Cutaneous Arsenical Exposure Induces Distinct Metabolic Transcriptional Alterations of Kidney Cells.

Arsenicals are deadly chemical warfare agents that primarily cause death through systemic capillary fluid leakage and hypovolemic shock. Arsenical exposure is also known to cause acute kidney injury, a condition that contributes to arsenical-associated death due to the necessity of the kidney in maintaining whole-body fluid homeostasis. Because of the global health risk that arsenicals pose, a nuanced understanding of how arsenical exposure can lead to kidney injury is needed. We used a nontargeted transcriptional approach to evaluate the effects of cutaneous exposure to phenylarsine oxide, a common arsenical, in a murine model. Here we identified an upregulation of metabolic pathways such as fatty acid oxidation, fatty acid biosynthesis, and peroxisome proliferator-activated receptor (PPAR)-α signaling in proximal tubule epithelial cell and endothelial cell clusters. We also revealed highly upregulated genes such as Zbtb16, Cyp4a14, and Pdk4, which are involved in metabolism and metabolic switching and may serve as future therapeutic targets. The ability of arsenicals to inhibit enzymes such as pyruvate dehydrogenase has been previously described in vitro. This, along with our own data, led us to conclude that arsenical-induced acute kidney injury may be due to a metabolic impairment in proximal tubule and endothelial cells and that ameliorating these metabolic effects may lead to the development of life-saving therapies. SIGNIFICANCE STATEMENT: In this study, we demonstrate that cutaneous arsenical exposure leads to a transcriptional shift enhancing fatty acid metabolism in kidney cells, indicating that metabolic alterations might mechanistically link topical arsenical exposure to acute kidney injury. Targeting metabolic pathways may generate promising novel therapeutic approaches in combating arsenical-induced acute kidney injury.

Microneedle-mediated transdermal delivery of N-acetyl cysteine as a potential antidote for lewisite injury.

Lewisite is a chemical warfare agent intended for use in World War and a potential threat to the civilian population due to presence in stockpiles or accidental exposure. Lewisite-mediated skin injury is characterized by acute erythema, pain, and blister formation. N-acetyl cysteine (NAC) is an FDA-approved drug for acetaminophen toxicity, identified as a potential antidote against lewisite. In the present study, we have explored the feasibility of rapid NAC delivery through transdermal route for potentially treating chemical warfare toxicity. NAC is a small, hydrophilic molecule with limited passive delivery through the skin. Using skin microporation with dissolving microneedles significantly enhanced the delivery of NAC into and across dermatomed human skin in our studies. Microporation followed by application of solution (poke-and-solution) resulted in the highest in vitro delivery (509.84 ± 155.04 µg/sq·cm) as compared to poke-and-gel approach (474.91 ± 70.09 µg/sq·cm) and drug-loaded microneedles (226.89 ± 33.41 µg/sq·cm). The lag time for NAC delivery through poke-and-solution approach (0.23 ± 0.04 h) was close to gel application (0.25 ± 0.02 h), with the highest for drug-loaded microneedles (1.27 ± 1.16 h). Thus, we successfully demonstrated the feasibility of rapid NAC delivery using various skin microporation approaches for potential treatment against lewisite-mediated skin toxicity.

An In Silico Investigation of Pharmacological Modulators and Inflammasomes in Glioblastoma Multiforme.

For the past decades, inflammatory signals have been considered a possible key for pharmacological interventions. There are several compounds and/or molecules that have been known as most promising medication against inflammation and its mediated chronic disorders. Inflammasomes could be recognized as a trigger by detrimental stimuli as pathogenic attack and endogenous signals mediated injury inside the cells. In addition, there has been an inflammatory key mechanism involved in cancers including glioblastoma multiforme (GBM). GBM has been considered the foremost aggressive primary brain tumors in adult stage. There is a scattered beam of light on both cellular and molecular links in inflammation and GBM. However, the immune response of GBM has been characterized extensively by macrophages and lymphocytes related to tumors, and some recent investigations have pinpointed the focus of inflammasomes on the progression of GBM. Nevertheless, risk factors linked with GBM are still debatable. In our study, the most considerable compounds and their bonded and/or targeted proteins have depicted the most promising highlights under in silico condition. Our in silico investigations have revealed a powerful pharmacological agents/compound against inflammasome-mediated GBM.

