Progestins provide safe, effective and cheap options for contraception as well as the treatment of a variety of gynaecological disorders. Episodes of irregular endometrial bleeding or breakthrough bleeding (BTB) are a major unwanted side effect of progestin treatment, such that BTB is the leading cause for discontinued use of an otherwise effective and popular medication. The cellular mechanisms leading to BTB are poorly understood. In this study, we make the novel finding that the large, dilated, thin walled vessels characteristic of human progestin-treated endometrium include both blood and lymphatic vessels. Increased blood and lymphatic vessel diameter are features of VEGF-D action in other tissues and we show by immunolocalisation and Western blotting that stromal cell decidualisation results in a significant increase in VEGF-D protein production, particularly of the proteolytically processed 21 kD form. Using a NOD/scid mouse model with xenografted human endometrium we were able to show that progestin treatment causes decidualisation, VEGF-D production and endometrial vessel dilation. Our results lead to a novel hypothesis to explain BTB, with stromal cell decidualisation rather than progestin treatment per se being the proposed causative event, and VEGF-D being the proposed effector agent. Progestins provide safe, effective and cheap options for contraception as well as the treatment of a variety of gynaecological disorders Understanding of the cellular mechanisms underlying BTB remains elusive because of both the range of hormonal conditions under which it can occur and the high degree of variability between women in terms of their endometrial response to exogenous hormones and their susceptibility to BTB. Thus, while administration of exogenous progestins will increase the incidence of BTB, the link between hormones and BTB is not direct. Numerous studies over the past 20 years have been undertaken with the aim of developing a better understanding of the local endometrial mechanisms responsible for BTB. Some of factors that have been proposed as playing a role in BTB include endometrial epithelial integrity Decidualisation is a hormonally regulated cellular differentiation process that occurs in the endometrial stroma of most mammals prior to and during placentation. It is accompanied by significant vascular remodelling of both blood and lymphatic vessels Endometrium exposed to progestins undergoes a well characterised series of morphological changes that includes stromal cell pseudo-decidualisation, epithelial cell regression The overall aim of this study was to investigate potential mechanisms by which progestin administration results in the formation of abnormally dilated and thin-walled endometrial vessels.We were able to demonstrate that a significant proportion of the dilated, thin walled vessels in progestin treated human endometrium are lymphatics and not blood vessels. Both lymphatic and blood vessels show marked increases in diameter following progestin treatment, while lymphatics in the functional layer are the only vessels to show a moderate increase in number. Increased blood and lymphatic vessel diameter, but not number, are features of VEGF-D action in other tissues Full-thickness endometrial sections from women with heavy menstrual bleeding who had either been treated with a levonorgestrel intrauterine system (LNG-IUS) (N = 16) or who were untreated controls (N = 16)  = 12.693, P = 0.001)  but not the basal layer . A similar analysis of the five largest lymphatic vessels showed a significant increase in area for LNG-IUS versus control treated tissues  = 19.569, P = 0.001)  and also the basal layer .Using 2-way ANOVA, the vessel cross sectional area of the 5 largest blood vessels was significantly greater in endometrium from women treated with LNG-IUS compared to controls (F= 0.001) A. Tukey= 0.001) B. This  = 1.08, P = 0.304 or lymph vessel density  = 2.384, P = 0.128) between control or LNG-IUS treated tissues  = 0.342, P = 0.562) or lymphatics  = 2.779, P = 0.101).In contrast to vessel cross sectional areas, there was no difference in endometrial blood vessel density  = 22.2, P = 0.001) with a significant variation among different isoforms  = 4.38, P = 0.02). Paired T-tests were significant after Bonferoni correction for the 53 kD  and 21 kD  isoforms, but not for the 41  and 29/31 kD (P = 0.26) isoforms. The increase in the 21 kD isoform is of particular relevance as following proteolytic processing this is the most biologically active isoform with the highest affinity for VEGF receptor-3 (VEGFR-3), which in the adult is found on lymphatic endothelial cells  = 25.7, P = 0.001), as well as a significant variation among different isoforms  = 4.93, P = 0.013). Paired T-tests for individual isoforms failed to reach significance after Bonferoni correction . Human endometrial stromal cells showed evidence by Western analysis for increased expression of both VEGF-C and VEGF-D after decidualisation in vitro . Up to sTo further explore the role of exogenous progestin in causing lymphatic vessel dilatation through decidualisation-dependent expression of VEGF-D, a human endometrial xenograft model in NOD/scid mice was utilised. A total of n = 4 human samples were subdivided and grafted into NOD/scid mice that were subsequently treated with estradiol valerate only, or estradiol valerate and medroxyprogesterone acetate (MPA). The estrogen component of this treatment maximizes xenograft establishment, vascularisation and survival, and the progestin recreates the hormonal status that exists in women using progestin contraceptives.There was a 100% recovery of graft tissue at the end of the experimental period. There were no obvious macroscopic differences between the treatment groups with all grafts becoming encapsulated by extracellular matrix. The endometrial vasculature was composed of both blood and lymphatic vessels which were predominantly of human origin (92±3%) with a small number of murine vessels also infiltrating the endometrial tissue (8±3%) A and 6B−4±5.0×10−5 vessels/mm2) when compared to the estrogen-only treated xenografts (1.1×10−3±1.0×10−4 vessels/mm2) t(6) = −5.873, P = 0.001) (2) had significantly reduced blood vessel density when compared to xenografts that received estrogen-only (406±44.3 vessels/mm2) for the same length of time (t(6) = 2.848, P = 0.029)  = 0.619, P = 0.559)  = −1.681, P = 0.179)  and control xenografts (n = 4) following immunostaining with antibodies to CD31 and D2-40. There was a significant increase in cross-sectional area of the 5 largest lymph vessels in the progestin treated xenografts (4.6×10= 0.001) D. Xenog= 0.029) A. There= 0.559) B. There= 0.179) C.In this study, we make the novel finding that the large, dilated vessels characteristic of progestin-treated endometrium include both blood and lymphatic vessels . That boProgestin-treated endometrium usually exhibits areas of decidualisation, especially in the first few months of exposure before endometrial regression has occurred While VEGF-D is a highly plausible candidate for causing the microvascular changes seen in progestin-exposed endometrium, decidual cells have upregulated expression of many other proteins, including connexin 43, prolactin, IL-6 and VEGF-A While the current study establishes a potential mechanistic link between progestins, decidualisation and endometrial microvessel dilatation, the link between dilated endometrial vessels and BTB remains more speculative. Further, if a relationship between dilated endometrial vessels and BTB does exist, we would assume that it is the blood vessels and not the lymphatic vessels that are the primary cause. There are several published studies that do support a link between vessel dilatation and BTB. Most significantly, dilated endometrial vessels, which are a common occurrence with systemic or intrauterine progestin exposure, have been shown to demonstrate increased fragility in hysteroscopic studies in vivo. Our results parallel a recent study that used LNG to treat human endometrial xenografts for 4 weeks in SCID mice In the current study we used a xenograft model to investigate the progestin-related changes to human endometrium caused by medroxyprogesterone acetate treatment The current study identifies both decidual cells and VEGF-D as potential targets for development of therapies for BTB. If decidual derived VEGF-D is the agent responsible for BTB, then a number of interventions might be possible. These include developing progestins that minimise or avoid decidualisation, developing agents that block progestin-induced decidualisation, and using inhibitors of VEGF-D to prevent endometrial microvascular dilatation.Despite BTB having been a significant clinical problem for many years, advances in understanding the mechanisms that cause BTB have been limited. The lack of a readily available animal model that reliably replicates progestin-induced BTB is probably the single most critical issue responsible for this lack of progress. The absence of an accepted model means that mechanistic studies can only be performed in humans, which limits significantly the research that can be undertaken. The xenograft model used the current study does not exhibit BTB, despite replicating many of the changes seen in human endometrium exposed to progestins. Future progress in understanding BTB is most likely to come from a combination of indirect mechanistic studies supported by observational and correlational data from human subjects with BTB.In conclusion, we have identified dilated lymphatics in progestin exposed human endometrium from women with BTB, which in conjunction with dilated blood vessels identifies VEGF-D as a potential causative agent. Further studies show that VEGF-D expression increases as endometrial stromal cells undergo decidualisation, and that this chain of events can be replicated in human endometrial xenografts in NOD/scid mice treated with progestin. Taken together these data implicate decidual cell-derived VEGF-D as a causative factor in progestin-induced BTB.n = 16, mean age 38.6 years) or control groups . LNG-IUS were in place for up to 156 days prior to surgery. For the xenograft study, uterine biopsies were obtained following written informed consent  from 4 patients aged 48.7±1 year undergoing hysterectomy for prolaspe or fibroids. Each sample was cut down to the endometrial myometrial boarder and divided into 8 grafts approximately 1×1×0.5 cm in size.Fixed and wax embedded human uterine tissue blocks from a previously published study n = 32; 6–8 weeks) received a single graft in a pocket created on the right side of the back flank  and approved by Monash Medical Centre Animal Ethics Committee A, approval No. 2008/14). Following closure of the wound site, mice received 1 µg 17β-estradiol valerate in oil (subcutaneous) every fourth day until experiment completion. After two weeks of graft establishment, mice were divided into two groups and received a subcutaneous silastic tube implant For xenograft studies, NOD/SCID female mice  . We found this approach to be a simple and statistically robust morphometric method for objectively demonstrating the existence of the abnormally dilated and thin-walled vessels found in progestin-exposed endometrium.In xenograft samples, vessel density was determined by calculating the average number of vessels from 4 separate endometrial areas. Statistical analysis was performed using SPSS for Windows, Version 11.0.0 . Vessel cross sectional area and vessel density of the functional layer and basal layer vessels in the endometrium of women +/− LNG-IUS was determined by 2-way ANOVA on logged data followed by Tukeys HSD post hoc analysis. Statistical analysis of xenograft +/− MPA vessel density and diameter was determined by Student t test. Values are presented as mean ± SE unless otherwise stated. A p value <0.05 was considered significant.Patient samples and xenografts were formalin fixed and paraffin embedded for immunohistological analysis. Tissue sections (5 µm) mounted on silane coated slides were dewaxed and rehydrated. Following antigen retrieval, endogenous peroxide block and protein block, immunolocalisation of human vascular endothelial cells , human lymphatic endothelial cells  −8 mol/l) and progesterone (10−7 mol/l) as previously described Primary isolated human endometrial stromal cells  were decidualised following incubation with estradiol 17β  VEGF-C and (B) VEGF-D protein expression in primary cultures of decidualised (Dec) and non-decidualised (ND) human endometrial stromal cells. White arrows indicate the reported isoforms including the C-VHD-N full length receptor , the VHD-N terminal , and the VHD (21 kD). Other bands may be alternatively processed forms including heterodimers, trimers and dimers, some of which also exhibit changes associated with decidualisation (black arrows).(TIF)Click here for additional data file.


