To investigate the relationship of human semen parameters with season, age and smoking status.The present study used data from subjects recruited into an ongoing cross-sectional study on the relationship between environmental agents and semen characteristics. Our population consisted of 306 patients who presented to the Vincent Memorial Andrology Laboratory of Massachusetts General Hospital for semen evaluation. Sperm concentration and motility were measured with computer aided sperm analysis (CASA). Sperm morphology was scored using Tygerberg Kruger strict criteria. Regression analyses were used to investigate the relationships between semen parameters and season, age and smoking status, adjusting for abstinence interval.p < 0.05). There was suggestive evidence of higher sperm motility and percent of sperm with normal morphology in the spring than in the other seasons. There were no statistically significant relationships between semen parameters and smoking status, though current smokers tended to have lower sperm concentration. We also did not find a statistically significant relationship between age and semen parameters.Sperm concentration in the spring was significantly higher than in winter, fall and summer (We found seasonal variations in sperm concentration and suggestive evidence of seasonal variation in sperm motility and percent sperm with normal morphology. Although smoking status was not a significant predictor of semen parameters, this may have been due to the small number of current smokers in the study. Male factor infertility has been identified as the main or secondary cause in >40% of infertile couples. The number of office visits each year for infertility in the United States has risen from 600,000 in 1968 to 2 million by the 1990's. Semen analysis is a central component in male infertility evaluation [et al. found a significant seasonal variation in sperm count, with highest sperm counts in late winter and early spring and lowest concentration in late summer [Seasonal variations in semen parameters have been reported in both fertile and infertile men -6. Sainte summer . There ae summer . Paulsone summer .et al. found an association between smoking and reduced semen quality [There is a consistent association between cigarette smoking and fertility in women -13. Howe quality  while ot quality .The present study explores the relationship of human semen parameters with season, age and smoking status, while controlling for abstinence interval. In an earlier publication, we reported on the relationship between semen parameters, season, and age using data from a retrospective review of an existing laboratory database at the Massachusetts General Hospital Vincent Memorial Andrology Laboratory . The preThe Human Subject Internal Review Board committees of the Harvard School of Public Health and the Massachusetts General Hospital approved the study (IRB #96-7545). Informed consent was obtained from each participant before entering the study.The study population consisted of patients (n = 306) presenting to the Vincent Memorial Andrology Laboratory of Massachusetts General Hospital (MGH) between January 2000 and January 2002 for semen analysis as a component of infertility evaluation. Of the 306 subjects, age was not available for five study subjects despite several attempts at follow up after the initial study visit. Sperm morphology was not performed on two subjects with azoospermia.Men were approached only if the referring physician had previously received a brief description of the study. The Andrologist approached patients and asked if they were willing to meet the research nurse to learn about the research study, noting that it was optional and not part of their clinic appointment. All patients approached had the opportunity to decide for themselves if they wanted to participate in the research study. Inclusion criteria for this study were: 1) scheduled for a routine semen analysis as a patient at the Vincent Memorial Andrology Laboratory, 2) either English or Spanish speaking, 3) age 18–54 years, 4) had not had a vasectomy, and 5) was not currently receiving hormone therapy.All subjects received an explanation of the study in a private setting prior to obtaining informed consent. Information on lifestyle, such as smoking history and medical history was obtained by nurse interview.From the date that semen specimen was produced, samples were classified by season as follows: winter = December, January, February; Spring = March, April, May; Summer = June, July, August; and Fall = September, October, November.Semen was collected by masturbation in a private room in the hospital. The semen specimen was allowed to liquefy for at least 20 minutes in an incubator at 37°C and was analyzed within 60 minutes after collection. A routine semen analysis was performed which included the following parameters: semen volume, pH, sperm concentration, sperm motility, and sperm morphology.Volume was determined and sample color and viscosity were recorded. Semen pH was measured within one hour of ejaculation .All fresh semen samples were analyzed for sperm concentration and motion parameters by the HTM-IVOS Semen analyzer (Hamilton-Thorn 10HTM-IVOS, Beverly, MA, USA). Setting parameters and the definition of measured sperm motion parameters for CASA were established by the manufacturer:  threshold: 80.0%; medium VAP cutoff: 25.0 um/s; and duration of tracking time: 0.38 sec). To measure both sperm concentration and motility, aliquots of semen samples (5 μl) were placed into a pre-warmed (37°C) Makler counting chamber . A minimum of 200 sperm from at least four different fields was analyzed from each specimen. Percent motile sperm was defined as World Health Organization (WHO) grade "a" sperm (rapidly progressive with velocity ≥ 25 μm/s at 37°C) plus "b" grade sperm (slow/sluggish progressive with velocity ≥ 5 μm/s but < 25 μm/s) .Using the "feathering" method at least two slides were derived from each fresh semen sample . The reset al. [Morphological assessment was performed at a magnification of 100X with an oil immersion using a Nikon microscope (Nikon Company, Tokyo, Japan). As the slide was scored, the normal and abnormal spermatozoa (head defects, midpiece defects and tail defects) were noted. Sperm morphology was determined using the strict criteria by Kruger et al. . From eaMultiple regression analyses (SAS version 8.2, SAS Institute, Cary, NC, USA) was performed to investigate whether there were differences in semen parameters with respect to season, age, and smoking status. Winter was used as the reference season. We also investigated month-to-month variation in semen parameters. For each semen parameter, a separate multiple regression was performed. Semen analysis parameters were entered into the models both untransformed and after square root transformation because of their skewed distribution. Since the square root transformed results were similar to the untransformed results, and are simpler to interpret, only the untransformed results are presented. To explore whether the semen parameters and age relationships were linear, age was used as both a continuous and categorical variable (less than 30 years, 30 to 40 years, and greater than 40 years old). Abstinence time was modeled as an ordinal five-category variable (2 or fewer days, 3, 4, 5, and 6 or more days).Subject's ages ranged from 18.2 to 54.3 years. The mean (± SD) age was 35.9 (± 5.6) years. The majority of the subjects (67%) were between 30 and 40 years old; only 11% were <30 years and 21% were >40 years old.Mean sperm concentration was above the WHO reference values . Averagep = 0.06).Seasonal variations in semen quality are shown in Table There were also seasonal variations in sperm morphology parameters. The mean percent normal morphology in Spring (7.5%) was greater than in Summer (6.7%), Fall (7.0%) or Winter (6.4%), though not statistically significant. These seasonal differences remained after adjusting for age and abstinence interval. Mean semen volume and pH were similar across season.There were no statistically significant relationships between age with semen volume, sperm concentration, percent motility, and percent normal morphology. However, there was little variability in age since the majority of men (> 65%) were between 30 and 40 years old.In the present study, only 22 men were current smokers and 57 were ex-smokers. After controlling for age and abstinence time, there was no statistically significant relationship between semen quality and smoking status, though current smokers tended to have lower mean sperm concentration (83 million/ml) than never smokers (104 million/ml).Although there are numerous previously published studies investigating the relationship among semen parameters and season, age and smoking status, the data are not entirely consistent. To determine if these associations are robust, replication is required. It is the accumulation of consistent observations from epidemiological studies that provides confidence in the findings. Therefore, we believe the present study adds to the literature since it provides replication of seasonal trends in semen parameters. In addition, the present study was conducted in men residing in the Northeast region of the United States, an area with distinct seasons and one that has not been well represented in the literature on this topic. Our study has several strengths. The center selected as the site of this study  has a large and readily accessible population of men seeking infertility evaluation. Based in a large tertiary care facility, the Andrology Laboratory draws patients from diverse backgrounds throughout the New England region of the United States, receiving referrals from physicians in the community and the medical center.Our study also had potential limitations. Since it is known that there is within-person variability in semen parameters, using a single sample to characterize an individual may introduce measurement error, likely to be random. Another potential limitation is that it is possible that if some men recently moved to the New England area this may introduce bias. However, of the men in the study, 96% of them lived in New England area for at least 3 months prior to their semen analysis, the period of sperm development. Therefore, the concern with recent immigration to the New England area would be minimal.In the present study, we found higher sperm concentrations, motility and percent normal morphology in the spring than in other seasons. This may partially explain seasonal patterns of births in United States, where there is a deficit of spring births , conceivet al. [Our data are in agreement with previous reports of seasonal variation in sperm concentration with spring having the highest concentration. Gyllenborg et al.  found hiet al. ,21. In oet al. . In our Seasonal variations in sperm morphology were recently explored by Centola and Eberly in a large sample of California men . AlthougEffects of temperature and hours of daylight may partially explain seasonal variations in semen quality. Sperm production in humans is known to decrease when testicular temperature is raised by experimental techniques . Normal et al. reported there were no significant month-to-month fluctuations in semen volume and sperm density among men who resided in the tropics [Spermatogenesis occurs over approximately three months and does not seem to vary in duration among men . Chia et tropics , where tet al. suggested that increased age was associated with a decline in semen volume, sperm motility, and sperm morphology but not with sperm concentration [While we did not find a relationship between age and semen parameters, there was little variability in age in our study population. In our earlier study we found inverse associations between age and sperm concentration, motility and morphology . Howeverntration .Studies have shown associations between cigarette smoking and infertility in women, time to conception, and risk of spontaneous abortion, as well as reduction in fecundity ,12. DataHowever, in contrast, in a cross-sectional study of men with proven fertility (n = 243), cigarette smoking was associated with significantly lower semen volume (but not other semen parameters) after adjusting for age and alcohol consumption . ResultsIn our study, only 22 men were current smokers and 57 were ex-smokers. After controlling for age and abstinence time, there was no significant relationship between semen quality and smoking status, though current smokers tended to have lower sperm concentration. The present study is ongoing and we will reexamine these relationships in a larger dataset. In addition, we are also collecting information on other lifestyle and personal factors, such as physical exercise, stress, and alcohol intake. Their relationship with semen parameters will be investigated in the future when we have a larger number of subjects.We found seasonal variations in sperm concentration and suggestive evidence of seasonal variation in sperm motility and percent sperm with normal morphology. Although smoking status was not a significant predictor of semen parameters, this may have been due to the small number of current smokers in the study.The authors declare that they have no competing interests.ZC, LG-B, IS, and RH all contributed equally to this work and reviewed the manuscript.


