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Due to the non-degradable and persistent nature of metal ions in the environment, they are released into water bodies, where they accumulate in fish. In order to assess pollution in fish, the enzyme, glucose 6-phosphate dehydrogenase (G6PD), has been employed as a biomarker due to sensitivity to various ions. This study investigates the kinetic properties of the G6PD enzyme in yellow catfish (Pelteobagrus fulvidraco), and analyzes the effects of these metal ions on the G6PD enzyme activity in the ovarian cell line (CCO) of channel catfish (Ictalurus punctatus). IC50 values and inhibition types of G6PD were determined in the metal ions Cu2+, Al3+, Zn2+, and Cd2+. While, the inhibition types of Cu2+ and Al3+ were the competitive inhibition, Zn2+ and Cd2+ were the linear mixed noncompetitive and linear mixed competitive, respectively. In vitro experiments revealed an inverse correlation between G6PD activity and metal ion concentration, mRNA levels and enzyme activity of G6PD increased at the lower metal ion concentration and decreased at the higher concentration. Our findings suggest that metal ions pose a significant threat to G6PD activity even at low concentrations, potentially playing a crucial role in the toxicity mechanism of metal ion pollution. This information contributes to the development of a biomonitoring tool for assessing metal ion contamination in aquatic species. [ABSTRACT FROM AUTHOR]

Purpose: This work characterizes the applications of CRISPR/Cas12 system, including nucleic acid detection, animal, plant and microbial genome editing. Methods: The literature on CRISPR/Cas12 system was collected and reviewed. Results: CRISPR/Cas system is an acquired immune system derived from bacteria and archaea, which has become the most popular technology around the world because of its outstanding contribution in genome editing. Type V CRISPR/Cas systems are distinguished by a single RNA-guided RuvC nuclease domain with single effector molecule. Cas12a, the first reported type V CRISPR/Cas system, targets double-stranded DNA (dsDNA) adjacent to PAM sequences and trans-cleaves single-stranded DNA (ssDNA). We present the applications of CRISPR/Cas12 system for nucleic acid detection and genome editing in animals, plants and microorganisms. Furthermore, this review also summarizes the applications of other Cas12 proteins, such as Cas12b, Cas12c, Cas12d, and so on, which further widen the application prospects of CRISPR/Cas12 system. Conclusions: Knowledge of the applications of CRISPR/Cas12 system is necessary for improving the understanding of the functional diversity of CRISPR/Cas12 system and also provides significant references for further research and utilization of CRISPR/Cas12 in other new fields. [ABSTRACT FROM AUTHOR]

Mineral dissolution and mechanical deformation of granite are two main mechanisms that affect permeability evolution of rock fracture. In this study, two water flow-through experiments with large granite fractures were conducted at 200 °C with a constant flow rate for 24 h, under confining pressures of 5 and 10 MPa, respectively. Water pressure and temperature were measured, fracture aperture and permeability were calculated, and chemical element concentrations in effluent water were tested for mechanism analysis. The permeability fluctuates up and down between 2.62 × 10−12 and 3.16 × 10−12 m2 at confining pressure of 5 MPa; while it decreased monotonously by 24% from 1.92 × 10−12 to 1.45 × 10−12 m2 at a confining pressure of 10 MPa. The heat transfer rates at both experiments stay stable at about 0.25 J/s. The mass concentration of Ca, Na, K, and Si in effluent water are between 5 to 23 mg/L, indicating slight dissolution of Ca-plagioclase, Na-plagioclase, and K-feldspar, as well as possible precipitation of minor amount of kaolinite or quartz. The total amount of free-face dissolution and pressure dissolution are similar at 5 and 10 MPa. The geochemical reaction counts for only small part of the aperture change, while the mechanical deformation counts the major part of the aperture change. [ABSTRACT FROM AUTHOR]

Background: Left bundle branch pacing (LBBP) is a novel near-physiological pacing method that still lacks quantitative criteria to guide the selection of lead-implanted sites to enhance the success likelihood of lead deployments. This study aimed to quantitatively analyze the relationships of LBBP success likelihood to the distribution of lead-implanted sites and the lead-localization-pacing electrocardiographic (ECG) features. Methods: All the lead-implanted sites in patients with finally successful LBBP were enrolled for analysis, including successful and failed sites. A novel coordinate system was invented to describe the sites' distribution as longitudinal distance (longit-dist) and lateral distance (lat-dist). Corrected distance parameters were generated to eliminate the cardiac dimension variations. The lead-localization-pacing ECG parameters were also collected, such as paced QRS duration (locat-QRSd), left ventricular activation time (locat-LVAT), LVAT/QRSd ratio (locat-LVAT/QRSd), and QRS directions. Results: A total of 94 patients with 105 successful sites and 93 failed sites were enrolled. Longit-dist and corrected longit-dist of successful sites were significantly longer, while locat-QRSd and locat-LVAT were shorter and locat-LVAT/QRSd was lower than failed sites. There was a positive dose–response relationship between LBBP success likelihood and corrected longit-dist with a cut-off of 26.95 mm, whereas there were negative dose–response relationships of LBBP success likelihood to locat-QRSd, locat-LVAT, and locat-LVAT/QRSd with the cut-offs of 142 ms, 92 ms, and 64.7%, respectively. Downward QRS direction in II/III ECG leads was also associated with successful LBBP. Conclusion: Longit-dist, locat-QRSd, locat-LVAT, and locat-LVAT/QRSd were quantitative parameters to guide the selection of lead-implanted sites during LBBP implantation. Quantitative distance and electrocardiographic parameters for lead-implanted site selection to enhance the success likelihood of left bundle branch pacing. LBBP, left bundle branch pacing; Longit-dist, longitudinal distance; CL-apex-dist, distance from contraction line to apex; LBBB, left bundle branch block; IVCD, intraventricular conduction delay; Locat-QRSd, lead-localization-pacing QRS duration; Locat-LVAT, lead-localization-pacing left ventricular activation time; Locat-LVAT/QRSd, lead-localization-pacing LVAT/QRSd ratio. [ABSTRACT FROM AUTHOR]

