Activated fibroblasts modify keratinocyte stem niche through TET1 and IL-6 to promote their rapid transformation in a mouse model of prenatal arsenic exposure.

Early life exposure to environmental pollutants such as arsenic (As) can increase the risk of cancers in the offspring. In an earlier study, we showed that only prenatal As exposure significantly increases epidermal stem cell proliferation and accelerates skin tumorigenesis in BALB/c mouse offspring. In the present work, we have examined the role of As-conditioned dermal fibroblasts (DFs) in creating pro-tumorigenic niches for Keratinocyte stem cells (KSCs) in the offspring. DFs isolated from prenatally exposed animals showed increased levels of activation markers (α-SMA, Fibronectin, Collagen IV), induction of ten-eleven translocation methylcytosine dioxygenase 1(TET1), and secreted high levels of niche modifying IL-6. This led to enhanced proliferation, migration, and survival of KSCs. Increased IL-6 production in As-conditioned fibroblast was driven through TET1 mediated 5-mC to 5-hmC conversion at -698/-526 and -856/-679 region on its promoter. IL-6 further acted through downstream activation of JAK2-STAT3 signaling, promoting epithelial-to-mesenchymal transition (EMT) in KSCs. Inhibition of pSTAT3 induced by IL-6 reduced the EMT process in KSCs resulting in a significant decrease in their proliferation, migration, and colony formation. Our results indicate that IL-6 produced by prenatally conditioned fibroblasts plays a major role in regulating the KSC niche and promoting skin tumor development in As-exposed offspring.

Prenatal arsenic exposure alters keratinocyte stem cell fate through persistent activation of IGF2R-MAPK cascade leading to aggravated skin carcinogenesis in mice offspring.

Chronic exposure to arsenic (As) promotes skin carcinogenesis in humans and potentially disturbs resident stem cell dynamics, particularly during maternal and early life exposure. In the present study, we demonstrate how only prenatal arsenic exposure disturbs keratinocyte stem cell (KSC) conditioning using a BALB/c mice model. Prenatal As exposure alters the normal stemness (CD34, KRT5), differentiation (Involucrin), and proliferation (PCNA) program in skin of offspring with progression of age as observed at 2, 10, and 18 weeks. Primary KSCs isolated from exposed animal at Day-2 showed increased survival (Bax:Bcl-xL, TUNEL assay), proliferation (BrdU), and differentiation (KRT5, Involucrin) potential through the activation of pro-carcinogenic IGF2R-MAPK cascade (IGF2R-G(α)q-MEK1-ERK1/2). This was associated with reduced enrichment of histone H3K27me3 and its methylase, EZH2 along with increased binding of demethylase, KDM6A at Igf2r promoter. Altered KSCs conditioning through disturbed Igf2r imprint contributed to impaired proliferation and differentiation and an aggravated tumor response in offspring.

Altered Igf2 imprint leads to accelerated adipogenesis and early onset of metabolic syndrome in male mice following gestational arsenic exposure.

Developmental exposure to environmental pollutants has been shown to promote adverse health outcomes in offspring. Exposure to heavy metals such as arsenic which also has endocrine-disrupting activity is being increasingly linked with cancers, diabetes, and lately with Metabolic Syndrome (MetS). In this work, we have assessed the effects of preconceptional plus gestational arsenic exposure on the developmental programming of MetS in offspring. In our study, only gestational arsenic exposure led to reduced birth weight, followed by catch-up growth, adiposity, elevated serum triglycerides levels, and hyperglycemia in male offspring. Significant adipocyte dysfunction was observed in offspring with increased hypertrophy, insulin resistance, and chronic inflammation in epididymal white adipose tissue. Adipose tissue regulates the metabolic health of individuals and its dysfunction resulted in elevated serum levels of metabolism-regulating adipokines (Leptin, Resistin) and pro-inflammatory cytokines (PAI-1, TNFα). The progenitor adipose-derived stem cells (AdSCs) from exposed progeny had increased proliferation and adipogenic potential with excess lipid accumulation. We also found increased activation of Akt, ERK1/2 & p38 MAPK molecules in arsenic-exposed AdSCs along with increased levels of phospho-Insulin-like growth factor-1 receptor (p-IGF1R) and its upstream activator Insulin-like growth factor-2 (IGF2). Overexpression of Igf2 was found to be due to arsenic-mediated DNA hypermethylation at the imprinting control region (ICR) located -2kb to -4.4 kb upstream of the H19 gene which caused a reduction in the conserved zinc finger protein (CTCF) occupancy. This further led to persistent activation of the MAPK signaling cascade and enhanced adipogenesis leading to the early onset of MetS in the offspring.

