Effect of dietary concentrate level on digestibility of nutrients in each region of the gastrointestinal tract and rumen fermentation in goats.

This study evaluated the effects of high concentrate diets (HCD) on the rumen fermentation and the digestibility of nutrients in different sites of the gastrointestinal tract (GIT) in goats. Four goats were used in a crossover design. The goats were fitted with a ruminal cannula and flexible T-cannulae proximal duodenum and terminal ileum. Treatments were as follows: low concentrate group (LCG) and high concentrate group (HCG). Duodenal flow and forestomach digestibility of starch were significantly higher in the HCG than those in the LCG (p < 0.05); There was no significant difference in ileum flow and digestibility of starch in the small intestine, large intestine and total GIT (p > 0.05). The digestibility of crude protein (CP) in the forestomach was significantly higher in the HCG than in the LCG (p < 0.05); the flow of the duodenum and ileum of CP, and the CP digestibility of the small intestine, large intestine and total GIT were not significantly different between groups (p > 0.05). The duodenal and ileal flow of neutral detergent fiber (NDF), the NDF digestibility of the different segments and total GIT were not significantly different between groups (p > 0.05). Compared to the LCG, the ruminal pH of the HCG was significantly lower (p < 0.05). The HCG concentrations of microbial crude protein, ammonia nitrogen and isovaleric acid were significantly higher (p < 0.05) than the LCG. The foam strength, foam production and viscosity of the rumen fluid in the HCG were higher than the LCG (p < 0.01). These results showed that when the goats were fed with HCD, the digestibility of nutrients was not significantly impaired, but the risk of frothy rumen bloat increased. ImplicationsDue to a serious shortage of high-quality roughage in China, producers commonly used a high-concentrate diet in ruminants, which can improve animal production performance.Gastrointestinal digestive function plays a vital role in the absorption of nutrients and the healthy growth of animals.Therefore, this research evaluated the digestibility of various nutrients in different segments of the gastrointestinal tract (GIT) under HCD feeding by using three-site cannula goats as experimental animals.The results indicated that the GIT of goats could fully digest nutrients such as starch and protein under HCD feeding conditions.

A submucosal lesion of the rectum: an unexpected MALT.

A 57-year-old female was found a 12 mm × 10 mm submucosal lesion in the rectum with a smooth mucosa and telangiectasia The lesion was considered as a neuroendocrine tumor, and removed by endoscopic submucosal dissection (ESD) It was finally diagnosed with mucosa-associated lymphoid tissue (MALT) lymphoma with negative margin by pathological examination and histopathological test. MALT lymphoma in the rectum is rare and difficult to diagnose without histopathological test. In this case, the characteristic of this case is telangiectasia on the surface of lesion. Therefore, our findings suggested small lesion in rectum but big in impact.

Epigenetic DNA methylation of Zbtb7b regulates the population of double-positive CD4+CD8+ T cells in ulcerative colitis.

BACKGROUND AND AIMS: Ulcerative colitis (UC) is a heterogeneous disorder with complex pathogenesis. Therefore, in the present study, we aimed to assess genome-wide DNA methylation changes associated explicitly with the pathogenesis of UC. METHODS: DNA methylation changes were identified by comparing UC tissues with healthy controls (HCs) from the GEO databases. The candidate genes were obtained and verified in clinical samples. Moreover, the underlying molecular mechanism related to Zbtb7b in the pathogenesis of UC was explored using the dextran sodium sulfate (DSS)-induced colitis model. RESULTS: Bioinformatic analysis from GEO databases confirmed that Zbtb7b, known as Th-inducing POZ-Kruppel factor (ThPOK), was demethylated in UC tissues. Then, we demonstrated that Zbtb7b was in a hypo-methylation pattern through the DSS-induced colitis model (P = 0.0357), whereas the expression of Zbtb7b at the mRNA and protein levels was significantly up-regulated in the inflamed colonic tissues of UC patients (qRT-PCR, WB, IHC: P < 0.0001, P = 0.0079, P < 0.0001) and DSS-induced colitis model (qRT-PCR, WB, IHC: P < 0.0001, P = 0.0045, P = 0.0004). Moreover, the expression of Zbtb7b was positively associated with the degree of UC activity. Mechanically, over-expression of Zbtb7b might activate the maturation of CD4+T cells (FCM, IF: P = 0.0240, P = 0.0003) and repress the differentiation of double-positive CD4+CD8+T (DP CD4+CD8+T) cells (FCM, IF: P = 0.0247, P = 0.0118), contributing to the production of inflammatory cytokines, such as TNF-α (P = 0.0005, P = 0.0005), IL-17 (P = 0.0014, P = 0.0381), and IFN-γ (P = 0.0016, P = 0.0042), in the serum and colonic tissue of DSS-induced colitis model. CONCLUSIONS: Epigenetic DNA hypo-methylation of Zbtb7b activated the maturation of CD4+T cells and repressed the differentiation of DP CD4+CD8+ T cells, resulting in the production of inflammatory cytokines and colonic inflammation in UC. Therefore, Zbtb7b might be a diagnostic and therapeutic biomarker for UC, and hypo-methylation might affect the biological function of Zbtb7b.

