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Objective To predict birth weight at various potential gestational ages of delivery based on data routinely available at the first antenatal visit.Design Individual participant data meta-analysis.Data sources Individual participant data of four cohorts (237 228 pregnancies) from the International Prediction of Pregnancy Complications (IPPIC) network dataset.Eligibility criteria for selecting studies Studies in the IPPIC network were identified by searching major databases for studies reporting risk factors for adverse pregnancy outcomes, such as pre-eclampsia, fetal growth restriction, and stillbirth, from database inception to August 2019. Data of four IPPIC cohorts (237 228 pregnancies) from the US (National Institute of Child Health and Human Development, 2018; 233 483 pregnancies), UK (Allen et al, 2017; 1045 pregnancies), Norway (STORK Groruddalen research programme, 2010; 823 pregnancies), and Australia (Rumbold et al, 2006; 1877 pregnancies) were included in the development of the model.Results The IPPIC birth weight model was developed with random intercept regression models with backward elimination for variable selection. Internal-external cross validation was performed to assess the study specific and pooled performance of the model, reported as calibration slope, calibration-in-the-large, and observed versus expected average birth weight ratio. Meta-analysis showed that the apparent performance of the model had good calibration (calibration slope 0.99, 95% confidence interval (CI) 0.88 to 1.10; calibration-in-the-large 44.5 g, −18.4 to 107.3) with an observed versus expected average birth weight ratio of 1.02 (95% CI 0.97 to 1.07). The proportion of variation in birth weight explained by the model (R2) was 46.9% (range 32.7-56.1% in each cohort). On internal-external cross validation, the model showed good calibration and predictive performance when validated in three cohorts with a calibration slope of 0.90 (Allen cohort), 1.04 (STORK Groruddalen cohort), and 1.07 (Rumbold cohort), calibration-in-the-large of −22.3 g (Allen cohort), −33.42 (Rumbold cohort), and 86.4 g (STORK Groruddalen cohort), and observed versus expected ratio of 0.99 (Rumbold cohort), 1.00 (Allen cohort), and 1.03 (STORK Groruddalen cohort); respective pooled estimates were 1.00 (95% CI 0.78 to 1.23; calibration slope), 9.7 g (−154.3 to 173.8; calibration-in-the-large), and 1.00 (0.94 to 1.07; observed v expected ratio). The model predictions were more accurate (smaller mean square error) in the lower end of predicted birth weight, which is important in informing clinical decision making.Conclusions The IPPIC birth weight model allowed birth weight predictions for a range of possible gestational ages. The model explained about 50% of individual variation in birth weights, was well calibrated (especially in babies at high risk of fetal growth restriction and its complications), and showed promising performance in four different populations included in the individual participant data meta-analysis. Further research to examine the generalisability of performance in other countries, settings, and subgroups is required.Trial registration PROSPERO CRD42019135045

Many wireless Internet-of-Things applications require extended battery life ranging from a few months to a few years. Such applications have motivated the recent developments in low power wide area networks, including the rise of Long Range (LoRa) technology. LoRa has a simple modulation scheme designed for extended converge, low battery consumption, and resistance to high interference levels. Thus LoRa is primarily targeted for shared spectrum applications where interference levels are typically higher than controlled spectrum applications where a single operator usually has a dominant control on the quality of service. As a result, it is of paramount importance to carefully design IoT networks while taking into account the impending impacts of interference and propagation environments. This paper presents a novel LoRa network design framework that utilizes a developed open-source emulator to provide a reliable network coverage estimation. The framework is tested in one of the largest open-access IoT network designs in Australia, which enabled the deployment of 294 sensors and 48 gateways. Both the framework and the emulator are implemented using MATLAB scripting, enabling integration with built-in and external radio planning tools. The framework leverages real interference measurements captured using software defined radio that records the spectrotemporal behavior of the existing traffic in the shared band.

