Changes of LncRNAs during the Process of Antioxidants Antagonize Cadmium-Induced Oxidative Damage in Islet ß Cells.

Long non-coding RNAs (LncRNAs) play important regulatory roles in oxidative damage. Resveratrol, curcumin, and cyanidin are phytogenic antioxidants widely existing in nature and they have been proved to antagonize certain heavy metal-induced oxidative damage in cells. However, can they antagonize oxidative damage induced by cadmium in islet ß cells? Are their mechanisms of antagonizing oxidative damage related to LncRNAs? In this study, we first detected the cell viability of each group by CCK8 assay. Next, reactive oxygen species (ROS) were detected by the fluorescent probe. The contents of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) were detected according to the instructions of corresponding kits. At last, the levels of LncRNAs were detected by fluorescence quantitative real-time polymerase chain reaction (qPCR). The results showed that resveratrol, curcumin and cyanidin were able to reverse the reduction of cell viability induced by cadmium (CdSO4). Further determination revealed that SOD activities of the resveratrol+CdSO4, curcumin+CdSO4, and cyanidin+CdSO4 treatment groups increased significantly, and ROS levels and MDA contents dramatically decreased when compared with single CdSO4-treated group. More importantly, the levels of three CdSO4-elevated LncRNAs (NONMMUT029382, ENSMUST00000162103, ENSMUST00000117235) were all decreased and levels of three CdSO4-inhibited LncRNAs (NONMMUT036805, NONMMUT014565, NONMMUT065427) were increased after the pretreatment of resveratrol, curcumin and cyanidin. In summary, resveratrol, curcumin and cyanidin may effectly reverse the cadmium-induced oxidative damage and suggest that phytogenic antioxidants may prevent cells from cadmium-induced oxidative damage through changing the levels of LncRNAs.

Single-cell low-pass whole genome sequencing accurately detects circulating tumor cells for liquid biopsy-based multi-cancer diagnosis.

Accurate detection of circulating tumor cells (CTCs) in blood and non-blood body fluids enables generation of deterministic cancer diagnosis and represent a less invasive and safer liquid biopsy approach. Although genomic alternations have been widely used in circulating tumor DNA (ctDNA) analysis, studies on cell-based genomic alternations profiling for CTC detection are rare due to major technical limitations in single-cell whole genome sequencing (WGS) including low throughput, low accuracy and high cost. We report a single-cell low-pass WGS-based protocol (scMet-Seq) for sensitive and accurate CTC detection by combining a metabolic function-associated marker Hexokinase 2 (HK2) and a Tn5 transposome-based WGS method with improved cell fixation strategy. To explore the clinical use, scMet-Seq has been investigated with blood and non-blood body fluids in diagnosing metastatic diseases, including ascites-based diagnosis of malignant ascites (MA) and blood-based diagnosis of metastatic small-cell lung cancer (SCLC). ScMet-Seq shows high diagnostic sensitivity (MA: 79% in >10 cancer types; metastatic SCLC: 90%) and ~100% of diagnostic specificity and positive predictive value, superior to clinical cytology that exhibits diagnostic sensitivity of 52% in MA diagnosis and could not generate blood-based diagnosis. ScMet-Seq represents a liquid biopsy approach for deterministic cancer diagnosis in different types of cancers and body fluids.

Clinical and epidemiological characteristics of 369 patients with pelvic fractures in Eastern Zhejiang Province of China: a retrospective study.

