Therapeutic strategies for miRNA delivery to reduce hepatocellular carcinoma.

Malignancies of hepatocellular carcinoma (HCC) are rapidly spreading and commonly fatal. Like most cancers, the gene expression patterns in HCC vary significantly from patient to patient. Moreover, the expression networks during HCC progression are largely controlled by microRNAs (miRNAs) regulating multiple oncogenes and tumor supressors. Therefore, miRNA-based therapeutic strategies altering these networks may significantly influence the cellular behavior enough for them to cure HCC. However, the most substantial challenges in developing such therapies are the stability of the oligos themselves and that of their delivery systems. Here we provide a comprehensive update describing various miRNA delivery systems, including virus-based delivery and non-viral delivery. The latter may be achieved using inorganic nanoparticles, polymer based nano-carriers, lipid-based vesicles, exosomes, and liposomes. Leaky vasculature in HCC-afflicted livers helps untargeted nanocarriers to accumulate in the tumor tissue but may result in side effects during higher dose of treatment. On the other hand, the strategies for actively targeting miRNA therepeutics to cancerous cells through nano-conjugates or vesicles by decorating their surface with antibodies against or ligands for HCC-specific antigens or receptors are more efficient in preventing damage to healthy tissue and cancer recurrence.

Transcriptome wide functional analysis of HBx expressing human hepatocytes stimulated with endothelial cell cross-talk.

Identification of genes dysregulated during the hepatitis B virus (HBV)-host cell interaction adds to the understanding of underlying molecular mechanisms and aids in discovering effective therapies to improve prognosis in hepatitis B virus (HBV)-infected individuals. Through bioinformatics analyses of transcriptomics data, this study aimed to identify potential genes involved in the cross-talk of human hepatocytes expressing the HBV viral protein HBx with endothelial cells. Transient transfection of HBV viral gene X (HBx) was performed in THLE2 cells using pcDNA3 constructs. Through mRNA Sequencing (RNA Seq) analysis, differentially expressed genes (DEGs) were identified. THLE2 cells transfected with HBx (THLE2x) were further treated with conditioned medium from cultured human umbilical vein derived endothelial cells (HUVEC-CM). Gene Ontology (GO) enrichment analysis revealed that interferon and cytokine signaling pathways were primarily enriched for the downregulated DEGs in THLE2x cells treated with HUVEC-CM. One significant module was selected following protein-protein interaction (PPI) network generation, and thirteen hub genes were identified from the module. The prognostic values of the hub genes were evaluated using Kaplan-Meier (KM) plotter, and three genes (IRF7, IFIT1, and IFITM1) correlated with poor disease specific survival (DSS) in HCC patients with chronic hepatitis. A comparison of the DEGs identified in HUVEC-stimulated THLE2x cells with four publicly available HBV-related HCC microarray datasets revealed that PLAC8 was consistently downregulated in all four HCC datasets as well as in HUVEC-CM treated THLE2x cells. KM plots revealed that PLAC8 correlated with worse relapse free survival and progression free survival in HCC patients with hepatitis B virus infection. This study provided molecular insights which may help develop a deeper understanding of HBV-host stromal cell interaction and open avenues for future research.

Divergent effect of Birinapant, and BV6 SMAC mimetic on TNFα induced NF-κB signaling and cell viability in activated hepatic stellate cells.

