Paricalcitol attenuates oxidative stress and inflammatory response in the liver of NAFLD rats by regulating FOXO3a and NFκB acetylation.

The lack of therapeutics along with complex pathophysiology made non-alcoholic fatty liver disease (NAFLD) a research hotspot. Studies showed that the deficiency of Vitamin D plays a vital role in NAFLD pathogenesis. While several research studies focused on vitamin D supplementation in NAFLD, there is still a need to understand the regulatory mechanism of direct vitamin D receptor activation in NAFLD. In the present study, we explored the role of direct Vitamin D receptor activation using paricalcitol in choline-deficient high-fat diet-induced NAFLD rat liver and its modulation on protein acetylation. Our results showed that paricalcitol administration significantly reduced the fat accumulation in HepG2 cells and the liver of NAFLD rats. Paricalcitol attenuated the elevated serum level of alanine transaminase, aspartate transaminase, insulin, low-density lipoprotein, triglyceride, and increased high-density lipoprotein in NAFLD rats. Paricalcitol significantly decreased the increased total protein acetylation by enhancing the SIRT1 and SIRT3 expression in NAFLD liver. Further, the study revealed that paricalcitol reduced the acetylation of NFκB and FOXO3a in NAFLD liver along with a decrease in the mRNA expression of IL1ß, NFκB, TNFα, and increased catalase and MnSOD. Moreover, total antioxidant activity, glutathione, and catalase were also elevated, whereas lipid peroxidation, myeloperoxidase, and reactive oxygen species levels were significantly decreased in the liver of NAFLD after paricalcitol administration. The study concludes that the downregulation of SIRT1 and SIRT3 in NAFLD liver was associated with an increased acetylated NFκB and FOXO3a. Paricalcitol effectively reversed hepatic inflammation and oxidative stress in NAFLD rats through transcriptional regulation of NFκB and FOXO3a, respectively, by inhibiting their acetylation.

Bone marrow stem cells incubated with ellipticine regenerate articular cartilage by attenuating inflammation and cartilage degradation in rabbit model.

BACKGROUND: Ellipticine (Ellip.) was recently reported to have beneficial effects on the differentiation of adipose-derived stem cells into mature chondrocyte-like cells. On the other hand, no practical results have been derived from the transplantation of bone marrow stem cells (BMSCs) in a rabbit osteoarthritis (OA) model. OBJECTIVES: This study examined whether autologous BMSCs incubated with ellipticine (Ellip.+BMSCs) could regenerate articular cartilage in rabbit OA, a model similar to degenerative arthritis in human beings. METHODS: A portion of rabbit articular cartilage was surgically removed, and Ellip.+BMSCs were transplanted into the lesion area. After two and four weeks of treatment, the serum levels of proinflammatory cytokines, i.e., tumor necrosis factor α (TNF-α) and prostaglandin E2 (PGE2), were analyzed, while macroscopic and micro-computed tomography (CT) evaluations were conducted to determine the intensity of cartilage degeneration. Furthermore, immuno-blotting was performed to evaluate the mitogen-activated protein kinases, PI3K/Akt, and nuclear factor-κB (NF-κB) signaling in rabbit OA models. Histological staining was used to confirm the change in the pattern of collagen and proteoglycan in the articular cartilage matrix. RESULTS: The transplantation of Ellip.+BMSCs elicited a chondroprotective effect by reducing the inflammatory factors (TNF-α, PGE2) in a time-dependent manner. Macroscopic observations, micro-CT, and histological staining revealed articular cartilage regeneration with the downregulation of matrix-metallo proteinases (MMPs), preventing articular cartilage degradation. Furthermore, histological observations confirmed a significant boost in the production of chondrocytes, collagen, and proteoglycan compared to the control group. Western blotting data revealed the downregulation of the p38, PI3K-Akt, and NF-κB inflammatory pathways to attenuate inflammation. CONCLUSIONS: The transplantation of Ellip.+BMSCs normalized the OA condition by boosting the recovery of degenerated articular cartilage and inhibiting the catabolic signaling pathway.