The biological basis of pain in chronic pancreatitis is poorly understood. Mast cells have been implicated in the pathogenesis of pain in other conditions. We hypothesized that mast cells play a role in the pain of chronic pancreatitis.We examined the association of pain with mast cells in autopsy specimens of patients with painful chronic pancreatitis. We explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS) -induced chronic pancreatitis in both wild type (WT) and mast cell deficient mice (MCDM).Archival tissues with histological diagnoses of chronic pancreatitis were identified and clinical records reviewed for presence or absence of reported pain in humans. Mast cells were counted.The presence of pain was assessed using von Frey Filaments (VFF) to measure abdominal withdrawal responses in both WT and MCDM mice with and without chronic pancreatitis.Humans with painful chronic pancreatitis demonstrated a 3.5-fold increase in pancreatic mast cells as compared with those with painless chronic pancreatitis.WT mice with chronic pancreatitis were significantly more sensitive as assessed by VFF pain testing of the abdomen when compared with MCDM.Humans with painful chronic pancreatitis have an increased number of pancreatic mast cells as compared with those with painless chronic pancreatitis. MCDM are less sensitive to mechanical stimulation of the abdomen after induction of chronic pancreatitis as compared with WT. Mast cells may play an important role in the pathogenesis of pain in chronic pancreatitis. Although pain is the presenting symptom of most patients with chronic pancreatitis, its neurobiological basis remains poorly understood . In the We hypothesized that mast cells are involved in the pathogenesis of pain in chronic pancreatitis. This hypothesis is based on the following observations. First, mast cells have been associated with human conditions in which pain is a predominant symptom. Interstitial cystitis and irritable bowel syndrome are both conditions in which pain is out of proportion to the objective pathological findings ,6. In boW/KitW-v mice, a strain deficient in mast cells (MCDM).Our first aim was to analyze the presence and distribution of mast cells in autopsy specimens of chronic pancreatitis and study the correlation, if any, with historical documentation of pain. We then explored our hypothesis further using an experimental model of trinitrobenzene sulfonic acid (TNBS)-induced chronic pancreatitis in both wild type and KitAutopsy records from the University of Texas Medical Branch from the years 1993 to 2000 were searched electronically for the term "pancreatitis." One-hundred sixty-six patients were identified of which 26 patients carried an autopsy diagnosis of chronic pancreatitis and 140 patients carried a diagnosis of acute pancreatitis. The medical charts from patients with an autopsy diagnosis of chronic pancreatitis were reviewed for documentation of a medical history of chronic pancreatitis. If no such documentation was present in the chart, patients were excluded from the study (12/26). Thus, 14/26 patients with both a documented history and an autopsy based diagnosis of chronic pancreatitis, were included in the study. Patients were categorized as painful chronic pancreatitis (8/26) when they fulfilled one of the following criteria: a documented history of chronic abdominal pain clinically attributed to chronic pancreatitis that required the use of narcotics, and/or frequent admissions for recurrent abdominal pain clinically attributed to chronic pancreatitis, and/or a surgical or endoscopic procedure for refractory abdominal pain clinically attributed to chronic pancreatitis. Patients were categorized as non-painful chronic pancreatitis (6/26) if patients did not fit any of the criteria listed under painful chronic pancreatitis. In addition, the following data were collected: demographic factors (age and race), cause of death, comorbidities, clinical history of pancreatitis, etiology of pancreatitis, diagnostic studies supporting a diagnosis of pancreatitis . Human pancreatic control tissue was obtained from 8 arbitrarily chosen patients of whom the autopsy records recorded acute myocardial infarction as the cause of death. Their medical records were reviewed to ensure that they did not have a clinical history of pancreatitis. Therefore there were three categories of patients: one with painful chronic pancreatitis, one with non-painful chronic pancreatitis and non-pancreatitis controls. A pathologist, blinded to the group assignment, verified all histological diagnoses and counted mast cells on a Giemsa stained tissue section (average of 10 high-power randomly chosen (40X) fields per specimen). The protocol was approved by the Institutional Review Board of the University of Texas Medical Branch.W/KitW-v (MCDM) and the respective littermate control mouse strain, KitW-v – +/+ (WT). The mice were 3 months of age at the onset of the experiment with body weights of 25–30 gram.All mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Male mice were used from the following strain: WBB6F1/j-KitExperimental protocols involving mice were approved by our Institutional Animal Care and Use Committee (IACUC) in accordance with the guidelines provided by the National Institutes of Health.Mice were anesthetized with sodium Nembutal (50 mg/kg body weigh, i.p.) Following a midabdominal laparotomy, a canula was introduced into the common pancreato-biliary duct; the duct was ligated proximally and distally to ensure perfusion into the pancreas and prevent entry of the injected substance into the liver or duodenum. 0.1 ml of 1% TNBS in phosphate buffered saline (PBS)-10% ethanol, pH 8, was infused into the pancreas (modified after Puig-Divi ). The caVFF hairs consist of a series of filaments of increasing diameter that produce increasing sensations of touch when applied to the skin. When the tip of a fiber of given length and diameter is pressed against the skin, the force of application increases until the fiber bends. After the fiber bends, continued advance creates more bend, but not more force of application. This principle makes it possible to apply a reproducible force to the skin surface. VFF testing is an established behavioral pain assay used to determine mechanical pain thresholds in somatic pain. More recently, VFF testing has been used as a surrogate marker for visceral pain ,15.Mice were placed in a cage with a mesh floor and habituated to the environment for 30–60 minutes. Measurements were taken from the abdomen and the plantar surface of both hindpaws over a period of three weeks prior to the surgery and for a total of three weeks after the surgery Figure . VFF fil2 at 4°C overnight. Sections from paraffin-embedded specimens were stained with hematoxyline and eosin and observed under a light microscope. Pathological changes were scored based on a scale described by Tito et al. by a pathologist blinded to the different treatment groups [Fresh specimens of the mouse pancreas were fixed in 10% formaldehyde in PBS pH 7.4 containing 1 mM MgClt groups .Comparisons of the number of mast cells in autopsy specimen were analyzed using the Mann-Whitney U test.For each behavioral experiment . This somatic sensitization can be quantified using VFF to stimulate the somatotopically appropriate abdominal region and measuring the abdominal withdrawal response. Thus, VFF testing of the anterior abdominal wall can be used as a surrogate marker for visceral pain. Although this is the first time that this technique has been used for the measurement of referred visceral hyperalgesia in a mouse model of chronic pancreatitis, this method has previously been described and validated to assess the severity of referred visceral pain for models of colonic hypersensitivity  as well Mast cells produce a variety of degranulation products in the setting of inflammation that may activate and/or sensitize primary nociceptive neurons. The neurotrophin growth factor (NGF) is one such product -22. NGF However, before concluding a definite role for mast cells from our experimental data, it should be noted that MCDM carry a spontaneous mutation for tyrosine kinase receptor c-kit which not only produces a deficiency of mast cells but may have an independent effect on the function of sensory neurons, which are known to express it . TherefoOur data should increase awareness of the importance of mast cells in the pathogenesis of painful inflammatory conditions such as chronic pancreatitis and encourage experimental studies for further testing of this hypothesis.TNBS – trinitrobenzene sulfonic acid, WT – wild type, MCDM – mast cell deficient mice, VFF – von Frey Filaments, IACUC – Institutional Animal Care and Use Committee, PBS – phosphate buffered saline, NGF – neurotrophin growth factor, TrkA – tyrosine kinase A, TRPV1 – Transient Receptor Potential Family V receptor 1 (previously known as VR1), PAR-2 – proteinase-activated receptor-2The author(s) declare that they have no competing interests.WAH conceived of the study, participated in its design and coordination and wrote the manuscript. KG completed the behavioral studies. SYX completed the histological analyses and mast cell counts. JHW provided technical assistance with the surgical procedures and participated in the data analyses and review of the manuscript. WDW provided support for the behavioral studies as well as their analyses. PJP provided financial support and participated in the review of the manuscript.The pre-publication history for this paper can be accessed here:


