Stepwise assembly and release of Tc toxins from Yersinia entomophaga.

Tc toxins are virulence factors of bacterial pathogens. Although their structure and intoxication mechanism are well understood, it remains elusive where this large macromolecular complex is assembled and how it is released. Here we show by an integrative multiscale imaging approach that Yersinia entomophaga Tc (YenTc) toxin components are expressed only in a subpopulation of cells that are 'primed' with several other potential virulence factors, including filaments of the protease M66/StcE. A phage-like lysis cassette is required for YenTc release; however, before resulting in complete cell lysis, the lysis cassette generates intermediate 'ghost' cells, which may serve as assembly compartments and become packed with assembled YenTc holotoxins. We hypothesize that this stepwise mechanism evolved to minimize the number of cells that need to be killed. The occurrence of similar lysis cassettes in diverse organisms indicates a conserved mechanism for Tc toxin release that may apply to other extracellular macromolecular machines.

Coupling Titanium and Chromium Catalysis in a Reaction Network for the Reprogramming of [BH4 ]- as Electron Transfer and Hydrogen Atom Transfer Reagent for Radical Chemistry.

We describe a unique catalytic system with an efficient coupling of Ti- and Cr-catalysis in a reaction network that allows the use of [BH4 ]- as stoichiometric hydrogen atom and electron donor in catalytic radical chemistry. The key feature is a relay hydrogen atom transfer from [BH4 ]- to Cr generating the active catalysts under mild conditions. This enables epoxide reductions, regiodivergent epoxide opening and radical cyclizations that are not possible with cooperative catalysis with radicals or by epoxide reductions via Meinwald rearrangement and ensuing carbonyl reduction. No typical SN 2-type reactivity of [BH4 ]- salts is observed.

Antibodies to SARS-CoV-2 in follicular fluids and their association with assisted reproduction.

Introduction: Every second woman suffering from infertility asks for medical help. There is public concern that vaccination-induced antibodies (Ab) are negatively associated with fertility. A recent study has demonstrated an association between SARS-CoV-2 vaccination and a lower pregnancy rate in the subsequent 60 days. Consequently, Ab could affect fertility success in assisted reproduction. Methods: To address this question, we compared fertilization outcomes of vaccinated (n=35) and nonvaccinated (n=34) women. Paired serum samples and multiple follicular fluids (FF) (up to 10 from the same donor) were collected during the course of assisted reproduction and characterized for oocyte quality, the presence of Ab and trace element concentrations. Results: The results showed a positive correlation of vaccination-induced neutralizing activity of SARS-CoV-2-Ab in serum and FF. On average, Ab concentrations in serum were higher than in the corresponding FF. However, wide variations in SARS-CoV-2 Ab titers were observed between different FF, correlating to trace element levels, even when retrieved from the same donor. Discussion: Overall, FF contents are highly variable, but no negative association was observed between Ab in serum or FF and fertilization success and oocyte development, supporting the safety of SARS-CoV-2 vaccination during assisted reproduction.

Detection of antibodies to SARS-CoV-2 after vaccination in seminal plasma and their association to sperm parameters.

There is a public concern that COVID-19 vaccination and SARS-CoV-2 antibodies (Abs) negatively affect male fertility. However, the evidence for the presence of SARS-CoV-2 Abs in seminal plasma (SP) is lacking. We examined whether Abs were detectable in SP after COVID-19 vaccination in 86 men using a direct Ab measurement and by quantification of their neutralizing activity. The results show the presence of SARS-CoV-2 Abs in SP, with a strong correlation to the serum Abs, increasing with the number of vaccinations. Furthermore, the Ab titers are correlating with the neutralization activity. The SARS-CoV-2 vaccination parameters showed no association with the markers of sperm quality. In conclusion, this study indicates substantial levels of Abs in SP after COVID-19 vaccination that correlate with serum Ab titers but do not associate with sperm quality.

SepN is a septal junction component required for gated cell-cell communication in the filamentous cyanobacterium Nostoc.

