Neoadjuvant chemotherapy before surgery improves survival compared with surgery alone for patients with oesophageal cancer. The OE05 trial assessed whether increasing the duration and intensity of neoadjuvant chemotherapy further improved survival compared with the current standard regimen.9 cells per L, platelet count at least 100 × 109 platelets per L, and a glomerular filtration rate at least 60 mL/min. Participants were randomly allocated (1:1) using a computerised minimisation program with a random element and stratified by centre and tumour stage, to receive two cycles of cisplatin and fluorouracil  or four cycles of epirubicin, cisplatin, and capecitabine  before surgery, stratified according to centre and clinical disease stage. Neither patients nor study staff were masked to treatment allocation. Two-phase oesophagectomy with two-field (abdomen and thorax) lymphadenectomy was done within 4–6 weeks of completion of chemotherapy. The primary outcome measure was overall survival, and primary and safety analyses were done in the intention-to-treat population. This trial is registered with the ISRCTN registry (number 01852072) and ClinicalTrials.gov (NCT00041262), and is completed.OE05 was an open-label, phase 3, randomised clinical trial. Patients with surgically resectable oesophageal adenocarcinoma classified as stage cT1N1, cT2N1, cT3N0/N1, or cT4N0/N1 were recruited from 72 UK hospitals. Eligibility criteria included WHO performance status 0 or 1, adequate respiratory, cardiac, and liver function, white blood cell count at least 3 × 10vs 101 [23%] of 441 people in the ECX group). The proportions of patients with postoperative complications  were similar between the two groups. One patient in the ECX group died of suspected treatment-related neutropenic sepsis.Between Jan 13, 2005, and Oct 31, 2011, 897 patients were recruited and 451 were assigned to the CF group and 446 to the ECX group. By Nov 14, 2016, 327 (73%) of 451 patients in the CF group and 302 (68%) of 446 in the ECX group had died. Median survival was 23·4 months (95% CI 20·6–26·3) with CF and 26·1 months (22·5–29·7) with ECX . No unexpected chemotherapy toxicity was seen, and neutropenia was the most commonly reported event . Alternative chemotherapy regimens and neoadjuvant chemoradiation are being investigated to improve outcomes for patients with oesophageal carcinoma.Cancer Research UK and Medical Research Council Clinical Trials Unit at University College London. Meta-analyses of randomised trials support the use of neoadjuvant chemotherapy or chemoradiotherapy before surgical resection of locally advanced oesophageal cancer to improve survival.24Evidence before this studyOn Jan 12 and 13, 2017, we searched PubMed and the abstracts of major conferences such as the American Society of Clinical Oncology for publications in English, with no publication date restrictions. The search terms used were “chemotherapy”, “neo-adjuvant (or neoadjuvant)”, and “oesophageal ”. We identified two meta-analyses comparing neoadjuvant chemotherapy with surgery alone in patients with oesophageal cancer. These meta-analyses showed a significant survival benefit after treatment with neoadjuvant chemotherapy, both overall and for the subset of patients with adenocarcinoma who were considered in this study. These meta-analyses were unable to assess the benefits of different neoadjuvant chemotherapy regimens, and we can identify no phase 3 randomised trials that have directly compared different regimens.Added value of this studyTo our knowledge, this is the largest randomised trial investigating whether an alternative neoadjuvant chemotherapy regimen might offer a survival benefit over the standard of two cycles of cisplatin and fluorouracil (CF). This trial showed that giving four cycles of epirubicin, cisplatin, and capecitabine (ECX) neoadjuvantly might increase the level of tumour regression, but does not lead to any survival benefit.Implications of all available evidenceFor patients with oesophageal adenocarcinoma, two cycles of CF should remain the standard choice of neoadjuvant chemotherapy regimen. Further research is ongoing into the use of neoadjuvant chemoradiotherapy, but little evidence exists that directly compares it to neoadjuvant chemotherapy.High-quality data on the effect of neoadjuvant chemotherapy or chemoradiotherapy on health-related quality of life (HRQL) are scarce; more information is needed for clinical decision making, where small survival benefits might only be achieved with detrimental effects on many aspects of HRQL.vs 34%; 9% increase [95% CI 3–14]), with a hazard ratio (HR) of 0·79 (95% CI 0·67–0·93), so two-cycle cisplatin with fluorouracil was used for the control group in this study. The randomised clinical MRC MAGIC trialThe results of the previous Medical Research Council (MRC) OE02 randomised clinical trialThe aims of the OE05 trial were therefore to investigate whether four cycles of preoperative ECX improves survival and HRQL compared with standard two-cycle cisplatin and fluorouracil (CF) in patients with locally advanced resectable adenocarcinoma of the oesophagus.9 cells per L, platelet counts at least 100 × 109 platelets per L, and the calculated or measured glomerular filtration rate at least 60 mL/min.The UK MRC OE05 study is an open-label, phase 3, randomised clinical trial done in 72 hospitals across the UK. Participants were of any age with surgically resectable histologically verified adenocarcinoma of the oesophagus  stage cT1N1, cT2N1, cT3N0/N1, or cT4N0/N1 where invasion was thought to be confined to diaphragm, crura, or mediastinal pleura and surgically resectable  was done on the basis of a multidisciplinary team discussion following endoscopy, endoscopic ultrasonography, CT, and laparoscopy if appropriate.Patients were ineligible if investigations indicated blood-borne metastases , peritoneal dissemination, local invasion involving the tracheobronchial tree, aorta, pericardium or lung, or abdominal para-aortic lymphadenopathy greater than 1 cm in diameter on CT scan or more than 6 mm in diameter on endoscopic ultrasonography. Patients were also excluded if they had received any previous treatment for oesophageal cancer, had Siewert type 3 cancer, a medical condition that was likely to compromise the proposed trial treatment. Uncontrolled angina pectoris, myocardial infarction in the 6 months before entry into the trial, heart failure, clinically significant uncontrolled cardiac arrhythmias, or any patient with a clinically significant abnormal ECG, as well as patients with abnormal left ventricular ejection fraction (LVEF) diagnosed on MUGA scan or echocardiography, including areas of abnormal contractility, were excluded. Patients with positive serology for HIV or hepatitis C, active hepatitis B, or were pregnant were also excluded.Participating centres were considered eligible if they had a multidisciplinary team structure, had experience of two-phase oesophagectomy with two-field lymphadenectomy , access to multislice CT and endoscopic ultrasonography equipment, and had pathologists who were experienced in reporting oesophageal cancer.The protocol was approved by the South West Multicentre Research Ethics Committee (04/6/005) and all patients gave written consent for participation in the study. Blood and tissue samples were collected for potential future translational studies (Trans-OE05). The protocol had five amendments during the course of the trial, predominantly for administrative reasons or to add clarity; amendments did not affect the eligibility or treatment of patients. The most recent version of the protocol (version 6.0) is available online.Eligible patients were enrolled by staff at participating centres who then called the randomisation line at the MRC Clinical Trials Unit at UCL . Patients were randomly assigned (1:1) to either two cycles of CF or four cycles of ECX. Allocation was done using a computerised minimisation program, with a random element, and stratified by centre and tumour stage. No masking to treatment allocation was done because of the difference in the number of chemotherapy cycles in the two groups.2) was infused intravenously on day 1 and fluorouracil (1 g/m2 per day) was administered intravenously on days 1–4 .The control group chemotherapy regimen was two 3-weekly cycles of cisplatin and fluorouracil. Cisplatin (80 mg/m2) and cisplatin (60 mg/m2) were given intravenously on day 1 and capecitabine (1250 mg/m2 daily) was given orally continuously over all four cycles .The investigational chemotherapy was four 3-weekly cycles of ECX. Epirubicin  lymphadenectomy. Centres were allowed to use a minimal access surgery approach after providing evidence that complication rates and lymph node yields were similar to an open surgery approach from at least 20 patients who had had minimal access oesophagectomy.The stomach was the preferred conduit for reconstruction based on the right gastric and right gastroepiploic vessels. Lymph node clearance in the abdomen was at the origin of the left gastric artery and along the hepatic and splenic arteries, the upper lesser curve, and at the right and left cardiac lymph node stations. Dissection at the diaphragm was used to minimise a positive radial or circumferential resection margin by inclusion of sufficient crural fibres and a cuff of diaphragm based on endoscopic ultrasonography and intraoperative appearances. Pyloroplasty, pyloromyotomy, the placement of a feeding jejunostomy, and drain insertions were all according to local practice.Either a right or left thoracic approach was permitted. Lymph node dissection involved para-oesophageal, subcarinal, and bronchial lymph node stations, and thoracic duct ligation just above the diaphragm was advised. The extent of proximal lymphadenectomy for the upper paraoesophageal nodes was determined by the extent of division of the oesophagus .Reconstruction was recommended above the aortic arch for the right-sided approach and just below it for the left, and preferably within 5 cm of the thoracic inlet. The anastomotic technique and method of chest drainage was according to local practice. Trans-hiatal surgery was not permitted.Postoperative complications, described as none, present but not life threatening, or life threatening, were recorded for each patient. These complications were reviewed clinically, and assigned to categories.Processing of the surgical resection specimens and histopathological assessment of all materials were done according to the recommendations of the Royal College of Pathologists:33Pathology data were collected from local pathologists at each centre, and used for the primary analyses of all pathology data. A second review of the Mandard primary TRG was done to assess data quality and the differences between local and central assessments of TRG. One of three experienced pathologists  independently reviewed all resection slides and provided a new Mandard TRG. If the reviewing pathologist's TRG agreed with the local pathologist assessment, this TRG was accepted as the true grade. If the reviewing pathologist disagreed, a second, and if necessary a third, pathologist reviewed the slides to reach a majority decision on the Mandard TRG. These centrally reviewed data form a secondary analysis of TRG.All participating patients were asked to complete the validated generic cancer questionnaire (European Organisation for Research and Treatment of Cancer [EORTC] QLQ-C30)The primary outcome measure of the trial was overall survival. Secondary outcomes were disease-free survival, effects on the primary tumour (as assessed by Mandard TRG), HRQL, and morbidity related to chemotherapy and surgery. Exploratory outcomes were progression-free survival , and an analysis of Mandard TRG using an amended responder definition.The sample size calculations were based on the primary outcome measure of overall survival. On the basis of the OE02 trial results,Although we anticipated that the required events would be obtained 2 years after the final patient was randomly assigned, they accrued far more slowly than expected. 