Search

Introduction While exposure to sulfur mustard (SM) is commonly associated with the production of vesicating dermal, ocular, and respiratory injuries, systemic damage to bone marrow and lymphatic tissue can decrease critical immune cell populations leading to higher susceptibility to life-threatening infection and septicemia. There are currently no approved medical countermeasures for SM-induced myelosuppression. An intravenous SM challenge model was developed in adult rats as a preliminary proof-of-principle platform to evaluate the efficacy of candidate immunostimulants. Materials and Methods Adult male and female Sprague Dawley rats were exposed to SM through tail vein injection. Toxicity progression was monitored through clinical observations, body weights, body temperatures, hematology, serum clinical chemistry, and flow cytometry of blood and bone marrow samples. Results Following SM exposure, overt toxicity progression was characterized by weight loss, changes in body temperature, and manifestation of toxic clinical signs (diarrhea, lethargy, hunched posture, rough hair coat, respiratory distress, and death). Drastic alterations in complete blood cell profiles included an early-onset lymphopenia followed by a delayed-onset neutropenia and thrombocytopenia. Only transient changes in serum clinical chemistry parameters were observed. Flow cytometry analysis of circulating blood revealed that B-cells were more predominantly affected by SM exposure than T-cells. Challenge with SM resulted in loss of hematopoietic and mesenchymal stem cell populations in the bone marrow. Conclusions The small animal model developed in this study replicates many key aspects of human SM exposures and should serve as a relevant, rapid, and cost-effective platform to screen candidate medical countermeasures for SM-induced hematologic toxicity.

Trichomes are hair-like structures that are essential for abiotic and biotic stress responses. Tomato Hair (H), encoding a C2H2 zinc finger protein, was found to regulate the multicellular trichomes on stems. Here, we characterized Solyc10g078990 (hereafter Hair2, H2), its closest homolog, to examine whether it was involved in trichome development. The H2 gene was highly expressed in the leaves, and its protein contained a single C2H2 domain and was localized to the nucleus. The number and length of type I trichomes on the leaves and stems of knock-out h2 plants were reduced when compared to the wild-type, while overexpression increased their number and length. An auto-activation test with various truncated forms of H2 using yeast two-hybrid (Y2H) suggested that H2 acts as a transcriptional regulator or co-activator and that its N-terminal region is important for auto-activation. Y2H and pull-down analyses showed that H2 interacts with Woolly (Wo), which regulates the development of type I trichomes in tomato. Luciferase complementation imaging assays confirmed that they had direct interactions, implying that H2 and Wo function together to regulate the development of trichomes. These results suggest that H2 has a role in the initiation and elongation of type I trichomes in tomato.

Parkinson’s disease (PD) is characterized pathologically by alpha-synuclein (α-Syn) aggregates and clinically by the motor as well as cognitive deficits, including impairments in sequence learning and habit learning. Using intracerebral injection of WT and A53T mutant α-Syn fibrils, we investigate the behavioral mechanism of α-Syn for procedure-learning deficit in PD by critically determining the α-Syn-induced effects on model-based goal-directed behavior, model-free (probability-based) habit learning, and hierarchically organized sequence learning. 1) Contrary to the widely held view of habit-learning deficit in early PD, α-Syn aggregates in the dorsomedial striatum (DMS) and dorsolateral striatum (DLS) did not affect acquisition of habit learning, but selectively impaired goal-directed behavior with reduced value sensitivity. 2) α-Syn in the DLS (but not DMS) and SNc selectively impaired the sequence learning by affecting sequence initiation with the reduced first-step accuracy. 3) Adenosine A2A receptor (A2AR) antagonist KW6002 selectively improved sequence learning by preferentially improving sequence initiation and shift of sequence learning as well as behavioral reactivity. These findings established a casual role of α-Syn in the SN-DLS pathway in sequence-learning deficit and DMS α-Syn in goal-directed behavior deficit and suggest a novel therapeutic strategy to improve sequence-learning deficit in PD with enhanced sequence initiation by A2AR antagonists.

Here we describe the development and application of miniature integrated microscopes (miniscopes) paired with microendoscopes that allow for the visualization and manipulation of neural circuits in superficial and subcortical brain regions in freely behaving animals. Over the past decade the miniscope platform has expanded to include simultaneous optogenetic capabilities, electrically-tunable lenses that enable multi-plane imaging, color-corrected optics, and an integrated data acquisition platform that streamlines multimodal experiments. Miniscopes have given researchers an unprecedented ability to monitor hundreds to thousands of genetically-defined neurons from weeks to months in both healthy and diseased animal brains. Sophisticated algorithms that take advantage of constrained matrix factorization allow for background estimation and reliable cell identification, greatly improving the reliability and scalability of source extraction for large imaging datasets. Data generated from miniscopes have empowered researchers to investigate the neural circuit underpinnings of a wide array of behaviors that cannot be studied under head-fixed conditions, such as sleep, reward seeking, learning and memory, social behaviors, and feeding. Importantly, the miniscope has broadened our understanding of how neural circuits can go awry in animal models of progressive neurological disorders, such as Parkinson’s disease. Continued miniscope development, including the ability to record from multiple populations of cells simultaneously, along with continued multimodal integration of techniques such as electrophysiology, will allow for deeper understanding into the neural circuits that underlie complex and naturalistic behavior.

Methods based on the evaluation of textural patterns in microscopy, such as the “gray-level co-occurrence matrix” (GLCM) analysis are modern and innovative computer and mathematical algorithms that can be used to quantify subtle structural changes in cells and their organelles. Potential application of GLCM method in the fields of psychophysiology and psychiatry to this date has not been systematically investigated. The main objective of our study was to test the existence and strength of the association between chromatin structural organization of peripheral blood neutrophils and levels of self-perceived mental stress. The research was done on a sample of 100 healthy student athletes, and the Depression, Anxiety, and Stress Scales (DASS-21) were used for the estimation of psychological distress. Chromatin textural homogeneity and uniformity were negatively correlated (p < 0.01) with mental distress and had relatively good discriminatory power in differentiating participants with normal and elevated stress levels. As an addition, we propose the creation of a machine learning model based on binomial logistic regression that uses these and other GLCM features to predict stress elevation. To the best of our knowledge, these results are one of the first to establish the link between neutrophil chromatin structural organization quantified by the GLCM method and indicators of normal psychological functioning.

Aims In recent years, microcalcifications identified in routine mammograms were found to be associated with cardiometabolic disease in women. Here, we aimed to systematically evaluate the association of microcalcifications and other mammographic features with cardiometabolic disease risk and mortality in a large screening cohort and to understand a potential genetic contribution. Methods and results This study included 57 867 women from a prospective mammographic screening cohort in Sweden (KARMA) and 49 583 sisters. Cardiometabolic disease diagnoses and mortality and medication were extracted by linkage to Swedish population registries with virtually no missing data. In the cardiometabolic phenome-wide association study, we found that a higher number of microcalcifications were associated with increased risk for multiple cardiometabolic diseases, particularly in women with pre-existing cardiometabolic diseases. In contrast, dense breasts were associated with a lower incidence of cardiometabolic diseases. Importantly, we observed similar associations in sisters of KARMA women, indicating a potential genetic overlap between mammographic features and cardiometabolic traits. Finally, we observed that the presence of microcalcifications was associated with increased cardiometabolic mortality in women with pre-existing cardiometabolic diseases (hazard ratio and 95% confidence interval: 1.79 [1.24–2.58], P = 0.002) while we did not find such effects in women without cardiometabolic diseases. Conclusions We found that mammographic features are associated with cardiometabolic risk and mortality. Our results strengthen the notion that a combination of mammographic features and other breast cancer risk factors could be a novel and affordable tool to assess cardiometabolic health in women attending mammographic screening.