BACKGROUND: Tumor necrosis factor-α (TNFα) is a pleiotropic cytokine involved in nuclear factor kappa B (NF-κB) mediated cell survival as well as cell death. High serum TNFα levels correlate with liver fibrosis and enhance hepatic stellate cell (HSC) viability. However, the regulatory role of cellular inhibitor of apoptosis-1/2 (cIAP1/2) during TNFα induced NF-κB signaling in activated HSCs is largely unknown. METHOD AND RESULTS: Activated HSCs were treated with cIAP1/2 inhbitiors i.e., SMAC mimetic BV6, and Birinapant in the presence of TNFα and macrophage conditioned media. TNFα cytokine increased cIAP2 expression and enhanced cell viability through the canonical NF-κB signaling in activated HSCs. cIAP2 inhibition via BV6 decreased the TNFα induced canonical NF-κB signaling, and reduced cell viability in activated HSCs. SMAC mimetic, Birinapant alone did not affect the cell viability but treatment of TNFα sensitized HSCs with Birinapant induced cell death. While BV6 mediated cIAP2 ablation was able to decrease the TNFα induced canonical NF-κB signaling, this effect was not observed with Birinapant treatment. Secreted TNFα from M1 polarized macrophages sensitized activated HSCs to BV6 or Birinapant mediated cell death. However, M2 polarized macrophage conditioned medium rescued the activated HSCs from BV6 mediated cytotoxicity. CONCLUSION: In this study, we describe the regulatory role of cIAP2 in TNFα induced NF-κB signaling in activated HSCs. Targeting cIAP2 may be a promising approach for liver fibrosis treatment via modulating NF-κB signaling in activated HSCs.

Context dependent role of p53 during the interaction of hepatocellular carcinoma and endothelial cells.

BACKGROUND: During the progression of hepatocellular carcinoma (HCC), several angiogenic factors are overexpressed in the hepatic microenvironment, which play a critical role in governing the phenotype of the endothelial cells. Mutation in the p53 gene (TP53) is a common event in HCC that may dysregulate the angiogenic signals. However, their functional messages remain largely unexplored at the onset of metastasis. METHODS: Role of p53 was studied by siRNA mediated silencing of p53 in HepG2 cells (WTp53), collecting and analyzing their conditioned medium, followed by indirect co-culture with endothelial cells (HUVECs). Gene and protein expression in HCC cells and endothelial cells was studied by RT-qPCR and western blotting respectively. ß-catenin protein expression and localization were analyzed by immunocytochemistry. RESULTS: We have studied a cell-to-cell interaction model to investigate the crosstalk of endothelial and hepatoma cells by either knocking down p53 or by using p53 null low metastatic HCC cell line. In the absence of p53, the HCC cells influence the migration and vascular network formation of endothelial cells through paracrine signaling of VEGF. Secretory VEGF activated the VEGF receptor-2 along with the survival signaling in endothelial cells. However, the ß-catenin signal is upregulated in endothelial cells only during interaction with metastatic set up irrespective of absence and presence of p53, indicating context-dependent participation of p53 during communication between hepatoma cells and endothelial cells. CONCLUSION: This study highlights that the role of p53 on cellular responses during interaction of hepatocellular carcinoma and endothelial cells is distinct to cell types and context.

Transition of amyloid/mutant p53 from tumor suppressor to an oncogene and therapeutic approaches to ameliorate metastasis and cancer stemness.

The tumor suppressor p53 when undergoes amyloid formation confers several gain-of-function (GOF) activities that affect molecular pathways crucial for tumorigenesis and progression like some of the p53 mutants. Even after successful cancer treatment, metastasis and recurrence can result in poor survival rates. The major cause of recurrence is mainly the remnant cancer cells with stem cell-like properties, which are resistant to any chemotherapy treatment. Several studies have demonstrated the role of p53 mutants in exacerbating cancer stemness properties and epithelial-mesenchymal transition in these remnant cancer cells. Analyzing the amyloid/mutant p53-mediated signaling pathways that trigger metastasis, relapse or chemoresistance may be helpful for the development of novel or improved individualized treatment plans. In this review, we discuss the changes in the metabolic pathways such as mevalonate pathway and different signaling pathways such as TGF-ß, PI3K/AKT/mTOR, NF-κB and Wnt due to p53 amyloid formation, or mutation. In addition to this, we have discussed the role of the regulatory microRNAs and lncRNAs linked with the mutant or amyloid p53 in human malignancies. Such changes promote tumor spread, potential recurrence, and stemness. Importantly, this review discusses the cancer therapies that target either mutant or amyloid p53, restore wild-type functions, and exploit the synthetic lethal interactions with mutant p53.

Novel thiophene Chalcones-Coumarin as acetylcholinesterase inhibitors: Design, synthesis, biological evaluation, molecular docking, ADMET prediction and molecular dynamics simulation.