Introduction Following the detection of fetal growth restriction, there is no consensus about the criteria that should trigger delivery in the late preterm period. The consequences of inappropriate early or late delivery are potentially important yet practice varies widely around the world, with abnormal findings from fetal heart rate monitoring invariably leading to delivery. Indices derived from fetal cerebral Doppler examination may guide such decisions although there are few studies in this area. We propose a randomised, controlled trial to establish the optimum method of timing delivery between 32 weeks and 36 weeks 6 days of gestation. We hypothesise that delivery on evidence of cerebral blood flow redistribution reduces a composite of perinatal poor outcome, death and short-term hypoxia-related morbidity, with no worsening of neurodevelopmental outcome at 2 years.Methods and analysis Women with non-anomalous singleton pregnancies 32+0 to 36+6 weeks of gestation in whom the estimated fetal weight or abdominal circumference is

For years we have investigated the presence and molecular evolution of tuberculosis in Pre Dynastic and Early Dynastic Egyptian mummy material from Abydos (c. 3500-2800 BC), Middle and New Kingdom until the Late Period in Thebes-West (c. 2050 – 500 BC). We have analysed 160 bone and soft tissue samples from different time periods and populations for the occurrence of M. tuberculosis complex DNA. All positive specimens were genetically characterised by spoligotyping and mutation analysis. Molecular analyses revealed excellent state of preservation of the specimens. Research showed a high incidence of M. tuberculosis during all time periods. We further detected specific MTB strain differences with M. africanum in some of the Middle Kingdom samples and “modern” M. tuberculosis strains in the New Kingdom to Late Period material. These results demonstrate that aDNA is excellently preserved in ancient Egyptian mummies allowing the reconstruction of occurrence, frequency, molecular evolution and spread of tuberculosis in Pharaonic populations.

DNA vectors have been widely used as a priming of poxvirus vaccine in prime/boost regimens. Whether the number of DNA impacts qualitatively or quantitatively the immune response is not fully explored. With the aim to reinforce T-cell responses by optimizing the prime-boost regimen, the multicentric EV03/ANRS VAC20 phase I/II trial, randomized 147 HIV-negative volunteers to either 3xDNA plus 1xNYVAC (weeks 0, 4, 8 plus 24; n = 74) or to 2xDNA plus 2xNYVAC (weeks 0, 4 plus 20, 24; n = 73) groups. T-cell responses (IFN-γ ELISPOT) to at least one peptide pool were higher in the 3xDNA than the 2xDNA groups (91% and 80% of vaccinees) (P = 0.049). In the 3xDNA arm, 26 (37%) recipients developed a broader T-cell response (Env plus at least to one of the Gag, Pol, Nef pools) than in the 2xDNA (15; 22%) arms (primary endpoint; P = 0.047) with a higher magnitude against Env (at week 26) (P

Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact.

ABSTRACT In a screen for compounds that inhibit infectivity of the obligate intracellular pathogen Chlamydia trachomatis, we identified the 2-pyridone amide KSK120. A fluorescent KSK120 analogue was synthesized and observed to be associated with the C. trachomatis surface, suggesting that its target is bacterial. We isolated KSK120-resistant strains and determined that several resistance mutations are in genes that affect the uptake and use of glucose-6-phosphate (G-6P). Consistent with an effect on G-6P metabolism, treatment with KSK120 blocked glycogen accumulation. Interestingly, KSK120 did not affect Escherichia coli or the host cell. Thus, 2-pyridone amides may represent a class of drugs that can specifically inhibit C. trachomatis infection. IMPORTANCE Chlamydia trachomatis is a bacterial pathogen of humans that causes a common sexually transmitted disease as well as eye infections. It grows only inside cells of its host organism, within a parasitophorous vacuole termed the inclusion. Little is known, however, about what bacterial components and processes are important for C. trachomatis cellular infectivity. Here, by using a visual screen for compounds that affect bacterial distribution within the chlamydial inclusion, we identified the inhibitor KSK120. As hypothesized, the altered bacterial distribution induced by KSK120 correlated with a block in C. trachomatis infectivity. Our data suggest that the compound targets the glucose-6-phosphate (G-6P) metabolism pathway of C. trachomatis, supporting previous indications that G-6P metabolism is critical for C. trachomatis infectivity. Thus, KSK120 may be a useful tool to study chlamydial glucose metabolism and has the potential to be used in the treatment of C. trachomatis infections.