Pacing-induced cardiomyopathy (PICM) or heart failure accompanied with chronic right ventricular pacing (CRVP-HF) has no established treatments. We aimed to carry out a meta-analysis of published studies about the therapeutic effects of the upgrade to cardiac resynchronization therapy (CRT) in patients of PICM/CRVP-HF. The PUBMED, EMBASE, MEDLINE, OVID databases, and Cochrane Library were systemically searched for relevant publications. Data about the improvements of left ventricular ejection fraction (LVEF), NYHA functional class (NYHA-FC), and the CRT response rate was extracted and synthesized. Mean difference (MD), odds ratio, and standard mean difference (SMD) with 95% confidence interval (CI) were calculated as the effect size by both fixed and random effect models. We included sixteen studies (four about PICM and twelve about CRVP-HF). The total sample size of PICM/CRVP-HF patients was 924. Upgrade to CRT improved the LVEF by 10.87% (95%CI, 8.90 to 12.84%) and reduce the NYHA-FC by around one class (MD, −1.25; 95%CI, −1.43 to −1.06) in PICM/CRVP-HF patients overall. Upgrade to CRT seemed to improve LVEF no less than de-novo CRT (SMD 0.24; 95%CI 0.05 to 0.43; P < 0.05). This meta-analysis suggested that upgrade CRT could improve the cardiac function in PICM/CRVP-HF patients. This strategy may be considered in these patients but require more evidence about the efficacy and procedure-related complications from prospective studies or randomized controlled trials. [ABSTRACT FROM AUTHOR]

Study objectives: This was a pilot study to evaluate the long-term variability and burden of respiratory disturbance index (RDI) detected by pacemaker and to investigate the relationship between RDI and atrial fibrillation (AF) event in patients with pacemakers. Methods: This was a prospective study enrolling patients implanted with a pacemaker that could calculate the night-to-night RDI. The mean follow-up was 348 ± 34 days. The RDI variability was defined as the standard deviation of RDI (RDI-SD). RDI burden was referred to as the percentage of nights with RDI ≥ 26. The patient with RDI ≥ 26 in more than 75% nights was considered to have a high sleep apnea (SA) burden. An AF event was defined as a daily AF duration > 6 h. Results: Among 30 patients, the mean RDI of the whole follow-up period was 24.5 ± 8.6. Nine (30%) patients were diagnosed with high SA burden. Patients with high SA burden had a higher BMI (26.7 ± 4.8 vs 23.2 ± 3.9, p = 0.036), a higher prevalence of hypertension (86% vs 39%, p = 0.031), and a larger left ventricular diastolic diameter (49.2 mm vs 46.7 mm, p = 0.036). The RDI-SD in patients with a higher burden was significantly greater than that in the patients with less burden (10.7 ± 4.9 vs 5.7 ± 1.4, p = 0.036). Linear regression showed that participants with a higher RDI tended to have a higher SD (R = 0.661; p < 0.001). The mean RDI (OR = 1.118, 95%CI 1.008–1.244, p = 0.044) was associated with AF occurrence. Conclusion: Using a metric such as burden of severe SA may be more appropriate to demonstrate a patient's true disease burden. [ABSTRACT FROM AUTHOR]

Aluminium, a substance found in large amounts in nature, has been widely used for various purposes, especially food additives. The effects of long-term and excessive exposure to aluminium on human health are receiving increasing attention. The extensive human use of aluminium food additives can also cause aluminium to enter the ecosystem, where it has significant impacts on insects. This study explored the cytotoxicity and changes in gene expression under aluminium potassium sulfate toward Spodoptera frugiperda 9 cells. We found that high concentrations of aluminium resulted in cell enlargement and cell membrane breakage, decreased cell vitality, and apoptosis. Through RNA-Seq transcriptomics, we found that aluminium ions may inhibit the expression of regulatory-associated protein of mTOR, tdIns-dependent protein kinase-1, and small heat shock proteins (heat shock 70 kDa protein and crystallin alpha B), leading to changes in mTOR-related pathways (such as the longevity regulation pathway and PI3K-Akt signalling pathway), and promoting cell apoptosis. On the other hand, aluminium ions lead to the overexpression of GSH S-transferase, prostaglandin-H2 D-isomerase and pyrimidodiazepine synthase, and induce intracellular oxidative damage, which ultimately affects cell growth and apoptosis through a series of cascade reactions. [ABSTRACT FROM AUTHOR]

Purpose: Magnetic resonance imaging (MRI) at 3.0 T is becoming more common, but there is a lack of sufficient evidence on the safety of a 3.0 T scan in patients with pacemakers. This study aimed to investigate the safety and practical concerns of 3.0 T scans for patients with MR-conditional pacemakers.Methods: Twenty consecutive patients were enrolled. A standardized protocol was developed by cardiologists, pacemaker engineers, and radiologists. Pacemaker interrogation was performed immediately before and after the scan. Scan-related adverse events were documented, and imaging quality was graded as level 1 to 4 by radiologists.Results: Twenty-three MRI scans of different body regions (brain = 13, lumbar spine = 4, cervical spine = 2, and heart = 4) were performed, and the average time of a scan was 25 ± 11 min. No significant changes in sensing amplitude (atrial 3.1 ± 1.1 mV vs. 2.9 ± 1.2 mV, P = 0.71; ventricular 9.3 ± 3.5 mV vs. 10.2 ± 3.4 mV, P = 0.46), lead impedances (atrial 647 ± 146 Ω vs. 627 ± 151 Ω, P = 0.7; ventricular: 780 ± 247 Ω vs.711 ± 226 Ω, P = 0.36), or pacing threshold (atrial 0.6 ± 0.2 V/0.4 ms vs. 0.6 ± 0.2 V/0.4 ms, P = 0.71; ventricular 0.7 ± 0.3 V/0.4 ms vs. 0.7 ± 0.2 V/0.4 ms, P = 0.85) were observed pre- and postscan. No adverse events were detected. Image quality review showed grade 1 quality in 16 patients and grade 2 quality in 4 patients with artifacts of pulse generators and leads in cardiac MRI scan and no impact on diagnostic value.Conclusion: Our initial data indicated that 3.0 T scanning might be feasible under a standardized protocol with good diagnostic imaging quality irrespective of body region in patients with MR-conditional pacemakers. [ABSTRACT FROM AUTHOR]