3 years after the final randomisation, a conditional survival analysis was done to assess the probability that continuing to wait for the full number of events would lead to a different trial conclusion. After discussion with the independent data monitoring committee and trial steering committee, a decision was reached that the current data were sufficiently robust for full analysis and dissemination, because the chance of obtaining a different conclusion with more events was less than 5%.All safety and primary analyses were done on an intention-to-treat basis. Overall survival was calculated from the date of group assignment to the date of death. Patients either lost to follow-up or still alive at the time of analysis were censored at the date they were last known to be alive.Because patients can only be deemed disease free after surgery, and to account for the different durations of preoperative chemotherapy, disease-free survival was analysed using a landmark analysis.2 test. The proportion of patients who survived and median survival times were estimated from a flexible parametric survival modelProgression-free survival was calculated from random assignment to the date of the event or, in event-free patients, the date last known to be alive and free from recurrence. Overall, disease-free, and progression-free survival were compared using the log-rank χTo analyse tumour regression, the original five-point Mandard scale was dichotomised into responders (Mandard TRG 1–3) and non-responders (Mandard TRG 4–5). A second, unplanned analysis was also done because emerging evidence suggested that a more appropriate dichotomisation was Mandard TRG 1–2 as responders and grades 3–5 as non-responders.Formal comparisons and summaries of HRQL function and symptom scales were restricted to a number of prespecified outcome measures. Global HRQL and prespecified symptoms related to the effects of chemotherapy and surgery  were compared immediately before surgery, and 3, 12, and 24 months after surgery, using an ANOVA with adjustment for baseline score. These timepoints were considered separately, rather than using a repeated measures method of analysis.2 test or Fisher's exact test, as appropriate. A p value of less than 0·05 was used as the significance threshold. For all analyses, no attempts were made to impute values for missing data. All analyses were done using Stata, version 14.Comparisons between chemotherapy toxicities, surgical complications, tumour regression, and resection were done using a χClinicalTrials.gov (NCT00041262).This trial is registered with the ISRCTN registry (registration number 01852072) and Cancer Research UK approved the study design, but had no role in data collection, data analysis, data interpretation, or writing of the report. Roche supplied capecitabine for use in the study, and had sight of the draft trial manuscript, but had no role in any other aspect of the study. MN had access to the raw data. The corresponding author had final responsibility for the decision to submit for publication.Between Jan 13, 2005, and Oct 31, 2011, 897 patients were recruited from 72 UK hospitals and randomly allocated to the CF group (n=451) or the ECX group n=446; . The medThree (4%) of 72 recruiting centres did not take part in the HRQL aspect of the trial for any of their patients, and HRQL assessment data were omitted at baseline for the patients from these centres . Baseline HRQL was also well balanced between the two groups. Mean values (SD) for the five prespecified domains of interest were: global HRQL 76·0 (18·34), appetite loss 37·4 (27·98), reflux 17·3 (24·05), pain 20·3 (20·50), and dysphagia 73·9 (25·7); data per treatment group are in the vs 363 [81%] of 446; p<0·0001), although a similar number of patients in the CF (435 [96%] of 451) and ECX (432 [97%] of 446) groups received at least two cycles. Of the 451 patients in the CF group, eight (2%) stopped chemotherapy because of toxicity and one (<1%) died, whereas in the ECX group, 46 (10%) of 446 patients stopped because of toxicity, and five (1%) died, one of which was thought to be related to chemotherapy toxicity , although the number receiving cisplatin dose reductions was similar between the groups (61 [14%] of 451 in the CF group and 53 [12%] of 446 in the ECX group).Details of the chemotherapy received are shown in toxicity . The numThe median time from randomisation to surgery was 71 days (IQR 66–80) in the CF group and 127 days (119–137) in the ECX group. The median time of surgery from the start of the last preoperative chemotherapy cycle was 44 days (39–52) for patients in the CF group and 57 days (52–64) for patients in the ECX group. Details of the surgical procedure are shown in 751 (84%) of 897 people had resection and reconstruction, whereas 47 (5%) of 897 patients were found to have progressive disease or to be inoperable during surgery . 588  of 897 patients were known to have died (327 [73%] of 451 in the CF group and 302 [68%] of 446 in the ECX group) and a further 12 had withdrawn consent for further follow-up. The median follow-up of the surviving patients was 6·4 years (IQR 4·8–8·2), and 250 (93%) of 268 patients had at least 3 years of follow-up assessments. The observed 3-year overall survival was 39% (95% CI 35–44) in the CF group, and 42% (37–47) in the ECX group . Median vs 316 events in the ECX group, based on a 6-month landmark analysis; Median disease-free survival (347 events in the CF group Chemotherapy toxicity data were not provided by three patients in each group and two in each group did not receive any chemotherapy. Grade 3 or 4 diarrhoea occurred in six (1%) of 446 people in the CF group and 36 (8%) of 441 in the ECX group (p<0·0001) and grade 3 or 4 neutropenia occurred in 74 (17%) of 446 in the CF group and 101 (23%) of 441 people in the ECX group (p=0·023); stomatitis occurred in 25 (6%) of 446 people in the CF group versus seven (2%) of 441 in the ECX group  reported serious adverse events over the course of the trial than in the CF group , although more people in the ECX group had respiratory complications (125 [33%] of 374) than in the CF group  after surgery and the number of patients categorised as ypN0 was also higher after ECX . The proportions of patients who achieved R0, R1, and R2 were similar in both groups.More specimens from the ECX group were classified by the local pathologist as ypT0 or T1 , distant metastases (94 [27%] of 343 people vs 78 [25%] of 313), local recurrence and distant metastases (87 [25%] of 343 vs 59 [19%] of 313), or death without confirmed progression (102 [30%] of 343 vs 130 [42%] of 313).In the exploratory analysis of progression-free survival, median progression-free survival  were low, and so this difference did not translate into an overall survival benefit.More patients in the ECX group had a good pathological response to chemotherapy (Mandard TRG 1 or 2), and consequently more were staged as ypT0 or 1 or ypN0 after surgery, than were those in the CF group. Results from the MRC gastro-oesophageal ST03The results of this trial raise the question of the optimal number of preoperative chemotherapy cycles. In the absence of a good pathological response of the tumour, giving more than two cycles might unnecessarily delay surgery. In the current absence of a reliable biomarker that enables prediction of responses either before or midway through preoperative treatment, the extent of preoperative chemotherapy relies on the judgement of the clinician after discussion with the patient, to balance the possibility of achieving a good response against the risk of not being able to proceed to surgery. The results from our OE05 trial favour the use of two cycles of a chemotherapy doublet if preoperative chemotherapy alone is offered. However, two cycles of CF preoperatively has not been directly compared with the perioperative approach assessed in the MAGIC trial,OE05 showed an improvement in overall survival of patients in the CF group (recruited 2005–11) compared with the same regimen in OE02 (recruited 1992–98), in which the median survival was 1·4 years compared with 2·0 years in OE05.A number of potential factors could explain why the survival of patients with oesophageal cancer who were treated with CF improved with time. Changes in referral patterns, moves towards centralisation of surgical services, the introduction of endoscopic ultrasonography, and the development of multidisciplinary teams are all events that occurred towards the end of recruitment to the OE02 study, and these changes might have led to better patient selection for attempted curative treatment by the time of this OE05 study. The introduction of routine PET scanning might also be an important factor for the absence of survival difference in this study. Post-hoc analyses indicated that ECX offered a survival benefit in the early years of the study when PET scans were rarely used, but offered no benefit during the later years when PET was used for nearly all patients. ECX might be more effective in treating small-volume, disseminated disease that was often missed before the routine use of PET scans. Patients with this type of disease are now usually identified and would have been ineligible for participation in the study.Neoadjuvant chemoradiotherapy has also led to improved survival compared with surgery alone for patients with oesophageal adenocarcinoma and squamous cell cancers, with a median survival of 48·6 months (95% CI 32·1–65·1) with adjuvant therapy compared with 24·0 months (95% CI 14·2–33·7) with surgery alone in the CROSS trial.The present study has a number of advantages over other oesophageal cancer trials. This study was confined to patients with adenocarcinomas of the oesophagus and gastro-oesophageal junction (Siewert type 1 and 2), specifically excluding squamous cell cancers and Siewert type 3 (gastric cancer). The study involved a large number of centres with a detailed protocol regarding pretreatment staging, the delivery of chemotherapy and surgery, and histopathological assessment. To our knowledge, our study is the only prospective randomised trial in neoadjuvant treatment and surgery for oesophageal cancer to have included a comprehensive prospective assessment of HRQL using validated generic and specific measures. Although no differences between trial groups were observed in HRQL, the data confirm findings from much smaller cohort studies.Data published in 2017 show a survival advantage for patients treated with docetaxel, oxaliplatin, and fluorouracil compared with epirubicin, cisplatin, and fluorouracil or ECX given perioperatively for junctional and gastric tumours, with a 3-year overall survival of 57% for the docetaxel-containing regimen compared with 48% with epirubicin, cisplatin, and fluorouracil or ECX .OE05 Clinical Trial Protocol Version 6.0 see http://www.ctu.mrc.ac.uk/research/documents/cancer_protocols/OE05_Protocol_Version_6_23_Dec_09.pdfFor the