BACKGROUND: Data on the epidemiological characteristics and prognostic factors of patients with pelvic fractures are lacking, particularly in China. This study aimed to summarise the clinical and epidemiological characteristics of patients with pelvic fractures in eastern Zhejiang Province, China, and to identify risk factors for poor prognosis. METHODS: The clinical data of 369 patients with pelvic fractures admitted to the Ningbo No. 6 Hospital between September 2020 and September 2021 were retrospectively analysed. Data on the demographic characteristics; fracture classification; injury time, cause, and site; treatment plan; and prognosis were collected using the Picture Archiving and Communication System and the Hospital Information System. Differences in constituent proportions were analysed using the chi-square test. Logistic regression analysis was used to identify factors affecting patient prognosis. Statistical significance was set at p ≤ 0.05. RESULTS: Among the 369 patients, there were 206 men and 163 women, at a ratio of 1.26:1, and the average age was 53.64 ± 0.78 years. More than 50% of patients were aged 41-65 years. The average length of hospital stay was 18.88 ± 1.78 days. The three most common causes of pelvic fractures were traffic accidents (51.2%), falls from height (31.44%), and flat-ground falls (14.09%). There were significant differences in the distribution of the three causes of injury depending on age (p < 0.001), sex (p < 0.001), and occupation (p < 0.0001). Most patients were manual workers (48.8%). Furthermore, most patients (n = 262, 71.0%) underwent surgical treatment for pelvic fractures. Postoperative complications occurred in 26 patients (7.05%), and infection was the main complication (73.08%). Age (p = 0.013), occupation (p = 0.034), cause of injury (p = 0.022), treatment options (p = 0.001), and complications (p < 0.0001) were independent factors affecting the prognosis of patients with pelvic fractures. One death (0.027%) occurred, which was due to severe blood loss. CONCLUSIONS: Age, occupation, cause of injury, treatment options and complications were factors affecting patient prognosis. In addition, changes in blood flow and prevention of infection warrant attention.

Claudin18.2 expression and clinicopathological features in cytology effusion specimens from gastric adenocarcinoma: A comparative study with tissue specimens.

BACKGROUND: Zolbetuximab (IMAB362) is under investigation for treating advanced gastrointestinal tumors because it targets Claudin18.2 (CLDN18.2). CLDN18.2 is a promising molecule along with the presence of human epidermal growth factor receptor 2 in gastric cancer. This study evaluated cell block (CB) preparations of serous cavity effusions for the feasibility for CLDN18.2 protein expression and compared the results with those of biopsy or resection specimens. The association of CLDN18.2 expression in effusion samples and the clinicopathological features were also investigated. METHODS: Cytological effusion specimens and matched surgical pathology biopsy or resection specimens of 43 gastric and gastroesophageal junctional cancer cases were stained for CLDN18.2 expression and quantified using immunohistochemistry based on the manufacturer's instructions. RESULTS: Positive staining was detected in 34 (79.1%) tissue and 27 (62.8%) effusion CB samples in this study. When "positivity" was defined as moderate-to-strong staining in ≥40% viable tumor cells, CLDN18.2 expression was observed in 24 (55.8%) tissue and 22 (51.2%) effusion CB samples. A cutoff of 40% for CLDN18.2 positivity was used to demonstrate high concordance (83.7%) between cytology CB and tissue specimens. The results showed that CLDN18.2 expression in effusion specimens correlated with tumor size (p = .021) but not with sex, age at diagnosis, primary tumor location, staging, Lauren phenotype, cytomorphologic features, or Epstein-Barr virus infection. Cytological effusions with or without CLDN18.2 expression did not significantly affect overall survival. CONCLUSIONS: This study's results show that serous body cavity effusions may be suitable for CLDN18.2 biomarker testing; however, discordant cases should be interpreted cautiously.

Peripheral Blood Levels of miR-448 and SIRT1 in Patients with Deep Venous Thrombosis and Their Relationship.

BACKGROUND: The goal of this study was to investigate the changes in peripheral blood levels of miR-448 and silent information regulator 1 (SIRT1) in patients with deep venous thrombosis (DVT) and to analyze their relationship. METHODS: A total of 112 patients treated from January 2019 to June 2020 were divided into DVT group (n = 40) and non-DVT group (n = 72). Fasting venous blood was extracted to separate serum and peripheral blood mononuclear cells (PBMCs). Enzyme-linked immunosorbent assay (ELISA) was employed to measure serum SIRT1 protein, and qPCR was utilized to detect miR-448 expression in PBMCs. The clinical data, serum indicators, and expressions of SIRT1 and miR-448 were compared, and the correlations of miR-448 and SIRT1 with DVT were analyzed using a multivariate Cox regression model. TargetScan Release 7.1 was used to predict the possibility of binding sites between miR-448 and SIRT1 mRNA 3'-untranslated region (3'-UTR), and dual-luciferase reporter assay was used to determine the targeting of miR-448 and SIRT1. HeLa cells were divided into overexpression, inhibition, and blank control groups. The cells were harvested 24 hours after transfection, followed by detection of SIRT1 mRNA expression by qPCR and measurement of supernatant SIRT1 protein expression by ELISA. RESULTS: Serum SIRT1 protein level was lower and miR-448 expression in PBMCs was higher in DVT group than those in non-DVT group (p < 0.05). DVT group had a larger number of patients with vascular diseases and history of venous thrombosis than that of non-DVT group (p < 0.05). miR-448 was an independent risk factor for postoperative DVT, and SIRT1 was a protective factor (p < 0.01). There were potential complementary base binding sites between miR-448 and SIRT1 mRNA 3'-UTR. Dual-luciferase reporter assay verified the targeted regulation between miR-448 and SIRT1. HeLa cell SIRT1 mRNA expression and supernatant SIRT1 protein expression were lower in overexpression group while higher in inhibition group than those in blank control group (p < 0.05). CONCLUSIONS: Serum SIRT1 protein level decreases while miR-448 expression in PBMCs increases in patients with DVT, and miR-448 inhibits SIRT1 expression through binding to SIRT1 mRNA 3'-UTR with complementary bases, thus inducing inflammatory response to participate in the formation of DVT. Targeting miR-448 to regulate cytokine expression may become an effective target and approach for the treatment of DVT. miR-488 combined with SIRT1 has a high predictive value for the occurrence of DVT.

