E3 ligase ATL5 positively regulates seed longevity by mediating the degradation of ABT1 in Arabidopsis.

Ubiquitination is a fundamental mechanism regulating the stability of target proteins in eukaryotes; however, the regulatory mechanism in seed longevity remains unknown. Here, we report that an uncharacterized E3 ligase, ARABIDOPSIS TÓXICOS EN LEVADURA 5 (ATL5), positively regulates seed longevity by mediating the degradation of ACTIVATOR OF BASAL TRANSCRIPTION 1 (ABT1) in Arabidopsis. Seeds in which ATL5 was disrupted showed faster accelerated aging than the wild-type, while expressing ATL5 in atl5-2 basically restored the defective phenotype. ATL5 was highly expressed in the embryos of seeds, and its expression could be induced by accelerated aging. A yeast two-hybrid screen identified ABT1 as an ATL5 interacting protein, which was further confirmed by bimolecular fluorescence complementary assay and co-immunoprecipitation analysis. In vitro and in vivo assays showed that ATL5 functions as an E3 ligase and mediates the polyubiquitination and degradation of ABT1. Disruption of ATL5 diminished the degradation of translated ABT1, and the degradation could be induced by seed ageing and occurred in a proteasome-dependent manner. Furthermore, disruption of ABT1 enhanced seed longevity. Taken together, our study reveals that ATL5 promotes the polyubiquitination and degradation of the ABT1 protein posttranslationally and positively regulates seed longevity in Arabidopsis.

Chrysophanol alleviates acute lung injury caused by Klebsiella pneumoniae infection by inhibiting pro-inflammatory cytokine production.

Acute lung injury (ALI) caused by acute bacterial infection remains a common life-threatening lung disease. An increased inflammatory response is the basis for the occurrence and development of ALI. Most antibiotics can only reduce the bacterial load but do not protect from lung damage because of an excessive immune response. Chrysophanol (chrysophanic acid, Chr), as a natural anthraquinone extracted from Rheum palmatum L., has various biological functions, including anti-inflammatory, anti-cancer activities, and ameliorative effects on cardiovascular diseases. Considering these properties, we investigated the effect of Chr in Klebsiella pneumoniae (KP)-induced ALI mice and its potential mechanism. Our results showed that Chr had protective effects against KP-infected mice, including increased survival rate, decreased bacterial burden, reduced recruitment of immune cells, and reduced reactive oxygen species level of lung macrophages. Chr reduced the expression of inflammatory cytokines by inhibiting the toll-like receptor 4/nuclear factor kappa-B (TLR4/NF-κB) signaling pathway and inflammasome activation and strengthening autophagy. Overactivation of the TLR4/NF-κB signaling pathway by the activator Neoseptin 3 led to Chr losing control of inflammatory cytokines in cells, resulting in increased cell death. Similarly, overactivation of the c-Jun N-terminal kinase signaling pathway using the activator anisomycin resulted in Chr losing its inhibitory effect on NOD-like receptor thermal protein domain associated protein 3 (NFRP3) inflammasome activation, and cell viability was reduced. In addition, autophagy was blocked by siBeclin1, so Chr could not reduce inflammatory factors, and cell viability was markedly inhibited. Collectively, this work unravels the molecular mechanism underpinning Chr-alleviated ALI via inhibiting pro-inflammatory cytokines. Thus, Chr is a potential therapeutic agent for KP-induced ALI.

High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease with manifestation of over-accumulation of fat in liver. Increasing evidences indicate that NAFLD may be in part caused by malfunction of very low density lipoprotein (VLDL) secretion. Hepatocyte nuclear factor 4α (HNF4α), a nuclear receptor protein, plays an important role in sustain hepatic lipid homeostasis via transcriptional regulation of genes involved in secretion of VLDL, such as apolipoprotein B (ApoB). However, the exact functional change of HNF4α in NAFLD remains to be elucidated. In the present study, we found that high fat diet (HFD) induced cytoplasmic retention of HNF4α in hepatocytes, which led to down-regulation of hepatic ApoB expression and its protein level in serum, as well as reduced secretion of VLDL. We further revealed that oxidative stress, elevated in fatty liver, was the key factor inducing the cytoplasmic retention of HNF4α in hepatocytes by activating protein kinase C (PKC)-mediated phosphorylation in HNF4α. Thus, our findings reveal a novel mechanism underlying HFD-induced fatty liver that oxidative stress impairs function of HNF4α on ApoB expression and VLDL secretion via PKC activation, eventually promoting fat accumulation in the liver. Therefore, oxidative stress/PKC/HNF4α pathway may be a novel target to treat diet-induced fatty liver.