Cryptobranchus alleganiensis alleganiensis have identified significant population declines and extirpations associated with habitat degradation, poor water quality and disease, leading to nomination as a candidate for listing under the Endangered Species Act. However, populations in the southern Appalachian region of the range have received less attention despite relatively high levels of watershed protection due to the establishment of federally protected National Forest and National Park public lands. These watersheds likely represent some of the best remaining available habitat, yet the lack of published studies make assessment of population stability and viability very difficult. Our objectives were to (1) conduct a capture-mark-recapture (CMR) demographic study and a point transect survey on the Hiwassee River in Tennessee which is designated a National Scenic River, and is largely contained within the Cherokee National Forest, (2) quantify the size structure of the population, (3) compare abundance, survival and recruitment with historic and contemporary hellbender populations across the range, (4) assess the importance of this population and the significance of National Forest and National Park lands in the context of hellbender population conservation in the southeastern United States. We detected all age classes present, with larval hellbenders comprising 21.5% of captures. Using a combination of static life table and CMR methods, we determined that survival rates during the first year were low (~10%), but were high (68–94%) for taggable sized hellbenders. Density of hellbenders at the study site was very high (84 taggable sized hellbenders per 100m of river) compared to recent demographic studies conducted in other regions of the range. We detected hellbenders over ~28 km of river, with a mean density of 23 taggable sized hellbenders per 100m of river, and a total population estimate of 6440 taggable hellbenders. National Forest and National Park lands are likely to continue to play a particularly important role in providing suitable habitat for hellbenders in the southern Appalachians. In fact, only six of 21 known hellbender locations in Tennessee appear to show consistent larval recruitment, all of which are located within or adjacent to National Forest or National Park land.Comparisons of recent and historic population demographic studies of eastern hellbenders  Cryptobranchus spp.) are giant aquatic salamanders (family Cryptobranchidae) with an extensive historic distribution in the USA [C. alleganiensis alleganiensis) ranges from southern New York to northern Georgia, Alabama and Mississipi, with disjunct Midwest populations in Missouri. The Ozark hellbender  range is limited to the Ozark region of Arkansas and Missouri. Across their range, hellbenders populations have declined significantly because of numerous factors including river impoundment, poor water quality and siltation, persecution, illegal collection, and disease [Hellbenders  within a section designated as a Tennessee State Scenic River, located near Reliance Tennessee . This seFor further information on the study area, see During 2004 to 2008, we conducted annual surveys of a 350 m section of the Hiwassee near Reliance, Tennessee using standard mask and snorkel rock lifting methods . TypicalFor every hellbender we captured, we recorded location (using GPS), mass, total and snout-vent length, and then injected a passive integrated transponder (PIT) tag subcutaneously at the base of the tail in all individuals >15 cm total length (TL). We classified individuals as larvae (<9 cm), subadults (9–29 cm) or adults (≥30 cm). We noted any injuries such as missing toes or legs and collected toe or tail tip tissue samples. In 2005–2006, we recorded shelter rock size . We were unable to reliably sex individuals caught before July because Hiwassee hellbenders mate in October (D. McGinnity pers. comm.) and males do not exhibit any cloacal swelling. However, for an additional 82 capture records during July to October 2004–2015, we were able to assign sex based on cloacal swelling.We used one-way ANOVA followed by Tukey’s honest significant difference test to determine differences between mean shelter rock size used by larvae, subadults, and adults. We conducted simple linear regression to investigate the relationship between shelter rock size and hellbender total length. The analyses were conducted using R version 3.3.1 base statistics package .We used a static life table approach to estimate the survival rate of hellbenders during their first year. From our field observations and previous studies ,19,32 onDuring the 2004–2008 CMR analysis, we only included captures from the Hiwassee core area that was surveyed from April 30 to June 4 ensuring the survey area was constant from year to year, and sampling occasions were effectively instantaneous .Ni), and survival (φi), capture (pi) and entry probabilities (bi) at time i. The POPAN version hypothesizes a “superpopulation” consisting of the total number of unique animals available for capture, with parameters bi which represent the probabilities that an animal from this hypothetical superpopulation enters the population between occasion i and i +1 and survive to the next sampling period [We used the POPAN parameterization of the Jolly-Seber (JS) model ,43 in prg period . We beli2 values for two tests: TEST 2 evaluates assumption 1 by determining if every marked animal present in the population is equally catchable at time (i+1) regardless of whether they were captured at time (i). Assumption 1 is sensitive to capture effects, or non-random temporary emigration. TEST 3 evaluates assumption 2 by determining if the probability that an individual is ever seen again depends on whether it was marked at or before occasion (i). Assumption 2 is sensitive to changes in mortality over the course of the study, or to differences in survival probabilities among individuals . Also, RELEASE provides a TEST 2 + TEST 3 overall summary χ2 test, which can be used to generate an estimate of dispersion (ĉ), given by ĉ = χ2/df, where ĉ should approximate to 1 if the data fit the model adequately.We performed goodness of fit tests of the JS POPAN model using program RELEASE which produced χφ and p, the log function for N, and the multinomial logit link function for b. A full parameterization of the JS model includes time dependence for survival and recapture probabilities, so we tested reduced parameter versions with constant survival and/or recapture probabilities. We ranked the models by Akaike’s information criterion AICc for low sample sizes [c weight to produce survival, recapture and entry probabilities, and estimates of abundance.We fit JS POPAN models using the logit link function for le sizes , and the2 = 3.2707, df = 8, p = 0.9162; TEST 2: χ2 = 2.329, df = 3, p = 0.5068; TEST 2 + TEST 3: χ2 = 5.6005, df = 11, p = 0.8986), low sample size warnings were returned for several test components, reducing our confidence in the model estimates. Therefore, we believe that pooling all captures maximized precision and reliability of the survival and recapture parameter estimates.It is possible that different survival probabilities or tendencies to move between subadults and adults could violate the assumptions and reduce the reliability of parameter estimates. Therefore, we performed the same POPAN JS and goodness of fit tests using two groups . We failed to detect substantial differences in survival and capture probabilities between subadults and adults, and although goodness of fit tests indicated no deviations from model assumptions . Points were mapped 500 m apart (powerhouse to Hwy 411) or 750 m apart (Hwy 411 to Patty Bridge) and picked randomly for sampling. We surveyed for 1 hour or for 100m, whichever occurred sooner and recorded CPU. Unlike the CMR study, we did not attempt to methodically turn every rock in the section because the intention was not to characterize the population size structure at each site, but rather to produce relative estimates of abundance of taggable sized hellbenders based on capture rate (hellbenders per person hour). Therefore, we limited our search to rocks >25 cm width. In August 2009, we surveyed the CMR site using this same survey methodology to generate a capture rate that would then be comparable to the other point survey sites. Because we have an estimate of the actual population density at the CMR site from the 2004–8 study, it is possible to use the mean capture rate from the point surveys to estimate the average population density over the entire river, assuming similar detectability at the point survey sites.http://nature.org/). Using qGIS version 2.14 we mapped contemporary (since 2000) hellbender populations and calculated the number of river kilometers that are currently likely to hold hellbenders. Also, we mapped historic hellbender locations that have not produced any recent (since 2000) occurrence observations, and are likely to be functionally extirpated. We used National Park Service, USDA National Forest Service, and TWRA Wildlife Management Areas boundary layers to qualitatively examine the landscape context of recent and historic hellbender occurrences.We investigated the importance of public lands for contemporary eastern hellbender populations by accessing all verified observations of eastern hellbenders across Tennessee from the Tennessee Wildlife Resource Agency State Wildlife (TWRA) Action Plan database maintained by the Tennessee chapter of the Nature Conservancy (http://www.asih.org/sites/default/files/documents/resources/guidelinesherpsresearch2004.pdf). To