Predicting the risk of hypertension using machine learning algorithms: A cross sectional study in Ethiopia.

BACKGROUND AND OBJECTIVES: Hypertension (HTN), a major global health concern, is a leading cause of cardiovascular disease, premature death and disability, worldwide. It is important to develop an automated system to diagnose HTN at an early stage. Therefore, this study devised a machine learning (ML) system for predicting patients with the risk of developing HTN in Ethiopia. MATERIALS AND METHODS: The HTN data was taken from Ethiopia, which included 612 respondents with 27 factors. We employed Boruta-based feature selection method to identify the important risk factors of HTN. The four well-known models [logistics regression, artificial neural network, random forest, and extreme gradient boosting (XGB)] were developed to predict HTN patients on the training set using the selected risk factors. The performances of the models were evaluated by accuracy, precision, recall, F1-score, and area under the curve (AUC) on the testing set. Additionally, the SHapley Additive exPlanations (SHAP) method is one of the explainable artificial intelligences (XAI) methods, was used to investigate the associated predictive risk factors of HTN. RESULTS: The overall prevalence of HTN patients is 21.2%. This study showed that XGB-based model was the most appropriate model for predicting patients with the risk of HTN and achieved the accuracy of 88.81%, precision of 89.62%, recall of 97.04%, F1-score of 93.18%, and AUC of 0. 894. The XBG with SHAP analysis reveal that age, weight, fat, income, body mass index, diabetes mulitas, salt, history of HTN, drinking, and smoking were the associated risk factors of developing HTN. CONCLUSIONS: The proposed framework provides an effective tool for accurately predicting individuals in Ethiopia who are at risk for developing HTN at an early stage and may help with early prevention and individualized treatment.

Lipoid Proteinosis: Identification of a Novel Nonsense Mutation c.1246C>T:p.R416X in ECM1 gene from Bangladesh.

Lipoid proteinosis is a rare multisystem genodermatosis inherited as autosomal recessive trait. We report a case of lipoid proteinosis in a 10-year-old boy born to first-degree consanguineous parents presented with marked hoarseness of voice, accelerated photoaging appearance, enlarged and erythematous tongue with restricted movement and widespread dermatoses. Biopsy of oral mucosa revealed Periodic acid-Schiff (PAS)-positive amorphous eosinophilic hyaline deposits. Mutational analysis revealed a homozygous nonsense mutation with C to T substitution at nucleotide position 1246(c.1246C>T) in exon-8 of the extracellular matrix protein 1 gene leading to a stop codon. Both the parents were unaffected heterozygous carriers. To our knowledge, this is the first case report of lipoid proteinosis with evidence of a novel nonsense genetic mutation from Bangladesh.

Bioinformatics screening of colorectal-cancer causing molecular signatures through gene expression profiles to discover therapeutic targets and candidate agents.