Clinical diagnostics of chronic polymicrobial infections, such as those found in chronic wounds, represent a diagnostic challenge for both culture and molecular methods. In the current retrospective study, the results of aerobic bacterial cultures and culture-free bacterial identification using DNA analyses were compared. A total of 168 chronic wounds were studied. The majority of bacteria identified with culture testing were also identified with molecular testing, but the majority of bacteria identified with the molecular testing were not identified with culture testing. Seventeen (17) different bacterial taxa were identified with culture, and 338 different bacterial taxa were identified with molecular testing. This study demonstrates the increased sensitivity that molecular microbial identification can have over culture methodologies, and previous studies suggest that molecular bacterial identification can improve the clinical outcomes of patients with chronic wounds. Chronic wounds, wounds that fail to progress through normal wound healing trajectory, are a large and growing problem for an already burdened healthcare system. The major chronic wounds  are all increasing at alarming rates due to sedentary lifestyles, obesity and, most importantly, double digit increases in diabetes each year. The total cost of chronic wounds is difficult to measure but has been estimated in over 25 billion dollars a year, but even more importantly is the patient impact resulting in loss of limb and eventually loss of life for far too many patients ,2.Wound care providers treat wounds as if the bioburden is one of the major barriers to wound healing. Debridement, antibiotics, and topical antiseptics are routinely used in managing the microbes that are universally found on chronic wounds. Yet, the only microbial diagnostic tool available to wound care providers until recently has been the routine clinical culture. The role of bacteria in chronic wounds and the appropriateness of using antimicrobial agents in the routine management of chronic wounds is a source of continual debate in the wound care community –5. It isIt is now recognized that the overwhelming majority of bacteria in their natural habit exist in a biofilm phenotype . ChronicBiofilms are inherently different from the bacterial clones that are cultivated in laboratories. In order to standardize testing of bacteria, the convention has been to examine free-floating clonal populations of a single species during its logarithmic growth phase. Unfortunately, biofilms are archetypically surface-associated groups, often comprised of multiple genera or kingdoms of organisms, held together by an extracellular matrix; and the growth conditions and population within the biofilm can be spatially and temporally variable . By defiMultiple bacteria genera comprise the biofilm in chronic wounds ,12,13, aThe debate over which organisms are harmless, helpful, or hateful to humans is continually being investigated and debated. A prerequisite assumption to this debate is that the presence of bacteria in or on humans is accurately being detected and identified. Traditionally, the presence of bacteria in or on humans during health or disease has been determined by growing the bacteria in culture. Indeed, Koch’s postulates and infectious disease dogma are anchored in culture technology. However, molecular biology has proven that culture methodologies can be markedly less sensitive to bacterial detection than DNA detection methodologies . Also, tMolecular technologies have enabled investigators to examine human microbiota more deeply and sensitively than culture testing has allowed ,17. In oIt is encouraging that these new molecular microbial diagnostic tests are enabling improvement in patient care, but it is also necessary to continue to formally compare these newer methods with traditional culture methods. Examining culture and molecular results in parallel is necessary in order to compare and contrast the methods’ abilities to detect bacteria in humans and to continue to evaluate the clinical relevance of molecular testing’s increased sensitivity. This study is an early attempt to compare clinical laboratory results of parallel testing of aerobic cultures and culture-free bacterial identification using 16S rDNA sequencing.This study was performed retrospectively on subjects who had undergone parallel bacterial analysis of wound samples. Initially, the results obtained by the Bacterial Identification Methods (2.3) were used clinically to guide patient care.Subjects were included in the study if they met the following criteria:The presence of a chronic wound ;Debridement required as part of standard of care wound management;The medical record revealed that parallel samples from the wound were sent for both, culture and molecular testing.Samples were collected from 168 subjects’ chronic wounds at the Southwest Regional Wound Care Center  in accordance with Western Institutional Review Board protocol number 20062347.Samples were tested using routine aerobic culture techniques in a non-hospital CLIA certified laboratory in Lubbock, Texas, USA.Samples were prepared and analyzed by PathoGenius Laboratory , a College of American Pathologists accredited laboratory.The debridement samples were centrifuged and then suspended in 500 μL RLT buffer  (with b-mercaptoethanol). A sterile 5 mm steel bead  and 500 μL sterile 0.1 mm glass beads  were added for complete bacterial lysis in a Qiagen TissueLyser , run at 30 Hz for 5 min. Samples were centrifuged briefly and 100 μL of 100% ethanol added to a 100 μL aliquot of the sample supernatant. This mixture was added to a DNA spin column, and DNA recovery protocols were followed as instructed in the QIAamp DNA Mini Kit  starting at step 5 of the Tissue Protocol. DNA was eluted from the column with 30 μL water and samples were diluted accordingly to a final concentration of 20 ng/μL.The modified 16S Eubacterial primers 28F, 5′-GAG TTT GAT CNT GGC TCA G-3′ and 519R, 5′-GTN TTA CNG CGG CKG CTG-3′ were used for amplifying the 500 bp region of 16S rRNA genes. The primer sets used for FLX-Titanium amplicon pyrosequencing were designed with adding linker A and 8 base pair subject-specific barcode sequence at the 5′ end of forward primers as follow: 28F-A, 5′-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-barcode-GAG TTT GAT CNT GGC TCA G-3′. The biotin and linker B sequence at the 5′ end of reverse primer 519R-B: 5′-Biotin-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG GTN TTA CNG CGG CKG CTG-3′. HotStarTaq Plus Master Mix Kit  was used for PCR under the following conditions: 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s; 54 °C for 40 s and 72 °C for 1 min, a final elongation step at 72 °C for 10 min was also included. The PCR products were cleaned by using Diffinity Rapid Tip  and pooled. The small fragments were removed by Agencourt Ampure Beads .Bacterial tag-encoded FLX-Titanium amplicon pyrosequencing (bTEFAP) was performed as described previously . In prepThe diagnostic panel for quantitative real-time PCR consists of Pseudomonas aeruginosa, Group B Streptococcus agalactiae (Group B Streptococcus), Streptococcus pyogenes (Group A Streptococcus), Staphylococcus aureus, Klebsiella pneumonia, Serratia marcescens, Candida albicans, Enterococcus faecalis, Enterococcus faecium. The enterococci were reported collectively in this paper as “Group D Enterococcus”. The quantitative real-time PCR was performed by a Roche Light Cycler 480 as described previously . The PCRThe concordance or discordance of molecular results with culture results were identified as “concordant”, “discordant”, or “partially concordant”. “Concordance” was defined as detecting the same bacterium/bacteria using molecular testing as were detected using culture testing. “Discordance” was defined as not detecting the bacterium/bacteria using molecular testing when the bacterium/bacteria was identified using culture testing. “Partial concordance” was possible when multiple bacteria were identified by culture. In partially concordant subjects’ samples, one or more of the bacteria identified by culture were also identified by molecular testing and one or more of the bacteria identified by culture were not identified by molecular testing.Staphylococcus aureus. In total, 17 different bacterial taxa were identified by culture. These organisms, the frequency of culture of each organism, and the corresponding frequency of detection by molecular testing are reported in Staphylococcus, Enterococcus, Serratia, and Pseudomonas. Molecular testing commonly identified Staphylococcus, Finegoldia, Corynebacterium, Anaerococcus, Bacteroides, and Serratia.One hundred sixty-eight (168) wounds from 168 subjects were analyzed by both culture and molecular testing including 24 decubitus ulcers, 40 diabetic extremity wounds, 23 non-healing surgical sites, 49 venous leg ulcers, and 32 wounds from trauma or an abscess. The most common results using both culture and molecular testing are reported by wound type in A subset of results are reported more exhaustively in From the culture-based diagnostics, a total of 11 (7%) chronic wounds were found to have 3 different bacterial taxa, 40 of the samples (24%) had 2 bacterial taxa, and 95 of the wounds (57%) had a single bacterium cultured. 22 of the wounds (13%) were found to have no culturable bacteria. Seventeen (17) different bacterial taxa were identified using culture based methods .Molecular testing identified 338 different bacterial taxa  in the 168 wounds. None of the chronic wounds were reported as negative based upon molecular diagnostics. It is of note that out of the top 20 most ubiquitous bacteria identified with molecular methods, 9 of these taxa are anaerobes . AnaerobBacteroides, Finegoldia, Peptoniphilus, Anaerococcus, Propionibacterium, Ruminococcus, Prevotella, Eubacterium, Streptococcus, Corynebacterium, Arthrobacter, Acinetobacter, Paracoccus, and Micrococcus. Many of these genera are obligate anaerobes.Of the 168 paired samples, 131 of the samples (78%) demonstrated some level of concordance between culture and molecular results. In 105 of the samples (63%), the molecular results were concordant with the culture results. Twenty-six (26) samples (15%) had partial concordance. Fifteen (15) samples (9%) were discordant. Twenty-two (22) samples (13%) were negative by culture testing but positive with molecular testing. In these 22 samples, molecular testing revealed these bacteria: Bacteroidetes (unknown genus), Of the 105 samples with complete concordance, 55 samples were in agreement between culture and molecular testing as to which bacteria were most prevalent in the sample. In 15 of the 105 samples, culture and molecular testing were not in agreement as to which bacteria were most prevalent in the sample, but the cultured organism(s) was determined to be one of the 3 most prevalent organisms by using molecular testing. In the other 35 samples, the cultured bacteria were identified as representing a minor population  when using molecular methods.Enterococcus spp. (19 times), Staphylococcus spp. (6 times), Pseudomonas spp. (4 times), Serratia marcescens (3 times), Enterobacter (3 times), Citrobacter (3 times), Proteus (1 time), Xanthamonas (1 time), and Klebsiella (1 time). Thirty-two (32) of these 41 samples were determined by molecular methods to have strong anaerobic contribution to the microbiota .Of the 41 samples with partial concordance or complete discordance, the bacteria that were cultured but not detected by molecular testing are were as follows: P. aeruginosa, but another pseudomonad species was detected by both culture and 16S sequencing. The only discrepancy between 16S sequencing and PCR is in Sample 1, in which S. marcescens and Group D