A series of around eight novel chalcone based coumarin derivatives (23a-h) was designed, subjected to in-silico ADMET prediction, synthesized, characterized by IR, NMR, Mass analytical techniques and evaluated as acetylcholinesterase (AChE) inhibitor for the treatment of Alzheimer's disease (AD). The results of predicted ADMET study demonstrated the drug-likeness properties of the titled compounds with developmental challenges in lipophilicity and solubility parameters. The in vitro assessment of the synthesized compounds revealed that all of them showed significant activity (IC50 ranging from 0.42 to 1.296 µM) towards AChE compared to the standard drug, galantamine (IC50 = 1.142 ±â€¯0.027 µM). Among these, compound 23e displayed the most potent inhibitory activity with IC50 value of 0.42 ±â€¯0.019 µM. Cytotoxicity of all compounds was tested on normal human hepatic (THLE-2) cell lines at three different concentrations using the MTT assay, in which none of the compound showed significant toxicity at the highest concentration of 1000 µg/ml compared to the control group. Based on the docking study against AChE, the most active derivative 23e was orientated towards the active site and occupied both catalytic anionic site (CAS) and peripheral anionic site (PAS) of the target enzyme. In-silico studies revealed tested showed better inhibition activity of AChE compared to Butyrylcholinesterase (BuChE). Molecular dynamics simulation explored the stability and dynamic behavior of 23e- AChE complex.

Z-Guggulsterone Is a Potential Lead Molecule of Dawa-ul-Kurkum against Hepatocellular Carcinoma.

An ancient saffron-based polyherbal formulation, Dawa-ul-Kurkum (DuK), has been used to treat liver ailments and other diseases and was recently evaluated for its anticancer potential against hepatocellular carcinoma (HCC) by our research team. To gain further insight into the lead molecule of DuK, we selected ten active constituents belonging to its seven herbal constituents (crocin, crocetin, safranal, jatamansone, isovaleric acid, cinnamaldehyde, coumaric acid, citral, guggulsterone and dehydrocostus lactone). We docked them with 32 prominent proteins that play important roles in the development, progression and suppression of HCC and those involved in endoplasmic reticulum (ER) stress to identify the binding interactions between them. Three reference drugs for HCC (sorafenib, regorafenib, and nivolumab) were also examined for comparison. The in silico studies revealed that, out of the ten compounds, three of them-viz., Z-guggulsterone, dehydrocostus lactone and crocin-showed good binding efficiency with the HCC and ER stress proteins. Comparison of binding affinity with standard drugs was followed by preliminary in vitro screening of these selected compounds in human liver cancer cell lines. The results provided the basis for selecting Z-guggulsterone as the best-acting phytoconstituent amongst the 10 studied. Further validation of the binding efficiency of Z-guggulsterone was undertaking using molecular dynamics (MD) simulation studies. The effects of Z-guggulsterone on clone formation and cell cycle progression were also assessed. The anti-oxidant potential of Z-guggulsterone was analyzed through DPPH and FRAP assays. qRTPCR was utilized to check the results at the in vitro level. These results indicate that Z-guggulsterone should be considered as the main constituent of DuK instead of the crocin in saffron, as previously hypothesized.

