A TRPC1 Protein-dependent Pathway Regulates Osteoclast Formation and Function

Ca²⁺ signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca²⁺ stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ϵ) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ϵ in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, mediating store-operated currents. TRPC1ϵ physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ϵ-Orai1 complex through TRPC1ϵ suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ϵ and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.     

The Effect of the PB2 Mutation 627K on Highly Pathogenic H5N1 Avian Influenza Virus Is Dependent on the Virus Lineage

Clade 2.2 Eurasian-lineage H5N1 highly pathogenic avian influenza viruses (HPAIVs) were first detected in Qinghai Lake, China, in 2005 and subsequently spread through Asia, Europe, and Africa. Importantly, these viruses carried a lysine at amino acid position 627 of the PB2 protein (PB2 627K), a known mammalian adaptation motif. Previous avian influenza virus isolates have carried glutamic acid in this position (PB2 627E), commonly described to restrict virus polymerase function in the mammalian host. We sought to examine the effect of PB2 627K on viral maintenance in the avian reservoir. Viruses constructed by reverse genetics were engineered to contain converse PB2 627K/E mutations in a Eurasian H5N1 virus (A/turkey/Turkey/5/2005 [Ty/05]) and, for comparison, a historical pre-Asian H5N1 HPAIV that naturally bears PB2 627E (A/turkey/England/50-92/1991 [50-92]). The 50-92 PB2 627K was genetically unstable during virus propagation, resulting in reversion to PB2 627E or the accumulation of the additional mutation PB2 628R and/or a synonymous mutation from an A to a G nucleotide at nucleotide position 1869 (PB2 A1869G). Intriguingly, PB2 628R and/or A1869G appeared to improve the genetic stability of 50-92 PB2 627K. However, the replication of 50-92 PB2 627K in conjunction with these stabilizing mutations was significantly restricted in experimentally infected chickens, where reversion to PB2 627E occurred. In contrast, no significant effects on viral fitness were observed for Ty/05 PB2 627E or 627K in in vitro or in vivo experiments. Our observations suggest that PB2 627K is supported in Eurasian-lineage viruses; in contrast, PB2 627K carries a significant fitness cost in the historical pre-Asian 50-92 virus.     

Microbiological Identification and Analysis of Swine Lungs Collected from Carcasses in Swine Farms, China

The primary objective of this 3 years study was to determine the prevalence of porcine pathogens of the lungs of swine in swine farms in southern China. A total of 5,420 samples were collected from 200 swine farms. The bacterium that was most commonly isolated was Streptococcus suis, with 10.24 % of the samples being positive, 114 lungs (2.1 %) were positive for pseudorabies virus and 263 (4.85 %) were positive for classical swine fever virus; much lower than positive for PRRSV (15.1 %, p = 0.023) and PCV2 (13.8 %, p = 0.038). lungs that were positive for PRRSV and/or PCV-2 have significantly increased odds of being positive for any of the S. suis (9.79 vs. 0.44 %, p = 0.003).     

Cloning and Expression of Genes Encoding F107-C and K88-1NT Fimbrial Proteins of Enterotoxigenic Escherichia coli from Piglets

We cloned two genes coding F107-C and K88-1NT fimbrial subunits from strains E. coli C and 1NT isolated from Thua Thien Hue province, Vietnam. The mature peptide of faeG gene from strain E. coli 1NT (called faeG-1NT) is 100 % similarity with faeG gene, while the CDS of fedA gene from strain C (called fedA-C) has a similarity of 97 % with the fedA gene. Expression of the faeG-1NT and fedA-C genes in E. coli BL21 Star™ (DE3) produced proteins of ~31 and 22 kDa, respectively. The effect of IPTG concentration on the K88-1NT and F107-C fimbriae production was investigated. The results showed that 0.5 mM IPTG is suitable for higher expression of K88-1NT subunit, while 0.75 mM IPTG strongly stimulated expression of F107-C subunit. The optimal induction time for expression was also examined. Generally, highest expression of K88-1NT subunit occurred after 6 h of induction, while that of F107-C subunit is after 14 h.     

Duration of sucrose preculture is critical for shoot regrowth of in vitro-grown apple shoot-tips cryopreserved by encapsulation-dehydration

A simple and efficient cryopreservation protocol using encapsulation-dehydration was established for in vitro-grown shoot-tips of apple ‘Gala’ (Malus × domestica Borkh.). Shoot-tips, of 2.0 mm in length and with 5–6 leaf primordia, excised from 4-week-old shoot stock cultures, without cold-hardening, were encapsulated into beads, each being about 5 mm in diameter and containing a single shoot-tip. The beads were precultured on MS medium containing 0.5 M sucrose for 7 days. The precultured beads were dehydrated by air-drying to reduce the water content of the beads to about 22–20 % in 5–7 h, followed by a direct immersion in liquid nitrogen for 1 h. Frozen shoot-tips were re-warmed in a water bath at 38 °C for 2 min and post-cultured on a recovery medium for shoot regrowth. This protocol was successfully applied to four Malus species and one hybrid, among which M. micromalus and M. robusta are wild species native to China. The highest and lowest shoot regeneration rates were found in ‘Gala’ (75 %) and ‘Wangshanhong’ (36 %), with a mean shoot regrowth rate of 61 % attained for the seven Malus genotypes tested. Histological studies revealed that shoots could be regenerated in cryopreserved shoot-tips only when many cells in the leaf primordia and most of the cells in the apical dome survived following cryopreservation. Morphologies of the regenerated plantlets were identical to those from the in vitro stock cultures. Therefore, the encapsulation-dehydration procedure developed in the present study should provide a technical support for setting-up Malus cryo-banking in China.     

Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology

Increasing concerns over limited petroleum resources and associated environmental problems are motivating the development of efficient cell factories to produce chemicals, fuels, and materials from renewable resources in an environmentally sustainable economical manner. Bacillus spp., the best characterized Gram-positive bacteria, possesses unique advantages as a host for producing microbial enzymes and industrially important biochemicals. With appropriate modifications to heterologous protein expression and metabolic engineering, Bacillus species are favorable industrial candidates for efficiently converting renewable resources to microbial enzymes, fine chemicals, bulk chemicals, and fuels. Here, we summarize the recent advances in developing Bacillus spp. as a cell factory. We review the available genetic tools, engineering strategies, genome sequence, genome-scale structure models, proteome, and secretion pathways, and we list successful examples of enzymes and industrially important biochemicals produced by Bacillus spp. Furthermore, we highlight the limitations and challenges in developing Bacillus spp. as a robust and efficient production host, and we discuss in the context of systems and synthetic biology the emerging opportunities and future research prospects in developing Bacillus spp. as a microbial cell factory.     

A metabonomic investigation of the effects of 60 days exposure of rats to two types of pyrethroid insecticides

Type I and II pyrethroid insecticides display different neurotoxicity. To investigate the long-term (60 days exposure) metabolic effect of the two types of pyrethroid insecticides deltamethrin and permethrin, ¹H nuclear magnetic resonance (NMR) spectroscopy-based metabonomics was used to analyze the biochemical composition of urine and serum samples from rats administrated daily with deltamethrin or permethrin for 60 consecutive days, and principal component analysis used to visualize similarities and differences in the resultant biochemical profiles. Rats treated with either deltamethrin or permethrin displayed increased levels of urinary acetate, dimethylamine, dimethylglycine, trimethylamine and serum free amino acids, and decreased urinary 2-oxoglutarate, all of which are indicative of kidney lesions and nephrotoxicity. The reduced excretion of tricarboxylic acid cycle intermediates, together with increased 3-D-hydroxybutyrate, acetate, and lactate in treated rats could suggest disturbance of the energy metabolism, including an increased rate of anaerobic glycolysis, enhanced fatty acid β-oxidation and ketogenesis. These results show that these two types of insecticides have similarities in the urine and serum spectra, indicating that similar metabolic pathways are perturbed by the insecticides, which induced hepatotoxicity and nephrotoxicity. This approach may lead to the discovery of novel biomarkers of pyrethroids toxicity and thereby provide new insights into the toxicological mechanisms of pesticides pyrethroids.     

Quantitative Imaging of Protein Secretions from Single Cells in Real Time

Protein secretions from individual cells create spatially and temporally varying concentration profiles in the extracellular environment, which guide a wide range of biological processes such as wound healing and angiogenesis. Fluorescent and colorimetric probes for the detection of single cell secretions have time resolutions that range from hours to days, and as a result, little is known about how individual cells may alter their protein secretion rates on the timescale of minutes or seconds. Here, we present a label-free technique based upon nanoplasmonic imaging, which enabled the measurement of individual cell secretions in real time. When applied to the detection of antibody secretions from single hybridoma cells, the enhanced time resolution revealed two modes of secretion: one in which the cell secreted continuously and another in which antibodies were released in concentrated bursts that coincided with minute-long morphological contractions of the cell. From the continuous secretion measurements we determined the local concentration of antibodies at the sensing array closest to the cell and from the bursts we estimated the diffusion constant of the secreted antibodies through the extracellular media. The design also incorporates transmitted light and fluorescence microscopy capabilities for monitoring cellular morphological changes and intracellular fluorescent labels. We anticipate that this technique can be adapted as a general tool for the quantitative study of paracrine signaling in both adherent and nonadherent cell lines.     

Halomonas nanhaiensis sp. nov., a halophilic bacterium isolated from a sediment sample from the South China Sea

A novel Gram-negative, aerobic, slightly halophilic, yellow-pigmented, oxidase-negative, Voges–Proskauer positive, non-spore-forming bacterium, designated YIM M 13059^T, was isolated from a sediment sample collected from the South China Sea at a depth of 310 m. Optimal growth was found to occur at 28–30 °C, pH 7.0 and in the presence of 3–4 % (w/v) NaCl. Cells were observed to be rod-shaped and motile by peritrichous flagella. The polar lipids of strain YIM M 13059^T were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a ninhydrin-positive phospholipid, one glycolipid and two unknown phospholipids. The predominant respiratory quinone was determined to be Q-9. The major fatty acids were identified as C_18:1 ω7c, C_16:1 ω6c/C_16:1 ω7c, C_16:0 and C_12:0 3-OH. The genomic DNA G+C content was determined to be 54.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate belongs to the genus Halomonas in the family Halomonadaceae. The 16S rRNA gene sequence similarities between strain YIM M 13059 ^T and the type strains of members of the genus Halomonas were in the range 93.3–98.3 %. However, the levels of DNA–DNA relatedness values between YIM M 13059 and the type strains of the most closely related species, Halomonas zhangjiangensis, Halomonas variabilis, Halomonas neptunia, Halomonas boliviensis and Halomonas sulfadieris were 50.2 ± 0.68 %, 46.8 ± 1.9 %, 28.5 ± 0.74 %, 42.9 ± 0.55 % and 37.1 ± 0.68 %, respectively. Based on phylogenetic, chemotaxonomic and phenotypic data, the strain YIM M 13059^T is proposed to represent a novel member of the genus Halomonas, with the name Halomonas nanhaiensis sp. nov. The type strain is YIM M 13059^T (=JCM 18142^T =CCTCC AB 2012911^T).     

IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells

Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.