Memento, the protagonist, Lenny, is plagued by a crippling neurological disorder that renders him incapable of storing memories for longer than fifteen minutes. He wants nothing more than to avenge the murder of his wife, but as another character in the film tells him, “Even if you get revenge, you're not going to remember it. You're not even going to know it happened.” Lenny has anterograde amnesia, a condition typically caused by stroke or other illness. (In the film, it's caused by a blow to the head.) Most forms of amnesia—including the more common retrograde amnesia, which involves the loss of long-term memory—are caused by some type of brain injury— particularly to the hippocampus.In the 2002 thriller Retrograde amnesia can arise from brain damage that interferes with memory storage, retrieval, or consolidation. And it is by studying retrograde amnesia that scientists have developed theories explaining where the brain stores memory traces and how it consolidates them into long-term memories. What ultimately causes amnesia—a failure to store memories or a failure to retrieve them—is not clear.A major challenge in resolving this question experimentally is being able to determine whether an animal has truly recovered a lost memory or has simply re-learned the task at hand. By refining a classic behavioral neuroscience experiment to test spatial learning and memory, Livia de Hoz, Stephen Martin, and Richard Morris have developed a novel protocol that distinguishes retrieval from new learning. The experiment, developed by Morris over fifteen years ago, takes advantage of the fact that rats are good swimmers but prefer life out of water. Rats are placed in a pool and trained to remember the location of an escape platform, hidden just under the surface. (The rats can't see it because the water is cloudy.)Here, de Hoz et al. trained rats to a particular platform location—their escape route—then gave them hippocampal lesions. One group received “sham” lesions, another partial lesions, and a third complete lesions. Partial lesions, the authors explain, should damage only a subset of the stored memory traces and thus weaken the rats' memory rather than completely disrupting it.The rats' postoperative memory was tested by first placing them in the pool with the platform hidden: as expected, the lesioned rats couldn't remember where it was. After a minute, the platform was raised above the water, to remind them that it existed and that they could escape by climbing onto it. The platform was revealed in the original location for half the animals and in a novel location—which could be construed as misleading information if you're a rat—for the other half. If raising the platform was facilitating re-learning, then animals capable of learning would look for the platform in the new location. But if raising the platform functioned as a reminder, then animals should gravitate toward the place they were trained to, regardless of whether the platform reminder was in the original location or a new one. And that's what happened to the rats with partial lesions.De Hoz et al. then repeated the experiments in a new environment. If the rats managed to escape in this situation, the authors explain, then the reminder treatment could be causing new learning rather than triggering recall of the original training experience. Not surprisingly, the rats with total lesions failed to learn in this new environment (or to be reminded in the previous experiment), while the control rats adapted to the new location. The group with partial lesions failed to learn.Since partially lesioned rats responded to the reminder treatment by recalling the original platform location, their initial memory failure couldn't have resulted from a storage failure. And since they did not realize they could escape from the new location, the reminder didn't induce new learning. Among the many questions these results raise is what role the hippocampus plays in memory storage and retrieval. It could be, as the authors propose, that essential components of spatial memory traces are either stored in the hippocampus or reactivated there, since only the partially lesioned rats responded to reminders. Whether that proves true, de Hoz et al. have contributed a much needed resource for investigating the neural basis of memory loss.


There are controversial results on the efficacy of the abandoned, intrathecal predominant methylprednisolone application in multiple sclerosis (MS) in contrast to the proven effectiveness in intractable postherpetic neuralgia.We performed an analysis of the efficacy of the application of 40 mg of the sustained release steroid triamcinolone acetonide (TCA). We intrathecally injected in sterile saline dissolved TCA six times within three weeks on a regular basis every third day in 161 hospitalized primary and predominant secondary progressive MS patients with spinal symptoms. The MS patients did not experience an acute onset of exacerbation or recent distinct increased progression of symptoms. We simultaneously scored the MS patients with the EDSS and the Barthel index, estimated the walking distance and measured somatosensory evoked potentials. Additionally the MS patients received a standardized rehabilitation treatment.EDSS score and Barthel index improved, walking distance increased, latencies of somatosensory evoked potentials of the median and tibial nerves shortened in all MS patients with serial evaluation . Side effects were rare, five patients stopped TCA application due to onset of a post lumbar puncture syndrome.Repeated intrathecal TCA application improves spinal symptoms, walking distance and SSEP latencies in progressive MS patients in this uncontrolled study. Future trials should evaluate the long-term benefit of this invasive treatment. There are controversial results on the efficacy of the nowadays still abandoned, intrathecal steroid application predominantly due to a missing detailed evaluation of patients' characteristics, careful monitoring and standardized outcome measurements in multiple sclerosis (MS) . InitialAccordingly, there was no convincing superiority over the efficacy of the systemic steroid treatment. Some enrolled MS patients experienced a recent deterioration of symptoms due to prior acute relapses and/or ongoing chronic progression. Moreover study participants were not classified according to the various subtypes of MS progression ,6. HowevWe only enrolled clinically well characterized, consecutively referred MS patients table , 2 with ®), tolperison , tizanidin  on the first and last day of the hospital stay, length of hospitalization in days  in a standardized fashion before start and at the end of the intraspinal TCA treatment within a prospective study design . A techns tables  &2. The Each participant gave written informed consent for the TCA treatment, which was approved by the local ethical committee. The consent form included a detailed description of all putative risks of lumbar puncture and intrathecal TCA application.Data showed a normal distribution according to the Kolmogorow-Smirnow test. As a result, we only performed parametric tests. We used ANCOVA with repeated measures design including MS duration, MS types, change of dosages of concomitant drugs against spasticity, length of hospital stay, sex and age as covariates. We computed SSEP results by adding both sides in order to reduce amount of calculations for comparisons. Then we calculated the differences of latencies between both timepoints of recordings according to the formula [Initial - End = Diff] for correlation analysis. We employed linear regression for correlation analysis. Level of significance of p-values were adjusted to 0.05 divided by the number of performed comparisons respectively correlations.EDSS score  and Barthel index (n = 68) improved and walking distance (n = 161) increased table . SSEP laThere were no significant relations between computed changes of EDSS scores, walking distances and SSEP results (results not shown). Diff P2 correlated with Diff P3 (R [correlation coefficient] = 0.71), Diff P2 with Diff N2 (R = 0.90), Diff P3 with Diff N2 (R = 0.74) of SSEP latencies of the tibial nerves. There were relations between Diff N2 and Diff P2 (R = 0.85) of SSEP latencies of the median nerves. P-values of these correlations were below 0.0001. Moreover Diff P2 of the tibial nerves correlated with Diff N2 of the median nerves (R = 0.28, p = 0.004, n = 100). There was a certain trend for a significant correlation between Diff P2 of the tibial nerve and Diff P2 of the median nerve (R = 0.22, p = 0.03). No other significant associations of SSEP data appeared (results not shown).Our results demonstrate and confirm the efficacy of repeated intraspinal TCA application in MS patients with spinal symptoms, which improved according to the EDSS outcomes and the results of assessed walking distance and determined SSEP latencies . We intrHowever our present study outcomes do not allow any conclusions on the duration of the achieved benefit and the impact of TCA treatment on progression of MS . TherefoWe cannot exclude a certain impact of physiotherapy, the standardized rehabilitation treatment and an beneficial effect of hospitalization in general with its resulting concomitant positive influence on activities of daily living ,13. HoweOur data demonstrate the efficacy and safety of repeated intrathecal TCA application in MS patients with predominant spinal symptoms, which markedly improved. Some MS patients experienced post lumbar puncture syndrome with a frequency within the normal range , but typThe author(s) declare that they have no competing interests.KH, FS, HP and TM designed, coordinated and carried out the study. TM performed statistical data analysis and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Emerging Themes in Epidemiology publishes a collection of articles on the theme of Epidemiology in conflict. Violent conflict is an issue of great sensitivity within public health, but more structured research and reasoned discussion will allow us to better mitigate the public health impacts of war, and place the public health community in a more informed position in discussions about possible