Targeted delivery of microRNA-199a-3p using self-assembled dipeptide nanoparticles efficiently reduces hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is an aggressive tumor with limited systemic and locoregional modalities of treatment. Although microRNA (miRNA) based therapies have significant potential, their targeted delivery remains a major challenge. miR-199a-3p functions as an important tumor suppressor in HCC, which regulates various cellular processes. Recently, peptide-based nanoparticles (NPs) have been developed to deliver oligonucleotides including miRNA. Here, we describe the synthesis and characterization of arginine α,ß-dehydrophenylalanine (RΔF) nanoparticles for the selective delivery of miR-199a-3p to restore dysregulated gene expression in HCC. Targeted delivery was achieved by conjugating lactobionic acid (LA) with RΔF NPs (RΔF-LA NPs), a ligand for the asialoglycoprotein receptor known to be overexpressed in HCC cell lines. RΔF-LA NPs condensed miR-199a-3p had an average size of ∼60nm and a zeta potential of ∼+2.54 mV. RΔF-LA/miR NPs were found to be stable in serum as well as against RNase attack. RΔF-LA/miR NPs showed an enhanced cellular uptake and an efficient delivery of miR-199a-3p leading to a significant increase in miR-199a-3p levels (over 500 fold). The increased miR-199a-3p levels remarkably suppressed cell proliferation and migration as well as induced cellular apoptosis and downregulation of the specific target gene (mTOR) in vitro. RΔF-LA/miR NPs showed high tumor/ low organ ratios after intravenous injection into HCC tumor bearing nude mice. RΔF-LA/miR NPs treated mice demonstrated>50% decline in tumor growth, which also corresponded well with suppression of mTOR protein expression, tumor cell proliferation and increased survival rate (P < 0.05). CONCLUSION: RΔF-LA/miR NPs showed significantly enhanced delivery of the miRNA which underscores their potential for further development as a therapeutic approach for HCC. (Hepatology 2018;67:1392-1407).

Dynamic Hyaluronan drives liver endothelial cells towards angiogenesis.

BACKGROUND: Angiogenesis, the formation of new blood vessels from pre-existing vasculature is essential in a number of physiological processes such as embryonic development, wound healing as well as pathological conditions like, tumor growth and metastasis. Hyaluronic acid (HA), a high molecular weight polysaccharide, major component of extracellular matrix is known to associate with malignant phenotypes in melanomas and various other carcinomas. Hyaluronic acid binding protein 1 (HABP1) has been previously reported to trigger enhanced cellular proliferation in human liver cancer cells upon its over-expression. In the present study, we have identified the HA mediated cellular behaviour of liver endothelial cells during angiogenesis. METHODS: Endothelial cells have been isolated from perfused liver of mice. Cell proliferation was studied using microwell plates with tetrazole dye. Cell migration was evaluated by measuring endothelial monolayer wound repair as well as through transwell migration assay. Alterations in proteins and mRNA expression were estimated by immunobloting and quantitative real time PCR using Applied Biosystems. The paraformaldehyde fixed endothelial cells were used for immuno- florescence staining and F-actin detection with conjugated antibodies. The images were captured by using Olympus florescence microscope (IX71). RESULTS: We observed that administration of HA enhanced cell proliferation, adhesion, tubular sprout formation as well as migration of liver endothelial cells (ECs). The effect of HA in the rearrangement of the actins confirmed HA -mediated cytoskeleton re-organization and cell migration. Further, we confirmed enhanced expression of angiogenic factors like VEGF-A and VEGFR1 in endothelial cells upon HA treatment. HA supplementation led to elevated expression of HABP1 in murine endothelial cells. It was interesting to note that, although protein levels of ß- catenin remained unaltered, but translocation of this protein from membrane to nucleus was observed upon HA treatment, suggesting its role not only in vessel formation but also its involvement in angiogenesis signalling. CONCLUSIONS: The elucidation of molecular mechanism (s) responsible for HA mediated regulation of endothelial cells and angiogenesis contributes not only to our understanding the mechanism of disease progression but also offer new avenues for therapeutic intervention.

Impact of inhibitor of apoptosis proteins on immune modulation and inflammation.

The routes leading to programmed cell death are as tightly regulated as those of cellular growth and proliferation, and a finely synchronized balance between the life and death of cells ensures proper organ size and function. Inhibitors of apoptosis (IAPs) proteins were initially characterized by their ability to directly bind and inhibit apoptotic caspases. However, recent studies have clarified that IAPs are much more functionally versatile, modulating a vast range of signaling pathways that have an impact on antimicrobial responses, tumorigenesis, metastasis and cellular migration. A significant contribution of IAPs in tumorigenesis is their inherent function as E3 ubiquitin ligases to modulate cellular signaling downstream of death receptors or certain pattern recognition receptors. In this review, we focus on modulation of the innate and adaptive immune systems, macrophage plasticity and inflammatory responses by IAP family members. We also explore the rationale to target IAPs pharmacologically for the treatment of a number of inflammatory diseases and cancer.

