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Copyright of West Indian Medical Journal is the property of West Indian Medical Journal (WIMJ) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Current N 6 -methyladenosine (m 6 A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m 6 A RNA immunoprecipitation and sequencing (picoMeRIP-seq) for studying m 6 A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m 6 A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos., (© 2023. The Author(s).)

The human extraembryonic mesoderm (EXM) is an important tissue in the postimplantation embryo which is specified before gastrulation in primates but not in rodents. EXM is mesenchymal and plays an important role in embryogenesis, including early erythropoiesis, and provides mechanical support to the developing embryo. Recently, it has been shown that self-renewing extraembryonic mesoderm cells (EXMCs) can be modeled in vitro by using human naive pluripotent stem cells. Here, we present a detailed step-by-step protocol to induce EXMCs from naive pluripotent stem cells in vitro., (© 2023. Springer Science+Business Media, LLC.)

A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here, we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. EXMCs are mesenchymal, can arise from an epiblast intermediate, and are capable of self-renewal. Thus, EXMCs arising via primate-specific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects., Competing Interests: Declaration of interests The Institute for Molecular Biotechnology, Austrian Academy of Sciences has filed patent application EP21151455.9 describing the protocols for human blastoid formation. H.K. and N.R. are the inventors of this patent. All other authors declare no competing interests. J.R. is a member of the Cell Stem Cell advisory board., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Establishment of the pluripotency regulatory network in somatic cells by introducing four transcription factors [octamer binding transcription factor 4 (OCT4; also known as POU5F1), sex determining region Y (SRY)-box 2 (SOX2), Kruppel-like factor 4 (KLF4) and cellular myelocytomatosis (c-MYC)] provides a promising tool for cell-based therapies in regenerative medicine. Nevertheless, the mechanisms at play when generating induced pluripotent stem cells from somatic cells are only partly understood. Here, we show that the RNA-specific N6-methyladenosine (m6A) demethylase ALKBH5 regulates somatic cell reprogramming in a stage-specific manner through its catalytic activity. Knockdown or knockout of Alkbh5 in the early reprogramming phase impairs reprogramming efficiency by reducing the proliferation rate through arresting the cells at G2/M phase and decreasing the upregulation of epithelial markers. On the other hand, ALKBH5 overexpression at the early reprogramming phase has no significant impact on reprogramming efficiency, whereas overexpression at the late phase enhances reprogramming by stabilizing Nanog transcripts, resulting in upregulated Nanog expression. Our study provides mechanistic insight into the crucial dynamic role of ALKBH5, mediated through its catalytic activity, in regulating somatic cell reprogramming at the post-transcriptional level. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Induction of pluripotency with defined factors (octamer-binding transcription factor 4 [Oct4], SRY (sex determining region Y)-box 2 [Sox2], Kruppel-like factor 4 [Klf4], c-Myc) raises hopes for successful clinical trials. Despite considerable efforts, the molecular mechanism of reprogramming remains poorly understood. The aim of the present study was to identify the role of calcineurin/nuclear factor of activated T cells (NFAT) in reprogramming. Our results demonstrated a biphasic role for calcineurin/NFAT signaling during reprogramming. In the early phase of reprogramming, calcineurin activity is required to maintain proper cell cycle division and for mesenchymal-epithelial transition. In the late phase, calcineurin exerts a negative effect that is mediated by NFATc2. NFATc2 interacts with Hdac3, Ezh2, and Suv39h1 to increase H3K9me3 and H3K27me3 over the Sox2 enhancer and Klf2 promoter, respectively, resulting in the downregulation of their expression. Moreover, Gαq was identified as a positive upstream regulator for calcineurin. The Gαq/calcineurin/NFATc2 axis negatively regulates the late step of reprogramming. By inhibiting NFATc2 or calcineurin, induced pluripotent stem cells could be established without exogenous Sox2. Thus, the present study revealed another regulatory level of reprogramming, and proposes a biological axis that could be useful for cancer therapy. Stem Cells 2017;35:1162-1175., (© 2017 AlphaMed Press.)