Enterococcus were detected by 16S sequencing and not detected by PCR. This contradiction in results is likely due to the high sensitivity of deep 16S sequencing and not due to poor specificity of 16S sequencing. Congruent with that explanation, 16S sequencing detected that S. marcescens and Group D Enterococcus comprised a very small portion of the total microbiota in Sample 1. Overall, the table demonstrates that deep 16S/18S sequencing detects organisms that are detected using other molecular methods (PCR) and culturing methods.More exhaustive results of a subset of 10 samples are reported in Culture results and 16S DNA sequencing results were compared more exhaustively in a subset of samples . QuantitIn the present study, the microbes that were both commonly predominant in wounds according to molecular testing and also not identified using culture methods were often bacteria that were obligate anaerobes. The results support the interpretation that aerobic cultures alone (as in this study) fail to resolve the complexity of the polymicrobial nature of chronic wounds. Although anaerobes were not attempted to be cultured, it is reasonable to estimate that the frequency with which anaerobes would have been detected would have been similar to the frequency with which aerobes were detected. As reported previously, although anaerobes are difficult to culture, they often comprise a large portion of the microbiota of chronic wounds ,16. The Bowler and Davies conducted one of the most comprehensive culture-based surveys of chronic wounds with methods specifically designed to enrich for and detect anaerobes, which revealed that anaerobes were highly prevalent ,31. In tEnterococcus and Escherichia coli  or molecular testing produces false positive results (poor specificity). Or, some combination of the two proposed explanations is possible. The verification of 16S sequencing results by PCR testing  helps toA potential limitation of molecular diagnostics is the inability to test for phenotypic antibiotic sensitivity. It is important to consider the method in which phenotypic antibiotic testing is performed. Culture sensitivities are performed using planktonic phenotype bacteria and not biofilm phenotype bacteria. Obtaining the phenotypic sensitivity of biofilms is much more cumbersome than traditional susceptibility testing –37. BiofMolecular diagnostics have proven useful at the Southwest Regional Wound Care Center in the past ,23. PublHere we evaluated 168 wounds with cultures and molecular diagnostics. Out of the 168 wounds a total of 17 different bacteria were cultured. Molecular methods identified 338 different bacterial taxa. The majority of these were detected in relatively low abundance (~1% of the wound microbiota). Culture identified up to 3 different bacteria in some wounds, and molecular testing identified individual wounds with up to 33 different bacteria. The majority of bacteria identified with culture were also identified with molecular testing, but the vast majority of bacteria identified with molecular methods were not identified with culture methods. Although not tested for by culture, anaerobes were commonly found to be a large component of the microbial population in these chronic wounds, which is consistent with previous studies. In several instances, the culture methods identified the predominant populations as determined by molecular testing, but in the majority of the samples, culture underreported the diversity of the wound microbiota and failed to detect the most abundant bacteria in the wound. Previous studies have demonstrated that using the molecular identification of bacteria to guide the management of chronic wounds can improve patient outcomes.


Ethiopia has the largest cattle population in Africa. The vast majority of the national herd is of indigenous zebu cattle maintained in rural areas under extensive husbandry systems. However, in response to the increasing demand for milk products and the Ethiopian government's efforts to improve productivity in the livestock sector, recent years have seen increased intensive husbandry settings holding exotic and cross breeds. This drive for increased productivity is however threatened by animal diseases that thrive under intensive settings, such as bovine tuberculosis (BTB), a disease that is already endemic in Ethiopia.Mycobacterium bovis. The comparative intradermal tuberculin test (CIDT), questionnaire survey, post-mortem examination, bacteriology, and molecular typing were used to get a better understanding of the BTB prevalence among dairy farms in the study area. Based on the CIDT, our findings showed that around 30% of 2956 tested dairy cattle from 88 herds were positive for BTB while the herd prevalence was over 50%. Post-mortem examination revealed gross tuberculous lesions in 34/36 CIDT positive cattle and acid-fast bacilli were recovered from 31 animals. Molecular typing identified all isolates as M. bovis and further characterization by spoligotyping and MIRU-VNTR typing indicated low strain diversity within the study area.An extensive study was conducted to: estimate the prevalence of BTB in intensive dairy farms in central Ethiopia; identify associated risk factors; and characterize circulating strains of the causative agent, M. bovis to human populations consuming dairy products coming from these farms. It is suggested that government and policy makers should work together with stakeholders to design methods for the control of BTB in intensive farms in Ethiopia.This study showed an overall BTB herd prevalence of 50% in intensive dairy farms in Addis Ababa and surroundings, signalling an urgent need for intervention to control the disease and prevent zoonotic transmission of  Bos indicus) managed under traditional husbandry systems (grazing in the field) in rural areas. However, in recent years the number of dairy cattle of highly productive exotic  and cross breeds has been on the rise, particularly in urban and peri-urban areas in response to the increasing demand for milk products and the Ethiopian government's effort to improve productivity in the livestock sector. The population of dairy cows accounts for 6.3 million animals  and the estimated total national milk production per year is 2.6 billion litres The population of Ethiopia has increased dramatically in the last two decades, growing from approximately 55 million people in 1992 to a current estimate of around 85 million Mycobacterium bovis, is a chronic and contagious disease of cattle and other domestic and wild animals th highest in the world In a country such as Ethiopia, where livestock are extremely important for people's livelihood, animal diseases can be a real threat to animal productivity and thus negatively impact on the agricultural sector and economic development. Bovine tuberculosis (BTB), caused by M. bovis strains circulating in this region of Ethiopia and discuss the importance of our findings for informing BTB control policies in Ethiopia.This study was designed to get a better understanding of the prevalence of BTB among intensive dairy farms in the urban and peri-urban belt of Addis Ababa , areas from where the vast majority of commercial dairy products in the country are produced. Furthermore, we show molecular typing results of Statistics from the Ethiopian Ministry of Agriculture and Rural Development (MoARD) as well as data from regional agricultural offices recognised a high density of intensive dairy farms in a 50 km radius region from Addis Ababa city centre. Most dairy farms in this region had access to one of the five main roads that lead into the capital. Therefore, the region was divided into six geographically defined study areas, including Addis Ababa city, Debre Zeit, Sebeta, Holeta, Sululta and Sendafa . The recM. avium positivity was defined as previously described All cattle older than six months within a herd, except clinically sick animals and cows one month pre-and post partum, were tested by CIDT: Two sites, 12 cm apart, horizontally of the mid-neck of the animal were shaved and the skin thickness was measured with a calliper. In calves with short neck, the sites were shaven vertically. Aliquots of 0.1 ml of 2500 IU/ml bovine purified protein derivative (PPD) and 0.1 ml of 2500 IU/ml avian PPD  were injected separately into the respective shaved site. The thickness of the skin at each injection site was measured again after 72 hours. The test results were interpreted in line with OIE recommendations (2004): if the difference between skin thickness at the bovine site of injection and the avian site of injection was <2 mm, between 2 mm and ≤4 mm, or >4 mm, the animal was classified as negative, doubtful (inconclusive), or positive for BTB, respectively. A herd with at least one positive reactor was considered as “PPD positive”. All cattle tested by CIDT were recorded for age, sex, breed, and given a body condition score . Selected CIDT positive cattle from farms of all six study areas were purchased and subjected to post mortem examination performed by an experienced veterinarian. The selection criteria for these purchased animals were based on strong PPD response, unproductive animal, and willingness of farmer to sell the animal. The pathology scoring system was based on the semi-quantitative procedure developed by Vordermeier et al. M. bovisDuring post-mortem, up to seven suspected visible TB-like lesions were collected per animal in individual 50 ml sterile universal tubes (containing sterile PBS buffer) and transported at 4°C to the AHRI for further processing. At AHRI, samples were stored at 4°C when processed within 48 hours or at −20°C if processed later than two days after sample collection. All samples were processed and cultured for mycobacteria as previously described 2O followed by incubation at 80°C for 1 hour and then stored at 4°C until used for molecular typing. AFB positive isolates were also prepared as 20% glycerol stocks and stored at −80°C. Heat-killed AFB positive isolates were investigated by multiplex PCR for the presence or absence of RD4 M. bovis. PCR amplification for RD4 typing was as follows: reactions were performed in a total volume of 20 µl consisting of 10 µl HotStarTaq Master Mix , 7.1 µl distilled H2O, 0.3 µl of each oligonucleotide primer (100 µM), and 2 µl DNA template . Isolates genetically typed as M. bovis were spoligotyped as previously described M. bovis isolates were typed for 24-loci MIRU-VNTR by Genoscreen, France Bacterial colonies from culture positive samples were Ziehl-Neelsen stained according to standardized protocol to identify Acid-Fast Bacilli (AFB). Slants with no growth at week 8 were considered negative. Heat-killed cells of each AFB positive isolate were prepared by mixing ∼2 loopfuls of cells (∼20 µl cell pellet) in 200 µl dHAll collected field and laboratory data were double entered into Microsoft Access database. Data validation was done with EpiInfo software (“Data compare” package). Data analysis was performed with STATA Version 10.1 . We used logistic regression models for the analysis of BTB prevalence and the univariate analysis of parameters associated with positive farms. A GEE model  with binary outcome was used to analyse potential risk factors for individual reactors, in which farms were used as random effect and farm size as fixed effect with adjusted odds ratio (OR). In both cases, results were presented with 95% confidence interval for OR and p-values.As shown in The vast majority (95%) of the investigated farms were privately owned while the remaining 5% were owned by a cooperative or the government. Seventeen of the 88 farms were owned by women. In our survey 80 out of these 88 farms answered our questionnaire . These 8The vast majority of milk produced in the investigated farms