BACKGROUND: Detection of appropriate receptor proteins and drug agents are equally important in the case of drug discovery and development for any disease. In this study, an attempt was made to explore colorectal cancer (CRC) causing molecular signatures as receptors and drug agents as inhibitors by using integrated statistics and bioinformatics approaches. METHODS: To identify the important genes that are involved in the initiation and progression of CRC, four microarray datasets (GSE9348, GSE110224, GSE23878, and GSE35279) and an RNA_Seq profiles (GSE50760) were downloaded from the Gene Expression Omnibus database. The datasets were analyzed by a statistical r-package of LIMMA to identify common differentially expressed genes (cDEGs). The key genes (KGs) of cDEGs were detected by using the five topological measures in the protein-protein interaction network analysis. Then we performed in-silico validation for CRC-causing KGs by using different web-tools and independent databases. We also disclosed the transcriptional and post-transcriptional regulatory factors of KGs by interaction network analysis of KGs with transcription factors (TFs) and micro-RNAs. Finally, we suggested our proposed KGs-guided computationally more effective candidate drug molecules compared to other published drugs by cross-validation with the state-of-the-art alternatives of top-ranked independent receptor proteins. RESULTS: We identified 50 common differentially expressed genes (cDEGs) from five gene expression profile datasets, where 31 cDEGs were downregulated, and the rest 19 were up-regulated. Then we identified 11 cDEGs (CXCL8, CEMIP, MMP7, CA4, ADH1C, GUCA2A, GUCA2B, ZG16, CLCA4, MS4A12 and CLDN1) as the KGs. Different pertinent bioinformatic analyses (box plot, survival probability curves, DNA methylation, correlation with immune infiltration levels, diseases-KGs interaction, GO and KEGG pathways) based on independent databases directly or indirectly showed that these KGs are significantly associated with CRC progression. We also detected four TFs proteins (FOXC1, YY1, GATA2 and NFKB) and eight microRNAs (hsa-mir-16-5p, hsa-mir-195-5p, hsa-mir-203a-3p, hsa-mir-34a-5p, hsa-mir-107, hsa-mir-27a-3p, hsa-mir-429, and hsa-mir-335-5p) as the key transcriptional and post-transcriptional regulators of KGs. Finally, our proposed 15 molecular signatures including 11 KGs and 4 key TFs-proteins guided 9 small molecules (Cyclosporin A, Manzamine A, Cardidigin, Staurosporine, Benzo[A]Pyrene, Sitosterol, Nocardiopsis Sp, Troglitazone, and Riccardin D) were recommended as the top-ranked candidate therapeutic agents for the treatment against CRC. CONCLUSION: The findings of this study recommended that our proposed target proteins and agents might be considered as the potential diagnostic, prognostic and therapeutic signatures for CRC.

Recent Advancement in Drug Design and Discovery of Pyrazole Biomolecules as Cancer and Inflammation Therapeutics.

Pyrazole, an important pharmacophore and a privileged scaffold of immense significance, is a five-membered heterocyclic moiety with an extensive therapeutic profile, viz., anti-inflammatory, anti-microbial, anti-anxiety, anticancer, analgesic, antipyretic, etc. Due to the expansion of pyrazolecent red pharmacological molecules at a quicker pace, there is an urgent need to put emphasis on recent literature with hitherto available information to recognize the status of this scaffold for pharmaceutical research. The reported potential pyrazole-containing compounds are highlighted in the manuscript for the treatment of cancer and inflammation, and the results are mentioned in % inhibition of inflammation, % growth inhibition, IC50, etc. Pyrazole is an important heterocyclic moiety with a strong pharmacological profile, which may act as an important pharmacophore for the drug discovery process. In the struggle to cultivate suitable anti-inflammatory and anticancer agents, chemists have now focused on pyrazole biomolecules. This review conceals the recent expansion of pyrazole biomolecules as anti-inflammatory and anticancer agents with an aim to provide better correlation among different research going around the world.

Human microbiome and cardiovascular diseases.

A number of microorganisms are co-evolved with the host, among which bacteria are the predominant organisms in the colonic site. The human microbiota contributes to various physiological functions, including the digestion and degradation of food components, harvesting of inaccessible nutrients, immune system regulation, maintenance of gut barrier function, and regulation of brain function and behavior. Microbes in the gut produce a wealth of metabolites from the exogenous dietary substances or endogenous metabolic compounds produced by the host and the resident microorganisms. These microbial-derived metabolites are the major factors in the host-microbiota cross-talk and influence the host's cardiometabolic health directly or indirectly depending on the structure and function of the microbial community. Evidence suggests that the perturbation in the composition and function of gut microbiota (referred to as gut dysbiosis) is associated with the development of several diseased conditions such as that of the gastrointestinal tract or colorectal cancer, metabolic diseases such as obesity, diabetes, immune disorders e.g. asthma, allergies, depression, anxiety and cardiometabolic disease. Several pathological conditions in the gastrointestinal tract may impair the intestinal barrier that allows translocation of bacteria and their metabolites to a remote organ such as the heart, which may ultimately be associated with systemic inflammation and the development of CVDs. In this chapter, we will discuss various gut microbiota-dependent metabolites, which have a significant role in cardiovascular diseases' pathologic processes and their risk factors. Finally, we will discuss the therapeutic potential of the gut-metabolite-heart axis as a novel target for the treatment of CVD and highlight the current updates and exciting directions for future research.

