Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.     

Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells

Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was beta-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9- and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells.     

Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis

The growth and morphological differentiation of dendrites are critical events in the establishment of proper neuronal connectivity and neural function. One extrinsic factor, BMP7, has been shown to specifically affect dendritic morphogenesis; however, the underlying mechanism by which this occurs is unknown. Here we show that LIM kinase 1 (LIMK1), a key downstream effector of Rho GTPases, colocalizes with the BMP receptor, BMPRII, in the tips of neurites and binds to BMPRII. This interaction is required for BMP-dependent induction of the dendritic arbor in cortical neurons. Furthermore, we demonstrate that the physical interaction of LIMK1 with BMPRII synergizes with the Rho GTPase, Cdc42, to activate LIMK1 catalytic activity. These studies thus define a Smad-independent pathway that directly links the BMP receptor to regulation of actin dynamics and provides insights into how extracellular signals modulate LIMK1 activity to permit fine spatial control over cytoskeletal remodelling during dendritogenesis.     

Pyrrolidine dithiocarbamate reduces coxsackievirus B3 replication through inhibition of the ubiquitin-proteasome pathway

Coxsackievirus B3 (CVB3) is one of the most common pathogens for viral myocarditis. The lack of effective therapeutics for CVB3-caused viral diseases underscores the importance of searching for antiviral compounds. Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and is recently reported to inhibit ubiquitin-proteasome-mediated proteolysis. Previous studies have shown that PDTC inhibits replication of rhinovirus, influenza virus, and poliovirus. In the present study, we report that PDTC is a potent inhibitor of CVB3. Coxsackievirus-infected HeLa cells treated with PDTC showed a significant reduction of CVB3 viral RNA synthesis, viral protein VP1 expression, and viral progeny release. Similar to previous observation that divalent ions mediate the function of PDTC, we further report that serum-containing copper and zinc are required for its antiviral activity. CVB3 infection resulted in massive generation of reactive oxygen species (ROS). Although PDTC alleviated ROS generation, the antiviral activity was unlikely dependent on its antioxidant effect because the potent antioxidant, N-acetyl-L-cysteine, failed to inhibit CVB3 replication. Consistent with previous reports that PDTC inhibits ubiquitin-proteasome-mediated protein degradation, we found that PDTC treatment led to the accumulation of several short-lived proteins in infected cells. We further provide evidence that the inhibitory effect of PDTC on protein degradation was not due to inhibition of proteasome activity but likely modulation of ubiquitination. Together with our previous findings that proteasome inhibition reduces CVB3 replication (H. Luo, J. Zhang, C. Cheung, A. Suarez, B. M. McManus, and D. Yang, Am. J. Pathol. 163:381-385, 2003), results in this study suggest a strong antiviral effect of PDTC on coxsackievirus, likely through inhibition of the ubiquitin-proteasome pathway.     

Retroviral restriction factor TRIM5alpha is a trimer

The retrovirus restriction factor TRIM5alpha targets the viral capsid soon after entry. Here we show that the TRIM5alpha protein oligomerizes into trimers. The TRIM5alpha coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5alpha mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5alpha, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5alpha to interact with threefold pseudosymmetrical structures on retroviral capsids.     

MicroRNA‐31a‐5p from aging BMSCs links bone formation and resorption in the aged bone marrow microenvironment

The alteration of age‐related molecules in the bone marrow microenvironment is one of the driving forces in osteoporosis. These molecules inhibit bone formation and promote bone resorption by regulating osteoblastic and osteoclastic activity, contributing to age‐related bone loss. Here, we observed that the level of microRNA‐31a‐5p (miR‐31a‐5p) was significantly increased in bone marrow stromal cells (BMSCs) from aged rats, and these BMSCs demonstrated increased adipogenesis and aging phenotypes as well as decreased osteogenesis and stemness. We used the gain‐of‐function and knockdown approach to delineate the roles of miR‐31a‐5p in osteogenic differentiation by assessing the decrease of special AT‐rich sequence‐binding protein 2 (SATB2) levels and the aging of BMSCs by regulating the decline of E2F2 and recruiting senescence‐associated heterochromatin foci (SAHF). Notably, expression of miR‐31a‐5p, which promotes osteoclastogenesis and bone resorption, was markedly higher in BMSCs‐derived exosomes from aged rats compared to those from young rats, and suppression of exosomal miR‐31a‐5p inhibited the differentiation and function of osteoclasts, as shown by elevated RhoA activity. Moreover, using antagomiR‐31a‐5p, we observed that, in the bone marrow microenvironment, inhibition of miR‐31a‐5p prevented bone loss and decreased the osteoclastic activity of aged rats. Collectively, our results reveal that miR‐31a‐5p acts as a key modulator in the age‐related bone marrow microenvironment by influencing osteoblastic and osteoclastic differentiation and that it may be a potential therapeutic target for age‐related osteoporosis.     

