Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells.

Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Endocytosis of red blood cell extracellular vesicles by macrophages leads to cytoplasmic heme release and prevents foam cell formation in atherosclerosis.

Extracellular vesicles (EVs) can be produced from red blood cells (RBCs) on a large scale and used to deliver therapeutic payloads efficiently. However, not much is known about the native biological properties of RBCEVs. Here, we demonstrate that RBCEVs are primarily taken up by macrophages and monocytes. This uptake is an active process, mediated mainly by endocytosis. Incubation of CD14+ monocytes with RBCEVs induces their differentiation into macrophages with an Mheme-like phenotype, characterized by upregulation of heme oxygenase-1 (HO-1) and the ATP-binding cassette transporter ABCG1. Moreover, macrophages that take up RBCEVs exhibit a reduction in surface CD86 and decreased secretion of TNF-α under inflammatory stimulation. The upregulation of HO-1 is attributed to heme derived from haemoglobin in RBCEVs. Heme is released from internalized RBCEVs in late endosomes and lysosomes via the heme transporter, HRG1. Consequently, RBCEVs exhibit the ability to attenuate foam cell formation from oxidized low-density lipoproteins (oxLDL)-treated macrophages in vitro and reduce atherosclerotic lesions in ApoE knockout mice on a high-fat diet. In summary, our study reveals the uptake mechanism of RBCEVs and their delivery of heme to macrophages, suggesting the potential application of RBCEVs in the treatment of atherosclerosis.

Effects of acute severe acute respiratory syndrome coronavirus 2 infection on male hormone profile, ACE2 and TMPRSS2 expression, and potential for transmission of severe acute respiratory syndrome coronavirus 2 in semen of Asian men.

OBJECTIVE: To confirm if severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen of men with acute coronavirus disease 2019 and if their male hormone profile (testosterone, follicle-stimulating hormone, luteinizing hormone, sex hormone binding globulin, and free androgen index) is adversely affected during the acute phase of infection and any relation to the ACE2 and/or TMPRSS2 expression in human semen. DESIGN: Clinical study. SETTING: National University Hospital, Singapore. PATIENTS: Asian men aged 21-55 years who were admitted to National University Hospital, Singapore, with a laboratory-confirmed diagnosis of SARS-CoV-2 infection via nasopharyngeal swab in the acute phase of the infection, within 2-14 days of the development of symptoms or contact history, were recruited for the study. INTERVENTIONS: Blood was collected in the morning to assess the male hormone profile. Human semen were obtained by masturbation and sent to the molecular diagnostic laboratories to detect the presence of SARS-CoV-2 RNA and assess the ACE2 and TMPRSS2 expression. MAIN OUTCOME MEASURES: Male hormone profile level and expression of SARS-CoV-2 RNA, ACE2, and TMPRSS2 in human semen. RESULTS: A total of 63 men of Asian ethnicities agreed to participate in the study. Subsequently, 65% of recruited men had completely normal levels of male hormone profile. Moreover, 27% were noted to have higher luteinizing hormone levels between 6.6 and 16.1 IU/L (normal range, 0.8-6.1 IU/L), and 10% had higher follicle-stimulating hormone levels between 13.6 and 41.6 IU/L (normal range, 1.5-12.4 IU/L); all had normal testosterone levels. No SARS-CoV-2 RNAs were detected in all human semen. The ACE2 and TMPRSS2 expression was undetectable in 26 samples, whereas 23 samples only had a detectable TMPRSS2 expression and 4 only had an ACE2 expression. The remaining 3 expressed both ACE2 and TMPRSS2. CONCLUSIONS: Severe acute respiratory syndrome coronavirus 2 could not be found in the semen of a cohort of young to middle-aged Asian men with mild acute SARS-CoV-2 infection. However, there was a detectable expression of ACE2 and TMPRSS2 in semen, although not causal, and it may be correlated to changes in male hormone profiles and male age.

Extracellular vesicles and lipoproteins - Smart messengers of blood cells in the circulation.

Blood cell-derived extracellular vesicles (BCEVs) and lipoproteins are the major circulating nanoparticles in blood that play an important role in intercellular communication. They have attracted significant interest for clinical applications, given their endogenous characteristics which make them stable, biocompatible, well tolerated, and capable of permeating biological barriers efficiently. In this review, we describe the basic characteristics of BCEVs and lipoproteins and summarize their implications in both physiological and pathological processes. We also outline well accepted workflows for the isolation and characterization of these circulating nanoparticles. Importantly, we highlight the latest progress and challenges associated with the use of circulating nanoparticles as diagnostic biomarkers and therapeutic interventions in multiple diseases. We spotlight novel engineering approaches and designs to facilitate the development of these nanoparticles by enhancing their stability, targeting capability, and delivery efficiency. Therefore, the present work provides a comprehensive overview of composition, biogenesis, functions, and clinical translation of circulating nanoparticles from the bench to the bedside.

SRSF3 maintains transcriptome integrity in oocytes by regulation of alternative splicing and transposable elements.

The RNA-binding protein SRSF3 (also known as SRp20) has critical roles in the regulation of pre-mRNA splicing. Zygotic knockout of Srsf3 results in embryo arrest at the blastocyst stage. However, SRSF3 is also present in oocytes, suggesting that it might be critical as a maternally inherited factor. Here we identify SRSF3 as an essential regulator of alternative splicing and of transposable elements to maintain transcriptome integrity in mouse oocyte. Using 3D time-lapse confocal live imaging, we show that conditional deletion of Srsf3 in fully grown germinal vesicle oocytes substantially compromises the capacity of germinal vesicle breakdown (GVBD), and consequently entry into meiosis. By combining single cell RNA-seq, and oocyte micromanipulation with steric blocking antisense oligonucleotides and RNAse-H inducing gapmers, we found that the GVBD defect in mutant oocytes is due to both aberrant alternative splicing and derepression of B2 SINE transposable elements. Together, our study highlights how control of transcriptional identity of the maternal transcriptome by the RNA-binding protein SRSF3 is essential to the development of fertilized-competent oocytes.

