ELM2-SANT Domain-Containing Scaffolding Protein 1 Regulates Differentiation and Maturation of Cardiomyocytes Derived From Human-Induced Pluripotent Stem Cells.

BACKGROUND: ELMSAN1 (ELM2-SANT domain-containing scaffolding protein 1) is a newly identified scaffolding protein of the MiDAC (mitotic deacetylase complex), playing a pivotal role in early embryonic development. Studies on Elmsan1 knockout mice showed that its absence results in embryo lethality and heart malformation. However, the precise function of ELMSAN1 in heart development and formation remains elusive. To study its potential role in cardiac lineage, we employed human-induced pluripotent stem cells (hiPSCs) to model early cardiogenesis and investigated the function of ELMSAN1. METHODS AND RESULTS: We generated ELMSAN1-deficient hiPSCs through knockdown and knockout techniques. During cardiac differentiation, ELMSAN1 depletion inhibited pluripotency deactivation, decreased the expression of cardiac-specific markers, and reduced differentiation efficiency. The impaired expression of genes associated with contractile sarcomere structure, calcium handling, and ion channels was also noted in ELMSAN1-deficient cardiomyocytes derived from hiPSCs. Additionally, through a series of structural and functional assessments, we found that ELMSAN1-null hiPSC cardiomyocytes are immature, exhibiting incomplete sarcomere Z-line structure, decreased calcium handling, and impaired electrophysiological properties. Of note, we found that the cardiac-specific role of ELMSAN1 is likely associated with histone H3K27 acetylation level. The transcriptome analysis provided additional insights, indicating maturation reduction with the energy metabolism switch and restored cell proliferation in ELMSAN1 knockout cardiomyocytes. CONCLUSIONS: In this study, we address the significance of the direct involvement of ELMSAN1 in the differentiation and maturation of hiPSC cardiomyocytes. We first report the impact of ELMSAN1 on multiple aspects of hiPSC cardiomyocyte generation, including cardiac differentiation, sarcomere formation, calcium handling, electrophysiological maturation, and proliferation.

Kupffer cells abrogate homing and repopulation of allogeneic hepatic progenitors in injured liver site.

BACKGROUND: Allogeneic hepatocyte transplantation is an emerging approach to treat acute liver defects. However, durable engraftment of the transplanted cells remains a daunting task, as they are actively cleared by the recipient's immune system. Therefore, a detailed understanding of the innate or adaptive immune cells-derived responses against allogeneic transplanted hepatic cells is the key to rationalize cell-based therapies. METHODS: Here, we induced an acute inflammatory regenerative niche (3-96 h) on the surface of the liver by the application of cryo-injury (CI) to systematically evaluate the innate immune response against transplanted allogeneic hepatic progenitors in a sustained micro-inflammatory environment. RESULTS: The resulting data highlighted that the injured site was significantly repopulated by alternating numbers of innate immune cells, including neutrophils, monocytes and Kupffer cells (KCs), from 3 to 96 h. The transplanted allo-HPs, engrafted 6 h post-injury, were collectively eliminated by the innate immune response within 24 h of transplantation. Selective depletion of the KCs demonstrated a delayed recruitment of monocytes from day 2 to day 6. In addition, the intrasplenic engraftment of the hepatic progenitors 54 h post-transplantation was dismantled by KCs, while a time-dependent better survival and translocation of the transplanted cells into the injured site could be observed in samples devoid of KCs. CONCLUSION: Overall, this study provides evidence that KCs ablation enables a better survival and integration of allo-HPs in a sustained liver inflammatory environment, having implications for rationalizing the cell-based therapeutic interventions against liver defects.

Neutralizing serum amyloid a protects against sinusoidal endothelial cell damage and platelet aggregation during acetaminophen-induced liver injury.