Optimization of production and characterization of a recombinant soluble human Cripto-1 protein inhibiting self-renewal of cancer stem cells.

Human Cripto-1 is a member of the epidermal growth factor (EGF)-Cripto-FRL-1-Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto-1 suppresses the self-renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto-1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His-tagged Cripto-1 protein. A concentrations of 0.2-0.4 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto-1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto-1 protein from a Ni-column in the smallest volume. Cation exchange column chromatography of the Cripto-1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto-1 exhibited high affinity to the anti-ALK-4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.

Ginsenoside-Rb1 prevents bone cartilage destruction through down-regulation of p-Akt, p-P38, and p-P65 signaling in rabbit.

BACKGROUND: Osteoarthritis (OA) is the most common joint complaint resulting in pain, disability, and loss of quality of life. On the other hand, ginsenoside-Rb1 is a plant product derived from ginseng that possesses immune-regulation and anti-inflammatory activities. However, it has been reported that different rout of administration but hydrogel-based Ginsenoside-Rb1 in an OA rabbit model has not been investigated. PURPOSE: The aim of this study was to investigate the potential effects of ginsenoside-Rb1 such as anti-arthritic activity in a rabbit knee OA model via NF- κB, PI3K/Akt, and P38/(MAPK) pathways. STUDY DESIGN: In the current study, rabbit osteoarthritis was induced by hollow trephine on the femur trochlea and the hydrogel-based Ginsenoside-Rb1 sheets were inserted on the rabbit knee to assess the anti-arthritis activity of ginsenoside-Rb1 which is sustained release. METHODS: After the hydrogel-based Rb1 sheet insert on the rabbit knee, macroscopic and micro CT was performed for investigation of chondroprotective effect. Matrix metalloproteinases (MMPs) and apoptotic expression were assessed through Immunohistochemistry and RT-PCR assay. In addition, the flow cytometry technique was used for the investigation of intracellular reactive oxygen species (ROS) production and histological changes were examined by HE, safranin O, and Masson trichrome staining method. Furthermore, the NF- κB, PI3K/Akt, and P38/(MAPK) pathways were investigated using Western blot analysis. RESULTS: Macroscopic and micro CT investigation of hydrogel-Rb1 treatment showed a dose-dependent chondroprotective effect. Immunohistochemistry and RT-PCR revealed that expression of matrix metalloproteinases (MMPs) and apoptotic markers TNF-α, caspase-3, and bax are down-regulated in a dose-dependent fashion following implantation of hydrogel-Rb. Higher levels of intracellular reactive oxygen species (ROS) were observed in the OA group. In histopathological investigation of hydrogel-Rb1 exhibited larger amounts of chondro cells, glycosaminoglycan's, and collagen compared to the defect group. Furthermore, the NF- κB, PI3K/Akt, and P38/(MAPK) pathways were downregulated by hydrogel-Rb1 while the disease model showed upstream. In the meantime, MMP expression level was considerably down-regulated. CONCLUSIONS: Our results indicate the protective effect of ginsenoside-Rb1 against OA pathogenesis through prevention of apoptosis with suppression of ROS production and activation of NF-κB signaling through downregulation of the MAPK and PI3K/Akt signaling pathways.

Design, Synthesis, Molecular Docking, and Biological Evaluation of Pyrazole Hybrid Chalcone Conjugates as Potential Anticancer Agents and Tubulin Polymerization Inhibitors.

