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Summary We introduce LongDat, an R package that analyzes longitudinal multivariable (cohort) data while simultaneously accounting for a potentially large number of covariates. The primary use case is to differentiate direct from indirect effects of an intervention (or treatment) and to identify covariates (potential mechanistic intermediates) in longitudinal data. LongDat focuses on analyzing longitudinal microbiome data, but its usage can be expanded to other data types, such as binary, categorical, and continuous data. We tested and compared LongDat with other tools (i.e., MaAsLin2, ANCOM, lgpr, and ZIBR) on both simulated and real data. We showed that LongDat outperformed these tools in accuracy, runtime, and memory cost, especially when there were multiple covariates. The results indicate that the LongDat R package is a computationally efficient and low-memory-cost tool for longitudinal data with multiple covariates and facilitates robust biomarker searches in high-dimensional datasets. Availability and Implementation The R package LongDat is available on CRAN (https://cran.r-project.org/web/packages/LongDat/) and GitHub (https://github.com/CCY-dev/LongDat). Supplementary information Supplementary data are available at Bioinformatics Advances online.

Junctional adhesion molecule 3 (JAM3) is involved in epithelial cell junction, cell polarity, and motility. The molecular mechanisms underlying the role of JAM3 in placental dysfunction remain unclear. We hypothesized that JAM3 expression regulates trophoblast fusion, differentiation, proliferation, and apoptosis. Our results revealed that JAM3 was expressed in the cytotrophoblasts and syncytiotrophoblasts of first-trimester and term placental villi. JAM3 expression in cell–cell junctions decreased with the formation of syncytiotrophoblasts. Using trophoblasts as an in vitro model, we observed that forskolin and JAM3 knockdown significantly reduced JAM3 expression and increased syncytium formation. JAM3 knockdown additionally inhibited trophoblast proliferation and increased the number of trophoblasts in the sub-G1 and G2/M phases, indicating cell-cycle disturbance and apoptosis. Cell-cycle arrest was associated with the engagement of checkpoint kinase 2-cell division cycle 25C-cyclin-dependent kinase 1/cyclin B1 signaling. Increased expression of BIM, NOXA, XAF1, cytochrome c, and cleaved caspase-3 further indicated trophoblast apoptosis. Overexpression of JAM3 or recombinant JAM3 protein enhanced trophoblast adhesion and migration, which were inhibited by JAM3 knockdown. JAM3 knockdown induced reactive oxygen species and syncytin 2 expression in trophoblasts. Furthermore, H2O2-induced oxidative stress reduced JAM3 expression in trophoblasts and cell culture supernatants. H2O2 simultaneously induced trophoblast apoptosis. JAM3 expression was significantly decreased in the plasmas and placentas of patients with early-onset severe preeclampsia. Thus, our results show that JAM3 may not only be a structural component of trophoblast cell junctions but also regulates trophoblast fusion, differentiation, proliferation, apoptosis, and motility. Dysregulated trophoblast JAM3 expression is crucial in preeclampsia development.

Background: W e investigated the combined impact of abdominal obesity and low skeletal muscle mass on cardiovascular and total mortality in an elderly Asian population. Methods: A total of 1,485 elderly individuals (≥65 years) from Elderly Nutrition and Health Survey in Taiwan (1999–2000) were enrolled, and their survival status was followed using data from the National Death Registry. Skeletal muscle mass index (SMMI) was calculated by dividing skeletal muscle mass (kg) by height squared (m 2 ). Low skeletal muscle mass was defined as the first quartile of SMMI. Abdominal obesity (high triglycerides plus waist circumference [HTGWC]) was defined as triglycerides ≥150 mg/dL and w aist circumference ≥90 cm (men) and ≥80 cm (women). The Cox proportional hazard model was used to evaluate the combined impact of abdominal obesity and low SMMI on cardiovascular and total mortality. Results: During follow -up (median 9.2 years), one third (n = 493) of subjects died from any cause, of which 34% (n = 168) were cardiovascular-related. Total and cardiovascular mortality were 4.2 and 1.4 per 100 person-years, respectively. Low SMMI and HTGWC were independently associated with total mortality in men, but only low SMMI was significantly associated in women. Those with both HTGWC and low SMMI had the highest mortality risk, with the cardiovascular mortality risk increased by >6.8-fold and 3.2-fold in men and women, respectively, compared with controls having normal SMMI and TGWC. Conclusions: Elderly indi viduals with abdominal obesity and low skeletal muscle mass have higher all-cause and cardiovascular mortality risk.

Reactive oxygen species may cause oxidative damage in the placenta, yet some mechanisms must exist to reduce or prevent such damage. We investigated whether oxidative injury to placental endothelial cells is inhibited by activation of antioxidant enzymes by paracrine factors secreted by human placental multipotent mesenchymal stromal cells (hPMSC). hPMSC-conditioned medium and umbilical endothelial cells were assayed for cytokines and cytokine receptor expression by immunoassay and real-time PCR. Endothelial cell survival was evaluated by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay and caspase 3 activity assay. tert-Butyl hydroperoxide was used to induce oxidative injury in endothelial cells, with fluorescent microscopy and flow cytometry used to detect intracellular peroxides and cell apoptosis. Western blot, real-time PCR, STAT3 DNA-binding activity assay, and STAT3 siRNA were used to assess endothelial cell antioxidant enzymes. hPMSC-conditioned medium supported endothelial cell survival and reduced endothelial cell intracellular peroxides and apoptosis. hPMSCs expressed the transcripts of the interleukin (IL) 6 cytokine family, including IL6 and leukemia-inhibitory factor. hPMSC-conditioned medium activated STAT3 expression in endothelial cells, which was inhibited by neutralizing antibody to interleukin 6 signal transducer (IL6ST) but not to IL6 or leukemia-inhibitory factor. STAT3 siRNA or manganese superoxide dismutase (SOD2) siRNA transfected into endothelial cells inhibited the antiapoptotic effect of conditioned medium. SOD2 was significantly upregulated in endothelial cells by conditioned medium via STAT3 activation that, in turn, was inhibited by IL6ST-neutralizing antibody or STAT3 siRNA. Paracrine factors secreted by hPMSCs support endothelial cell survival. STAT3 activation and SOD2 production protect against oxidative stress-induced endothelial cell damage.

