MicroRNA-1 Regulates Smooth Muscle Cell Differentiation by Repressing Kruppel-Like Factor 4

The role of microRNA-1 (miR-1) has been studied in cardiac and skeletal muscle differentiation. However, it remains unexplored in vascular smooth muscle cells (SMCs) differentiation. The aim of this study was to uncover novel targets of and shed light on the function of miR-1 in the context of embryonic stem cell (ESC) differentiation of SMCs in vitro. miR-1 expression is steadily increased during differentiation of mouse ESC to SMCs. Loss-of-function approaches using miR-1 inhibitors uncovered that miR-1 is required for SMC lineage differentiation in ESC-derived SMC cultures, as evidenced by downregulation of SMC-specific markers and decrease of derived SMC population. In addition, bioinformatics analysis unveiled a miR-1 binding site on the Kruppel-like factor 4 (KLF4) 3' untranslated region (3-UTR), in a region that is highly conserved across species. Consistently, miR-1 mimic reduced KLF4 3-UTR luciferase activity, which can be rescued by mutating the miR-1 binding site on the KLF4 3-UTR in the reporter construct. Additionally, repression of the miR-1 expression by miR-1 inhibitor can reverse KLF4 downregulation during ESC-SMC differentiation, which subsequently inhibits SMC differentiation. We conclude that miR-1 plays a critical role in the determination of SMC fate during retinoid acid-induced ESC/SMC differentiation, which may indicate that miR-1 has a role to promote SMC differentiation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90488/1/scd-2E2010-2E0283.pd

HDAC3 maintains oocyte meiosis arrest by repressing amphiregulin expression before the LH surge.

It is known that granulosa cells (GCs) mediate gonadotropin-induced oocyte meiosis resumption by releasing EGF-like factors in mammals, however, the detailed molecular mechanisms remain unclear. Here, we demonstrate that luteinizing hormone (LH) surge-induced histone deacetylase 3 (HDAC3) downregulation in GCs is essential for oocyte maturation. Before the LH surge, HDAC3 is highly expressed in GCs. Transcription factors, such as FOXO1, mediate recruitment of HDAC3 to the amphiregulin (Areg) promoter, which suppresses AREG expression. With the LH surge, decreased HDAC3 in GCs enables histone H3K14 acetylation and binding of the SP1 transcription factor to the Areg promoter to initiate AREG transcription and oocyte maturation. Conditional knockout of Hdac3 in granulosa cells in vivo or inhibition of HDAC3 activity in vitro promotes the maturation of oocytes independent of LH. Taking together, HDAC3 in GCs within ovarian follicles acts as a negative regulator of EGF-like growth factor expression before the LH surge

Comprehensive analysis of the association between inflammation indexes and complications in patients undergoing pancreaticoduodenectomy

BackgroundDuring clinical practice, routine blood tests are commonly performed following pancreaticoduodenectomy (PD). However, the relationship between blood cell counts, inflammation-related indices, and postoperative complications remains unclear.MethodWe conducted a retrospective study, including patients who underwent PD from October 2018 to July 2023 at the First Hospital of Chongqing Medical University, and compared baseline characteristics and clinical outcomes among different groups. Neutrophil count (NC), platelet count (PLT), lymphocyte count (LC), systemic immune-inflammation index (SII), platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), and the product of platelet count and neutrophil count (PPN) were derived from postoperative blood test results. We investigated the association between these indicators and outcomes using multivariable logistic regression and restricted cubic spline analysis. The predictive performance of these indicators was assessed by the area under the curve (AUC) of the receiver operating characteristic (ROC) curve and decision curve analysis (DCA).ResultA total of 232 patients were included in this study. Multivariate logistic regression and restricted cubic spline analysis showed that all indicators, except for PLT, were associated with clinical postoperative pancreatic fistula (POPF). SII, NLR, and NC were linked to surgical site infection (SSI), while SII, NLR, and PLR were correlated with CD3 complication. PLT levels were related to postoperative hemorrhage. SII (AUC: 0.729), NLR (AUC: 0.713), and NC (AUC: 0.706) effectively predicted clinical POPF.ConclusionIn patients undergoing PD, postoperative inflammation-related indices and blood cell counts are associated with various complications. NLR and PLT can serve as primary indicators post-surgery for monitoring complications

