Overexpression of a delayed early gene hlg1 of temperate mycobacteriophage L1 is lethal to both M. smegmatis and E. coli

Two genes of temperate mycobacteriophage L5, namely, gp63 and gp64, were hypothesized to be toxic to M. smegmatis. An identical L5 gp64 ortholog (designated hlg1) was cloned from homoimmune mycobacteriophage L1 and characterized at length here. As expected, hlg1 affected the growth of M. smegmatis when overexpressed from a resident plasmid. HLG1 (the protein encoded by hlg1) in fact caused growth retardation of M. smegmatis and the region encompassing its 57-114 C-terminal amino acid residues was found indispensable for its growth-retardation activity. Both nucleic acid and protein biosynthesis were severely impaired in M. smegmatis expressing HLG1. Interestingly, HLG1 also affected E. coli almost similarly. This putative delayed early lipoprotein did not participate in the lytic growth of L1.     

The Association of Pet Ownership and Attachment with Perceived Stress among Chinese Adults

Pet ownership is associated with both positive and negative emotions. Given that not everyone responds to pet care in a stressful way, perceptions of companion animals may play a role in how owners respond to them. In this study, we explored the relationship between pet ownership and perceived stress among Chinese pet owners. We also examined the effect of care practices and perceptions of pets on perceived stress. A total of 288 Hong Kong Chinese, adult pet owners, aged 18 to 70 years, were recruited. Participants were surveyed using a self-administered questionnaire covering demographics, pet ownership background, pet attachment, and perceived stress. Regression analyses were performed to examine pet attachment in different demographic groups and its relationship with perceived stress. Moderation and mediation analyses were performed to elucidate their underlying mechanisms. After adjusting for demographics and pet ownership background, greater attachment to a pet was associated with lower stress in owners. The perception of pets as family members minimized the stressful burden of pet care. More time spent caring for a pet increased attachment to that animal which in turn reduced stress in owners. The findings suggest attachment to companion animals and the perception of them as family members can help manage and reduce stress in owners.     

The presence of serotonin in cigarette smoke – a possible mechanistic link to 5-HT-induced airway inflammation

We previously reported the involvement of serotonin (5-HT) metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo. Here, we report cigarette smoke as a source of serotonin (5-HT) to the airways and aim at investigating the effects of 5-HT on oxidative stress and inflammation in human bronchial epithelial cells (BEAS-2B). A 5-HT analog was identified to be present in aqueous phase cigarette smoke using the LC-MS/MS approach, which was later confirmed by a 5-HT enzyme-linked immune assay (EIA). Furthermore, exposure to 5-HT caused a time-dependent elevation of intracellular ROS level, which was blocked in the presence of apocynin (a NOX inhibitor). In support, the immunoblot analysis indicated that there was an increase in the expression of NOX2 time-dependently. 5-HT-induced elevation of IL-8 at both mRNA and protein levels was observed, which was inhibited by TEMPOL (a free radical scavenger), and inhibitors for p38 MAPK (SB203580) and ERK (U0126), in line with the time-dependent phosphorylation of p38 MAPK and ERK. In conclusion, our findings suggest that 5-HT presented in bronchial epithelium of smokers may be involved in cigarette smoke-induced oxidative stress and inflammation via activation of p38 MAPK and ERK pathway after the formation of free radicals.     

Proteomic characterization of Phragmites communis in ecotypes of swamp and desert dune

Phragmites communis Trin. (common reed) is a recognized model plant for studying its adaptation to contrasting and harsh environments. To understand the inherent molecular basis for its remarkable resistance to combined stresses, we performed a comprehensive proteomic analysis of the leaf proteins from two ecotypes, i.e. swamp and desert dune, naturally growing in the desert region of northwestern China. First, a proteome reference map of Phragmites was established based on the swamp ecotype. Proteins were resolved by 2‐D/SDS‐PAGE and identified by MALDI‐TOF/TOF MS. In total, 177 spots were identified corresponding to 51 proteins. The major proteins identified are proteins involved in photosynthesis, glutathione and ascorbic acid metabolism as well as protein synthesis and quality control. Second, the 2‐DE profiles of the two ecotypes were compared quantitatively via DIGE analysis. Compared with swamp ecotype, 51 proteins spots are higher‐expressed and 58 protein spots are lower‐expressed by twofold or more in desert dune ecotype. Major differences were found for the proteins involved in light reaction of photosynthesis, protein biosynthesis and quality control and antioxidative reactions. The physiological significance of such differences is discussed in the context of a flow of complex events in relation to plant adaptation to combined environmental stresses.     

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow,umbilical cord,and placenta: Implication in the migration

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC‐ and P‐MSC possess immunophenotypic and functional characteristics similar to BM‐MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM‐ and P‐MSC was found 5.9‐ and 3.2‐folds higher than that of UC‐MSC, respectively. By the use of 2‐DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC‐MSC when compared with those in BM‐ and P‐MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor‐1 (PAI‐1) and manganese superoxide dismutase, higher expression was found in the UC‐MSC. We also showed that the overexpression of the PAI‐1 impaired the migration capacity of BM‐ and P‐MSC while silencing of PAI‐1 enhanced the migration capacity of UC‐MSC. Our study indicates that PAI‐1 and other migration‐related proteins are pivotal in governing the migration capacity of MSC.     