interventions in future conflicts.In this first special theme issue,  For allBritish Medical Journal editorial: to do nothing is as much a political decision as to challenge an issue head-on [The issue of war is particularly sensitive in the field of public health, some might even say taboo. It is not seen as appropriate to openly denounce war. Perhaps we feel that becoming involved in what is largely perceived to be a political issue constitutes a threat to that most precious of our ideals, that of objectivity. Or maybe we are uncomfortable with the thought of being associated with military activities. Specifically, the opportunistic use of military language in public health issues has not been particularly welcome. Political calls for war on the societal problems of drugs and cancer have been accused of victimizing individuals and setting implausible goals for their prevention and control -6. Ironi head-on .Epidemiology in conflict. Our reason for doing so is simple: regardless of the political context, war is bad for your health. The politicization of war within public health is unfortunate; re-framing public health questions within a political context prevents us from conducting informed discussion and finding rational solutions to them. Here, we draw a parallel with the early efforts to communicate the link between smoking and lung cancer. In his reflections on the subject [Given such an environment, readers may find it surprising that we should devote our first special issue to the subject of  subject , Ernst W...the consensus of opinion among experts is not sufficient to create action unless such consensus is translated into preventive or control measures.... Scientists and physicians cannot be content with discoveries until their beneficial or protective outcome for the population has been fully realized. This means that the members of the scientific and medical community must become more proactive in public health matters [" matters ."The issue of consensus is important, but difficult. Some will argue that war is inherently bad for public health and should always be opposed. Others will consider some wars justifiable if they address gross injustice or human rights abuses. And yet others, in line with the Geneva Conventions, will see wars, just or not, as inevitable and will want to focus on mitigation of human suffering.Regardless of one's viewpoint, each of these positions needs to be supported by an evidence base with answers to the questions of when, how and why war is bad for public health, as well as how the adverse health effects of war may be prevented. Therein lies the greatest challenge for epidemiologists. Conflict situations deny us access to data and dissolve the health infrastructures on which we rely for the collection of epidemiologic information. In the face of such challenges, the authors of the articles in this special issue deal with a broad range of issues of great relevance to epidemiologists.et al. [Mock et al.  argue thet al. [McDonnell et al.  evaluateRoberts and Hofmann  place thEpidemiology in conflict, so that we may be kept informed of developments in the field and promote the public health perspective in discussions about future conflicts.These papers provide a foundation on which we hope authors will continue to build so that a comprehensive range of relevant topics on this subject may be compiled. There remain many issues to be addressed by the epidemiology community (see figure). Some of these issues involve challenges so great that we have perhaps not even begun to think about how we might start tackling them. We encourage authors to continue submitting articles on the theme of We hope readers intending to take up these challenges will be informed, inspired and provoked into action. This is a call to arms, not against the barriers of physical inactivity and excessive caloric intake, the adaptability of infectious agents or the subtleties of gene-environment interactions, but against the sheer brutality of human beings killing each other. We encourage readers to research and discuss the humanitarian and public health consequences of this social disease. The knowledge gained will allow us to better mitigate the public health impacts of war, and place the public health community in a more informed position in discussions about possible interventions in future conflicts. The pen may yet prove to be mightier than the sword, but only as long as it keeps writing.


ATP channels in rat insulinoma cells, by the same mechanism as leptin.3-guanidinopropionic acid derivatives reduce body weight in obese, diabetic mice. We have assessed whether one of these analogues, the aminoguanidine carboxylate BVT.12777, opens KATP channels. In contrast, BVT.12777 did not activate heterologously expressed pancreatic β-cell KATP subunits directly. Although BVT.12777 stimulated phosphorylation of MAPK and STAT3, there was no effect on enzymes downstream of PI3K. Activation of KATP in CRI-G1 cells by BVT.12777 was not dependent on MAPK or PI3K activity. Confocal imaging showed that BVT.12777 induced a re-organization of cellular actin. Furthermore, the activation of KATP by BVT.12777 in CRI-G1 cells was demonstrated to be dependent on actin cytoskeletal dynamics, similar to that observed for leptin.BVT.12777 hyperpolarized CRI-G1 rat insulinoma cells by activation of KATP channels in insulinoma cells. Unlike leptin, BVT.12777 activates KATP channels in a PI3K-independent manner, but, like leptin, channel activation is dependent on actin cytoskeleton remodelling. Thus, BVT.12777 appears to act as a leptin mimetic, at least with respect to KATP channel activation, and may bypass up-stream signalling components of the leptin pathway.This study shows that BVT.12777, like leptin, activates K KATP channels are comprised of two proteins, derived from the sulphonylurea receptor (SUR) family and an inwardly rectifying K+ channel (Kir6.x family), the exact composition of these being dependent upon tissue [ATP channels consisting of Kir6.2 and SUR1 subunits [ATP channels are present in numerous tissues and are the target for drugs that inhibit or increase channel activity [ATP channel activity in pancreatic β-cells has profound effects on insulin secretion and glucose homeostasis [ATP channel activation induces hyperglycaemia in animals and man [ATP channels in many other tissues, notably muscle and central neurons, has stimulated interest in the development of novel, selective KATP channel openers for the treatment of various diseases [ATP-sensitive Kn tissue ,2. For esubunits . KATP chactivity ,5. The aannel Kir.x familyeostasis . In cont and man . This la and man . It has  and man . A simil and man . The prediseases ,11.ob gene product leptin has been demonstrated to activate KATP channels in pancreatic β-cells [ATP channels of hypothalamic glucose-responsive neurones [ATP channels may also be an important target for drug manipulation with respect to centrally driven control of glucose and energy homeostasis. The aminoguanidine carboxylate, BVT.12777  mice [ATP channels in the CRI-G1 insulin secreting cell line, a useful model for pancreatic β-cells [ATP channels [The  β-cells  and insu β-cells , consist β-cells . One of  β-cells . Interesneurones ,17 indicneurones . These lob) mice . Here we β-cells , and forchannels ,22,23.ATP channels in the closed state, the mean resting potential was -38.7 ± 1.7 mV (n = 10), similar to values reported in previous studies [k of -84 mV in this system, indicating increased K+ conductance. CRI-G1 cells responded to BVT.12777 in an all or none manner, with cells undergoing full hyperpolarization and increase in conductance, at all concentrations (100 – 300 μM) examined. Such an effect has also been reported for leptin on CRI-G1 cells [ATP channel inhibitor, tolbutamide (100 μM) during BVT.12777 exposure  resulted in activation of single KATP channel currents , normalised to the control for each recording, plotted against time of exposure to BVT.12777. BVT.12777 activation of KATP channels was demonstrated to be reversibly inhibited by 100 μM tolbutamide .Under current clamp conditions with 5 mM ATP in the pipette solution to maintain K studies ,22 underG1 cells . Removale Figure  completes Figure ; n = 7. ATP channel activity in excised membrane patches was also examined. Recordings were made from inside-out patches in symmetrical (140 mM KCl in pipette and bath solutions) K+ at a membrane potential of -40 mV. KATP channels were identified by inhibition of channel activity following application of 100 μM MgATP to the inner membrane aspect of the patch, which reduced normalised NfPo from 1.0 to 0.23 ± 0.05 . Subsequent application of 100 μM BVT.12777, in the continued presence of MgATP, induced a gradual increase in KATP channel activity .The effect of BVT.12777 on KATP channels is PI3K dependent [ATP channels in CRI-G1 cells by direct (like diazoxide) or indirect (like leptin) mechanisms.Leptin and diazoxide hyperpolarized CRI-G1 cells, in a manner similar to that of BVT.12777 (data not shown). Leptin (10 nM) induced a hyperpolarization from a mean membrane potential of -47.6 ± 1.6 mV to -68.5 ± 1.9 mV , and application of tolbutamide (100 μM) reversed this action, returning the membrane potential to -47.5 ± 1.9 mV (n = 4). Diazoxide (200 μM) rapidly hyperpolarized CRI-G1 cells from a mean membrane potential of -49.9 ± 1.7 mV to -74.0 ± 1.5 mV , with tolbutamide (100 μM) also reversing this action, returning membrane potential to -46.9 ± 3.8 mV (n = 6). Leptin, but not diazoxide activation of CRI-G1 Kependent ,23. ThusATP channels .Pre-incubation of CRI-G1 cells (20 min) with inhibitors of PI 3-kinase, wortmannin (10 nM) or LY294002 (10 μM) had no significant effect on the mean resting membrane potential or slope conductance of CRI-G1 cells and did not prevent BVT.12777 from causing hyperpolarization and increased