Anti-NSCLC activity in vitro of Hsp90N inhibitor KW-2478 and complex crystal structure determination of Hsp90N-KW-2478.

KW-2478 is a promising anti-cancer lead compound targeting to the molecular chaperone heat shock protein 90 N (Hsp90N). Absence of complex crystal structure of Hsp90N-KW-2478, however, hampered further structure optimization of KW-2478 and understanding on the molecular interaction mechanism. Herein, a high-resolution complex crystal structure of Hsp90N-KW-2478 was determined by X-ray diffraction (XRD, resolution limit: 1.59 Å; PDB ID: 6LT8) and their molecular interaction was analyzed in detail, which suggested that KW-2478 perfectly bound in the N-terminal ATP-binding pocket of Hsp90 to disable its molecular chaperone function, therefore suppressed or killed cancer cells. The results from thermal shift assay (TSA, ΔTm, 18.82 ± 0.51 °C) and isothermal titration calorimetry (ITC, Kd, 7.30 ± 2.20 nM) suggested that there is an intense binding force and favorable thermodynamic changes during the process of KW-2478 binding with Hsp90N. Additionally, KW-2478 exhibited favorable anti-NSCLC activity in vitro, as it inhibited cell proliferation (IC50, 8.16 µM for A549; 14.29 µM for H1975) and migration, induced cell cycle arrest and promoted apoptosis. Thirty-six novel KW-2478 derivatives were designed, based on the complex crystal structure and molecular interaction analysis of Hsp90N-KW-2478 complex. Among them, twenty-two derivatives exhibited increased binding force with Hsp90N evaluated by molecular docking assay. The results would provide new guidance for anti-NSCLC new drug development based on the lead compound KW-2478.

Tight Covalent Organic Framework Membranes for Efficient Anion Transport via Molecular Precursor Engineering.

Fabricating covalent organic frameworks (COFs) membranes with tight structure, which can fully utilize well-defined framework structure and thus achieve superior conduction performance, remains a grand challenge. Herein, through molecular precursor engineering of COFs, we reported the fabrication of tight COFs membrane with the ever-reported highest hydroxide ion conductivity over 200 mS cm-1 at 80 °C, 100 % RH. Six quaternary ammonium-functionalized COFs were synthesized by assembling functional hydrazides and different aldehyde precursors. In an organic-aqueous reaction system, the impact of the aldehyde precursors with different size, electrophilicity and hydrophilicity on the reaction-diffusion process for fabricating COFs membranes was elucidated. Particularly, more hydrophilic aldehydes were prone to push the reaction zone from the interface region to the aqueous phase of the reaction system, the tight membranes were thus fabricated via phase-transfer polymerization process, conferring around 4-8 times the anion conductivity over the loose membranes via interfacial polymerization process.

Scalable Fabrication of Crystalline COF Membranes from Amorphous Polymeric Membranes.