Leucocyte Membrane-Coated Janus Microcapsules for Enhanced Photothermal Cancer Treatment.

Polyelectrolyte multilayer (PEM) capsules are promising candidates for many kinds of cancer detection and treatment but are usually intended to deliver cargo to specific sites or to destroy cancer cells based on photothermal effects from the outside. In this publication we prove that it is possible to kill cancer cells from the inside based on phagocytosed PEM capsules. In addition we show how to open the cells and bring the PEM capsules to the surface of cancer cells based on photothermal effects and rapid evaporation of water. Diffusion-based temperature determinations of the photothermal effect up to the evaporation temperature of water are presented.

Discovery of pyrido[2,3-d]pyrimidine-based inhibitors of HCV NS5A.

Efforts to improve the genotype 1a potency and pharmacokinetics of earlier naphthyridine-based HCV NS5A inhibitors resulted in the discovery of a novel series of pyrido[2,3-d]pyrimidine compounds, which displayed potent inhibition of HCV genotypes 1a and 1b in the replicon assay. SAR in this system revealed that the introduction of amides bearing an additional 'E' ring provided compounds with improved potency and pharmacokinetics. Introduction of a chiral center on the amide portion resulted in the observation of a stereochemical dependence for replicon potency and provided a site for the attachment of functional groups useful for improving the solubility of the series. Compound 21 was selected for administration in an HCV-infected chimpanzee. Observation of a robust viral load decline provided positive proof of concept for inhibition of HCV replication in vivo for the compound series.

Self-propelled predator-prey of swarming Janus micromotors.

It is a long-standing challenge to accomplish bionic microrobot that acts in a similar way of white blood cell, chasing bacteria in complex environment. Without an effective external control field, most swarming microrobots systems are usually unable to perform directional movement and redirect their motion to capture the target. Here we report the predatory-prey dynamics of self-propelled clusters of Janus micromotors. The active cluster generates an oxygen bubbles cloud around itself by decomposing H2O2, which levitated it above the substrate, enhancing its mobility in solution to wander around to devour other clusters. The fast decomposition of H2O2 also induced a tubular low-concentration zone that bridges two clusters far separated from each other, resulting in a diffusio-osmotic pressure that drives the two clusters to meet. This predatory-prey phenomena mimic white blood cells chasing bacteria and swarming flocks in nature, shedding light on emergent collective intelligence in biology.

Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells.

OBJECTIVE: To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. METHODS: RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1ß), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured. RESULTS: The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1ß and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity. CONCLUSION: EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

Investigations of Fish Assemblages Using Two Methods in Three Terminal Reservoirs of the East Route of South-to-North Water Transfer Project, China.

The terminal reservoirs of water transfer projects directly supply water for domestic, agricultural, and industrial applications, and the water quality of these reservoirs produce crucial effects on the achievement of project targets. Typically, fish assemblages are monitored as indicators of reservoir water quality, and can also be regulated for its improvement. In the present study, we compared traditional fish landing (TFL) and environmental DNA (eDNA) metabarcoding methods for monitoring fish assemblages in three terminal reservoirs of the East Route of the South-to-North Water Transfer Project, China. Results of TFL and eDNA showed similar assemblage structures and patterns of diversity and spatial distribution with obvious differences in fish composition across three examined reservoirs. Demersal and small fish were dominant in all reservoirs. In addition, a strong association between water transfer distance and assemblages and distribution of non-native fish was found. Our findings highlight the necessity of the fish assemblage monitoring and managing for water quality and revealed the impact of water diversion distance on the structure of fish assemblages and dispersal of alien species along the water transfer project.

Synergistic activity of ceftobiprole and vancomycin in a rat model of infective endocarditis caused by methicillin-resistant and glycopeptide-intermediate Staphylococcus aureus.