The STAT3 Target Mettl8 Regulates Mouse ESC Differentiation via Inhibiting the JNK Pathway.

The capacity of embryonic stem cells (ESCs) to differentiate into all lineages of mature organism is precisely regulated by cellular signaling factors. STAT3 is a crucial transcription factor that plays a central role in maintaining ESC identity. However, the underlying mechanism by which STAT3 directs differentiation is still not completely understood. Here, we show that STAT3 positively regulates gene expression of methyltransferase-like protein 8 (Mettl8) in mouse ESCs. We found that METTL8 is dispensable for pluripotency but affects ESC differentiation. Subsequently, we discovered that METTL8 interacts with Mapkbp1's mRNA, which is an intermediate factor in c-Jun N-terminal kinase (JNK) signaling, and inhibits the translation of the mRNA. Thereby, METTL8 prohibits the activation of JNK signaling and enhances the differentiation of mouse ESCs. Collectively, our study uncovers a STAT3 target, Mettl8, which regulates mouse ESC differentiation via JNK signaling.

Stella modulates transcriptional and endogenous retrovirus programs during maternal-to-zygotic transition.

The maternal-to-zygotic transition (MZT) marks the period when the embryonic genome is activated and acquires control of development. Maternally inherited factors play a key role in this critical developmental process, which occurs at the 2-cell stage in mice. We investigated the function of the maternally inherited factor Stella (encoded by Dppa3) using single-cell/embryo approaches. We show that loss of maternal Stella results in widespread transcriptional mis-regulation and a partial failure of MZT. Strikingly, activation of endogenous retroviruses (ERVs) is significantly impaired in Stella maternal/zygotic knockout embryos, which in turn leads to a failure to upregulate chimeric transcripts. Amongst ERVs, MuERV-L activation is particularly affected by the absence of Stella, and direct in vivo knockdown of MuERV-L impacts the developmental potential of the embryo. We propose that Stella is involved in ensuring activation of ERVs, which themselves play a potentially key role during early development, either directly or through influencing embryonic gene expression.

A genetic and developmental pathway from STAT3 to the OCT4-NANOG circuit is essential for maintenance of ICM lineages in vivo.

Although it is known that OCT4-NANOG are required for maintenance of pluripotent cells in vitro, the upstream signals that regulate this circuit during early development in vivo have not been identified. Here we demonstrate, for the first time, signal transducers and activators of transcription 3 (STAT3)-dependent regulation of the OCT4-NANOG circuitry necessary to maintain the pluripotent inner cell mass (ICM), the source of in vitro-derived embryonic stem cells (ESCs). We show that STAT3 is highly expressed in mouse oocytes and becomes phosphorylated and translocates to the nucleus in the four-cell and later stage embryos. Using leukemia inhibitory factor (Lif)-null embryos, we found that STAT3 phosphorylation is dependent on LIF in four-cell stage embryos. In blastocysts, interleukin 6 (IL-6) acts in an autocrine fashion to ensure STAT3 phosphorylation, mediated by janus kinase 1 (JAK1), a LIF- and IL-6-dependent kinase. Using genetically engineered mouse strains to eliminate Stat3 in oocytes and embryos, we firmly establish that STAT3 is essential for maintenance of ICM lineages but not for ICM and trophectoderm formation. Indeed, STAT3 directly binds to the Oct4 and Nanog distal enhancers, modulating their expression to maintain pluripotency of mouse embryonic and induced pluripotent stem cells. These results provide a novel genetic model of cell fate determination operating through STAT3 in the preimplantation embryo and pluripotent stem cells in vivo.

Upregulation of secretory connective tissue growth factor (CTGF) in keratinocyte-fibroblast coculture contributes to keloid pathogenesis.

Connective tissue growth factor (CTGF) plays a critical role in keloid pathogenesis by promoting collagen synthesis and deposition. Previous work suggested epithelial-mesenchymal interactions as a plausible factor affecting the expression of various growth factors and cytokines by both the epithelial and dermal mesenchymal cells. The aim of this study is to explore the role of epithelial-mesenchymal interactions in modulating CTGF expression. Immunohistochemistry was employed to check CTGF localization in skin tissue. Western blot assay was performed on total protein extracts from skin tissue, cell lysates and conditioned media to detect the basal/expression levels of CTGF. Study groups were subjected to serum stimulation (fibroblast-single cell culture) and pharmacological inhibitors targeted against mTOR (Rapamycin), Sp1 (WP631 and Mitoxanthrone), Smad3 (SB431542), and PI3K (LY294002). Increased localization of CTGF in the basal layer of keloid epidermis and higher expression of CTGF was observed in the keloid tissue extract. Interestingly, lower basal levels of CTGF was observed in fibroblast cell lysates cocultured with keloid keratinocytes compared to normal keratinocytes, while the conditioned media from the former culture consistently demonstrated a higher expression of secreted CTGF as compared to the latter group. These results demonstrate an important role of epithelial-mesenchymal interactions in the regulation of CTGF expression. Fibroblasts treated with inhibitors against mTOR, Sp1, Smad3, and PI3K demonstrated a reduced expression of CTGF, suggesting these signaling pathways to be important in the regulation of CTGF expression. Thus, revealing the therapeutic potentials for inhibitors that are selective for these factors in controlling CTGF expression in fibrotic conditions.