Covalent organic framework (COF) membranes hold potential for widespread applicability, but scalable fabrication is challenging. Here, we demonstrate the disorder-to-order transformation from amorphous polymeric membrane to crystalline COF membrane via monomer exchange. Solution processing is used to prepare amorphous membrane and the replacing monomer is selected based on the chemical and thermodynamical stability of the final framework. Reversible imine bonds allow the extraneous monomers to replace the pristine monomers within amorphous membrane, driving the transformation from disordered network to ordered framework. Incorporation of intramolecular hydrogen bonds enables the crystalline COF to imprint the amorphous membrane morphology. The COF membranes harvest proton conductivity up to 0.53 S cm-1 at 80 °C. Our strategy bridges amorphous polymeric and crystalline COF membranes for large-scale fabrication of COF membranes and affords guidance on materials processing.

smCounter2: an accurate low-frequency variant caller for targeted sequencing data with unique molecular identifiers.

MOTIVATION: Low-frequency DNA mutations are often confounded with technical artifacts from sample preparation and sequencing. With unique molecular identifiers (UMIs), most of the sequencing errors can be corrected. However, errors before UMI tagging, such as DNA polymerase errors during end repair and the first PCR cycle, cannot be corrected with single-strand UMIs and impose fundamental limits to UMI-based variant calling. RESULTS: We developed smCounter2, a UMI-based variant caller for targeted sequencing data and an upgrade from the current version of smCounter. Compared to smCounter, smCounter2 features lower detection limit that decreases from 1 to 0.5%, better overall accuracy (particularly in non-coding regions), a consistent threshold that can be applied to both deep and shallow sequencing runs, and easier use via a Docker image and code for read pre-processing. We benchmarked smCounter2 against several state-of-the-art UMI-based variant calling methods using multiple datasets and demonstrated smCounter2's superior performance in detecting somatic variants. At the core of smCounter2 is a statistical test to determine whether the allele frequency of the putative variant is significantly above the background error rate, which was carefully modeled using an independent dataset. The improved accuracy in non-coding regions was mainly achieved using novel repetitive region filters that were specifically designed for UMI data. AVAILABILITY AND IMPLEMENTATION: The entire pipeline is available at https://github.com/qiaseq/qiaseq-dna under MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

UBE2T promotes hepatocellular carcinoma cell growth via ubiquitination of p53.

Deregulation of Ubiquitin-conjugating enzyme E2T (UBE2T) contributes to the progression of human cancers. However, its clinical significance and role in hepatocellular carcinoma (HCC) remain unclear. Here, we show that UBE2T is up-regulated in HCC and exerts oncogenic activities via ubiquitination of p53. High UBE2T expression was correlated with higher pathological grade, advanced TNM stage, tumor vascular invasion, and poor overall and disease-free survivals in two independent cohorts containing 827 patients with HCC. UBE2T was further identified as an independent factor for overall survival by multivariate analyses. Luciferase reporter assays confirmed that UBE2T was directly targeted by miR-543 which was down-regulated in HCC. In vitro experiments demonstrated that UBE2T overexpression promoted, whereas UBE2T knockdown inhibited HCC cell growth. Ectopic expression of UBE2T resulted in the decreases of p53, p21 and Noxa. Further studies revealed that UBE2T facilitated the degradation of p53 protein via enhancing its ubiquitination. Collectively, our findings suggest UBE2T serves as a promising prognostic factor for HCC and functions as an oncogene. The newly identified miR-543/UBE2T/p53 axis may represent a new potential therapeutic target for HCC intervention.

Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes.

BACKGROUND: PCR amplicon sequencing has been widely used as a targeted approach for both DNA and RNA sequence analysis. High multiplex PCR has further enabled the enrichment of hundreds of amplicons in one simple reaction. At the same time, the performance of PCR amplicon sequencing can be negatively affected by issues such as high duplicate reads, polymerase artifacts and PCR amplification bias. Recently researchers have made some good progress in addressing these shortcomings by incorporating molecular barcodes into PCR primer design. So far, most work has been demonstrated using one to a few pairs of primers, which limits the size of the region one can analyze. RESULTS: We developed a simple protocol, which enables the use of molecular barcodes in high multiplex PCR with hundreds of amplicons. Using this protocol and reference materials, we demonstrated the applications in accurate variant calling at very low fraction over a large region and in targeted RNA quantification. We also evaluated the protocol's utility in profiling FFPE samples. CONCLUSIONS: We demonstrated the successful implementation of molecular barcodes in high multiplex PCR, with multiplex scale many times higher than earlier work. We showed that the new protocol combines the benefits of both high multiplex PCR and molecular barcodes, i.e. the analysis of a very large region, low DNA input requirement, very good reproducibility and the ability to detect as low as 1% mutations with minimal false positives (FP).