Context A gluten-free diet (GFD) is the recommended treatment for gluten-dependent disease. In addition, gluten withdrawal is popular and occasionally is suggested as a treatment for other autoimmune diseases (ADs). Objective The current systematic review summarizes those entities and discusses the logic behind using a GFD in classical non–gluten-dependentADs. Data Sources A search for medical articles in PubMed/MEDLINE, Web of Sciences, LILACS, and Scielo published between 1960 and 2020 was conducted, using the key words for various ADs and GFDs. Data Exxtraction Eight-three articles were included in the systematic review (using PRISMA guidelines). Data Analysis Reduction in symptoms of ADs after observance of a GFD was observed in 911 out of 1408 patients (64.7%) and in 66 out of the 83 selected studies (79.5%). The age of the patients ranged from 9 months to 69 years. The duration of the GFD varied from 1 month to 9 years. A GFD can suppress several harmful intraluminal intestinal events. Potential mechanisms and pathways for the action of GFD in the gut – remote organs’ axis have been suggested. Conclusion A GFD might represent a novel nutritional therapeutic strategy for classical non–gluten-dependent autoimmune conditions.

Characterizing the nature of genetic differentiation among individuals and populations and its distribution across the genome is increasingly important to inform both conservation and management of exploited species. Atlantic Halibut (Hippoglossus hippoglossus) is an ecologically and commercially important fish species, yet knowledge of population structure and genomic diversity in this species remains lacking. Here, we use restriction-site associated DNA sequencing and a chromosome-level genome assembly to identify over 86 000 single nucleotide polymorphisms mapped to 24 chromosome-sized scaffolds, genotyped in 734 individuals across the Northwest Atlantic. We describe subtle but significant genome-wide regional structuring between the Gulf of St. Lawrence and adjacent Atlantic continental shelf. However, the majority of genetic divergence is associated with a large putative chromosomal rearrangement (5.74 megabases) displaying high differentiation and linkage disequilibrium, but no evidence of geographic variation. Demographic reconstructions suggest periods of expansion coinciding with glacial retreat, and more recent declines in Ne. This work highlights the utility of genomic data to identify multiple sources of genetic structure and genomic diversity in commercially exploited marine species.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility; however, whether women with PCOS and spontaneous or drug-induced ovulatory cycles have the same reproductive potential as non-PCOS controls is a matter of debate. In the present opinion paper, the author takes the opportunity to summarize the collective evidence supporting the hypothesis of reduced fertility potential in women with PCOS, regardless of ovulatory status, and speculate that reduced reproductive potential may be caused by altered oocytes, embryo and endometrial competence, and infertility-related co-morbidities as well as an increased risk of pregnancy complications.

With the dramatic development of single-cell RNA sequencing (scRNA-seq) technologies, the systematic decoding of cell-cell communication has received great research interest. To date, several in-silico methods have been developed, but most of them lack the ability to predict the communication pathways connecting the insides and outsides of cells. Here, we developed CellCall, a toolkit to infer inter- and intracellular communication pathways by integrating paired ligand-receptor and transcription factor (TF) activity. Moreover, CellCall uses an embedded pathway activity analysis method to identify the significantly activated pathways involved in intercellular crosstalk between certain cell types. Additionally, CellCall offers a rich suite of visualization options (Circos plot, Sankey plot, bubble plot, ridge plot, etc.) to present the analysis results. Case studies on scRNA-seq datasets of human testicular cells and the tumor immune microenvironment demonstrated the reliable and unique functionality of CellCall in intercellular communication analysis and internal TF activity exploration, which were further validated experimentally. Comparative analysis of CellCall and other tools indicated that CellCall was more accurate and offered more functions. In summary, CellCall provides a sophisticated and practical tool allowing researchers to decipher intercellular communication and related internal regulatory signals based on scRNA-seq data. CellCall is freely available at https://github.com/ShellyCoder/cellcall.

When it emerged in late 2019, COVID-19 was carried via travelers to Germany, France and Italy, where freedom of movement accelerated its transmission throughout Europe. However, effective non-pharmaceutical interventions introduced by European governments led to containment of the rapid increase in cases within European nations. Electronic searches were performed to obtain the number of confirmed cases, incident rates and non-pharmaceutical government measures for each European country. The spread and impact of non-pharmaceutical interventions throughout Europe were assessed and visualized. Specifically, heatmaps were used to represent the number of confirmed cases and incident rates for each of the countries over time. In addition, maps were created showing the number of confirmed cases and incident rates in Europe on three different dates (15 March, 15 April and 15 May 2020), which allowed us to assess the geographic and temporal patterns of the disease.

Amphotericin B (AmB) is used to treat cryptococcal meningoencephalitis. However, the mortality rate remains high. Higher doses of AmB in deoxycholate buffer (AmBd) are toxic to human red blood cells (hRBC) and have no effect on brain organism load in mice. Here we show that while AmBd lysed 96% of hRBC, AmB complexed with gold nanoparticles (AuNP-SA-AmB) lysed only 27% of hRBC. In vitro growth of C. neoformans was inhibited by 0.25 μg/ml AmBd and 0.04 μg/ml of AuNP-SA-AmB. In mice infected with C. neoformans, five daily treatments with AuNP-SA-AmB containing 0.25 mg/kg AmB significantly lowered the fungal burden in the brain tissue compared to either untreated or treatment with 0.25 mg/kg of AmBd. When a single dose of AmBd was injected intravenously into BALB/c mice, 81.61% of AmB cleared in the α-phase and 18.39% cleared in the β-phase at a rate of 0.34% per hour. In contrast, when AuNP-SA-AmB was injected, 49.19% of AmB cleared in the α-phase and 50.81% of AmB cleared in the β-phase at a rate of 0.27% per hour. These results suggest that AmB complexed with gold nanoparticles is less toxic to hRBC, is more effective against C. neoformans and persists longer in blood when injected into mice resulting in more effective clearing of C. neoformans from the brain tissue. Lay summary Amphotericin B (AmB) was complexed with gold nanoparticles (AuNP-SA-AmB) to improve brain delivery. AuNP-SA-AmB was more effective than AmB alone in clearing of Cryptococcus neoformans from the brain tissue of infected mice. This may be due to longer plasma half-life of AmB as AuNP-SA-AmB.

Mixed β(1,3;1,4)-linkage glucan (MLG) is commonly found in the monocot lineage, at particularly high levels in the Poaceae family, but also in the evolutionally distant genus, Equisetum. MLG has several properties that make it unique from other plant cell wall polysaccharides. It consists of β1,4-linked polymers of glucose interspersed with β1,3-linkages, but the presence of β1,3-linkages provides quite different physical properties compared to its closest form of the cell wall component, cellulose. The mechanisms of MLG biosynthesis have been investigated to understand whether single or multiple enzymes are required to build mixed linkages in the glucan chain. Currently, MLG synthesis by a single enzyme is supported by mutagenesis analyses of cellulose synthase-like F6, the major MLG synthase, but further investigation is needed to gather mechanistic insights. Because of transient accumulation of MLG in elongating cells and vegetative tissues, several hypotheses have been proposed to explain the role of MLG in the plant cell wall. Studies have been carried out to identify gene expression regulators during development and light cycles as well as enzymes involved in MLG organization in the cell wall. A role of MLG as a storage molecule in grains is evident, but the role of MLG in vegetative tissues is still not well understood. Characterization of a cell wall component is difficult due to the complex heterogeneity of the plant cell wall. However, as detailed in this review, recent exciting research has made significant impacts in the understanding of MLG biology in plants.

To contribute to what is known about involvement of vegetation dynamics in Neotropical speciation, we used the Epidendrum latilabre complex, a taxonomically well-defined species group, to investigate past connections between Amazonian (AM) and Atlantic (AF) forests and address the following topics: (1) divergence times between sister species currently distributed in AM and AF; (2) distribution patterns of ancestral species of the E. latilabre complex and (3) potential routes connecting ancestral ranges between AM and AF. We developed a robust phylogenetic estimate for species of the E. latilabre complex by sequencing two nuclear and six plastid loci. Then, we combined divergence time estimation, ancestral range reconstruction and ecological niche modelling. Our biogeographic reconstruction exhibits a complex pattern of connections among tropical forests east of the Andes in South America. The AM and AF species of the E. latilabre complex are intermixed in the results, and climatic shifts during the Pleistocene (Chibanian) are suggested here as a major force promoting speciation. Sister species tend to be ecologically distinct in their climate niche spaces, and vicariance and peripheral isolation are reconstructed as the main drivers of speciation. There is evidence to suggest that the south-east/north-west bridge and the northern/north-eastern coastal route have been occupied by the ancestors of the E. latilabre complex, and alternative routes across the South American dry diagonal were unlikely. Further studies on Neotropical epiphytic taxa are still necessary for understanding the dynamics of historical connections between AM and AF.