The therapeutic activity of ceftobiprole medocaril, the prodrug of ceftobiprole, was compared to that of vancomycin, daptomycin, and the combination of a subtherapeutic dose of ceftobiprole and vancomycin in a rat model of infective endocarditis due to methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300) or glycopeptide-intermediate Staphylococcus aureus (GISA) (NRS4 and HIP 5836) strains. The minimum bactericidal concentrations of ceftobiprole, vancomycin, and daptomycin at bacterial cell densities similar to those encountered in the cardiac vegetation in the rat endocarditis model were 2, >64, and 8 µg/ml, respectively, for MRSA ATCC 43300 and 4, >64, and 8 µg/ml, respectively, for the GISA strain. Ceftobiprole medocaril administered in doses of 100 mg/kg of body weight given intravenously (i.v.) twice a day (BID) every 8 h (q8h) (equivalent to a human therapeutic dose of ceftobiprole [500 mg given three times a day [TID]) was the most effective monotherapy, eradicating nearly 5 log(10) CFU/g MRSA or 6 log(10) CFU/g GISA organisms from the cardiac vegetation and had the highest incidence of sterile vegetation compared to the other monotherapies in the endocarditis model. In in vitro time-kill studies, synergistic effects were observed with ceftobiprole and vancomycin on MRSA and GISA strains, and in vivo synergy was noted with combinations of subtherapeutic doses of these agents for the same strains. Additionally, sterile vegetations were achieved in 33 and 60%, respectively, of the animals infected with MRSA ATCC 43300 or GISA NRS4 receiving ceftobiprole-vancomycin combination therapy. In summary, ceftobiprole was efficacious both as monotherapy and in combination with vancomycin in treating MRSA and GISA infections in a rat infective endocarditis model and warrants further evaluation.

Novel hepatitis C virus replicon inhibitors: synthesis and structure-activity relationships of fused pyrimidine derivatives.

The synthesis of several pyrido[2,3-d]pyrimidine and pyrimido[4,5-d]pyrimidine analogs is described with one such analog possessing subnanomolar potency in both genotype 1a and 1b cell culture HCV replicon assays.

Diagnostic and Prognostic Value of Dysregulated miR-10a-3p in Patients with Severe Pneumonia.

Purpose: Previous studies have shown that microRNA is involved in regulating a variety of human inflammatory diseases. The purpose of this study was to investigate the expression of miR-10a-3p in the blood of patients with severe pneumonia and evaluate its value in the diagnosis and prognosis of severe pneumonia. Patients and Methods: Seventy patients with severe pneumonia and 75 healthy individuals were included in this study. Venous blood of all subjects was obtained for RT-qPCR analysis to obtain the relative expression level of miR-10a-5p. The diagnostic accuracy of miR-10a-5p for severe pneumonia was assessed by ROC curve. After standardized treatment, the prognosis of patients with severe pneumonia was analyzed by a 28-day follow-up method. Kaplan-Meier curve and multivariate Cox regression analysis were used to determine the basic factors influencing the prognosis of patients. Results: Compared with healthy control, serum miR-10a-3p expression in patients with severe pneumonia was distinctly upregulated (P < 0.001). Besides, ROC analysis showed that miR-10a-3p had high diagnostic accuracy for severe pneumonia, with an AUC of 0.881, sensitivity and specificity of 75.7% and 84.0%, respectively. Kaplan-Meier curve exhibited that high miR-10a-3p expression group had a higher probability of death than those with low miR-10a-3p expression. Multivariate Cox regression analysis demonstrated that miR-10a-3p and CRP were independent risk factors affecting the prognosis of patients. Conclusion: The expression of miR-10a-3p was increased in patients with severe pneumonia, and abnormally expressed miR-10a-3p has the potential to be used as a diagnostic and prognostic marker for severe pneumonia, which provides a new biological direction for the early detection and risk assessment of severe pneumonia.

Artificial Intelligence Algorithm-Based Computerized Tomography Image Features Combined with Serum Tumor Markers for Diagnosis of Pancreatic Cancer.

The objective of this study was to analyze the value of artificial intelligence algorithm-based computerized tomography (CT) image combined with serum tumor markers for diagnoses of pancreatic cancer. In the study, 68 hospitalized patients with pancreatic cancer were selected as the experimental group, and 68 hospitalized patients with chronic pancreatitis were selected as the control group, all underwent CT imaging. An image segmentation algorithm on account of two-dimensional (2D)-three-dimensional (3D) convolution neural network (CNN) was proposed. It also introduced full convolutional network (FCN) and UNet network algorithm. The diagnostic performance of CT, serum carbohydrate antigen-50 (CA-50), serum carbohydrate antigen-199 (CA-199), serum carbohydrate antigen-242 (CA-242), combined detection of tumor markers, and CT-combined tumor marker testing (CT-STUM) for pancreatic cancer were compared and analyzed. The results showed that the average Dice coefficient of 2D-3D training was 84.27%, which was higher than that of 2D and 3D CNNs. During the test, the maximum and average Dice coefficient of the 2D-3D CNN algorithm was 90.75% and 84.32%, respectively, which were higher than the other two algorithms, and the differences were statistically significant (P < 0.05). The penetration ratio of pancreatic duct in the experimental group was lower than that in the control group, the rest were higher than that in the control group, and the differences were statistically significant (P < 0.05). CA-50, CA-199, and CA-242 in the experimental group were 141.72 U/mL, 1548.24 U/mL, and 83.65 U/mL, respectively, which were higher than those in the control group, and the differences were statistically significant (P < 0.05). The sensitivity, specificity, positive predictive value, and authenticity of combined detection of serum tumor markers were higher than those of CA-50, CA-199, and CA-242, and the differences were statistically significant (P < 0.05). The results showed that the proposed algorithm 2D-3D CNN had good stability and image segmentation performance. CT-STUM had high sensitivity and specificity in diagnoses of pancreatic cancer.