Survivin inhibition ameliorates liver fibrosis by inducing hepatic stellate cell senescence and depleting hepatic macrophage population.

Persistent activation of hepatic stellate cells (HSCs) in the injured liver leads to the progression of liver injury from fibrosis to detrimental cirrhosis. In a previous study, we have shown that survivin protein is upregulated during the early activation of HSCs, which triggers the onset of liver fibrosis. However, the therapeutic potential of targeting survivin in a fully established fibrotic liver needs to be investigated. In this study, we chemically induced hepatic fibrosis in mice using carbon tetrachloride (CCl4) for 6 weeks, which was followed by treatment with a survivin suppressant (YM155). We also evaluated survivin expression in fibrotic human liver tissues, primary HSCs, and HSC cell line by histological analysis. αSMA+ HSCs in human and mice fibrotic liver tissues showed enhanced survivin expression, whereas the hepatocytes and quiescent (qHSCs) displayed minimal expression. Alternatively, activated M2 macrophage subtype induced survivin expression in HSCs through the TGF-ß-TGF-ß receptor-I/II signaling. Inhibition of survivin in HSCs promoted cell cycle arrest and senescence, which eventually suppressed their activation. In vivo, YM155 treatment increased the expression of cell senescence makers in HSCs around fibrotic septa such as p53, p21, and ß-galactosidase. YM155 treatment in vivo also reduced the hepatic macrophage population and inflammatory cytokine expression in the liver. In conclusion, downregulation of survivin in the fibrotic liver decreases HSC activation by inducing cellular senescence and modulating macrophage cytokine expression that collectively ameliorates liver fibrosis.

Mechanistic Insight into the Autophagic and Apoptotic Activity of Kaempferol on Liver Cancer Cells.

Background: The accumulation of poorly folded protein in the endoplasmic reticulum (ER) promotes ER stress and contributes to the pathogenesis of hepatocellular carcinoma (HCC). Current therapies have various adverse effects, therefore, laying the need for an alternative approach. Kaempferol (KP), a naturally occurring flavonoid, possesses potent anti-proliferative properties against various cancer cells. Nevertheless, its involvement in HCC remains relatively unexplored, particularly regarding its influence on apoptosis and autophagy pathways. Methods: The effect of KP on cell viability, and motility of Hep3B cells was evaluated by MTT, and scratch assay, respectively. Hoechst staining and FACS analysis were done to check the effect of KP on apoptosis and cell cycle progression. qRTPCR was used to evaluate the expression of several apoptosis and autophagy-related genes. KP was docked with several ER stress-related proteins involved in HCC to gain further insights into molecular mechanisms. The results of docking studies were validated with MD simulation and in vitro studies. Results: Treatment with KP at different time intervals showed dose- and time-dependent growth inhibition of liver cancer cells. KP decreased motility and arrested the cell cycle at the G0/G1 phase in Hep3B cells. Additionally, in the context of HCC, the relationship between KP, apoptosis, and autophagy is significant. It induced apoptosis and autophagy in Hep3B cells by downregulating the expression of Bcl-2 and upregulated Bax and Bid, Caspase-3, Beclin-1, and LC3. KP showed a better binding affinity with Nrf2, PERK, and IRE1α among all selected proteins. Further, it reversed the protective effect of 4-PBA (ER Stress inhibitor) by inducing apoptosis and autophagy in Hep3B cells. Conclusion: The study suggested KP as a potential chemopreventive agent for managing HCC by effectively inducing apoptosis and autophagy in Hep3B cells.

Long-Term Follow-Up of Abatacept, Post-Transplantation Cyclophosphamide, and Sirolimus-Based Haploidentical Transplantation in Younger Patients with Nonmalignant Diseases.