ObjectivesThis systematic review aimed to evaluate the evidence for air and surface contamination of workplace environments with SARS-CoV-2 RNA and the quality of the methods used to identify actions necessary to improve the quality of the data.MethodsWe searched Web of Science and Google Scholar until 24th December 2020 for relevant articles and extracted data on methodology and results.ResultsThe vast majority of data come from healthcare settings, with typically around 6 % of samples having detectable concentrations of SARS-CoV-2 RNA and almost none of the samples collected had viable virus. There were a wide variety of methods used to measure airborne virus, although surface sampling was generally undertaken using nylon flocked swabs. Overall, the quality of the measurements was poor. Only a small number of studies reported the airborne concentration of SARS-CoV-2 virus RNA, mostly just reporting the detectable concentration values without reference to the detection limit. Imputing the geometric mean air concentration assuming the limit of detection was the lowest reported value, suggests typical concentrations in health care settings may be around 0.01 SARS-CoV-2 virus RNA copies/m3. Data on surface virus loading per unit area were mostly unavailable.ConclusionThe reliability of the reported data is uncertain. The methods used for measuring SARS-CoV-2 and other respiratory viruses in work environments should be standardised to facilitate more consistent interpretation of contamination and to help reliably estimate worker exposure.Key messagesWhat is already known about this subject?Low level contamination of air and surfaces in hospitals with SARS-CoV-2 RNA have been reported during the Covid-19 pandemic.Limited data have published from non-healthcare settings.What are the new findings?Typically, around 6% of air and surface samples in hospitals were positive for SARS-COV-2 RNA, although there is very limited data for non-healthcare settings.The quality of the available measurement studies is generally poor, with little consistency in the sampling and analytical methods used.Few studies report the concentration of SARS-CoV-2 in air or as surface loading of virus RNA, and very few studies have reported culture of the virus.The best estimate of typical air concentrations in health care settings is around 0.01 SARS-CoV-2 virus RNA copies/m3How might this impact on policy or clinical practice in the foreseeable future?There should be concerted efforts to standardise the methods used for measuring SARS-CoV-2 and other respiratory viruses in work environments.

Thalidomide causes teratogenic effects in several animal species and in humans. Accordingly, the World Health Organization banned thalidomide when mothers who took thalidomide during pregnancy delivered abnormal fetuses. After four decades, thalidomide underwent drug “re-purposing” since its antiangiogenic and immunomodulatory effects were therapeutic for multiple myeloma. There are no reports of thalidomide’s effects on prokaryotes, but it showed teratogenic effects in Arabidopsis thaliana, an ancestor of the plant kingdom. This proof of concept study clearly shows that thalidomide caused a significant and reproducible decrease in germination rate, nitric oxide (NO) production, and chlorophyll content of fennel plantlets. Thalidomide also induced the formation of abnormal fennel plantlets with stunting, wrinkling, and curling of fennel shoots and leaves. Notably, quantitative analysis showed that thalidomide caused a 50% increase in the formation of abnormal fennel plantlets and that these negative effects of thalidomide showed a 2.50- to 4-fold decrease when fennel seeds were co-incubated with an NO donor (Spermine NoNoate) or a stable cGMP analog 8-bromo Guanosine 3′,5′-cyclic monophosphate (8-Bromo-cGMP). This study is important because it confirms that thalidomide’s negative effects on fennel seed germination and growth are mediated by attenuation of NO and disruption of NO signaling. This reproducible model of thalidomide-induced, NO-dependent damage in a plant system can be used to further investigate the molecular mechanisms of thalidomide action in plants. Importantly, this study establishes a link between the evolution of development of higher plants and mammals.

ITGB1 (Integrin β1, CD29) is a member of the integrin family and has a role as a major adhesion receptor. Gastric cancer (GC) is an important cause of mortality worldwide, especially in China. As a potential cancer enhancer, the role ITGB1 plays in GC progression remains unclear. In the current study, our assay on the databases of tumoassociated gene expression and interaction found that the high expression of ITGB1 was closely correlated with the poor prognosis of GC patients. To explore the roles, ITGB1 plays in GC progression, and an ITGB1-deleted cell line (ITGB1−/−SGC7901) was generated using the CRISPR/Cas9 method. The tumor malignancy-associated cell behaviors and microstructures were detected, imaged, and analyzed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), wound healing, transwell, scanning electron microscopy, laser scanning confocal microscopy, and others. The results indicated that ITGB1 deletion decreased the GC cell proliferation and motility, and inhibited motility-relevant microstructures, such as pseudopodia and filopodia, markedly in ITGB1-deleted SGC7901 cells. The analysis of STRING database and western blots indicated that ITGB1 contributes to the malignancy of GC mediated by Src-mediated FAK/PI3K/Akt signaling pathways. Taken together, the results showed that ITGB1 may be a potential targeting marker for GC diagnosis and therapy in the future.

Background Insecticide resistance has become a widespread problem causing a decline in the effectiveness of vector control tools in sub-Saharan Africa. In this situation, ongoing monitoring of vector susceptibility to insecticides is encouraged by the WHO to guide national malaria control programmes. Our study was conducted from April to November 2018 in Tchonka (Sud-Kivu, Democratic Republic of the Congo) and reported primary data on the resistance status of Anopheles funestus and Anopheles gambiae. Methods Insecticide susceptibility bioassays were performed on wild populations of A. funestus and A. gambiae using WHO insecticide-impregnated papers at discriminating concentration. In addition, PCR was performed to identify mosquito species and to detect kdr and ace-1R mutations involved in insecticide resistance. Results Bioassay results show resistance to all tested insecticides except pirimiphos-methyl, propoxur, fenitrothion and malathion with a mortality rate ranging from 95.48 to 99.86%. The addition of piperonyl butoxide (PBO) increased the susceptibility of vectors to deltamethrin and alpha-cypermethrin by exhibiting a mortality ranging from 91.50 to 95.86%. The kdr mutation was detected at high frequencies (approximately 0.98) within A. gambiae while ace-1R was not detected. Conclusions This study provides useful data on the insecticide resistance profiles of malaria vector populations to better manage vector control. Our results highlight that, despite the high level of resistance, organophosphorus compounds and pyrethroids + PBO remain effective against the vectors.

Cervical cancer (CC) is a type of pelvic malignant tumor that severely threatens women's health. Current evidence suggests that IER5, as a potential radiosensitizer, promotes irradiation-induced apoptosis in CC tissues in patients undergoing chemoradiotherapy. IER5 has been shown to be involved in the G2/M-phase transition. In the present study, we used Cdc25B as the breakthrough point to explore the underlying mechanism of IER5 in the cell cycle regulation of radiation-damaged HeLa cells. IER5 was evidently upregulated after irradiation, but Cdc25B was significantly downregulated. In monoclonal IER5-silenced HeLa cells, irradiation-induced downregulation of Cdc25B was attenuated. The effect of irradiation on Cdc25B promoter activity was determined by dual-luciferase reporter assays. The response elements on the Cdc25B promoter related to irradiation were predicted by JASPAR. These conserved sequences were mutated individually or in combination by splicing-by-overlap extension PCR, and their function was confirmed by dual-luciferase reporter assays. The enrichment efficiency of transcription factors after irradiation was determined by chromatin immunoprecipitation (ChIP) assay. Both Sp1/Sp3 and NF-YB binding sites were involved in irradiation-mediated regulation of Cdc25B. IER5 was involved in irradiation-mediated regulation of Cdc25B through the NF-YB binding site. Furthermore, ChIP assays showed that IER5 bound to the Cdc25B promoter, and the binding of IER5 to the Cdc25B promoter region in irradiation-induced HeLa cells induced the release of the coactivator p300 through interaction with NF-YB. Taken together, these findings indicate that IER5 is the transcriptional repressor that accelerates the downregulation of Cdc25B expression after irradiation.