Antistaphylococcal activities of the new fluoroquinolone JNJ-Q2.

The new broad-spectrum fluoroquinolone JNJ-Q2 displays in vitro activity against Gram-negative and Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant MRSA isolates. Tested with isogenic methicillin-susceptible S. aureus (MSSA) and MRSA strains bearing quinolone-resistant target mutations, JNJ-Q2 displayed MICs ≤ 0.12 µg/ml, values 16- to 32-fold lower than those determined for moxifloxacin. Overexpression of the NorA efflux pump did not impact JNJ-Q2 MICs. Inhibition of S. aureus DNA gyrase and DNA topoisomerase IV enzymes demonstrated that JNJ-Q2 was more potent than comparators against wild-type enzymes and enzymes carrying quinolone-resistant amino acid substitutions, and JNJ-Q2 displayed equipotent activity against both enzymes. In serial-passage studies comparing resistance selection in parallel MRSA cultures by ciprofloxacin and JNJ-Q2, ciprofloxacin readily selected for mutants displaying MIC values of 128 to 512 µg/ml, which were observed within 18 to 24 days of passage. In contrast, cultures passaged in the presence of JNJ-Q2 displayed MICs ≤ 1 µg/ml for a minimum of 27 days of serial passage. A mutant displaying a JNJ-Q2 MIC of 4 µg/ml was not observed until after 33 days of passage. Mutant characterization revealed that ciprofloxacin-passaged cultures with MICs of 256 to 512 µg/ml carried only 2 or 3 quinolone resistance-determining region (QRDR) mutations. Cultures passaged with JNJ-Q2 selection for up to 51 days displayed MICs of 1 to 64 µg/ml and carried between 4 and 9 target mutations. Established in vitro biofilms of wild-type or ciprofloxacin-resistant MRSA exposed to JNJ-Q2 displayed greater decreases in bacterial counts (7 days of exposure produced 4.5 to >7 log(10) CFU decreases) than biofilms exposed to ciprofloxacin, moxifloxacin, rifampin, or vancomycin.

Longitudinal survey of carbapenem resistance and resistance mechanisms in Enterobacteriaceae and non-fermenters from the USA in 2007-09.

BACKGROUND: Antibiotic resistance is problematic in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, and is often associated with serious infections. Carbapenems are often one of the few remaining therapeutic options, so it is important to monitor carbapenem activity against these pathogens and to identify resistance mechanisms. METHODS: Carbapenem susceptibilities were determined for 14 359 Enterobacteriaceae, 3614 P. aeruginosa and 994 A. baumannii from the USA (2007-09). Klebsiella pneumoniae with doripenem MICs ≥2 mg/L (n = 88), and P. aeruginosa (n = 452), A. baumannii (n = 349) and other enterics (n = 13) with doripenem MICs ≥4 mg/L were screened for carbapenem resistance mechanisms. RESULTS: Doripenem/meropenem and imipenem susceptibilities for Enterobacteriaceae were >99% and 89%, respectively. Doripenem susceptibility (2007-09) for P. aeruginosa was 87.4%-84.1%; comparable to meropenem and higher than imipenem. For A. baumannii, doripenem susceptibility (2007-09) was 63%-58.2%; lower than imipenem and meropenem. Resistant K. pneumoniae had KPC and lacked porins OmpK35/OmpK36. In 2009, 3.4% of all K. pneumoniae possessed KPC. Five other enterics and one P. aeruginosa possessed KPC. Resistance mechanisms in P. aeruginosa were loss of porin OprD (90%), efflux (55%) and elevated AmpC activity (25%). Acquired carbapenemases OXA-23/-24 were present in 48% of resistant A. baumannii. VIM metallo-ß-lactamases were present in three P. aeruginosa and one A. baumannii isolates. CONCLUSIONS: Doripenem and meropenem were more active than imipenem against Enterobacteriaceae and P. aeruginosa from the USA. Carbapenem resistance mechanisms included serine carbapenemases, elevated AmpC activity, efflux and porin deficiencies occurring mostly in P. aeruginosa. Metallo-ß-lactamases were found in <0.1% of isolates.