A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells. • A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture. • The preparation method of shrimp cell growth medium is successfully established. • Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days

A novel function prediction approach using protein overlap networks

Background: Construction of a reliable network remains the bottleneck for network-based protein function prediction. We built an artificial network model called protein overlap network (PON) for the entire genome of yeast, fly, worm, and human, respectively. Each node of the network represents a protein, and two proteins are connected if they share a domain according to InterPro database.Results: The function of a protein can be predicted by counting the occurrence frequency of GO (gene ontology) terms associated with domains of direct neighbors. The average success rate and coverage were 34.3% and 43.9%, respectively, for the test genomes, and were increased to 37.9% and 51.3% when a composite PON of the four species was used for the prediction. As a comparison, the success rate was 7.0% in the random control procedure. We also made predictions with GO term annotations of the second layer nodes using the composite network and obtained an impressive success rate (\u3e30%) and coverage (\u3e30%), even for small genomes. Further improvement was achieved by statistical analysis of manually annotated GO terms for each neighboring protein.Conclusions: The PONs are composed of dense modules accompanied by a few long distance connections. Based on the PONs, we developed multiple approaches effective for protein function prediction

Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner

Improvement and applications of adeno-associated virus 2 (AAV-2) system for efficient gene transfer and expression in penaeid shrimp cells

Adeno-associated virus 2 (AAV-2)-mediated expression system is a well-developed and widely used gene transfer and expression tool in mammalian cells, but no attempt has been done to examine its infection and expression efficiency in shrimp cells. In this study, using eGFP as a reporter gene, we found that unmodified AAV-2 could infect primarily cultured shrimp hemolymph cells, but with an extremely low infection efficiency of up to 0.0067%. However, all the improvements of AAV-2 expression system tried in this study including the insertion of shrimp virus (IHHNV)-sourced P2 promoter into the AAV-2 vector (i.e. pAAV-CMV-P2-eGFP) and the inclusion of shrimp virus (IHHNV)-sourced nucleocapsid protein （IHCP） into or association of shrimp virus (WSSV)-sourced tegument protein (VP26) with the capsid of the improved AAV-2 s, respectively, could significantly increase the infection and expression efficiencies of this expression system in the primarily cultured shrimp hemolymph cells in comparison with wild-type AAV-2. Moreover, from the viewpoint of improving the infection and expression efficiency, the insertion of P2 promoter did best, followed by the association of WSSV tegument protein of VP26, and the last was the inclusion of IHHNV nucleocapsid protein of IHCP. In conclusion, an improved AAV-2 expression system which consists of four expression plasmids of pAAV-CMV-P2-eGFP, pAAV-RC, pHelper and pcDNA3.1-VP26 and one packaging cell line of HEK293T has been successfully developed in this study. This improved AAV-2 expression system will provide us a useful tool for efficient gene transfer and expression in shrimp cells

Long-term exposure of two plasticizers di (2-ethylhexyl) phthalate (DEHP) and acetyl tributyl citrate (ATBC): toxic effects on gonadal development and reproduction of zebrafish (“<em>Danio rerio</em>”)

789-797Present study investigated the exposure effects of two plasticizers, di (2-ethylhexyl) phthalate (DEHP) and acetyl tributyl citrate (ATBC) on the gonadal development and reproduction in zebrafish (Danio rerio) at environmentally relevant concentration (0.5 μg/L) for six months by comparing with the control group. We observed the significant adverse effects of two plasticizers in the embryos and larvae of zebrafish resulting inhibition of body length, weight and gonado-somatic index (GSI) in both exposed male and female zebrafish with impaired fecundity. Histological analysis revealed the apparent inhibition of ovaries in zebrafish after six months DEHP exposure. Present study reveals that the DEHP phthalate can adversely affect the reproduction and functioning in the male and female zebrafish compared to the ATBC exposure which showed relatively low effects