We hypothesize that intracellular trafficking pathways are altered in chlamydial infected cells to maximize the ability of Chlamydia to scavenge nutrients while not overtly stressing the host cell. Previous data demonstrated the importance of two eukaryotic SNARE proteins, VAMP4 and syntaxin 10 (Stx10), in chlamydial growth and development. Although, the mechanism for these effects is still unknown. To interrogate whether chlamydial infection altered these proteins\' networks, we created BirA*-VAMP4 and BirA*-Stx10 fusion constructs to use the BioID proximity labeling system. While we identified a novel eukaryotic protein-protein interaction between Stx10 and VAPB, we also identified caveats in using the BioID system to study the impact of infection by an obligate intracellular pathogen on SNARE protein networks. The addition of the BirA* altered the localization of VAMP4 and Stx10 during infection with Chlamydia trachomatis serovars L2 and D and Coxiella burnetii Nine Mile Phase II. We also discovered that BirA* traffics to and biotinylates Coxiella-containing vacuoles and, in general, has a propensity for labeling membrane or membrane-associated proteins. While the BioID system identified a novel association for Stx10, it is not a reliable methodology to examine intracellular trafficking pathway dynamics during infection with intracellular pathogens.

The Pictet-Spengler (PS) reaction, i.e., the acid-catalyzed condensation between β-arylethylamine and an aldehyde or a ketone and the subsequent ring closure, is an important reaction in organic chemistry. A number of enzymes (called Pictet-Spenglerases, PSases) have been identified to catalyze this reaction, usually with very high enantioselectivity, making these enzymes of potential value in biocatalysis. PSases catalyze the key step in the biosynthesis of indole and benzylisoquinoline alkaloids of plant origin, some of which have pharmacological importance. However, the reaction mechanisms and the origins of the selectivity are not fully understood. Herein, we report a quantum chemical investigation of the mechanism and enantioselectivity of norcoclaurine synthase (NCS), an enzyme that catalyzes the PS condensation between dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA). A large model of the active site is designed on the basis of a recent crystal structure, and it is used to calculate the detailed energy profile of the reaction. Good agreement is obtained between the calculated energies and available experimental information. Both the \"dopamine-first\" and the \"HPAA-first\" binding modes of the substrates reported in the literature are shown to be energetically accessible in the enzyme-substrate complex. However, it is demonstrated that only the dopamine-first pathway is associated with feasible energy barriers. Key active site residues are identified, and their roles in the catalysis are discussed and compared to site-directed mutagenesis experiments. Very importantly, the calculations are able to reproduce and rationalize the observed enantioselectivity of NCS. A detailed analysis of the geometries of the intermediates and transition states helps to pinpoint the main factors controlling the selectivity.

An alternative molecular marker with respect to the 16S rRNA gene demonstrating better identification and phylogenetic parameters has not been designed for the whole Bifidobacteriaceae family, which includes the genus Bifidobacterium and scardovial genera. Therefore, the aim of the study was to find such a gene in available genomic sequences, suggest appropriate means and conditions for asmplification and sequencing of the desired region of the selected gene in various strains of the bacterial family and verify the importance in classification and phylogeny. Specific primers flanking the variable region (~800 pb) within the pyrG gene encoding the CTP synthetase were designed by means of gene sequences retrieved from the genomes of strains belonging to the family Bifidobacteriaceae. The functionality and specificity of the primers were subsequently tested on the wild (7) and type strains of bifidobacteria (36) and scardovia (7). Comparative and phylogenetic studies based on obtained sequences revealed actual significance in classification and phylogeny of the Bifidobacteriaceae family. Gene statistics (percentages of mean sequence similarities and identical sites, mean number of nucleotide differences, P- and K-distances) and phylogenetic analyses (congruence between tree topologies, percentages of bootstrap values >50 and 70%) indicate that the pyrG gene represents an alternative identification and phylogenetic marker exhibiting higher discriminatory power among strains, (sub)species, and genera than the 16S rRNA gene. Sequences of the particular gene fragment, simply achieved through specific primers, enable more precisely to classify and evaluate phylogeny of the family Bifidobacteriaceae including, with some exceptions, health-promoting probiotic bacteria.