Haploidentical (haplo) hematopoietic cell transplantation (HCT) for nonmalignant disease (NMD) carries inherent challenges of both alloreactivity and graft failure. Building on promising results from pilot studies in which abatacept was combined with post-transplantation cyclophosphamide (PTCy) and sirolimus (AbaCyS) in younger NMD patients undergoing haplo-HCT, we present the long-term outcomes of this protocol. On the back of uniform disease-specific conditioning regimens containing antithymocyte globulin 4.5 mg/kg from day -9 to day -7, GVHD prophylaxis with AbaCyS consisted of abatacept administered on days 0, +5, +20, +35, and monthly until 180 days with PTCy and sirolimus. The patients were followed up with longitudinal assessment of immune reconstitution, growth, and reproductive development and quality of life (QoL) analyses. Among 40 patients (aplastic anemia, n = 24; hemoglobinopathies, n = 14; and primary immunodeficiencies, n = 2) with a median age of 10 years (range, 2 to 25 years), 95% achieved sustained engraftment. Post-transplantation hemophagocytic syndrome was detected in 3 patients, leading to graft failure in 2 cases. The incidence of acute graft-versus-host disease (GVHD) was 2.6%, and that of chronic GVHD (cGVHD) was 14.3%. Cytomegalovirus, adenovirus, and Epstein-Barr virus infections were observed in 45%, 5%, and 0% respectively. Rates of nonrelapse mortality, overall survival, event-free survival, and GVHD-free, event-free survival were 5%, 95%, 90%, and 82%, respectively, at a median follow-up of 4.6 years. Absence of cGVHD correlated with younger patient age and early sustained recovery of regulatory T cells and mature natural killer cells, which in turn was associated with improved QoL and lack of late infections. The AbaCyS protocol was associated with excellent long-term survival, with attenuation of both early and late alloreactivity in >80% of younger patients undergoing haplo-HCT for NMD. This study sheds light on predispositions to cGVHD and its impact on QoL, warranting further optimization of this approach.

Variation in the anti-oxidant, anti-obesity, and anti-cancer potential of different polarity extracts of saffron petals.

The aim of the present study is to explore the anti-cancer, anti-oxidant, and anti-obesity potential of saffron petal extract (SPE) prepared through the hydro-alcoholic extraction method. Further partitioning was done with a series of polar and non-polar solvents to find out the most potent fraction of SPE against HCC. Organoleptic characterization depicted the color, odor, taste, and texture of the sub-fractions of SPE. Phytochemical, and pharmacognostic screening of these fractions revealed the presence of alkaloids, flavonoids, carbohydrates, glycosides, and phenols. The quantitative assessment demonstrated that the n-butanol fraction showed maximum phenolic (60.8 mg GAE eq./mg EW), and flavonoid (23.3 mg kaempferol eq./mg EW) content. The anti-oxidant study revealed that the n-butanol fraction exhibited the highest radical scavenging activity, as assessed through DPPH and FRAP assay. The results of the comparative cytotoxic potential also showed n-butanol as the best against liver cancer cells (Huh-7), as it has the least IC50 value (462.8 µg/ml). While other extracts viz., chloroform, n-hexane, ethyl acetate, and aqueous fractions have IC50 values as 1088, 733.9, 1043, and 1245 µg/ml, respectively. Additionally, the n-butanol fraction exerted the highest inhibitory potential against α-amylase (92.5%) and pancreatic lipase enzymes (78%), indicating its anti-adipogenesis property. Based on the current finding, we can deduce that the n-butanol fraction of SPE has better cytotoxic, anti-oxidant, and anti-obesity potential than the other fractions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03669-x.

Augmenting Vaccine Efficacy against Delta Variant with 'Mycobacterium-w'-Mediated Modulation of NK-ADCC and TLR-MYD88 Pathways.

Mycobacterium-w (Mw) was shown to boost adaptive natural killer (ANK) cells and protect against COVID-19 during the first wave of the pandemic. As a follow-up of the trial, 50 healthcare workers (HCW) who had received Mw in September 2020 and subsequently received at least one dose of ChAdOx1 nCoV-19 vaccine (Mw + ChAdOx1 group) were monitored for symptomatic COVID-19 during a major outbreak with the delta variant of SARS-CoV-2 (April-June 2021), along with 201 HCW receiving both doses of the vaccine without Mw (ChAdOx1 group). Despite 48% having received just a single dose of the vaccine in the Mw + ChAdOx1 group, only two had mild COVID-19, compared to 36 infections in the ChAdOx1 group (HR-0.46, p = 0.009). Transcriptomic studies revealed an enhanced adaptive NK cell-dependent ADCC in the Mw + ChAdOx1 group, along with downregulation of the TLR2-MYD88 pathway and concomitant attenuation of downstream inflammatory pathways. This might have resulted in robust protection during the pandemic with the delta variant.