Cellulose is one of the most abundant biopolymers on Earth. It provides mechanical support to growing plant cells and important raw materials for paper, textiles and biofuel feedstocks. Cellulose biosynthesis inhibitors (CBIs) are invaluable tools for studying cellulose biosynthesis and can be important herbicides for controlling weed growth. Here, we review CBIs with particular focus on the most widely used CBIs and recently discovered CBIs. We discuss the effects of these CBIs on plant growth and development and plant cell biology and summarize what is known about the mode of action of these different CBIs.

STUDY QUESTION What are the differences in ease of use between two different embryo transfer (ET) techniques: the preload direct approach and the afterload approach. SUMMARY ANSWER The afterload technique seems to reduce the rate of difficult ETs. WHAT IS KNOWN ALREADY Numerous published trials now document that the ET procedure has an impact on pregnancy and delivery rates after IVF. Difficult transfers should be avoided, as they reduce implantation and pregnancy rates. Preload direct ETs with soft catheters under ultrasound guidance is currently considered the best procedure. However, when using soft catheters, it is not known which technique is preferable or which one should be implemented to reduce the operator factor. STUDY DESIGN, SIZE, DURATION This prospective randomised unblinded controlled clinical trial, included 352 ultrasound-guided ETs assigned to either direct ET or afterload ET, between September 2017 and March 2019. The sample size was calculated based on the historical rate of difficult ETs encountered between 2014 and 2015 with a direct ET procedure. PARTICIPANTS/MATERIALS, SETTING, METHODS The inclusion criteria were women 18–38 years old, with BMI between 18 and 28, receiving a single-thawed blastocyst transfer. The exclusion criteria were use of testicular sperm and preimplantation genetic testing (PGT) cycles. The primary outcome was the rate of difficult or suboptimal transfers defined as: advancement of the outer sheath (specific for the direct transfer), multiple attempts, use of force, required manipulation, use of a stylet or tenaculum, dilatation, or use of a different catheter. The secondary outcome was clinical pregnancy rate. MAIN RESULTS AND THE ROLE OF CHANCE A total of 352 frozen ETs were randomised, with 176 patients in each group. The two arms were homogeneous for female and male age, female BMI, duration of infertility, secondary infertility, previous deliveries or miscarriages, myomas, previous surgery to the uterine cavity, cycle day at ovulation trigger, freeze all cycles, first transfers, indication for treatment, endometrial preparation protocol and duration, endometrial thickness, and blastocyst grade at vitrification. Across the entire population, 85 (24.1%) ETs were defined as difficult. The rate of difficult transfers was significantly higher in the direct ET group than in the afterload group: 68 (38.6%) versus 17 (9.7%), respectively (OR 0.17, 95% CI 0.09–0.30, P LIMITATIONS, REASONS FOR CAUTION There were 18 experienced operators who participated in the trial. Conclusions about the pregnancy rate should not be generalised, since the sample analysis was not performed on this outcome and, although clinically relevant, the difference was not significantly different. WIDER IMPLICATIONS OF THE FINDINGS The rate of difficult transfers was significantly higher in the direct ET group compared with the afterload ET group, although a wide variation was observed among operators. Further studies regarding the association between transfer technique and ART outcomes are required. STUDY FUNDING/COMPETING INTEREST(S) No specific funding was sought and there are no competing interests. TRIAL REGISTRATION NUMBER NCT03161119. TRIAL REGISTRATION DATE 5 April 2017. DATE OF FIRST PATIENT'S ENROLMENT 26 September 2017.

Woody cells generate lignocellulosic biomass, which is a promising sustainable bioresource for wide industrial applications. Woody cell differentiation in vascular plants, including the model plant poplar (Populus trichocarpa), is regulated by a set of NAC family transcription factors, the VASCULAR-RELATED NAC-DOMAIN (VND), NAC SECONDARY CELL WALL THICKENING PROMOTING FACTOR (NST)/SND, and SOMBRERO (SMB) (VNS)-related proteins, but the precise contributions of each VNS protein to wood quality are unknown. Here, we performed a detailed functional analysis of the poplar SMB-type VNS proteins PtVNS13–PtVNS16. PtVNS13–PtVNS16 were preferentially expressed in the roots of young poplar plantlets, similar to the Arabidopsis thalianaSMB gene. PtVNS13 and PtVNS14, as well as the NST-type PtVNS11, suppressed the abnormal root cap phenotype of the Arabidopsis sombrero-3 mutant, whereas the VND-type PtVNS07 gene did not, suggesting a functional gap between SMB- or NST-type VNS proteins and VND-type VNS proteins. Overexpressing PtVNS13–PtVNS16 in Arabidopsis seedlings and poplar leaves induced ectopic xylem-vessel-like cells with secondary wall deposition, and a transient expression assay showed that PtVNS13–16 transactivated woody-cell-related genes. Interestingly, although any VNS protein rescued the pendant stem phenotype of the Arabidopsis nst1-1 nst3-1 mutant, the resulting inflorescence stems exhibited distinct cell wall properties: poplar VNS genes generated woody cell walls with higher enzymatic saccharification efficiencies compared with Arabidopsis VNS genes. Together, our data reveal clear functional diversity among VNS proteins in woody cell differentiation and demonstrate a novel VNS-based strategy for modifying woody cell wall properties toward enhanced utilization of woody biomass.

Background Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). Methods In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. Results All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. Conclusions In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

This article comments on:Krizek BA, Bantle AT, Heflin JM, Han H, Freese NH, Loraine AE. 2021. AINTEGUMENTA and AINTEGUMENTA-LIKE6 directly regulate floral homeotic, growth, and vascular development genes in young Arabidopsis flowers. Journal of Experimental Botany 72, 5478–5493.

Background Multiple tissue reservoirs are established soon after HIV infection, and some tissues may also be pharmacological sanctuaries. Parenteral administration of antiretroviral (ARV) drugs for treatment and prevention of HIV infection is an active area of drug development. The influence of route of administration on ARV tissue pharmacokinetics is not known. Objectives To investigate ARV pharmacokinetics in lymphatic and select non-lymphatic tissues (e.g. brain and testes) after intramuscular and subcutaneous administration compared with oral in BALB/c mice. Methods Tissue concentrations of cobicistat, efavirenz, elvitegravir, maraviroc, rilpivirine, tenofovir alafenamide and tenofovir disoproxil fumarate were determined. The tissue penetration ratio (TPR) was the primary measure for comparison; a change in TPR arises from factors affecting tissue distribution controlling for changes in systemic bioavailability. Results Intramuscular and subcutaneous delivery increased TPRs in the lymph node and spleen for 27 of 28 (96%) drug administration events. Decreased TPRs, however, were found in some tissues such as the brain and testes. Conclusions These results demonstrate a change in route of drug administration from oral to intramuscular or subcutaneous can change tissue uptake. This has implications for HIV pharmacotherapy. For example, HIV persists in lymphoid tissues despite long-term oral ARV therapy, and low ARV concentrations have been found in lymphoid tissues. The improved ARV lymphatic tissue bioavailability with intramuscular and subcutaneous administration allows future studies to investigate these routes of drug administration as a therapeutic manoeuvre to limit viral persistence and eliminate viral sanctuaries in the lymphatic tissues, which is a prerequisite for eradication of HIV.

The methylation of cytosine residues that precede adenine/thymine or other cytosine nucleotides instead of guanine in DNA is known as non-CpG methylation. It is a pronounced epigenetic modification with a central role in gene regulation similar to CpG methylation. Due to technological limitations, the locus-specific role of non-CpG methylation was scarcely understood. At present, high-throughput analyses and improved enrichment methods can elucidate the role of genome-wide non-CpG methylation distributions. Although the functional basis of non-CpG methylation in regulating gene expression control is known, its role in cancer development is yet to be ascertained. This review sheds light on the possible mechanism of non-CpG methylation in embryos and developed tissues with a special focus on cancer development and progression. In particular, the maintenance and alteration of non-CpG methylation levels and the crucial factors that determine this level of non-CpG methylation and its functional role in cancer are discussed.

