Modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the TRIM5alpha B-box 2 domain

An intact B-box 2 domain is essential for the antiretroviral activity of TRIM5alpha. We modeled the structure of the B-box 2 domain of TRIM5alpha based on the existing three-dimensional structure of the B-box 2 domain of human TRIM29. Using this model, we altered the residues predicted to be exposed on the surface of this globular structure. Most of the alanine substitutions in these residues exerted little effect on the antiretroviral activity of human TRIM5alphahu or rhesus monkey TRIM5alpharh. However, alteration of arginine 119 of TRIM5alphahu or the corresponding arginine 121 of TRIM5alpharh diminished the abilities of the proteins to restrict retroviral infection without affecting trimerization or recognition of the viral capsid. The abilities of these functionally defective TRIM5alpha proteins to accelerate the uncoating of the targeted retroviral capsid were abolished. Removal of the positively charged side chain from B-box 2 arginines 119/120/121 resulted in diminished proteasome-independent turnover of TRIM5alpha and the related restriction factor TRIMCyp. However, testing of an array of mutants revealed that the rapid turnover and retroviral restriction functions of this B-box 2 region are separable.     

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.     

Brassinosteroids promote development of rice pollen grains and seeds by triggering expression of Carbon Starved Anther,a MYB domain protein

Transport of photoassimilates from leaf tissues (source regions) to the sink organs is essential for plant development. Here, we show that a phytohormone, the brassinosteroids (BRs) promotes pollen and seed development in rice by directly promoting expression of Carbon Starved Anther (CSA) which encodes a MYB domain protein. Over‐expression of the BR‐synthesis gene D11 or a BR‐signaling factor OsBZR1 results in higher sugar accumulation in developing anthers and seeds, as well as higher grain yield compared with control non‐transgenic plants. Conversely, knockdown of D11 or OsBZR1 expression causes defective pollen maturation and reduced seed size and weight, with less accumulation of starch in comparison with the control. Mechanically, OsBZR1 directly promotes CSA expression and CSA directly triggers expression of sugar partitioning and metabolic genes during pollen and seed development. These findings provide insight into how BRs enhance plant reproduction and grain yield in an important agricultural crop.     

Coexistence of Anhedonia and anxiety-independent increased novelty-seeking behavior in the chronic mild stress model of depression

Previous research demonstrated excessive decreases in reward sensitivity and increases in harm avoidance in depressed individuals. These results straightly lead to a hypothesis that depressed patients should avoid novelty or express reduced novelty-seeking behavior. Nevertheless, literature in this regard is inconsistent. Furthermore, whether the potentially altered novelty-associated behavior is dependent on changed anxiety/fear or related to altered goal-directed approaching tendency is unclear. Here, we tested novel object-approaching behavior in a free-exploration paradigm in chronic mild stress (CMS)-induced anhedonic and stress-resistant rats respectively. Other CMS-induced, emotional behaviors were also examined in a battery of behavioral tests including novel cage, exploration, locomotor activity and elevated plus maze (EPM). We found that compared with controls, stress-resistant rats who consistently showed lower anxiety level in EPM (time in open arms) and, open-field (OF) test (time in central area) showed no sign of enhanced novel object approaching behavior. To the contrary, the anhedonic ones who did not express any sign of reduced anxiety showed paradoxically intensified novelty-approaching behavior. We concluded that reduced anxiety would not necessarily lead to enhanced novelty-seeking behavior; anhedonia coexists with anxiety-independent, increased novelty-seeking behavior. The salient paradox of coexistence of anhedonia and increased novelty-seeking behavior was critically discussed.     

Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining

Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5′ resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5′-overhanging DSBs (5′ DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3′ DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5′ DSBs were rejoined more efficiently than 3′ DSBs, consistent with a robust recruitment of NHEJ proteins to 5′ DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5′ DSBs and facilitated protection of 3′ DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5′ DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3′ and 5′ DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5′ DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5′ DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes.     

Modulation of DNA end joining by nuclear proteins

DNA double strand breaks in mammalian cells are primarily repaired by homologous recombination and non-homologous end joining (NHEJ). NHEJ may either be error-free or mutagenic with deletions or insertions at the joint. Recent studies showed that DNA ends can also be joined via microhomologous sequences flanking the break point especially when proteins responsible for NHEJ, such as Ku, are absent. Microhomology-mediated end joining (MHEJ) is always accompanied by a deletion that spans one of the two homologous sequences and the intervening sequence, if any. In this study we evaluated several factors affecting the relative contribution of MHEJ to DNA end joining using nuclear extracts and DNA substrates containing 10-bp repeats at the ends. We found that the occurrence of MHEJ is determined by the relative abundance of nuclear proteins. At low DNA/protein ratios, an error-free end-joining mechanism predominated over MHEJ. As the DNA/protein ratio increased, MHEJ became predominant. We show that the nuclear proteins that contribute to the inhibition of the error-prone MHEJ include Ku and histone H1. Treatment of extracts with flap endonuclease 1 antiserum significantly reduced MHEJ. Addition of a 17-bp intervening sequence between the microhomologous sequences significantly reduced the efficiency of MHEJ. Thus, this cell-free assay provides a platform for evaluating factors modulating end joining.     

Temporal dynamics of soil bacterial network regulate soil resistomes

Soil bacteria are diverse and form complicated ecological networks through various microbial interactions, which play important roles in soil multi-functionality. However, the seasonal effects on the bacterial network, especially the relationship between bacterial network topological features and soil resistomes remains underexplored, which impedes our ability to unveil the mechanisms of the temporal-dynamics of antibiotic resistance genes (ARGs). Here, a field investigation was conducted across four seasons at the watershed scale. We observed significant seasonal variation in bacterial networks, with lower complexity and stability in autumn, and a wider bacterial community niche in summer. Similar to bacterial communities, the co-occurrence networks among ARGs also shift with seasonal change, particularly with respect to the topological features of the node degree, which on average was higher in summer than in the other seasons. Furthermore, the nodes with higher betweenness, stress, degree, and closeness centrality in the bacterial network showed strong relationships with the 10 major classes of ARGs. These findings highlighted the changes in the topological properties of bacterial networks that could further alter antibiotic resistance in soil. Together, our results reveal the temporal dynamics of bacterial ecological networks at the watershed scale, and provide new insights into antibiotic resistance management under environmental changes.     

Identification of novel antipoxviral agents: mitoxantrone inhibits vaccinia virus replication by blocking virion assembly

The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.