Biotinylation has been extensively used for antibody tagging, affinity-based purification, and in protein/DNA-protein interaction studies. Here we describe the use of biotinylation to study the turn-over of proteins in cells. We use the prokaryotic biotin ligase (BirA) to biotinylate the human leukocyte antigen (HLA)-A2 (A2) heavy chain (HC), which was engineered to contain a biotin acceptor peptide (BAP). Controlled availability of biotin in combination with visualization using streptavidin-conjugated peroxidase made it possible to detect biotinylated BAP-A2. Further, we exploited the effects of human cytomegalovirus (HCMV) unique short (US) proteins US2 and US11 on the turn-over of BAP-A2 HC. The full-length BAP-A2 HC and its mutants lacking either the cytosolic tail (tail-less) or both the transmembrane and cytosolic regions (soluble) were expressed via recombinant adenoviruses (rAd). The effect of US2, US11 and a control HCMV protein US9, also expressed via rAd, on each of the BAP- A2 forms was assessed. Experiments using this system showed that US2 and US11 cause proteasome-mediated degradation of full-length BAP-A2 HC but only US2 could cause degradation of tail-less BAP-A2. The results demonstrate that the technique of biotinylation can be used to study protein turn-over in cells.

Hepatocellular carcinoma (HCC) is the sixth common cancer and the third common cause of cancer mortality worldwide. However, the exact molecular mechanism of HCC remains uncertain. Many enzymes are involved in one-carbon metabolism (OCM), and single nucleotide polymorphisms (SNPs) in the corresponding genes may play a role in liver carcinogenesis. In this study, we enrolled 1500 HCC patients and 1500 cancer-free controls, which were frequency-matched by age, gender, and HBV infection status. Then eight SNPs from seven OCM genes (MTHFR, MTR, MTRR, FTHFD, GART, SHMT, and CBS) were evaluated. Results showed that six SNPs (MTHFR rs1801133, MTRR rs2287780, MTRR rs10380, FTHFD rs1127717, GART rs8971, and SHMT rs1979277) were significantly associated with HCC risk in Chinese population, with P values range from 2.26 × 10(-4) to 0.035). The most significant association was detected for GART rs8971. Compared with individuals with the TT genotype, the age- and sex-adjusted odds ratio (OR) for developing HCC was 1.44 (95% confidence interval (CI): 1.03-2.02) among those with the CC genotype and 1.30 (95% CI: 1.10-1.53) for those with CT genotype. Under the log-additive model, each additional copy of minor allele C was associated with a 1.28-fold increased risk of HCC (OR = 1.28, 95% CI: 1.12-1.45). These findings indicated that genetic variants in OCM genes might contribute to HCC susceptibility.

OBJECTIVE: To study the feasibility of using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) for the detection of DNA methylation in placenta tissue.
METHODS: For blood cells from 13 non-pregnant women and 9 euploid placenta, the ratios of DNA methylation were evaluated for 4 genes including CGI149, CGI113, HLCS and ACTB with MS-MLPA and bisulfite sequencing, respectively.
RESULTS: The methylation ratio of the ACTB gene was 0-0.1 for the blood cells when the digestion control was completely digested. The cutoff value for the methylation ratio of MS-MLPA has been determined as 0.1. For the 9 placenta samples, results of MS-MLPA and bisulfite sequencing were concordant for all of the four genes.
CONCLUSIONS: MS-MLPA is an effective alternative to bisulfite sequencing for the assessment of methylation ratios in placental tissues.