protect hellbenders from illegal collection and disturbance [This study was performed with the approval of the Institutional Review Board of Lee University. We received permission from the USDA National Forest Service to conduct fieldwork in the Cherokee National Forest. Animals were collected under permit from the Tennessee Wildlife Resource Agency (# 1505), processed and released unharmed at the capture site in accordance with the Guidelines for Use of Live Amphibian and Reptiles in Field and Laboratory Research . The estimate of dispersion ĉ = 1.01, indicating good model fit.A total of 197 individual hellbenders provided capture records that met the criteria for consistent survey area and time . We detep(.),b(t), where survival varied with time, and recapture probability was constant  hellbenders per 100 m2 of suitable habitat. There were approximately 12745 m2 of suitable habitat within the 350 m study section, providing an estimated total mean abundance of 294 hellbenders (95% CI = 190–400), or 84 (95% CI = 54–114) hellbenders per 100 m of river length. These estimates are conservative because hellbenders that are too small to tag comprised 21.5% of all captures at the study site.The best supported model was φ(t),constant ; howeververaging  providedr strips  given byHellbenders were encountered at 19 of 23 sites over 28 river km, and capture rates varied from 0 to 4 individuals per person hour . The 2009 point survey capture rate at the CMR site was 4.6 hellbenders per person hour. Using the actual density estimate of 84 hellbenders per 100 m of river length calculated above from the 2004–8 CMR site, the average density between the Apalachia powerhouse and Patty Bridge was (84/4.6)*1.28 = 23 (95% CI = 15–31) hellbenders of taggable size per 100 m, indicating a total population of 6440 (95% CI = 4200–9240) taggable sized hellbenders over 28 river km.We identified a minimum of 54 separate rivers and streams with hellbender occurrence records since 1915 . HoweverOur study indicates the Hiwassee River has a substantial population of hellbenders with consistent successful reproduction each year. On average, gilled larvae represented 21.5% of all captures which is likely to be an underestimate given their small size and lower detectability compared to subadults and adults. Reproduction in the Hiwassee occurs in October, and larval hellbenders remain under the nest rock guarded by a male until apparently dispersing in late April . The next size class (9–12 cm) only made up 2% of captures. Assuming there was no net emigration of larval hellbenders within the study site over the first year, mortality rates of larvae post dispersal are ~ 90%, which is consistent with estimates for combined egg/larval survival provided by Unger et al . Also, tSize class frequencies remained similar until growth rates slowed until around 30 cm where cohorts become indistinguishable and size class frequencies increase see  suggestiThere are several factors that contributed to our success in detecting larval hellbenders. First, there appears to be consistent successful reproduction each year in this population. Second, the Hiwassee water clarity is excellent, and the shallow cobblestone substrates are relatively amenable to systematic searches. Third, our surveys were conducted primarily in May within weeks of emergence from the nest rock, giving little time for dispersion and predation to reduce abundance. Conversely, most previous studies did not detect larval hellbenders, presumably because of significant differences in recruitment rates, substrate type, or survey technique. It is possible that declines had already commenced by the mid 1970’s and the lack of larval captures was symptomatic of poor recruitment. However, substrate type is a more likely explanation: Nickerson et al  argued t2 of suitable habitat or 840 hellbenders per river km, with an average density of 230 hellbenders per river km. Our population estimates are derived from five consecutive years of survey data. Five years is significantly longer than all other published hellbender CMR studies, which were conducted over either one or two years. Therefore, our estimates should be less sensitive to errors caused by stochastic events, which might temporarily influence local abundance, or affect search efficiency and encounter probabilities [2 of suitable habitat respectively. However, they defined habitable spaces as estimated square meters of cover rock in the study area, and thus only 2–10% and 1–6% respectively of the streambed was considered suitable habitat. Nickerson and Mays [2 riffle and estimated 50–60% as “suitable diurnal habitat” giving “one hellbender per 8–10 m2” (10–13 hellbenders per 100 m2). Similar densities were observed during surveys at the same site a decade later [2 total area [We were interested in comparing the density of hellbenders in the Hiwassee with other pre- and post-decline populations see . We detebilities . Previoubilities  and Fostbilities  used cloand Mays  reportedtal area , and thetal area  because tal area , illustrtal area  state thtal area , with fo2  in the Blue River in Indiana, with a CPU of 0.05 hellbenders per hour which represents a significant decline compared to Kern [2 in a tributary of the New River in West Virginia [Our capture rates and density estimates of eastern hellbenders in the Hiwassee appear to be much higher than those observed in recent post-decline studies of other populations across their range. Burgmeier et al.  detected to Kern  who estiVirginia , and capVirginia ,33 and tVirginia  respectiOur study provides an important reference point for long term monitoring of eastern hellbenders in Tennessee. Without baseline data from 1970 to 1990, it would have been difficult to document the decline of hellbenders in the Ozarks ,12,17. MWe believe that National Forest and National Park lands are extremely important to the ongoing management and recovery of hellbenders in Tennessee. Historic occurrence records support the statement by Gentry  that easOne noticeable feature is the absence of hellbenders from numerous Tennessee Valley rivers on public lands that appear to have excellent water quality and suitable habitat. We believe these rivers are impacted by historic and contemporary land use practices at multiple hierarchical levels within the watershed. Impoundment and impairment of lower-mid elevation mainstem river sections has caused extensive fragmentation and isolation of populations on public lands, which tend to be close to the upper elevation limit for hellbenders. Prior to the establishment of the Cherokee NF and GSMNP, these populations experienced severely destructive practices such as mining and large scale clear-cutting including splash dam logging , causingS1 Appendix(DOCX)Click here for additional data file.S1 FigPhotograph illustrates a portion of the CMR study site comprising of shallow cobblestone glides (foreground) interspersed with deeper runs and riffles containing large shelter rocks.(DOCX)Click here for additional data file.


MX1, IFIT1, and ISG15) and used to analyze a prospective cohort at the Colorado ARDS Network site. One hundred twenty ARDS patients from four urban hospitals were enrolled within 72 hours of initiation of mechanical ventilation. Circulating neutrophils were isolated from patients and expression of ISG determined by PCR. Samples were stratified by standard deviation from the mean into High (n = 21), Mid, (n = 82) or Low (n = 17) ISG expression. Clinical outcomes were compared between patients with High or Low ISG expression to those with Mid-range expression. At enrollment, there were no differences in age, gender, co-existing medical conditions, or type of physiologic injury between cohorts. After adjusting for age, race, gender and BMI, patients with either High or Low ISG expression had significantly worse clinical outcomes than those in the Mid for number of 28-day ventilator- and ICU-free days (P = 0.0006 and 0.0004), as well as 90-day mortality and 90-day home with unassisted breathing (P = 0.02 and 0.004). These findings suggest extremes of ISG expression by circulating neutrophils from ARDS patients recovered early in the syndrome are associated with poorer clinical outcomes.Acute Respiratory Distress Syndrome (ARDS) severity may be influenced by heterogeneity of neutrophil activation. Interferon-stimulated genes (ISG) are a broad gene family induced by Type I interferons, often as a response to viral infections, which evokes extensive immunomodulation. We tested the hypothesis that over- or under-expression of immunomodulatory ISG by neutrophils is associated with worse clinical outcomes in patients with ARDS. Genome-wide transcriptional profiles of circulating neutrophils isolated from patients with sepsis-induced ARDS (n = 31) and healthy controls (n = 19) were used to characterize ISG expression. Hierarchical clustering of expression identified 3 distinct subject groups with Low, Mid and High ISG expression. ISG accounting for the greatest variability in expression were identified ( Extensive variability in severity and survival is a common feature of acute respiratory distress syndrome (ARDS), and identification of mechanisms that regulate this variability may lead to more personalized treatment. Age, race, cigarette smoking, and alcoholism impair the immune system, and are linked to increased prevalence or worse ARDS outcomes –9. SpeciViral infections can modify the immune response to subsequent bacterial infections –34, and Based on findings that ISG expression can modify the immune response, we hypothesized that both elevated and suppressed ISG expression could be associated with more severe outcomes in ARDS. Herein, we analyzed the range of neutrophil ISG expression in ARDS patients, and the correlation with clinical outcomes. Hierarchical clustering of neutrophil ISG demonstrated the presence of 3 distinct cohorts of subjects with High, Mid, or Low ISG expression. Three ISG with the greatest variability in expression were selected for expression analysis, and the association of these ISG expression levels with clinical outcome was prospectively tested at the onset of ARDS.The study protocol was approved by the Colorado Multiple Institutional Review Board (COMIRB) committee and the National Jewish Health Institutional Review Board. All subjects, or an appropriate proxy, gave written informed consent. The study was conducted in accordance with the Declaration of Helsinki.Volunteers (n = 40) were administered an 18-point questionnaire to verify they were not experiencing any acute symptoms of a viral illness, did not have any recently diagnosed medical problems, vaccinations, underlying chronic conditions, or had recently received blood products. In addition, these healthy subjects were verified to have negative screening results for hepatitis B