The subfamily Potamotrygoninae, the only extant clade of elasmobranchs exclusive to freshwater environments, encompasses four genera and 38 species distributed across almost every major South American river basin. Despite their importance in the ornamental fish trade, the taxonomy and evolutionary relationships within potamotrygonines have not yet been resolved. Here, we present a comprehensive molecular phylogeny for the Neotropical freshwater stingrays, based on extensive species and population sampling (35 species and > 350 individuals from drainages across South America). Our phylogeny corroborates the monophyly of the genera Paratrygon and Heliotrygon and the monophyly of the Potamotrygon + Plesiotrygon clade. Within the Potamotrygon + Plesiotrygon clade, we identify a core Potamotrygon clade characterized by short branches, low nodal support and incongruence with current species-level taxonomy. In the core Potamotrygon clade, specimens of widespread species, such as Potamotrygon motoro and Potamotrygon orbignyi, do not form monophyletic lineages; instead, specimens from these species are often closely related to those of other species from the same river basins. These patterns could be caused by inaccurate taxonomy, hybridization, incomplete lineage sorting and rapid diversification. We discuss the conservation of Neotropical freshwater stingrays from a phylogenetic perspective and suggest ways to prioritize potamotrygonid conservation efforts with respect to endemism and evolutionary distinctiveness.

Objectives Acute lung injury (ALI) is a pulmonary manifestation of an acute systemic inflammatory response, which is associated with high morbidity and mortality. Accordingly, from the perspective of treating ALI, it is important to identify effective agents and elucidate the underlying modulatory mechanisms. β-Caryophyllene (BCP) is a naturally occurring bicyclic sesquiterpene that has anti-cancer and anti-inflammatory activities. However, the effects of BCP on ALI have yet to be ascertained. Methods ALI was induced intratracheally, injected with 5 mg/kg LPS and treated with BCP. The bone marrow-derived macrophages (BMDMs) were obtained and cultured then challenged with 100 ng/ml LPS for 4 h, with or without BCP pre-treatment for 30 min. Key findings BCP significantly ameliorates LPS-induced mouse ALI, which is related to an alleviation of neutrophil infiltration and reduction in cytokine production. In vitro, BCP was found to reduce the expression of interleukin-6, interleukin-1β and tumour necrosis factor-α, and suppresses the MAPK signalling pathway in BMDMs, which is associated with the inhibition of TAK1 phosphorylation and an enhancement of MKP-1 expression. Conclusions Our data indicate that BCP protects against inflammatory responses and is a potential therapeutic agent for the treatment of LPS-induced acute lung injury.

Objectives Epithelial–mesenchymal transition (EMT) plays a role in cancer metastasis as well as in drug resistance through various mechanisms, including increased drug efflux mediated by P-glycoprotein (P-gp). In this study, we investigated the activation mechanism of P-gp, including its regulatory factors, during EMT in hepatoblastoma-derived HepG2 cells. Methods HepG2 cells were transfected with SNAI1 using human adenovirus serotype 5 vector. We quantified mRNA and protein expression levels using qRT-PCR and western blot analysis, respectively. P-gp activity was evaluated by uptake assay, and cell viability was assessed by an MTT assay. Key findings P-gp protein expression on plasma membrane was higher in SNAI1-transfected cells than in Mock cells, although there was no difference in P-gp protein level in whole cells. Among the scaffold proteins such as ezrin, radixin and moesin (ERM), only radixin was increased in SNAI1-transfected cells. Uptake of both Rho123 and paclitaxel was decreased in SNAI1-transfected cells, and this decrease was blocked by verapamil, a P-gp inhibitor. The reduced susceptibility of SNAI1-transfected cells to paclitaxel was reversed by elacridar, another P-gp inhibitor. Conclusions Increased expression of radixin during SNAI1-induced EMT leads to increased P-gp membrane expression in HepG2 cells, enhancing P-gp function and thereby increasing drug resistance.

Mammals typically process food items more extensively in their oral cavities than do other vertebrates. Dental morphology, jaw-muscle activity patterns, mandibular movements, and tongue manipulation work to facilitate oral fragmentation of dietary items. While processing mechanically challenging foods, mammals modulate mandibular movements and bite forces via recruitment of greater jaw-adductor muscle forces and protracted biting or chewing. Because jaw-loading patterns are influenced by magnitude; frequency; and duration of muscular, bite, and reaction forces during routine feeding behaviors, relatively larger jaws are thought to be more characteristic of mammals that experience higher masticatory loads due to the processing of mechanically challenging foods. The ease of food fracture during post-canine biting and chewing is mainly determined by food stiffness and toughness. Such foods have been associated with increased loading magnitude and/or greater amounts of cyclical loading (i.e., chewing duration). Dietary properties are thought to modulate cyclical loading through changes in chewing frequency and chewing investment. On the other hand, chewing frequency has been found to be independent of dietary properties in rabbits and primates; however, little evidence exists regarding the influence of dietary properties on these parameters in a broader range of mammals. Here, we assessed chewing behavior in seven adult llamas (Llama glama) processing foods with a wide range of mechanical properties (grain, hay, carrots, and dried corn). Each subject was filmed at 60 frames/s, with video slowed for frame-by-frame computer analysis to obtain length of feeding bout and number of chewing cycles for each food type. These parameters were used to calculate chewing frequency (chews/s), chewing investment (chews/g), and chewing duration (s/g). Chewing frequency was not significantly related to mechanical properties of food, but chewing investment and chewing duration were significantly related to dietary stiffness and toughness. Therefore, cyclical loading is positively influenced by stiff and tough foods. This suggests that variation in jaw morphology in extinct and extant mammals is positively related to dietary stiffness and toughness, which requires greater chewing investment and increased chewing duration.

Objectives Tonkinensine B, a novel compound with cytisine–pterocarpan skeleton isolated from the root of Sophora tonkinensis Gagnep, was reported to have a significant antitumor effect. The effect and intrinsic mechanism of tonkinensine B on tumour need to be further investigated. Methods With the help of cell cytotoxicity, the effect of tonkinensine B on MDA-MB-231 cells was investigated. By observing mitochondrial function changes, the intrinsic mechanism was further studied. The levels of key apoptosis-associated proteins Bcl-2, Bax, caspase-9, caspase-3 and AKT in MDA-MB-231 cells were analysed to determine whether tonkinensine B caused apoptosis via the mitochondrial pathway. Key findings After treated with tonkinensine B, MDA-MB-231 cells multiplication was repressed, and the decreased mitochondrial membrane potential, loss of ATP synthesis and elevated ROS generation were detected. Furthermore, the proportions of Bax/Bcl-2, cleaved caspase-3 and caspase-9 proteins production were up-regulated, indicating that tonkinensine B acted on intrinsic mitochondrial-mediated apoptosis pathway. In addition, tonkinensine B also reduced phosphorylation levels of AKT, and thus the activation of apoptosis might likewise be correlated with the inhibition of the PI3K/AKT pathway. Conclusions Tonkinensine B may be a hopeful candidate for human triple-negative breast cancer, and further structural optimization is expected to improve its anti-tumour activity.

Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies.

Malaria and leishmaniasis are the public health problems in southern Iran. The main activity of vector control is indoor residual spraying using pyrethroids, using different insecticides as larviciding and impregnated bednets. The aim of study was to evaluate the biological assays of bendiocarb wettable powder (WP) at different surfaces of wall. The residual effect of bendiocarb WP80 at 400 milligram/meter square (mg/m2) was evaluated on various local surfaces of rooms such as mud and plaster as well as thatch roofs and wooden. World Health Organization standard cones using contact bioassays were carried out using laboratory reared sugar-fed, 48–72 h old females of Anopheles stephensi. Contact bioassays were carried out on sprayed surfaces for 150 days. Contact bioassay on surfaces treated with bendiocarb WP80 at different surfaces was estimated about 2 months. Fumigant tests of bendiocarb WP80 at 400 mg/m2 revealed 50–93.83% mortality with 1 month persistency. The results showed that carbamate insecticide could be used as rotation with pyrethroids for malaria vector control. Monitoring and evaluation of environmental toxicology of pesticides is important for decision making for choosing appropriate pesticides for disease vector control.