cell conductance Figure . In the s Figure . Mean chATP activation, as described previously [ATP current . Consequently, we utilized an alternative expression system, the HEK 293 cell line [Oocytes injected with Kir6.2 and SUR1 cRNAs were challenged with sodium azide (3 mM) to elicit a reversible increase in current, which was completely blocked by 1 μM glibenclamide or 0.5 mM tolbutamide, indicating that the current was due to Keviously ,25. In oATP channels. However, activation of MAPK has been implicated as a significant intermediate for both insulin and leptin signalling pathways in various cell types [ATP channels. Application of UO126 (25 μM) inhibited approximately 90 % of KATP channel activity in cell-attached or inside-out recordings, whereas 1–10 μM UO126, concentrations that suppresses activation of MAPKK [f.Po was 1.86 ± 1.45 (n = 3) and 2.44 ± 2.00 (n = 3), at 5 and 15 minutes respectively; P < 0.05). In addition, increasing UO126 to 10 μM had no effect on BVT.12777 induced KATP channel activation.Exposure of CRI-G1 cells to BVT.12777 (100 μM) for up to 30 minutes had no consistent effect on the phosphorylation of enzymes downstream of PI3K (PKB and its downstream target, GSK3), but did increase the phosphorylation of STAT3 (n = 4) and MAPK . These data are in agreement with the lack of BVT.12777 sensitivity to PI3K inhibitors on activation of Kll types -29. ThusATP channels in the CRI-G1 cell line is dependent upon reorganisation of the cytoskeleton, a process downstream from PI3K activation [ATP channels occurs through alteration of actin filament dynamics. For this series of experiments the heptapeptide mushroom toxin phalloidin [ATP channel activation by BVT.12777 in the inside-out isolated patch configuration  and subsequent addition of 10 μM phalloidin had no further effect, as reported previously [ATP channel activity, with mean Nf.Po values of 0.06 ± 0.05 and 0.03 ± 0.01 in the absence and presence of BVT.12777 respectively . In contrast, the direct KATP channel opener, diazoxide activates KATP channels in the presence of phalloidin. In whole-cell experiments  contributes to this action. Leptin activation of KATP channel currents in CRI-G1 cells has previously been shown to be independent of MAPK, but prevented by the inhibitors of PI3K [ATP in this cell line. These data led us to suspect that BVT.12777, irrespective of its ability to initiate various signalling cascades in this cell line, increased KATP channel activity by a more direct effect on the channel subunits in a manner analogous to diazoxide, which is purported to interact directly with the SUR1 subunit [ATP channels, Kir6.2 and SUR1, in Xenopus oocytes, a commonly utilised expression system for electrophysiological studies of these recombinant channels [ATP channel currents. Thus we explored this question further by utilising a second heterologous expression system for Kir6.2-SUR1, HEK293 cells. Recordings from inside-out patches demonstrated that BVT.12777 did not activate Kir6.2-SUR1 currents in the presence of Mg-ATP, in contrast to diazoxide [ATP channel subunits, Kir6.2 and SUR1 are insufficient per se to bring about sensitivity to BVT.12777, and indicate that this opener may activate this channel type by an indirect mechanism (which is not available in oocytes or HEK cells).Leptin, via activation of the main signalling form of the leptin receptor (ObRb), has been shown to increase the phosphorylation of STAT3, MAPK and to stimulate PI3K pathways in various peripheral tissues, cell lines , and in  of PI3K . However subunit ,10. Thischannels ,25. Howeiazoxide  or sodiuiazoxide . OverallATP channels by leptin in CRI-G1 cells is PI3-kinase dependent the lipid products of this enzyme system, such as PtdIns(3,4,5)P3 also do not interact directly with KATP channels [3 increase KATP channel activity indirectly, through changes in cytoskeletal dynamics [ATP channel activity in cardiac myocytes [ATP channel currents in whole cell and inside out recording configurations indicating that this molecule likely causes the opening of KATP channels by a membrane delimited alteration of cytoskeletal dynamics. This mechanism of action is identical to that proposed for leptin and PtdIns(3,4,5)P3 activation of KATP in this cell line [ATP channel activity by both leptin and BVT.12777. The lack of effect of diazoxide on the actin filament structure is also supportive of this opener acting directly on the KATP channel subunits.Although the activation of Kchannels . Recent dynamics . It is wdynamics  and recedynamics . For examyocytes  and CRI-myocytes . Indeed,myocytes . Actin fmyocytes . The hepmyocytes  binds toell line . FluoresATP channels in CRI-G1 cells was evident regardless of whether it was applied to the external or internal surface of the cell. BVT.12777 signalling to KATP channels is not mediated by PI 3-kinase or MAPK, but does appear to depend on actin filament re-modelling. As leptin hyperpolarizes a sub-population of hypothalamic neurones by opening KATP channels [per se to be useful anti-obesity agents as they display hepatotoxicity [BVT.12777 activation of Kchannels , it is fchannels . Thus, atoxicity , understCells from the insulin secreting cell line, CRI-G1, and the human embryonic kidney cell line, HEK 293, were grown as described previously ,35. The Xenopus laevis frogs  following killing of the animal by destruction of the brain. The use of animals was in accordance with the Home Office Animals (Scientific Procedures) Act (1986) and approved by the local ethics committee. Separation and selection of oocytes and the preparation and injection of cRNAs were performed as described by [Ovarian lobes were removed from mature female ribed by .2 1, CaCl2, 1, HEPES 10 with glucose 10 (pH 7.4) were treated with BVT.12777 (100 μM) for 0, 1, 5, 15 or 30 minutes and whole-cell extracts were prepared as described [CRI-G1 cells, in normal saline . Cells were then fixed, permeabilised, stained with rhodamine-conjugated phalloidin (2.66 U ml-1) and visualised using a BioRad Microradiance, confocal imaging system as described by [t test. p < 0.05 was considered significant.CRI-G1 cells were gently washed in normal saline (containing in mM): NaCl 135, KCl 5, MgClribed by . The int2 1.8, HEPES 5 (pH 7.4 with KOH). Working concentrations of drugs were prepared in KD96 and superfused into the bath. Whole-cell current-clamp recordings with excursions to voltage clamp mode were used to monitor membrane potential and macroscopic currents from CRI-G1 cells. Cell-attached and excised inside-out recordings were made from CRI-G1 cells and HEK cells expressing Kir6.2 and SUR1 to examine single channel responses as described previously [f.Po; where Nf is the number of functional channels in the patch and Po is the open probability) as described previously [t test. P < 0.05 was considered significant. Recording electrodes were pulled from borosilicate glass and had resistances of 2–5 MΩ for whole cell recordings and 7–10 MΩ for cell-attached and inside-out experiments when filled with electrolyte solution. The pipette solution for whole-cell recordings comprised (in mM): KCl 140, MgCl2 0.6, CaCl2 2.73, Mg-ATP 5.0, EGTA 10, HEPES 10, pH 7.2 (free [Ca2+] of 100 nM), whereas for single channel recordings the pipette solution contained (in mM): KCl 140, CaCl2 1, MgCl2 1, HEPES 10, pH 7.2. The bath solution for whole-cell and cell-attached recordings was normal saline whereas for inside-out patches the bath solution contained (in mM): KCl 140, MgCl2 1, CaCl2 2, EGTA 10, HEPES 10, pH 7.2 (free [Ca2+] of 30 nM). All solution changes were achieved by superfusing the bath with a gravity feed system at a rate of 10 ml min-1, which allowed complete exchange within 2 min. All experiments were performed at room temperature (22–25°C).Whole cell currents from Xenopus oocytes were measured using a two-electrode voltage clamp technique as described by . Recordieviously ,35. Singeviously . All dat-1) and LY 294002 (10 mM) were stored as stock solutions in 1% methanol at -20°C. Cytochalasin B was stored as a 10 mM stock solution, and diazoxide and tolbutamide as 100 mM solutions, all in DMSO at 2–4°C. Mg-ATP was stored at -20°C as a 100 mM solution in 10 mM HEPES (pH 7.2). Wortmannin and UO126 were stored as 10 mM stock solutions in Me2SO at -20°C.Anti-PKB, which recognises all three isoforms of PKB, and the phospho-specific PKB (Thr308), GSK3α/β (Ser21/9), STAT3 (Tyr705) and p44/42 MAPK (Thr202/Tyr204) antibodies were obtained from Cell Signalling Technology Inc. Recombinant human leptin, wortmannin and LY 294002 were obtained from Novachem-Calbiochem and BVT.12777 ([2-(hydrazinoiminomethyl) hydrazino] acetic acid) was a gift from Biovitrum (Stockholm, Sweden). Tolbutamide, Mg-ATP, diazoxide, sodium azide, phalloidin and cytochalasin B were obtained from Sigma. Rhodamine-conjugated phalloidin was obtained from Molecular Probes and UO126 from Promega. BVT.12777 was prepared as a 100 mM stock solution in normal saline and stored at -70°C prior to use. Leptin was prepared as a 10 μM stock solution in normal saline containing 0.2 % bovine serum albumin as carrier. Rhodamine-conjugated phalloidin  receptor-b; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5 tris-phosphate; STAT3, signal transducer and activator of transcription-3; SUR, sulphonylurea receptorCRI-G1, Cambridge Rat Insulinoma-G1; GSK3, glycogen synthase kinase-3; HEK293, human embryonic kidney 293; JAK, janus kinase; KJK carried out the majority of the electrophysiology and cytoskeletal fluorescence studies. HL carried out the western blot experiments. TT and JH participated in the electrophysiological experiments. CS participated in the design and implementation of the western blot experiments. MA conceived of the study, participated in its design and co-ordination and drafted the manuscript. All authors read and approved the final manuscript.