Comparison of somatic mutation calling methods in amplicon and whole exome sequence data.

BACKGROUND: High-throughput sequencing is rapidly becoming common practice in clinical diagnosis and cancer research. Many algorithms have been developed for somatic single nucleotide variant (SNV) detection in matched tumor-normal DNA sequencing. Although numerous studies have compared the performance of various algorithms on exome data, there has not yet been a systematic evaluation using PCR-enriched amplicon data with a range of variant allele fractions. The recently developed gold standard variant set for the reference individual NA12878 by the NIST-led "Genome in a Bottle" Consortium (NIST-GIAB) provides a good resource to evaluate admixtures with various SNV fractions. RESULTS: Using the NIST-GIAB gold standard, we compared the performance of five popular somatic SNV calling algorithms (GATK UnifiedGenotyper followed by simple subtraction, MuTect, Strelka, SomaticSniper and VarScan2) for matched tumor-normal amplicon and exome sequencing data. CONCLUSIONS: We demonstrated that the five commonly used somatic SNV calling methods are applicable to both targeted amplicon and exome sequencing data. However, the sensitivities of these methods vary based on the allelic fraction of the mutation in the tumor sample. Our analysis can assist researchers in choosing a somatic SNV calling method suitable for their specific needs.

Clinical and endoscopic characteristics and management of 220 cases with serrated polyps.

BACKGROUND: Serrated polyps are considered the precursor lesions of colorectal cancer through the serrated pathway. In the present study, we aimed to evaluate and discuss the clinical and endoscopic characteristics and management of serrated polyps. METHODS: The data of 220 cases with serrated polyps between September 2018 and November 2021 in Shenzhen People's Hospital were retrospectively analyzed. RESULTS: Of all these cases, 32 were hyperplastic polyps, 36 were traditional serrated adenomas, 126 were sessile serrated lesions, 25 were SSLs with dysplasia, and one was an unclassified serrated adenoma. Although most patients were males aged ≥50 years and most serrated polyps were located in the distal colon and rectum with a size of 6-10 mm and the shape of type 0-Is, there was no significant difference (P > 0.05). Serrated polyps of ≤5 mm in size and type 0-IIa were mostly removed by cold biopsy forceps. Cold snare polypectomy was primarily used for those of 6-10 mm in size. Endoscopic mucosal resection was used for those of 6-20 mm, and endoscopic submucosal dissection was used for those of ≥20 mm (P < 0.05). All complications occurred in SSL patients with or without dysplasia (P < 0.05). CONCLUSIONS: Clinical and endoscopic characteristics were beneficial for distinguishing and diagnosing serrated polyps. In addition, management options were crucial to prevent recurrence and progression. However, the detection rate of serrated polyps was relatively low. Therefore, prospective multicenter studies with large samples are necessary to better assess colorectal serrated polyps.

Ligation-assisted endoscopic submucosal resection following unroofing technique for small esophageal subepithelial lesions originating from the muscularis propria.

BACKGROUND: The majority of esophageal subepithelial lesions originating from the muscularis propria (SEL-MPs) are benign in nature, although a subset may exhibit malignant characteristics. Conventional endoscopic resection techniques are time-consuming and lack efficacy for small SEL-MPs. AIM: To evaluate the efficacy and safety of ligation-assisted endoscopic submucosal resection (ESMR-L) following unroofing technique for small esophageal SEL-MPs. METHODS: From January 2021 to September 2023, 17 patients diagnosed with esophageal SEL-MPs underwent ESMR-L following unroofing technique at the endoscopy center of Shenzhen People's Hospital. Details of clinicopathological characteristics and clinical outcomes were collected and analyzed. RESULTS: The mean age of the patients was 50.12 ± 12.65 years. The mean size of the tumors was 7.47 ± 2.83 mm and all cases achieved en bloc resection successfully. The average operation time was 12.2 minutes without any complications. Histopathology identified 2 Lesions (11.8%) as gastrointestinal stromal tumors at very low risk, 12 Lesions (70.6%) as leiomyoma and 3 Lesions (17.6%) as smooth muscle proliferation. No recurrence was found during the mean follow-up duration of 14.18 ± 9.62 months. CONCLUSION: ESMR-L following roofing technique is an effective and safe technique for management of esophageal SEL-MPs smaller than 20 mm, but it cannot ensure en bloc resection and may require further treatment.

