Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse

Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC’s undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.     

G protein-coupled receptor kinase 2 modifies the cellular reaction to cisplatin through interactions with NADPH oxidase 4

G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells—a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.

     

Overexpression of SoACLA-1 Gene Confers Drought Tolerance Improvement in Sugarcane

Drought is one of the most severe stresses which limit sugarcane production in China. ATP citrate lyase (ACL) is a major enzyme responsible for the production of acetyl-CoA in cytoplasm and plays an important role in plant metabolism and stress response. In this study, sugarcane ACL gene SoACLA-1 was cloned. The plant overexpression vector of SoACLA-1 was built and transformed into sugarcane calli by Agrobacterium-mediated transformation, and PCR analysis confirmed that SoACLA-1 gene had been stably present in the T0, T1, and T2 generations of the transgenic sugarcane. In order to evaluate the drought resistance of the transgenic lines and verify the function of SoACLA-1 gene in the transgenic sugarcane, T1 generation of the SoACLA-1 transgenic sugarcane lines was used as the material to investigate the physiological and biochemical characteristics at 0 day, 3 days, 6 days, and 9 days after water stress and rewatering for 3 days. Comprehensive evaluation of four indicators (chlorophyll, malondialdehyde, proline, soluble sugar) related to drought resistance was done with membership fuzzy function method. The results showed that the drought resistance of five transgenic sugarcane lines from strong to weak, in turn, was RT2?>?RT4?>?RT3?>?RT1?>?WT, and the recovery ability after drought, in turn, was RT1?>?RT2?>?RT4?>?RT3?>?WT. The T2 generation of the SoACLA-1 transgenic sugarcane lines was used to analyze the physiological and biochemical changes and the expression of drought-related genes under water stress. The results showed that the transgenic sugarcane lines were more tolerant to drought as compared with the wild-type plants. Our findings indicated that SoACLA-1 gene plays an important role as a positive factor in response to water stress, and overexpression of SoACLA-1 can enhance drought tolerance in transgenic sugarcane plants.

     

HIV Infection,Risk Factors,and Preventive Services Utilization among Female Sex Workers in the Mekong Delta Region of Vietnam

Background

Risk behaviors among female sex workers (FSW) are considerable drivers of HIV infections in Vietnam, especially transmission between high-risk and low-risk groups. We assessed HIV prevalence and its correlates among FSWs, and the use of preventive services among this community in the Mekong Delta region, southern Vietnam.

Methods

A cross-sectional survey of 1,999 FSWs was carried out in five provinces including Ben Tre, Hau Giang, Kien Giang, Tien Giang, and Vinh Long between June, 2006 and June, 2007. We interviewed participants face-to-face in order to elicit information about their lives and potential risk factors, and we tested their sera to determine their HIV status. We then performed multivariate logistic regression analyses to investigate factors associated with HIV infection.

Results

Seventeen percent of the participating FSWs were street-based sex workers (SSWs) and the rest (83%) were entertainment establishment-based sex workers (ESWs). Unprotected sex with regular and casual clients in the past month was frequent among study participants (40.5% and 33.5% respectively). However, few respondents (1.3%) had ever injected drugs. Only 2.1% (95% confidence interval (CI): 1.6%–2.8%) of FSWs were found to be infected with HIV. HIV prevalence among SSWs was greater than among ESWs (3.8% vs. 1.8%, p = 0.02, respectively). Increased risk for HIV infection was significantly associated with the number of clients per month (adjusted odd ratio (aOR) = 2.65, 95% CI: 1.26–5.59).

Conclusions

Interventions to reduce unsafe sex and drug injection, and to increase uptake of HIV testing among FSWs are necessary. Differences in HIV prevalence and its correlates by type of sex work emphasize the importance of constrained contexts in shaping risk behaviors among FSWs; that should be considered in designing HIV prevention programs.     

Global and Regional Associations of Smaller Cerebral Gray and White Matter Volumes with Gait in Older People

Background

Gait impairments increase with advancing age and can lead to falls and loss of independence. Brain atrophy also occurs in older age and may contribute to gait decline. We aimed to investigate global and regional relationships of cerebral gray and white matter volumes with gait speed, and its determinants step length and cadence, in older people.