The adaptive radiations of cichlid fishes in East Africa are well known for their spectacular diversity and their astonishingly fast rates of speciation. About 80% of all 2,500 cichlid species in East Africa, and virtually all cichlid species from Lakes Victoria (~500 species) and Malawi (~1,000 species) are haplochromines. Here, we present the most extensive phylogenetic and phylogeographic analysis so far that includes about 100 species and is based on about 2,000 bp of the mitochondrial DNA.sensu lato are relatively species poor, albeit widely distributed in Africa, whereas a fourth newly defined lineage – the 'modern haplochromines' – contains an unparalleled diversity that makes up more than 7% of the worlds' ~25,000 teleost species. The modern haplochromines' ancestor, most likely a riverine generalist, repeatedly gave rise to similar ecomorphs now found in several of the species flocks. Also, the Tanganyikan Tropheini are derived from that riverine ancestor suggesting that they successfully re-colonized Lake Tanganyika and speciated in parallel to an already established cichlid adaptive radiation. In contrast to most other known examples of adaptive radiations, these generalist ancestors were derived from highly diverse and specialized endemics from Lake Tanganyika. A reconstruction of life-history traits revealed that in an ancestral lineage leading to the modern haplochromines the characteristic egg-spots on anal fins of male individuals evolved.Our analyses revealed that all haplochromine lineages are ultimately derived from Lake Tanganyika endemics. We find that the three most ancestral lineages of the haplochromines We conclude that Lake Tanganyika is the geographic and genetic cradle of all haplochromine lineages. In the ancestors of the replicate adaptive radiations of the 'modern haplochromines', behavioral , morphological (egg-spots) and sexually selected (color polymorphism) key-innovations arose. These might be – together with the ecological opportunity that the habitat diversity of the large lakes provides – responsible for their evolutionary success and their propensity for explosive speciation. H. Greenwood (1983)With estimated numbers of about 1,000 and 500 species respectively the assemblages of cichlid fishes of East African lakes Malawi (LM) and Victoria (LV) are, by far, the most species-rich species flocks . In Lakee.g., [Not all haplochromines are lacustrine, however, and some 200 species inhabit rivers and occur in northern-, eastern-, southern- and central-Africa but are virtually absent from West Africa ,12 , discrete adaptive radiations of non-haplochromines are comprised by about a dozen species at most. Thus, which evolutionary novelties might be causally related to the explosive speciation that distinguish the haplochromines from all other cichlid lineages remains a crucial question for the understanding of the explosive patterns of cichlid evolution in East Africa. Haplochromines furthermore represent prime examples for parallel evolution, and it is particularly the lacustrine haplochromine species flocks that independently evolved morphologies and color-patterns that are convergent between species of different lakes ,18.In order to gain a deeper understanding of the adaptive radiations of cichlids in general and the formation of East African cichlid species flocks in particular, several crucial questions still remain to be answered: (i) What evolutionary lineages from which geographic ranges make up the diversity of haplochromines? (ii) Which were the founding lineages of the lacustrine adaptive radiations and were they riverine generalists? (iii) Can particular behavioral and/or morphological key-innovations be identified that might be causally related with the diversification of haplochromines? With the aim of addressing these questions, we conducted the most extensive phylogenetic and phylogeographic study of haplochromine cichlids so far, analyzing a portion of up to 2,000 bp of the mitochondrial genome of about 100 species. We included representatives of relevant cichlid tribes from LT ,9,15 as The neighbor-joining analysis based on 304 complete mitochondrial control region sequences Fig.  could noLimnochromis auritus, Triglachromis otostigma) plus Cyphotilapia frontosa, and all remaining taxa. Among these, a clade that includes four LT tribes (Cyprichromini, Ectodini, Limnochromini, Perissodini) plus the Orthochromis species from the Malagarasi drainage, which had so far been considered to belong to the Haplochromini, was recovered as sister group to the remaining haplochromine representatives. The latter, the haplochromines sensu lato, clustered into four distinct groups, with a Congolese/South-African lineage (CSA) sister to the Pseudocrenilabrus-, the Astatoreochromis-lineage, and the 'modern haplochromines' . Bayesian inference revealed the same branching order for the different lineages. In the strict consensus topology of the 78,617 most parsimonious trees , the Eretmodini were again resolved as sister group to the Lamprologini, the Limnochromini plus C. frontosa, four LT tribes plus the Malagarasi Orthochromis and the haplochromines sensu lato. Here, the Astatoreochromis-lineage was resolved as most ancestral lineage, and as sister group to a clade comprised by the Pseudocrenilabrus- and the CSA lineage, and the modern haplochromines. Also, the neighbor-joining analysis recovered a tree (not shown) with the Eretmodini as sister-group to the Lamprologini, the Limnochromini plus C. frontosa, the four LT tribes including the Malagarasi River Orthochromis species, and the haplochromines sensu lato. In the neighbor-joining tree, the Pseudocrenilabrus-lineage occupied the most ancestral branch in the haplochromines sensu lato, followed by the Congolese/South-African clade, the Astatoreochromis-lineage and the modern haplochromines. A Shimodaira-Hasegawa test [sensu lato received support greater than 50%.In the maximum likelihood tree of the dataset including 100 taxa Fig.  the LT Eawa test  revealedawa test  none of sensu lato. The most recent common ancestor in the CSA-clade was estimated to have lived about 2.0 MYA (1.15 – 3.89 MYA), the split of the CSA lineage from the common ancestor with the Pseudocrenilabrus-, Astatoreochromis-, and modern haplochromine lineage was dated to about 2.4 MYA (1.22 MYA – 4.02 MYA). The test for shifts in the probabilities of speciation in the haplochromines sensu lato according to [pc < 0.01) along three branches in the maximum likelihood, the Bayesian inference and the neighbor-joining trees: (i) the branch leading to Astatoreochromis-lineage plus the modern haplochromines; (ii) the branch leading to the modern haplochromines; and (iii) the branch leading to the LV region superflock  sequence data have a long and successful history in the study of East African cichlid evolution (see e.g., ,17,24,25, [e.g., ), or bec, [e.g., -29). Nev, [e.g., ; for phy, [e.g., ,31; and , [e.g., . Also, b, [e.g., ).The different phylogenetic algorithms, with which we analyzed our data, revealed largely congruent results. In all analyses, and in agreement with previous results , we founsensu lato. These are further divided into four distinct groups, a Congolese/Southern African lineage (CSA), the genera Pseudocrenilabrus (Ps.) and Astatoreochromis (As.), and the modern haplochromines (MH). While the respective monophyly of these four lineages was supported by high bootstrap values and Bayesian posterior probabilities, our analyses could not unambiguously resolve the exact relationships between these four lineages [maximum likelihood and Bayesian inference  ancestral to Southern African genera (Serranochromis, Sargochromis, Pharyngochromis) was resolved as sister group to a clade comprised by solely Congo drainage taxa (Orthochromis, Haplochromis sp. nov., Cyclopharynx and Thoracochromis). Within the Pseudocrenilabrus- and Astatoreochromis-clades, branch lengths were relatively short and a more detailed phylogeographic sampling would be necessary to resolve the relationships between the different geographic lineages.The CSA lineage is composed of several widespread and moderately species-rich groups of the Congo drainage and Southern Africa and consists of two main clades: A clade with a species from the Congo drainage  sister-group to a clade comprised by the entire species flock of LM (~1,000 species) and several East African riverine and lacustrine lineages (~200 species) plus the LV region superflock (~600 species). With approximately 1,800 species this – here phylogenetically defined – monophyletic lineage makes up about 7% of all known teleost fish.e.g., [Astatotilapia emerges as polyphyletic genus, with representatives assigned to both the East African riverine clade in the modern haplochromines and the LV region superflock.Our phylogenies show that several genera are in fact polyphyletic, and major taxonomic revisions will be required in the future to take our phylogenetic results into consideration. For example, and in agreement with previous studies (see e.g., ,14,33), Thoracochromis, represented in our analysis by T. brauschi, has also been shown to be polyphyletic before [T. brauschi from the Congo drainage as a more ancestral lineage, and T. petronius and T. pharyngealis from the Nile drainage with affinities to the LV region superflock . Further analyses including nuclear DNA sequence data and more taxa assigned