Effect of Nursing in Operating Room Combined with Intraoperative Heat Preservation Intervention on Prevention of Incision Infection and Improvement of Hemodynamics in Patients with Anterior Cruciate Ligament Injury and Reconstruction under Knee Arthroscopy.

Objective: To explore the effect of nursing in operating room combined with intraoperative heat preservation intervention on preventing incision infection and improving hemodynamics in patients with anterior cruciate ligament (ACL) injury and reconstruction under knee arthroscopy. Methods: About 200 patients with knee arthroscopic ACL reconstruction in our hospital from January 2019 to July 2021 were enrolled. The patients were randomly assigned into two groups: the control group and the study group. The former group received nursing care in the operating room operating room and the latter group received nursing care in operating room combined with intraoperative heat preservation intervention. Nursing satisfaction, incidence of incision infection, knee joint VAS score, knee joint range of motion, knee joint Lysholm score, and hemodynamic indexes were compared. Results: First of all, we compared the nursing satisfaction, the study group was very satisfied in 78 cases, satisfactory in 20 cases, and general in 2 cases, and the satisfaction rate was 100.00%, while in the control group, 445 cases were very satisfied, 20 cases were satisfied, 15 cases were general, and 8 cases were dissatisfied. The satisfaction rate was 82.00%. The nursing satisfaction of the study group was higher compared to the control group (P < 0.05). Secondly, we compared the incidence of incision infection. The incidence of incision infection in the study group was lower compared to the control group (P < 0.05). With regard to the knee joint VAS score, the knee joint VAS score of the study group was lower compared to the control group at 2 weeks, 4 weeks, 8 weeks, and 12 weeks after operation (P < 0.05). In terms of the range of motion of the knee joint, the range of motion of the knee joint in the study group was higher compared to the control group at 2 weeks, 4 weeks, 8 weeks, and 12 weeks after operation (P < 0.05). Regarding the knee joint Lysholm score, the knee joint Lysholm score of the study group was higher compared to the control group at 2 weeks, 4 weeks, 8 weeks, and 12 weeks after operation (P < 0.05). Finally, we compared the hemodynamic indexes. Before nursing, there exhibited no significant difference (P > 0.05). During and after nursing, the indexes of HR and MAP in the study group fluctuated little (P < 0.05). Conclusion: During the perioperative period of patients with ACL injury and reconstruction under knee arthroscopy, standardized and necessary operating room combined with intraoperative thermal insulation intervention measures should be given, attention should be paid to the management of operating room, and intraoperative thermal insulation intervention should be strengthened. It includes preoperative visit, psychological nursing of patients, strict application of antibiotics before operation, monitoring of air quality in operating room, disinfection and sterilization of surgical instruments, shortening operation time, maintaining body temperature during operation, and paying attention to hand hygiene of medical staff. It plays a supervisory role in promoting the attention of medical staff to the prevention of wound infection, which is beneficial to the healing of surgical wounds of patients. It plays a positive role in enhancing hemodynamic indexes. Comprehensive nursing intervention on the risk factors of each link can effectively prevent postoperative wound infection and strengthen the prognosis and quality of life of patients.

Misdiagnosis of unroofed coronary sinus syndrome as an ostium primum atrial septal defect by echocardiography: A case report.