Mechanism underlying follicular hyperproliferation and oncogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a skin disorder that causes chronic painful inflammation and hyperproliferation, often with the comorbidity of invasive keratoacanthoma (KA). Our research, employing high-resolution immunofluorescence and data science approaches together with confirmatory molecular analysis, has identified that the 5'-cap-dependent protein translation regulatory complex eIF4F is a key factor in the development of HS and is responsible for regulating follicular hyperproliferation. Specifically, eIF4F translational targets, Cyclin D1 and c-MYC, orchestrate the development of HS-associated KA. Although eIF4F and p-eIF4E are contiguous throughout HS lesions, Cyclin D1 and c-MYC have unique spatial localization and functions. The keratin-filled crater of KA is formed by nuclear c-MYC-induced differentiation of epithelial cells, whereas the co-localization of c-MYC and Cyclin D1 provides oncogenic transformation by activating RAS, PI3K, and ERK pathways. In sum, we have revealed a novel mechanism underlying HS pathogenesis of follicular hyperproliferation and the development of HS-associated invasive KA.

NLRP3 inflammasome in rosmarinic acid-afforded attenuation of acute kidney injury in mice.

Cisplatin (CP) is a well-known anticancer drug used to effectively treat various kinds of solid tumors. CP causes acute kidney injury (AKI) and unfortunately, there is no therapeutic approach in hand to prevent AKI. Several signaling pathways are responsible for inducing AKI which leads to inflammation in proximal convoluted tubule cells in the kidney. Furthermore, the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome is involved in the CP-induced AKI. In this study, we investigated therapeutic effects of rosmarinic acid (RA) against inflammation-induced AKI. RA was orally administered at the dose of 100 mg/kg for two consecutive days after 24 h of a single injection of CP at the dose of 20 mg/kg administered intraperitoneally in Swiss albino male mice. Treatment of RA inhibited the activation of NLRP3 signaling pathway by blocking the activated caspase-1 and downstream signal molecules such as IL-1ß and IL18. CP activated HMGB1-TLR4/MyD88 axis was also found to be downregulated with the RA treatment. Activation of nuclear factor-κB and elevated protein expression of cyclooxygenase-2 (COX-2) were also found to be downregulated in RA-treated animals. Alteration of early tubular injury biomarker, kidney injury molecule-1 (KIM-1), was found to be subsided in RA-treated mice. RA has been earlier reported for antioxidant and anti-inflammatory properties. Our findings show that blocking a critical step of inflammasome signaling pathway by RA treatment can be a novel and beneficial approach to prevent the CP-induced AKI.

5'-Cap‒Dependent Translation as a Potent Therapeutic Target for Lethal Human Squamous Cell Carcinoma.

Skin squamous cell carcinomas (SCCs) are a major cause of death in patients who have undergone or will undergo organ transplantation. Moreover, these neoplasms cause significant disease and economic burden and diminish patients' life quality. However, no effective treatment or intervention strategies are available. In this study, we investigated the pathologic role of 5'-cap translation, which is regulated by the formation of a ternary initiation factor complex involving eIF4E, eIF4G, and eIF4A1. We detected increased expression of phosphorylated eIF4E, eIF4G, and eIF4A1 in human and murine skin SCCs. The increase in these ternary initiation factor complex proteins was associated with enhanced eIF4E translation targets cyclin D1 and c-Myc. Conversely, small interfering RNA-mediated depletion of eIF4E in human SCC cells (A431 and SCC-13) reduced eIF4G and proteins that regulate the cell cycle and proliferation. Notably, inhibition of Raf/MAPK/extracellular signal-regulated kinase signaling decreased eIF4E and phosphorylated eIF4E accumulation and significantly diminished cell-cycle gene expression and tumor volume of A431-derived xenograft tumors. Furthermore, disrupting the eIF4E with an allosteric inhibitor of eIF4E and eIF4G binding, 4EGI-1, decreased the eIF4E/eIF4G expression and reduced the proliferation. Finally, combined inhibition of the Raf/MAPK/extracellular signal-regulated kinase axis and eIF4E impaired 5'-cap‒dependent translation and abrogated tumor cell proliferation. These data demonstrate that 5'-cap‒dependent translation is a potential therapeutic target for abrogating lethal skin SCCs in patients who have undergone or will undergo organ transplantation.

