Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study

Background

Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.

Methods and Findings

The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001).

Conclusions

The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.     

ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth

Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.     

Human U87 astrocytoma cell invasion induced by interaction of βig-h3 with integrin α5β1 involves calpain-2

It is known that βig-h3 is involved in the invasive process of many types of tumors, but its mechanism in glioma cells has not been fully clarified. Using immunofluorescent double-staining and confocal imaging analysis, and co-immunoprecipitation assays, we found that βig-h3 co-localized with integrin α5β1 in U87 cells. We sought to elucidate the function of this interaction by performing cell invasion assays and gelatin zymography experiments. We found that siRNA knockdowns of βig-h3 and calpain-2 impaired cell invasion and MMP secretion. Moreover, βig-h3, integrins and calpain-2 are known to be regulated by Ca(2+), and they are also involved in tumor cell invasion. Therefore, we further investigated if calpain-2 was relevant to βig-h3-integrin α5β1 interaction to affect U87 cell invasion. Our data showed that βig-h3 co-localized with integrin α5β1 to enhance the invasion of U87 cells, and that calpain-2, is involved in this process, acting as a downstream molecule.     

Analysis of Drug Resistance Determinants in Klebsiella pneumoniae Isolates from a Tertiary-Care Hospital in Beijing, China

Background

The rates of multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) isolates among Enterobacteriaceae isolates, particularly Klebsiella pneumoniae, have risen substantially worldwide.

Methodology/Principal Findings

To better understand the molecular mechanisms of drug resistance in K. pneumoniae, we analyzed the drug resistance determinants for K. pneumoniae isolates collected from the 306 Hospital, a tertiary-care hospital in Beijing, China, for the period of September 1, 2010-October 31, 2011. Drug susceptibility testing, PCR amplification and sequencing of the drug resistance determinants were performed. Conjugation experiments were conducted to examine the natural ability of drug resistance to disseminate among Enterobacteriaceae strains using a sodium azide-resistant Escherichia coli J53 strain as a recipient. Among the 223 consecutive non-repetitive K. pneumoniae isolates included in this study, 101 (45.3%) were extended-spectrum beta-lactamases (ESBLs) positive. The rates of MDR, XDR, and PDR isolates were 61.4% (n = 137), 22.0% (n = 49), and 1.8% (n = 4), respectively. Among the tested drug resistance-associated genes, the following ones were detected at relatively high rates bla _CTX-M-10 (80, 35.9%), aacC2 (73, 32.7%), dhfr (62, 27.8%), qnrS (58, 26.0%), aacA4 (57, 25.6%), aadA1 (56, 25.1%). Results from conjugation experiments indicate that many of the drug resistance genes were transmissible.

Conclusions/Significance

Our data give a “snapshot” of the complex genetic background responsible for drug resistance in K. pneumoniae in China and demonstrate that a high degree of awareness and monitoring of those drug resistance determinants are urgently needed in order to better control the emergence and transmission of drug-resistant K. pneumoniae isolates in hospital settings.     

Identification of genes related to white and black plumage formation by RNA-Seq from white and black feather bulbs in ducks

To elucidate the genes involved in the formation of white and black plumage in ducks, RNA from white and black feather bulbs of an F(2) population were analyzed using RNA-Seq. A total of 2,642 expressed sequence tags showed significant differential expression between white and black feather bulbs. Among these tags, 186 matched 133 annotated genes that grouped into 94 pathways. A number of genes controlling melanogenesis showed differential expression between the two types of feather bulbs. This differential expression was confirmed by qPCR analysis and demonstrated that Tyr (Tyrosinase) and Tyrp1 (Tyrosinase-related protein-1) were expressed not in W-W (white feather bulb from white dorsal plumage) and W-WB (white feather bulb from white-black dorsal plumage) but in B-B (black feather bulb from black dorsal plumage) and B-WB (black feather bulb from white-black dorsal plumage) feather bulbs. Tyrp2 (Tyrosinase-related protein-2) gene did not show expression in the four types of feather bulbs but expressed in retina. C-kit (The tyrosine kinase receptor) expressed in all of the samples but the relative mRNA expression in B-B or B-WB was approximately 10 fold higher than that in W-W or W-WB. Additionally, only one of the two Mitf isoforms was associated with plumage color determination. Downregulation of c-Kit and Mitf in feather bulbs may be the cause of white plumage in the duck.     