BACKGROUND: Unroofed coronary sinus syndrome (UCSS) is a rare congenital heart disease, which has variable morphologic features and is strongly associated with persistent left superior vena cava (PLSVC). However, it is often difficult to visualize the left-to-right shunt pathway through the CS by transthoracic echocardiography (TTE). CASE SUMMARY: A 37-year-old female was admitted to the hepatological surgery department of a hospital with complaint of subxiphoid pain that had started 1 wk prior. Physical examination revealed a grade 3/6 systolic murmur at the left margin of the sternum, between the 2nd and 3rd intercostal cartilage. The patient underwent echocardiography and was diagnosed with ostium primum atrial septal defect (ASD); thus, she was subsequently transferred to the cardiovascular surgery department. A second TTE evaluation before surgery showed type IV UCSS with secundum ASD. Right-heart contrast echocardiography (RHCE) showed that the right atrium and right ventricle were immediately filled with microbubbles, but no microbubble was observed in the CS. Meanwhile, negative filling was observed at the right atrium orifice of the CS and right atrium side of the secundum atrial septal. RHCE identified UCSS combined with secundum ASD but without PLSVC in this patient. CONCLUSION: This rare case of UCSS highlights the value of TTE combined with RHCE in confirming UCSS with ASD or PLSVC.

Activation of the NLRP3 inflammasome by RAC1 mediates a new mechanism in diabetic nephropathy.

OBJECTIVE: Inflammation is central to the development and progression of diabetic nephropathy (DN). Although the exact mechanisms of inflammation in the kidney have not been well elucidated, pyrin domain containing 3 (NLRP3) inflammasome activation is involved in the onset and progression of DN. Here, we investigated the underlying regulatory mechanisms of hyperglycaemia-induced NLRP3 inflammasome activation in the kidney. METHODS: HEK293T cells received high glucose, and the cell proliferation and apoptosis were detected. Biochemical indicators in db/db mice were tested by kits, and the morphological changes in the kidney were observed using staining methods and transmission electron microscopy. The interaction of Ras-related C3 botulinum toxin substrate 1 (RAC1) and NLRP3 inflammasome in cells and in mice was assessed by co-immunoprecipitation (Co-IP) and immunofluorescence. Expression of all proteins was examined by western blotting and immunohistochemistry. In additional, the directly combination of RAC1 and NLRP3 was evaluated by GST Pulldown. RESULTS: High-glucose and hyperglycaemia conditions resulted in Ras-related C3 botulinum toxin substrate 1 (RAC1) and NLRP3 inflammasome interactions in cells and in mice. Additionally, RAC1 promoted NLRP3 inflammasome activation and then induced cell damage, and morphological and functional abnormalities in the kidney. We also observed that RAC1 activates the NLRP3 inflammasome by directly binding to NLRP3. CONCLUSION: In the present study, we confirmed that RAC1 binding to NLRP3 is sufficient to activate the NLRP3 inflammasome in the kidney and accelerate DN pathological processes. These results elucidate the upstream cellular and molecular mechanisms of NLRP3 inflammasome activation and provide new therapeutic strategies for the treatment of DN.

Ras-Related C3 Botulinum Toxin Substrate 1 Combining With the Mixed Lineage Kinase 3- Mitogen-Activated Protein Kinase 7- c-Jun N-Terminal Kinase Signaling Module Accelerates Diabetic Nephropathy.

Ras-related C3 botulinum toxin substrate 1 (RAC1) activation plays a vital role in diabetic nephropathy (DN), but the exact mechanism remains unclear. In this study, we attempted to elucidate the precise mechanism of how RAC1 aggravates DN through cellular and animal experiments. In this study, DN was induced in mice by intraperitoneal injection of streptozotocin (STZ, 150mg/kg), and the RAC1 inhibitor NSC23766 was administered by tail vein injection. Biochemical indicators, cell proliferation and apoptosis, and morphological changes in the kidney were detected. The expression of phosphorylated c-Jun N-terminal kinase (p-JNK), nuclear factor-κB (NF-κB), and cleaved caspase-3 and the interaction between RAC1 and the mixed lineage kinase 3 (MLK3)-mitogen-activated protein kinase 7 (MKK7)-JNK signaling module were determined. Furthermore, the colocalization and direct co-interaction of RAC1 and MLK3 were confirmed. Our results showed that RAC1 accelerates renal damage and increases the expression of p-JNK, NF-κB, and cleaved caspase-3. However, inhibition of RAC1 ameliorated DN by downregulating p-JNK, NF-κB, and cleaved caspase-3. Also, RAC1 promoted the assembly of MLK3-MKK7-JNK, and NSC23766 blocked the interaction between RAC1 and MLK3-MKK7-JNK and inhibited the assembly of the MLK3-MKK7-JNK signaling module. Furthermore, RAC1 was combined with MLK3 directly, but the RAC1 Y40C mutant inhibited the interaction between RAC1 and MLK3. We demonstrated that RAC1 combining with MLK3 activates the MLK3-MKK7-JNK signaling module, accelerating DN occurrence and development, and RAC1 Y40 is an important site for binding of RAC1 to MLK3. This study illustrates the cellular and molecular mechanisms of how RAC1 accelerates DN and provides evidence of DN-targeted therapy.