Objectives To investigate the prevalence of high-risk human papillomavirus (HPV)–negative cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma (ICC) and to analyze the distribution of other genotypes in this subset. Methods In total, 431 women who underwent excisional surgical treatment for CIN or ICC at the European Institute of Oncology, Milan, Italy, from January 2016 to December 2017 were retrospectively analyzed. The Linear Array HPV genotyping test (Roche Diagnostics) was performed on a postaliquot from high-risk-HPV–negative liquid-based cervical specimens, when available. Patient characteristics and the prevalence of high-risk-HPV–negative CIN grade 2 or worse (CIN2+) were tabulated. We used t tests to compare age between high-risk-HPV–positive and high-risk-HPV–negative patients. Results Overall, 8.9% of CIN2+ and 7.5% of ICC cases were high-risk HPV negative. There was no age difference between high-risk-HPV–negative CIN2+ women (mean [SD], 41.3 [8.7] years) and high-risk-HPV–positive women (mean [SD], 39.5 [9.0] years) (P = .28). The Linear Array result was available in 22 cases. Most high-risk-HPV–negative patients were positive for a single other genotype infection (32.6%). HPV 73 was the most prevalent genotype, followed by HPV 53 and HPV 84. HPV 26 was detected in 1 case of ICC. Conclusions Our results showed a not-negligible proportion of high-risk-HPV–negative CIN2+, suggesting that cotesting would not miss these cases.

Target enrichment (such as Hyb-Seq) is a well-established high throughput sequencing method that has been increasingly used for phylogenomic studies. Unfortunately, current widely used pipelines for analysis of target enrichment data do not have a vigorous procedure to remove paralogs in target enrichment data. In this study, we develop a pipeline we call Putative Paralogs Detection (PPD) to better address putative paralogs from enrichment data. The new pipeline is an add-on to the existing HybPiper pipeline, and the entire pipeline applies criteria in both sequence similarity and heterozygous sites at each locus in the identification of paralogs. Users may adjust the thresholds of sequence identity and heterozygous sites to identify and remove paralogs according to the level of phylogenetic divergence of their group of interest. The new pipeline also removes highly polymorphic sites attributed to errors in sequence assembly and gappy regions in the alignment. We demonstrated the value of the new pipeline using empirical data generated from Hyb-Seq and the Angiosperms353 kit for two woody genera Castanea (Fagaceae, Fagales) and Hamamelis (Hamamelidaceae, Saxifragales). Comparisons of data sets showed that the PPD identified many more putative paralogs than the popular method HybPiper. Comparisons of tree topologies and divergence times showed evident differences between data from HybPiper and data from our new PPD pipeline. We further evaluated the accuracy and error rates of PPD by BLAST mapping of putative paralogous and orthologous sequences to a reference genome sequence of Castanea mollissima. Compared to HybPiper alone, PPD identified substantially more paralogous gene sequences that mapped to multiple regions of the reference genome (31 genes for PPD compared with 4 genes for HybPiper alone). In conjunction with HybPiper, paralogous genes identified by both pipelines can be removed resulting in the construction of more robust orthologous gene data sets for phylogenomic and divergence time analyses. Our study demonstrates the value of Hyb-Seq with data derived from the Angiosperms353 probe set for elucidating species relationships within a genus, and argues for the importance of additional steps to filter paralogous genes and poorly aligned regions (e.g., as occur through assembly errors), such as our new PPD pipeline described in this study. [Angiosperms353; Castanea; divergence time; Hamamelis; Hyb-Seq, paralogs, phylogenomics.]

The mammalian cerebral cortex is characterized by a 6-layer structure, and proper neuronal migration is critical for its formation. Cyclin-dependent kinase 5 (Cdk5) has been shown to be a critical kinase for neuronal migration. Several Cdk5 substrates have been suggested to be involved in ordered neuronal migration. However, in vivo loss-of-function studies on the function of Cdk5 phosphorylation substrates in neuronal migration in the developing cerebral cortex have not been reported. In this study, we demonstrated that Cdk5-mediated phosphorylation of collapsing mediator protein (CRMP) 2 is critical for neuronal migration in the developing cerebral cortex with redundant functions of CRMP1 and CRMP4. The cerebral cortices of triple-mutant CRMP1 knock-out (KO); CRMP2 knock-in (KI)/KI; and CRMP4 KO mice showed disturbed positioning of layers II–V neurons in the cerebral cortex. Further experiments using bromodeoxyuridine birthdate-labeling and in utero electroporation implicated radial migration defects in cortical neurons. Ectopic neurons were detected around the CA1 region and dentate gyrus in CRMP1 KO; CRMP2 KI/KI; and CRMP4 KO mice. These results suggest the importance of CRMP2 phosphorylation by Cdk5 and redundancy of CRMP1 and CRMP4 in proper neuronal migration in the developing cerebral cortex and hippocampus.

The staining procedure is critical for investigating intra- and extra-cellular ultrastructure of microorganisms by transmission electron microscopy (TEM). Here, we propose a new ultra-low lead staining (ULLS) technique for preparing the ultrathin sections for TEM analysis. Sections of Enterobacter sp. (bacteria), Aspergillus niger (filamentous fungi), Rhodotorula mucilaginosa (fungi), and Chlamydomonas reinhardtii (microalgae) were tested. Compared with the sections prepared by the typical double-staining technique, ULLS-based sections showed evident advantages: (i) the staining process only required the addition of Pb(NO3)2; (ii) the Pb level during incubation was set as low as 1 mg/L, which had negligible toxicity to most microbial cells; (iii) the Pb cations were added during microbial culture, which avoided complicated sample preparation as in typical double staining. Taking C. reinhardtii as an example, the ULLS technique allowed fine investigation of microbial ultrastructure, e.g., starch granule, mitochondrion, Golgi apparatus, vacuole, and vesicle. Meanwhile, the physiological processes of the cells such as cell lysis and exocytosis were successfully captured, with relatively high contrast. This study hence shows a bright future on preparation of the high-quality ultrathin sections of microbial cells by the ULLS technique.

Background Antibiotic tolerance allows bacteria to overcome antibiotic treatment transiently and potentially accelerates the emergence of resistance. However, our understanding of antibiotic tolerance at the genetic level during adaptive evolution of Staphylococcus aureus remains incomplete. We sought to identify the mutated genes and verify the role of these genes in the formation of vancomycin tolerance in S. aureus. Methods Vancomycin-susceptible S. aureus strain Newman was used to induce vancomycin-tolerant isolates in vitro by cyclic exposure under a high concentration of vancomycin (20× MIC). WGS and Sanger sequencing were performed to identify the genetic mutations. The function of mutated genes in vancomycin-tolerant isolates were verified by gene complementation. Other phenotypes of vancomycin-tolerant isolates were also determined, including mutation frequency, autolysis, lysostaphin susceptibility, cell wall thickness and cross-tolerance. Results A series of vancomycin-tolerant S. aureus (VTSA) strains were isolated and 18 mutated genes were identified by WGS. Among these genes, pbp4, htrA, stp1, pth and NWMN_1068 were confirmed to play roles in VTSA formation. Mutation of mutL promoted the emergence of VTSA. All VTSA showed no changes in growth phenotype. Instead, they exhibited reduced autolysis, decreased lysostaphin susceptibility and thickened cell walls. In addition, all VTSA strains were cross-tolerant to antibiotics targeting cell wall synthesis but not to quinolones and lipopeptides. Conclusions Our results demonstrate that genetic mutations are responsible for emergence of phenotypic tolerance and formation of vancomycin tolerance may lie in cell wall changes in S. aureus.