Multicellular organisms require controlled intercellular communication for their survival. Strains of the filamentous cyanobacterium Nostoc regulate cell-cell communication between sister cells via a conformational change in septal junctions. These multi-protein cell junctions consist of a septum spanning tube with a membrane-embedded plug at both ends, and a cap covering the plug on the cytoplasmic side. The identities of septal junction components are unknown, with exception of the protein FraD. Here, we identify and characterize a FraD-interacting protein, SepN, as the second component of septal junctions in Nostoc. We use cryo-electron tomography of cryo-focused ion beam-thinned cyanobacterial filaments to show that septal junctions in a sepN mutant lack a plug module and display an aberrant cap. The sepN mutant exhibits highly reduced cell-cell communication rates, as shown by fluorescence recovery after photobleaching experiments. Furthermore, the mutant is unable to gate molecule exchange through septal junctions and displays reduced filament survival after stress. Our data demonstrate the importance of controlling molecular diffusion between cells to ensure the survival of a multicellular organism.

Confirmation of human ovulation in assisted reproduction using an adhesive axillary thermometer (femSense®).

Objective: Timing for sexual intercourse is important in achieving pregnancy in natural menstrual cycles. Different methods of detecting the fertile window have been invented, among them luteinization hormone (LH) to predict ovulation and biphasic body basal temperature (BBT) to confirm ovulation retrospectively. The gold standard to detect ovulation in gynecology practice remains transvaginal ultrasonography in combination with serum progesterone. In this study we evaluated a wearable temperature sensing patch (femSense®) using continuous body temperature measurement to confirm ovulation and determine the end of the fertile window. Methods: 96 participants received the femSense® system consisting of an adhesive axillary thermometer patch and a smartphone application, where patients were asked to document information about their previous 3 cycles. Based on the participants data, the app predicted the cycle length and the estimated day of ovulation. From these predictions, the most probable fertile window and the day for applying the patch were derived. Participants applied and activated the femSense® patch on the calculated date, from which the patch continuously recorded their body temperature throughout a period of up to 7 days to confirm ovulation. Patients documented their daily urinary LH test positivity, and a transvaginal ultrasound was performed on day cycle day 7, 10, 12 and 14/15 to investigate the growth of one dominant follicle. If a follicle reached 15 mm in diameter, an ultrasound examination was carried out every day consecutively until ovulation. On the day ovulation was detected, serum progesterone was measured to confirm the results of the ultrasound. The performance of femSense® was evaluated by comparing the day of ovulation confirmation with the results of ovulation prediction (LH test) and detection (transvaginal ultrasound). Results: The femSense® system confirmed ovulation occurrence in 60 cases (81.1%) compared to 48 predicted cases (64.9%) with the LH test (p = 0.041). Subgroup analysis revealed a positive trend for the femSense® system of specific ovulation confirmation within the fertile window of 24 h after ovulation in 42 of 74 cases (56.8%). Cycle length, therapy method or infertility reason of the patient did not influence accuracy of the femSense® system. Conclusions: The femSense® system poses a promising alternative to the traditional BBT method and is a valuable surrogate marker to transvaginal ultrasound for confirmation of ovulation.

Structure of a thylakoid-anchored contractile injection system in multicellular cyanobacteria.

Contractile injection systems (CISs) mediate cell-cell interactions by phage tail-like structures, using two distinct modes of action: extracellular CISs are released into the medium, while type 6 secretion systems (T6SSs) are attached to the cytoplasmic membrane and function upon cell-cell contact. Here, we characterized a CIS in the multicellular cyanobacterium Anabaena, with features distinct from extracellular CISs and T6SSs. Cryo-electron tomography of focused ion beam-milled cells revealed that CISs were anchored in thylakoid membrane stacks, facing the cell periphery. Single particle cryo-electron microscopy showed that this unique in situ localization was mediated by extensions of tail fibre and baseplate components. On stress, cyanobacteria induced the formation of ghost cells, presenting thylakoid-anchored CISs to the environment. Functional assays suggest that these CISs may mediate ghost cell formation and/or interactions of ghost cells with other organisms. Collectively, these data provide a framework for understanding the evolutionary re-engineering of CISs and potential roles of these CISs in cyanobacterial programmed cell death.

Investigation of Non-invasive Continuous Body Temperature Measurements in a Clinical Setting Using an Adhesive Axillary Thermometer (SteadyTemp®).