Extreme Li-Mg selectivity via precise ion size differentiation of polyamide membrane.

Achieving high selectivity of Li+ and Mg2+ is of paramount importance for effective lithium extraction from brines, and nanofiltration (NF) membrane plays a critical role in this process. The key to achieving high selectivity lies in the on-demand design of NF membrane pores in accordance with the size difference between Li+ and Mg2+ ions, but this poses a huge challenge for traditional NF membranes and difficult to be realized. In this work, we report the fabrication of polyamide (PA) NF membranes with ultra-high Li+/Mg2+ selectivity by modifying the interfacial polymerization (IP) process between piperazine (PIP) and trimesoyl chloride (TMC) with an oil-soluble surfactant that forms a monolayer at oil/water interface, referred to as OSARIP. The OSARIP benefits to regulate the membrane pores so that all of them are smaller than Mg2+ ions. Under the solely size sieving effect, an exceptional Mg2+ rejection rate of over 99.9% is achieved. This results in an exceptionally high Li+/Mg2+ selectivity, which is one to two orders of magnitude higher than all the currently reported pressure-driven membranes, and even higher than the microporous framework materials, including COFs, MOFs, and POPs. The large enhancement of ion separation performance of NF membranes may innovate the current lithium extraction process and greatly improve the lithium extraction efficiency.

Insufficient Plasma Melatonin and Its Association With Neuropsychiatric Impairments in Patients With T2DM.

Purpose: Type 2 diabetes mellitus (T2DM) is associated with multiple neuropsychiatric impairments, including cognitive dysfunction, and melatonin (MLT) plays a crucial role in maintaining normal neuropsychiatric functions. This study is aimed at investigating the change in plasma MLT levels and its association with neuropsychiatric impairments in T2DM patients. Methods: One hundred twenty-six T2DM patients were recruited, and their demographics and clinical data were collected. Apart from the plasma glycated hemoglobin (HbA1c) levels and other routine metabolic indicators, the plasma concentrations of MLT, C-reactive protein (CRP), Interleukin 6 (IL-6), soluble myeloid triggered receptor 1 (sTREM 1), and receptor 2 (sTREM 2) were measured. Moreover, the executive function and depressive tendency were evaluated via the Behavior Rating Inventory of Executive Function-Adult Version (BRIEF-A) and the Epidemiological Research Center Depression Scale (CES-D), respectively. Result: Compared with the low HbA1c group, the T2DM patients in the high HbA1c group presented lower plasma MLT levels but higher plasma concentrations of inflammatory biomarker levels, together with higher scores in the BRIEF-A and CES-D scales. Moreover, results of the Pearson correlation test showed that the plasma MLT levels were negatively correlated with the BRIEF-A and CES-D scores, as well as plasma concentrations of HbA1c and inflammatory indications, indicating that MLT may mediate their neuroinflammation and neuropsychiatric impairments. Furthermore, the ROC curve results indicated that plasma MLT levels have a predictive effect on executive impairment and depressive status in T2DM patients. Conclusion: MLT levels decreased in patients with T2DM and were associated with neuropsychiatric impairments and inflammatory status, and MLT might be developed as a therapeutic agent and predictive indicator for T2DM-associated executive impairment and depression status.

Differential toxic and antiepileptic features of Vigabatrin raceme and its enantiomers.

