Pregnenolone sulfate induces transcriptional and immunoregulatory effects on T cells.

Pregnenolone sulfate is a steroid metabolite of the steroidogenesis precursor, pregnenolone, with similar functional properties, including immunosuppression. We recently reported an elevation in serum levels of pregnenolone sulfate in children with malaria, contributing to an immunosuppressed state. Yet, the molecular mechanisms in which this steroid exerts its immunoregulatory functions are lacking. In this study, we examined the effects of pregnenolone sulfate on T cell viability, proliferation and transcriptome. We observed a pregnenolone sulfate dose-dependent induction of T cell death and reduction in proliferation. RNA sequencing analysis of pregnenolone sulfate-treated T cells for 2 and 24 h revealed the downregulation of pro-inflammatory genes and the upregulation of the steroid nuclear receptor superfamily, NR4A, as early-response genes. We also report a strong activation of the integrated stress response mediated by the upregulation of EIF2AK3. These results contribute to the knowledge on transcriptional regulation driving the immunoregulatory effects of pregnenolone sulfate on T cells.

DNA and protein methyltransferases inhibition by adenosine dialdehyde reduces the proliferation and migration of breast and lung cancer cells by downregulating autophagy.

Protein and DNA methylation is involved in various biological functions such as signal transmission, DNA repair, and gene expression. Abnormal regulation of methyltransferases has been linked to multiple types of cancer, but its link to autophagy and carcinogenesis in breast and lung cancer is not fully understood. We utilized UALCAN, a web tool, to investigate breast and lung cancer database from The Cancer Genome Atlas. We found that 17 methyltransferases are upregulated in breast and/or lung cancer. We investigated the effect of methylation inhibition on two breast cancer cell lines (MDA-MB-231 and MCF-7) and two lung cancer cell lines (H292 and A549) by treating them with the indirect methyltransferase inhibitor adenosine dialdehyde (AdOx). We found that the migration ability of all cell lines was decreased, and the growth rate of MDA-MB-231, MCF-7 and H292 was also decreased after AdOx treatment. These results were correlated with an inhibition of the autophagy in MDA-MB-231, MCF-7 and H292 cell lines, since AdOx treatment induced a decreased expression of ATG7, a reduced ratio LC3-II/LC3-I and an increased p62 level. These findings suggest that inhibiting cells' methylation ability could be a potential target for breast and lung cancer treatment.

Association between visual impairment and sleep duration in college students: A study conducted in UAE and Lebanon.

Objective: To examine whether self-reported sleep duration and visual impairment were associated among College students. Participants: Students (n = 1002, age 17-35 years) from Lebanon and the United Arab Emirates. Methods: Students were asked to complete a validated questionnaire between October 2018 and May 2019. The questions were related to sociodemographics, lifestyle characteristics, visual impairment status, sleeping pattern, mobile-phone use and chronic conditions. Results: 18.3% of the respondents reported to suffer from visual impairment. Among them, 72.7% were females (p < .001), 65% admitted to frequently use mobile phones before sleeping (p < .001), 54.6% reported to sleep less than 7 h (p = .008) and 71.6% reported to suffer from sleep disturbances (p = .05). Visual impairment was associated with poor sleep quality (p < .001), mobile phone use before sleeping (p < .01) and daily stress (p < .05). Conclusion: Visual impairment in college students is associated with short sleep duration, mobile phone use before sleeping and stress level.

Expired medications and disposal practices in Arab households.

OBJECTIVES: Over the past few decades, the accumulation of expired and unused medications in households has become a concern. Most people are unaware of how to properly dispose of unused and/or expired medicines. Our objective was to inspect the extent of expired medications within Arab households in United Arab Emirates (UAE), to determine which therapeutic groups yield greater amounts of unused medications, and evaluate drugs' disposal practices. METHODS: This descriptive study was written in accordance with the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) checklist for cross-sectional studies. It was conducted among Arab households in UAE (n = 503) using an online questionnaire between November 2020 and January 2021. Questions were related to participants' socio-demographics, the prevalence of expired medications in households and their disposal. KEY FINDINGS: Around 58% of the respondents had expired medications in their houses and 74% had drugs that were never used. The most common medicines left unused were analgesics (34%) followed by cosmetics (27%) and antibiotics (26%). More than 42% of expired medications were in solid dosage forms, 28% were semisolid and 24% were liquid dosage forms. The predominant disposal method among the surveyed participants was throwing medications into the garbage (86%). CONCLUSIONS: Large quantities of expired medications in Arab households exist with a high prevalence of analgesics, antibiotics and cosmetics. Arab households are unaware of the proper drug disposal procedures. Therefore, community pharmacists are recommended to offer training on proper medication disposal practices and to encourage the public to return medications to pharmacies.