Food additives are widely used in various food products to preserve the taste, color, and other qualities. However, if they are used improperly or exceed the standard, they will cause damage to the human body. Sulfite is a commonly used food additive to prevent oxidation from deteriorating the nutrients in foods, it has been widely used as a bleaching agent in the food industry for a long time. In this study, human hepatocytes L02 cells were used as a model cell line to evaluate the toxicity of sodium sulfite. The cell morphology and cell proliferation were affected by sodium sulfite treatment, and apoptosis was detected. Transcriptome sequencing showed 97 differentially expressed genes (DEGs) between the experimental group (IC50) and the control group (MOCK), and 27 differentially expressed genes related to cell apoptosis, metabolism and inflammation were selected for validation by qPCR. Among them, 13 significantly upregulated genes and 14 significantly downregulated genes were identified by qPCR. The results showed that with increase of sodium sulfite concentration, the morphology of L02 changed, cell proliferation and activity were inhibited, and sodium sulfite caused apoptosis in a concentration- and time-dependent manner. The resulting toxic mechanism inhibits proliferation, damages the mitochondrial integrity, and promotes apoptosis. [ABSTRACT FROM AUTHOR]

Purpose: Rapid-rate non-sustained ventricular tachycardia (RR-NSVT) that meets detection criteria but terminates itself before the delivery of implantable cardioverter-defibrillator (ICD) therapy is not rare in routine ICD interrogation. Whether sustained ventricular tachycardia/fibrillation will occur in a short time after RR-NSVT has not been fully elucidated.Methods: Clinical features and follow-up data of 828 ICD patients with home monitoring were retrospectively collected. RR-NSVT characteristics and time interval between the first episode of RR-NSVT and subsequent appropriate ICD therapy were analyzed.Results: During a mean follow-up of 44.75 ± 20.87 months, 335 episodes of RR-NSVT were documented in 145 patients. A total of 119 patients had both RR-NSVT and appropriate ICD therapy. In multivariate COX regression models, RR-NSVT was an independent predictor of appropriate ICD therapy (HR 7.599, 95%CI 5.926-9.745, P < 0.001), appropriate shock (HR 6.222, 95%CI 4.667-8.294, P < 0.001), and all-cause mortality (HR 2.156, 95%CI 1.499-3.099, P < 0.001). Appropriate ICD therapy was administered after the first RR-NSVT episode in 101 patients, with a median interval of 21 days. Compared to RR-NSVT with appropriate ICD therapy occurring beyond 21 days, RR-NSVT within 21 days prior to appropriate ICD therapy had a longer median duration time (14 s vs. 12 s, P = 0.013), but without significant difference in mean RR interval at initial detection and mean RR interval after episode termination.Conclusions: Rapid-rate non-sustained VT was an independent predictor of appropriate ICD therapy and all-cause mortality. The presence of RR-NSVT should be considered a possible herald of more serious cardiac events in ICD patients. [ABSTRACT FROM AUTHOR]

Background: Pacing patients was revealed with a high prevalence of sleep disorder, but mostly undiagnosed. The pacemaker with transthoracic impedance sensor and novel algorithm could identify sleep apnea (SA) event. This study aimed to evaluate accuracy of pacemaker in sleep apnea diagnosis. Methods: This is a prospective study which enrolled patients implanted with pacemakers integrated with transthoracic impedance sensor and SA-identified algorithm (AP Scan). All patients underwent a polysomnography (PSG). The apnea and hypopnea index (AHI) of the PSG (PSG-AHI) and the respiratory disturbance index (RDI) of the pacemaker (PM-RDI) were recorded on the same night. The correlation between two methods was evaluated by the kappa coefficient, receiver operating characteristic (ROC) curves, and Bland and Altman statistics. Results: Sixty-four patients were enrolled, who had never been diagnosed with SAS or underwent PSG exam. After PSG examination, 76.4% patients were diagnosed as combining with SA (20% severe, 18.2% moderate, and 38.2% mild). RDI calculated by PM has a strong positive correlation with PSG-AHI (r = 0.76, p < 0.001, 95% CI 0.61–0.85). The optimal cutoff value of PM-RDI for advanced SAS (PSG-AHI ≥ 15) diagnosis was 26, with AUC of 0.89 (95% CI 0.77 to 0.96, p < 0.001). The best cutoff value for severe SA (PSG-AHI ≥ 30) identification was 41, with a sensitivity of 81.6%, a specificity of 88.6%. Conclusions: Pacemaker patients present a high prevalence of undiagnosed SA. Detection of SA by pacemaker is feasible and accurate in SA screening and monitoring. [ABSTRACT FROM AUTHOR]

Ag nanoparticles (AgNPs), a widely used non-antibiotic, antibacterial material, have shown toxic and other potentially harmful effects in mammals. However, the deleterious effects of AgNPs on insects are still unknown. Here, we studied the effects of AgNPs on the model invertebrate organism Bombyx mori. After feeding silkworm larvae different concentrations of AgNPs, we evaluated the changes of B. mori body weights, survival rates, and proteomic differences. The results showed that low concentrations (<400 mg/L) of AgNPs promoted the growth and cocoon weights of B. mori. Although high concentrations (≥800 mg/L) of AgNPs also improved B. mori growth, they resulted in silkworm death. An analysis of fat body proteomic differences revealed 13 significant differences in fat body protein spots, nine of which exhibited significantly downregulated expression, while four showed significantly upregulated expression. Reverse transcription-polymerase chain reaction results showed that at an AgNP concentration of 1600 mg/L, the expression levels of seven proteins were similar to the transcription levels of their corresponding genes. Our results suggest that AgNPs lowered the resistance to oxidative stress, affected cell apoptosis, and induced cell necrosis by regulating related protein metabolism and metabolic pathways in B. mori. [ABSTRACT FROM AUTHOR]

Both environmental agents and spontaneous cellular events cause serious DNA damage, threatening the integrity of the genome. In response to replication stress or genotoxic agents triggered DNA damage, degradation of p12 subunit of DNA polymerase delta (Pol δ) results in an inter-conversion between heterotetramer (Pol δ4) and heterotrimer (Pol δ3) forms and plays a significant role in DNA damage response in eukaryotic cells. In this work, we used mass spectrometry-based proteomic approach to identify those cellular stress response protein changes corresponding to the degradation of p12 in DNA-damaged HeLa cells by the treatment with hydroxyurea (HU). A total of 736 ± 13 proteins in non-treated control group and 741 ± 19 protein spots in HU-treated cells were detected, of which 34 proteins (17 up-regulated and 17 down-regulated) exhibited significantly altered protein expression levels. Their physiological roles are mainly associated with cellular components, molecular functions, and biological processes by gene ontology analysis, among which 21 proteins were mapped to KEGG pathways. They are involved in 5 primary pathways with the subsets involving 16 secondary pathways by further KEGG analysis. More interestingly, the up-regulation of translationally controlled tumor protein was further identified to be associated with p12 degradation by Western blot analysis. Our works may enlarge and broaden our view for deeply understanding how global cellular stress responds to DNA damage, which could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability. [ABSTRACT FROM AUTHOR]

