Hi-C profiling in tissues reveals 3D chromatin-regulated breast tumor heterogeneity and tumor-specific looping-mediated biological pathways.

Current knowledge in three-dimensional (3D) chromatin regulation in normal and disease states was mostly accumulated through Hi-C profiling in in vitro cell culture system. The limitations include failing to recapitulate disease-specific physiological properties and often lacking clinically relevant disease microenvironment. In this study, we conduct tissue-specific Hi-C profiling in a pilot cohort of 12 breast tissues comprising of two normal tissues (NTs) and ten ER+ breast tumor tissues (TTs) including five primary tumors (PTs), and five tamoxifen-treated recurrent tumors (RTs). We find largely preserved compartments, highly heterogeneous topological associated domains (TADs) and intensively variable chromatin loops among breast tumors, demonstrating 3D chromatin-regulated breast tumor heterogeneity. Further cross-examination identifies RT-specific looping-mediated biological pathways and suggests CA2, an enhancer-promoter looping (EPL)-mediated target gene within the bicarbonate transport metabolism pathway, might play a role in driving the tamoxifen resistance. Remarkably, the inhibition of CA2 not only impedes tumor growth both in vitro and in vivo , but also reverses chromatin looping. Our study thus yields significant mechanistic insights into the role and clinical relevance of 3D chromatin architecture in breast cancer endocrine resistance.

The role of DRP1 mediated mitophagy in HT22 cells apoptosis induced by silica nanoparticles.

Silica nanoparticles (SiNPs) are widely used in the biomedical field and can enter the central nervous system through the blood-brain barrier, causing damage to hippocampal neurons. However, the specific mechanism remains unclear. In this experiment, HT22 cells were selected as the experimental model in vitro, and the survival rate of cells under the action of SiNPs was detected by MTT method, reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and adenosine triphosphate (ATP) were tested by the kit, the ultrastructure of the cells was observed by transmission electron microscope, membrane potential (MMP), calcium ion (Ca2+) and apoptosis rate were measured by flow cytometry, and the expressions of mitochondrial functional protein, mitochondrial dynein, mitochondrial autophagy protein as well as apoptosis related protein were detected by Western blot. The results showed that cell survival rate, SOD, CAT, GSH-Px, ATP and MMP gradually decreased with the increase of SiNPs concentration, while intracellular ROS, Ca2+, LDH and apoptosis rate increased with the increase of SiNPs concentration. In total cellular proteins,the expressions of mitochondrial functional proteins VDAC and UCP2 gradually increased, the expression of mitochondrial dynamic related protein DRP1 increased while the expressions of OPA1 and Mfn2 decreased. The expressions of mitophagy related proteins PINK1, Parkin and LC3Ⅱ/LC3Ⅰ increased and P62 gradually decreased, as well as the expressions of apoptosis related proteins Apaf-1, Cleaved-Caspase-3, Caspase-3, Caspase-9, Bax and Cyt-C. In mitochondrial proteins, the expressions of mitochondrial dynamic related proteins DRP1 and p-DRP1 were increased, while the expressions of OPA1 and Mfn2 were decreased. Expressions of mitochondrial autophagy associated proteins PINK1, Parkin, LC3II/LC3I increased, P62 decreased gradually, as well as the expressions of apoptosis related proteins Cleaved-Caspase-3, Caspase-3, and Caspase-9 increased, and Cyt-C expressions decreased. To further demonstrate the role of ROS and DRP1 in HT22 cell apoptosis induced by SiNPs, we selected the ROS inhibitor N-Acetylcysteine (NAC) and Dynamin-related protein 1 (DRP1) inhibitor Mdivi-1. The experimental results indicated that the above effects were remarkably improved after the use of inhibitors, further confirming that SiNPs induce the production of ROS in cells, activate DRP1, cause excessive mitochondrial division, induce mitophagy, destroy mitochondrial function and eventually lead to apoptosis.

VDAC1 Protein Regulation of Oxidative Damage and Mitochondrial Dysfunction-Mediated Cytotoxicity by Silica Nanoparticles in SH-SY5Y Cells.