Fine mapping of the Bsr1 barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon

The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25 °C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F(6:7) recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F(2) population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.     

Halohasta litorea gen. nov. sp. nov., and Halohasta litchfieldiae sp. nov., isolated from the Daliang aquaculture farm, China and from Deep Lake, Antarctica, respectively

Two halophilic archaeal strains, R30^T and tADL^T, were isolated from an aquaculture farm in Dailing, China, and from Deep Lake, Antarctica, respectively. Both have rod-shaped cells that lyse in distilled water, stain Gram-negative and form red-pigmented colonies. They are neutrophilic, require >120?g/l NaCl and 48–67?g/l MgCl₂ for growth but differ in their optimum growth temperatures (30?°C, tADL^T vs. 40?°C, R30^T). The major polar lipids were typical for members of the Archaea but also included a major glycolipid chromatographically identical to sulfated mannosyl glucosyl diether (S-DGD-1). The 16S rRNA gene sequences of the two strains are 97.4?% identical, show most similarity to genes of the family Halobacteriaceae, and cluster together as a distinct clade in phylogenetic tree reconstructions. The rpoB′ gene similarity between strains R30^T and tADL^T is 92.9?% and less to other halobacteria. Their DNA G?+?C contents are 62.4–62.9?mol?% but DNA–DNA hybridization gives a relatedness of only 44?%. Based on phenotypic, chemotaxonomic and phylogenetic properties, we describe two new species of a novel genus, represented by strain R30^T (=?CGMCC 1.10593^T?=?JCM 17270^T) and strain tADL^T (=?JCM 15066^T?=?DSMZ 22187^T) for which we propose the names Halohasta litorea gen. nov., sp. nov. and Halohasta litchfieldiae sp. nov., respectively.     

Protective effects of garlic oil on hepatocarcinoma induced by N-nitrosodiethylamine in rats

To investigate the protective effects and the possible mechanisms of garlic oil (GO) against N-nitrosodiethylamine (NDEA)-induced hepatocarcinoma in rats, Wistar rats were gavaged with GO (20 or 40 mg/kg) for 1 week, and then were gavaged with GO and NDEA (10 mg/kg) for the next 20 weeks. The changes of morphology, histology, the biochemical indices of serum, and DNA oxidative damage of liver were examined to assess the protective effects. Lipid peroxidation (LPO), antioxidant defense system, and apoptosis-related proteins were measured to investigate potential mechanisms. At the end of the study (21 weeks), GO administration significantly inhibited the increase of the nodule incidence and average nodule number per nodule-bearing liver induced by NDEA, improved hepatocellular architecture, and dramatically inhibited NDEA-induced elevation of serum biochemical indices (alanine aminotransferase , aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase) and hepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in a dose-dependent manner. The mechanistic studies demonstrated that GO counteracted NDEA-induced oxidative stress in rats illustrated by the restoration of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) levels, and the reduction of the malondialdehyde (MDA) levels in liver. Furthermore, the mRNA and protein levels of Bcl-2, Bcl-xl, andβ-arrestin-2 were significantly decreased whereas those of Bax and caspase-3 were significantly increased. These data suggest that GO exhibited significant protection against NDEA-induced hepatocarcinogenesis, which might be related with the enhancement of the antioxidant activity and the induction of apoptosis.     

丹参2 酮戊二酸依赖性双加氧酶基因克隆及表达分析

以商洛紫花丹参为材料,对其转录组序列SRA020132进行Blast分析,采用PCR技术克隆得到丹参2-酮戊二酸依赖性双加氧酶基因Sm2-ODD1,GenBank登录号为JN935923。Sm2-ODD1基因全长1 365bp,包含3个外显子和2个内含子;cDNA全长1 189bp,读码框951bp,编码316个氨基酸残基;预测的编码蛋白具有2-酮戊二酸依赖性双加氧酶中结合2-酮戊二酸和亚铁离子的"H-T-D"、"H-X"和"R-Y-S"保守基序以及"果冻状"空间结构。表达分析显示,Sm2-ODD1在丹参各个器官都表达,但表达水平具有组织特异性,在根中表达量最高,在叶中表达量最低;该基因表达明显受到MeJA、GA3和ABA的诱导,可能参与了丹参萜类代谢下游途径。