was either sold , or partly sold and partly used for own consumption (50%). The remaining 5% of the farms produced milk for their own consumption only. Thirty-five farms sold their milk to processing plants, 23 to cafés and restaurants, 19 to intermediate caterers, and 16 directly to private individuals.The number of people living on a farm varied between one and 53. However, the majority (81%) of farms accommodated less than ten people while half had only between one and four people living on the farm. The majority (86%) of the farm owners did not drink raw milk and 71% knew that BTB is an animal and a zoonotic disease.Over a period of 13 months (January 2009 to January 2010), a total of 2956 cattle from 88 dairy farms were tuberculin tested by CIDT. The vast majority (94%) of these cattle were female and either of Holstein-Frisian breed (37%) or cross breeds thereof (62%) with local zebu cattle . The remThe result of the tuberculin testing is presented in More detailed analysis of small and medium sized farms indicated that the areas of Addis Ababa and Sendafa  had the The results obtained from the analysis of large dairy farms showed a somewhat different picture. The individual BTB prevalence in any tested large farm varied from 0% and up to 93%. By study area, Holeta showed the lowest prevalence with only two CIDT positive animals between the two large farms tested. The individual prevalence in all other study areas varied between 23.5–90%, . OverallMycobacterium avium complex (MAC) or other environmental mycobacteria. In this study 318 out of the 2956 tested animals (∼11%) reacted positive to PPD-A. The PPD-A response was lowest in Holeta  and highest in Sululta   , regardl5; 18.5)  and the The univariate analysis  showed tThere was no significant difference in BTB status amongst farms with different sanitation status and amongst the type of cattle housing (data not shown).M. bovis and to get a better understanding of the population structure of the causative agent, we purchased and slaughtered a minimum of three PPD positive animals per study area. In total 36 cattle from 19 farms  were purchased and investigated by post-mortem and each animal was given a pathology score based on disease progression . The geographical distribution of these four spoligotypes suggested three main clusters; SB0134 was the most dispersed and found in all six study areas, SB1176 was represented in all areas but Sendafa, while SB0133 was only found in Sululta, Sendafa and Debre Zeit. The one isolate of type SB1477 was collected in a farm from Addis Ababa.Heat-killed cells of 67 AFB isolates originating from 31 animals were enrolled in a typing scheme initiated by RD4 deletion typing. The result showed that all isolates could be identified as M. bovis isolates. MIRU-VNTR results were generated for 27 isolates of the four different spoligotypes  breeds are more resistant to BTB than Bos taurus (European breeds). The vast majority of the dairy cattle included in this study were cross breeds (62%) and Holstein-Frisian (34%), however the BTB prevalence did not differ significantly between these two categories  but seemed unidirectional; dairy cattle were not purchased but only sold to other regions across Ethiopia. The response from farmers in this survey also suggested that some animals were sold because they were weak or diseased. As no official test-and-movement policy is in place, selling of infected animals from this highly affected region of Ethiopia may consequently contribute to spreading BTB to other parts of the country.M. bovis and the CIDT test was confirmed by culture in 86% of the slaughtered cattle. This culturing yield can be compared with a 90% yield normally seen in slaughterhouse cases in the UK from animals with visible lesions and of which the vast majority were caused by M. bovisM. tuberculosis was isolated in our study in contrast to several other Ethiopian studies M. tuberculosis as CIDT testing do not distinguish between M. tuberculosis and M. bovis. However, the dairy cattle of this study were managed under intensive husbandry systems with animals staying in separate barns, as compared to management systems common in the rural areas where domestic animals often sleep in the same house as people. It has previously been suggested that human-to-cattle transmission of M. tuberculosis is possible in settings where farm members in high-burden TB areas are in close contact with their animals The mycobacterial strains isolated from cattle in this study were solely M. bovis strains isolated from the slaughtered animals identified four different spoligotype patterns which previously have been shown as common in several regions of Ethiopia M. bovis African 2 (Af2) so far only found in East Africa M. bovis isolates with spoligotype SB0134 has been found also in Europe. However, the phylogenetic relationship between the African and European strains of SB0134 is not known. Future chromosomal sequencing may shed light over their relationship.Molecular typing of the 31 M. bovis isolates performed by MIRU-VNTR typing suggested low diversity within each isolated spoligotype. This is in agreement with other studies as in high prevalence settings the genetic diversity of sampled strains is usually low, reflecting on-going local transmission events (due to high transmission rate) M. bovis strains that were collected from other parts of Ethiopia about 2–3 years earlier M. bovis strains of SB0134 isolated in central Ethiopia (this study). Overall, based on these molecular typing results of our M. bovis collection, epidemiological links can be suggested for strains collected within the North/Central Ethiopian highlands and through the rift valley down to Negelle, while strains isolated from Jinka and Ghimbi in the south/west of Ethiopia diverged. This may reflect on past and ongoing cattle movements and trading patterns in the country. The low strain frequency from some collection sites means that these results should be interpreted with care. Nevertheless, this genotype comparison gives an indication of the diversity and the epidemiology of M. bovis in Ethiopia. Future studies may give better clarification on this subject. The 24-loci MIRU-VNTR typing employed for this study showed no diversity in at least 14 of the 24 loci  from cattle-to-human should also be considered as a potential risk factor. Ethiopian milk consumers generally prefer raw milk (as compared to treated milk) because of its taste, availability and lower price. Therefore the demand for fresh whole milk in rural areas is and will probably remain high, and in general, the demand from these consumers is likely to be satisfied by home production or by purchasing from neighbouring producers. On the other hand, the markets for surplus milk are mainly found in the urban population, of which currently ∼65% is accounted for by the capital Addis Ababa and its neighbouring districts The zoonotic risk of BTB is often associated with consumption (ingestion) of dairy products based on unpasteurised milk infected with The annual total milk supply to Addis Ababa has been estimated at 65 million litres and the major sources are from small private farms and smallholder urban dairies in and around the city that own upgraded breeds M. bovis causing TB in humans M. bovis is currently below 1%. However, we concluded that areas where BTB prevalence in cattle is high  could generate an increase of BTB in the human population in such areas. It is important to note that existing human TB control methods relying on the directly observed treatment short course (DOTS) strategy will not directly impact on BTB transmission. The high rate of BTB in the dairy farms of this study is therefore likely to pose a serious risk to public health and deserves a targeted intervention as early as possible.In a recent Ethiopian study of the rate of This study suggests that the overall prevalence of BTB in intensive dairy farms in Addis Ababa and its surroundings is very high and requires urgent intervention to control the disease. To comprehensively control BTB regardless of the method chosen, a robust system of individual animal and herd identification is required. The powers to restrict movements to prevent disease dissemination and the ability to trace movements to catch up with translocation are essential components of an eradication strategy. Most developed countries where BTB is a problem have introduced a test-and-slaughter policy to control/eradicate the disease. The CIDT, although not 100% sensitive, is the standard test widely used in developed countries for this purpose. Most importantly, infected animals have to be detected and removed at an early stage to minimise further transmission. As such policy is considered costly it has rarely been implemented in developing countries. No compensation scheme for elimination of infected animals is currently in place in Ethiopia and due to financial constraints changes toward such policy might not be feasible in the near future. However, a test-and-segregation policy of tuberculin positive animals can be suggested and pursued and the milk of infected animals strictly and consequently pasteurized before selling and consumption. Additional interventions by the government authorities could be to encourage farmers to regularly test their animals for BTB and create incentives to keep their herds free from BTB. It could be compulsory for dairy farms that are supplying milk to the public to test their cattle for BTB, and create incentives to TB free herds – “good milk certificate” – that could lead to better economic values for farmers. Also dairy plants should be engaged and could e.g. pay a higher rate for milk from herds certified as being BTB free. Pasteurization at a bigger scale has to be promoted, either directly at farm level before selling the milk or at household level through boiling the milk before consumption (e.g. awareness campaigns).As the dairy industry in Ethiopia has expanded in recent years and is expected to continue doing so, significant number of high productive exotic and cross breed animals are likely to be traded from the urban areas around the capital to the rural areas where dairy cattle numbers are still relatively low. Such movement is linked with increasing financial well-being of farmers and amelioration of artificial insemination technique/delivery. However, without any control strategy the risk of spreading BTB by such movements is high and may create new hot-spots of BTB in other parts of the country. Several recommendations can be made to minimize the risk of spreading the disease between farms and regions. First and most important is to spread knowledge about BTB and make farmers and people involved in trading of cattle aware of the risks involved in trading animals. On a smaller scale, our field work and specific training to the farmers aimed on this, informing them in how to handle cattle infected with BTB and other diseases, and encouraging them not to trade with diseased animals. Another way to decrease risk of transmission is to encourage farmers who are restocking by purchasing animals to request that the animals should be tested for BTB. The CIDT test is relatively inexpensive and would reduce the risk to the farmer of purchasing an infected animal. A possibility for Ethiopia to in part meet the demand for cattle of upgraded breeds could be to establish farms with dairy cattle free of BTB and from which farmers could restock their farms without the risk of introducing the disease into their herds. This might imply scaling up of existing artificial insemination centres to improve productivity in the national dairy herd and possibly purchase of new stocks of exotic breeds from abroad.Questionnaire S1(PDF)Click here for additional data file.