and HIV within 6 months of the blood collection. Vital signs checked on the day of the collection were within the normal range.Peripheral blood neutrophils isolated from patients with sepsis-induced ARDS (n = 31) were used to establish ISG expression levels. Neutrophils were isolated within 24 hours of meeting criteria for ARDS criteria. Gene expression analysis from a subset of this cohort were previously reported in an analysis of HMGB1 and LPS induced patterns of gene expression in ARDS .7 cells in 1 ml of RNAlater , then stored at –20°C. Subsequent isolation with Trizol  and purification with RNEasy MinElute columns  was performed following the manufacturer’s protocol. Between 1 and 5 μg of total RNA was used for microarray target labeling using standard methods for reverse transcription and one round of in vitro transcription [< 3). This procedure insures that each of the arrays in the group can be directly compared, and that the input mRNA was intact. Single channel hybridization was performed per sample. The internal quality control for each hybridization included comparison of overall intensity across all arrays (intensity consistently varied within two standard deviations of the median intensity) and the integrity of the labeled target as determined by the ratio of hybridization intensity to 3’ and 5’ regions of GAPD and β-actin . RNA quality was assessed by spectrophotometry (A260/A280> 1.8) and Agilent Bioanalyzer (28S/18S rRNA intensity > 1.5). Affymetrix Eukaryotic Hybridization Control mixture was employed as external control. The complete set of gene expression data has been deposited in the GEO database .Samples were obtained from 31 patients and 19 healthy volunteers. Briefly, total RNA was stabilized in freshly isolated neutrophils by resuspension of 2x10cription . HG-U133cription . Individcription  by: (a) Mx1, ISG15, IFIT1, and IFIT3 as genes whose expression differences were the greatest between subjects. A three-gene panel including MX1, ISG15, and IFIT1 was used in subsequent analyses; IFIT3 was eliminated from further analyses because of the high similarity, known co-expression, and co-evolutionary origin between it and IFIT1.Gene expression analysis was performed using modules within MeV 4.6 . Genes iTable 1). None of the interventions tested in the parent studies resulted in a treatment benefit [2/FiO2<271 –adjusted for Denver altitude of 1600m), bilateral infiltrates by chest radiograph, and requirement for intubation and positive pressure ventilation in the absence of evidence for left-sided cardiac failure or other exclusionary criteria[Circulating neutrophils of ARDS patients (n = 120) were isolated from patients enrolled into one of four NHLBI ARDS Network studies conducted at the University of Colorado affiliated hospitals, each with identical enrollment criteria. The parent studies were: Drug Study of Albuterol to Treat ALI , Early V benefit –63. The  criteria. All pat criteria. Periphe6 isolated neutrophils in the absence of ex vivo stimulation using TRIzol reagent [Neutrophils were isolated from peripheral blood using the plasma Percoll method  in an id reagent .MX1, IFIT1, and ISG15 relative to GAPDH by the ΔCt method using standard conditions. Primers and probes were obtained from Applied BioSystems  and Roche  as previously described [5 to convert all values greater than 1, and the mean of the log2 transformed expression values for each subject was calculated  using an ELISA from PBL Laboratories, as directed by the manufacturer. Plasma was isolated during the course of neutrophil isolation.Because hierarchical clustering is less reliable for grouping samples when the number of variables is small, we divided groups based on deviation from the mean. Other methods to model high versus low ISG expression in ARDS patients were tested , but were found to have worse model fit by Akaike information criterion comparison (not shown). Overall group comparisons were made using ANOVA or Kruskal-Wallis tests for continuous variables and Fisher’s exact tests for categorical variables, as indicated in the text. Pairwise comparisons were made between the High and Mid-range and Low and Mid-range ISG groups when the corresponding overall test was significant. Ventilator-free and ICU-free days  were anaFig 1).Genome-wide transcriptional profiles of circulating neutrophils isolated from patients with sepsis-induced ARDS (n = 31) and healthy controls (n = 19) was used to characterize ISG expression. Genes identified as the “Type I Interferon-mediated Signaling Pathway Cluster” by the Gene Ontology Consortium (GO:0060337) yielded 66 unique genes with available expression data. Significance Analysis for Microarrays determined that 31 genes were significantly changed between subjects. Using hierarchical clustering of expression, 3 distinct subject groups were identified. Both healthy controls and patients were found distributed in clusters of High and Mid ISG expression, while only ARDS patients were found within a cluster of Low ISG expression . Alternatively, when gene expression was examined by Principal Component Analysis to discern changes in variation, the same four previously identified genes were identified as a distinct cluster . PC1 (accounting for 91.6% of the variability) appears to generally represent average expression across subjects (high expression at the low end). PC2 (accounting for 4.5% of the variability) appears to distinguish within gene variability, where genes with the highest variability in expression are at the low end.A test for variance was used to identify ISG that were representative of variability between subjects. When these genes were ranked for extent of variance of expression levels using relative size of the standard deviation between subjects, the genes with the largest variance were identified as MX1, ISG15, and IFIT1) identified as having the most variable expression  was used as a marker of neutrophil ISG expression in ARDS. IFIT3 was not further analyzed due to its high similarity, co-regulation, nearby chromosomal location, and co-evolutionary origin with IFIT1. We hypothesized that the variable expression of these genes would be associated with changes in clinical outcomes in ARDS. Relative expression level of each gene was determined prospectively by quantitative PCR of neutrophils isolated from ARDS patients (n = 120). The means of the transformed values of gene expression were normally distributed . Samples greater than one standard deviation above mean expression were designated as the “High” ISG expression cohort (n = 21), and the corresponding group of samples with ISG expression less than one standard deviation below the mean was designated as the “Low” ISG expression cohort (n = 17). Subjects falling within one standard deviation of the mean were designated as “Mid” ISG expression (n = 82) . ARDS patients in the test set were compared with healthy control subjects (n = 40) analyzed in an identical fashion. As with the first cohort, ISG expression of healthy controls was found to fall only within the High and Mid-ranges , while the Low-expressing cohort was confined to subjects with ARDS.A three-gene panel . No differences in race or ethnicity were identified in the Low ISG cohort compared to Mid. BMI was significantly lower in the Low ISG cohort compared to the Mid cohort (pairwise P = 0.006). However, the Low ISG cohort did not have an increased frequency of identified pre-existing medical conditions. Patients with at least one identified underlying medical condition ranged from 63.4% to 71.4% across the three groups (Fisher’s test P = 0.80).Demographic features and underlying medical conditions for ARDS patients were quite similar across cohorts at the time of enrollment (Table 3). The most common presenting injury was pneumonia, with a prevalence ranging from 71.9% to 76.5% between the three groups (Fisher’s test P = 0.95), followed by secondary sepsis . No patient had primary trauma. Study enrollment occurred 2.4±1.1 days (mean±S.D.) after meeting ARDS criteria. Clinical and physiologic assessment made at this early time point demonstrated a divergence in disease severity (Table 4). While gas exchange as measured by the PaO2 to FiO2 ratio was not different between the three groups, the Glasgow Coma score (GCS) and the APACHE III score differed significantly, with the Mid group averaging a less severe score. GCS represented the major contributor to the observed difference within the APACHE III score.Identified risk factors for ARDS at presentation were also not different between the study groups . The fraction of patients discharged to home with unassisted breathing was significantly lower at 90 days for High ISG compared to Mid . Mortality was higher by 90 days for High ISG, with 33.3% reported as dead prior to discharge to home with unassisted breathing for High ISG, compared to 12.2% for the Mid ISG group .Outcomes for patients with High or Low ISG expression were compared with the Mid ISG cohort. High ISG expression was associated with worse outcomes, with fewer ventilator-free and ICU-free days over the first 28 days than the Mid cohort . The fraction of patients discharged to home with unassisted breathing was lower at 90 days for Low ISG compared to Mid . Mortality was significantly increased at 28 days , and by 90 days, with 35.3% reported as dead prior to discharge to home with unassisted breathing for Low ISG .Patients with Low ISG expression also demonstrated worse outcomes, similar in severity to the High ISG expression cohort, with fewer ICU-free days over the first 28 days compared to the Mid cohort . Similarly, after adjusting for covariates, both High and Low ISG expression cohorts had significantly fewer days ICU-free compared to the Mid ISG cohort . Multivariate analysis was not performed for APACHE III score, as it measures elements of disease severity likely associated with extremes in ISG expression.After adjusting for gender, race, age and BMI, both High and Low ISG expression cohorts had significantly fewer days ventilation-free compared to the Mid ISG cohort . Patients’ 90 day mortality was significantly different for High and Low ISG expression cohorts, compared to the Mid cohort . In addition, time to discharge to home with unassisted breathing was significantly delayed in the High and Low cohorts compared to the Mid cohort . A survival analysis of time to mortality by day 90 was also significantly different for High and Low ISG expression cohorts, compared to the Mid cohort  .Patients’ status of being discharged home with unassisted breathing by day 90 was significantly different for High and Low ISG expression cohorts, compared to the Mid cohort (P = 0.23). We measured serum IFNα in patients as a possible surrogate to detect viral infection. At enrollment into this protocol, 52.4% of patients in the High ISG cohort had detectable IFNα in