Baculovirus expression vector systems (BEVSs) have been widely used for recombinant protein production. Many studies have tried to improve the recombinant protein production by either modification of BEVSs vectors or by cell line selection. In this study, using a modified shRNA vector, we established a stable Bombyx mori cell line that significantly inhibits expression of caspase-1 after selection. We further compared cell proliferation and viability between caspase-1-suppressed cells and control cells, and found that there is no significant difference in these stable cell lines. We utilized these cell lines to analyze recombinant protein production after infection with recombinant baculovirus. We found that both intracellular and extracellular recombinant protein production significantly increased when expression of caspase-1 is inhibited in BmN cells. These data indicate that blocking the apoptotic pathway is a promising way to enhance recombinant protein production in BEVSs. [ABSTRACT FROM AUTHOR]

Necroptosis is a form of caspase-independent programmed cell death which is mediated by the RIP1-RIP3 complex. Although phagocytosis of apoptotic cells has been extensively investigated, how necroptotic cells are engulfed has remained elusive. Here, we investigated how necroptotic cells attracted and were engulfed by macrophages. We found that necroptotic cells induced the migration of THP-1 cells in a transwell migration assay. Further analysis showed that ATP released from necroptotic cells acted as a find-me signal that induced the migration of THP-1 cells. We also found that Annexin V blocked phagocytosis of necroptotic cells by macrophages. Furthermore, necroptotic cells were shown to be silently cleared by macrophages without any proinflammatory cytokine production. These data uncover an evolutionarily conserved mechanism of the find-me signal in different types of cell death and immunological consequences between apoptotic and necroptotic cells during phagocytosis. [ABSTRACT FROM AUTHOR]

The aim of the study was to exam the prediction of ventricular arrhythmia events in ischemic heart disease patients with implantable cardioverter-defibrillators (ICD). A total of 123 consecutive patients confirmed ischemia heart disease with ICD were examined. After device implantation, the occurrence of appropriate ICD therapy was noted. Patients were divided into two groups according to the ventricular arrhythmia occurrence. Patients with ventricular arrhythmia occurrence had a significantly great incidence of atrial fibrillation history compare to the no-ventricular arrhythmia occurrence group (8 vs. 39 %, P = 0.02). The level of high-sensitive C-reactive protein (hsCRP) baseline was also significantly higher in the ventricular arrhythmia group than in the no ventricular arrhythmia (3.78 ± 1.1 vs. 0.94 ± 0.7, P < 0.01). The taking of β blocker is not common in ventricular arrhythmia group patients than no ventricular arrhythmia group (5 vs. 29 %, P = 0.03). By univariate comparison, male sex, the history of atrial fibrillation, and a high level of hsCRP were significant predictors for ventricular arrhythmia occurrence. By multivariate analysis, the atrial fibrillation burden, and had a high level of hsCRP were significant for incidence of ventricular arrhythmia occurrence in ischemic heart disease patients. β-block were more likely to be free from ventricular arrhythmia occurrence. The high level of hsCRP, and the atrial fibrillation burden were strong predictor of ventricular arrhythmia occurrence in secondary prevention ICD recipients with ischemic heart disease. Taking β-blockers was free from ventricular arrhythmia occurrence. [ABSTRACT FROM AUTHOR]

Lipases expressed in microbial hosts have great commercial value, but their applications are restricted by the high costs of production and harsh conditions used in industrial processes, such as high temperature and alkaline environment. In this study, an Escherichia coli- Bacillus subtilis shuttle vector (pHS-cotB-Tm1350) was constructed for the spore surface display of the lipase Tm1350 from hyperthermophilic bacterium Thermotoga maritima MSB8. Successful display of the CotB-Tm1350 fusion protein on spore surface was confirmed by Western blot analysis and activity measurements. The optimal catalytic temperature and pH of the spore surface-displayed Tm1350 were 80 °C and 9, respectively, which were higher than non-immobilized Tm1350 (70 °C and pH 7.5). Analysis of thermal and pH stability showed that spore surface-displayed Tm1350 retained 81 or 70 % of its original activity after 8 h of incubation at pH 8 or pH 9 (70 °C), which were 18 % higher than the retained activity of the non-immobilized Tm1350 under the same conditions. Meanwhile, recycling experiments showed that the recombinant spores could be used for up to three reaction cycles without a significant decrease in the catalytic rate (84 %). These results suggested that enzyme display on the surface of the B. subtilis spore could serve as an effective approach for enzyme immobilization, which has potential applications in the harsh biochemical industry. [ABSTRACT FROM AUTHOR]

Acetobacter pasteurianus plays an important role in the process of traditional vinegar production and is also essential for the fermentation of Zhenjiang aromatic vinegar. In this study, we utilized the proteomic approach to analyze the proteomic profile of A. pasteurianus HSZ3-21, and 258 proteins were successfully identified by MALDI-TOF-MS and database search. The hydropathy and GO analyse combined with COG results of the identified proteins revealed the molecular biological characteristics of A. pasteurianus proteins, that is, most proteins of A. pasteurianus were related to metabolic process, binding, catalytic or cellular response. Meanwhile, our results also showed that some proteins of A. pasteurianus may be responsible for acetic acid tolerance, thermotolerance, and stress response. Therefore, the identification of 258 proteins not only deciphers protein composition and functional classification of A. pasteurianus, but also provides useful information for improving quality of Zhenjiang aromatic vinegar. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV) VD1-ORF4 consists of 3,318 nucleotides, which codes for a predicted protein with molecular weight of about 127 kDa. However, the authentic proteins encoded by VD1-ORF4 in silkworm midguts infected with BmBDV and their interacting proteins are still unclear. In this study, Western blot analysis revealed that a 127-kDa protein was confirmed to be translated from the VD1-ORF4 transcript using polyclonal antibodies and monoclonal antibodies against VD1-ORF4 deduced amino acid. Moreover, four smaller proteins with molecular weight of about 70, 60, 53, and 42 kDa were also examined in the infected midguts. Transient expression assay indicated that the expression amount of VD1-ORF4 fused with egfp was at least 30-fold lower than that of egfp gene, and immunofluorescence staining result indicated that these proteins encoded by VD1-ORF4 were located in both the cytoplasm and nucleus. Co-immunoprecipitation result showed that Aminopeptidase and Heat shock protein 90 can be captured by these proteins encoded by VD1-ORF4. In conclusion, multiple proteins were produced from the transcripts of VD1-ORF4 gene by an uncertain expression strategy, which may play important roles in viral replication and assembly. [ABSTRACT FROM AUTHOR]