Many helminthic and protozoal infections have been implicated in iron deficiency anemia (IDA) but few reports have suggested a link between Blastocystis sp. infection and IDA. Herein, we investigated the frequency and the association of the Blastocystis sp. genotype with IDA. Two-hundred and six stool samples were examined for Blastocystis sp. Samples were obtained from 96 cases with a confirmed diagnosis of IDA and 110 matched non-anemic controls. The prevalence of the parasite was significantly higher in the IDA group (54.2%) when compared to controls (17.3%) and was 34.5% in all study subjects. Thus, a relationship between Blastocystis sp. infection and IDA was confirmed. PCR amplification of isolates from cases with IDA and controls using subtype-specific sequenced-tagged site primers found that subtype 3 was the most common (83.3%), followed by subtype 1 (16.7%), and both had similar prevalence in both groups. Therefore, there was no correlation found between the Blastocystis sp. genotype and the occurrence of IDA.

Knowledge of the prevalence of Toxoplasma gondii infection in pregnant women would be a valuable approach for planning appropriate preventive strategies. However, no enough data currently exist as to the magnitude of T. gondii infection among pregnant women in Egypt. In view of paucity of reports on this issue, the current cross-sectional study aimed to determine the rate of T. gondii infection and maternal-fetal transmission in antenatal population in Menoufia governorate, Egypt. Out of 323 pregnant women who were screened for Toxoplasma-specific IgG and IgM antibodies with ELFA, 218 samples were IgG seropositive, of which, 9 samples were IgM seropositive. Therefore, seropositivity rates of 67.5% (95% CI: 62.39%, 72.61%), and 2.8% (95% CI: 1%, 4.6%) for IgG and IgM, respectively were found. Analysis of serological patterns revealed that a large proportion of subjects were immune to Toxoplasma infection (IgG+/IgM-), as prevalence of chronic infection was 64.7% (209 cases), and 32.5% of cases were susceptible to primary infection (IgG-/IgM-). Meanwhile, the rate of probable acute Toxoplasma infection (IgG+/IgM+) was 2.8%, with one case (0.3%) confirmed for recent infection, as she had low avidity index with positive amniotic fluid analysis with both PCR and mouse inoculation. Significant relations were seen between Toxoplasma-specific IgG and knowledge about transmission modes, consumption of milk/milk products and unwashed raw vegetable/fruit, hand hygiene, contact with soil and farm animals. In conclusion, we reported high prevalence for T. gondii infection among antenatal population in Menoufia governorate. Thus, the susceptibility for toxoplasmosis is relatively low. Sources of infection revealed herein might represent potential threats for primary infection in seronegative women. Accordingly, there is urgent need for implementation of health education programs as an appropriate approach for prevention., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Intestinal parasitic infection increases the risk of developing iron deficiency anemia (IDA) during pregnancy. The objective of this study was to assess Blastocystis hominis as a contributing risk factor for development of IDA in pregnant women. A total of 200 fecal specimens from 120 pregnant women with IDA (mean Hb = 9.6 g/dl), and 80 non-anemic controls were examined for Blastocystis. Fecal specimens were examined by the formalin/ethyl-acetate concentration technique, iron hematoxylin staining, modified Ziehl-Neelsen acid-fast staining, and by the in vitro cultivation technique for Blastocystis. Frequency of Blastocystis infection, detected microscopically and by the in vitro culture technique, was significantly higher in IDA study group (n = 48; 40%) compared to non-anemic controls (n = 5; 6.3%; P < 0.0001), and 26.5% (n = 53) in all study subjects. Among the 48 cases, Blastocystis without other intestinal parasitic infections was detected in 41 cases (34.2%), while seven cases (5.8%) with Blastocystis were coinfected with other intestinal parasites which included Giardia and Cryptosporidium (1.7% each), and Entamoeba sp., Ascaris, and Trichuris (0.8% each). The mean Hb level of the 48 Blastocystis-infected cases was 9.2 g/dl (mild anemia). While the other 72 IDA cases with no infection had mean Hb of 10.0 g/dl (mild anemia), with a significant difference in mean Hb level between Blastocystis-infected and the non-infected IDA cases (P < 0.0001). Furthermore, among the 48 Blastocystis-infected IDA cases, the mean Hb of the 41 Blastocystis-infected cases without other intestinal parasitic co-infection was 9.1 g/dl (mild anemia), while the mean Hb level of the 7 Blastocystis-infected cases with other intestinal parasitic co-infection was 8.7 g/dl (moderate anemia). Findings of the current study showed that B. hominis infection contributes to the development of IDA in pregnant women. Hence, parasitological diagnostic tests are recommended in routine examination at all antenatal clinics.