to the genus will be necessary to address this problem.The genus c before , with T.romis of  are consromis in ). The plogeny of  seems topport in . Also, tuaudi in ) as singuaudi in  indicateOrthochromis lineage, which is confined to the Malagarasi River system and two isolated rivers, East of LT [Orthochromis are biparental caregivers [sensu lato are all maternal mouthbrooders. Thus, the Orthochromis-species from the Malagarasi area, a group of exclusively riverine fish, should be placed into its own tribe. The name Orthochromis is, however, also used for riverine species from the Congo drainage. In our analyses, Orthochromis polyacanthus and O. stormsi from the Congo River system fall – according to their distribution – into the CSA-clade leaving also the genus Orthochromis polyphyletic  for the Malagarasi area species, since this name was repeatedly used for some species of that complex . We sugescribed , and Schee e.g., ,34,35).e.g., [sensu lato, and about 2 MYA (1.15 – 3.89 MYA) for the first branching events within the CSA clade. The spread of Congo drainage taxa into southern river systems occurred at a later stage, most likely at the relatively shallow watershed between upper branches of the Congo River and the Zambezi River – a scenario that is also supported by the placement of Serranochromis sp. (from Lake Mweru in the upper Congo) as sister group to the Zambezi/Southern African genera Sargochromis and Pharyngochromis in our phylogenies. However, further sampling in that area would be necessary to reconstruct the southward spread of the CSA lineage.We note that the application of a molecular clock for estimating divergence times has the potential of not being without problems for several reasons (see e.g., ,38). Howe.g.,   and LM (Pseudocrenilabrus) region – the genera Astatoreochromis and Pseudocrenilabrus never underwent considerable speciation. The three described Astatoreochromis species occur in the LV region including Lakes Edward and George , in the rivers Rusizi and Lukuga (A. straeleni), and in the Malagarasi River (A. vanderhorsti). The three species of Pseudocrenilabrus occur from the Nile system to the LV region (P. multicolor), in Eastern- and Southern Africa including LM (P. philander), and in the central Congo basin (P. nicholsi). All analyzed representatives are relatively closely related suggesting a recent spread of these lineages in East Africa. However, we did not include P. nicholsi, which is morphologically different from P. multicolor and P. philander and would – if it really belonged to the Pseudocrenilabrus-lineage – represent the only haplochromine in the Congo drainage that is not a member of the CSA lineage.At essentially the same time as the CSA lineage, the ancestors of the The most recent common ancestor of the modern haplochromines was dated to have existed about 1.8 MYA 0.66 – 3.78 MYA) Fig. . This an.78 MYA FHaplochromis gracilior form Lake Kivu [H. paludinosus that occurs in the Malagarasi , as welested by ,42. Thissensu lato is the particular type of cranial apophysis for the upper pharyngeal bones [sensu lato are maternal mouthbrooders with the females alone incubating the eggs in their buccal cavities [maternal mouthbrooding behavior displayed by haplochromines is believed to being a derived character state [One of only few synapomorphies of the haplochromines al bones . The disal bones , is charal bones ,3,10,43.al bones  might fucavities . Mouthbrcavities -46, has cavities ,47,48. Ter state ,46,47. Me.g., some deep-water lineages of LM, have lost their egg-spots secondarily. In mimicking real eggs to attract females, these egg-spots function as natural releasers [ocelli) [Astatoreochromis-lineage and the modern haplochromines. There are other cichlid species in which males display yellow or red marks on their pelvic, dorsal or anal fins, but only in these lineages true egg-spots on the males' anal fins with a yellow, orange or red center and a colorless/transparent outer ring [Astatoreochromis-lineage and the modern haplochromines is the one with a pronounced potential for an increased rate of speciation  ,23 to thter ring ,22 are fsee Fig. . Based osee Fig.  it seemsAnother innovation that further distinguishes the exceptionally species-rich modern haplochromines from all other cichlids is the occurrence of numerous color morphs, often accompanied by sexual color dimorphism. Inter- and intra-specific polychromatism combined with maternal mouthbrooding involving egg-spots as releasers can be hypothesized to being permissive for sexual selection through female choice and, hence, the haplochromines' propensity for species formation, as sexual selection is probably a major causal factor in the origin of isolating mechanisms and the maintenance of reproductive isolation ,49-53. TAstatoreochromis, Astatotilapia, Pseudocrenilabrus, and Haplochromis (e.g., bloyeti), are ancestral to the adaptive radiations of the Tropheini of LT, and/or the radiations of LM and the LV region superflock. These genera are widely distributed and not confined to Eastern Africa, and they are the only ones that could inhabit the waterways that – over geological time spans – connected the lakes of Eastern Africa.A common feature of many adaptive radiations is that their founders are believed to have had a more generalist's lifestyle, while adaptive radiations themselves are defined by being composed of highly specialized species with narrower niche widths ,55. TheoThe phylogeny presented here Fig.  reveals i.e., generalists arose from highly specialized lineages, yet, apparently retained their high propensity for speciation and level of evolvability (see [In an apparent contrast to most other known examples of adaptive radiations  is the fity (see ). The faity see ,9,58), b,58, bi.eity , yetAntilles ,12,13,17It has been noted before that lineages of LT origin have left the lake secondarily  ,58. HereOrthochromis species of the Malagarasi area apparently do not belong to the radiation of the haplochromine cichlids and should be placed into a new tribe, we defined four new lineages within the haplochromines sensu lato: A clade combining Congolese and South-African genera (CSA-lineage), the Pseudocrenilabrus-, the Astatoreochromis-lineage, and the exceptionally species-rich modern haplochromines. The ca. 1,800 species of modern haplochromines are comprised of the entire haplochromine species flocks of Lake Malawi and the Lake Victoria region, some 200 riverine and lacustrine species, as well as the Tanganyikan Tropheini, which are likely to have evolved from a riverine ancestor and secondarily colonized Lake Tanganyika. This proposed "out of Tanganyika" scenario of haplochromine evolution is in agreement with the geological and palaeo-geological history of East Africa. Based on a character-state reconstruction from this new phylogeny, we were able to discover the evolution of several key-innovations that arose in the lineage leading to the modern haplochromines. These character reconstructions suggest that a combination of behavioral  and morphological characteristics  as well as ecological opportunities (after the colonization of large lakes) might have predestined this particular lineage to give rise to replicate adaptive radiations and, therefore, be causally related to the extraordinary success of these particular cichlid fish radiations.Our phylogenetic analyses that include representatives of all major haplochromine lineages show that all haplochromines are derived from Lake Tanganyika cichlids. While the For this study, a total of 304 specimens were analyzed. We combined all available GenBank entries from previous studies ,15,25,62Sample preparation, polymerase chain reaction (PCR) amplification and DNA sequencing have been performed as described elsewhere  for bothThe complete sequences of the mitochondrial control region (895 bp) and the ND2 gene (1,047 bp) were aligned using the computer program Clustal W ; alignmeTylochromis, which is ancestral to the Tilapiini). We did not include representatives of three LT tribes – the Bathybatini, Trematocarini and Tylochromini -, as these ancient lineages were shown to have existed before the primary radiation of mouthbrooders in LT [10 rearrangements. For neighbor joining, we used the HKY model and conducted a bootstrap analysis with 5,000 replicates. Bootstrap analyses for maximum likelihood were performed with 100 replicates and for maximum parsimony with 1,000 replicates. We also applied Bayesian inference of phylogeny with Mr. Bayes 3.0b4 [sensu lato into four groups according to the four lineages that were recovered from the phylogenetic analyses (CSA-, Pseudocrenilabrus-, Astatoreochromis-lineage, and modern haplochromines).For phylogenetic reconstruction we performed maximum likelihood, maximum parsimony and neighbor-joining methods in parallel using the computer program PAUP* 4.0b10 . Two taxrs in LT ,15,67. Ars in LT . For thees 3.0b4  running es 3.0b4  under a es 3.0b4 . To esties 3.0b4 ,70 in whpc [versus lacustrine), breeding behavior, and the occurrence of true egg-spots on the maximum likelihood tree using MacClade 4.0 (Sinauer, Sunderland, MA) for maximum parsimony character state reconstructions and Mesquite [To test whether or not shifts in the probabilities of speciation occurred on certain branches of the obtained phylogeny, we quantitatively tested the fit of this tree to a Markovian null model in which the probability of speciation is equally distributed along branches. Therefore, we calculated the cumulative probability pc  for releMesquite  for maxiMesquite  applyingMesquite . ConfideMesquite ,75, the Mesquite ,4,25 as Mesquite ,77. An eMesquite ,76-79. TAstatoreochromis; bp, base pairs; CSA, Congo/South Africa; LM, Lake Malawi; LT, Lake Tanganyika; LV, Lake Victoria; MH, modern haplochromines; mtDNA, mitochondrial DNA; MYA, million years ago; ND2, NADH Dehydrogenase Subunit II; Ps., Pseudocrenilabrus.As., WS, EV, and AM designed the study and were involved in sampling. WS and TM carried out the molecular work and the analyses. All authors contributed to the preparation of the manuscript. They read and approved the final version.Table 1 – Specimen information, geographic origin and GenBank accession numbers of all taxa included in this study. This table lists the species and tribe names, geographic origin, source of specimens, names of collectors and collection numbers (if available), and GenBank accession numbers for both mitochondrial gene segments. The taxa that are included in Fig. Click here for file