Some (E)-3-(3-(4-(benzyloxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)-1-phenylprop-2-en-1-one conjugates 5a-r were designed; synthesized; characterized by 1H, 13C NMR, and ESI-MS; and evaluated for tubulin polymerization inhibitory activity and in vitro cytotoxicity against breast (MCF-7), cervical (SiHa), and prostate (PC-3) cancer cell lines, as well as a normal cell line (HEK-293T). The compounds were also tested to determine their binding modes at the colchicine-binding site of tubulin protein (PDB ID-3E22), for in silico ADME prediction, for bioactivity study, and for PASS prediction studies. Among all the synthesized conjugates, compound 5o exhibited excellent cytotoxicity with an IC50 value of 2.13 ± 0.80 µM (MCF-7), 4.34 ± 0.98 µM (SiHa), and 4.46 ± 0.53 µM (PC-3) against cancer cell lines. The compound did not exhibit significant toxicity to the HEK cells. Results of the in silico prediction revealed that the majority of the conjugates possessed drug-like properties.

Signaling Inhibitors Accelerate the Conversion of mouse iPS Cells into Cancer Stem Cells in the Tumor Microenvironment.

Cancer stem cells (CSCs) are a class of cancer cells characterized by self-renewal, differentiation and tumorigenic potential. We previously established a model of CSCs by culturing mouse induced pluripotent stem cells (miPSCs) for four weeks in the presence of a conditioned medium (CM) of cancer cell lines, which functioned as the tumor microenvironment. Based on this methodology of developing CSCs from miPSCs, we assessed the risk of 110 non-mutagenic chemical compounds, most of which are known as inhibitors of cytoplasmic signaling pathways, as potential carcinogens. We treated miPSCs with each compound for one week in the presence of a CM of Lewis lung carcinoma (LLC) cells. However, one-week period was too short for the CM to convert miPSCs into CSCs. Consequently, PDO325901 (MEK inhibitor), CHIR99021 (GSK-3ß inhibitor) and Dasatinib (Abl, Src and c-Kit inhibitor) were found to confer miPSCs with the CSC phenotype in one week. The tumor cells that survived exhibited stemness markers, spheroid formation and tumorigenesis in Balb/c nude mice. Hence, we concluded that the three signal inhibitors accelerated the conversion of miPSCs into CSCs. Similarly to our previous study, we found that the PI3K-Akt signaling pathway was upregulated in the CSCs. Herein, we focused on the expression of relative genes after the treatment with these three inhibitors. Our results demonstrated an increased expression of pik3ca, pik3cb, pik3r5 and pik3r1 genes indicating class IA PI3K as the responsible signaling pathway. Hence, AKT phosphorylation was found to be up-regulated in the obtained CSCs. Inhibition of Erk1/2, tyrosine kinase, and/or GSK-3ß was implied to be involved in the enhancement of the PI3K-AKT signaling pathway in the undifferentiated cells, resulting in the sustained stemness, and subsequent conversion of miPSCs into CSCs in the tumor microenvironment.

Paclitaxel and Sorafenib: The Effective Combination of Suppressing the Self-Renewal of Cancer Stem Cells.

"Combination therapy", which is a treatment modality combining two or more therapeutic agents, is considered a cornerstone of cancer therapy. The combination of anticancer drugs, of which functions are different from the other, enhances the efficiency compared to the monotherapy because it targets cancer cells in a synergistic or an additive manner. In this study, the combination of paclitaxel and sorafenib in low concentration was evaluated to target cancer stem cells, miPS-BT549cmP and miPS-Huh7cmP cells, developed from mouse induced pluripotent stem cells. The synergistic effect of paclitaxel and sorafenib on cancer stem cells was assessed by the inhibition of proliferation, self-renewal, colony formation, and differentiation. While the IC50 values of paclitaxel and sorafenib were approximately ranging between 250 and 300 nM and between 6.5 and 8 µM, respectively, IC50 of paclitaxel reduced to 20 and 25 nM, which was not toxic in a single dose, in the presence of 1 µM sorafenib, which was not toxic to the cells. Then, the synergistic effect was further assessed for the potential of self-renewal of cancer stem cells by sphere formation ability. As a result, 1 µM of sorafenib significantly enhanced the effect of paclitaxel to suppress the number of spheres. Simultaneously, paclitaxel ranging in 1 to 4 nM significantly suppressed not only the colony formation but also the tube formation of the cancer stem cells in the presence of 1 µM sorafenib. These results suggest the combination therapy of paclitaxel and sorafenib in low doses should be an attractive approach to target cancer stem cells with fewer side effects.

