malley S: Chorda tympani nerve function after middle ear surgery. Otol Neurotol. 2007; 28(3): 335–40. PubMed Abstract \| Publisher Full Text\n\nCheal M, Oakley B: Regeneration of fungiform taste buds: temporal and spatial characteristics. J Comp Neurol. 1977; 172(4): 609–26. PubMed Abstract \| Publisher Full Text\n\nGuagliardo NA, Hill DL: Fungiform taste bud degeneration in C57BL/6J mice following chorda-lingual nerve transection. J Comp Neurol. 2007; 504(2): 206–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBartel DL: Glial responses after chorda tympani nerve injury. J Comp Neurol. 2012; 520(12): 2712–29. PubMed Abstract \| Publisher Full Text\n\nNin T, Sakagami M, Sone-Okunaka M, et al.: Taste function after section of chorda tympani nerve in middle ear surgery. Auris Nasus Larynx. 2006; 33(1): 13–7. 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PubMed Abstract\n\nStephens MK, Riley AL: Naloxone-precipitated conditioned taste aversions in morphine-dependent Fischer (F344) and Lewis rat strains. Pharmacol Biochem Behav. 2009; 92(1): 60–7. PubMed Abstract \| Publisher Full Text\n\nAgwuh KN, MacGowan A: Pharmacokinetics and pharmacodynamics of the tetracyclines including glycylcyclines. J Antimicrob Chemother. 2006; 58(2): 256–65. PubMed Abstract \| Publisher Full Text\n\nAndes D, Craig WA: Animal model pharmacokinetics and pharmacodynamics: a critical review. Int J Antimicrob Agents. 2002; 19(4): 261–8. PubMed Abstract \| Publisher Full Text\n\nFagan SC, Edwards DJ, Borlongan CV, et al.: Optimal delivery of minocycline to the brain: implication for human studies of acute neuroprotection. Exp Neurol. 2004; 186(2): 248–51. PubMed Abstract \| Publisher Full Text\n\nMiyake K: Innate immune sensing of pathogens and danger signals by cell surface Toll-like receptors. Semin Immunol. 2007; 19(1): 3–10. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "821", "date": "08 Mar 2013", "name": "Odd-Geir Berge", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 results are interesting and worth publishing. I would like to make a few points, however. Reporting of methods could have been more complete, there are, for instance, no mentioning of whether any measures were taken to randomize animals to treatment groups or blind the observer to treatments. Neither is there much description of how the animals recovered after the various procedures. Applying one of several recently published guidelines aimed at improving experimental design and reporting, e.g. “Kilkenny, C., Browne, W.J., Cuthill, I.C., Emerson, M., Altman, D.G. (2010) Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research PLoS Biol. 8(6)”, might have been useful. An estimate of the power of the design to detect a reasonable effect of the treatments would have been warranted given the relatively small number of animals in each experimental group. Finally, the authors consistently refer to neuropathic model readouts as pain. This is common practice but nevertheless controversial. The models nearly exclusively measure evoked responses to stimuli that may or may not provoke pain and changes in sensory thresholds or perception are not well correlated to clinical pain. A more stringent vocabulary would make interaction between different professions easier.", "responses": [] }, { "id": "883", "date": "08 Apr 2013", "name": "Lynnette Phillips McCluskey", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 investigate whether microglial responses to gustatory nerve injury parallel activation in neuropathic pain. This context was helpful as a guide to the experimental progression, as well as a model for their hypotheses.The experiments were conducted appropriately. Though group sizes were small, the results appeared to be straightforward and could be replicated. A brief rationale for the different behavioural schedules would be useful in that regard.\n\nThe idea that the discrepancy in afferent activity after nerve sectioning (vs. the absence of activity in P2X KO mice) contributes to microglial responses is intriguing and will be interesting to pursue. Distinct receptor subtypes, such as P2X4, may also be involved in microglial responses in the NTS.", "responses": [] }, { "id": "907", "date": "23 Apr 2013", "name": "Susan Travers", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 an interesting follow-up to the first author’s recent report (JCN, 2012) that chorda tympani (CT) nerve section causes a robust increase in the number and functional activation of microglia in the rostral nucleus of the solitary tract (rNST), an increase largely confined to the terminal field of that nerve. In the initial paper, the authors established that the mechanism of microglial responses in rNST was distinct from the