The Discovery of Potent SHP2 Inhibitors with Anti-Proliferative Activity in Breast Cancer Cell Lines.

Despite available treatments, breast cancer is the leading cause of cancer-related death. Knowing that the tyrosine phosphatase SHP2 is a regulator in tumorigenesis, developing inhibitors of SHP2 in breast cells is crucial. Our study investigated the effects of new compounds, purchased from NSC, on the phosphatase activity of SHP2 and the modulation of breast cancer cell lines' proliferation and viability. A combined ligand-based and structure-based virtual screening protocol was validated, then performed, against SHP2 active site. Top ranked compounds were tested via SHP2 enzymatic assay, followed by measuring IC50 values. Subsequently, hits were tested for their anti-breast cancer viability and proliferative activity. Our experiments identified three compounds 13030, 24198, and 57774 as SHP2 inhibitors, with IC50 values in micromolar levels and considerable selectivity over the analogous enzyme SHP1. Long MD simulations of 500 ns showed a very promising binding mode in the SHP2 catalytic pocket. Furthermore, these compounds significantly reduced MCF-7 breast cancer cells' proliferation and viability. Interestingly, two of our hits can have acridine or phenoxazine cyclic system known to intercalate in ds DNA. Therefore, our novel approach led to the discovery of SHP2 inhibitors, which could act as a starting point in the future for clinically useful anticancer agents.

Linking Alzheimer's Disease and Type 2 Diabetes: Characterization and Inhibition of Cytotoxic Aß and IAPP Hetero-Aggregates.

The cytotoxic self-aggregation of ß-amyloid (Aß) peptide and islet amyloid polypeptide (IAPP) is implicated in the pathogenesis of Alzheimer's disease (AD) and Type 2 diabetes (T2D), respectively. Increasing evidence, particularly the co-deposition of Aß and IAPP in both brain and pancreatic tissues, suggests that Aß and IAPP cross-interaction may be responsible for a pathological link between AD and T2D. Here, we examined the nature of IAPP-Aß40 co-aggregation and its inhibition by small molecules. In specific, we characterized the kinetic profiles, morphologies, secondary structures and toxicities of IAPP-Aß40 hetero-assemblies and compared them to those formed by their homo-assemblies. We demonstrated that monomeric IAPP and Aß40 form stable hetero-dimers and hetero-assemblies that further aggregate into ß-sheet-rich hetero-aggregates that are toxic (cell viability <50%) to both PC-12 cells, a neuronal cell model, and RIN-m5F cells, a pancreatic cell model for ß-cells. We then selected polyphenolic candidates to inhibit IAPP or Aß40 self-aggregation and examined the inhibitory effect of the most potent candidate on IAPP-Aß40 co-aggregation. We demonstrated that epigallocatechin gallate (EGCG) form inter-molecular hydrogen bonds with each of IAPP and Aß40. We also showed that EGCG reduced hetero-aggregate formation and resulted in lower ß-sheets content and higher unordered structures in IAPP-Aß40-EGCG samples. Importantly, we showed that EGCG is highly effective in reducing the toxicity of IAPP-Aß40 hetero-aggregates on both cell models, specifically at concentrations that are equivalent to or are 2.5-fold higher than the mixed peptide concentrations. To the best of our knowledge, this is the first study to report the inhibition of IAPP-Aß40 co-aggregation by small molecules. We conclude that EGCG is a promising candidate to prevent co-aggregation and cytotoxicity of IAPP-Aß40, which in turn, contribute to the pathological link between AD and T2D.

In vivo oral insulin delivery via covalent organic frameworks.

With diabetes being the 7th leading cause of death worldwide, overcoming issues limiting the oral administration of insulin is of global significance. The development of imine-linked-covalent organic framework (nCOF) nanoparticles for oral insulin delivery to overcome these delivery barriers is herein reported. A gastro-resistant nCOF was prepared from layered nanosheets with insulin loaded between the nanosheet layers. The insulin-loaded nCOF exhibited insulin protection in digestive fluids in vitro as well as glucose-responsive release, and this hyperglycemia-induced release was confirmed in vivo in diabetic rats without noticeable toxic effects. This is strong evidence that nCOF-based oral insulin delivery systems could replace traditional subcutaneous injections easing insulin therapy.

Lipopolysaccharide Induces GFAT2 Expression to Promote O-Linked ß-N-Acetylglucosaminylation and Attenuate Inflammation in Macrophages.

Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a reversible posttranslational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway. This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate the protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amidotransferase (GFAT), the enzyme that catalyzes the rate-limiting step of the hexosamine biosynthesis pathway. More specifically, we observed that LPS potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired the LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered a new LPS-inducible gene involved in regulation of protein O-GlcNAcylation, which permits limited exacerbation of inflammation upon macrophage activation.