A novel model of liver cancer stem cells developed from induced pluripotent stem cells.

BACKGROUND: Liver cancer is the second most common cause of cancer-related death. Every type of tumours including liver cancer contains cancer stem cells (CSCs). To date, the molecular mechanism regulating the development of liver CSCs remains unknown. METHODS: In this study, we tried to generate a new model of liver CSCs by converting mouse induced pluripotent stem cells (miPSCs) with hepatocellular carcinoma (HCC) cell line Huh7 cells conditioned medium (CM). miPSCs treated with CM were injected into the liver of BALB/c nude mice. The developed tumours were then excised and analysed. RESULTS: The primary cultured cells from the malignant tumour possessed self-renewal capacity, differentiation potential and tumorigenicity in vivo, which were found rich in liver cancer-associated markers as well as CSC markers. CONCLUSIONS: We established a model of liver CSCs converting from miPS and showed different stages of stemness during conversion process. Our CSC model will be important to assess the molecular mechanisms necessary to develop liver CSCs and could help in defeating liver cancer.

Catestatin reverses the hypertrophic effects of norepinephrine in H9c2 cardiac myoblasts by modulating the adrenergic signaling.

Catestatin (CST) is a catecholamine release-inhibitory peptide secreted from the adrenergic neurons and the adrenal glands. It regulates the cardiovascular functions and it is associated with cardiovascular diseases. Though its mechanisms of actions are not known, there are evidences of cross-talk between the adrenergic and CST signaling. We hypothesized that CST moderates the adrenergic overdrive and studied its effects on norepinephrine-mediated hypertrophic responses in H9c2 cardiac myoblasts. CST alone regulated the expression of a number of fetal genes that are induced during hypertrophy. When cells were pre-treated CST, it blunted the modulation of those genes by norepinephrine. Norepinephrine (2 µM) treatment also increased cell size and enhanced the level of Troponin T in the sarcomere. These effects were attenuated by the treatment with CST. CST attenuated the immediate generation of ROS and the increase in glutathione peroxidase activity induced by norepinephrine treatment. Expression of fosB and AP-1 promoter-reporter constructs was used as the endpoint readout for the interaction between the CST and adrenergic signals at the gene level. It showed that CST largely attenuates the stimulatory effects of norepinephrine and other mitogenic signals through the modulation of the gene regulatory modules in a characteristic manner. Depending upon the dose, the signaling by CST appears to be disparate, and at 10-25 nM doses, it primarily moderated the signaling by the ß1/2-adrenoceptors. This study, for the first time, provides insights into the modulation of adrenergic signaling in the heart by CST.

Identification of AcrAB-TolC Efflux Pump Genes and Detection of Mutation in Efflux Repressor AcrR from Omeprazole Responsive Multidrug-Resistant Escherichia coli Isolates Causing Urinary Tract Infections.

Antimicrobial resistance poses a threat in the treatment of infectious diseases in Bangladesh as well as in the world. Multidrug-resistant (MDR) Enterobacteriaceae, the most common cause of one such infectious disease, urinary tract infection (UTI), has contributed to the escalating problem of selecting empiric antibiotics against UTIs. The aim of this study was to investigate the presence of the efflux pump in MDR Escherichia coli isolates from UTI in the North-East region of Bangladesh, to isolate and characterize the AcrAB-TolC efflux pump genes of these locally isolated strains and to do mutation analysis of the efflux pump repressor AcrR gene to understand the AcrAB-TolC efflux pump mechanism. In the presence of omeprazole, an efflux pump inhibitor, every MDR E. coli isolate showed increased susceptibility to at least 1 of the 7 antibiotics investigated, indicating that efflux pump might be involved in their antibiotic resistance. Omeprazole decreased the minimum inhibitory concentration of every antibiotics being investigated by 2- to 8-fold. DNA and the deduced amino acid sequences of the polymerase chain reaction (PCR) products analyzed by bioinformatics tools revealed that the chromosomal AcrAB-TolC and AcrR genes were present in all MDR and antibiotic-susceptible E. coli isolates. However, the deduced amino acid sequences of the amplification refractory mutation system (ARMS) PCR product of the AcrR gene revealed that the substitution of arginine to cysteine at position 45 of AcrR was observed only in the MDR E. coli whose antibiotic susceptibility increased in the presence of omeprazole. Data reported herein support the notion that the increased antibiotic susceptibility of the MDR E. coli isolates in the presence of omeprazole might be due to efflux pump(s) inhibition and the AcrAB-TolC efflux pump might be a contributor to antibiotic resistance when the mutation of arginine to cysteine occurs at position 45 of AcrR.