Silica nanoparticles (SiNPs) have been widely used in industry, electronics, and pharmaceutical industries. In addition, it is also widely used in medicine, tumor treatment and diagnosis, as well as other biomedical and biotechnology fields. The opportunities for people to contact SiNPs through iatrogenic, occupational, and environmental exposures are gradually increasing. The damage and biological effects of SiNPs on the nervous system have attracted widespread attention in the field of toxicology. Central nerve cells are rich in mitochondria. It is suggested that the effects of SiNPs on mitochondrial damage of nerve cells may involve the maintenance of neuronal membrane potential, the synthesis and operation of neurotransmitters, and the transmission of nerve pulses, and so on. We established an experimental model of SH-SY5Y cells to detect the cell survival rate, apoptosis, changes of reactive oxygen species and mitochondrial membrane potential, and the expression of mitochondrial function-related enzymes and proteins, so as to reveal the possible mechanism of SiNPs on neuronal mitochondrial damage. It was found that SiNPs could cause oxidative damage to cells and mitochondria, destroy some normal functions of mitochondria, and induce apoptosis in SH-SY5Y cells. The voltage-dependent anion channel 1(VDAC1) protein inhibitor DIDS could effectively reduce intracellular oxidative stress, such as the reduction of ROS content, and could also usefully restore some functional proteins of mitochondria to normal levels. The inhibition of VDAC1 protein may play an important role in the oxidative damage and dysfunction of neuronal mitochondria induced by SiNPs.

Modeling of and Experimenting with Concentric Tube Robots: Considering Clearance, Friction and Torsion.

Concentric tube robots (CTRs) are a promising prospect for minimally invasive surgery due to their inherent compliance and ability to navigate in constrained environments. Existing mechanics-based kinematic models typically neglect friction, clearance, and torsion between each pair of contacting tubes, leading to large positioning errors in medical applications. In this paper, an improved kinematic modeling method is developed. The effect of clearance on tip position during concentric tube assembly is compensated by the database method. The new kinematic model is mechanic-based, and the impact of friction moment and torsion on tubes is considered. Integrating the infinitesimal torsion of the concentric tube robots eliminates the errors caused by the interaction force between the tubes. A prototype is built, and several experiments with kinematic models are designed. The results indicate that the error of tube rotations is less than 2 mm. The maximum error of the feeding experiment does not exceed 0.4 mm. The error of the new modeling method is lower than that of the previous kinematic model. This paper has substantial implications for the high-precision and real-time control of concentric tube robots.

Feasibility of Enhanced Recovery After Surgery Protocols Implemented Perioperatively in Endoscopic Submucosal Dissection for Early Gastric Cancer: A Single-Center Retrospective Study.

Background: Endoscopic submucosal dissection (ESD) for early gastric cancer (EGC) has advantages over traditional radical gastrectomy. We investigated whether enhanced recovery after surgery (ERAS) protocols are appropriate in the ESD perioperative period. Materials and Methods: We screened 129 consecutive patients, and 12 were excluded. All study patients underwent ESD for EGC. Of the 117 included patients, 57 received traditional perioperative care between January 2017 and December 2018, and 60 patients received perioperative care according to ERAS protocols between January 2019 and September 2020. The primary study endpoint was ESD-related complications. Secondary endpoints included the following postoperative parameters: anal exhaust time, incidence of nausea or vomiting, length of hospitalization, fever rate, abdominal pain on the visual analog scale (VAS), and reported perioperative satisfaction. Results: Complications were comparable between the 2 groups. In the ERAS group, no patients experienced delayed bleeding or perforation. One traditional group patient bled, and one perforated. Postoperative anal exhaust time, nausea or vomiting incidence, hospitalization, fever rate, and VAS pain scores were significantly lower, and perioperative satisfaction rate was significantly higher in the ERAS group. Conclusions: ERAS protocols are both feasible and safe for patients undergoing ESD. ERAS protocols enhance the advantages of ESD for EGC without increasing complications.

Distal pancreatectomy with En bloc celiac axis resection for locally advanced pancreatic body/tail cancer: A systematic review and meta-analysis.