Mycobacterium bovis – in cattle and the Kafue lechwe antelopes (Kobus leche Kafuensis) has been reported in the Kafue basin. Consumption of unpasteurised milk and meat products from infected animals poses a risk of transmitting zoonotic tuberculosis to people living at the human-animal interface. Despite the reported high prevalence of BTB in both livestock and wildlife, information on the proportion of human patients infected with M. bovis is unknown in Zambia. This paper reviews the available information in English on human, livestock and wildlife TB in Zambia with the purpose of assessing the burden of animal infections with M. tuberculosis complex and its public health implications.Zambia’s estimated incidence of all forms of human tuberculosis (TB) is 707/100,000. High prevalence of bovine tuberculosis (BTB) – infection with  Please see Additional file Mycobacterium tuberculosis complex (MTC), which includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium caprae, Mycobacterium microti, Mycobacterium pinnipedii and Mycobacterium canettii[Human tuberculosis (TB), although an ancient disease, has re-emerged with devastating consequences on global public health and is currently one of the most widespread infectious diseases. In addition, it is the leading cause of death due to a single infectious agent among human adults in the world . Tubercu canettii. Approxi canettii. The TB  canettii.Mycobacterium tuberculosis is usually transmitted to a human by inhalation of aerosol droplets containing tubercle bacilli which are expectorated from infected individuals with open pulmonary TB [The World Health Organization (WHO) estimates that the incidence of all forms of TB in Zambia stands at 707/100,000 . Mycobaconary TB .Mycobacterium bovis (M. bovis), the bovine tubercle bacilli, is the cause of bovine tuberculosis . It has a wide range of host animal species, which includes cattle, goats, bisons, antelopes, humans and non-human primates, and can cause disease in susceptible hosts [le hosts .Kobus leche Kafuensis) has been reported at the wildlife-livestock interface in the Kafue basin [M. bovis transmission between species sharing the same environment , zoonotic tuberculosis remains significantly underrepresented as causal agents of extra pulmonary and pulmonary TB in developing countries, especially in rural regions at the human-animal interface [High prevalence of BTB in cattle and the Kafue lechwe antelopes  has been reported to be endemic in the Zambian traditional cattle sector with a high herd prevalence of 49.8% recorded from areas within, and adjacent to, the Kafue basin as far back as 1947 ,17. RepoKobus leche Kafuensis) is a medium-sized, semi-aquatic antelope with a population of 44,000 that is endemic to the Kafue flats [Syncerus caffer) is present in the Kafue basin. In 2011, a study reported that no BTB positive reactors were found in the comparative intradermal tuberculin test (CIDT), suggesting an absence of M. bovis infection in buffaloes [The Kafue lechwe , where M. tuberculosis was isolated from milk samples obtained from cattle in India, suggesting that infections may spill back to humans through consumption of unpasteurised milk [entified . It has entified -26. The sed milk .M. bovis and that 0.4-10% of sputum isolates from patients in African countries may be M. bovis. This is despite the fact that M. bovis is mainly associated with extra pulmonary disease in humans [M. bovis in Zambia and other developing countries is limited, owing to technical problems posed by identification of this species, such as trained personnel and laboratory facilities [Mycobacterium[M. bovis leading rapidly to disease [In 1998, the WHO reported that 3.1% of tuberculosis cases in humans worldwide are attributable to n humans . Data oncilities ,29. Indebacterium. HoweverIn a more recent study, Gumi et al. (2012) documented, by using molecular tools, an epidemiological link in the zoonotic transmission between livestock and pastoralists of south-east Ethiopia . ZoonotiLastly, the Kafue lechwe is hunted for meat, trophies and hides. It is estimated that approximately 80% of lechwe carcasses hunted for meat may be infected with BTB. However, poaching levels specific to lechwe are speculated at 50% of the official annual hunting quota . This plM. tuberculosis in animals exists.This review has identified the knowledge gaps in the management of BTB in Zambia. No molecular epidemiological information linking the observed BTB in cattle and wildlife is available. In addition, the review noted that information on the incidences of zoonotic TB is also unavailable despite the reported cultural practices by the people living at the interface. Finally, the review noted that no molecular epidemiological study has been conducted on TB patients living at the interface area and that no information on the presence of M. bovis infections in humans, cattle and Kafue lechwe in the Kafue basin. This could be done by molecular characterisation of isolates obtained from these three species , as well as from milk and the environment [•There is a need to document epidemiological links of ironment ,37.M. tuberculosis infection has not yet been reported from cattle in Zambia, it has been documented elsewhere [M. tuberculosis, and to evaluate its epidemiological significance in transmission to humans.•Although lsewhere . This hiM. bovis.•The interaction of cattle, lechwe and other wildlife, such as buffaloes, at water and grazing points has been documented in the Kafue Basin. Therefore, there is need to conduct BTB field and molecular studies in order to draw epidemiological links between BTB in the different species and to study the risk factors for the transmission of M. bovis suggests that environmental contamination is not the preferential route by which wildlife gets contaminated [M. bovis in the Kafue basin are warranted in order to clarify the most important transmission routes at the livestock/wildlife interface.•The fact that buffaloes are not infected with aminated . Moreove•Given the high prevalence of BTB in traditional cattle, it is important that surveillance is conducted in communities to ascertain the impact of zoonotic tuberculosis.Mycobacterium tuberculosis complex; OIE: World Organisation for Animal Health; TB: Tuberculosis; WHO: World Health Organization.ABF: Acid-fast bacilli; BTB: Bovine tuberculosis; CIDT: Comparative intradermal tuberculin test; MTC: The authors declared that they have no competing interests.SM contributed to the conception and drafting of the manuscript, JBM and JG contributed to the conception and writing of the manuscript. All authors have read and approved the final manuscript.Multilingual abstracts in the six official working languages of the United Nations.Click here for file


This Research Topic covers issues in psychology, behavioral economics, and cognitive neuroscience investigating the neural structures and mechanisms underlying approach, and avoidance behavior in the face of rewards and punishments. The objective is to understand the nature of critical differences and asymmetries between the ways that appetitive and aversive outcomes are processed by the brain. A number of topics are covered, such as the development of economic models integrating costs and benefits into a single value, neuroimaging approaches of appetitive and aversive conditioning, reward-punishment interactions, pain and defensive behavior, the role of dopamine neurons in aversive conditioning, and the interactions between serotonin and dopamine in punishment, pain, and aversion. The neural bases of reward-punishment interactions are of great interest to a broad readership because of the fundamental role of dopamine and serotonin in a number of motivational and decision processes, and because of their theoretical and clinical implications for understanding dysfunctions of these two systems. Findings in this research field are also important to basic neuroscientists interested in the computational processes of pain and aversive learning and cognitive psychologists working on conditioning/reinforcement. Punishment-based decision making and reward processing cover a wide range of topics and levels of analysis, from basic neural mechanisms and computational models of appetitive and aversive conditioning, to the system neuroscience level. The contributions to this Frontiers Research Topic in Decision Neuroscience are forward-looking assessments of the current and future issues faced by researchers.Porcelli et al.  investigIn a second study, Singh and Khan  studied In the third article, Rigoli et al.  studied Barberini et al.  focus onIn a review article, Kobayashi  extends Wiech and Tracey  review tIn a mini review, Ilango et al.  examine In another paper, McCutcheon et al.  review dFinally, Talmi and Pine  review bWe anticipate that while some readers may read this Frontiers Research Topic from the first to the last chapter, other readers may read only one or more chapters at a time, and not necessarily in the order presented in this e-book. This is why we encouraged an organization of this volume whereby each chapter can stand alone, while making references to others and minimizing redundancies across the e-book.Given the consistent acceleration of advances in the different approaches described in this Research Topic, we hope that you will enjoy these new stages of an exciting era in neuroscience research on the interactions between appetitive and aversive systems.