Identification of de novo copy number variants associated with human disorders of sexual development

Disorders of sexual development (DSD), ranging in severity from genital abnormalities to complete sex reversal, are among the most common human birth defects with incidence rates reaching almost 3%. Although causative alterations in key genes controlling gonad development have been identified, the majority of DSD cases remain unexplained. To improve the diagnosis, we screened 116 children born with idiopathic DSD using a clinically validated array-based comparative genomic hybridization platform. 8951 controls without urogenital defects were used to compare with our cohort of affected patients. Clinically relevant imbalances were found in 21.5% of the analyzed patients. Most anomalies (74.2%) evaded detection by the routinely ordered karyotype and were scattered across the genome in gene-enriched subtelomeric loci. Among these defects, confirmed de novo duplication and deletion events were noted on 1p36.33, 9p24.3 and 19q12-q13.11 for ambiguous genitalia, 10p14 and Xq28 for cryptorchidism and 12p13 and 16p11.2 for hypospadias. These variants were significantly associated with genitourinary defects (P = 6.08×10⁻¹²). The causality of defects observed in 5p15.3, 9p24.3, 22q12.1 and Xq28 was supported by the presence of overlapping chromosomal rearrangements in several unrelated patients. In addition to known gonad determining genes including SRY and DMRT1, novel candidate genes such as FGFR2, KANK1, ADCY2 and ZEB2 were encompassed. The identification of risk germline rearrangements for urogenital birth defects may impact diagnosis and genetic counseling and contribute to the elucidation of the molecular mechanisms underlying the pathogenesis of human sexual development.     

Are special read alignment strategies necessary and cost-effective when handling sequencing reads from patient-derived tumor xenografts?

Background

Patient-derived tumor xenografts in mice are widely used in cancer research and have become important in developing personalized therapies. When these xenografts are subject to DNA sequencing, the samples could contain various amounts of mouse DNA. It has been unclear how the mouse reads would affect data analyses. We conducted comprehensive simulations to compare three alignment strategies at different mutation rates, read lengths, sequencing error rates, human-mouse mixing ratios and sequenced regions. We also sequenced a nasopharyngeal carcinoma xenograft and a cell line to test how the strategies work on real data.

Results

We found the "filtering" and "combined reference" strategies performed better than aligning reads directly to human reference in terms of alignment and variant calling accuracies. The combined reference strategy was particularly good at reducing false negative variants calls without significantly increasing the false positive rate. In some scenarios the performance gain of these two special handling strategies was too small for special handling to be cost-effective, but it was found crucial when false non-synonymous SNVs should be minimized, especially in exome sequencing.

Conclusions

Our study systematically analyzes the effects of mouse contamination in the sequencing data of human-in-mouse xenografts. Our findings provide information for designing data analysis pipelines for these data.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1172) contains supplementary material, which is available to authorized users.     

Identification of Critical Regions and Candidate Genes for Cardiovascular Malformations and Cardiomyopathy Associated with Deletions of Chromosome 1p36

Cardiovascular malformations and cardiomyopathy are among the most common phenotypes caused by deletions of chromosome 1p36 which affect approximately 1 in 5000 newborns. Although these cardiac-related abnormalities are a significant source of morbidity and mortality associated with 1p36 deletions, most of the individual genes that contribute to these conditions have yet to be identified. In this paper, we use a combination of clinical and molecular cytogenetic data to define five critical regions for cardiovascular malformations and two critical regions for cardiomyopathy on chromosome 1p36. Positional candidate genes which may contribute to the development of cardiovascular malformations associated with 1p36 deletions include DVL1, SKI, RERE, PDPN, SPEN, CLCNKA, ECE1, HSPG2, LUZP1, and WASF2. Similarly, haploinsufficiency of PRDM16–a gene which was recently shown to be sufficient to cause the left ventricular noncompaction–SKI, PRKCZ, RERE, UBE4B and MASP2 may contribute to the development of cardiomyopathy. When treating individuals with 1p36 deletions, or providing prognostic information to their families, physicians should take into account that 1p36 deletions which overlie these cardiac critical regions may portend to cardiovascular complications. Since several of these cardiac critical regions contain more than one positional candidate gene–and large terminal and interstitial 1p36 deletions often overlap more than one cardiac critical region–it is likely that haploinsufficiency of two or more genes contributes to the cardiac phenotypes associated with many 1p36 deletions.     

Inhibitor-Induced Conformational Stabilization and Structural Alteration of a Mip-Like Peptidyl Prolyl cis-trans Isomerase and Its C-Terminal Domain

FKBP22, an Escherichia coli-encoded PPIase (peptidyl-prolyl cis-trans isomerase) enzyme, shares substantial identity with the Mip-like pathogenic factors, caries two domains, exists as a dimer in solution and binds some immunosuppressive drugs (such as FK506 and rapamycin) using its C-terminal domain (CTD). To understand the effects of these drugs on the structure and stability of the Mip-like proteins, rFKBP22 (a chimeric FKBP22) and CTD⁺ (a CTD variant) have been studied in the presence and absence of rapamycin using different probes. We demonstrated that rapamycin binding causes minor structural alterations of rFKBP22 and CTD⁺. Both the proteins (equilibrated with rapamycin) were unfolded via the formation of intermediates in the presence of urea. Further study revealed that thermal unfolding of both rFKBP22 and rapamycin-saturated rFKBP22 occurred by a three-state mechanism with the synthesis of intermediates. Intermediate from the rapamycin-equilibrated rFKBP22 was formed at a comparatively higher temperature. All intermediates carried substantial extents of secondary and tertiary structures. Intermediate resulted from the thermal unfolding of rFKBP22 existed as the dimers in solution, carried an increased extent of hydrophobic surface and possessed relatively higher rapamycin binding activity. Despite the formation of intermediates, both the thermal and urea-induced unfolding reactions were reversible in nature. Unfolding studies also indicated the considerable stabilization of both proteins by rapamycin binding. The data suggest that rFKBP22 or CTD⁺ could be exploited to screen the rapamycin-like inhibitors in the future.