Design, synthesis, anti-acetylcholinesterase evaluation and molecular modelling studies of novel coumarin-chalcone hybrids.

The major enzyme responsible for the hydrolytic breakdown of the neurotransmitter acetylcholine (ACh) is acetylcholinesterase (AChE). Acetylcholinesterase inhibitors (AChEIs) are the most prescribed class of medications for the treatment of Alzheimer's disease (AD) and dementia. The limitations of available therapy, like side effects, drug tolerance, and inefficacy in halting disease progression, drive the need for better, more efficacious, and safer drugs. In this study, a series of fourteen novel chalcone-coumarin derivatives (8a-n) were designed, synthesized and characterized by spectral techniques like FT-IR, NMR, and HR-MS. Subsequently, the synthesized compounds were tested for their ability to inhibit acetylcholinesterase (AChE) activity by Ellman's method. All tested compounds showed AChE inhibition with IC50 value ranging from 0.201 ± 0.008 to 1.047 ± 0.043 µM. Hybrid 8d having chloro substitution on ring-B of the chalcone scaffold showed relatively better potency, with IC50 value of 0.201 ± 0.008 µM compared to other members of the series. The reference drug, galantamine, exhibited an IC50 at 1.142 ± 0.027 µM. Computational studies revealed that designed compounds bind to the peripheral anionic site (PAS), the catalytic active site (CAS), and the mid-gorge site of AChE. Putative binding modes, ligand-enzyme interactions, and stability of the best active compound are studied using molecular docking, followed by molecular dynamics (MD) simulations. The cytotoxicity of the synthesised derivatives was determined using the MTT test at three concentrations (100 g/mL, 500 g/mL, and 1 mg/mL). None of the chemicals had a significant effect on the body at the highest dose of 1 mg/mL.Communicated by Ramaswamy H. Sarma.

New immune horizons in therapeutics and diagnostic approaches to Preeclampsia.

Hypertensive disorders of pregnancy (HDP) are one of the commonest maladies, affecting 5%-10% of pregnancies worldwide. The American College of Obstetricians and Gynecologists (ACOG) identifies four categories of HDP, namely gestational hypertension (GH), Preeclampsia (PE), chronic hypertension (CH), and CH with superimposed PE. PE is a multisystem, heterogeneous disorder that encompasses 2%-8% of all pregnancy-related complications, contributing to about 9% to 26% of maternal deaths in low-income countries and 16% in high-income countries. These translate to 50 000 maternal deaths and over 500 000 fetal deaths worldwide, therefore demanding high priority in understanding clinical presentation, screening, diagnostic criteria, and effective management. PE is accompanied by uteroplacental insufficiency leading to vascular and metabolic changes, vasoconstriction, and end-organ ischemia. PE is diagnosed after 20 weeks of pregnancy in women who were previously normotensive or hypertensive. Besides shallow trophoblast invasion and inadequate remodeling of uterine arteries, dysregulation of the nonimmune system has been the focal point in PE. This results from aberrant immune system activation and imbalanced differentiation of T cells. Further, a failure of tolerance toward the semi-allogenic fetus results due to altered distribution of Tregs such as CD4+FoxP3+ or CD4+CD25+CD127(low) FoxP3+ cells, thereby creating a cytotoxic environment by suboptimal production of immunosuppressive cytokines like IL-10, IL-4, and IL-13. Also, intracellular production of complement protein C5a may result in decreased FoxP3+ regulatory T cells. With immune system dysfunction as a major driver in PE pathogenesis, it is logical that therapeutic targeting of components of the immune system with pharmacologic agents like anti-inflammatory and immune-modulating molecules are either being used or under clinical trial. Cholesterol synthesis inhibitors like Pravastatin may improve placental perfusion in PE, while Eculizumab (monoclonal antibody inhibiting C5) and small molecular inhibitor of C5a, Zilucoplan are under investigation. Monoclonal antibody against IL-17(Secukinumab) has been proposed to alter the Th imbalance in PE. Autologous Treg therapy and immune checkpoint inhibitors like anti-CTLA-4 are emerging as new candidates in immune horizons for PE management in the future.