Involvement of endoplasmic reticulum stress in amyloid ß (1-42)-induced Alzheimer's like neuropathological process in rat brain.

Amyloid-ß (Aß) accumulation in the brain is a pathological hallmark of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress has been implicated in aetiology of neurodegenerative disorders. We studied the involvement of ER stress in Aß-induced neuronal degeneration in rat brain to correlate it with cellular and molecular modifications in Aß-induced Alzheimer's like neuropathological process. Aß (1-42) (5 µg) was administered by bilateral intracerebroventricular (icv) injection in the brain of adult male Wistar rats. Acetylcholinesterase (AChE) activity and histological alterations were observed in different brain regions. ER stress-associated proteins- glucose regulated protein-78 (GRP78), eukaryotic translation initiation factor-2α (eIF2α) and growth arrest and DNA damage-inducible protein-153 (GADD153), neuronal marker- microtubule associated protein-2 (MAP-2) and microglial protein- ionized calcium binding adaptor molecule-1 (Iba-1) were measured by western blot. Reduced glutathione (GSH), nitrite level and levels of caspase-12 and caspase-3 were also measured. ER stress inhibitor, salubrinal (1 mg/kg, intraperitoneally, ip) was used to assess the specific role of ER stress. Aß (1-42)-induced increase in AChE activity, GRP78 and GADD protein levels, dephosphorylation of eIF2-α and caspase-12 and caspase-3 levels and decrease in GSH and MAP-2 levels were attenuated by salubrinal. Increase in Iba-1 protein and nitrite levels after Aß (1-42) administration were partially attenuated by salubrinal. Aß (1-42)-induced histological alterations were correlated with findings of ER stress. Results of present study implicate ER stress as a potential molecular mechanism in Aß-induced Alzheimer's like neuropathology which could serve as surrogate biomarker for study of AD progression and efficacy of therapeutic interventions for AD management.

Protective role of HO-1 against acute kidney injury caused by cutaneous exposure to arsenicals.

Lewisite and many other similar arsenicals are warfare vesicants developed and weaponized for use in World Wars I and II. These chemicals, when exposed to the skin and other epithelial tissues, cause rapid severe inflammation and systemic damage. Here, we show that topically applied arsenicals in a murine model produce significant acute kidney injury (AKI), as determined by an increase in the AKI biomarkers NGAL and KIM-1. An increase in reactive oxygen species and ER stress proteins, such as ATF4 and CHOP, correlated with the induction of these AKI biomarkers. Also, TUNEL staining of CHOP-positive renal tubular cells suggests CHOP mediates apoptosis in these cells. A systemic inflammatory response characterized by a significant elevation in inflammatory mediators, such as IL-6, IFN-α, and COX-2, in the kidney could be the underlying cause of AKI. The mechanism of arsenical-mediated inflammation involves activation of AMPK/Nrf2 signaling pathways, which regulate heme oxygenase-1 (HO-1). Indeed, HO-1 induction with cobalt protoporphyrin (CoPP) treatment in arsenical-treated HEK293 cells afforded cytoprotection by attenuating CHOP-associated apoptosis and cytokine mRNA levels. These results demonstrate that topical exposure to arsenicals causes AKI and that HO-1 activation may serve a protective role in this setting.

AgNPs from Nigella sativa Control Breast Cancer: An In Vitro Study.