Background Randomized trials showed the survival benefits of the combined use of androgen receptor axis-targeted agents with androgen deprivation therapy in metastatic hormone-sensitive prostate cancer (mHSPC), regardless of the risk. However, treating patients with low-risk mHSPC with such intensive treatment is still debatable. Methods This retrospective study included 155 low-risk patients among 467 mHSPC patients treated in our affiliated institutions. The association between predictive factors and treatment outcomes was estimated using the Kaplan–Meier method and log-rank test. Predictive factors for castration resistant prostate cancer (CRPC)-free survival were investigated using Cox regression analyses. Results During the median follow-up of 39 months, 38.7% of patients developed CRPC and 14.2% died. In the multivariate analyses, a presence of Gleason pattern 5 (hazard ratio [HR] 2.04), high alkaline phosphatase (HR 1.007) and high lactate dehydrogenase (HR 1.009) were significant predictive factors for shorter CRPC-free survival. Finally, 155 patients were stratified into favorable- and unfavorable-risk groups based on the numbers of the predictive factors. The overall survival (OS) in the unfavorable-risk group (total scores: 2–3) was significantly worse than that of the favorable-risk group (total score: 0–1) (P = 0.02). This prognostic model was assessed with 50 low-risk mHSPC patients from the external validation dataset and found both the time to CRPC, and the OS in the unfavorable-risk group was significantly worse than that of the favorable-risk group (P Conclusions The combination of Gleason pattern 5, high alkaline phosphatase and lactate dehydrogenase can predict those with worse OS in low-risk mHSPC patients.

During buzz pollination, bees use vibrations to remove pollen from flowers. Vibrations at the natural frequency of pollen-carrying stamens are amplified through resonance, resulting in higher amplitude vibrations. Because pollen release depends on vibration amplitude, bees could increase pollen removal by vibrating at the natural frequency of stamens. However, few studies have characterized the natural frequencies of stamens and compared them to the frequencies of buzz-pollinating bees. We use laser Doppler vibrometry to characterize natural frequencies of stamens of six morphologically diverse, buzz-pollinated, heterantherous Solanum taxa and compare the frequency of bumblebee buzzes produced on two Solanum spp. with different natural frequencies. We found that stamen morphology and plant identity explain variation in their natural frequency. The natural frequencies of the stamens in the studied Solanum taxa fell between 45 and 295 Hz; in five out of six taxa the frequencies were < 190 Hz, which only partly overlaps floral vibrations of buzz-pollinating bees. We show that captive bumblebees produce vibrations at a frequency of 345 Hz and do not change their floral vibrations to match the natural frequency of the visited flowers. Our results suggest that pollen release induced by vibrating stamens at their natural frequencies might only play a role in a subset of buzz pollination interactions.

Endosymbiotic bacteria can induce parthenogenesis in many haplo-diploid species, but little is known about their role in asexual reproduction in diplodiploids. Bacteria are essential for reproduction in the asexual lineages of springtails and booklice, and possibly some weevils, but whether they are the causative agent of parthenogenesis remains to be proven. This would require comparing unisexual forms and their closely related bisexual species, but such model species are very rare. Empoasca leafhoppers from the Madeira Archipelago provide an excellent case to test whether bacterial infections can cause parthenogenesis. Here we examined the diversity of the sex-distorting bacteria, Wolbachia, Cardinium, Rickettsia, and Arsenophonus, in three apomictic morphotypes (A, B, C) and three bisexual relatives of Empoasca. Wolbachia of group B was present in all unisexual and bisexual species. Rickettsia were the only bacteria restricted to the three unisexual forms, suggesting an association between infection and asexual reproduction. In addition, we found Asaia for the third time in the Cicadellidae, although this may have little to do with the reproductive mode of its host. Adult females of morphotype C reared on plants watered with tetracycline solution did not result in the production of male offspring. To our knowledge, this is the first study to suggest a possible association between bacterial infection and parthenogenesis in apomictic XO/XX diplodiploid bisexual species based on a range of sex ratio-distorting bacteria.

The causes and consequences of interspecific variation in sex-specific contributions to animal parental care are relatively well understood during pregnancy or incubation and during offspring provisioning, but comparative patterns of sex-biased investment during nest-, den-, or other shelter-building have been almost completely overlooked. This is surprising because birthing shelters’ protective properties have important fitness consequences for both parents and offspring. Here, we address this gap in our knowledge by testing predictions concerning sex-specific contributions to avian nest building in more than 500 species of Western Palearctic birds in relation to the time available to breed and sex-specific reproductive effort, while also examining correlates with nesting site and nest structure. Using multivariate phylogenetic comparative and path analysis approaches, we found that, opposite to what had been predicted, species in which females build nests alone have shorter breeding seasons and breed at higher latitudes. In addition, species in which females lay larger clutch sizes and incubate eggs alone are more likely to have nests built by females alone, again countering predictions that reproductive contributions are not traded-off between the sexes. Finally, however, sex-specific nest building contributions were predictably related to nest site and structure, as species in which females built nests alone were more likely to have open cup nests relative to enclosed, domed nests of species in which both parents build. Our study provides important new insights, and generates several new questions for experimental research into the adaptive dynamics of sex-specific contributions prior or at the onset of parental care.

Some leafmining fly species are pests of agricultural and ornamental plants but they also include many species with little economic importance. The taxonomy of leafmining flies is often challenging because of putative cryptic species. Following new outbreaks of Liriomyza (Diptera:Agromyzidae) in Australia, we here characterize 13 dipteran leafminer species collected from Australia based on cytochrome c oxidase subunit 1 (COI) DNA barcodes, and we compare these with overseas data. We confirm barcodes of nine species from the Agromyzidae (Liriomyza sativae, L. huidobrensis, L. trifolii, L. bryoniae, L. chinensis, L. brassicae, L. chenopodii, Phytomyza plantaginis and P. syngenesiae) and one species from the Drosophilidae (Scaptomyza flava); we describe new haplotypes for some of these species. Furthermore, we provide the first DNA barcodes for two species (Cerodontha milleri and Phytoliriomyza praecellens) from the Agromyzidae and one species (Scaptomyza australis) from the Drosophilidae. These findings provide a baseline for DNA-based identification of pest Liriomyza incursions spreading across the Australian east coast in relation to other species already present in Australia.

Black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), larvae are suspected to inhibit house flies, Musca domestica (L.) (Diptera: Muscidae), seeking oviposition sites. However, the mechanisms regulating these interactions are not well known. In this study, a Y-tube olfactometer was used to determine whether black soldier fly eggs, larvae, puparia (without pupae inside), adult carcasses, or resulting frass of black soldier fly decrease attractiveness of adult house flies to an oviposition substrate (i.e., sugar and wheat bran diet, used as the control). Secondly, two symbiotic bacteria associated were isolated, identified, and tested for house fly attractiveness with a Y-tube olfactometer. Thirdly, volatile organic compounds produced by swine feed before and after black soldier fly, or house fly larval digestion were analyzed with gas chromatography–mass spectrometry (GC–MS). Results indicate feed mixed with black soldier fly larval frass, and symbiotic bacteria, Paenalcaligenes sp. associated with black soldier fly, were less attractive to house flies than the control. However, another symbiotic bacterium Lysinibacillus sp. associated with black soldier fly pupae was attractive. In total, 52 volatile organic compounds were identified from treatments previously described. Quantitative differences in volatile profiles across treatments potentially explain the mechanism regulating house fly substrate choices in the presence, or absence, of black soldier fly larvae.

Taxa are frequently labeled incertae sedis when their placement is debated at ranks above the species level, such as their subgeneric, generic, or subtribal placement. This is a pervasive problem in groups with complex systematics due to difficulties in identifying suitable synapomorphies. In this study, we propose combining DNA barcodes with a multilocus backbone phylogeny in order to assign taxa to genus or other higher-level categories. This sampling strategy generates molecular matrices containing large amounts of missing data that are not distributed randomly: barcodes are sampled for all representatives, and additional markers are sampled only for a small percentage. We investigate the effects of the degree and randomness of missing data on phylogenetic accuracy using simulations for up to 100 markers in 1000-tips trees, as well as a real case: the subtribe Polyommatina (Lepidoptera: Lycaenidae), a large group including numerous species with unresolved taxonomy. Our simulation tests show that when a strategic and representative selection of species for higher-level categories has been made for multigene sequencing (approximately one per simulated genus), the addition of this multigene backbone DNA data for as few as 5¿10% of the specimens in the total data set can produce high-quality phylogenies, comparable to those resulting from 100% multigene sampling. In contrast, trees based exclusively on barcodes performed poorly. This approach was applied to a 1365-specimen data set of Polyommatina (including ca. 80% of described species), with nearly 8% of representative species included in the multigene backbone and the remaining 92% included only by mitochondrial COI barcodes, a phylogeny was generated that highlighted potential misplacements, unrecognized major clades, and placement for incertae sedis taxa. We use this information to make systematic rearrangements within Polyommatina, and to describe two new genera. Finally, we propose a systematic workflow to assess higher-level taxonomy in hyperdiverse groups. This research identifies an additional, enhanced value of DNA barcodes for improvements in higher-level systematics using large data sets., This work was funded by projects PID2019-107078GB-I00/AEI/10.13039/501100011033 and 2017-SGR-991 (Generalitat de Catalunya) to R.V. and G.T., by the Committee for Research and Exploration of the National Geographic Society [grant WW1300R18 to G.T.], by the Putnam Expeditionary Fund of the Museum of Comparative Zoology (to all authors), by the U.S. National Science Foundation [DEB-0447244, and DEB-1541560 to N.E.P], and by the Ramón y Cajal programme of the Spanish Ministry of Science and Innovation [RYC2018-025335-I to G.T.]. Taxonomic studies and descriptions of new genera and subgenera were supported by the Russian Science Foundation [19-14-00202] to the Zoological Institute of the Russian Academy of Sciences to V.L., Materials and MethodsResultsDiscussionConclusionsSupplementary MaterialAcknowledgmentsFundingReferences