Distal pancreatectomy with En-bloc celiac axis resection (DP-CAR) is a challenging procedure that has yielded certain clinical efficacy in the treatment of locally advanced pancreatic body/tail cancer, especially in patients with invasion of abdominal vessels. However, the clinical efficacy and safety of DP-CAR remain controversial. The study aimed to systematically review efficacy and safety of DP-CAR in the treatment of locally advanced pancreatic body/tail cancer. We systematically searched PubMed, EMBASE, Cochrane Library, and Web of Science databases from inception to 1 October 2020. Two studiers independently accomplished the study selection, data extraction, and quality assessment. Initially, of 1032 studies were searched, among which 11 high quality studies including 1072 patients were finally identified. The pooled results showed that DP-CAR versus Distal pancreatectomy (DP), the rate of R0 resection (RR = 0.76; 95%CI: 0.66 to 0.88; p = 0.0002) and 3-year survival (RR = 0.65; 95%CI: 0.43 to 0.98; p = 0.04) was lower, postoperative mortality (RR = 2.48; 95%CI: 1.02 to 6.03; p = 0.04) was higher, the operation time (MD = 104.67; 95%CI: 84.70 to 124.64; p < 0.001) and hospital stay (MD = 3.94, 95% CI 1.35 to 6.53; p = 0.003) were longer. There was no statistical difference between the DP-CAR and DP group in 1-year, 2-year survival rate (RR = 0.84; 95%CI: 0.57 to 1.23; p = 0.37) (RR = 0.70; 95%CI: 0.45 to 1.10; p = 0.12). In conclusion, compared with DP, DP-CAR has worse efficacy and prognosis survival and is more dangerous, but it can obtain better survival benefit and quality of life than palliative treatment. We suggest that DP-CAR can be carefully attempted for effective margin-negative resection. However, surgeons and patients need to know its potential perioperative risk.

Mitochondrial ATP synthase subunit d, a component of the peripheral stalk, is essential for growth and heat stress tolerance in Arabidopsis thaliana.

As rapid changes in climate threaten global crop yields, an understanding of plant heat stress tolerance is increasingly relevant. Heat stress tolerance involves the coordinated action of many cellular processes and is particularly energy demanding. We acquired a knockout mutant and generated knockdown lines in Arabidopsis thaliana of the d subunit of mitochondrial ATP synthase (gene name: ATPQ, AT3G52300, referred to hereafter as ATPd), a subunit of the peripheral stalk, and used these to investigate the phenotypic significance of this subunit in normal growth and heat stress tolerance. Homozygous knockout mutants for ATPd could not be obtained due to gametophytic defects, while heterozygotes possess no visible phenotype. Therefore, we used RNA interference to create knockdown plant lines for further studies. Proteomic analysis and blue native gels revealed that ATPd downregulation impairs only subunits of the mitochondrial ATP synthase (complex V). Knockdown plants were more sensitive to heat stress, had abnormal leaf morphology, and were severely slow growing compared to wild type. These results indicate that ATPd plays a crucial role in proper function of the mitochondrial ATP synthase holoenzyme, which, when reduced, leads to wide-ranging defects in energy-demanding cellular processes. In knockdown plants, more hydrogen peroxide accumulated and mitochondrial dysfunction stimulon (MDS) genes were activated. These data establish the essential structural role of ATPd and support the importance of complex V in normal plant growth, and provide new information about its requirement for heat stress tolerance.

BdFAR4, a root-specific fatty acyl-coenzyme A reductase, is involved in fatty alcohol synthesis of root suberin polyester in Brachypodium distachyon.

Suberin is a complex hydrophobic polymer of aliphatic and phenolic compounds which controls the movement of gases, water, and solutes and protects plants from environmental stresses and pathogenic infection. The synthesis and regulatory pathways of suberin remain unknown in Brachypodium distachyon. Here we describe the identification of a B. distachyon gene, BdFAR4, encoding a fatty acyl-coenzyme A reductase (FAR) by a reverse genetic approach, and investigate the molecular relevance of BdFAR4 in the root suberin synthesis of B. distachyon. BdFAR4 is specifically expressed throughout root development. Heterologous expression of BdFAR4 in yeast (Saccharomyces cerevisiae) afforded the production of C20:0 and C22:0 fatty alcohols. The loss-of-function knockout of BdFAR4 by CRISPR/Cas9-mediated gene editing significantly reduced the content of C20:0 and C22:0 fatty alcohols associated with root suberin. In contrast, overexpression of BdFAR4 in B. distachyon and tomato (Solanum lycopersicum) resulted in the accumulation of root suberin-associated C20:0 and C22:0 fatty alcohols, suggesting that BdFAR4 preferentially accepts C20:0 and C22:0 fatty acyl-CoAs as substrates. The BdFAR4 protein was localized to the endoplasmic reticulum in Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf epidermal cells. BdFAR4 transcript levels can be increased by abiotic stresses and abscisic acid treatment. Furthermore, yeast one-hybrid, dual-luciferase activity, and electrophoretic mobility shift assays indicated that the R2R3-MYB transcription factor BdMYB41 directly binds to the promoter of BdFAR4. Taken together, these results imply that BdFAR4 is essential for the production of root suberin-associated fatty alcohols, especially under stress conditions, and that its activity is transcriptionally regulated by the BdMYB41 transcription factor.