Changes and relationship of N6-methyladenosine modification and long non-coding RNAs in oxidative damage induced by cadmium in pancreatic ß-cells.

N6-methyladenosine (m6A) modification and m6A-modified Long non-coding RNAs (LncRNAs) play crucial roles in various pathological processes, yet their changes and relationship in cadmium-induced oxidative damage are largely unknown. Here, five m6A-modified LncRNAs (LncRNA-TUG1, LncRNA-PVT1, LncRNA-MALAT1, LncRNA-XIST, LncRNA-NEAT1), which have been evidenced to involve in oxidative damage, were selected and their binding proteins were submitted to bioinformatics analysis. Our analysis results showed that these five m6A-modified LncRNAs bound to different regulatory proteins of m6A modification, implicating that m6A modification on LncRNAs may synergistically control by multiple regulatory proteins. Furthermore, the detection data revealed that levels of m6A modification, methyltransferase-like 3 (METTL3) and fat mass and obesity-associated protein (FTO) were all significantly decreased in CdSO4-induced oxidative damage, which was demonstrated by increasing ROS accumulation and MDA contents as well as decreasing SOD activities. More importantly, LncRNA-MALAT1 and LncRNA-PVT1 indicated downward trend and showed positive relationship with m6A modification. Collectively, our results showed that m6A modification and m6A-modified LncRNAs may involve in oxidative damage induced by cadmium.

Inpatient satisfaction with nursing care in a backward region: a cross-sectional study from northwestern China.

OBJECTIVES: The aim of the study was to examine the level of patient satisfaction with nursing care and identify the factors affecting satisfaction from the inpatient's perspective in a backward region of China. DESIGN: This was a cross-sectional study. SETTING: The study was conducted at a tertiary hospital located in northwest China. PARTICIPANTS: Patients admitted to the ward for at least 48 hours were chosen to participate in the survey. PRIMARY OUTCOME MEASURE: The Newcastle Satisfaction with Nursing Care Scale was used. Data were collected from 219 patients. RESULTS: The overall inpatient satisfaction with nursing care was 78.15±4.74. Patients were more satisfied with nurses who respected their privacy and treated them as individuals (67.7%). Patients were least satisfied with the type of information nurses gave them (11.7%) and with the sufficient awareness of their needs. Patients who were married, had a history of hospitalisation, surgery and were taken charge of by junior nurses had higher satisfaction. CONCLUSIONS: The overall level of patient satisfaction was moderate. Patient-centred individualised care and providing sufficient information model of care are needed. There was a need for nurses to be aware of patients' individualised care needs and to provide them with more information. This study may suggest/urge hospital administrators, policymakers and nurses to be more sensitive with patients' married status, history of hospitalisation and surgery, the professional title of in charged nurses when care is provided. Ultimately to achieve better outcome of patients' hospitalisation.

Glycemic variability indices determined by self-monitoring of blood glucose are associated with ß-cell function in Chinese patients with type 2 diabetes.