better-studied response in nociceptive regions of the CNS because of the lack of immune cell migration from bone marrow. The current study provides additional data suggesting that the microglial response in the NST is distinct—it is attenuated only marginally following minocycline treatment and does not rely on the toll-like 4 receptor, again in contrast to what occurs after injury of nociceptive afferents. It is also noteworthy that the microglial response persists in P2X2/P2X3 double knockout mice, suggesting that a lack of gustatory-driven activity does not drive the effect. The methods, including the analytical approach, were thoroughly explained or suitable references provided. The experimental design was appropriate and the methods used seemed adequate to test the hypotheses advanced. Because the outcomes were essentially negative, however, it would have been more compelling if an example of a positive control had been presented to show that these authors could replicate the attenuation of the microglial response in the pain system, the system from which these techniques were adapted. On the other hand, the fact that 2 different approaches were used to probe the role of TLR-4 is reassuring. The authors’ conclusions are based on the data, which are quite clear, presented in detail, and discussed in an appropriate and scholarly fashion.", "responses": [] } ]	1	https://f1000research.com/articles/2-65
https://f1000research.com/articles/2-63/v1	27 Feb 13	{ "type": "Research Article", "title": "Architecture of the superintegron in Vibrio cholerae: identification of core and unique genes", "authors": [ "Michel A Marin", "Ana Carolina P Vicente", "Ana Carolina P Vicente" ], "abstract": "Background: Vibrio cholerae, the etiologic agent of cholera, is indigenous to aquatic environments. The V. cholerae genome consists of two chromosomes; the smallest of these harbors a large gene capture and excision system called the superintegron (SI), of ~120 kbp. The flexible nature of the SI that results from gene cassette capture, deletion and rearrangement is thought to make it a hotspot of V. cholerae diversity, but beyond the basic structure it is not clear if there is a core genome in the SI and if so how it is structured. The aim of this study was to explore the core genome structure and the differences in gene content among strains of V. cholerae.Methods: From the complete genomes of seven V. cholerae and one Vibrio mimicus representative strains, we recovered the SI sequences based on the locations of the structural gene IntI4 and the V. cholerae repeats. Analysis of the pangenome, including cluster analysis of functional genes, pangenome profile analysis, genetic variation analysis of functional genes, strain evolution analysis and function enrichment analysis of gene clusters, was performed using a pangenome analysis pipeline in addition to the R scripts, splitsTree4 and genoPlotR.Results and conclusions: Here, we reveal the genetic architecture of the V. cholerae SI. It contains eight core genes when V. mimicus is included and 21 core genes when only V. cholerae strains are considered; many of them are present in several copies. The V. cholerae SI has an open pangenome, which means that V. cholerae may be able to import new gene cassettes to SI. The set of dispensable SI genes is influenced by the niche and type species. The core genes are distributed along the SI, apparently without a position effect.", "keywords": [ "Vibrio cholerae", "superintegron", "core genes", "unique genes" ], "content": "Introduction\n\nVibrio cholerae is a diverse, environmental, gram-negative bacterial species that can be pathogenic and can cause cholera, a severe diarrheal disease that occurs most frequently in epidemic form1,2. The V. cholerae genome consists of two chromosomes. The largest chromosome of 2.96 Mbp encodes most essential genes. The 1.07 Mbp small chromosome contains few essential genes and the superintegron (SI), a large gene capture and excision system of ~120 kbp2 (Figure 1). The SI is characterized by a site-specific integrase gene (IntI4) closely associated with a cognate recombination site attI and a promoter Pc followed by a large array of gene cassettes. Within the SI, the gene cassettes generally consist of a promoterless open reading frame (ORF) flanked by two recombination sites termed V. cholerae repeats (VCRs)3. Cassettes can be excised from any position in the array through VCR × VCR recombination mediated by the integrase. The resulting circular intermediate can then be integrated, preferentially through attI × VCR recombination by the integrase, bringing the cassette under control of Pc4,5. Since gene cassettes are usually promoterless, only the first few cassettes are expressed by Pc and the rest of the array can be seen as a reservoir of standing genetic variation5.