Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase

Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed.     

Testin regulates the blood-testis barrier via disturbing occludin/ZO-1 association and actin organization

The blood-testis barrier (BTB) separates the seminiferous epithelium into the apical and basal compartments. The BTB has to operate timely and accurately to ensure the correct migration of germ cells, meanwhile maintaining the immunological barrier. Testin was first characterized from primary Sertoli cells, it is a secretory protein and a sensitive biomarker to monitor junctions between Sertoli and germ cells. Till now, the functions of testin on BTB dynamics and the involving mechanisms are unknown. Herein, testin acts as a regulatory protein on BTB integrity. In vitro testin knockdown by RNAi caused significant damage to the Sertoli cell barrier with no apparent changes in the protein levels of several major tight junction (TJ), adhesion junction, and gap junction proteins. Also, testin RNAi caused the diffusion of two TJ structural proteins, occludin and ZO-1, diffusing away from the Sertoli cell surface into the cytoplasm. Association and colocalization between ZO-1 and occludin were decreased after testin RNAi, examined by Co-IP and coimmunofluorescent staining, respectively. Furthermore, testin RNAi induced a dramatic disruption on the arrangement of actin filament bundles and a reduced F-actin/G-actin ratio. The actin regulatory protein ARP3 appeared at the Sertoli cell interface after testin RNAi without its protein level change, whereas overexpressing testin in Sertoli cells showed no effect on TJ barrier integrity. The above findings suggest that besides as a monitor for Sertoli-germ cell junction integrity, testin is also an essential molecule to maintain Sertoli–Sertoli junctions.     

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71

Enterovirus A71(EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease(HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43(TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis(ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3 C between the residues 331 Q and332 S, while mutated TDP-43(Q331 A) was not cleaved. In addition, mutated 3 C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2 A rather than 3 C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71 infection.     

Intensive aquaculture selects for increased virulence and interference competition in bacteria

Although increased disease severity driven by intensive farming practices is problematic in food production, the role of evolutionary change in disease is not well understood in these environments. Experiments on parasite evolution are traditionally conducted using laboratory models, often unrelated to economically important systems. We compared how the virulence, growth and competitive ability of a globally important fish pathogen, Flavobacterium columnare, change under intensive aquaculture. We characterized bacterial isolates from disease outbreaks at fish farms during 2003–2010, and compared F. columnare populations in inlet water and outlet water of a fish farm during the 2010 outbreak. Our data suggest that the farming environment may select for bacterial strains that have high virulence at both long and short time scales, and it seems that these strains have also evolved increased ability for interference competition. Our results are consistent with the suggestion that selection pressures at fish farms can cause rapid changes in pathogen populations, which are likely to have long-lasting evolutionary effects on pathogen virulence. A better understanding of these evolutionary effects will be vital in prevention and control of disease outbreaks to secure food production.     

Quantitative diffusion tensor MRI-based fiber tracking of human skeletal muscle.

Diffusion-tensor magnetic resonance imaging (DT-MRI) offers great potential for understanding structure-function relationships in human skeletal muscles. The purposes of this study were to demonstrate the feasibility of using in vivo human DT-MRI fiber tracking data for making pennation angle measurements and to test the hypothesis that heterogeneity in the orientation of the tibialis anterior (TA) muscle's aponeurosis would lead to heterogeneity in pennation angle. Eight healthy subjects (5 male) were studied. T(1)-weighted anatomical MRI and DT-MRI data were acquired of the TA muscle. Fibers were tracked from the TA's aponeurosis by following the principal eigenvector. The orientations of the aponeurosis and muscle fiber tracts in the laboratory frame of reference and the orientation of the fiber tracts with respect to the aponeurosis [i.e., the pennation angle (theta)] were determined. The muscle fiber orientations, when expressed relative to the laboratory frame of reference, did not change as functions of superior-to-inferior position. The sagittal and coronal orientations of the aponeurosis did not change in practically significant manners either, but the aponeurosis' axial orientation changed by approximately 40 degrees . As a result, the mean value for theta decreased from 16.3 (SD 6.9) to 11.4 degrees (SD 5.0) along the muscle's superior-to-inferior direction. The mean value of theta was greater in the deep than in the superficial compartment. We conclude that pennation angle measurements of human muscle made using DT-MRI muscle fiber tracking are feasible and reveal that in the foot-head direction, there is heterogeneity in the pennation properties of the human TA muscle.     