BACKGROUND: The study aimed to investigate the potential pharmacological and toxicological differences between Vigabatrin (VGB) and its enantiomers S-VGB and R-VGB. The researchers focused on the toxic effects and antiepileptic activity of these compounds in a rat model. METHODS: The epileptic rat model was established by intraperitoneal injection of kainic acid, and the antiepileptic activity of VGB, S-VGB, and VGB was observed, focusing on the improvements in seizure latency, seizure frequency and sensory, motor, learning and memory deficits in epileptic rats, as well as the hippocampal expression of key molecular associated with synaptic plasticity and the Wnt/ß-catenin/GSK 3ß signaling pathway. The acute toxic test was carried out and the LD50 was calculated, and tretinal damages in epileptic rats were also evaluated. RESULT: The results showed that S-VGB exhibited stronger antiepileptic and neuroprotective effects with lower toxicity compared to VGB raceme. These findings suggest that S-VGB and VGB may modulate neuronal damage, glial cell activation, and synaptic plasticity related to epilepsy through the Wnt/ß-catenin/GSK 3ß signaling pathway. The study provides valuable insights into the potential differential effects of VGB enantiomers, highlighting the potential of S-VGB as an antiepileptic drug with reduced side effects. CONCLUSION: S-VGB has the highest antiepileptic effect and lowest toxicity compared to VGB and R-VGB.

Pharmacological Research Progress of Novel Antihypertensive Drugs.

Cardiovascular disease stands as the leading cause of death globally, with hypertension emerging as an independent risk factor for its development. The worldwide prevalence of hypertension hovers around 30%, encompassing a staggering 1.2 billion patients, and continues to escalate annually. Medication plays a pivotal role in managing hypertension, not only effectively regulating blood pressure (BP) but also substantially mitigating the occurrence of cardiovascular and cerebrovascular diseases. This review comprehensively outlines the categories, mechanisms, clinical applications, and drawbacks of conventional antihypertensive drugs. It delves into the five primary pharmacological classifications, namely ß-receptor blockers, calcium channel blockers (CCBs), angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and diuretics. The emphasis is placed on elucidating the mechanisms, advantages, and research progress of novel antihypertensive drugs targeting emerging areas. These include mineralocorticoid receptor antagonists (MRAs), atrial natriuretic peptides (ANPs), neutral endopeptidase inhibitors (NEPIs), sodium-dependent glucose transporter 2 inhibitors (SGLT-2Is), glucagon-like peptide-1 receptor agonists (GLP-1RAs), endothelin receptor antagonists (ERAs), soluble guanylate cyclase (sGC) agonists, brain aminopeptidase A inhibitors (APAIs), and small interfering ribonucleic acids (siRNAs) targeting hepatic angiotensinogen. Compared to conventional antihypertensive drugs, these novel alternatives exhibit favorable antihypertensive effects with minimal adverse reactions. This review serves as a valuable reference for future research and the clinical application of antihypertensive drugs.

Application of a novel and green temperature-responsive deep eutectic solvent system to simultaneously extract and separate different polar active phytochemicals from Schisandra chinensis (Turcz.) Baill.

In the present study, a novel and green temperature-responsive deep eutectic solvent (TRDES) system was developed and applied for the simultaneous extraction and separation of different polar active phytochemicals from Schisandra chinensis (Turcz.) Baill. The TRDES, consisting of amino alcohols and phenolic compounds, was chosen as the switching medium, and an upper critical solution temperature (UCST) type switchable solvent was obtained by adding an inorganic salt solution. The switchable phase diagram was plotted based on the relationship between the phase change temperature, the concentration and the amount of sodium chloride solution. Under optimal parameters, the yields with TRDES for different polar active phytochemicals (lignanoids and polysaccharides) from the dried fruit of Schisandra chinensis (DFSC) were 1.62 âˆ¼ 1.17-fold and 1.39-fold to those with conventional solvents. Also, the TRDES system was still effective on extraction of DFSC lignanoids and polysaccharides after four cycles of extraction. The separated polysaccharides and lignanoids both had strong antioxidant activities with IC50 values of 1.92 mg/ mL and 0.10 mg/ mL against 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS), respectively. The extraction mechanism of TRDES was postulated by Density functional theory (DFT) calculations the hydrogen bonding in TRDES was the main factor to the higher extraction yield. This temperature-responsive deep eutectic solvent could be widely used for the efficient extraction and separation of multi-polar components. As a green and recyclable solvents, TRDES has great potential for the lower cost production from plants.

Cell individuality: the bistable gene expression of the type III secretion system in Dickeya dadantii 3937.

Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.

[Study on effects of microRNA-21 antisense oligonucleotide in vivo and in vitro on bionomics of human cervical squamous carcinoma cell lines SiHa].