Methods

In a population-based study, participants aged >60 years without Parkinson''s disease or brain infarcts underwent magnetic resonance imaging and gait measurements using a computerized walkway. Linear regression was used to study associations of total gray and white matter volumes with gait, adjusting for each other, age, sex, height and white matter hyperintensity volume. Other covariates considered in analyses included weight and vascular disease history. Voxel-based morphometry was used to study regional relationships of gray and white matter with gait.

Results

There were 305 participants, mean age 71.4 (6.9) years, 54% male, mean gait speed 1.16 (0.22) m/s. Smaller total gray matter volume was independently associated with poorer gait speed (p = 0.001) and step length (p<0.001), but not cadence. Smaller volumes of cortical and subcortical gray matter in bilateral regions important for motor control, vision, perception and memory were independently associated with slower gait speed and shorter steps. No global or regional associations were observed between white matter volume and gait independent of gray matter volume, white matter hyperintensity volume and other covariates.

Conclusion

Smaller gray matter volume in bilaterally distributed brain networks serving motor control was associated with slower gait speed and step length, but not cadence.     

Phylogenetic and Functional Diversity of Microbial Communities Associated with Subsurface Sediments of the Sonora Margin,Guaymas Basin

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.     

Structural Characteristics of Allomyrina Dichotoma Beetle＇s Hind Wings for Flapping Wing Micro Air Vehicle

In this study, we present a complete structural analysis ofAllomyrina dichotoma beetle＇s hind wings by investigating their static and dynamic characteristics. The wing was subjected to the static loading to determine its overall flexural stiffness. Dy- namic characteristics such as natural frequency, mode shape, and damping ratio of vibration modes in the operating frequency range were determined using a Bruel ＆ Kjaer fast Fourier transform analyzer along with a laser sensor. The static and dynamic characteristics of natural Allomyrina dichotoma beetle＇s hind wings were compared to those of a fabricated artificial wing. The results indicate that natural frequencies of the natural wing were significantly correlated to the wing surface area density that was defined as the wing mass divided by the hind wing surface area. Moreover, the bending behaviors of the natural wing and artificial wing were similar to that of a cantilever beam. Furthermore, the flexural stiffness of the artificial wing was a little higher than that of the natural one whereas the natural frequency of the natural wing was close to that of the artificial wing. These results provide important information for the biomimetic design of insect-scale artificial wings, with which highly ma- neuverable and efficient micro air vehicles can be designed.     

Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG5 in complex with PE25–PPE41 dimer

The growth or virulence of Mycobacterium tuberculosis bacilli depends on homologous type VII secretion systems, ESX‐1, ESX‐3 and ESX‐5, which export a number of protein effectors across membranes to the bacterial surface and environment. PE and PPE proteins represent two large families of highly polymorphic proteins that are secreted by these ESX systems. Recently, it was shown that these proteins require system‐specific cytoplasmic chaperones for secretion. Here, we report the crystal structure of M. tuberculosis ESX‐5‐secreted PE25–PPE41 heterodimer in complex with the cytoplasmic chaperone EspG₅. EspG₅ represents a novel fold that is unrelated to previously characterized secretion chaperones. Functional analysis of the EspG₅‐binding region uncovered a hydrophobic patch on PPE41 that promotes dimer aggregation, and the chaperone effectively abolishes this process. We show that PPE41 contains a characteristic chaperone‐binding sequence, the hh motif, which is highly conserved among ESX‐1‐, ESX‐3‐ and ESX‐5‐specific PPE proteins. Disrupting the interaction between EspG₅ and three different PPE target proteins by introducing different point mutations generally affected protein secretion. We further demonstrate that the EspG₅ chaperone plays an important role in the ESX secretion mechanism by keeping aggregation‐prone PE–PPE proteins in their soluble state.     

Functional genomics of soybean for improvement of productivity in adverse conditions

Global soybean production is frequently impacted by various stresses, including both abiotic and biotic stresses. To develop soybean plants with enhanced tolerance to different stressors, functional genomics of soybean and a comprehensive understanding of available biotechnological resources and approaches are essential. In this review, we will discuss recent advances in soybean functional genomics which provide unprecedented opportunities to understand global patterns of gene expression, gene regulatory networks, various physiological, biochemical, and metabolic pathways as well as their association with the development of specific phenotypes. Soybean functional genomics, therefore, will ultimately enable us to develop new soybean varieties with improved productivity under adverse conditions by genetic engineering.