Quantitative genetics of temperature performance curves of Neurospora crassa

Earth's temperature is increasing due to anthropogenic CO emissions; and organisms need either to adapt to higher temperatures, migrate into colder areas, or face extinction. Temperature affects nearly all aspects of an organism's physiology via its influence on metabolic rate and protein structure, therefore genetic adaptation to increased temperature may be much harder to achieve compared to other abiotic stresses. There is still much to be learned about the evolutionary potential for adaptation to higher temperatures, therefore we studied the quantitative genetics of growth rates in different temperatures that make up the thermal performance curve of the fungal model system Neurospora crassa. We studied the amount of genetic variation for thermal performance curves and examined possible genetic constraints by estimating the G -matrix. We observed a substantial amount of genetic variation for growth in different temperatures, and most genetic variation was for performance curve elevation. Contrary to common theoretical assumptions, we did not find strong evidence for genetic trade-offs for growth between hotter and colder temperatures. We also simulated short-term evolution of thermal performance curves of N. crassa, and suggest that they can have versatile responses to selection.     

胎脑提取液对衰老小鼠肝细胞酶活性影响的实验研究

目的：观察胎脑提取液对衰老小鼠肝细胞琥珀酸脱氢酶(SDH)和酸性磷酸酶(ACP)活性的影响,探讨胎脑提取液的抗衰老作用。方法：选用健康昆明种小白鼠30只,随机分为3组;采用D-半乳糖制备亚急性衰老模型;酶组织化学染色观察各组小鼠肝细胞SDH和ACP的活性。结果：衰老模型组与正常对照组相比,小鼠肝细胞SDH活性明显降低,ACP活性明显升高;给药组与衰老模型组相比,小鼠肝细胞SDH活性明显升高,ACP活性明显降低。结论：胎脑提取液可以延缓肝细胞的衰老进程,具有一定的抗衰老作用。     

部分肝病患者和健康人血清中HBV基因型分析

确定了内蒙古海拉尔地区乙型肝炎病毒 (HBV)感染者HBV基因型的分布情况。采用基因型特异引物的巢式聚合酶链式反应方法对 38例无症状HBV携带者 ,3例急性肝炎 ,41例慢性肝炎 ,3例肝硬化 ,3例肝癌患者和38例健康人血清中HBV基因型进行分析。结果显示 ,12 6例中有 91例检测到HBVDNA ,其中 ,HBVB型 ,C型分别为 11例 ( 12 .1% )和 6 8例 ( 74.7% ) ,同时检测到B型和C型者有 8例 ( 8.8% ) ,有 4例 ( 4.4% )尚未确定基因型别。可见 ,在海拉尔地区HBV感染同时存在HBVB型和C型 ,而且C型是这一地区流行的主要基因型。     

DOES ENVIRONMENTAL ROBUSTNESS PLAY A ROLE IN FLUCTUATING ENVIRONMENTS?

Fluctuating environments are expected to select for individuals that have highest geometric fitness over the experienced environments. This leads to the prediction that genetically determined environmental robustness in fitness, and average fitness across environments should be positively genetically correlated to fitness in fluctuating environments. Because quantitative genetic experiments resolving these predictions are missing, we used a full‐sib, half‐sib breeding design to estimate genetic variance for egg‐to‐adult viability in Drosophila melanogaster exposed to two constant or fluctuating temperatures that were above the species’ optimum temperature, during development. Viability in two constant environments (25°C or 30°C) was used to estimate breeding values for environmental robustness of viability (i.e., reaction norm slope) and overall viability (reaction norm elevation). These breeding values were regressed against breeding values of viability at two different fluctuating temperatures (with a mean of 25°C or 30°C). Our results based on genetic correlations show that average egg‐to‐adult viability across different constant thermal environments, and not the environmental robustness, was the most important factor for explaining the fitness in fluctuating thermal environments. Our results suggest that the role of environmental robustness in adapting to fluctuating environments might be smaller than anticipated.