Fresh biomass of Aspergillus oryzae ( A. oryzae) CGMCC5992 can effectively remove gallic acid from aqueous solution. To improve the removal rate of gallic acid, this study first identified the important factors affecting the removal rate of gallic acid with univariate analysis, and then used four-factor and three-level Box-Behnken design (BBD) with the removal rate of gallic acid as response value, to obtain the optimum conditions for the removal of gallic acid as follows: 6.95 h treatment time, pH 3.70, 7.07 g/L mycelium volume, and 120.64 mg/L initial concentration of gallic acid. Under such optimized condition, the removal rate of gallic acid approached 99.21 %. HPLC-MS analysis proved that the gallic acid in aqueous solution was completely removed by A. oryzae, rather than being metabolized into its derivatives. Scanning electron microscopy (SEM) indicated that the biomass morphology and surface structure of A. oryzae changed after the adsorption of gallic acid. Thus, the present study has provided an optimal condition for A. oryzae removal of gallic acid in water. [ABSTRACT FROM AUTHOR]

In our previous study, Orf101 ( Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle. [ABSTRACT FROM AUTHOR]

Data normalization is essential for reliable output of quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assays, as the unsuitable choice of reference gene(s), whose expression might be influenced by exogenous treatments in plant tissues, could cause misinterpretation of results. To date, no systematic studies on reference genes have been performed in stressed Brassica napus. In this study, we investigated the expression variations of nine candidate reference genes in 40 samples of B. napus leaves subjected to various exogenous treatments. Parallel analyses by geNorm and NormFinder revealed that optimal reference genes differed across the different sets of samples. The best-ranked reference genes were PP2A and TIP41 for salt stress, TIP41 and ACT7 for heavy metal (Cr) stress, PP2A and UBC21 for drought stress, F- box and SAND for cold stress, F- box and ZNF for salicylic acid stress, TIP41, ACT7, and PP2A for methyl jasmonate stress, TIP41 and ACT7 for abscisic acid stress, and TIP41, UBC21, and PP2A for Sclerotinia sclerotiorum stress. Two newly employed reference genes, TIP41 and PP2A, showed better performances, suggesting their suitability in multiple conditions. To further validate the suitability of the reference genes, the expression patterns of BnWRKY40 and BnMKS1 were studied in parallel. This study is the first systematic analysis of reference gene selection for qRT-PCR normalization in B. napus, an agriculturally important crop, under different stress conditions. The results will contribute toward more accurate and widespread use of qRT-PCR in gene analysis of the genus Brassica. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV) is a new designated species of the new genus Bidensovirus in the new family Bidnaviridae, which contains two single-stranded linear DNAs (VD1 and VD2) and causes the chronic densonucleosis disease of silkworm. Previous researches revealed that VD1-ORF3 encodes the major structural proteins VPs. In this work, through western blot, we found that VPs expressed from 48 h post-inoculation and kept increasing until 120 h post-inoculation in midgut of Bombyx mori. In order to further investigate the translation of vp gene, the ORFs ( vp1 and vp2) of the VP started just up-stream of the first two candidate initiation codons were expressed in Sf9 cells by a baculovirus expression system. The expression products were purified by gradient density centrifugation and analyzed by Western blot and electron microscopy. The results showed that the expressions of vp1 yielded three proteins (VP1, VP1′, and VP2), which are the same with the viral VPs expression in midgut of Bombyx mori, and vp2 generated two VPs with the molecular weights of about 51 kDa (VP2) and 37 kDa. The observation by electron microscopy indicated that these VPs can auto-assemble into virus-like particles that could not be distinguished from virus particles. These findings will provide materials for studying the structure of BmBDV and be helpful in the studies on BmBDV-based disease in silkworms. [ABSTRACT FROM AUTHOR]

Orf101 ( Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope. [ABSTRACT FROM AUTHOR]

Degradation performance of silk fibroin is an important property for its medical applications. Herein we constructed a shortened silk fibroin heavy chain protein fused with a matrix metalloproteinase cleavage site (SSFH-MMP) along with a glutathione S-transferase tag ahead. The digestion assay shows it can be cut by matrix metalloproteinase-2 (MMP-2) at its MMP cleavage site. Furthermore, we introduced the SSFH-MMP into silk fibroin by genetic modification of silkworms in order to increase the degradation rate of the silk fibroin. After acquisition of a race of transgenic silkworms with the coding sequence of the MMP cleavage site in their genomic DNA, we tested some properties of their silk fibroin designated TSF-MMP. The results show that the TSF-MMP has MMP cleavage sites and yields a quicker degradation rate during dilution in MMP-2 enzyme buffer or implantation into tumor tissues compared with that of normal silk fibroin. Moreover, the TSF-MMP is in vitro non-toxic to human bone marrow mesenchymal stem cells (hBM-MSCs) indicating that the TSF-MMP may become a biomaterial with a quicker degradation rate for its medical applications. [ABSTRACT FROM AUTHOR]

The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication. [ABSTRACT FROM AUTHOR]

Human growth hormone (hGH) is the major and important hormone component of human being. At present, hGH for clinical uses is mostly produced in Escherichia coli, which requires costly denaturation and refolding to recover functionality. To obtain long-term bioactive hormone, we used hGH as a foreign gene and constructed a recombinant plasmid pJS700-hGH which carries a recombinant gene cotC- hgh with an enterokinase site under the control of cotC promoter. Plasmid pJS700-hGH was transformed into Bacillus subtilis by double crossover and an amylase-inactivated mutant was produced. After spore formation, Western blot and fluorescence immunoassay were used to monitor hGH surface expression on spores. Oral administration to silkworm with spores displaying hGH further showed that the recombinant spores may have potential ability to be digested and absorbed into the silkworm's hemolymph due to both the resistant characters of spores and the addition of enterokinase site. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus ( BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase ( BmNPV- pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV- pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV- pol (co- BmNPV- pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co- BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co- BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL. [ABSTRACT FROM AUTHOR]