Chromosome-level genome assembly of the Chinese longsnout catfish Leiocassis longirostris.

The Chinese longsnout catfish ( Leiocassis longirostris Günther) is one of the most economically important freshwater fish in China. As wild populations have declined sharply in recent years, it is also a valuable model for research on sexual dimorphism, comparative biology, and conservation. However, the current lack of high-quality chromosome-level genome information for the species hinders the advancement of comparative genomic analysis and evolutionary studies. Therefore, we constructed the first high-quality chromosome-level reference genome for L. longirostris. The total genome was 703.19 Mb, with 389 contigs and contig N50 length of 4.29 Mb. Using high-throughput chromosome conformation capture (Hi-C) data, the genome sequences (685.53 Mb) were scaffolded into 26 chromosomes ranging from 17.36 to 43.97 Mb, resulting in a chromosomal anchoring rate for the genome of 97.44%. In total, 23 708 protein-coding genes were identified in the genome. Phylogenetic analysis indicated that L. longirostris and its closest related species P. fulvidraco diverged approximately 26.6 million years ago. This high-quality reference genome of L. longirostris should pave the way for future genomic comparisons and evolutionary research.

Affinity of ceftobiprole for penicillin-binding protein 2b in Streptococcus pneumoniae strains with various susceptibilities to penicillin.

Wild-type penicillin-binding protein (PBP) 2b from penicillin-susceptible Streptococcus pneumoniae had high affinity for ceftobiprole and penicillin (50% inhibitory concentrations [IC(50)s] of ≤0.15 µg/ml) but not ceftriaxone (IC(50) of >8 µg/ml). In clinical isolates, ceftobiprole and PBP 2b affinities were reduced 15- to 30-fold with a Thr-446-Ala substitution and further still with an additional Ala-619-Gly PBP 2b substitution. Ceftobiprole remained active (MICs of ≤1 µg/ml) against all strains tested and behaved more like penicillin than ceftriaxone with respect to PBP 2b binding.

In vitro antibacterial activities of JNJ-Q2, a new broad-spectrum fluoroquinolone.

JNJ-Q2, a novel fluorinated 4-quinolone, was evaluated for its antibacterial potency by broth and agar microdilution MIC methods in studies focused on skin and respiratory tract pathogens, including strains exhibiting contemporary fluoroquinolone resistance phenotypes. Against a set of 118 recent clinical isolates of Streptococcus pneumoniae, including fluoroquinolone-resistant variants bearing multiple DNA topoisomerase target mutations, an MIC(90) value for JNJ-Q2 of 0.12 microg/ml was determined, indicating that it was 32-fold more potent than moxifloxacin. Against a collection of 345 recently collected methicillin-resistant Staphylococcus aureus (MRSA) isolates, including 256 ciprofloxacin-resistant strains, the JNJ-Q2 MIC(90) value was 0.25 microg/ml, similarly indicating that it was 32-fold more potent than moxifloxacin. The activities of JNJ-Q2 against Gram-negative pathogens were generally comparable to those of moxifloxacin. In further studies, JNJ-Q2 exhibited bactericidal activities at 2x and 4x MIC levels against clinical isolates of S. pneumoniae and MRSA with various fluoroquinolone susceptibilities, and its activities were enhanced over those of moxifloxacin. In these studies, the activity exhibited against strains bearing gyrA, parC, or gyrA plus parC mutations was indicative of the relatively balanced (equipotent) activity of JNJ-Q2 against the DNA topoisomerase target enzymes. Finally, determination of the relative rates or frequencies of the spontaneous development of resistance to JNJ-Q2 at 2x and 4x MICs in S. pneumoniae, MRSA, and Escherichia coli were indicative of a lower potential for resistance development than that for current fluoroquinolones. In conclusion, JNJ-Q2 exhibits a range of antibacterial activities in vitro that is supportive of its further evaluation as a potential new agent for the treatment of skin and respiratory tract infections.

Full-length transcript sequencing accelerates the transcriptome research of Gymnocypris namensis, an iconic fish of the Tibetan Plateau.