\n\nThe functional platform of the SI consists of an integrase gene, a cassette promoter (Pc), and a primary recombination site (attI). The system maintains an array of several cassettes, which generally consist of a promoterless ORF flanked by two recombination sites termed VCR (V. cholerae repeats).\n\nThe functions of the majority of the SI genes are unknown; however, a few genes have been characterized and it has been suggested that they are involved in adaptive functions such as toxin-antitoxin (TA) loci. TA loci consist of two genes in an operon encoding a ‘toxin’ and an ‘antitoxin’. The expression of the toxins reduces cell growth and prevents colony formation, thus exerting a bacteriostatic rather than bacteriocidal condition. However, cell viability can be rescued by later overproduction of the cognate antitoxins6.\n\nThe pangenome describes the complete repertoire of genes in a bacterial species, which includes the \"core genome\" containing genes present in all strains, a \"dispensable genome\" containing genes present in two or more strains, and \"unique genes\" specific to single strains7. Previous phylogeographic analysis, considering V. cholerae strains and its sister species Vibrio metecus8, showed that, in contrast to the core genome, the SI displays strong geographical differentiation, and cassettes from the V. cholerae group cluster with those of V. metecus from the same place rather than with cassettes from geographically distinct V. cholerae. It suggested that SI structure is influenced by geographic boundaries and in response to environmental conditions. The flexible nature of the SI that results from gene cassette capture, deletion and rearrangement is thought to make it a hotspot of V. cholerae diversity, but beyond the basic structure it is not clear if there is a core genome in the SI and if so how it is structured. The aim of this work was to explore the core genome structure and the differential gene content among strains of V. cholerae.\n\n\nMethodology\n\nBased on the complete genomes of seven V. cholerae and one V. mimicus representative strains (Table 1), we searched repeats above 10 nucleotides and used one VCR sequence (AAC AAA CGC CTC AAG AGG GAC TGT CAA CGC GTG GCG TTT CCA GTC CCA TTG AGC CGT GGT GGT TTC GGT TGT TGT GTT TGA GTT TAG TGT TAT GCG TTG TCA GCC CCT TAG GCG GGC G) to search for sequences with more of 45% nucleotide identity. The SI sequences were recovered using the locations of the structural gene IntI4 and VCRs identified with the UGENE software9. Cluster analysis of functional genes was performed using the pangenome analysis pipeline10, which searches for homologs or orthologs among multiple genomes using the MultiParanoid (MP) method (based on a 90% nucleotide identity threshold). For each pair of genes in the same cluster, the local matched region is no less than 25% of the longer protein coding sequence and the global matched region is no less than 50% of the longer protein coding sequence. The minimum score value and E-value in BLAST are 50 and 1e-810. The gene content was converted to a presence/absence (0/1) matrix and then the core, dispensable and unique genes were identified by in-house R scripts. The phylogenetic tree based in gene content and split network for gene content were constructed with SplitsTree411 using the GeneContentDistance method12. The SI structure and comparison of seven V. cholerae and, their sister species, V. mimicus were performed using genoPlotR13.\n\nNucleotide position on the chromosome. ND, not determined.\n\n\nResults and discussion\n\nSI regions were extracted from the seven V. cholerae and one V. mimicus genomes (Table 1). The 1285 genes recovered were clustered and a total of 408 clusters were detected (Figure 2A; Table S1). The pangenome of the SI of Vibrio strains evaluated was 408 genes, of which eight correspond to core genes, 196 are distributed or dispensable genes and 204 are unique genes. Six of the eight core genes are present in many copies (Table 2). The pangenome profile analysis shows that the cluster numbers of core genome are almost the same, when the SI considered reaches nine, while the pangenome is still increasing (Figure 2A). We infer that the V. cholerae SI has an open pangenome, which means that V. cholerae may have the ability to import new SI gene cassettes, which affect its plasticity and diversity. On the other hand, the set of SIs, from clinical and environmental lineages, used in this study are apparently representative of this species because allowed to establish that the core genome is close to being completed.