In this study, we report formation of weblike fibrous nanostructure and nanoparticles of magnetic neodymium-iron-boron (NdFeB) via femtosecond laser radiation at MHz pulse repetition frequency in air at atmospheric pressure. Scanning electron microscopy (SEM) analysis revealed that the nanostructure is formed due to aggregation of polycrystalline nanoparticles of the respective constituent materials. The nanofibers diameter varies between 30 and 70 nm and they are mixed with nanoparticles. The effect of pulse to pulse separation rate on the size of the magnetic fibrous structure and the magnetic strength was reported. X-ray diffraction (XRD) analysis revealed metallic and oxide phases in the nanostructure. The growth of magnetic nanostructure is highly recommended for the applications of magnetic devices like biosensors and the results suggest that the pulsed-laser method is a promising technique for growing nanocrystalline magnetic nanofibers and nanoparticles for biomedical applications. Nanomaterials field is of current interest because it studies materials with morphological features on the nanoscale. Nanosized materials show distinctive properties compared with bulk materials -3. In paThe growth of nanofibers using ultrafast laser offers advantages of high resolution, high throughput, uniformity, localized heating, simplicity, and reproducibility -8. The tMagnetic neodymium-iron-boron (NdFeB) nanofibers and nanoparticles have become one of the hotspots in the research field of magnetic materials to meet the demand for miniaturization of electronic components in recent years, and have been successfully prepared by various routes like the sol-gel auto-combustion method , co-precIn the present study a magnetic weblike fibrous nanostructure is formed due to the agglomeration of the bulk quantity of nanoparticles created during laser ablation at mega hertz pulse frequency. A distinct characteristic of the fibrous nanostructures is that particles are fused and the agglomeration shows certain degree of organization, unlike the random stacking of particles observed at femtosecond laser ablation at pulse frequency in kilohertz and hertz regime. The effect of pulse repetition rate on the nanofibers size and hence the magnetization was also investigated. The nanostructures were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX), X-ray diffraction (XRD), and magnetic force microscopy (MFM). The mechanism of formation is explained by the well-established theory of vapor condensation induced by ultrafast laser ablation. Also, the fibrous nanostructures have relatively uniform diameters (30-90 nm) and did not observe a wide range of variation in size distribution. This agrees with the characteristics of nanoparticle formation through homogenous nucleation, which tends to generate monosized nanoparticles.2Fe14B crystal structure has exceptionally high uniaxial magnetocrystalline anisotropy. This gives the compound the potential to have high coercivity. To generate magnetic nanofibers, the first piece of the magnetic specimen was irradiated with laser using 1040 nm wavelength with 15 W power and a pulse repetition rate of 4 MHz. The experiment was repeated to generate nanofibers on the specimen using the same power and wavelength with frequencies of 8, 13, and 26 MHz. The irradiated sample was characterized using SEM, TEM, EDX, and XRD analysis.The laser source is a diode-pumped Yb-doped fiber oscillator/amplifier system (Clark MXR Inc.) capable of producing an average power of 15.5 W with pulse repetition frequency between 200 kHz and 25 MHz. A neodymium-iron-boron magnetic specimen of 1" ± 0.008" length by 1" ± 0.008"; width by 0.1" ± 0.005" thickness was cut into four pieces of same size. The tetragonal NdThe energy of the femtosecond laser is delivered into the material in a short time scale that absorption occurs at nearly solid-state. The energy is first deposited in the electronic subsystem within a layer of thickness of tens of nanometer. Enough energy is absorbed to produce macroscopic ablation when the density of the free electrons exceeds a certain threshold . The ionThe laser pulse repetition rate plays a critical role in the formation of nanofibrous like structure. Figure During ablation, the ionized material is removed away from the surface in the form of expanding high pressure plasma. The temperature of the plasma is above the melting temperature and hence Curie temperature of the magnet. Thus the irradiated spot and the surrounding area where temperatures above 400°C will be demagnetized while the rest of the sample remains magnet. The plasma remains confined close to the specimen surface at atmospheric pressure. Condensation of vapor in the plume leads to the generation of nanoparticles which move in the direction where the paramagnetization potential energy is minimized . The nanThe laser pulse repetition rate plays a critical role in the formation of magnetic nanofibrous structure . In ordeCharacterization was performed using XRD as a function of femtosecond laser pulse repetition rate. Figure Figure r is the nanofiber size, λ is the X-ray wavelength, B is the full width at half maximum of the peak (FWHM), and θ is the diffraction angle. From the diffraction peaks in Figure where 2Fe14B or from the amorphous phase produced at grain boundaries during the laser ablation process.The metastable Nd-rich phase is a grain-boundary phase which has an FCC structure. This grain boundary phase exhibits a characteristic contrast which is similar to a metastable high-pressure phase observed previously as FCC γNd . The strM as a function of laser repetition rate for the NdFeB nanofibers grown at room temperature. The thickness of the generated fibers layer in all of the four pieces were the same [M of the nanofibrous structure get lower due to the presence of an abundant amorphous phase which also shows lower coercivity. The relatively large coercivities of nanofibrous structures were due primarily to their specific morphology. Theory has predicted that a system containing magnetic dipoles that are arranged into a linear chain will exhibit an increase in coercivity [Figure the same , howeverercivity . Our resWe introduced synthesis of NdFeB magnetic fibrous nanostructure and nanoparticle on bulk substrate using femtosecond laser radiation under ambient conditions. The phase structures and microstructures have been investigated using XRD, SEM and EDX analysis. The magnetic nanofibers were grown in the order of few nanometers and organized themselves in weblike structures. Magnetic nanoparticles with diameter in the order of few nanometers were attached to the nanofibrous structure. Increasing the repetition rate of the femtosecond laser results in increasing the number of pulses and hence decreases size of the generated magnetic nanofibers. Increasing repetition rate of the femtosecond laser results in generating smaller size magnetic nanofibers. The magnetic strength of the generated nanofibers can be controlled by changing the repetition rate of the femtosecond laser. These magnetic nanofibers may be utilized in many applications, such as magnetic devices, carriers, tissue engineering materials, and drug delivery.EDX: energy-dispersive X-ray; MFM: magnetic force microscopy; NdFeB: neodymium-iron-boron; SEM: scanning electron microscopy; TEM: transmission electron microscopy; XRD: X-ray diffraction.The authors declare that they have no competing interests.MA carried out laser processing of the samples, characterisation and drafted the manuscript. KV conceived of the study, and participated in its design and coordination. BT conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Molecular phylogenies have been used to study the diversification of many clades. However, current methods for inferring diversification dynamics from molecular phylogenies ignore the possibility that clades may be decreasing in diversity, despite the fact that the fossil record shows this to be the case for many groups. Here we investigate the molecular phylogenetic signature of decreasing diversity using the most widely used statistic for inferring diversity dynamics from molecular phylogenies, the γ statistic. We show that if a clade is in decline its molecular phylogeny may show evidence of the decrease in the diversification rate that occurred between its diversification and decline phases. The ability to detect the change in diversification rate depends largely on the ratio of the speciation rates of the diversification and decline phases, the higher the ratio the stronger the signal of the change in diversification rate. Consequently, molecular phylogenies of clades in relative rapid decline do not carry a signature of their decreasing diversification. Further, the signal of the change in diversification rate, if present, declines as the diversity drop. Unfortunately, the molecular signature of clades in decline is the same as the signature produced by diversity dependent diversification. Given this similarity, and the inability of current methods to detect declining diversity, it is likely that some of the extant clades that show a decrease in diversification rate, currently interpreted as evidence for diversity dependent diversification, are in fact in decline. Unless methods can be developed that can discriminate between the different modes of diversification, specifically diversity dependent diversification and declining diversity, we will need the fossil record, or data from some other source, to distinguish between these very different diversity trajectories. Understanding the controls of biodiversity is one of the main goals of ecology and evolutionary biology. In recent decades this endeavor has been revitalized by the use of molecular phylogenies to study the diversification of many clades Currently, these tools have been used to distinguish between clades that are diversifying exponentially from those that might be undergoing diversity dependent diversification, and are at or approaching an equilibrium carrying capacity However, molecular phylogenies, by the virtue of only considering extant species, carry the perceptual bias of increasing diversity Unfortunately, none of the current methods used for deducing diversity trajectories from molecular phylogenies incorporate this very real possibility of negative diversification rates The fossil record clearly shows that clades rise and fall in diversity The specific simulation approach used here was motivated by importance of diversity dependent diversification reported in the literature For each set of simulations a total of 100 replicates were run, a sample size large enough to capture the stochastic variation in the models used. We used the R package TreeSim Several different tools are available for using molecular phylogenies to infer diversity dynamics The γ statistic has some other advantages. First, it is easily understood . Second, it is widely used, especially in the literature on diversity-dependent diversification The γ statistic does have its limitations, primarily an appreciable type II error rate. Specifically, it has low discriminating power between exponential growth and the early phases of diversity dependent diversification The rise and fall of simulated clades was modeled by exponential growth followed by exponential decline. We chose this way of simulating the waxing and waning of diversity because preliminary data suggested that the resulting phylogenies would give the appearance of diversity dependence, and so we wanted to make sure that we did not confound the interpretation of our results by having the initial diversification process be diversity dependent. We chose a range of realistic speciation and extinction rates To characterize the molecular signature of the declining diversity as it unfolds, we calculated the γ statistic at different points in time, a procedure we term “time traveling” see . The molThe reason time-traveling analysis is required is that in the absence of a good fossil record it is virtually impossible to know, even if you knew your clade was in decline, how long ago peak diversity was reached, and what its peak diversity was Our simulations show that clades in the initial phases of their decline typically result in molecular phylogenies with the most negative γ values . The strtatistic . When th, when in fact the clade is in exponential decline. We wished to understand why the γ values become negative in the first place, and why γ then becomes more positive as the clades continue to decline in diversity, and so we ran additional simulations to explore these questions.Under-sampling species will generate a predictable bias in the diversification signature of molecular phylogenies by artificially increasing the relative importance of early splitting events To test this supposition, we ran a set of simulations with simple exponential growth followed by different degrees of under-sampling after a pre-assigned fixed diversity had been achieved. For these simulations we used the same speciation and extinction rates used in the initial diversification phase of our clades-in-decline simulations . For theWhile under-sampling made the γ statistic more negative than the fully sampled tree, γ only became significantly negative for the 760 species scenario  – under Given that the loss of terminals, per se, is not responsible for the strongly negative γ values seen in our simulations of clades in decline, what could be responsible? Our second guess was based on the realization that one way to achieve strongly negative γ values is to simply extend the lengths of the terminal branches, without adding or subtracting branches. Thus, we ran a set of “pure-aging” simulations, initiated with exponential growth with the same rates of initial diversification used in the clades-in-decline simulations . Then, iAs expected, the clades in stasis produce highly negative γ values . Rates of speciation and extinction used here were chosen to produce the same diversification rates in the rise  and decline  phases as used in the scenario shown in (TIF)Click here for additional data file.