Gymnocypris namensis, the only commercial fish in Namtso Lake of Tibet in China, is rated as nearly threatened species in the Red List of China's Vertebrates. As one of the highest-altitude schizothorax fish in China, G. namensis has strong adaptability to the plateau harsh environment. Although being an indigenous economic fish with high value in research, the biological characterization, genetic diversity, and plateau adaptability of G. namensis are still unclear. Here, we used Pacific Biosciences single molecular real time long read sequencing technology to generate full-length transcripts of G. namensis. Sequences clustering analysis and error correction with Illumina-produced short reads to obtain 319,044 polished isoforms. After removing redundant reads, 125,396 non-redundant isoforms were obtained. Among all transcripts, 103,286 were annotated to public databases. Natural selection has acted on 42 genes for G. namensis, which were enriched on the functions of mismatch repair and Glutathione metabolism. Total 89,736 open reading frames, 95,947 microsatellites, and 21,360 long non-coding RNAs were identified across all transcripts. This is the first study of transcriptome in G. namensis by using PacBio Iso-seq. The acquisition of full-length transcript isoforms might accelerate the transcriptome research of G. namensis and provide basis for further research.

Effect of MexXY overexpression on ceftobiprole susceptibility in Pseudomonas aeruginosa.

Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus broad-spectrum cephalosporin, has activity (MIC for 50% of strains tested, < or =4 microg/ml) against many Pseudomonas aeruginosa strains. A common mechanism of P. aeruginosa resistance to beta-lactams, including cefepime and ceftazidime, is efflux via increased expression of Mex pumps, especially MexAB. MexXY has differential substrate specificity, recognizing cefepime but not ceftazidime. In ceftobiprole clinical studies, paired isolates of P. aeruginosa from four subjects demonstrated ceftobiprole MICs of 2 to 4 microg/ml at baseline but 16 microg/ml posttreatment, unrelated to beta-lactamase levels. Within each pair, the level of mexXY RNA, but not mexAB, mexCD, and mexEF, increased by an average of 50-fold from baseline to posttreatment isolates. Sequencing of the negative regulatory gene mexZ indicated that each posttreatment isolate contained a mutation not present at baseline. mexXY expression as a primary ceftobiprole and cefepime resistance mechanism was further examined in isogenic pairs by using cloned mexXY and mexZ. Expression of cloned mexXY in strain PAO1 or in a baseline isolate increased the ceftobiprole MIC to that for the posttreatment isolate. In contrast, in posttreatment isolates, lowering mexXY expression via introduction of cloned mexZ decreased the ceftobiprole MIC to that for the baseline isolates. Similar changes were observed for cefepime. A spontaneous mutant selectively overexpressing mexXY displayed a fourfold elevation in its ceftobiprole MIC, while overexpression of mexAB, -CD, and -EF had a minimal effect. These data indicate that ceftobiprole, like cefepime, is an atypical beta-lactam that is a substrate for the MexXY efflux pump in P. aeruginosa.

Knockdown of TRIM28 inhibits PDGF-BB-induced vascular smooth muscle cell proliferation and migration.

Atherosclerosis is a common type of cardiovascular disease (CVD), remaining one of the leading causes of global death. Tripartite motif-containing 28 (TRIM28) is a member of TRIM family that has been found to be involved in atherosclerosis. However, the role of TRIM28 in atherosclerosis remains unknown. This study aimed to investigate the effects of TRIM28 on the phenotypic switching of human aortic smooth muscle cells (HASMCs), which is considered as a fundamental event during the development of atherosclerosis. The results showed that TRIM28 was highly expressed in human atherosclerotic tissues, as well in cultured HASMCs stimulated by platelet-derived growth factor subunit B homodimer (PDGF-BB). Knockdown of TRIM28 by transfection with siRNA targeting TRIM28 (si-TRIM28) significantly suppressed the PDGF-BB-induced cell proliferation and migration of HASMCs. Besides, knockdown of TRIM28 inhibited the expressions of matrix metalloproteinase (MMP)-2 and MMP-9. The VSMC markers including α-smooth muscle actin (α-SMA), calponin and SM22α were upregulated in TRIM28 knocked down HASMCs. Furthermore, knockdown of TRIM28 blocked PDGF-BB-induced NF-κB activation in HASMCs. Collectively, knockdown of TRIM28 prevented PDGF-BB-induced phenotypic switching of HASMCs, which might be mediated by the regulation of NF-κB signaling pathway.