Bacillus subtilis have always been regarded as one of the most important functional bacteria for flavor formation in liquor industry. During the flavor formation process, B. subtilis facilitate the conversion of ethanol into aroma compounds by esterification and Maillard reactions mainly due to their vigorous enzymatic secretion and metabolic systems. However, high concentrations of ethanol could be a significant stressor to inhibit Bacillus spp. propagation and thus impair their effective flavor-forming ability. Therefore, in order to obtain improved B. subtilis strains with enhanced ethanol tolerance for liquor industrial potential, we adopted spore surface display strategy in the present study. Acetobacter pasteurianus alcohol dehydrogenase A ( adhA) gene was employed as foreign gene, and recombinant integrative plasmid pJS700- adhA was constructed by using B. subtilis spore coat protein CotC as a fusion partner. A combination of amylase activity assay, site-directed PCR identification, Western blot analysis, and immunofluorescence microscopy sequentially demonstrated that AdhA was successfully expressed and displayed on recombinant B. subtilis mutant spore surface. In addition, ethanol tolerance assay showed that the recombinant mutant exhibited enhanced ethanol resistance than its wild-type form when confronted with high concentrations of ethanol, indicating both of its validity in methodology and application potential for liquor industry. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV), which had been assigned to Densovirinae in Parvoviridae previously, replicates mainly in silkworm midgut olumnar cells and causes the fatal flacheric disease. In contrast to parvovirus, this virus possesses two single-stranded DNA genome segments and encodes a putative protein-primed DNA polymerase. The accumulating evidence suggests that it has unique characteristics different to parvovirus and adopts its own mechanisms for replication. So far, little is known about the replication mechanisms of BmBDV. In this review, we focus on the pathology associated with this virus and the viral biology such as viral genome structure, viral genes, and viral replication and expression strategies. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV), which had been assigned to Densovirinae in Parvoviridae previously, replicates mainly in silkworm midgut olumnar cells and causes the fatal flacheric disease. In contrast to parvovirus, this virus possesses two single-stranded DNA genome segments and encodes a putative protein-primed DNA polymerase. The accumulating evidence suggests that it has unique characteristics different to parvovirus and adopts its own mechanisms for replication. So far, little is known about the replication mechanisms of BmBDV. In this review, we focus on the pathology associated with this virus and the viral biology such as viral genome structure, viral genes, and viral replication and expression strategies. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus ( BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase ( Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10 9 infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance. [ABSTRACT FROM AUTHOR]

Peptide-mediated interactions are crucial to a variety of functions in the living cell and are estimated to be involved in up to 40 % of all cellular processes. Fast and reliable inference of such interactions is fundamentally important for our understanding and, then, reconstruction of complete virtual interactomics involved in a specific cell, tissue or organism. In the current study, we performed structure-level characterization, modeling and prediction of protein-peptide recognition specificity and stability in a high-throughput manner. To achieve this, the classical chemometrics methodology quantitative structure-activity relationship (QSAR), which is traditionally applied to small-molecule entities such as drug compounds and environmental chemicals, was employed to statistically correlate structure features with binding affinities for a panel of structure-solved, affinity-known protein-peptide complexes compiled from the PDB database and literatures. In the standard QSAR procedure, various structural descriptors including physicochemical, geometrical and constitutional parameters that characterize diverse aspects of protein-peptide interaction property were derived from the biomacromolecular complex structure architecture, and these descriptors were then correlated with experimentally measured affinities by using the partial least squares (PLS) regression and Gaussian process (GP) in conjunction with genetic algorithm (GA) variable selection. The nonlinear GA/GP method was found to perform much well as compared to linear GA/PLS modeling, suggesting that the protein-peptide interaction system is highly complicated that may involve strong noise and interactive effect. The optimal GA/GP model revealed that the interface size and solvent effect play a critical role in protein-peptide binding, and other properties such as peptide length and flexibility also contribute significantly to the binding. A further test on 2,018 human amphiphysin SH3 domain-binding peptides demonstrated that the purposed QSAR modeling procedure is very fast and effective, which can thus be readily used to perform proteome-wide inference of peptide-mediated interactions. [ABSTRACT FROM AUTHOR]

Helix-loop-helix (bHLH) proteins play essential regulatory roles in a variety of biological processes. These highly conserved proteins form a large transcription factor superfamily, and are commonly identified in large numbers within animal, plant, and fungal genomes. The bHLH domain has been well studied in many animal species, but has not yet been characterized in non-avian reptiles. In this study, we identified 102 putative bHLH genes in the genome of the green anole lizard, Anolis carolinensis. Based on phylogenetic analysis, these genes were classified into 43 families, with 43, 24, 16, 3, 10, and 3 members assigned into groups A, B, C, D, E, and F, respectively, and 3 members categorized as 'orphans'. Within-group evolutionary relationships inferred from the phylogenetic analysis were consistent with highly conserved patterns observed for introns and additional domains. Results from phylogenetic analysis of the H/E(spl) family suggest that genome and tandem gene duplications have contributed to this family's expansion. Our classification and evolutionary analysis has provided insights into the evolutionary diversification of animal bHLH genes, and should aid future studies on bHLH protein regulation of key growth and developmental processes. [ABSTRACT FROM AUTHOR]

It is difficult to obtain intact embryos, especially intact early embryos, from insect eggs because of their small sizes. Based on the means traditionally used to get silkworm embryos and the previous approaches used for getting Drosophila embryos, we established a novel method of silkworm embryo preparation. The new method is straightforward and easy to operate. Silkworm embryos could be prepared without severe damage in large quantities by this new protocol. In addition, the novel method of silkworm embryo preparation is quite suitable for immunohistochemistry. [ABSTRACT FROM AUTHOR]

A new yeast antagonist, Pichia caribbica, isolated in our laboratory from the soil collected from unsprayed orchards, was evaluated for its biocontrol capability against Rhizopus stolonifer on peaches and the possible mechanisms involved. The decay incidence and lesion diameter of Rhizopus decay of peaches treated by P. caribbica were significantly reduced compared with the control fruits, and the higher the concentration of P. caribbica, the better the efficacy of the biocontrol. Rapid colonization of the yeast in peach wounds stored at 25 °C was observed. In peaches, the activities of peroxidase (POD), catalase (CAT), and phenylalanine ammonia-lyase (PAL) were significantly induced by P. caribbica treatment compared to those of the control fruits. All these results indicated that P. caribbica has a great potential for the development of commercial formulations to control postharvest Rhizopus decay of peaches. Its modes of action were based on competition for space and nutrients with pathogens, inducement of activities of defense-related enzymes such as POD, CAT, and PAL of peaches. [ABSTRACT FROM AUTHOR]