The supramolecular hydrogels were characterized by transmission electron micrograph (TEM) and fluorescence spectroscopy, and the results obtained by both techniques correlate well with their rheological properties. Notably, compound 6 can undergo cis/trans-isomerization upon UV irradiation.We report herein the use of an aromatic–aromatic interaction to produce small molecule hydrogelators that self-assemble in water and form molecular nanofibers in the resulting hydrogels. Among these hydrogelators, a hydrogelator ( Gels formed by three-dimensional, elastic networks to encapsulate a liquid , have maN-(4-pyridyl)isonicotinamide (MW = 199.21 g/mol) can act as a non-polymeric hydrogelator at the concentration of 0.37 wt %, which further suggests the possibility of peptide-based hydrogelators that are smaller than the aroyl L-cystine noted above.However, despite the progress noted above, an important question still remains to be answered: What is the minimal set of aromatic–aromatic interactions and hydrogen bonding for molecular self-assembly in water to produce supramolecular nanofibers and hydrogels? About a decade ago, Menger  and cowoHere, we reported the systematic synthesis and examination of a series of phenylalanine derivatives and the identification of the lowest molecular weight peptide-based hydrogelator (MW = 295.33 g/mol) produced to date. Since phenylalanine derivatives belong to a class of insulin absorption promoters , this wo1, 3, 4 and 6 form hydrogels. Compared with compound 3, compound 2 fails to form a gel due to the conjugation between the carbonyl group and the naphthyl group, which partial reduces the rotational freedom of the naphthyl group that is necessary for self-assembly. As shown in I, II, III and IV formed by compounds 1, 3, 4 and 6, respectively) are thermally and pH reversible. For example, heating the gel formed by 0.3 wt % of compound 1 in water to 56 °C or changing its pH from 6.6 to 9.0 leads to a gel–sol phase transition. The gel forms again after restoring the previous conditions, and this cycle can be repeated several times.IV exists in two phases  isomerism.We noted that the gel o phases  when it o phases . In ordee 1H NMR , a new scitation . Since t6 also exhibits another interesting phenomenon. On acidification by 1N HCl solution, the solution of 6  and loss modulus (G’’) for gels I to IV at a concentration of 1.0 wt %. The values of the dynamic storage moduli (G’) of all gels are higher than those of their dynamic loss moduli (G”), indicating that all samples behave as viscoelastic materials. The values of G’ of the hydrogels exhibit little dependence on the frequency (from 0.1 to 200 rad/s), suggesting that the matrices of gels have good tolerance to external shear force. Gel IV, which formed by the lowest molecular weight hydrogelator among the phenylalanine derivatives prepared, has the lowest storage modulus. Gel I, which was formed by a hydrogelator with the highest molecular weight among compounds 1–6, exhibits the strongest mechanical strength among the gels reported in this work. This is in agreement with the fact that the interactions between fluorenyl and phenyl groups are stronger than the interactions among phenyl groups. G’ and G’’)-strain profiles of gels I to IV, which provide the maximum G’ values in the linear region and values of critical strain. The G’ remained almost constant when the strain increased and then suddenly decreased, indicating the loss of crosslinking within the gel network. Obviously, the critical strain of gel IV is greater than the others. These results suggest that gel IV can form a more complex network structure, which agrees with the morphology as revealed by TEM , suggesting that the interactions between naphthyl groups and phenyl groups in compounds 3 and 4 result in less overlap than that of fluorenyl and phenyl groups.Based on emission spectra of the hydrogels , aromaticis/trans-isomerization offers a novel pathway for achieving well-dispersed nanofibers, which could be used in drug separation, drug release and other fields.In summary, we have demonstrated that an aromatic group  covalently attached to phenylalanine gives rise to a series of new low molecular weight hydrogelators and the identification of the lowest molecular weight peptide-based hydrogelator prepared to date. Obviously, the cinnamoyl group is the minimum structure motif to provide sufficient aromatic–aromatic interactions for a phenylalanine derivative to be a hydrogelator. It not only provides a useful experimental system for further elucidating the relationship between hydrogelation and molecular structure, but also offers a small molecular building block for the development of enzyme based molecular self-assembly. Furthermore, File 1Experimental part.


Transient nausea and, to a lesser extent, vomiting are common adverse effects of exenatide that can be mitigated by dose titration and usually do not result in treatment discontinuation. This retrospective analysis of data from a phase 1, open-label, parallel-group, single-dose study in healthy subjects evaluated the effect of oral anti-emetics on exenatide-associated nausea and vomiting and on the pharmacokinetics of exenatide.2). Incidences of nausea and vomiting were compared between 60 subjects premedicated with two oral anti-emetics 30 min before the exenatide dose and 60 non-premedicated subjects. Similarly, the area under the concentration-time curve (AUC) and the maximum observed concentration (Cmax) of plasma exenatide concentrations over 8 h post-dose were compared.A single subcutaneous dose (10 μg) of exenatide was administered to 120 healthy subjects ], mild to moderate nausea was the most frequent adverse event after exenatide dosing. Vomiting was also observed. Subjects premedicated with anti-emetics experienced significantly less nausea and vomiting  vs. non-premedicated subjects . The mean area under the concentration-time curve and the maximum observed concentration AUC and Cmax of plasma exenatide concentrations during 8 h post-dose were not significantly different between groups.Among all subjects [61% male, 32 ± 12 years, body mass index (BMI) 29.1 ± 3.4 kg/mAdministration of oral anti-emetics before a single 10-μg exenatide dose was associated with significant reductions in treatment-emergent nausea and vomiting, with no discernible effect on the pharmacokinetics of exenatide. Use of anti-emetic therapy may provide a short-term strategy to minimize the nausea and vomiting associated with exenatide treatment. Exenatide, a first-in-class glucagon-like peptide 1 (GLP-1) receptor agonist, is approved as an adjunct to diet and exercise and/or to some oral anti-diabetic therapies for the treatment of Type 2 diabetes mellitus. Exenatide improves glycaemic control by enhancing glucose-dependent insulin secretion by pancreatic B-cells, suppressing inappropriately elevated glucagon secretion, slowing gastric emptying, and reducing food intake  and maximum observed concentration from time zero to time of last quantifiable sample [Cmax(0–tlast)] of plasma exenatide concentrations over 8 h following exenatide dosing were determined using the non-compartmental method and summarized descriptively for intent-to-treat subjects with valid exenatide pharmacokinetic profiles by anti-emetic treatment group (premedicated and non-premedicated subjects).The intent-to-treat population was defined as all enrolled subjects who received at least one dose of study medication. Adverse events were coded using the Medical Dictionary for Regulatory Activities (MedDRA®) version 11.0  and Cmax(0–tlast) of plasma exenatide concentrations over 8 h post-dose were compared between groups to evaluate the potential effects of the anti-emetics on the pharmacokinetics of exenatide. P-values were calculated using Student's t-test.A 2 (mean ± sd). Demographic and baseline characteristics in subjects who did and did not receive anti-emetics were comparable.All 120 enrolled subjects were included in the intent-to-treat population. Subjects were: 78% Caucasian, 9% Black, 8% Hispanic, 3% Native American, 1% Asian and 2% other ethnicities; 61% male; age 32 ± 12 years, with a screening BMI of 29.1 ± 3.4 kg/mP-value < 0.0001 for both nausea and vomiting)  .In the group that did not receive anti-emetics, 21 of the 37 subjects who experienced nausea also vomited; in the group premedicated with anti-emetics, three of the 10 subjects who experienced nausea also vomited. In addition to receiving prophylactic anti-emetic medications prior to the exenatide dose, one of the 60 premedicated subjects was administered a second dose (4 mg) of ondansetron hydrochloride on the day of the exenatide dose to treat treatment-emergent nausea. Likewise, one subject in the non-premedicated group was given one dose (4 mg) of ondansetron hydrochloride to treat treatment-emergent vomiting.A higher incidence of somnolence was reported in the group premedicated with anti-emetics (8.3%) compared with the non-premedicated group 1.7%) (data not shown). Somnolence has been reported with exenatide treatment post-approval [.7% , leaving a total of 116 intent-to-treat subjects evaluable for pharmacokinetic analysis.max of plasma exenatide concentrations during the 8 h following exenatide dosing between subjects who did and did not receive anti-emetics  higher than previously observed in studies of exenatide monotherapy or in combination with anti-diabetic therapy (8–44% for nausea and 4–13% for vomiting) [Several limitations of the study design and current analysis warrant consideration with regard to extrapolating these results beyond the context of this study. The study used a single, fixed dose of exenatide, with oral anti-emetics administered at a prespecified time (30 min prior to dosing). Therefore, the study did not address the effect of repeated administration of anti-emetics with continued exenatide treatment or the optimal time of anti-emetic administration relative to exenatide dosing. Additionally, all subjects in the premedicated group received both metoclopramide and ondansetron hydrochloride. It is not known whether administration of either anti-emetic agent alone would suffice in mitigating nausea and/or vomiting.  Additional potential limitations of the study design include the subject population, which comprised healthy individuals whose tolerance to exenatide may be different from that in patients with diabetes, and the open-label trial design, which inherently invites the potential for bias because it can affect patient expectations. Notwithstanding the aforementioned limitations, the results of this analysis provide evidence that prophylactic oral anti-emetics significantly reduced nausea and vomiting following administration of exenatide and may be relevant to clinical practice.1c, fasting plasma/serum glucose concentrations, body weight  and postprandial plasma glucose concentrations [Implementing strategies to address the potential nausea and vomiting adverse events associated with exenatide treatment could promote patient's adherence to the treatment regimen and facilitate the achievement of glycaemic targets . Exenatitrations ,12, as wGradual dose escalation at initiation of exenatide therapy is recommended, having been shown to be effective at mitigating nausea and vomiting with no loss of glucoregulatory activity . In clinThere is also anecdotal evidence of additional strategies that have been employed to reduce nausea associated with exenatide . These iAlthough nausea is mostly of mild to moderate intensity and its incidence and intensity generally decrease after the first 8 weeks of exenatide treatment ,3–6, pro