INTRODUCTION: Glycemic control plays an important role in diabetes management, and self-monitoring of blood glucose (SMBG) is critical to achieving good glycemic control. However, there are few studies about the relationship between SMBG-estimated glycemic indices and ß-cell function. Here we investigated the association between glucose variation indices estimated by SMBG and ß-cell function among Chinese patients with type 2 diabetes mellitus (T2DM). METHODS: In this cross­sectional study, 397 patients with T2DM were recruited from February 2015 to October 2016. ß-cell function was monitored using the Homeostasis Model Assessment 2 (HOMA2)-%ß index. The parameters evaluated by SMBG were the mean blood glucose (MBG), standard deviation of MBG (SDBG), largest amplitude of glycemic excursions (LAGE), and postprandial glucose excursion (PPGE). RESULTS: HOMA2-%ß was negatively correlated with SDBG, LAGE, PPGE, and MBG (r = -0.350, -0.346, -0.178, and -0.631, respectively; all p < 0.01). After adjusting for confounding characteristics (diabetic duration, triglyceride, total cholesterol, fasting C-peptide, HOMA2-insulin resistance index, hypoglycemia, and diabetic treatments) and glycated hemoglobin A1c on a continuous scale, odds ratios of SDBG, LAGE, PPGE, and MBG between the patients in the lowest and highest HOMA2-%ß quartiles were 2.02 (1.14-3.57), 1.24 (1.04-1.49), 1.13 (0.86-1.51), and 2.26 (1.70-3.00). HOMA2-%ß was independently associated with SDBG, LAGE, and MBG. CONCLUSIONS: Increased SDBG and LAGE assessed by SMBG are associated with ß-cell dysfunction in Chinese patients with T2DM.

Emerging Roles of MicroRNAs and Long Noncoding RNAs in Cadmium Toxicity.

Metal cadmium (Cd) and its compounds are ubiquitous industrial and environmental pollutants and they have been believed to exert severe damage to multiple organs and tissues. MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are the two most common noncoding RNAs and have pivotal roles in various cellular and physiological processes. Since the importance of miRNAs and lncRNAs in Cd toxicity has been widely recognized, we focus our interests on the current researches of miRNAs and lncRNAs as well as their regulation roles in Cd toxicity. In this paper, the keywords "cadmium" in combination with "miRNA" or "LncRNA" or "noncoding RNA" was used to retrieve relevant articles in PubMed, EMbase, CNKI, Wan Fang, and CBM databases. The literatures which contained the above keywords and carried out in animals (in vivo and in vitro) have been collected, collated, analyzed, and summarized. Our summary results showed that hundreds of miRNAs and lncRNAs are involved in the Cd toxicity, which have been demonstrated as multiple organ injury, reproductive toxicity, malignant transformation, and abnormal repair of DNA damage. In this paper, we also discussed the blank in present research field of Cd toxicity as well as suggested some ideas for future study in Cd toxicity.

Streptococcus suis synthesizes deoxyadenosine and adenosine by 5'-nucleotidase to dampen host immune responses.

Streptococcus suis is a major porcine bacterial pathogen and emerging zoonotic agent. S. suis 5'-nucleotidase is able to convert adenosine monophosphate to adenosine, resulting in inhibiting neutrophil functions in vitro and it is an important virulence factor. Here, we show that S. suis 5'-nucleotidase not only enables producing 2'-deoxyadenosine from 2'-deoxyadenosine monophosphate by the enzymatic assay and reversed-phase high performance liquid chromatography (RP-HPLC) analysis in vitro, but also synthesizes both 2'-deoxyadenosine and adenosine in mouse blood in vivo by RP-HPLC and liquid chromatography with tandem mass spectrometry analyses. Cellular cytotoxicity assay and Western blot analysis indicated that the production of 2'-deoxyadenosine by 5'-nucleotidase triggered the death of mouse macrophages RAW 264.7 in a caspase-3-dependent way. The in vivo infection experiment showed that 2'-deoxyadenosine synthesized by 5'-nucleotidase caused monocytopenia in mouse blood. The in vivo transcriptome analysis in mouse blood showed the inhibitory effect of 5'-nucleotidase on neutrophil functions and immune responses probably mediated through the generation of adenosine. Taken together, these findings indicate that S. suis synthesizes 2'-deoxyadenosine and adenosine by 5'-nucleotidase to dampen host immune responses, which represents a new mechanism of S. suis pathogenesis.

Streptococcus suis serotype 9 strain GZ0565 contains a type VII secretion system putative substrate EsxA that contributes to bacterial virulence and a vanZ-like gene that confers resistance to teicoplanin and dalbavancin in Streptococcus agalactiae.