\n\n(A) Pangenome plot of the SI region considering seven V. cholerae and one V. mimicus genomes. 1285 total genes, 408 pangenome clusters and eight core clusters were identified. (B) Word clouds of cluster function enrichment comparison according to clusters of orthologous groups (COG) for whole and core clusters identified are shown at the top and bottom, respectively. Clusters that are not assigned in the COG classification were excluded from the figure.\n\nThe table shows the clusters, conservation level between genomes, the functional categories, gene description and the corresponding locus tag in the reference N16961 genome.\n\nCOG: Cluster of Orthologous Groups; \"-\" depicts no COG assignation.\n\nFunction enrichment analysis of gene clusters were performed according to description of gene annotation (File S1) supplied to the pangenome analysis pipeline10. From the 408 clusters, 329 were unclassified by the function enrichment analysis. Following the categorization of Cluster of Orthologous Groups (COG), the characterized clusters were rich in the following categories: translation, ribosomal structure and biogenesis, transcription, replication, recombination and repair, cell cycle control, cell division, chromosome partitioning, defense mechanisms, cell wall/membrane/envelope biogenesis and posttranslational modification, protein turnover, chaperones, amino acid transport and metabolism, nucleotide transport and metabolism, lipid transport and metabolism, secondary metabolites biosynthesis, transport and catabolism (Figure 2B).\n\nIn the SI, random excisions occur throughout the cassette array to form nonreplicative circular intermediates containing one or several cassettes; integration events preferentially occur at the attI site5 and are subjected to selection. It is expected that SI core genes would be arranged and stay together; however, we found the core genes are distributed along the SI (Figure 3), apparently without any position effect.\n\nWe identified 204 unique genes, 94 belonging to V. mimicus MB451, nine to LMA3984, 45 to O395, nine to 2010EL1786, 14 to MJ1236, seven to IEC224, 20 to M66, and six to N16961 (Figure 3; Table S1). Considering only the V. cholerae SI, there are 21 core genes, most of them present in many copies and rich in the transcription, replication, recombination and repair, translation, ribosomal structure and biogenesis categories.\n\nThe core, dispensable and unique genes are indicated by red, cream and blue arrows, respectively. Vertical blocks between sequences indicate regions with more than 1 kb of shared similarity shaded according to BLASTn. A phylogenetic tree based on gene content of the SI is shown on the left.\n\nPandey and Gerdes14 identified 13 TA loci within the SI of the N16961 strain. Here we identified six TA genes as part of core SI genes (Table 2), of which the relB genes (VCA0349 and VCA0504) were present in all V. cholerae strains (including V. mimicus) SIs. The parE (VCA0359), relB (VCA0477) and relE (VCA0489) genes were present in all V. cholerae SIs. Moreover, we also identified two higBA loci (VCA0469 and VCA468), which encode mRNA cleaving enzymes and can stabilize plasmids6, as well as SI genes. The previous authors14 also identified higBA-1 TA loci (VCA0392 and VCA0391); in our results, these two TA loci are present in all clinical V. cholerae strains (Table S1). These results suggest that V. cholerae TA loci function as essential stress response elements that help cells survive6, as well as act to stabilize the massive arrays of SI cassettes, as reported previously15.\n\nA previous study suggested that SI structure is influenced by geographic boundaries in response to environmental conditions8. Here, we found that the clinical nature of the V. cholerae and V. mimicus strains evaluated were not grouped together by the analyses performed. Therefore, the ability of V. cholerae to cause disease must be explained by other virulence factors found outside the SI region.\n\nThere are 199 clusters involved with indel or mutation events (Table S2). As for the non-synonymous/synonymous substitution (dN/dS) ratio, we found that 30 clusters were suffering positive selection pressure (dN/dS > 1). At the same time, we could also select those variable clusters as the markers for different strains. Based on pangenome profiles and single nucleotide polymorphism (SNP) information, gene content and phylogenetic trees were constructed (Figure 4). The SNP information from SI was useful for separating V. cholerae from V. mimicus, but nevertheless lacked the resolution to distinguish between the different lineages of V. cholerae. However, using gene content information (Figure 4), a good resolution was reached that was coherent with the evolution of the species and the environmental or clinical nature of the strains. These results indicate that the evolution of V. cholerae into different lineages is reflected in the diversity of the SI, which would be also influenced by horizontal gene transfer in these region, as proposed elsewhere8,16,17.