Endoplasmic Reticulum Stress and Unfolded Protein Response in Neurodegenerative Diseases.

The endoplasmic reticulum (ER) is an important organelle involved in protein quality control and cellular homeostasis. The accumulation of unfolded proteins leads to an ER stress, followed by an adaptive response via the activation of the unfolded protein response (UPR), PKR-like ER kinase (PERK), inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) and activating transcription factor 6 (ATF6) pathways. However, prolonged cell stress activates apoptosis signaling leading to cell death. Neuronal cells are particularly sensitive to protein misfolding, consequently ER and UPR dysfunctions were found to be involved in many neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and prions diseases, among others characterized by the accumulation and aggregation of misfolded proteins. Pharmacological UPR modulation in affected tissues may contribute to the treatment and prevention of neurodegeneration. The association between ER stress, UPR and neuropathology is well established. In this review, we provide up-to-date evidence of UPR activation in neurodegenerative disorders followed by therapeutic strategies targeting the UPR and ameliorating the toxic effects of protein unfolding and aggregation.

An updated view on the functions of caspases in inflammation and immunity.

The binary classification of mammalian caspases as either apoptotic or inflammatory is now obsolete. Emerging data indicate that all mammalian caspases are intricately involved in the regulation of inflammation and immunity. They participate in embryonic and adult tissue homeostasis, control leukocyte differentiation, activation and effector functions, and mediate innate and adaptive immunity signaling. Caspases also promote host resistance by regulating anti-oxidant defense and pathogen clearance through regulation of phagosomal maturation, actin dynamics and phagosome-lysosome fusion. Beyond apoptosis, they regulate inflammatory cell death, eliciting rapid pyroptosis of infected cells, while inhibiting necroptosis-mediated tissue destruction and chronic inflammation. In this review, we describe the cellular and molecular mechanisms underlying non-apoptotic functions of caspases in inflammation and immunity and provide an updated view of their functions as central regulators of tissue homeostasis and host defense.

Non-canonical Wnt induces chondrocyte de-differentiation through Frizzled 6 and DVL-2/B-raf/CaMKIIα/syndecan 4 axis.

Dysregulation of Wnt signaling has been implicated in developmental defects and in the pathogenesis of many diseases such as osteoarthritis; however, the underlying mechanisms are poorly understood. Here, we report that non-canonical Wnt signaling induced loss of chondrocyte phenotype through activation of Fz-6/DVL-2/SYND4/CaMKIIα/B-raf/ERK1/2 cascade. We show that in response to Wnt-3a, Frizzled 6 (Fz-6) triggers the docking of CaMKIIα to syndecan 4 (SYND4) and that of B-raf to DVL-2, leading to the phosphorylation of B-raf by CaMKIIα and activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling, which leads to chondrocyte de-differentiation. We demonstrate that CaMKIIα associates and phosphorylates B-raf in vitro and in vivo. Our study reveals the mechanism by which non-canonical Wnt activates ERK1/2 signaling that induces loss of chondrocyte phenotype, and demonstrates a direct functional relationship between CaMKIIα and B-raf during chondrocyte de-differentiation. The identification of Fz-6, SYND4, and B-raf as novel physiological regulators of chondrocyte phenotype may provide new potential anti-osteoarthritic targets.

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription.

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5'LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.

Regulation of xylosyltransferase I gene expression by interleukin 1ß in human primary chondrocyte cells: mechanism and impact on proteoglycan synthesis.

Xylosyltransferase I (XT-I) is an essential enzyme of proteoglycan (PG) biosynthesis pathway catalyzing the initial and rate-limiting step in glycosaminoglycan chain assembly. It plays a critical role in the regulation of PG synthesis in cartilage; however, little is known about underlying mechanism. Here, we provide evidence that, in human primary chondrocytes, IL-1ß regulates XT-I gene expression into an early phase of induction and a late phase of down-regulation. Based on promoter deletions, the region up to -850 bp was defined as a major element of XT-I gene displaying both constitutive and IL-1ß-regulated promoter activity. Point mutation and signaling analyses revealed that IL-1ß-induced promoter activity is achieved through AP-1 response elements and mediated by SAP/JNK and p38 signaling pathways. Transactivation and chromatin immunoprecipitation assays indicated that AP-1 is a potent transactivator of XT-I promoter and that IL-1ß-induced activity is mediated through increased recruitment of AP-1 to the promoter. Finally, we show that Sp3 is a repressor of XT-I promoter and bring evidence that the repressive effect of IL-1ß during the late phase is mediated through Sp3 recruitment to the promoter. This suggests that modulation of Sp3 in cartilage could prevent IL-1ß inhibition of PG synthesis and limit tissue degradation.