Metastasis of Cancer Stem Cells Developed in the Microenvironment of Hepatocellular Carcinoma.

Metastasis develops when cancer cells spread from the primary site of a malignant tumor to the surrounding and distant tissues, and it is the most critical problem in cancer treatment. Our group developed cancer stem cells (CSCs) from induced pluripotent stem cells (iPSCs) in the presence of a conditioned medium (CM) of cancer-derived cells. The CSCs were characterized by the formation of malignant tumors in vivo, followed by metastasis. In this study, CSCs converted from mouse iPSCs in the presence of CM from hepatocellular carcinoma (HCC) cell line Huh7 cells. These converted cells (miPS-Huh7cm cells) were established as the metastatic cells. The generated CSCs were injected into the liver or spleen of nude mice. Almost one month after transplantation, the tumors were excised, and the primary cultured cells derived from the malignant tumors and metastatic nodules were evaluated by stemness and metastatic markers to compare their differences. The miPS-Huh7cm cells exhibited metastatic potential, and efficiently formed malignant tumors with lung and/or liver lesions in vivo, whereas the injected miPS formed teratoma. The primary cultured cells derived from the malignant tumors and metastatic nodules sustained the expression of stemness markers, such as Nanog, Klf4 and c-Myc, and acquired cancer stem markers, such as CD90, CD44 and ALDH1. Simultaneously, the expression of metastatic markers, such as Slug, Twist1 and vimentin, in primary cells derived from the malignant tumors, was higher than in metastatic nodules. The CSCs derived from iPSCs, forming malignant tumors and displaying high metastasis, will provide a good animal model to study the mechanisms of metastasis.

Concurrent decreases in the prevalence of wheezing and Ascaris infection among 5-year-old children in rural Bangladesh and their regulatory T cell immunity after the implementation of a national deworming program.

INTRODUCTION: Epidemiological research on the prevalence of asthma and helminthic infections in various countries has led to the hypothesis that helminthic infections protect against asthma by suppressing the host's immune response. This study was conducted to elucidate whether decreased Ascaris infection following a national deworming program was associated with increased recurrent wheezing among rural Bangladeshi children and to test their anti-inflammatory immunity. METHODS: This nested case-control study was conducted from December 2015 to October 2016 in the rural service area of the International Centre for Diarrhoeal Disease Research, Bangladesh. Of the 1800 5-year old children randomly selected for the study, informed consent was obtained from the guardians of 1658 children. Data were collected using a semistructured questionnaire adopted from the International Study of Asthma and Allergies in Childhood and blood samples for the analysis of regulatory T (Treg) cell immune responses and the balance between Th1 and Th2 immunity in Ascaris infections. RESULTS: A total of 145 children were found to have wheezing, yielding a prevalence rate of 8.7%, which was significantly lower than the rate found in 2001 (16.2%, P < .001); Ascaris infection also decreased from 2001 to 2016. The 127 wheezing children who agreed to participate further were compared to 114 randomly selected never-wheezing children. Wheezing had a significant positive association with antibiotic use, history of pneumonia, parents' history of asthma, and Ascaris infection; children with Ascaris infection were twice as likely to have wheezing (adjusted odds ratio = 2.31, P = .053). Flow cytometry found no significant differences in the rates of Th1, Th2, and CD4 + CD25 + CD127low cells by the wheezing group. CONCLUSIONS: Ascaris infection had a positive rather than a negative association with wheezing and the rates of wheezing and Ascaris infections both decreased from 2001 to 2016. These findings undermines the hypothesis that such infections provide protection against asthma.