Serum amyloid A (SAA) is an acute response protein that mainly produced by hepatocytes, and it can promote endothelial dysfunction via a pro-inflammatory and pro-thrombotic effect in atherosclerosis and renal disease. Overdose of Acetaminophen (APAP) will cause hepatotoxicity accompany with hepatocyte necrosis, liver sinusoidal endothelial cells (LSECs) damage and thrombosis in liver. However, whether SAA plays a role in APAP-induced liver toxicity remains unclear. Here, we evaluated the Saa1/2 expression in APAP-induced liver injury, and found that Saa1/2 production was significantly increased in an autocrine manner in APAP injury model. Moreover, we used neutralizing antibody (anti-SAA) to block the function of serum Saa1/2. We found that neutralizing serum Saa1/2 protected against APAP-induced liver injuries and increased the survival rate of mice that were treated with lethal dose APAP. Further investigations showed that blocking Saa1/2 reduced APAP-induced sinusoidal endothelium damage, hemorrhage and thrombosis. In addition, in vitro experiments showed that Saa1/2 augmented the toxic effect of APAP on LSECs, and Saa1/2 promoted platelets aggregation on LSECs cell membrane. Taken together, this study suggests that Saa1/2 may play a critical role in APAP-induced liver damages through platelets aggregation and sinusoidal damage. Therefore, we conceptually demonstrate that inhibition of SAA may be a potential intervention for APAP-directed acute liver injuries.

Hepatic DKK1-driven steatosis is CD36 dependent.

Nonalcoholic fatty liver disease (NAFLD) is prevalent worldwide; about 25% of NAFLD silently progress into steatohepatitis, in which some of them may develop into fibrosis, cirrhosis and liver failure. However, few drugs are available for NAFLD, partly because of an incomplete understanding of its pathogenic mechanisms. Here, using in vivo and in vitro gain- and loss-of-function approaches, we identified up-regulated DKK1 plays a pivotal role in high-fat diet-induced NAFLD and its progression. Mechanistic analysis reveals that DKK1 enhances the capacity of hepatocytes to uptake fatty acids through the ERK-PPARγ-CD36 axis. Moreover, DKK1 increased insulin resistance by activating the JNK signaling, which in turn exacerbates disorders of hepatic lipid metabolism. Our finding suggests that DKK1 may be a potential therapeutic and diagnosis candidate for NAFLD and metabolic disorder progression.

Inhibition of Hedgehog Delays Liver Regeneration through Disrupting the Cell Cycle.

Liver regeneration is a complicated biological process orchestrated by various liver resident cells. Hepatic cell proliferation and reconstruction of the hepatic architecture involve multiple signaling pathways. It has been reported that the Hh signal is involved in liver regeneration. However, the signal transduction pathways and cell types involved are ill studied. This study aimed to investigate hedgehog signal response cell types and the specific molecular mechanism involved in the process of liver regeneration. Partial hepatectomy (PH) of 70% was performed on ICR (Institute of Cancer Research) mice to study the process of liver regeneration. We found that the hedgehog signal was activated significantly after PH, including hedgehog ligands, receptors and intracellular signaling molecules. Ligand signals were mainly expressed in bile duct cells and non-parenchymal hepatic cells, while receptors were expressed in hepatocytes and some non-parenchymal cells. Inhibition of the hedgehog signal treated with vismodegib reduced the liver regeneration rate after partial hepatectomy, including inhibition of hepatic cell proliferation by decreasing Cyclin D expression and disturbing the cell cycle through the accumulation of Cyclin B. The current study reveals the important role of the hedgehog signal and its participation in the regulation of hepatic cell proliferation and the cell cycle during liver regeneration. It provides new insight into the recovery of the liver after liver resection.

Utilization of information and communication technology (ICT) among undergraduate health science students: a cross-sectional study.

BACKGROUND: We already know that incorporating information and Communication technology (ICT) into every aspect of human activity result in significant change and makes tasks easier to complete. It can help in areas of healthcare systems and medical education. Therefore, this study aimed to assess utilization ICT and its associated factors among Arba Minch University College Medicine and Health Science students. METHODS: A cross sectional study design was conducted in June through August 2021 among under graduate students in college of medicine and health science at Arba Minch University, Ethiopia. A self-administered questionnaire was used to collect information on the students' socio-demographic factors as well as the utilization ICT. The data entry form was prepared with Epi-data 3.1 versions software and STATA version 14 software was used to analyze the data. RESULTS: A total of 355 participants enrolled in the study, with a response rate of 98.34%. The percentage of students who used ICT was 55.77% [95% CI, 0.50, 0.60]. Regarding of field of study, health informatics students (84%) used the most ICT, while midwifery students (52%) used the least. Urban resident [AOR = 1.85, 95% CI = 1.08, 3.16], ICT knowledge [AOR = 3.8, 95% CI = 2.25, 6.40], having formal training of ICT [AOR = 1.9, 95% CI = 1.06,3.48], having IT in current course study [AOR = 2.2, 95% CI = 1.23, 3.84], and had good IT skill [AOR = 2.4, 95% CI = 1.34, 4.23] revealed a significant and positive correlation with the use of ICT. CONCLUSION: In the current study previous residence, ICT knowledge, having formal training, having IT in current courses, and IT skill were significantly associated with student ICT utilization. Therefore, the university should continue to invest in professional development in order to improve teaching and student performance, as well as provide the college with student-centered ICT computer labs to encourage students to use technology.