their circulation, which trended greater than patients in the Mid ISG cohort (26.3%) and the Low ISG cohort . The quantity of IFNα in circulation was highly variable, but greater in the High ISG cohort  compared to the Mid ISG cohort .Screening of patients for viral infection was not systemically performed. However, acute or chronic viral infections were identified in 15 subjects during clinical care, including H1N1 influenza (n = 6), HIV (n = 4), Hepatitis B (n = 1), Hepatitis C (n = 3), or H1N1 combined with Hepatitis C (n = 1). While 24% of the High ISG expression cohort had a confirmed viral infection, compared to 11% in the Mid ISG cohort and 5.9% in the Low ISG cohort, these differences were not significant  are prototypical ISGs, previously shown to be highly expressed in stimulated neutrophils [This single-center prospective study is the first known exploration of the clinical significance of systemic ISG expression in ARDS. In a preliminary set of patients (n = 31) we identified the ISGs that account for the greatest variability in expression. These genes , most of the High ISG group were not known to have a co-existing viral infection. Conversely, patients with proven viral infections were also found (in lower percentages) in the Mid and Low ISG expression cohorts, underscoring the relatively poor correlation between clinically evident viral infections and systemic ISG expression. Although IFNα/β and ISG expression is indicative for viral infections, we have no evidence outside the confirmed cases that viral infection was responsible for ISG expression. IFNα/β is released into the circulation early after viral infections, and may be undetectable or present in very low quantities, even in experimental settings [Occurrence of elevated ISG expression in a subgroup of ARDS patients and healthy controls is not surprising. Acute and chronic viral infections are common within the population , and maysettings , a findisettings . ISG expsettings , and evesettings  as part settings . Similarsettings , and a sS. aureus, which included reduced p38 MAP kinase activation, attenuation of O2- generation and IL8 secretion, increased apoptosis, and impaired bacterial killing [vs 2.4%, P = 0.015). African Americans have been found to experience worse outcomes from ARDS [The association of worse clinical outcomes with High ISG expression in ARDS is supported by clinical observations and animal models that demonstrate viral infections increase susceptibility to secondary bacterial infections –34. Prim killing . Others  killing –47. No dP = 0.006). However, the prevalence of pre-existing medical conditions was not greater in the Low ISG subgroup, and differences in BMI within this range are not associated with changes in outcomes from ARDS [The presence of a distinct subpopulation of ARDS patients with relatively inhibited ISG expression was seen in both cohorts of patients used in this study. Outcomes in this cohort were also worse than the Mid ISG group, without differences in demographic or clinical features. The Low ISG group had a normal BMI, which was significantly lower than the Mid group, who were on average overweight Click here for additional data file.


Approximately 1% of the general population and 10% of patients with migraine suffer from vestibular migraine (VM). However, this condition remains relatively unknown; therefore, it is often underdiagnosed despite the recent adoption of international diagnostic criteria for VM. The diagnosis of VM is based on the symptoms, degree, frequency, and duration of the vestibular episodes, a history of migraine, the temporal association of migraine symptoms with vestibular episodes in at least 50% of cases, and the exclusion of other causes. Physical examination and laboratory findings are usually normal in patients with VM but can be used to rule out other vestibular disorders with similar symptoms. The pathophysiology of VM remains incompletely understood; however, several mechanisms link the trigeminal system, which is activated during migraine attacks, and the vestibular system. Because few controlled trials have specifically investigated VM, the treatment options for this order are largely the same as those for migraine and include antiemetics for severe acute attacks, pharmacological migraine prophylaxis, and lifestyle changes. Therefore, it appears that there is increasing awareness of this emerging entity. As a result, the present review aimed to describe the diagnostic criteria and epidemiology of VM, the current understanding of its pathogenesis, and its treatment options, with a focus on clinical features.Since the initial report describing the association between migraine and vertigo , a numbeβ, which lists new disorders in need of further research for validation [β criteria for the diagnosis of VM require that a patient has current or previous migraines with or without aura, a patient has had at least five vestibular episodes of moderate or severe intensity lasting 5 min to 72 h, and at least 50% of these episodes involved 1 or more migrainous symptoms. Although probable VM is not included in the ICHD-3β, studies have shown that approximately half of probable VM cases ultimately progress to VM. If future studies support these findings, then probable VM may be included in later versions of the ICHD [Various terms have been used to describe the relationship between migraine and vestibular symptoms including migrainous vertigo, migraine-associated vertigo or dizziness, migraine-related vestibulopathy, and benign recurrent vertigo –6. The dlidation . Followilidation . The ICHthe ICHD , 12. Diagnostic Criteria for Vestibular MigraineVMAt least 5 episodes fulfilling criteria (C) and (D)A current or past history of 1.1 migraine without aura or 1.2 migraine with auraVestibular symptoms of moderate or severe intensity, lasting between 5 min and 72 hAt least 50% of episodes associated with at least 1 of the following 3 migrainous featuresHeadache with at least 2 of the following 4 characteristics: unilateral headache, pulsating quality, moderate to severe intensity, or aggravation by routine physical activityPhotophobia and phonophobiaVisual auraβ diagnosis or by another vestibular disorderNot better accounted for by another ICHD-3Probable VMAt least 5 episodes with vestibular symptoms of moderate or severe intensity, lasting between 5 min and 72 hOnly 1 of criteria (B) and (D) for VM fulfilled (migraine history or migraine features during the episode)β diagnosis or by another vestibular disorderNot better accounted for by another ICHD-3β criteria and the rate of probable VM to be 2.5% based on the consensus diagnostic criteria agreed upon by the International Headache Society and the Bárány Society [The reported prevalence of VM varies according to the different diagnostic criteria and study populations used; its prevalence is reported in the range of 4.3–29.3%, while the prevalence of probable VM is 4–5.7% –15. Prio Society . A commu Society . It has  Society . Therefo Society . Despite Society .VM is 1.5–5 times more frequent in females than in males , 5, 22 aMigraine attacks can be replaced by independent vertigo, dizziness, or a transient feeling of disequilibrium in elderly patients, especially postmenopausal women . AdditioIn VM, rotational or nonrotational vertigo may occur spontaneously or in association with position changes. A large population-based survey conducted using telephone interviews found that the rate of spontaneous rotatory vertigo is 67% while that of positional vertigo is 24% . FurtherThe duration of an attack can vary from a few seconds 10%) to several minutes 30%) or several hours (30%) and even up to a few days (30%) [0% to sev0% or sevVM has triggering factors that are similar to those of typical migraines, including menstruation, stress, lack of sleep, and diet. Additionally, vertigo induced by vestibular stimulation  may induce a migraine attack and can act as a specific migraine trigger . AuditorPatients with VM have poor sleep quality, high levels of depression, and a low overall quality of life , 35. AppA variety of diseases that may cause episodic vertigo are included in the differential diagnosis of VM . In particular, the symptoms of VM may mimic those of Meniere's disease (MD) and BPPV . BPPV shDifferential Diagnosis for VMMeniere's disease (MD)Benign paroxysmal positional vertigo (BPPV)Migraine with brainstem auraTransient ischemia of the vertebrobasilar circulationCentral positional vertigoSyncope and orthostatic hypotensionMotion sicknessVascular compression or schwannoma of the 8th nerveEpisodic ataxia type 2Somatoform vertigoβ; a single episode of dizziness is not included in these criteria to increase specificity. The five vestibular symptoms necessary for a diagnosis of VM are spontaneous vertigo, positional vertigo that occurs when changing the position of the head, visually induced vertigo induced by a complex or large moving visual stimulation, head motion-induced vertigo that occurs during head motion, and head motion-induced dizziness with nausea. The duration of an episodes is defined as the total period during which short attacks occur repeatedly during head motion and visual stimulation or after changes of head position [VM can be diagnosed based on the criteria described in “Diagnostic Criteria for Vestibular Migraine.” This diagnosis should be made after taking a full medical history of the patient that assesses the main vestibular symptoms, the intensity (severity), duration (5 min to 72 h), and frequency (5 times or more) of attacks, history of migraine, time association between migrainous symptoms and vestibular episodes, and the exclusion of other diseases . There aposition , 10. Composition . TherefoNumerous studies have reported that there are no disease-specific findings indicative of VM on physical examinations or laboratory tests. Additionally, neurotological findings between attacks are usually normal, although abnormalities are found in some patients . On the other hand, mild clinical signs of central vestibular dysfunction, such as gaze-induced nystagmus, central positional nystagmus, and spontaneous nystagmus, have been reported . FurtherReports of Clinical Signs in Patients with VMNeurotological FindingsBetween Attacks Gaze-induced nystagmus (27%) and spontaneous nystagmus (11%)  Persistent positional nystagmus and positional nystagmus 12→28%) [→28% 2,  Vertical 48%) and/or horizontal (22%) saccadic pursuit [8% and/or Subtle saccadic pursuit 20→63% in follow-up study over 9 years  Unilateral canal paresis (8–22%) , 57, 58 Bilateral vestibular failure 11%) [1% 1, 2,,2, 58 Low-frequency, mild cochlear loss 3–12%) [2% 1, 2, Mild bilateral sensorineural hearing loss (18%) in follow-up study over 9 years During Attacks Spontaneous nystagmus   Low-velocity, sustained, central positional nystagmus (100%)  Pathologic nystagmus with