OhsR acts as an organic peroxide-sensing transcriptional activator using an <Emphasis Type="Italic">S</Emphasis>-mycothiolation mechanism in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Background

Corynebacterium glutamicum is a well-known producer of various l-amino acids in industry. During the fermenting process, C. glutamicum unavoidably encounters oxidative stress due to a specific reactive oxygen species (ROS) produced by consistent adverse conditions. To combat the ROS, C. glutamicum has developed many common disulfide bond-based regulatory devices to control a specific set of antioxidant genes. However, nothing is known about the mixed disulfide between the protein thiol groups and the mycothiol (MSH) (S-mycothiolation)-based sensor. In addition, no OhrR (organic hydroperoxide resistance regulator) homologs and none of the organic hydroperoxide reductase (Ohr) sensors have been described in the alkyl hydroperoxide reductase CF-missing C. glutamicum, while organic hydroperoxides (OHPs)-specific Ohr was a core detoxification system.

Results

In this study, we showed that the C. glutamicum OhsR acted as an OHPs sensor that activated ohr expression. OhsR conferred resistance to cumene hydroperoxide (CHP) and t-butyl hydroperoxide but not H₂O₂, hypochlorous acid, and diamide; this outcome was substantiated by the fact that the ohsR-deficient mutant was sensitive to OHPs but not inorganic peroxides. The DNA binding activity of OhsR was specifically activated by CHP. Mutational analysis of the two cysteines (Cys125 and Cys261) showed that Cys125 was primarily responsible for the activation of DNA binding. The oxidation of Cys125 produced a sulfenic acid (C125-SOH) that subsequently reacted with MSH to generate S-mycothiolation that was required to activate the ohr expression. Therefore, OhsR regulated the ohr expression using an S-mycothiolation mechanism in vivo.

Conclusion

This is the first report demonstrating that the regulatory OhsR specifically sensed OHPs stress and responded to it by activating a specific ohr gene under its control using an S-mycothiolated mechanism.

     

Mapping QTL for Omega-3 Content in Hybrid Saline Tilapia

Tilapia is one of most important foodfish species. The low omega-3 to omega-6 fatty acid ratio in freshwater tilapia meat is disadvantageous for human health. Increasing omega-3 content is an important task in breeding to increase the nutritional value of tilapia. However, conventional breeding to increase omega-3 content is difficult and slow. To accelerate the increase of omega-3 through marker-assisted selection (MAS), we conducted QTL mapping for fatty acid contents and profiles in a F₂ family of saline tilapia generated by crossing red tilapia and Mozambique tilapia. The total omega-3 content in F₂ hybrid tilapia was 2.5 ± 1.0 mg/g, higher than that (2.00 mg/g) in freshwater tilapia. Genotyping by sequencing (GBS) technology was used to discover and genotype SNP markers, and microsatellites were also genotyped. We constructed a linkage map with 784 markers (151 microsatellites and 633 SNPs). The linkage map was 2076.7 cM long and consisted of 22 linkage groups. Significant and suggestive QTL for total lipid content were mapped on six linkage groups (LG3, -4, -6, -8, -13, and -15) and explained 5.8–8.3% of the phenotypic variance. QTL for omega-3 fatty acids were located on four LGs (LG11, -18, -19, and -20) and explained 5.0 to 7.5% of the phenotypic variance. Our data suggest that the total lipid and omega-3 fatty acid content were determined by multiple genes in tilapia. The markers flanking the QTL for omega-3 fatty acids can be used in MAS to accelerate the genetic improvements of these traits in salt-tolerant tilapia.