Chitosan/alginate nanogel potentiate berberine uptake and enhance oxidative stress mediated apoptotic cell death in HepG2 cells.

Biopolymer-based nanoscale drug delivery systems have become a promising approach to overcome the limitations associated with conventional chemotherapeutics used for cancer treatment. Herein, we reported to develop a hydrophilic nanogel (NG) composed of Chitosan (Chi) and sodium alginate (Alg) using the ion gelation method for delivering Berberine hydrochloride (BBR), an alkaloid obtained from Berberis aristata roots. The use of different nanocarriers for BBR delivery has been reported previously, but the bioavailability of these carriers was limited due to phagocytic uptake and poor systemic delivery. The developed NG showed enhanced stability and efficient entrapment of BBR ∼92 %, resulting in a significant increase in bioavailability. The pH-dependent release behavior demonstrated sustained and effective release of ∼86 %, ∼74 % and, ∼53 % BBR at pH 5.5, 6.6, and 7.4 respectively after 72h, indicating its potential as a drug carrier. Additionally, the cellular uptake of BBR was significantly higher ∼19 % in the BBR-NG (25 µM) than in bulk BBR (100 µM), leading to enhanced ROS generation, mitochondrial depolarisation, and inhibition of cell proliferation and colony formation in HepG2 cells. In summary, the results suggest that the Chi/Alg biopolymer-based nano-formulation could be an effective approach for delivering BBR and enhancing its cellular uptake, efficacy, and cytotoxicity.

Assessment of anti-arthritic activity of lipid matrix encased berberine in rheumatic animal model.

The purpose of this study was to evaluate the drug delivery and therapeutic potential of berberine (Br) loaded nanoformulation in rheumatoid arthritis (RA)-induced animal model. The Br-loaded NLCs (nanostructured lipid carriers) were prepared employing melt-emulsification process, and optimised through Box-Behnken design. The prepared NLCs were assessed for in-vitro and in-vivo evaluations. The optimised NLCs exhibited a mean diameter of 180.2 ± 0.31 nm with 88.32 ± 2.43% entrapment efficiency. An enhanced anti-arthritic activity with reduced arthritic scores to 0.66 ± 0.51, reduction in ankle diameter to 5.80 ± 0.27 mm, decline in paw withdrawal timing, and improvements in walking behaviour were observed in the Br-NLCs treated group. The radiographic images revealed a reduction in bone and cartilage deformation. The Br-NLCs showed promising results in the management of RA disease, can be developed as an efficient delivery system at commercial levels, and may be explored for clinical application after suitable experiments in the future.

Downregulation of pro-inflammatory markers IL-6 and TNF-α in rheumatoid arthritis using nano-lipidic carriers of a quinone-based phenolic: an in vitro and in vivo study.

Rheumatoid arthritis (RA) is a joint ailment with multi-factorial immune-mediated degenerative pathogenesis, including genetic and environmental defects. Resistance to disease-modifying anti-rheumatic drugs (DMARDs) happens due to excessive drug efflux over time, rendering the concentration insufficient to elicit a response. Thymoquinone (TQ) is a quinone-based phenolic compound with antioxidant and anti-inflammatory activities that downregulate numerous pro-inflammatory cytokines. However, its pharmaceutical importance and therapeutic utility are underexplored due to intrinsic physicochemical characteristics such as inadequate biological stability, short half-life, low hydrophilicity, and less systemic availability. Tamanu oil-stabilised nanostructured lipid carriers (TQ-NLCs) were prepared and optimised using Box-Behnken design (BBD) with the size of 153.9 ± 0.52 nm and surface charge of -30.71 mV. The % entrapment efficiency and drug content were found to be 84.6 ± 0.50% and 14.75 ± 0.52%, respectively. Furthermore, the TQ-loaded NLCs (TQ-NLCs) assayed for skin permeation for transdermal delivery which significantly (p < 0.05) increased skin enhancement ratio 14.6 times compared to the aqueous solution of TQ. Tamanu oil displayed the synergistic anti-inflammatory potential with TQ in comparison to pure TQ, as evidenced against carrageenan (CRG)-induced paw oedema model and Freund's adjuvant-induced arthritic model. The arthritic and X-ray scores significantly (p < 0.05) reduced in TQ-NLCs and standard formulation-treated groups. Moreover, serum pro-inflammatory marker TNF-α and IL-6 levels were also significantly (p < 0.05) reduced in TQ-NLCs gel-treated group compared to the arthritic control group.