OBJECTIVE: To explore the effect of microRNA-21 (miR-21) antisense oligonucleotide on the biological characteristics of human cervical squamous carcinoma cell lines SiHa in vivo and in vitro. METHODS: Specific phosphorothioate antisense oligodeoxynucleotides targeting miR-21 were synthesized and transfected into cervical cancer cells in vitro. Expression of miR-21 in SiHa after transfection was detected by real-time RT-PCR. The cell proliferation was evaluated by MTT assay and colony formation experiment. The cell apoptosis was analyzed by annexin V-FITC/PI analysis. The inhibitory effect of miR-21 antisense oligonucleotide on tumor growth was evaluated by tumor growth curves and immunohistochemistry (MaxVision method). H-E staining was used to document morphological changes and fluorometric TUNEL assay was to detect the apoptotic activity. RESULTS: After the transfection of antisense miR-21, the expression of miR-21 decreased along with an obvious growth inhibition, compared with that of the control groups (P < 0.05). Colony formation of both cell lines was markedly inhibited with antisense miR-21 (55.6% ± 1.4%), as compared with that in the negative group (98.3% ± 2.0%, P < 0.05). Flow cytometry assay showed that antisense miR-21 expression significantly enhanced the cell apoptosis (6.7% ± 1.3% and 29.4% ± 1.7%, P < 0.05). The tumor-forming rates of miR-21 transfected group, and negative control groups were 3/8 and 6/8, respectively (P < 0.05). Ki-67 proliferative marker staining decreased significantly (42% vs 90%) in the transfected group compared with negative control groups. Extensive dead tumor cells were seen in the miR-21 transfected cells along with a marked increase of apoptosis (P < 0.05). CONCLUSION: Targeted antisense oligonucleotide miR-21 effectively suppresses the growth of cervical carcinoma SiHa cells both in vitro and in vivo through an induction of apoptosis.

[MicroRNA383 regulates expression of PRDX3 in human medulloblastomas].

OBJECTIVE: To investigate the effects of microRNA-383 (miR-383) on PRDX3 gene expression, cell proliferation and apoptosis of human medulloblastma. METHODS: PRDX3 and miR-383 RNA expression was detected by real-time quantitative RT-PCR in human medulloblastoma tumor tissue samples, Daoy cell line and normal brain tissue samples. Western blot was used to detect protein expression of PRDX3. Synthetic miR-383 mimics were transfected into Daoy cells by lipofectamine. Using Cell Counting Kit-8 (CCK-8) method, flow cytometry was used to investigate the cell proliferation and apoptosis, cells reactive oxgen species(ROS), mitochondrial membrane potential changes in each experimental groups. RESULTS: Of 15 cases of human medulloblastoma tumor, 13 cases had miR-383 expression levels significantly lower than that of normal brain tissue, and 14 had PRDX3 mRNA expression levels significantly higher than that of normal brain tissue. The expression levels of miR-383 and PRDX3 in Daoy cells were 0.353 and 1.315 times than those of normal brain tissue, respectively. The protein expression levels of PRDX3 were higher in human medulloblatoma tumors and Daoy cells than that of normal brain tissue. Transfected miR-383 mimics increased the expression level of miR-383 after 24 h and 48 h was significantly higher than that of the control. In contrast, PRDX3 gene mRNA and protein expression levels were significantly decreased at 48 h compared with the control group. Using CCK-8 assay, the cell proliferation rate in the experimental group was significantly lower than that of the control group (P < 0.05). Annexin V-FITC assay demonstrated that early apoptosis rate of the experimental group (11.60 ± 0.30)% was significantly higher than those of the control group (2.3 ± 0.20)% and negative control group (10.37 ± 0.25)% (P = 0.000) after 48 h of transfection. The intracellular ROS levels after transfection at 24 and 48 h significantly increased than those of the control group. Mitochondrial membrane potential level at 24 h after transfection significantly decreased, comparing with the blank control group and the negative control group. CONCLUSIONS: Compared with normal brain tissue, decreased expression of miR-383 but elevated expression of PRDX3 are medulloblastoma tumour and Daoy cell lines. Up-regulation of miR-383 knockdowns the expression of PRDX3, inhibits proliferation and promotes apoptosis of Daoy cells, leading to increased intracellular ROS and decreased levels of mitochondrial membrane potential.