Due to slow diagnostics, physicians must optimize antibiotic therapies based on clinical evaluation of patients without specific information on causative bacteria. We have investigated metabolomic analysis of blood for the detection of acute bacterial infection and early differentiation between ineffective and effective antibiotic treatment. A vital and timely therapeutic difficulty was thereby addressed: the ability to rapidly detect treatment failures because of antibiotic-resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) were used in vitro and for infecting mice, while natural MSSA infection was studied in humans. Samples of bacterial growth media, the blood of infected mice and of humans were analyzed with combined Gas Chromatography/Mass Spectrometry. Multivariate data analysis was used to reveal the metabolic profiles of infection and the responses to different antibiotic treatments. In vitro experiments resulted in the detection of 256 putative metabolites and mice infection experiments resulted in the detection of 474 putative metabolites. Importantly, ineffective and effective antibiotic treatments were differentiated already two hours after treatment start in both experimental systems. That is, the ineffective treatment of MRSA using cloxacillin and untreated controls produced one metabolic profile while all effective treatment combinations using cloxacillin or vancomycin for MSSA or MRSA produced another profile. For further evaluation of the concept, blood samples of humans admitted to intensive care with severe sepsis were analyzed. One hundred thirty-three putative metabolites differentiated severe MSSA sepsis (n = 6) from severe Escherichia coli sepsis (n = 10) and identified treatment responses over time. Combined analysis of human, in vitro, and mice samples identified 25 metabolites indicative of effective treatment of S. aureus sepsis. Taken together, this study provides a proof of concept of the utility of analyzing metabolite patterns in blood for early differentiation between ineffective and effective antibiotic treatment in acute S. aureus infections.

Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU(CC). Here we show that depletion of calcium induces intramolecular dissociation of YscU(CC) from YscU followed by secretion of the YscU(CC) polypeptide. Thus, YscU(CC) behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscU(CC)in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscU(CC) dissociation for Yop secretion. We propose that YscU(CC) orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

Many gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated β1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with a series of malignant tumors. These include lymphomas (Burkitt’s lymphoma, Hodgkin’s disease, T/NK-cell lymphoma, post-transplant lymphoproliferative disease, AIDS-associated lymphoma, X-linked lymphoproliferative syndrome), carcinomas (nasopharyngeal carcinoma, gastric carcinoma, carcinomas of major salivary glands, thymic carcinoma, mammary carcinoma) and a sarcoma (leiomyosarcoma). The latent EBV genomes persist in the tumor cells as circular episomes, co-replicating with the cellular DNA once per cell cycle. The expression of latent EBV genes is cell type specific due to the strict epigenetic control of their promoters. DNA methylation, histone modifications and binding of key cellular regulatory proteins contribute to the regulation of alternative promoters for transcripts encoding the nuclear antigens EBNA1 to 6 and affect the activity of promoters for transcripts encoding transmembrane proteins (LMP1, LMP2A, LMP2B). In addition to genes transcribed by RNA polymerase II, there are also two RNA polymerase III transcribed genes in the EBV genome (EBER 1 and 2). The 5’ and internal regulatory sequences of EBER 1 and 2 transcription units are invariably unmethylated. The highly abundant EBER 1 and 2 RNAs are not translated to protein. Based on the cell type specific epigenetic marks associated with latent EBV genomes one can distinguish between viral epigenotypes that differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Whereas latent EBV genomes are regularly targeted by epigenetic control mechanisms in different cell types, EBV encoded proteins may, in turn, affect the activity of a set of cellular promoters by interacting with the very same epigenetic regulatory machinery. There are EBNA1 binding sites in the human genome. Because high affinity binding of EBNA1 to its recognition sites is known to specify sites of DNA demethylation, we suggest that binding of EBNA1 to its cellular target sites may elicit local demethylation and contribute thereby to the activation of silent cellular promoters. EBNA2 interacts with histone acetyltransferases, and EBNALP (EBNA5) coactivates transcription by displacing histone deacetylase 4 from EBNA2-bound promoter sites. EBNA3C (EBNA6) seems to be associated both with histone acetylases and deacetylases, although in separate complexes. LMP1, a transmembrane protein involved in malignant transformation, can affect both alternative systems of epigenetic memory, DNA methylation and the Polycomb-trithorax group of protein complexes. In epithelial cells LMP1 can up-regulate DNA methyltransferases and, in Hodgkin lymphoma cells, induce the Polycomb group protein Bmi-1. In addition, LMP1 can also modulate cellular gene expression programs by affecting, via the NF-κB pathway, levels of cellular microRNAs miR-146a and miR-155. These interactions may result in epigenetic dysregulation and subsequent cellular dysfunctions that may manifest in or contribute to the development of pathological changes (e.g. initiation and progression of malignant neoplasms; autoimmune phenomena; immunodeficiency). Thus, Epstein-Barr virus, similarly to other viruses and certain bacteria, may induce pathological changes by epigenetic reprogramming of host cells. Elucidation of the epigenetic consequences of EBV-host interactions (within the framework of the emerging new field of patho-epigenetics) may have important implications for therapy and disease prevention, because epigenetic processes are reversible and continuous silencing of EBV genes contributing to patho-epigenetic changes may prevent disease development.

Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with a series of malignant tumors. These include lymphomas (Burkitt’s lymphoma, Hodgkin’s disease, T/NK-cell lymphoma, post-transplant lymphoproliferative disease, AIDS-associated lymphoma, X-linked lymphoproliferative syndrome), carcinomas (nasopharyngeal carcinoma, gastric carcinoma, carcinomas of major salivary glands, thymic carcinoma, mammary carcinoma) and a sarcoma (leiomyosarcoma). The latent EBV genomes persist in the tumor cells as circular episomes, co-replicating with the cellular DNA once per cell cycle. The expression of latent EBV genes is cell type specific due to the strict epigenetic control of their promoters. DNA methylation, histone modifications and binding of key cellular regulatory proteins contribute to the regulation of alternative promoters for transcripts encoding the nuclear antigens EBNA1 to 6 and affect the activity of promoters for transcripts encoding transmembrane proteins (LMP1, LMP2A, LMP2B). In addition to genes transcribed by RNA polymerase II, there are also two RNA polymerase III transcribed genes in the EBV genome (EBER 1 and 2). The 5’ and internal regulatory sequences of EBER 1 and 2 transcription units are invariably unmethylated. The highly abundant EBER 1 and 2 RNAs are not translated to protein. Based on the cell type specific epigenetic marks associated with latent EBV genomes one can distinguish between viral epigenotypes that differ in transcriptional activity in spite of having an identical (or nearly identical) DNA sequence. Whereas latent EBV genomes are regularly targeted by epigenetic control mechanisms in different cell types, EBV encoded proteins may, in turn, affect the activity of a set of cellular promoters by interacting with the very same epigenetic regulatory machinery. There are EBNA1 binding sites in the human genome. Because high affinity binding of EBNA1 to its recognition sites is known to specify sites of DNA demethylation, we suggest that binding of EBNA1 to its cellular target sites may elicit local demethylation and contribute thereby to the activation of silent cellular promoters. EBNA2 interacts with histone acetyltransferases, and EBNALP (EBNA5) coactivates transcription by displacing histone deacetylase 4 from EBNA2-bound promoter sites. EBNA3C (EBNA6) seems to be associated both with histone acetylases and deacetylases, although in separate complexes. LMP1, a transmembrane protein involved in malignant transformation, can affect both alternative systems of epigenetic memory, DNA methylation and the Polycomb-trithorax group of protein complexes. In epithelial cells LMP1 can up-regulate DNA methyltransferases and, in Hodgkin lymphoma cells, induce the Polycomb group protein Bmi-1. In addition, LMP1 can also modulate cellular gene expression programs by affecting, via the NF-κB pathway, levels of cellular microRNAs miR-146a and miR-155. These interactions may result in epigenetic dysregulation and subsequent cellular dysfunctions that may manifest in or contribute to the development of pathological changes (e.g. initiation and progression of malignant neoplasms; autoimmune phenomena; immunodeficiency). Thus, Epstein-Barr virus, similarly to other viruses and certain bacteria, may induce pathological changes by epigenetic reprogramming of host cells. Elucidation of the epigenetic consequences of EBV-host interactions (within the framework of the emerging new field of patho-epigenetics) may have important implications for therapy and disease prevention, because epigenetic processes are reversible and continuous silencing of EBV genes contributing to patho-epigenetic changes may prevent disease development.

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+) cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

Nucleotide oligomerisation domain 2 (NOD2) is a component of the innate immunity known to be involved in the homeostasis of Peyer patches (PPs) in mice. However, little is known about its role during gut infection in vivo. Yersinia pseudotuberculosis is an enteropathogen causing gastroenteritis, adenolymphitis and septicaemia which is able to invade its host through PPs. We investigated the role of Nod2 during Y. pseudotuberculosis infection. Death was delayed in Nod2 deleted and Crohn's disease associated Nod2 mutated mice orogastrically inoculated with Y. pseudotuberculosis. In PPs, the local immune response was characterized by a higher KC level and a more intense infiltration by neutrophils and macrophages. The apoptotic and bacterial cell counts were decreased. Finally, Nod2 deleted mice had a lower systemic bacterial dissemination and less damage of the haematopoeitic organs. This resistance phenotype was lost in case of intraperitoneal infection. We concluded that Nod2 contributes to the susceptibility to Y. pseudotuberculosis in mice.

Background: Little is known about fetal outcome after Listeria monocytogenes septicemia in the first trimester of pregnancy.