Since the human body reacts to a variety of different diseases with elevated body temperature, measurement of body temperature remains relevant in clinical practice. The absolute temperature value for fever definition is still arbitrary and depends on the measuring site, as well as underlying disease and individual factors. Hence, a simple threshold for fever definition is outdated and a definition which relies on the relative changes in the individual seems reasonable as it takes these individual factors into account. In this prospective multicentric study we validate an adhesive axillary thermometer (SteadyTemp®) which allows continuous non-invasive temperature measurements. It consists of a patch to measure temperature and a smartphone application to process and visualize gathered data. This article provides information of the new diagnostic possibilities when using this wearable device and where it could be beneficial. Furthermore, it discusses how to interpret the generated data and when it is not practical to use, based on its characteristics and physiological phenomena.

Pronounced Trace Element Variation in Follicular Fluids of Subfertile Women Undergoing Assisted Reproduction.

Female subfertility is a growing concern, especially in view of an increasing prevalence of polycystic ovary syndrome (PCOS). Assisted reproductive technologies (ART) offer a perspective for pregnancy, but the outcome rate is still suboptimal. The trace elements (TE), copper (Cu), selenium (Se), and zinc (Zn) are essential for fertility and development. We hypothesized that TE concentrations are related to oocyte quality and growth and affect pregnancy outcomes in women undergoing ART. Concentrations of TE were measured by total reflection X-ray fluorescence. Extracellular glutathione peroxidase 3 (GPX3) and selenoprotein P (SELENOP) were determined as additional Se biomarkers. Corresponding serum and follicular fluid (FF) samples were available from women with (n = 20) and without (n = 20) PCOS diagnosis undergoing hormone treatment within the ART procedure, respectively, and FF samples were classified into five groups based on morphological assessment. Serum showed higher TE concentrations than FF, and TE levels correlated positively between both matrices. Individual FF from the same women showed surprisingly high variability in TE concentration, and follicles without oocytes displayed the lowest TE concentrations. Both Se biomarkers GPX3 and SELENOP were present in FF and correlated positively to Se concentrations. Some notable relationships were observed between morphokinetic parameters, TE concentrations, and GPX3 activity. A slightly depressed serum Zn concentration was observed in PCOS. Our results indicate a direct relationship between TE in serum and FF, positive correlations between the three Se biomarkers in FF, and high variability between the FF from the same woman with the lowest TE concentrations in the follicles with the poorest quality. The differences observed in relation to PCOS diagnoses appear relatively minor. Collectively, the data support the notion that TE assessment of follicles may contribute to optimal oocyte selection and subsequently influence ART success.

Effect of probiotics on vaginal Ureaplasma parvum in women suffering from unexplained infertility.

RESEARCH QUESTION: Does oral probiotic supplementation influence the relative abundance of different vaginal microbiota in women experiencing infertility? DESIGN: A prospective, monocentric randomized controlled trial. To study the influence of probiotics on infertility, 80 patients with primary or secondary infertility were included. Patients were assigned to either a probiotic treatment or a control group. Participants in the treatment group (n = 40) took one sachet (2 g) a day of a defined probiotic supplement limiting Lactobacillus strains. Patients in the control group did not receive any additional probiotic supplements. Vaginal samples were taken on day 20 of the menstrual cycle and 4 weeks later, on day 20, of the consecutive cycle. Subsequently, 16s rRNA gene analysis of the vaginal samples was conducted. RESULTS: After the intervention phase, no effects on alpha diversity resulting from treatment could be observed. The between sample diversity of different women (beta diversity) at baseline had no effects of age, treatment group or body mass index. Primary or secondary sterility, however, had a significant effect on community. Three clusters (Lactobacillus crispatus, Lactobacillus iners and Lactobacillus gasseri) were identified as the leading representatives. Furthermore, patients treated with probiotics showed limited growth of Ureaplasma parvum compared with the control group (P = 0.021). CONCLUSIONS: This study points to a possible protective effect of probiotic supplements on the vaginal microbiota. It is tempting to speculate that this effect assists in containing the growth of non-beneficial bacteria and helps to prevent or cure a dysbiotic vaginal flora.

Structural Determinants and Their Role in Cyanobacterial Morphogenesis.