The association between infertility and sperm disomy is well documented. Results vary but most report that men with severely compromised semen parameters have a significantly elevated proportion of disomic sperm. The relationship between individual semen parameters and segregation of specific chromosome pairs is however less well reported as is the variation of disomy levels in individual men.In order to address these questions the technique of fluorescent in-situ hybridisation (FISH) was utilised to determine the disomy levels of chromosomes X, Y and 21 in 43 sperm samples from 19 infertile males. The results generated from this study were analysed using logistic regression.In this study we compared levels of sperm concentration, motility and morphology with levels of sperm disomy for chromosome 21 and the sex chromosomes. Our results suggest that there is considerable variation in disomy levels for certain men. They also suggest that oligozoospermic males have significantly elevated levels of sex chromosome disomy but not disomy 21; they suggest that severe asthenozoospermic males have significantly elevated levels of disomy 21 but not sex chromosome disomy. Surprisingly, severe teratozoopsermic males appeared to have significantly lower levels of sperm disomy for both the sex chromosomes and chromosome 21.We suggest that the association between sex chromosome disomy and oligozoospermia may be due to reduced recombination in the XY pairing region and discuss the relevance of our findings for the correlations between sperm disomy and sperm motility and morphology. The relationship between male infertility and elevated proportions of sperm with extra or missing chromosomes in any given ejaculate is now extensively documented. There have been over 30 studies that have investigated this effect e.g. -7], and , and 7]]A number of authors ,11,19-21In most of the above studies either semen parameters and or aneuploidies for individual chromosome pairs were grouped together and thus not considered individually. Moreover, cases where males have given multiple samples are rare and thus there are few occasions where the individual specific parameters have been compared on a sample-by-sample basis. Establishing chromosome-specific and parameter-specific correlations between male infertility and percentage of aneuploid sperm in an ejaculate is a preliminary step towards understanding the mechanisms of the association between male infertility and chromosome segregation. In this study, our results provide evidence for a variation in rates of disomy for individual men and a correlation between specific semen parameters and individual chromosome disomies.A series of males undergoing infertility treatment with a range of andrological phenotypes were assessed for conventional semen parameters and for sperm disomy. All patients were attending IVF clinics in the central London area. Semen samples were taken, with patients' informed consent, from 19 different men on 43 occasions from infertility clinics in central London. None had known constitutional karyotypic abnormalities or Y chromosome deletions. We received 1 sample each from 12 men, 2 samples from one man, 3 samples from 2 men, 4 samples from one man, 6 samples from 2 men and 7 samples from one man. In some men one, two or three of the semen parameters measured (concentration, motility and morphology) were within the normal range; these were hence placed in a control group. In other cases (test group) individual parameters were in the abnormal range . Given that some samples were taken from individual patients on several occasions, sometimes males appeared in the control group for some samples and in the test group for others. We restricted our molecular cytogenetic studies to chromosome 21 and the sex chromosomes for three reasons. First, according to previous studies -28 theseMen were required to abstain from ejaculating for between 2 and 5 days prior to providing a sample for the study. Samples were produced on site into sterile 60 ml containers and kept at room temperature for up to 60 minutes to allow for liquefaction. Semen parameters were then analysed according to guidelines defined by WHO . PatientIndividual samples were then placed in three occasions into a "test" or "control" group. On the first occasion, the control group had a sperm concentration of ≥ 20 million/ml and the test group <20 million/ml. On the second occasion, the control group had forward motility of ≥ 20% and the test group <20%. On the third occasion the control group had a normal morphology of ≥ 4% and the test group <4%. The cut-off points for considering individual samples as being in the test or control groups were based on WHO guidelines for oligozoospermia, severe asthenozoospermia, and severe teratozoospermia and were comparable to those used in other studies for sperm disomy [e.g. [FISH analysis was performed according to Griffin et al . BrieflyApproximately 5,000 sperm were scored per patient by two or more independent observers. The proportion of aneuploid sperm per sample for each chromosome was noted.The hypotheses of interest were whether the rate of disomy was significantly different for the test and control groups in terms of sperm concentration, morphology or motility. To this end, in the first case, all oligospermic patients designated "O" . The total rate of disomy for the sex chromosomes and chromosome 21 was found to be 0.3% (633/209,188) and 0.19% (398/209,188) respectively. Disomy levels ranged from 0.02% – 2.00% for the sex chromosomes and 0.00% – 1.46% for chromosome 21. For the patients who gave multiple samples, individual disomy rates were surprisingly varied: For instance patient 9 (who gave 6 samples) had sex chromosome disomy frequencies ranging from 0.1% and 0.73%. The results of each individual sample are presented in Table To the best of our knowledge, this is the first report that demonstrates a relationship between individual clinically defined semen parameters and segregation of specific chromosome pairs. The relationship between sex chromosome disomy and the failure of spermatocytes to complete spermatogenesis is manifested by significantly higher sex chromosome disomy levels in oligozoospermic men. These results are similar to those of Rives et al. , VegettiOur results also suggest a significant association between asthenozoospermia (poor motility) and non-disjunction of chromosome 21. This is similar to reports by Vegetti et al.  but in tThe apparent inverse association between sperm morphology and chromosome segregation was surprising and it is, again, possible that this is a statistical anomaly. Indeed although a number of studies have found no significant correlation between morphology and disomy ,34-36, oIn conclusion we provide evidence that sperm disomy levels can vary considerably between samples from the same man, the reasons for this are unclear but one possible explanation is the involvement of extrinsic factors or lifestyle changes. Such differences provide hope for possible treatment regimes to improve disomy rates. The evidence of correlations between individual semen parameters and increased disomy of individual chromosome pairs, while statistically significant, warrants further investigation. Closer correlations of disomy rates in men with defects in only one of the three criteria used to measure semen quality will form the basis of our future investigations. In future studies it is also likely that we would include a second control group of normal, fertile donors not attending fertility clinics. Ethical considerations precluded this in this case. Through these studies, a closer understanding of the mechanistic basis of the relationship between chromosome segregation and infertility will be achieved.HGT- performed the majority of FISH experiments, scoring of semen samples, collected data generated within this study and assisted in drafting the manuscript. SH- performed all semen assessments. MD and DC- performed FISH experiments and acted as independent scorers of semen samples. DW- performed statistical analysis. XPZ- provided patient samples with signed informed consent. DKG- conceived the study and participated in its design and drafted the manuscript. All authors have read and approved the final manuscript.