Monosodium Glutamate Perturbs Human Trophoblast Invasion and Differentiation through a Reactive Oxygen Species-Mediated Pathway: An In-Vitro Assessment.

The overproduction of reactive oxygen species (ROS) has been associated with various human diseases. ROS exert a multitude of biological effects with both physiological and pathological consequences. Monosodium glutamate (MSG), a sodium salt of the natural amino acid glutamate, is a flavor-enhancing food additive, which is widely used in Asian cuisine and is an ingredient that brings out the "umami" meat flavor. MSG consumption in rats is associated with ROS generation. Owing to its consumption as part of the fast-food culture and concerns about its possible effects on pregnancy, we aimed to study the impact of MSG on placental trophoblast cells. MSG exposure influenced trophoblast invasion and differentiation, two of the most critical functions during placentation through enhanced production of ROS. Similar findings were also observed on MSG-treated placental explants, as confirmed by elevated Nrf2 levels. Ultrastructural studies revealed signs of subcellular injury by MSG exposure. Mechanistically, MSG-induced oxidative stress with endoplasmic reticulum stress pathways involving Xbp1s and IRE1α was observed. The effect of MSG through an increased ROS production indicates that its long-term exposure might have adverse health effect by compromising key trophoblast functions.

A role for proapoptotic Bax and Bak in T-cell differentiation and transformation.

Proapoptotic Bax and Bak are the key B-cell lymphoma-2 family members mediating apoptosis through the intrinsic pathway. Cells doubly deficient for Bax and Bak are profoundly resistant to apoptotic stimuli originating from multiple stimuli. Here we describe mice in which Bax and Bak have been deleted specifically in T-cells using Lck-Cre. In these T cell-specific BaxBak-deficient mice, early T-cell progenitors accumulate in the thymus, with relative depletion of more mature T cells. In addition, bone marrow progenitor cells fail to progress to the double positive stage when cultured on OP9 stromal cells expressing the Notch ligand Delta-like 1, consistent with a critical role for Bax and Bak in early T-cell development. Over time, T cell-specific BaxBak-deficient mice progress to an aggressive T-cell lymphoblastic leukemia/lymphoma. Interestingly, quantitative real-time polymerase chain reaction analysis of BaxBak-deficient T-cell lymphomas does not display amplification of the Notch signal transduction pathway, commonly activated in T-cell leukemia in both mouse and man. Bax and Bak, key regulators of the intrinsic pathway of apoptosis, are thus required to prevent T-cell malignancy, and for normal T-cell differentiation, regulating early T-cell development at the stage of early T-lineage progenitor cells.

Environmental sensing and signal transduction pathways regulating morphopathogenic determinants of Candida albicans.

Candida albicans is an opportunistic fungal pathogen that is found in the normal gastrointestinal flora of most healthy humans. However, under certain environmental conditions, it can become a life-threatening pathogen. The shift from commensal organism to pathogen is often correlated with the capacity to undergo morphogenesis. Indeed, under certain conditions, including growth at ambient temperature, the presence of serum or N-acetylglucosamine, neutral pH, and nutrient starvation, C. albicans can undergo reversible transitions from the yeast form to the mycelial form. This morphological plasticity reflects the interplay of various signal transduction pathways, either stimulating or repressing hyphal formation. In this review, we provide an overview of the different sensing and signaling pathways involved in the morphogenesis and pathogenesis of C. albicans. Where appropriate, we compare the analogous pathways/genes in Saccharomyces cerevisiae in an attempt to highlight the evolution of the different components of the two organisms. The downstream components of these pathways, some of which may be interesting antifungal targets, are also discussed.