Syncytin is an envelope protein of the human endogenous retrovirus family W (HERV-W). Syncytin is specifically expressed in the human placenta and mediates trophoblast cell fusion into the multinucleated syncytiotrophoblast layer. It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. Functional characterization of syncytin protein can aid understanding of the molecular mechanism underlying syncytin-mediated cell fusion. In this report, we studied the structure-function relationship of syncytin in 293T and HeLa cells transiently expressing wild-type syncytin or syncytin mutants generated by linker scanning and deletion mutagenesis. Of the 22 linker-inserted mutants, mutants InS51, InV139, InE156, InS493, InA506, and InL529 were fusogenic, suggesting that regions around amino acids S51, V139, and E156 in the SU subunit and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation. Of the 17 deletion mutants, nine mutants with deletions in the region from amino acids 479 to 538 were fusogenic. The deletion mutant DelI480, containing only the first four amino acid residues in the cytoplasmic domain, had enhanced fusogenic activity in comparison with the wild-type. In addition, two heptad repeat regions (HRA and B) were defined in the TM subunit of syncytin. A peptide inhibitor derived from the C-terminal heptad repeat region (HRB) was shown to potently inhibit syncytin-mediated cell fusion. Our results suggest that the cytoplasmic domain of syncytin is not essential for syncytin-mediated fusion but may play a regulatory role, and an intramolecular interaction between HRA and B is involved in the fusion process.

We studied the smooth muscle cell differentiation capability of human placental multipotent mesenchymal stromal cells (hPMSCs) and identified how endothelial cells recruit hPMSCs participating in vessel formation. hPMSCs from term placentas were induced to differentiate into smooth muscle cells under induction conditions and different matrix substrates. We assessed endothelial cells from umbilical veins for platelet-derived growth factor (PDGF)-BB expression and to induce hPMSC PDGFR-beta and STAT3 activation. Endothelial cells were co-cultured with hPMSCs for in vitro angiogenesis. Cell differentiation ability was then further assessed by mouse placenta transplantation assay. hPMSCs can differentiate into smooth muscle cells; collagen type I and IV or laminin support this differentiation. Endothelial cells expressed significant levels of PDGF-BB and activated STAT3 transcriptional activity in hPMSCs. Endothelial cell-conditioned medium induced hPMSC migration, which was inhibited by STAT3 small interfering RNA transfection or by pretreatement with PDGFR-beta-blocking antibody but not by PDGFR-alpha-blocking antibody or isotype immunoglobulin G (IgG; P < 0.001). hPMSCs can incorporate into endothelial cells with tube formation and promote endothelial cells, forming capillary-like networks than endothelial cells alone (tube lengths: 12 024.1 ± 960.1 vs. 9404.2 ± 584.7 pixels; P < 0.001). Capillary-like networks were significantly reduced by hPMSCs pretreated with PDGFR-beta-blocking antibody but not by PDGFR-alpha-blocking antibody or isotype IgG (P < 0.001). Transplantation of hPMSCs into mouse placentas revealed incorporation of the hPMSCs into vessel walls, which expressed alpha-smooth muscle actin, calponin, and smooth muscle myosin (heavy chain) in vivo. In conclusion, endothelial cell-hPMSC interactions occur during vessel development of placenta. Placental endothelial cell-derived PDGF-BB recruits hPMSCs involved in vascular development via PDGFR-beta/STAT3 activation.

The X-linked DEAD-box RNA helicase DDX3 (DDX3X) is a multifunctional protein that has been implicated in gene regulation, cell cycle control, apoptosis, and tumorigenesis. However, the precise physiological function of Ddx3x during development remains unknown. Here, we show that loss of Ddx3x results in an early post-implantation lethality in male mice. The size of the epiblast marked by Oct3/4 is dramatically reduced in embryonic day 6.5 (E6.5) Ddx3x-/Y embryos. Preferential paternal X chromosome inactivation (XCI) in extraembryonic tissues of Ddx3x heterozygous (Ddx3x-/+) female mice with a maternally inherited null allele leads to placental abnormalities and embryonic lethality during development. In the embryonic tissues, Ddx3x exhibits developmental- and tissue-specific differences in escape from XCI. Targeted Ddx3x ablation in the epiblast leads to widespread apoptosis and abnormal growth, which causes embryonic lethality in the Sox2-cre/+;Ddx3xflox/Y mutant around E11.5. The observation of significant increases in γH2AX and p-p53Ser15 indicates DNA damage, which suggests that loss of Ddx3x leads to higher levels of genome damage. Significant upregulation of p21WAF1/Cip1 and p15Ink4b results in cell cycle arrest and apoptosis in Ddx3x-deficient cells. These results have uncovered that mouse Ddx3x is essential for both embryo and extraembryonic development.