Hcmv-miR-UL148D regulates the staurosporine-induced apoptosis by targeting the Endoplasmic Reticulum to Nucleus signaling 1(ERN1).

The propensity of viruses to co-opt host cellular machinery by reprogramming the host's RNA-interference machinery has been a major focus of research, however, regulation of host defense mechanisms by virus-encoded miRNA, is an additional regulatory realm gaining momentum in the arena of host-viral interactions. The Human Cytomegalovirus (HCMV) miRNAs, regulate many cellular pathways alone or in concordance with HCMV proteins, thereby paving a conducive environment for successful infection in the human host. We show that HCMV miRNA, hcmv-miR-UL148D inhibits staurosporine-induced apoptosis in HEK293T cells. We establish that ERN1 mRNA is a bonafide target of hcmv-miR-UL148D and its encoded protein IRE1α is translationally repressed by the overexpression of hcmv-miR-UL148D resulting in the attenuation of apoptosis. Unlike the host microRNA seed sequence (6-8 nucleotides), hcmv-miR-UL148D has long complementarity to 3' UTR of ERN1 mRNA resulting in mRNA degradation. The repression of IRE1α by the hcmv-miR-UL148D further downregulates Xbp1 splicing and c-Jun N-terminal kinase phosphorylation thus regulating ER-stress and ER-stress induced apoptotic pathways. Strikingly, depletion of ERN1 attenuates staurosporine-induced apoptosis which further suggests that hcmv-miR-UL148D functions through regulation of its target ERN1. These results uncover a role for hcmv-miR-UL148D and its target ERN1 in regulating ER stress-induced apoptosis.

Gestational exposure to silver nanoparticles enhances immune adaptation and protection against streptozotocin-induced diabetic nephropathy in mice offspring.

Silver nanoparticles (AgNPs) possess unique antimicrobial properties. As a result, they are being increasingly used in a wide range of applications. Several studies have shown detrimental effects of AgNPs exposure, including inflammation, accumulation, and cellular damage to different organs. However, the effect of AgNPs exposure during gestation, a critical and susceptible period of human development, on pregnant females and its long-term effects on offspring's health has not been studied. Therefore, we conducted a long-term study where we assessed the effect of gestational AgNPs exposure on pregnant mice and followed their offspring until the age of 12 months. Gestational exposure to AgNPs induced systemic inflammation in the pregnant mice at gestational day (GD) 18. Interestingly, developing fetuses exposed to AgNPs, showed anti-inflammatory conditions as indicated by reduced expression of inflammatory genes in fetal organs at GD 18 and reduced serum levels of TNF-α, IFN-γ, IL-17A, IL-6, and MCP-1 in AgNPs exposed pups at postnatal day (PD) 2. Surprisingly, post-weaning, AgNPs exposed offspring showed a heightened immune activation as shown by upregulation of inflammatory cytokines at PD 28, which persisted till late in life. Moreover, we observed metabolic alterations which persisted until adulthood in mice. To understand the impact of long-term immunometabolic changes on the progression of diabetes and kidney diseases under stressed conditions, we exposed offspring to streptozotocin which revealed a protective role of low-dose gestational AgNPs exposure against streptozotocin-induced diabetes and associated nephropathy.

Prenatal exposure to arsenic promotes sterile inflammation through the Polycomb repressive element EZH2 and accelerates skin tumorigenesis in mouse.

Prenatal and postnatal life stress could be a potent programmer of phenotype or disease state of an individual in the later life. Prenatal arsenic exposure has been shown to promote developmental defects, low birth weight, immunotoxicity and is associated with various cancers including skin cancer in adulthood. To investigate the association between prenatal arsenic exposure and adult life skin carcinogenesis, we used a two-stage cutaneous carcinogenesis model in which BALB/c mice were prenatally exposed to 0.04 mg/kg and 0.4 mg/kg arsenic (As). Exposure to arsenic was sufficient to shorten the tumor latency period and promote epidermal hyperplasia in the offspring upon challenge with dimethylbenz[a]/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA). The levels of inflammatory and tissue microenvironment remodeling factors such as IL-1α and TNF-α were persistently elevated in the skin, and their inhibition through diacerein led to a significant decrease in the tumor response, suggesting their role in tumorigenesis. While there was overexpression of multiple epigenetic regulators at tissue level, we found decreased enrichment of Polycomb repressive complex 2 (PRC2) member EZH2 and H3K27me3 mark at the upstream of the affected inflammatory genes. The higher expression of the inflammatory genes suggests the gene specific selective nature of EZH2 repression which was also associated with increased binding of the activator KDM6a (demethylase). Further, arsenic conditioned basal keratinocytes cells (BKCs) showed increased migration and proliferation along with higher expression of tumor associated cytokines. Inhibition of EZH2 in the BKCs lead to their further upregulation suggesting that BKCs might be the potential cell type for the interaction of EZH2 and inflammatory cytokines. The present study provides new evidence for the role of PRC2 group regulators in inflammatory conditioning and development of skin cancer in offspring prenatally exposed to arsenic.