\n\nTop: Phylogenetic trees for the V. cholerae SI based on SNPs constructed by the Maximum Likelihood (A), Neighbor-Joining (B) and UPGMA (C) methods. The numbers indicate the bootstrap values. Bottom: Split network for gene content based on the 408 genes in seven V. cholerae and one V. mimicus genomes. The network was constructed with SplitsTree4 using the GeneContentDistance method12.\n\n\n\n\n\n\n\n\n\n\nConclusions\n\nIn this study, we have revealed the genetic architecture of the V. cholerae SI, which contains eight core genes, many of them present in many copies. The V. cholerae SI has an open pangenome, which means that V. cholerae may have the ability to import new gene cassettes into the SI. The set of the dispensable SI gene cassettes is influenced by the niche and type species. The core genes are distributed along the SI, apparently without a position effect.", "appendix": "Author contributions\n\n\n\nMAM and ACPV designed the study. MAM and ACPV analyzed the data. MAM and ACPV wrote the article. All authors have approved the final manuscript for publication.\n\n\nCompeting interests\n\n\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) doctoral fellowship to MAM.\n\n\nSupplementary tables\n\nTable S1. Orthologs clusters identification among SIs from V. cholerae and V. mimicus genomes. These clusters were identified using the pangenome analysis pipeline10, strains without genes in the cluster are marked with \"-\".\n\nTable S2. Clusters involved with indel or mutation events. The 1st column is the Cluster ID, which is consistent with the ID in Table S1. The 2nd column is the cluster conservation of current cluster. The 3rd column is the variation position, which counts according to the alignment result of protein sequences in this cluster. For indel events, the position is an integer. For synonymous mutation and non synonymous mutation, the position is a floating number, in which the integer part marks the position of the amino acid in the alignment result of protein sequences, while the decimal part mark the position of codon. The 4th column shows the amino acid types on current position. The 5th column shows the nucleotide types on current position, indel is marked with \"-\". The 6th column shows all gene nucleotide profile in current position (for indel, amino acid will be listed). The 7th column shows the variation type (indel, synonymous and non synonymous). The CDS.variation.analysis spreadsheet shows the summary result for CDS.variation.\n\n\nReferences\n\nBlokesch M, Schoolnik GK: Serogroup conversion of Vibrio cholerae in aquatic reservoirs. PLoS Pathog. 2007; 3(6): e81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeidelberg JF, Eisen JA, Nelson WC, et al.: DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature. 2000; 406(6795): 477–83. PubMed Abstract \| Publisher Full Text\n\nMazel D: Integrons: agents of bacterial evolution. Nat Rev Microbiol. 2006; 4(8): 608–20. PubMed Abstract \| Publisher Full Text\n\nRowe-Magnus DA, Guérout AM, Mazel D: Super-integrons. Res Microbiol. 1999; 150(9–10): 641–51. PubMed Abstract \| Publisher Full Text\n\nCambray G, Guerout AM, Mazel D: Integrons. Annu Rev Genet. 2010; 44: 141–66. PubMed Abstract \| Publisher Full Text\n\nChristensen-Dalsgaard M, Gerdes K: Two higBA loci in the Vibrio cholerae superintegron encode mRNA cleaving enzymes and can stabilize plasmids. Mol Microbiol. 2006; 62(2): 397–411. PubMed Abstract \| Publisher Full Text\n\nMedini D, Donati C, Tettelin H, et al.: The microbial pan-genome. Curr Opin Genet Dev. 2005; 15(6): 589–94. PubMed Abstract \| Publisher Full Text\n\nBoucher Y, Cordero OX, Takemura A, et al.: Local mobile gene pools rapidly cross species boundaries to create endemicity within global Vibrio cholerae populations. MBio. 2011; 2(2): e00335–10. PubMed Abstract \| Publisher Full Text\n\nOkonechnikov K, Golosova O, Fursov M: Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012; 28(8): 1166–7. PubMed Abstract \| Publisher Full Text\n\nZhao Y, Wu J, Yang J, et al.: PGAP: pan-genomes analysis pipeline. Bioinformatics. 2012; 28(3): 416–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuson DH, Bryant D: Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006; 23(2): 254–67. PubMed Abstract \| Publisher Full Text\n\nHuson DH, Steel M: Phylogenetic trees based on gene content. Bioinformatics. 2004; 20(13): 2044–9. PubMed Abstract \| Publisher Full Text\n\nGuy L, Kultima JR, Andersson SG: genoPlotR: comparative gene and genome visualization in R. Bioinformatics. 