Targeting Ovarian Cancer Cells Overexpressing CD44 with Immunoliposomes Encapsulating Glycosylated Paclitaxel.

Paclitaxel (PTX) is one of the front-line drugs approved for the treatment of ovarian cancer. However, the application of PTX is limited due to the significant hydrophobicity and poor pharmacokinetics. We previously reported target-directed liposomes carrying tumor-selective conjugated antibody and encapsulated glycosylated PTX (gPTX-L) which successfully overcome the PTX limitation. The tubulin stabilizing activity of gPTX was equivalent to that of PTX while the cytotoxic activity of gPTX was reduced. In human ovarian cancer cell lines, SK-OV-3 and OVK18, the concentration at which cell growth was inhibited by 50% (IC50) for gPTX range from 15⁻20 nM, which was sensitive enough to address gPTX-L with tumor-selective antibody coupling for ovarian cancer therapy. The cell membrane receptor CD44 is associated with cancer progression and has been recognized as a cancer stem cell marker including ovarian cancer, becoming a suitable candidate to be targeted by gPTX-L therapy. In this study, gPTX-loading liposomes conjugated with anti-CD44 antibody (gPTX-IL) were assessed for the efficacy of targeting CD44-positive ovarian cancer cells. We successfully encapsulated gPTX into liposomes with the loading efficiency (LE) more than 80% in both of gPTX-L and gPTX-IL with a diameter of approximately 100 nm with efficacy of enhanced cytotoxicity in vitro and of convenient treatment in vivo. As the result, gPTX-IL efficiently suppressed tumor growth in vivo. Therefore gPTX-IL could be a promising formulation for effective ovarian cancer therapies.

Exogenous Cripto-1 Suppresses Self-Renewal of Cancer Stem Cell Model.

Cripto-1 is a glycophosphatidylinositol (GPI) anchored signaling protein of epidermal growth factor (EGF)-Cripto-1-FRL1-Cryptic (CFC) family and plays a significant role in the early developmental stages and in the different types of cancer cells, epithelial to mesenchymal transition and tumor angiogenesis. Previously, we have developed cancer stem cells (miPS-LLCcm) from mouse iPSCs by culturing them in the presence of conditioned medium of Lewis Lung Carcinoma (LLC) cells for four weeks. Nodal and Cripto-1 were confirmed to be expressed in miPS-LLCcm cells by quantitative reverse transcription PCR (rt-qPCR) implying that Cr-1 was required in maintaining stemness. To investigate the biological effect of adding exogenous soluble CR-1 to the cancer stem cells, we have prepared a C-terminally truncated soluble form of recombinant human CR-1 protein (rhsfCR-1), in which the GPI anchored moiety was removed by substitution of a stop codon through site-directed mutagenesis. rhsfCR-1 effectively suppressed the proliferation and sphere forming ability of miPS-LLCcm cells in a dose-dependent manner in the range of 0 to 5 µg/mL, due to the suppression of Nodal-Cripto-1/ALK4/Smad2 signaling pathway. Frequency of sphere-forming cells was dropped from 1/40 to 1/69 by rhsfCR-1 at 1 µg/mL. Moreover, rhsfCR-1 in the range of 0 to 1 µg/mL also limited the differentiation of miPS-LLCcm cells into vascular endothelial cells probably due to the suppression of self-renewal, which should reduce the number of cells with stemness property. As demonstrated by a soluble form of exogenous Cripto-1 in this study, the efficient blockade would be an attractive way to study Cripto-1 dependent cancer stem cell properties for therapeutic application.

Up-Regulation of PI 3-Kinases and the Activation of PI3K-Akt Signaling Pathway in Cancer Stem-Like Cells Through DNA Hypomethylation Mediated by the Cancer Microenvironment.

Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.