Cells have to erect and sustain an organized and dynamically adaptable structure for an efficient mode of operation that allows drastic morphological changes during cell growth and cell division. These manifold tasks are complied by the so-called cytoskeleton and its associated proteins. In bacteria, FtsZ and MreB, the bacterial homologs to tubulin and actin, respectively, as well as coiled-coil-rich proteins of intermediate filament (IF)-like function to fulfil these tasks. Despite generally being characterized as Gram-negative, cyanobacteria have a remarkably thick peptidoglycan layer and possess Gram-positive-specific cell division proteins such as SepF and DivIVA-like proteins, besides Gram-negative and cyanobacterial-specific cell division proteins like MinE, SepI, ZipN (Ftn2) and ZipS (Ftn6). The diversity of cellular morphologies and cell growth strategies in cyanobacteria could therefore be the result of additional unidentified structural determinants such as cytoskeletal proteins. In this article, we review the current advances in the understanding of the cyanobacterial cell shape, cell division and cell growth.

Architecture and function of human uromodulin filaments in urinary tract infections.

Uromodulin is the most abundant protein in human urine, and it forms filaments that antagonize the adhesion of uropathogens; however, the filament structure and mechanism of protection remain poorly understood. We used cryo-electron tomography to show that the uromodulin filament consists of a zigzag-shaped backbone with laterally protruding arms. N-glycosylation mapping and biophysical assays revealed that uromodulin acts as a multivalent ligand for the bacterial type 1 pilus adhesin, presenting specific epitopes on the regularly spaced arms. Imaging of uromodulin-uropathogen interactions in vitro and in patient urine showed that uromodulin filaments associate with uropathogens and mediate bacterial aggregation, which likely prevents adhesion and allows clearance by micturition. These results provide a framework for understanding uromodulin in urinary tract infections and in its more enigmatic roles in physiology and disease.

Fully automated, sequential focused ion beam milling for cryo-electron tomography.

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

Endometriosis accelerates synchronization of early embryo cell divisions but does not change morphokinetic dynamics in endometriosis patients.

OBJECTIVE: The pathology of endometriosis and its impact on embryo development is still a black box in reproductive medicine. In this time-lapse study we investigated the influence of endometriosis on morphokinetic parameters of embryo development, taking variables of dynamic monitoring into account. Furthermore we evaluated reproductive medicine treatment outcome such as fetal heartbeat and live birth rate. METHODS: 1148 embryos (control: n = 596, endometriosis: n = 552) were retrospectively analyzed. Patients were stimulated with GnRH antagonist protocol. After fertilization, embryos were incubated in a time-lapse system (EmbryoScope). RESULTS: The mixed-model analysis revealed a significant main effect of time (p<0.001), with post-hoc tests showing that any time needed to reach a specific developmental stage was significantly different from all the others (all p<0.001). Embryos of endometriosis patients showed the same absolute morphokinetic time parameters as the control group, however, synchronization of early embryo cell divisions (s2) was faster in endometriosis patients compared to the control group. CONCLUSION: In general, endometriosis does not induce changes in early embryo morphokinetics. However, observed acceleration in cell cycle synchronization of embryo cleavage patterns might be a missing explanation for contradicting results in literature regarding the impairments in reproductive medicine treatment outcome of endometriosis patients.

Structure and Function of a Bacterial Gap Junction Analog.

Multicellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood, and it is unknown whether they are capable of controlling intercellular communication. Here, we resolved the in situ architecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacterial filaments. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a FraD-containing plug, which was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. Gating was accompanied by a reversible conformational change of the septal junction cap. We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange in multicellular organisms.

Sense classification of shallow discourse relations with focused RNNs.

Understanding the sense of discourse relations between segments of text is essential to truly comprehend any natural language text. Several automated approaches have been suggested, but all rely on external resources, linguistic feature engineering, and their processing pipelines are built from substantially different models. In this paper, we introduce a novel system for sense classification of shallow discourse relations (FR system) based on focused recurrent neural networks (RNNs). In contrast to existing systems, FR system consists of a single end-to-end trainable model for handling all types and senses of discourse relations, requires no feature engineering or external resources, is language-independent, and can be applied at the word and even character levels. At its core, we present our novel generalization of the focused RNNs layer, the first multi-dimensional RNN-attention mechanism for constructing text/argument embeddings. The filtering/gating RNN enables downstream RNNs to focus on different aspects of the input sequence and project it into several embedding subspaces. These argument embeddings are then used to perform sense classification. FR system has been evaluated using the official datasets and methodology of CoNLL 2016 Shared Task. It does not fall a lot behind state-of-the-art performance on English, the most researched and supported language, but it outperforms existing best systems by 2.5% overall results on the Chinese blind dataset.