With the continuous improvement of the living standards of human society, the number of diabetics worldwide is growing rapidly. To date, the main effective therapy for diabetic is intravenous injection of insulin, which is accompanied by a lot of shortage such as high cost and side effects. To obtain long-term bioactive anti-diabetic drug for oral administration, we used human proinsulin ( hpi) as a foreign gene to construct a recombinant plasmid pJS700-HPI with an enterokinase site Asp-Asp-Asp-Asp-Lys suitable for digestion. Plasmid pJS700-HPI was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spores formation, western blot was used to monitor HPI surface expression on spores. Oral administration to the silkworms with spores implied that the HPI protein displayed on recombinant spores may be digested and absorbed into the silkworm's hemolymph due to the resistant characters of spores and the addition of enterokinase site. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV) VD1-ORF4 (open reading frame 4, ORF4) consists of 3,318 nucleotides, which codes for a predicted 1,105-amino acid protein containing a conserved DNA polymerase motif. However, its functions in viral propagation remain unknown. In the current study, the transcription of VD1-ORF4 was examined from 6 to 96 h postinfection (p.i.) by RT-PCR, 5′-RACE revealed the transcription initiation site of BmBDV ORF4 to be −16 nucleotides upstream from the start codon, and 3′-RACE revealed the transcription termination site of VD1-ORF4 to be +7 nucleotides downstream from termination codon. Three different proteins were examined in the extracts of BmBDV-infected silkworms midguts by Western blot using raised antibodies against VD1-ORF4 deduced amino acid, and a specific protein band about 53 kDa was further detected in purified virions using the same antibodies. Taken together, BmBDV VD1-ORF4 codes for three or more proteins during the viral life cycle, one of which is a 53 kDa protein and confirmed to be a component of BmBDV virion. [ABSTRACT FROM AUTHOR]

The aim of this study was to isolate indigenous ethanol-tolerant acetic acid bacteria (AAB) from industrial fermented vinegar. As routine phenotypic methods for AAB isolation and identification appear to be very time-consuming and not so accurate, we adopted a two-step isolation strategy in the present study. In the preliminary screening step, GYEC agar plates with 3-10 % (v/v) ethanol as selective stress were utilized to recover potential AAB strains with ethanol tolerance from vinegar samples. In the rescreening process, acetic acid bacterial genus' specific adhA gene was amplified as an effective DNA target for directional detection of real AAB so that non-AAB isolates could be eliminated rapidly. In this way, an AAB pure isolate named ET-7-3 with 7 % (v/v) ethanol tolerance was successfully isolated and further identified to Acetobacter pasteurianus according to 16S rDNA sequencing and phylogenetic analysis. Summarizing, by the use of ethanol as selective stress combined with molecular identification method, ethanol-tolerant AAB could be isolated from industrial fermented vinegar with both efficiency and accuracy. [ABSTRACT FROM AUTHOR]

Orf65 ( Bm65) of Bombyx mori nucleopolyhedrovirus ( BmNPV) is a highly conserved gene that encodes an unknown 104-amino acid protein. In the present study, we have shown the role of Bm65 in the baculovirus life cycle. 5′-RACE analysis showed that the transcription start site of Bm65 was 14 nucleotides upstream of the start codon ATG. The transcription profile of Bm65 was detected from 6 to 72 h postinfection (p. i.) by RT-PCR. A Bm65-knockout bacmid was constructed by homologous recombination to characterize the role of Bm65 in viral life cycle. Fluorescence microscopy showed that Bm65-knockout virus was unable to generate infectious budded virus in BmN cells. Furthermore, quantitative real-time PCR analysis demonstrated that Bm65 deletion did not affect the viral DNA replication. To conclude, Bm65 is essential for the propagation of BmNPV, but is unnecessary for the replication of viral DNA. [ABSTRACT FROM AUTHOR]

Open reading frame 17 ( Bm17) gene of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this report, transient-expression and superinfection assays indicated that BM17 localized in the nucleus and cytoplasm of infected BmN cells. To determine the role of Bm17 in baculovirus life cycle, we constructed a Bm17 knockout virus and characterized its properties in cells. Analysis of the production and infection of budded virions, the level of viral DNA replication revealed showed that there was no significant difference among the mutant, the control, and the Bm17 repaired virus strains. These results suggest that BM17 is not essential for virus replication in cultured cells. [ABSTRACT FROM AUTHOR]

The vacuolar protein sorting 4 (Vps4) protein is essential for the multivesicular body (MVB) pathway, virus budding process and cytokinesis. Vps4 has been identified and characterized from many species, but not from silkworm Bombyx mori. In this study, we firstly identified and cloned the silkworm homologous gene for VPS4, expressed it in Escherichia coli, purified and characterized the protein designated as BmVps4. The BmVps4 cDNA contains an open reading frame of 1,314 bp, and encodes a protein of 438 amino acid residues. BmVps4 is of high sequence-similarity to Vps4 proteins from other species. The recombinant BmVps4 shows ATPase activity, which can be stimulated by Mg and inhibited by dominant mutations. Together, our data suggest BmVps4 is the genuine silkworm homologue of Vps4. To our knowledge, this is the first-time characterization of any silkworm MVB proteins. This study will facilitate further investigation of silkworm MVB pathway and its possible roles in the infection and budding of B. mori nuclear polyhedrosis virus (BmNPV), which is one of the most common and severe pathogens for silkworms. The cloned BmVps4 sequence is deposited in GenBank (Accession number GQ995504). [ABSTRACT FROM AUTHOR]