Hypomorphic ASGR1 modulates lipid homeostasis via INSIG1-mediated SREBP signaling suppression.

A population genetic study identified that the asialoglycoprotein receptor 1 (ASGR1) mutation carriers had substantially lower non-HDL-cholesterol (non-HDL-c) levels and reduced risks of cardiovascular diseases. However, the mechanism behind this phenomenon remained unclear. Here, we established Asgr1-knockout mice that represented a plasma lipid profile with significantly lower non-HDL-c and triglyceride (TG) caused by decreased secretion and increased uptake of VLDL/LDL. These 2 phenotypes were linked with the decreased expression of microsomal triglyceride transfer protein and proprotein convertase subtilisin/kexin type 9, 2 key targeted genes of sterol regulatory element-binding proteins (SREBPs). Furthermore, there were fewer nuclear SREBPs (nSREBPs) on account of more SREBPs being trapped in endoplasmic reticulum, which was caused by an increased expression of insulin-induced gene 1 (INSIG1), an anchor of SREBPs. Overexpression and gene knockdown interventions, in different models, were conducted to rescue the ASGR1-deficient phenotypes, and we found that INSIG1 knockdown independently reversed the ASGR1-mutated phenotypes with increased serum total cholesterol, LDL-c, TG, and liver cholesterol content accompanied by restored SREBP signaling. ASGR1 rescue experiments reduced INSIG1 and restored the SREBP network defect as manifested by improved apolipoprotein B secretion and reduced LDL uptake. Our observation demonstrated that increased INSIG1 is a critical factor responsible for ASGR1 deficiency-associated lipid profile changes and nSREBP suppression. This finding of an ASGR1/INSIG1/SREBP axis regulating lipid hemostasis may provide multiple potential targets for lipid-lowering drug development.

Toll-like receptor 2 signaling in liver pathophysiology.

Chronic liver diseases (CLD) are among the major cause of mortality and morbidity worldwide. Despite current achievements in the area of hepatitis virus, chronic alcohol abuse and high-fat diet are still fueling an epidemic of severe liver disease, for which, an effective therapy has yet not been discovered. In particular, the therapeutic regimens that could prevent the progression of fibrosis and, in turn, aid cirrhotic liver to develop a robust regenerative capability are intensively needed. To this context, a better understanding of the signaling pathways regulating hepatic disease development may be of critical value. In general, the liver responds to various insults with an orchestrated healing process involving variety of signaling pathways. One such pathway is the TLR2 signaling pathway, which essentially regulates adult liver pathogenesis and thus has emerged as an attractive target to treat liver disease. TLR2 is expressed by different liver cells, including Kupffer cells (KCs), hepatocytes, and hepatic stellate cells (HSCs). From a pathologic perspective, the crosstalk between antigens and TLR2 may preferentially trigger a distinctive set of signaling mechanisms in these liver cells and, thereby, induce the production of inflammatory and fibrogenic cytokines that can initiate and prolong liver inflammation, ultimately leading to fibrosis. In this review, we summarize the currently available evidence regarding the role of TLR2 signaling in hepatic disease progression. We first elaborate its pathological involvement in liver-disease states, such as inflammation, fibrosis, and cirrhosis. We then discuss how therapeutic targeting of this pathway may help to alleviate its disease-related functioning.

SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury.

Hepatic stellate cells (HSCs) are crucial for liver injury repair and cirrhosis. However, the mechanism of chemotactic recruitment of HSCs into injury loci is still largely unknown. Here, we demonstrate that serum amyloid A1 (SAA1) acts as a chemokine recruiting HSCs toward injury loci signaling via TLR2, a finding proven by gene manipulation studies in cell and mice models. The mechanistic investigations revealed that SAA1/TLR2 axis stimulates the Rac GTPases through PI3K-dependent pathways and induces phosphorylation of MLC (pSer19). Genetic deletion of TLR2 and pharmacological inhibition of PI3K diminished the phosphorylation of MLCpSer19 and migration of HSCs. In brief, SAA1 serves as a hepatic endogenous chemokine for the TLR2 receptor on HSCs, thereby initiating PI3K-dependent signaling and its effector, Rac GTPases, which consequently regulates actin filament remodeling and cell directional migration. Our findings provide novel targets for anti-fibrosis drug development.