Streptococcus suis (SS), an important pathogen for pigs, is not only considered as a zoonotic agent for humans, but is also recognized as a major reservoir of antimicrobial resistance contributing to the spread of resistance genes to other pathogenic Streptococcus species. In addition to serotype 2 (SS2), serotype 9 (SS9) is another prevalent serotype isolated from diseased pigs. Although many SS strains have been sequenced, the complete genome of a non-SS2 virulent strain has been unavailable to date. Here, we report the complete genome of GZ0565, a virulent strain of SS9, isolated from a pig with meningitis. Comparative genomic analysis revealed five new putative virulence or antimicrobial resistance-associated genes in strain GZ0565 but not in SS2 virulent strains. These five genes encode a putative triacylglycerol lipase, a TipAS antibiotic-recognition domain protein, a putative TetR family transcriptional repressor, a protein containing a LPXTG domain and a G5 domain, and a type VII secretion system (T7SS) putative substrate (EsxA), respectively. Western blot analysis showed that strain GZ0565 can secrete EsxA. We generated an esxA deletion mutant and showed that EsxA contributes to SS virulence in a mouse infection model. Additionally, the antibiotic resistance gene vanZSS was identified and expression of vanZSS conferred resistance to teicoplanin and dalbavancin in Streptococcus agalactiae. We believe this is the first experimental demonstration of the existence of the T7SS putative substrate EsxA and its contribution to bacterial virulence in SS. Together, our results contribute to further understanding of the virulence and antimicrobial resistance characteristics of SS.

Streptococcus suis small RNA rss04 contributes to the induction of meningitis by regulating capsule synthesis and by inducing biofilm formation in a mouse infection model.

Streptococcus suis (SS) is an important pathogen for pigs, and it is also considered as a zoonotic agent for humans. Meningitis is one of the most common features of the infection caused by SS, but little is known about the mechanisms of SS meningitis. Recent studies have revealed that small RNAs (sRNAs) have emerged as key regulators of the virulence in several bacteria. In the previous study, we reported that SS sRNA rss04 was up-regulated in pig cerebrospinal fluid and contributes to SS virulence in a zebrafish infection model. Here, we show that rss04 facilitates SS invasion of mouse brain and lung in vivo. Label-free quantitation mass spectrometry analysis revealed that rss04 regulates transcriptional regulator CcpA and several virulence factors including LuxS. Transmission electron microscope and Dot-blot analyses indicated that rss04 represses capsular polysaccharide (CPS) production, which in turn facilitates SS adherence and invasion of mouse brain microvascular endothelial cells bEnd.3 in vitro and activates the mRNA expression of TLR2, CCL2, IL-6 and TNF-α in mouse brain in vivo at 12h post-infection. In addition, rss04 positively regulates SS biofilm formation. Survival analysis of infected mice showed that biofilm state in brain contributes to SS virulence by intracranial subarachnoidal route of infection. Together, our data reveal that SS sRNA rss04 contributes to the induction of meningitis by regulating the CPS synthesis and by inducing biofilm formation, thereby increasing the virulence in a mouse infection model. To our knowledge, rss04 represents the first bacterial sRNA that plays definitive roles in bacterial meningitis.

A Streptococcus suis LysM domain surface protein contributes to bacterial virulence.

Streptococcus suis (SS) is a major swine pathogen, as well as a zoonotic agent for humans. Numerous factors contribute to SS virulence, but the pathogenesis of SS infection is poorly understood. Here, we show that a novel SS surface protein containing a LysM at the N-terminus (SS9-LysM) contributes to SS virulence. Homology analysis revealed that the amino acid sequence of SS9-LysM from the SS strain GZ0565 shares 99.8-68.7% identity with homologous proteins from other SS strains and 41.2% identity with Group B Streptococcal protective antigen Sip. Immunization experiments showed that 7 out of 30 mice immunized with recombinant SS9-LysM were protected against challenge with the virulent GZ0565 strain, while all of the control mice died within 48h following bacterial challenge. In mouse infection model, the virulence of the SS9-LysM deletion mutant (ΔSS9-LysM) was reduced compared with the wild-type (WT) strain GZ0565 and SS9-LysM complemented strain. In addition, ΔSS9-LysM was significantly more sensitive to killing by pig blood ex vivo and mouse blood in vivo compared with the WT strain and SS9-LysM complemented strain. In vivo transcriptome analysis in mouse blood showed that the WT strain reduced the expression of host genes related to iron-binding by SS9-LysM. Moreover, the total free iron concentration in blood from infected mice was significantly lower for the ΔSS9-LysM strain compared with the WT strain. Together, our data reveal that SS9-LysM facilitates SS survival within blood by releasing more free iron from the host. This represents a new mechanism of SS pathogenesis.