Aims Smokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms. Methods and results We assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed tomography (µCT) scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; P Conclusion In this study, we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.

MicroRNAs (miRNAs) are small non-coding RNAs that play key roles in tumorigenesis as modulators of cell signaling pathways. miRNA expression has been found to be dysregulated in several human and canine tumors, but data are not yet available on canine meningioma. In this study, we analyzed the expression of 12 miRNAs (i.e. miR-335, miR-200a, miR-98, miR-96, miR-190a, miR-29c, miR-219-5p, miR-155, miR-146a, miR-145, miR-136, miR-451) by RT-qPCR in a series of 41 formalin-fixed, paraffin-embedded canine meningiomas, and normal arachnoid samples. We identified 8 dysregulated miRNAs that might be involved in canine meningioma pathogenesis. Five miRNAs (i.e. miR-96, miR-145, miR-335, miR-200a, miR-29c), were downregulated in tumor samples and 3 (i.e. miR-136, miR-155, miR-146a) were upregulated. Moreover, miR-200a was overexpressed in grade III compared to grade I and grade II meningiomas, suggesting that it might have a dual role in tumor initiation and progression. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses suggest that dysregulated miRNAs might influence cellular processes and pathways mainly involved in tumor cell migration, extracellular matrix interactions, cell proliferation, and inflammatory responses. The characterization of miRNA functions in canine meningiomas is needed to assess their potential clinical utility, also in view of the relevance of the dog as a potential spontaneous animal model of human disease.

Aims Heart rate (HR) is a critical indicator of cardiac performance that is determined by sinoatrial node (SAN) function and regulation. Natriuretic peptides, including C-type NP (CNP), have been shown to modulate ion channel function in the SAN when applied exogenously. CNP is the only NP that acts as a ligand for natriuretic peptide receptor-B (NPR-B). Despite these properties, the ability of CNP and NPR-B to regulate HR and intrinsic SAN automaticity in vivo, and the mechanisms by which it does so, are incompletely understood. Thus, the objective of this study was to determine the role of NPR-B signalling in regulating HR and SAN function. Methods and results We have used NPR-B deficient mice (NPR-B+/−) to study HR regulation and SAN function using telemetry in conscious mice, intracardiac electrophysiology in anaesthetized mice, high-resolution optical mapping in isolated SAN preparations, patch-clamping in isolated SAN myocytes, and molecular biology in isolated SAN tissue. These studies demonstrate that NPR-B+/− mice exhibit slow HR, increased corrected SAN recovery time, and slowed SAN conduction. Spontaneous AP firing frequency in isolated SAN myocytes was impaired in NPR-B+/− mice due to reductions in the hyperpolarization activated current (If) and L-type Ca2+ current (ICa,L). If and ICa,L were reduced due to lower cGMP levels and increased hydrolysis of cAMP by phosphodiesterase 3 (PDE3) in the SAN. Inhibiting PDE3 or restoring cGMP signalling via application of 8-Br-cGMP abolished the reductions in cAMP, AP firing, If, and ICa,L, and normalized SAN conduction, in the SAN in NPR-B+/− mice. NPR-B+/− mice did not exhibit changes in SAN fibrosis and showed no evidence of cardiac hypertrophy or changes in ventricular function. Conclusions NPR-B plays an essential physiological role in maintaining normal HR and SAN function by modulating ion channel function in SAN myocytes via a cGMP/PDE3/cAMP signalling mechanism.

Runcinida is a small heterobranch order of sea slugs with 61 known species distributed worldwide across temperate and tropical latitudes with two established families (Ilbiidae and Runcinidae). Little is known about the phylogenetic relationships within Runcinida. Here, we present the first molecular phylogeny of the order with an emphasis on European species and we discuss the taxonomic status of the type species Runcina coronata. Molecular phylogenetics based on the mitochondrial genes cytochrome c oxidase subunit I and 16S rRNA and nuclear gene histone H3 is used to investigate relationships between species. Detailed morpho-anatomical worked was additionally employed to study Runcina coronata. Our results suggest the monophyly of Runcinida and showed that Runcina coronata is a complex of four species, namely: R. coronata proper, R. aurata and two new species here formally described (R. caletensis sp. nov. and R. tingensis sp. nov.).

STUDY QUESTION Is it possible to establish a new in-vitro activation (IVA) protocol for infertility treatment? SUMMARY ANSWER A new IVA procedure is an efficient and easily performed approach for infertility treatment of patients with diminished ovarian reserve (DOR). WHAT IS KNOWN ALREADY IVA of primordial follicles with or without stimulators has been developed to treat patients with primary ovarian insufficiency (POI) successfully. However, the efficiency of the procedure is still very low. There is a requirement to optimize the protocol with increased efficiency for clinical application. STUDY DESIGN, SIZE, DURATION Newborn mouse ovaries were used to establish a new 1-h IVA protocol with the mechanistic target of rapamycin (mTOR) stimulator phosphatidic acid (PA, 200 µM) and the phosphatidylinositol-3-kinase (PI3K) stimulator 740Y-P (250 µg/ml); a prospective observational cohort study in POI patients was performed on 15 POI patients and 3 poor ovarian response (POR) patients in three different centers of reproductive medicine in China. PARTICIPANTS/MATERIALS, SETTING, METHODS One-third of ovarian cortex was removed and processed into bigger strips (1 × 1 cm2, 1–2 mm thickness). Strips were then sutured back after treatment. The new approach only requires one laparoscopic surgery. MAIN RESULTS AND THE ROLE OF CHANCE Follicular activation and development increased in cultured mouse and human ovarian tissues after 1 h of stimulator treatment. Compared with tiny ovarian cortex pieces (1 × 1 mm2), large ovarian strips (1 × 1 cm2) showed the lowest apoptotic signals after incubation. We applied the orthotropic transplantation procedure with large strips in the clinic, and 9 of 15 POI patients showed at least one-wave follicular growth during the monitoring period. One patient was reported with one healthy delivery after natural conception and another patient with a healthy singleton delivery after IVF. All the contacted patients (n = 13) responded with no side effects on their health 2–4 years after IVA procedure. LIMITATIONS, REASONS FOR CAUTION Further clinical trials with a large number of well-defined patients are required to compare different IVA protocols. A long-term follow-up system should be set up to monitor patient’s health in the future cohort study. WIDER IMPLICATIONS OF THE FINDINGS By using stimulators, the findings in the study provide a more efficient IVA protocol for the treatment of patients with DOR. It requires only one laparoscopic surgery and thus minimizes patients’ discomfort and costs. This strategy could be useful for patients diagnosed with POI and desire pregnancy as soon as possible after the operation. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Key Research and Development Program of China (2018YFC1003703 and 2018YFC1004203); the National Natural Science Foundation of China (81871221); Co-construction of Provincial Department (201601006). The authors have no conflict of interest to disclose. TRIAL REGISTRATION NUMBER ChiCTR2000030872.