PRRX1 promotes lymph node metastasis of gastric cancer by regulating epithelial-mesenchymal transition.

BACKGROUND: Gastric cancer has multiple metastasis pathways, of which lymph node metastasis plays a dominant role. However, the specific mechanism of lymph node metastasis is still not unclear. METHODS: The bioinformatics technology was utilized to mine gene chip data related to gastric cancer and Epithelial-Mesenchymal Transition (EMT) in a high-throughput gene expression database (Gene Expression Omnibus, GEO), we screened out all genes that have differential expression levels in gastric cancer tissues and in adjacent normal gastric mucosa tissues. The corresponding function package of R language software were performed for gene annotation and cluster analysis, then enrichment analysis of genes with differential expression and protein interaction network diagram for correlation analysis were performed, we finally screened out the paired related homeobox 1 gene (PRRX1) related to EMT. Next, we collected 65 metastatic lymph node samples and 93 gastric cancer tissue samples. The expression levels of PRRX1 and EMT-related protein E-cadherin (E-ca) and vimentin (Vim) in gastric cancer tissues and metastatic lymph node tissues were determined by immunohistochemistry (IHC) staining of streptavidin-peroxidase (SP). The expression differences of PRRX1, E-ca and Vim in gastric cancer tissues and metastatic lymph node tissues as well as the correlation were analyzed by the experimental data, and the clinical significance was analyzed in combination with the clinicopathological data. RESULTS: The PRRX1 expression levels in gastric cancer tissues are significantly higher than that in adjacent normal gastric mucosa tissues. The positive expression rates of PRRX1, Vim and E-ca in gastric cancer and in metastatic lymph node tissues were significantly different. Comparing with that in gastric cancer, expression of PRRX1 and Vim was significantly down-regulated, and E-ca expression was significantly up-regulated in metastatic lymph nodes. CONCLUSION: PRRX1 may promote lymph node metastasis of gastric cancer by regulating EMT, and then affect the prognosis of patients. PRRX1 may be used as a new biological indicator to predict or prevent lymph node metastasis in gastric cancer.

Salicylic Acid Effects on Flue-Cured Tobacco Quality and Curing Characteristics During Harvesting and Curing in Cold-Stressed Fields.

Salicylic acid (SA) can induce plants to actively enhance abiotic stress resistance. Spraying SA to prevent cold stress in flue-cured tobacco fields can provide theoretical support and practical guidance for the actual protection from cold stress in fields at high altitude in Yunnan. The experiment was performed in Jianchuan County Yunnan Province, China. Honghuadajinyuan, a flue-cured tobacco variety with cold resistance, was used as the research object. SA was tested at two concentrations (0.05 [SA-1] and 0.1 [SA-1] mol L-1) relative to an untreated control (Control) to compare the quality of fresh tobacco leaves, curing characteristics, enzyme activity of antioxidants, and quality of the first-cured tobacco leaves. The tissue structure thickness, SPAD, and plastid pigment content of fresh tobacco leaves were least in the control; there was no significant difference between SA-1 and SA-2. The change of moisture content during curing was SA-1 > SA-2 > Control, and the water loss rate was Control > SA-2 > SA-1, and both varied greatly at 38-48°C. In each curing stage, the carbon and nitrogen metabolites and polyphenols changed most rapidly at 38°C, and the sugar metabolites changed as follows: Control > SA-1 > SA-2. The activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase in fresh tobacco leaves were SA-1 > SA-2 > Control. Malondialdehyde content and the inactivation rate of antioxidant enzymes during curing was Control > SA-2 > SA-1. The economic character and sensory smoking quality of flue-cured tobacco leaves were SA-1 > SA-2 > Control. In high-altitude tobacco planting areas prone to cold stress in the field, early warning weather forecast and field spraying 0.05 mol L-1 SA are beneficial to protect and improve the quality of fresh tobacco leaves, curing characteristics, antioxidant system enzyme activities, and the quality of flue-cured tobacco leaves.

TaCER1-1A is involved in cuticular wax alkane biosynthesis in hexaploid wheat and responds to plant abiotic stresses.