In our current study, we synthesized silver nanoparticles (AgNPs) from an aqueous seed extract of Nigella sativa. The seed extract contains phytochemical compounds including phenols, terpenoids, and flavonoids that may act as reducing agents and are able to convert metal ions to metal nanoparticles. The formation of synthesized AgNPs was characterized using UV-visible spectroscopy, Fourier transform infra-red spectroscopy (FT-IR), scanning electron microscopy (SEM) and energy dispersive analysis of X-rays (EDX). The efficacy of N-AgNPs against human breast cancer (MCF-7) cells was tested. The synthesized AgNPs displayed dose-dependent cytotoxicity (1-200 µg/mL) against MCF-7 cells. Morphological alterations of the cells also appeared as bright field images. Treatment of synthesized AgNPs altered the expression of Bax and Bcl-2 (apoptotic proteins) and COX-2 (inflammatory marker) in MCF-7 cells. To our knowledge, this is the first report demonstrating that N-AgNPs from Nigella sativa can induce apoptosis in MCF-7 cells.

Combined mTORC1/mTORC2 inhibition blocks growth and induces catastrophic macropinocytosis in cancer cells.

The mammalian target of rapamycin (mTOR) pathway, which plays a critical role in regulating cellular growth and metabolism, is aberrantly regulated in the pathogenesis of a variety of neoplasms. Here we demonstrate that dual mTORC1/mTORC2 inhibitors OSI-027 and PP242 cause catastrophic macropinocytosis in rhabdomyosarcoma (RMS) cells and cancers of the skin, breast, lung, and cervix, whereas the effects are much less pronounced in immortalized human keratinocytes. Using RMS as a model, we characterize in detail the mechanism of macropinocytosis induction. Macropinosomes are distinct from endocytic vesicles and autophagosomes in that they are single-membrane bound vacuoles formed by projection, ruffling, and contraction of plasma membranes. They are positive for EEA-1 and LAMP-1 and contain watery fluid but not organelles. The vacuoles then merge and rupture, killing the cells. We confirmed the inhibition of mTORC1/mTORC2 as the underpinning mechanism for macropinocytosis. Exposure to rapamycin, an mTORC1 inhibitor, or mTORC2 knockdown alone had little or reduced effect relative to the combination. We further demonstrate that macropinocytosis depends on MKK4 activated by elevated reactive oxygen species. In a murine xenograft model, OSI-027 reduced RMS tumor growth. Molecular characterization of the residual tumors was consistent with the induction of macropinocytosis. Furthermore, relative to the control xenograft tumors, the residual tumors manifested reduced expression of cell proliferation markers and proteins that drive the epithelial mesenchymal transition. These data indicate a role of mTORC2 in regulating tumor growth by macropinocytosis and suggest that dual inhibitors could help block refractory or recurrent RMS and perhaps other neoplasms and other cancer as well.

Biogenic AgNPs synthesized via endophytic bacteria and its biological applications.

In this present study, the endophytic bacteria were isolated from the drought-tolerant ornamental plant Pennisetum setaceum. The biomass of endophytic bacteria was utilized for the biogenic synthesis of silver nanoparticles (AgNPs). The synthesis of AgNPs was confirmed by UV-Visible and FTIR spectroscopy followed by SEM analysis. The antibacterial studies were performed through MIC, MBC, and biofilm assays. Efficacy of AgNPs against the human breast cancer (MCF-7) cells was also tested, and the IC50 was determined by MTT assay. In our study, we have observed that the synthesized AgNPs exhibited a dose-dependent cytotoxicity (1-100 µg/mL) against MCF-7 cells and morphological alterations of the cells were also visualized and the IC50 was observed at 50 µg/mL. The treatment of synthesized AgNPs altered the expression of apoptotic proteins including Bax, Bcl-2, and inflammatory marker COX-2 in MCF-7 cells. To the best of our knowledge, this is the first report that demonstrates the AgNPs from endophytic bacteria isolated from the plant Pennisetum setaceum can induce apoptosis in human breast cancer MCF-7 cells. Our results suggest that AgNPs used in this study can be utilized to control human pathogens and can also be utilized to induce apoptosis in breast cancer cells.