Over-expression of CYP78A98, a cytochrome P450 gene from Jatropha curcas L, increases seed size of transgenic tobacco

Background: Jatropha curcas L. (further referred to as Jatropha), as a rapidly emerging biofuel crop, has attracted worldwide interest. However, Jatropha is still an undomesticated plant, the true potential of this shrub has not yet been fully realized. To explore the potential of Jatropha, breeding and domestication are needed. Seed size is one of the most important traits of seed yield and has been selected since the beginning of agriculture. Increasing the seed size is a main goal of Jatropha domestication for increasing the seed yield, but the genetic regulation of seed size in Jatropha has not been fully understood. Results: We cloned CYP78A98 gene from Jatropha,a homologue of CYP78A5 in Arabidopsis.Wefound that CYP78A98 was highly expressed in male flower, female flower, stem apex, leaf and developing seed. However, its transcripts were hardly detected in root and stem. CYP78A98 protein localized in endoplasmic reticulum (ER) and the hydrophobic domain at the N-terminus was essential for the correct protein localization. Furthermore, INNER NO OUTER promoter (pINO) drove specific overexpression of CYP78A98 in transgenic tobacco seeds resulted in increased seed size and weight, as well as improved seed protein and fatty acid content. Conclusions: The results indicated that CYP78A98 played a role in Jatropha seed size control. This may help us to better understand the genetic regulation of Jatropha seed development, and accelerate the breeding progress of Jatropha.

Anti-hepatitis B virus activities of friedelolactones from Viola diffusa Ging.

BACKGROUND: Hepatitis B virus (HBV) infection is the major factor of causing hepatitis B, cirrhosis and liver cancer. Interferon and nucleoside drugs, the main drugs to treat HBV infection, have disadvantages of scavenge difficulty and drug resistance respectively. Viola diffusa Ging is used as a traditional Chinese herbal medicine for the treatment of hepatitis. PURPOSE: The aim of the study is to investigate the chemical constituents of Viola diffusa Ging and their anti-HBV activity. METHODS: Chemical constituents were extracted and purified by ethanol reflux extraction and chromatographic separation technology including D-101 Macroporous resin, silica gel, Sephadex LH-20 and preparative thin-layer chromatography. Their structures were elucidated on the basis of extensive NMR and MS data. Cytotoxicity and inhibiting effects on HBsAg and HBeAg secretion of HepG2.2.15 of all compounds except 10 were studied by MTT method and ELISA method. RESULTS: Three friedelolactones with naturally occurring seco-ring-A friedelane triterpenoids, 2ß-hydroxy-3, 4-seco-friedelolactone-27-oic acid (1), 2ß, 28ß-dihydroxy-3,4-seco-friedelolactone-27-oic acid (2) and 2ß, 30ß-dihydroxy-3,4-seco-friedelolactone-27-lactone (3), and a stigmastane, stigmast-25-ene-3ß,5α,6ß-triol (11) together with nine known compounds were isolated from the whole plant of Viola diffusa G. (Violaceae). Compounds 1-3, 9, 11, 12 exhibited significant activities of blocking both HBsAg and HBeAg secretion, and compound 4, 6, 7, 8 selectively inhibited HBeAg secretion while compound 13 selectively inhibited HBsAg secretion. IC50 values of compounds 1 and 2, 26.2 µM and 33.7 µM for HBsAg, 8.0 µM and 15.2 µM for HBeAg, was significantly lower than that of positive control lamivudine. CONCLUSION: Compounds 1-3, 11 are new compounds never reported before and the promising results demonstrate the potential of compound 1-3, 9, 11, 12 for the treatment of HBV infection.

[Extraction of lung electrical impedance character points based on wavelet transformation].

Lung electrical impedance signal carries the information of hemodynamics such as pulmonary blood supply intensity, vessel elasticity, blood flow resistance and so on. It can be used to diagnose and distinguish various kinds of heart diseases and to judge cardiac functions. The character points of lung impedance are the main basis to analyze the information of hemodynamics. This article is based on wavelet transformation to extract the character points of lung impedance. First we used the scale waveform of character points of lung impedance to make the template. Then we got wavelet ratio wave form from lung impedance by wavelet transformation. Finally we used the wavelet ratio wave form to do matching operation with the template in order to locate character points. The result of experiment demonstrates that it is an efficient and feasible method to locate character points by wavelet transformation because of its strong real time and high detection efficiency.