Bombyx mori Bidensovirus ( BmBDV), a bipartite virus possesses two single-stranded linear DNAs (VD1 and VD2) and shows high pathogenic ability to Bombyx mori. Previous research found that the genes of nonstructural protein ns1 and ns2 were in the same transcript. To investigate the mechanism of transcriptional regulation of ns1 and ns2 genes, the 5′-flanking sequence (289 nt) of ns1 gene, encompasses the regions of the common terminal sequence (CTS) and the predicted P5 promoter from the 5′-terminus of the viral genome to the transcription initiation site of the ns1 gene was cloned and fused to the upstream of the luciferase reporter gene. The luciferase reporter assay showed that the 53 nt CTS of VD1 and VD2 can downregulate the activity of P5 by 13.3 %. The comparison in different cell lines showed that P5 possessed high promoter activity in BmN and Hi5 cell lines. Interestingly, P5 also had high activity in Hela cells, a kind of cancer cell of human. Subsequent truncated promoter analysis showed that the 31 nt (−236 to −206 nt) sequence is very important to P5 for the activity down to 36.5 % after deletion of it. While the activity also remained 26.5 % after the deletion of the TATA box, suggesting that the promoter is TATA independent. Moreover, in order to further understand the activity intensity of P5, a comparison with other three promoters, B. mori actin3 ( Bm-actin3), B. mori nuclear polyhedrosis virus ( BmNPV) immediate early 1 gene promoter ( BmNPV-ie-1), and a synthetic promoter (3xP3) was carried out, the result indicated that the activity of P5 was weaker than that of anyone of them. [ABSTRACT FROM AUTHOR]

Human serum albumin (HSA) is the major protein component of human plasma. To date, HSA for clinical uses is mostly produced by fractionation of human whole blood, which is accompanied by a lot of limitations. To obtain long-term bioactive albumin, we used hsa as a foreign gene and constructed a recombinant plasmid pJS700-HSA which carries a recombinant gene cotC- hsa under the control of cotC promoter. Plasmid pJS700-HSA was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spore formation, western blot and fluorescence immunoassay were used to monitor HSA surface expression on spores. We estimated that HSA displayed on the spore accounted for 0.135 % of the total spore proteins and about 0.023 fg HSA were exposed on the surface of each spore. Oral administration to mice with spores displaying HSA implied that the recombinant spores may have potential ability to increase the serum albumin level in vivo due to the resistant characters of spores. [ABSTRACT FROM AUTHOR]

Natural wild-type strains of Bacillus subtilis spore is regarded as a non-pathogenic for both human and animal, and has been classified as a novel food which is currently being used as probiotics added in the consumption. To identify B. subtilis spore proteins, we have accomplished a preliminary proteomic analysis of B. subtilis spore, with a combination of two-dimensional electrophoretic separations and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF-MS). In this article, we presented a reference map of 158 B. subtilis spore proteins with an isoelectric point (pI) between 4 and 7. Followed by mass spectrometry (MS) analysis, we identified 71 B. subtilis spore proteins with high level of confidence. Database searches, combined with hydropathy analysis and GO analysis revealed that most of the B. subtilis spore proteins were hydrophilic proteins related to catalytic function. These results should accelerate efforts to understand the resistance of spore to harsh conditions. [ABSTRACT FROM AUTHOR]

Bombyx mori parvo-like virus ( BmPLV) has two complementary single-stranded DNA genome (VD1 and VD2) and owns a self-encoding DNA polymerase motif, but its replication mechanism is unclear . In our previous research, a protein encoded by VD1-ORF1 was indentified in the midgut of BmPLV China Zhenjiang isolate-( BmPLV-Z) infected silkworm larvae via two-dimensional gel electrophoresis (2-DE). This protein was named as non-structural protein 2 (NS2), which showed no similarity to that of parvoviruses. To date, little is known about it. In this study, sequence alignment results showed that NS2 shared homology with some chromosomal replication initiator protein dnaA and DNA-binding response regulators. The ns2 was cloned and expressed in E. coli, and then a polyclonal antibody of the NS2 protein was prepared successfully. The data from real-time quantitative PCR displayed that the transcription of VD1-ORF1 from BmPLV-Z-infected midguts started from 28-h post inoculation (h p.i.) in low amounts, but in high amounts at late stages of infection. Immunofluorescence showed that NS2 ultimately concentrated on the nuclear membrane in BmN cells at late stages, indicating that NS2 might be associated with integral membrane protein. [ABSTRACT FROM AUTHOR]

HIV-1 co-receptor tropism is central for understanding the transmission and pathogenesis of HIV-1 infection. We performed a genome-wide comparison between the adaptive evolution of R5 and X4 variants from HIV-1 subtypes B and C. The results showed that R5 and X4 variants experienced differential evolutionary patterns and different HIV-1 genes encountered various positive selection pressures, suggesting that complex selection pressures are driving HIV-1 evolution. Compared with other hypervariable regions of Gp120, significantly more positively selected sites were detected in the V3 region of subtype B X4 variants, V2 region of subtype B R5 variants, and V1 and V4 regions of subtype C X4 variants, indicating an association of positive selection with co-receptor recognition/binding. Intriguingly, a significantly higher proportion (33.3% and 55.6%, P<0.05) of positively selected sites were identified in the C3 region than other conserved regions of Gp120 in all the analyzed HIV-1 variants, indicating that the C3 region might be more important to HIV-1 adaptation than previously thought. Approximately half of the positively selected sites identified in the env gene were identical between R5 and X4 variants. There were three common positively selected sites (96, 113 and 281) identified in Gp41 of all X4 and R5 variants from subtypes B and C. These sites might not only suggest a functional importance in viral survival and adaptation, but also imply a potential cross-immunogenicity between HIV-1 R5 and X4 variants, which has important implications for AIDS vaccine development. [ABSTRACT FROM AUTHOR]

This paper develops a GIS-based integrated approach to risk assessment in natural hazards, with reference to bushfires. The challenges for undertaking this approach have three components: data integration, risk assessment tasks, and risk decision-making. First, data integration in GIS is a fundamental step for subsequent risk assessment tasks and risk decision-making. A series of spatial data integration issues within GIS such as geographical scales and data models are addressed. Particularly, the integration of both physical environmental data and socioeconomic data is examined with an example linking remotely sensed data and areal census data in GIS. Second, specific risk assessment tasks, such as hazard behavior simulation and vulnerability assessment, should be undertaken in order to understand complex hazard risks and provide support for risk decision-making. For risk assessment tasks involving heterogeneous data sources, the selection of spatial analysis units is important. Third, risk decision-making concerns spatial preferences and/or patterns, and a multicriteria evaluation (MCE)-GIS typology for risk decision-making is presented that incorporates three perspectives: spatial data types, data models, and methods development. Both conventional MCE methods and artificial intelligence-based methods with GIS are identified to facilitate spatial risk decision-making in a rational and interpretable way. Finally, the paper concludes that the integrated approach can be used to assist risk management of natural hazards, in theory and in practice. [ABSTRACT FROM AUTHOR]