Generation of a TLR2 homozygous knockout human embryonic stem cell line WAe001-A-64 using CRISPR/Cas9 editing.

Toll-like receptor 2 (TLR2) is a pattern recognition receptor which plays an important role in innate immune system. In humans it's encoded by the TLR2 gene and also been designated as CD282. Using CRISPR/Cas9 gene editing technology, we have established a TLR2 mutant WAe001-A-64 cell line from the original embryonic stem cell line H1. It has adopted two biallelic deletions in exon 3 of TLR2 which resulted in a frame shift and early termination in the translation of TLR2. Moreover, WAe001-A-64 has maintained the normal karyotype, pluripotent phenotype, parental cell morphology and the ability to differentiate into three germ layers.

Robust expansion and functional maturation of human hepatoblasts by chemical strategy.

BACKGROUND: Chemically strategies to generate hepatic cells from human pluripotent stem cells (hPSCs) for the potential clinical application have been improved. However, producing high quality and large quantities of hepatic cells remain challenging, especially in terms of step-wise efficacy and cost-effective production requires more improvements. METHODS: Here, we systematically evaluated chemical compounds for hepatoblast (HB) expansion and maturation to establish a robust, cost-effective, and reproducible methodology for self-renewal HBs and functional hepatocyte-like cell (HLC) production. RESULTS: The established chemical cocktail could enable HBs to proliferate nearly 3000 folds within 3 weeks with preserved bipotency. Moreover, those expanded HBs could be further efficiently differentiated into homogenous HLCs which displayed typical morphologic features and functionality as mature hepatocytes including hepatocyte identity marker expression and key functional activities such as cytochrome P450 metabolism activities and urea secretion. Importantly, the transplanted HBs in the injured liver of immune-defect mice differentiated as hepatocytes, engraft, and repopulate in the injured loci of the recipient liver. CONCLUSION: Together, this chemical compound-based HLC generation method presents an efficient and cost-effective platform for the large-scale production of functional human hepatic cells for cell-based therapy and drug discovery application.

Kupffer cells mediate the recruitment of hepatic stellate cells into the localized liver damage.

Currently, there is a growing interest in understanding the cellular and molecular events of immune-cell trafficking and recruitment of hepatic stellate cells (HSCs) in liver diseases. Aberrant activation of HSCs is the key event leading to chronic liver fibrosis. However, the underlying mechanisms of the recruitment of HSCs in a locally injured liver are not clearly understood. Here, we report a new experimental approach for the study of inflammatory responses as well as the recruitment of HSCs into the localized cryolesion. We observed a significant liver damage accompanied by the up-regulation of plasma ALT and AST. In addition, we also found increased levels of MCP-1, IL-6 and IL-10 cytokines. The peak cytokine levels were detected at 8 h after injury, followed by intrahepatic infiltration of neutrophils and monocytes into the injury site (from 8 h to day 3), while the kupffer cells (KCs) and HSCs were mainly detected on day 3 after injury. Interestingly, the depletion of KCs, but not neutrophils, reduced the directional recruitment and accumulation of HSCs at the injury site. Moreover, the combinatorial recruitment of KCs and HSCs resulted in the gradual restoration of fibrotic area to almost typical histological appearance on day 14 post-injury. In conclusion, our data demonstrated a localized infiltration and accumulation of neutrophils and monocytes at a "predefined loci", and further revealed that KCs are critical for the recruitment of HSCs during injury, and thus, may play an important role in tissue repair.

Ascorbate protects liver from metabolic disorder through inhibition of lipogenesis and suppressor of cytokine signaling 3 (SOCS3).