Prenatal arsenic exposure induces immunometabolic alteration and renal injury in rats.

Arsenic (As) exposure is progressively associated with chronic kidney disease (CKD), a leading public health concern present worldwide. The adverse effect of As exposure on the kidneys of people living in As endemic areas have not been extensively studied. Furthermore, the impact of only prenatal exposure to As on the progression of CKD also has not been fully characterized. In the present study, we examined the effect of prenatal exposure to low doses of As 0.04 and 0.4 mg/kg body weight (0.04 and 0.4 ppm, respectively) on the progression of CKD in male offspring using a Wistar rat model. Interestingly, only prenatal As exposure was sufficient to elevate the expression of profibrotic (TGF-ß1) and proinflammatory (IL-1α, MIP-2α, RANTES, and TNF-α) cytokines at 2-day, 12- and 38-week time points in the exposed progeny. Further, alteration in adipogenic factors (ghrelin, leptin, and glucagon) was also observed in 12- and 38-week old male offspring prenatally exposed to As. An altered level of these factors coincides with impaired glucose metabolism and homeostasis accompanied by progressive kidney damage. We observed a significant increase in the deposition of extracellular matrix components and glomerular and tubular damage in the kidneys of 38-week-old male offspring prenatally exposed to As. Furthermore, the overexpression of TGF-ß1 in kidneys corresponds with hypermethylation of the TGF-ß1 gene-body, indicating a possible involvement of prenatal As exposure-driven epigenetic modulations of TGF-ß1 expression. Our study provides evidence that prenatal As exposure to males can adversely affect the immunometabolism of offspring which can promote kidney damage later in life.

Human Cytomegalovirus miR-UL70-3p Downregulates the H2O2-Induced Apoptosis by Targeting the Modulator of Apoptosis-1 (MOAP1).

Human Cytomegalovirus (HCMV) is a prototypic beta herpesvirus, causing persistent infections in humans. There are medications that are used to treat the symptoms; however, there is no cure yet. Thus, understanding the molecular mechanisms of HCMV replication and its persistence may reveal new prevention strategies. HCMV evasive strategies on the antiviral responses of the human host largely rely on its significant portion of genome. Numerous studies have highlighted the importance of miRNA-mediated regulation of apoptosis, which is an innate immune mechanism that eradicates virus-infected cells. In this study, we explore the antiapoptotic role of hcmv-miR-UL70-3p in HEK293T cells. We establish that hcmv-miR-UL70-3p targets the proapoptotic gene Modulator of Apoptosis-1 (MOAP1) through interaction with its 3'UTR region of mRNA. The ectopic expression of hcmv-miR-UL70-3p mimic significantly downregulates the H2O2-induced apoptosis through the translational repression of MOAP1. Silencing of MOAP1 through siRNA also inhibits the H2O2-induced apoptosis, which further supports the hcmv-miR-UL70-3p mediated antiapoptotic effect by regulating MOAP1 expression. These results uncover a role for hcmv-miR-UL70-3p and its target MOAP1 in regulating apoptosis.

Original article submission: Platelet stress accumulation analysis to predict thrombogenicity of an artificial kidney.

An implantable artificial kidney using a hemofilter constructed from an array of silicon membranes to provide ultrafiltration requires a suitable blood flow path to ensure stable operation in vivo. Two types of flow paths distributing blood to the array of membranes were evaluated: parallel and serpentine. Computational fluid dynamics (CFD) simulations were used to guide the development of the blood flow paths. Pressure data from animal tests were used to obtain pulsatile flow conditions imposed in the transient simulations. A key consideration for stable operation in vivo is limiting platelet stress accumulation to avoid platelet activation and thrombus formation. Platelet stress exposure was evaluated by CFD particle tracking methods through the devices to provide distributions of platelet stress accumulation. The distributions of stress accumulation over the duration of a platelet lifetime for each device revealed that stress accumulation for the serpentine flow path exceeded levels expected to cause platelet activation while the accumulated stress for the parallel flow path was below expected activation levels.