2010; 26(18): 2334–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPandey DP, Gerdes K: Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res. 2005; 33(3): 966–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRowe-Magnus DA, Guerout A, Biskri L, et al.: Comparative analysis of superintegrons: engineering extensive genetic diversity in the Vibrionaceae. Genome Res. 2003; 13(3): 428–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGao Y, Pang B, Wang HY, et al.: Structural variation of the superintegron in the toxigenic Vibrio cholerae O1 El Tor. Biomed Environ Sci. 2011; 24(6): 579–92. PubMed Abstract \| Publisher Full Text\n\nFeng L, Reeves PR, Lan R, et al.: A recalibrated molecular clock and independent origins for the cholera pandemic clones. PLoS One. 2008; 3(12): e4053. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "937", "date": "09 May 2013", "name": "Thandavarayan Ramamurthy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript the authors have explored the pattern of the superintegrons (SIs) in V. cholerae using the published DNA sequences of relevant strains. This study has shown the dynamic nature of V. cholerae O1 and genetic relatedness of SIs at the biotype level.Comments:Abstract: Results and Conclusions: replace the word ‘reveal’ with “describe”Introduction: first paragraph: replace the word ‘standing’ with “standby”Page 6. The role of TA gene should be validated with non-toxigenic strains of V. cholerae (e.g., sequence comparison with non-O1, non-O139 strains of V. cholerae", "responses": [] }, { "id": "3764", "date": "14 Mar 2014", "name": "Yan Boucher", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article, although it has a very narrow focus, investigates an interesting question about integron regions in V. cholerae that scientists have so far mostly applied to whole genome sequences: what is their pan-genome and core genome? The scope of the question is quite narrow, as it focuses on a single species (V. cholerae), but is certainly novel.The methods and approach used are sound and the results generally well presented. I have two major issues with the analysis:Why use only seven genomes when >200 are available? This is quite puzzling to me, as no data should be excluded if it is available. Some of the >200 V. cholerae genomes available are relatively redundant (they all belong to the 7th pandemic group) and present little gene content/SNP diversity, but the variations they present are crucial in understanding small scale variability. Many available environmental V. cholerae genomes were also not included and should have been. The article is mostly descriptive and needs to make more informative statements about the results. What is the significance of such a small core genome? How does the pan-genome of the integron overlap with the rest of the genome? What is the meaning of the specific subset of functions found in the integron? How are sites under positive selection distributed in the integron? What genes are under positive/ negative selection? Much more can be said about integrons from the analyses performed.Basically, the manuscript is interesting but could really benefit from a broader (more genomes) analysis and a more in-depth look at the results to infer hypotheses about integrons in V. cholerae.", "responses": [] }, { "id": "4479", "date": "14 Apr 2014", "name": "Nur Hasan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Marin and Vicente investigated the genomic diversity of V. cholerae Super Integron (SI) with the aim to identify a set of orthologous genes that are conserved and unique among and in between V. cholerae and V. mimicus SI’s. While one must appreciate the efforts that have gone into the analyses, unfortunately, given the known diversity of V. cholerae SI, the number of genomes analyzed was very limited, and was not a good representative of all major phyletic lineages of V. cholerae either. Yet, the manuscript provides some valuable information about the diversity and repository of SI genes and their biological functions. The core genes estimates for SI (21 and 8, among V. cholerae and V. cholerae-V. mimicus respectively) reported in this study may not be very meaningful as the core genome might diminish or at least reduced further if additional genomes from distinct lineages are included. I strongly recommend inclusion of additional genomes at least from the major phylogenetic lineages of V. cholerae O1. The function of TA loci as an essential stress response element needs to be supported by some experimental data.", "responses": [] } ]	1	https://f1000research.com/articles/2-63

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