BACKGROUND: Fatty liver is a reversible status, but also an origin stage to develop to other metabolic syndromes, such as diabetes and heart disease that threatens public health worldwide. Ascorbate deficiency is reported to be correlated with increasing risks for metabolic syndromes, but whether ascorbate has a therapeutic effect is unknown. Here, we investigated if ascorbate treatment alone could work on protecting from the development of steatosis and mechanisms beyond. METHODS: Guinea pigs were fed with a chow diet or a high palm oil diet (HPD) respectively. HPD induced animals were administered different concentrations of ascorbate in different time intervals through water. Besides, hepatocyte-like cells derived from human embryonic stem cells and HepG2 cells were treated with palmitic acid (PA) to induce lipid accumulation for molecular mechanism study. RESULTS: We find that ascorbate rescues HPD and PA induced steatosis and insulin tolerance in vivo and in vitro. We demonstrate that ascorbate changes cellular lipid profiles via inhibits lipogenesis, and inhibits the expression of SOCS3 via STAT3, thus enhances insulin signal transduction. Overexpression of SOCS3 abolishes the ascorbate rescue effects on insulin signal and lipid accumulation in hepatic cells. CONCLUSIONS: Ascorbate ameliorates hepatic steatosis and improves insulin sensitivity through inhibiting lipogenesis and SOCS3.

Direct Conversion of Human Urine Cells to Neurons by Small Molecules.

Transdifferentiation of other cell type into human neuronal cells (hNCs) provides a platform for neural disease modeling, drug screening and potential cell-based therapies. Among all of the cell donor sources, human urine cells (hUCs) are convenient to obtain without invasive harvest procedure. Here, we report a novel approach for the transdifferentiation of hUCs into hNCs. Our study demonstrated that a combination of seven small molecules (CAYTFVB) cocktail induced transdifferentiation of hUCs into hNCs. These chemical-induced neuronal cells (CiNCs) exhibited typical neuron-like morphology and expressed mature neuronal markers. The neuronal-like morphology revealed in day 1, and the Tuj1-positive CiNCs reached to about 58% in day 5 and 38.36% Tuj1+/MAP2+ double positive cells in day 12. Partial electrophysiological properties of CiNCs was obtained using patch clamp. Most of the CiNCs generated using our protocol were glutamatergic neuron populations, whereas motor neurons, GABAergic or dopaminergic neurons were merely detected. hUCs derived from different donors were converted into CiNCs in this work. This method may provide a feasible and noninvasive approach for reprogramming hNCs from hUCs for disease models and drug screening.

Construction of a GLI3 compound heterozygous knockout human embryonic stem cell line WAe001-A-20 by CRISPR/Cas9 editing.

The human GLI3 protein has a dual function as a transcriptional activator or repressor of hedgehog signaling, depending on the proteolytic processing forms of GLI3. In this study, we established a compound heterozygous GLI3 mutant human embryonic stem cell line (WAe001-A-20) through CRISPR/Cas9 editing. The WAe001-A-20 cells carried two deletions on two different alleles of exon 2 of GLI3, respectively, which resulted in a frame shift and early termination in the translation of GLI3. Moreover, WAe001-A-20 maintains a normal karyotype, parental cell morphology, pluripotent phenotype and the ability to differentiate into three germ layers. Resource table.

Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material.

BACKGROUND: Polyhydroxybutyrates (PHBs) are macromolecules synthesized by bacteria. They are inclusion bodies accumulated as reserve materials when the bacteria grow under different stress conditions. Because of their fast degradability under natural environmental conditions, PHBs are selected as alternatives for production of biodegradable plastics. The aim of this work was to isolate potential PHB producing bacteria, evaluate PHB production using agro-residues as carbon sources. RESULT: Among fifty bacterial strains isolated from different localities, ten PHB accumulating strains were selected and compared for their ability to accumulate PHB granules inside their cells. Isolate Arba Minch Waste Water (AWW) identified as Bacillus spp was found to be the best producer. The optimum pH, temperature, and incubation period for best PHB production by the isolate were 7, 37 °C, and 48 h respectively at 150 rpm. PHB production was best with glucose as carbon source and peptone as nitrogen source. The strain was able to accumulate 55.6, 51.6, 37.4 and 25% PHB when pretreated sugar cane bagasse, corn cob, teff straw (Eragrostis tef) and banana peel were used as carbon sources respectively. Fourier transform-infrared authentication results of the extracted and purified PHB identified its functional units as C-H, CH2, C=O and C-O groups. UV-Vis spectrophotometric analysis and biodegradability test confirmed the similarity of the extract with standard PHB and its suitability for bioplastic production. CONCLUSION: The isolated Bacillus sp can be used for feasible production of PHB using agro-residues especially sugarcane bagasse which can reduce the production cost in addition to reducing the disposal problem of these substrates. The yield of PHB can further be boosted by optimization of production parameters as substrates.