Impact of polar body biopsy on embryo morphokinetics-back to the roots in preimplantation genetic testing?

PURPOSE: Polar body biopsy (PBB) is a common technique in preimplantation genetic testing (PGT) to assess the chromosomal status of the oocyte. Numerous studies have been implemented to investigate the impact of biopsies on embryo development; however, information on embryo morphokinetics is still lacking. Hence, we investigated the impact of PBB on morphokinetic parameters in early embryo development. METHODS: Four hundred four embryos (202 PBB, 202 control) were retrospectively analyzed. Patients were stimulated with a gonadotropin-releasing hormone antagonist ovarian hyperstimulation protocol. After fertilization check, embryos were incubated in a time-lapse incubator. The groups were matched for maternal age at time of oocyte retrieval. RESULTS: Mean group times for reaching specific developmental time points showed no significant difference comparing embryos with PBB conducted and without. Likewise, further subdivision of the PBB group in euploid and aneuploid embryos revealed no differences in the early embryo morphokinetic development compared to the control group. Aneuploidy testing revealed a high prevalence of chromosomal aberrations for chromosomes 21, 4, 16, and 19. CONCLUSIONS: In conclusion, PBB does not impact the morphokinetic parameters of the embryo development. PBB can be safely applied without the risk of impairing the reproductive potential of the embryo and can be highly recommended as safe and practicable PGT approach, especially in countries with prevailing restrictions regarding PGT analysis.

Robust workflow and instrumentation for cryo-focused ion beam milling of samples for electron cryotomography.

Electron cryotomography is able to visualize macromolecular complexes in their cellular context, in a frozen-hydrated state, and in three dimensions. The method, however, is limited to relatively thin samples. Cryo-focused ion beam (FIB) milling is emerging as a powerful technique for sample thinning prior to cryotomography imaging. Previous cryo-FIB milling reports utilized custom-built non-standard equipment. Here we present a workflow and the required commercially available instrumentation to either implement the method de novo, or as an upgrade of pre-existing dual beam milling instruments. We introduce two alternative protocols and the respective sample holders for milling. The "bare grid holder" allows for milling on plain grids, having the advantage of enabling relatively shallow milling angles for wedge geometries. The "Autogrid holder" is designed for milling grids clamped into a mechanical support ring (Autogrid), resulting in increased stability for lamella geometries. We applied the workflow to prepare samples and record high-quality tomograms of diverse model organisms, including infected and uninfected HeLa cells, amoebae, yeast, multicellular cyanobacteria, Pseudomonas aeruginosa and Escherichia coli cells. The workflow will contribute to the dissemination of electron cryotomography of cryo-FIB milled samples in the biological sciences.

In situ architecture, function, and evolution of a contractile injection system.

Contractile injection systems mediate bacterial cell-cell interactions by a bacteriophage tail-like structure. In contrast to extracellular systems, the type 6 secretion system (T6SS) is defined by intracellular localization and attachment to the cytoplasmic membrane. Here we used cryo-focused ion beam milling, electron cryotomography, and functional assays to study a T6SS in Amoebophilus asiaticus The in situ architecture revealed three modules, including a contractile sheath-tube, a baseplate, and an anchor. All modules showed conformational changes upon firing. Lateral baseplate interactions coordinated T6SSs in hexagonal arrays. The system mediated interactions with host membranes and may participate in phagosome escape. Evolutionary sequence analyses predicted that T6SSs are more widespread than previously thought. Our insights form the basis for understanding T6SS key concepts and exploring T6SS diversity.

In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography.

The unique property of electron cryotomography (ECT) is its capability to resolve the structure of macromolecular machines in their cellular context. The integration of ECT data with high-resolution structures of purified subcomplexes and live-cell fluorescence light microscopy can generate pseudo-atomic models that lead to a mechanistic understanding across size and time scales. Recent advances in electron detection, sample thinning, data acquisition, and data processing have significantly enhanced the applicability and performance of ECT. Here we describe a detailed workflow for an ECT experiment, including cell culture, vitrification, data acquisition, data reconstruction, tomogram analysis, and subtomogram averaging. This protocol provides an entry point to the technique for students and researchers and indicates the many possible variations arising from specific target properties and the available instrumentation.