To protect above-ground plant organs from excessive water loss, their surfaces are coated by waxes. The genes involved in wax formation have been investigated in detail in Arabidopsis but scarcely in crop species. Here, we aimed to isolate and characterize a CER1 enzyme responsible for formation of the very long-chain alkanes present in high concentrations especially during late stages of wheat development. On the basis of comparative wax and transcriptome analyses of various wheat organs, we selected TaCER1-1A as a primary candidate and demonstrated that it was located to the endoplasmic reticulum, the subcellular compartment for wax biosynthesis. A wheat nullisomic-tetrasomic substitution line lacking TaCER1-1A had significantly reduced amounts of C33 alkane, whereas rice plants overexpressing TaCER1-1A showed substantial increases of C25 -C33 alkanes relative to wild type control. Similarly, heterologous expression of TaCER1-1A in Arabidopsis wild type and the cer1 mutant resulted in increased levels of unbranched alkanes, iso-branched alkanes and alkenes. Finally, the expression of TaCER1-1A was found activated by abiotic stresses and abscisic acid treatment, resulting in increased production of alkanes in wheat. Taken together, our results demonstrate that TaCER1-1A plays an important role in wheat wax alkane biosynthesis and involved in responding to drought and other environmental stresses.

Engineering a novel protease-based Exendin-4 derivative for type 2 antidiabetic therapeutics.

To develop an effective long-acting antidiabetic agent, we designed a novel Exendin-4 derivative (termed LEx4) containing an albumin-binding domain (ABD), a protease-cleavable linker and a native Exendin-4. Here, we present the LEx4 with balanced glucoregulatory activity and prolonged in vivo activity. As a first step, the LEx4 with purity more than 99% was prepared. Microscale thermophoresis (MST) results demonstrated that LEx4 associates with rat and monkey serum albumin with high-affinity (Ka = 1.26 × 106 M-1 and 1.52 × 106 M-1, respectively). Then the stability test in vitro showed the enhanced antiproteolytic ability of LEx4 in rat and human plasma compared to native Exendin-4. Oral glucose tolerance test (OGTT) in type 2 diabetic mice showed the glucose-lowering efficacy of LEx4 was clearly dosage-dependent within 25-250 nmol/kg. In addition, the protracted antidiabetic effects of LEx4 were further confirmed by both multiple OGTTs and hypoglycemic efficacies test in type 2 diabetic mice. In Sprague Dawley (SD) rats, LEx4 also showed 3.3-fold longer elimination half-life (t1/2) than native Exendin-4. Furthermore, once daily injection of LEx4 to db/db mice achieved long-term beneficial effects on body weight, blood biochemical values, glucose tolerance and pancreatic tissue. We believe LEx4 has superior pharmaceutical potential as a therapeutic drug to against type-2 diabetes mellitus (T2DM) based on these results. This strategy of albumin binding is also applicable to other bioactive peptides for development of long-acting therapeutic drugs.

Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus

Abstract Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13 × 3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13 × 3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.

[Study of plasmid-mediated 16S rRNA methylase genes and drug-resistant transferability of Acinetobacter baumannii isolated from burn ward].

OBJECTIVE: To investigate the drug-resistance of Acinetobacter baumannii (Ab) isolated from patients in burn ward, and study the incidence of 16S rRNA methylase genes mediated high-level aminoglycoside drug-resistance and its mechanism of transfer. METHODS: A total of 40 Ab clinical isolates were collected from burn ward in Gansu Province People's Hospital from May 2006 to Dec. 2007. The sensitivity of Ab for 20 antibiotics were determinated by K-B agar diffusion. The minimal inhibitory concentrations (MIC) of amikacin, gentamicin, tobramycin, netilmicin, isepamicin and kanamycin against Ab strains were determinated by agar dilution. Five kinds of 16S rRNA methylase genes including armA, rmtA, rmtB, rmtC, rmtD were amplified by PCR, the positive PCR-products were purified and sequenced, and the plasmid were extracted by alkaline lysis. The transferability of drug-resistance were determinated by conjugation and plasmid transformation tests. RESULTS: The drug-resistance rates of Ab against six aminoglycosides antibiotics was 72.5%, 72.5%, 70.0%, 67.5%, 70.0%, 70.0%, respectively. Twenty five strains were resistant to six aminoglycosides antibiotics (62.5%), among which 10 isolates were armA-positive (40.0%); rmtA, rmtB, rmtC and rmtD-positive isolates were not found. Ten transformants and 10 conjugates showed high-level resistance against aminoglycosides antibiotics, all of which the value of MIC > or = 256 microg/mL carried armA gene. CONCLUSIONS: The drug-resistance of Ab clinical isolates have high drug-resistance. 16S rRNA methylases gene exists in Ab and locates in plasmid chromosome.