spontaneous or positional nystagmus (70%) Central vestibular dysfunction (50%)Peripheral vestibular dysfunction (15%)Unclear mixture (35%)Various abnormalities on laboratory tests, but not pathognomonic findings, have been associated with VM. Approximately 25% of VM cases exhibit a unilateral reduction of peripheral vestibular function and about 50% of VM cases show vestibuloocular asymmetry . AdditioAt present, the neural mechanisms of VM remain unknown and require further study; a majority of the current hypotheses are based on knowledge of migraine itself. It has been proposed that the reciprocal connections between brainstem vestibular nuclei and the structures that modulate trigeminal nociceptive inputs may underlie the pathophysiology of VM. Furthermore, different clinical findings from the periods between and during attacks refer to an interaction between the vestibular system and the mechanisms underlying migraine at various levels . CorticaVM is related to migraine and exhibits a familial trend . A genetThe vestibular and pain pathways are neurochemically similar and may share a central mechanism involved in the perception of sensations . Based oRecent functional neuroimaging findings have suggested that the dysmodulation of multimodal sensory integration and the processing of vestibular and nociceptive information could be factors related to VM. Positron emission tomography (PET) studies revealed an increase in metabolism in the temporoparietoinsular areas and bilateral thalami during VM attacks, which indicates that there was an activation of the vestibulothalamocortical pathway . PatientTreatment for VM is based on expert opinions because few randomized controlled trials have been carried out to identify optimal treatment strategies . Two ranProphylactic medications are mainstays for the management of VM. Because relatively few controlled trials have been conducted to date, most of the drugs used for VM are also used for the prevention of migraine headaches . A revieNonpharmacological treatment options for VM such as avoiding triggering factors, getting regular sleep and meals, and exercising may also be helpful for VM and should be considered as preventative measures, as with general migraine. Behavioral modifications can be tried. One retrospective study showed that 14% of 38 patients enrolled reported improvement in symptoms after caffeine cessation . Furtherβ, future studies addressing this issue will aid in the characterization of this disorder. To identify VM, detailed physical examination, the assessment of a patient's medical history regarding vestibular symptoms, and consideration of differential diagnoses to exclude other diseases are necessary. Additionally, in clinical practice, the possibility of VM in patients with a history of migraine or who currently suffer from migraine headaches should always be considered.VM is a common cause of recurrent episodic vertigo but it is likely underdiagnosed due to the absence of a unified set of diagnostic criteria. However, with the recent addition of new diagnostic criteria in the appendix of the ICHD-3


Saccharomyces cerevisiae as a biofilm model, overexpression screens identified DIG1, SFL1, HEK2, TOS8, SAN1, and ROF1/YHR177W as regulators of biofilm formation. Subsequent RNA-seq analysis of biofilm and nonbiofilm-forming strains revealed that all of the overexpression strains, other than DIG1 and TOS8, were adopting a single differential expression profile, although induced to varying degrees. TOS8 adopted a separate profile, while the expression profile of DIG1 reflected the common pattern seen in most of the strains, plus substantial DIG1-specific expression changes. We interpret the existence of the common transcriptional pattern seen across multiple, unrelated overexpression strains as reflecting a transcriptional state, that the yeast cell can access through regulatory signaling mechanisms, allowing an adaptive morphological change between biofilm-forming and nonbiofilm states.Biofilm formation by microorganisms is a major cause of recurring infections and removal of biofilms has proven to be extremely difficult given their inherent drug resistance . Understanding the biological processes that underlie biofilm formation is thus extremely important and could lead to the development of more effective drug therapies, resulting in better infection outcomes. Using the yeast  Many opportunistic human pathogens form highly structured, multicellular communities called biofilms, which are a key factor in persistent infections . BiofilmSaccharomyces cerevisiae is an established model for the study of biofilm formation. While most S. cerevisiae strains form smooth, unstructured colonies on solid media, some strains are able to form highly structured “fluffy” colonies. Fluffy S. cerevisiae colonies not only visually resemble bacterial and fungal biofilms, but also share their structural and functional characteristics, including the presence of an extracellular matrix  whose modest overexpression was sufficient to cause the fluffy-to-smooth transition, identified DIG1  in all the pools. Transformation using a negative vector control, AB352 , did not produce any smooth colonies.With an assumed 1000 plasmids per pool, screening 4600 transformants was expected to give us a 99% chance of recovering each plasmid at least once. Based on this, we screened five bioassay trays (YPD with 2% glucose and G418 for plasmid selection) per pool, plating 1000–1500 CFU per tray. Transformations were performed using standard LiAc/DMSO methods. Colony morphology was examined after 4 d of colony growth. Transformants with completely smooth or intermediate, Plasmid DNA was prepared from the smooth strains by standard methods  and the vs. fluffy colonies resulting from transformation with this singleton pool would help distinguish if the false positive rate was high or if the screen was undersaturated. The transformation plates were almost entirely fluffy with <1% smooths in ∼3000 CFU. This suggested that the singletons observed in the screen were largely false positives, rather than reflecting screen undersaturation.Among the final set of plasmids, only 18 were identified more than once, leaving 241 singletons. The large proportion of single hits could have been due to a high false positive rate and/or failure to saturate the screen. To distinguish between these possibilities, we constructed a new smaller pool (a “singleton” pool) that contained 91 plasmids that came up in the list of single hits. The relative number of smooth SAN1, TOS8, YHR177W, HEK2, and SFL1. This included all three of the genes isolated >2 times, but only 2 of the 15 genes observed twice.The 18 plasmids observed more than once in the initial screen were retested by individual transformations of each plasmid into F45, followed by screening for colony morphology. Plasmids that induced a phenotype change were further verified to ensure they contained the correct ORF, based on restriction digest pattern and size confirmation by PCR. Only five genes were verified as positive: After 4 d of growth on YPD (with 2% glucose plus G418 to maintain plasmid selection) plates at 30°, whole colonies, arrayed in a “checkerboard” pattern , with the FASTA and GFF files extended to include noncoding RNAs (ncRNAs) and genes present in F45, but absent in S288c, as described in Materials and Methods). The table of counts was split into two subtables, with the first consisting of ORFs present in the S288c reference genome (genes with systematic names beginning with “Y”) and the second consisting of the novel F45 genes and ncRNAs. Library sizes were normalized using calcNormFactors, and dispersion parameters were estimated using the estimateGLMTrendedDisp and estimateGLMTagwiseDisp commands. To identify genes differentially expressed between overexpression strains and the F45 empty-vector control, we conducted pairwise testing using the glmFit and glmLRT commands, with a P-value cutoff of 0.01 . Only nuclear-encoded ORFs, present in the reference genome, with median basal expression of at least 1 read per million reads in the F45 empty-vector control were included when plotting log2 fold changes on scatterplots, carrying out factor analysis, and calculating regression parameters or correlation coefficients.Analysis of differential gene expression was carried out using edgeR [v. 3.6.8] (S. cerevisiae gene lists was performed using g:Profiler (http://biit.cs.ut.ee/gprofiler/). Holm-Bonferroni corrected enrichment P-values <0.05 were accepted, with moderate hierarchical filtering.Functional enrichment of DIG1 = 0.731, SAN1 = 0.883, TOS8 = 0.206, ROF1/YHR177W = 0.812, SFL1 = 0.894, and HEK2 = 0.918. Significant nonzero values on the common factor were identified using the general linear model functionality of edgeR . Significant nonzero values for the residual DIG1 profile were identified as the residual from a linear regression between the DIG1 profile and the common factor. The “contrast” terms for the common factor, multiplied by the regression coefficient, were subtracted from the contrast terms used to calculate the full DIG1 profile to give the contrast terms producing the residual. The variance explained by the common factor for each overexpression profile was calculated as the variance of the overexpression profile times the communality value from the factor analysis. The communality values were: DIG1 = 0.534, SAN1 = 0.780, TOS8 = 0.042, ROF1/YHR177W = 0.659, SFL1 = 0.799, and HEK2 = 0.842.Factor analysis was carried out using the fa command in the “psych” package of R (version 3.1.1 ), with aThe datasets generated during and/or analyzed in the current study are available in the Gene Expression Omnibus (GEO) under GSE98079.DIG1. To identify such modulators, we performed a large-scale overexpression screen for genes that reduced or eliminated the complex structure of the fluffy colonies. Briefly, we transformed the fluffy, euploid version of F45 from our previous study  we proceeded with only the multiply-hit plasmids. The high frequency of false positives may result from the gain of additional copies of chromosomes, a phenomenon known to occur at high frequency in the F45 background and which often results in loss of colony morphology , through intermediate , to weak (TOS8). These results . Because of the weak phenotypes associated with overexpressing some of these genes, we further tested their role in colony morphology by deleting them in a different strain background, F13 . HEK2 encodes an RNA-binding protein which regulates the flocculin FLO11, first via regulation of the ASH1 mRNA transcript, which specifies differential gene expression in mother vs. daughter cells, and also through a second, posttranscriptional mechanism  with a single amino acid change in the RING domain that inactivates the protein’s ubiquitin ligase activity without altering