In this work, we studied a copper complex-based dye, which is proposed for potential photovoltaic applications and is named Cu (I) biquinoline dye. Results of electron affinities and ionization potentials have been used for the correlation between different levels of calculation used in this study, which are based on The Density Functional Theory (DFT) and time-dependent (TD) DFT. Further, the maximum absorption wavelengths of our theoretical calculations were compared with the experimental data. It was found that the M06/LANL2DZ + DZVP level of calculation provides the best approximation. This level of calculation was used to find the optimized molecular structure and to predict the main molecular vibrations, the molecular orbitals energies, dipole moment, isotropic polarizability and the chemical reactivity parameters that arise from Conceptual DFT. Global average atmospheric CO2 has increased from 280 ppm in the 1750s to 389 ppm in 2010 2+-based  photosensitizer, the dyes reported in the literature have predominantly been ruthenium(II) complexes. We have recently become interested in the study and optimization of copper(I)-based DSSC, literature reports of which are scarce. Sauvage and co-workers [2]+  which is shown in Ruthenium(II) complexes as dyes have been extensively used as sensitizers in DSSCs owing to their strong absorption in the visible range and relatively long-lived excited states . The str-workers  discover-workers . Copper and the ionization potential (I) were obtained by energy calculations , taking into account the ground state geometry optimization. The aim of this was to establish the correlation of results between the different levels of calculation, since there are no experimental results reported for this system; this is a contribution of our research. max); according to the levels of theory selected, the UV-Vis spectrum and the λmax were calculated. The results of λmax are shown in Another fundamental property as a potential sensitizer for DSSC is the maximum absorption wavelength  model.The optimized structure of Cu(I) biquinoline dye is shown in A selection of geometric parameters was made to clearly visualize how the geometric structure has a very small structural variation of the atoms bonded with respect to the characteristic lengths and angles reported in the literature . Table 4et al. 2011 [et al. in 2008 [Another very interesting research by X. Lu al. 2011  presents in 2008 . Conside−1. The O–H bond stretching vibration appears at 2533 cm−1, while another peak due to the vibration of C=O is present at 1707 cm−1. The vibration of the C–C bonds occurs at 1403 cm−1 and the bending of C–H in the rings is shown at 1277 cm−1. At 1100 cm−1, the corresponding vibration appears for the bending of the O–H bond. The out-of-plane bending vibration associated with the aromatic rings is observed at 807 cm−1. All signals are described in the typical ranges. This calculation, besides checking the minimum energy structure, also helps to elucidate the infrared spectra because this has not been reported in the literature up to now.In infrared (IR) spectral calculation , the vibThe charge transfer efficiency from the dye molecule to the nanocrystalline oxide is extremely important in the solar cell design. Since the crucial electronic excitations occur from the highest occupied molecular orbitals (HOMO) to the lowest unoccupied molecular orbitals (LUMO), it is therefore important to form efficient charge-separated states with the HOMO localized on the donor subunit and the LUMO on the acceptor subunit. In this molecular system, −) and chlorine (Cl−). The diagram in The HOMO-LUMO molecular orbitals’ energetic position is another factor to consider, as the dye LUMO level must be greater than the conduction band level of nanocrystalline oxides that are commonly used in such devices . Further3, respectively. Chemical reactivity parameters were obtained by energy calculations , taking into account the ground state geometry optimization. These parameters are the electronegativity (χ = 7.30 eV), chemical hardness (η = 2.50 eV) and electrophilic index (ω = 10.64 eV). These results are of great importance, since they can be used during synthesis to determine the solubility and chemical reactivity of the molecule, and they can also be employed in photovoltaics, as reported in different studies [The values of the total dipole moment and the isotropic polarizability at the fundamental state obtained at the M06/LANL2DZ + DZVP level of calculation are 5.63 debye and 838.56 bohr studies –46.Molecular calculations were carried out with the Gaussian09 code. In this study, we have tested the M06 family of density functionals –49 basedMolecular structure calculations of the ground state were obtained by the established technique in Gaussian 09W. The strength constants and vibrational frequencies were determined via analytic frequency calculations on stationary points obtained after geometry optimization. Both calculations were carried out at the same theory level. Ultraviolet-Visible spectra (UV-Vis) were simulated using the TD-DFT approach; solvent effects sdfd taken into account by an implicit approach, namely the polarizable continuum model (PCM) , using tIn this work we calculated the total dipole moment (μ) and the isotropic polarizability (α). The molecular dipole moment is an experimental measure of the charge distribution in a molecule. It is difficult to accurately evaluate the global electron distribution in a molecule because it involves all the multipoles. The polarizability contributes in a significant way to the understanding of the response of the system facing an external field. On the other hand, using the DFT framework makes it possible to find the chemical reactivity descriptor values, such as electron affinity, ionization potential, electronegativity, hardness and electrophilic index. All these values were obtained using system energy calculations.In this work, we studied a copper complex with a polypyridine-type ligand. A set of methodologies suitable for the study of this type of molecular systems was applied, and based on a comparison of experimental data with theoretical calculations, we elected the M06/LANL2DZ + DZVP level of calculation, which was found to be the better approach. The molecular system characterization includes the geometry optimization, the calculation of vibrations of functional groups, total dipole moment, isotropic polarizability, molecular orbitals, and chemical reactivity parameters. The molecular orbital energy indicates an energy gap of 3.146 eV in the Cu biquinoline dye. The influence of anion species such as hexafluorophosphate and chlorine does not contribute to the energy levels. Analyzing the data makes it possible to find potential applications for this dye in photovoltaic devices.The M06/LANL2DZ + DZVP level of calculation can be used as a useful tool for studying the molecular structure and electronic properties of Cu biquinoline dye, as well as other structures derived from it.


Molecular studies have not wavered as the numbers of genes and species have increased, but morphologists have been reluctant to embrace the molecular tree.The position of turtles among amniotes remains in dispute, with morphological and molecular comparisons giving different results. Morphological analyses align turtles with either lizards and their relatives, or at the base of the reptile tree, whereas molecular analyses, including a recent study by Chiari Please see Research article www.biomedcentral.com/1741-7007/10/65 The tree of life is rapidly coming into focus as hundreds of molecular phylogenies are published each year. For the most part, trees from morphology and molecules have agreed, but there are some notable exceptions, with one being the position of turtles. Classically, the absence of temporal openings in the skull of turtles, the anapsid condition, has been used as evidence to place turtles at the bottom of the amniote tree, after the single-holed (synapsid) mammals split off but before the double-holed (diapsid) reptiles diversified  Figure . Those dThis dispute is similar to other major controversies in amniote evolution, such as the relationships of squamate reptiles   and Afriet al. [The study of Chiari et al.  raises tet al. . These tIn systematics there is usually a trade-off between the number of characters  and taxa , and even these two large molecular studies could be viewed as having too few taxa. The authors of both studies encountered some systematic biases during their analyses, likely attributable to limited taxonomic sampling. This means that there is room for improvement in the future. For example, the earliest-branching lepidosaurs (tuataras) and birds  would be important additions to large molecular data sets, to help stabilize the phylogenies.One molecular study using micro-RNAs, published recently, stands out among all others in concluding that turtles are most closely related to lepidosaurs, thus supporting morphology . At faceOn the morphological side, there is mostly agreement that turtles are diapsids , albeit Fortunately, major conflicts between molecules and morphology, like this one involving turtles, are not common in evolutionary biology. They are usually resolved relatively quickly as new data are collected and analyzed, or independent evidence such as biogeography is brought to bear on the issue. In this case, determining which result is correct will require additional evidence, such as greater taxon-sampling in molecular data sets and greater scrutiny of the morphological and fossil evidence. In the end, having a stable amniote tree that includes turtles and fossil groups will be of tremendous value in understanding the ecological, physiological, and biogeographic history of amniotes and their adaptive radiation on land and in the seas.