protein structure  identified in our overexpression screen are known or putative transcription factors, differences in RNA expression levels could help explain their influence on colony morphology. To investigate this, we began by comparing the mRNA expression patterns of each of the smooth overexpression strains to the fluffy progenitor (F45). Briefly, we isolated total RNA from single colonies growing on solid medium and prepared libraries to measure stranded mRNA using the Illumina TruSeq method . Each smooth overexpression strain showed a substantial number of genes that were significantly differentially expressed relative to the fluffy F45 vector-alone control  . Notably, significant differential expression was seen in the SAN1 and HEK2 overexpression strains, despite the fact that these genes do not encode transcription factors.Because four of the six genes  and the SAN1 and ROF1 strains (R = 0.84) were very similar, despite the fact that genes in each pair encode one transcription factor and one protein with a different function. One possible explanation for these correlated pairs is that overexpression of the nontranscription factor leads to increased expression of the transcription factor, resulting in the same transcriptional profile as when the transcription factor is directly overexpressed from the plasmid. However, we found that for both pairs of genes with highly similar expression profiles (HEK2-SFL1 and SAN1-ROF1), the expression level of one gene in the pair was essentially unperturbed by the overexpression of the other . Factor analysis seeks to explain correlations between observed variables  in terms of a potentially smaller number of unobserved variables called factors or latent variables. Each observed variable is explained as a linear combination of these latent factors plus residual effects unique to the observed variable (including noise).To further explore the observed correlations between the RNA expression profiles of many of our overexpression strains, we carried out common factor analysis . This factor explained most of the variance in the ROF1, HEK2, SFL1, and SAN1 strains and left a similar level of residual variance in each strain, suggesting this residual variance might largely reflect a common level of noise rather than substantial strain-specific effects on expression (DIG1 strain (variance explained = 53%), leaving a substantially higher degree of residual variance for DIG1 than for the ROF1, HEK2, SFL1, and SAN1 overexpression strains . Taken together, these data suggest that overexpression of ROF1, HEK2, SFL1, and SAN1 induces a single expression profile (the common factor), although to differing extents, as the slopes of linear regressions between the common factor and the transcriptional profiles of these strains varied from 0.24 for ROF1 to 0.39 for HEK2  (CIS3 deletion (cis3Δ) provided the cleanest readout of the smooth colony environment, and that this environment does not substantially alter gene expression. Consistent with this result, correlations between the cis3Δ and our overexpression profiles were very poor  and strongly (>1.5-fold) induced and repressed in each profile (Table S2 and Table S3). This analysis identified several overlapping features of GO term and pathway enrichment. In both profiles, cell periphery genes were overrepresented among induced genes, and genes encoding anchored components of the membrane and reproductive genes were overrepresented among repressed genes. In both the TOS8 and common factor profiles, MAPK signaling genes were significantly overrepresented among genes repressed in the smooth colonies, as were genes involved in mating. One of the pathways known to induce complex colony morphology is the filamentation MAPK cascade, and the components of this pathway overlap to a large extent with the mating/pheromone-response pathway  term, transcription factor target, and metabolic/regulatory pathway enrichment among the sets of genes significantly   differentially expressed in both profiles tended to show a consistent direction of effect . Among 400 such genes, 153 were induced and 173 were repressed in both profiles. In contrast, only 36 genes were induced in the common factor and repressed by TOS8 overexpression, and only 38 were repressed in the common factor and induced by TOS8 overexpression.We hypothesized that the genes consistently upregulated or consistently downregulated in both the common factor and the  profile . Similar = 0.04) , genes tTOS8 overexpression and common factor profiles were enriched for several GO terms including “transmembrane transport” , “cell periphery” , and “extracellular region”  (Table S4). Similarly, enrichment for several GO terms was seen in the set of genes commonly repressed in both the TOS8 overexpression and common factor profiles, including “conjugation with cellular fusion” , “cell periphery” , and “anchored component of membrane”  (Table S4). Repressed genes were also enriched for targets of Tec1 (P = 3.35e−6), a transcriptional inducer of complex colony morphology that acts in the filamentation MAPK pathway (Table S4). TEC1 itself was also one of the genes commonly repressed, as were the flocculin genes FLO10 and FLO11 that are known to be effectors of complex colony phenotypes  and targets of Tec1 (P = 1.85e−5) were overrepresented among the genes repressed in the DIG1-residual profile (Table S5). Therefore, the residual DIG1 profile appears to reflect repression of the filamentation MAPK pathway.The  strains . The traTOS8 profile are genes in the filamentation MAPK pathway. Consistent with this, many of these genes (80/173) were also downregulated in the DIG1 residual profile, a highly significant enrichment . This further supports the hypothesis that a substantial component of the overlap between the TOS8 and common factor profiles reflects repression of the filamentation MAPK pathway. In the DIG1 overexpression strain these genes are repressed as part of the common factor and then further repressed by specific effects of DIG1 overexpression . This double signaling through the filamentation MAPK pathway may explain why the DIG1 overexpression strain has such a strong smooth phenotype . Similar to reference mRNAs, substantial differential expression was observed among these transcripts in all overexpression strains (Table S6). The same pattern of correlations between overexpression strains that was seen with reference genes . This implies that the three underlying transcriptional profiles that we identified from analysis of reference genes extend to include effects on the expression of substantial numbers of nonreference genes and noncoding transcripts.Finally, we extended our analysis to the expression levels of a large number of noncoding RNAs (ncRNAs) and a small number of genes present in strain F45, but absent from the S288c reference genome  is the master regulator of the white-opaque switch and ectopic expression of WOR1 can drive an entire population of white cells to become opaque  of the regulated switching between the fluffy and smooth transcriptional states produces an adaptive change in colony morphology, and the expression profiles that we observe reflect transcriptional states that S. cerevisiae may access in response to environmental conditions favoring the biofilm or nonbiofilm states of the microbial community. We previously demonstrated that the smooth and fluffy (biofilm) states of the F45 background used in this study are each adaptive in different environmental conditions  is sufficient to cause a fluffy-to-smooth transition in the same genetic background . This was true for the putative transcription factor Rof1, which we named based on its phenotype in this study, and the SFL1, SAN1, ROF1, and HEK2) produced a single transcriptional state, the common factor, consistent with them operating in a single transcriptional regulatory pathway. This common factor is also a large component of the transcriptional profile observed when a fifth gene, DIG1, is overexpressed.We expected that perturbation of genes operating in the same regulatory pathway would produce the same transcriptional response. Overexpression of four of the six genes that we identified (SFL1) and the filamentation MAPK pathway (including DIG1)  transcriptional nodes in the signaling network, while HEK2 and SAN1 might regulate elements of the signaling pathways at the RNA- and protein-abundance levels. For these four genes, the signal produced by overexpression would, after signal integration across the network, have the same final effect in terms of which transcriptional state the cell/colony adopts. That is, the transcriptional state corresponding to the common factor may be one that emerges from any one of a range of perturbations.The “single regulatory pathway” identified by these genes is probably better understood as a signaling network, as it encompasses both the cAMP-PKA pathway (including ng DIG1) . Such a TOS8 did not induce the common factor transcriptional state. This result suggests that Tos8 operates in a different regulatory pathway than the products of the other genes. This, in turn, suggests that the TOS8 overexpression profile may represent a distinct transcriptional state that cells can adopt under environmental conditions that differ from those producing the common profile.In contrast to the other genes in our study, overexpression of TOS8 transcriptional profiles are essentially uncorrelated and only a small proportion of genes are significantly differentially expressed in both. However, among the overlap between these two profiles, there is a statistically significant overrepresentation of genes showing differential expression with the same direction of effect, i.e., induced or repressed in both profiles, compared to genes that have opposite directions of effect. This result suggests the existence of a differentially expressed subset of genes that are involved in the common fluffy–smooth morphological change associated with both profiles. Because the overexpression strains appear to be operating through gene expression regulatory mechanisms, the commonly differentially expressed genes may represent modules of coregulated genes that respond as a group to environmental signals.The common factor and TOS8 and common factor profiles were also significantly repressed in the residual DIG1-expression profile, i.e., the gene expression changes seen in the DIG1 overexpression strain, after accounting for the effect of the common factor. It therefore appears that DIG1 and the filamentation MAPK pathway are particularly important in control of the critical gene module(s), repressing genes whose expression is required for fluffy colony formation.A high proportion of the genes commonly repressed in both the www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.042440/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.