The development of congenital heart defects (CHDs) involves a complex interplay between genetic variants, epigenetic variants, and environmental exposures. Previous studies have suggested that susceptibility to CHDs is associated with maternal genotypes, fetal genotypes, and maternal-fetal genotype (MFG) interactions. We conducted a haplotype-based genetic association study of obstructive heart defects (OHDs), aiming to detect the genetic effects of 877 SNPs involved in the homocysteine, folate, and transsulfuration pathways. Genotypes were available for 285 mother-offspring pairs with OHD-affected pregnancies and 868 mother-offspring pairs with unaffected pregnancies. A penalized logistic regression model was applied with an adaptive least absolute shrinkage and selection operator (lasso), which dissects the maternal effect, fetal effect, and MFG interaction effects associated with OHDs. By examining the association between 140 haplotype blocks, we identified 9 blocks that are potentially associated with OHD occurrence. Four haplotype blocks, located in genes MGMT, MTHFS, CBS, and DNMT3L, were statistically significant using a Bayesian false-discovery probability threshold of 0.8. Two blocks in MGMT and MTHFS appear to have significant fetal effects, while the CBS and DNMT3L genes may have significant MFG interaction effects.

In this report, we describe a novel method for producing mature and biologically active mono-biotinylated nerve growth factors (mBtNGF) that can be used for single molecule studies of real-time movement of neurotrophins within axons of neurons. We inserted an AviTag sequence into the C-terminal of the full length mouse preproNGF cDNA and cloned the fusion construct into a pcDNA3.1 mammalian expression vector. We also subcloned the Escherichia coli biotin ligase, BirA, into a pcDNA3.1 vector. These two plasmids were then transiently co-expressed in HEK293FT cells. As a result, the AviTag located in the C-terminal of preproNGF was selectively ligated to a single biotin by BirA. The prepro sequence of NGF was subsequently cleaved within the cell. Mature mono-biotinylated NGF (mBtNGF) was secreted into cell culture media and was purified using Ni resin. We carried out activity assays and our results showed that mBtNGF retained biological activities that were comparable to normal NGF purified from mouse sub maxillary glands. We further verified the biotinylation efficiency of mBtNGF and the level of non-biotinylated NGF was virtually undetectable in the final preparation. Finally, by conjugating to quantum-dot streptavidin, mBtNGF was successfully used for single molecule study of axonal NGF trafficking in neurons.

BACKGROUND: Aminoacyl tRNA synthetases (aaRSs) constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains.
RESULTS: We utilized the Common Peptides (CPs) framework, based on extracted deterministic motifs from all aaRSs, to study family-specific properties. We identified novel aaRS-class related signatures that may supplement the current classification methods and provide a basis for identifying functional regions specific to each aaRS class. We exploited the space spanned by the CPs in order to identify similarities between aaRS families that are not observed using sequence alignment methods, identifying different inter-aaRS associations across different kingdom of life. We explored the evolutionary history of the aaRS families and evolutionary origins of the mitochondrial aaRSs. Lastly, we showed that prevalent CPs significantly overlap known catalytic and binding sites, suggesting that they have meaningful functional roles, as well as identifying a motif shared between aaRSs and a the Biotin-[acetyl-CoA carboxylase] synthetase (birA) enzyme overlapping binding sites in both families.
CONCLUSIONS: The study presents the multitude of ways to exploit the CP framework in order to extract meaningful patterns from the aaRS super-family. Specific CPs, discovered in this study, may play important roles in the functionality of these enzymes. We explored the evolutionary patterns in each aaRS family and tracked remote evolutionary links between these families.

BACKGROUND: Migraine is a common disorder that often coexists with depression. While a functional polymorphism in methyleneterahydrofolate reductase gene (MTHFR C677T) has been implicated in depression; the evidence to support an association of MTHFR with migraine has been inconclusive. We aim to investigate the effect of this variant on propensity for migraine and to perform a systematic review and meta-analysis of studies of MTHFR and migraine to date.
METHODS: Individuals with migraine (n = 447) were selected from the Depression Case Control (DeCC) study to investigate the association between migraine and MTHFR C677T single nucleotide polymorphism (SNP) rs1801133 using an additive model compared to non-migraineurs adjusting for depression status. A meta-analysis was performed and included 15 studies of MTHFR and migraine.
RESULTS: MTHFR C677T polymorphism was associated with migraine with aura (MA) (OR 1.31, 95% CI 1.01-1.70, p = 0.039) that remained significant after adjusting for age, sex and depression status. A meta-analysis of 15 case-control studies showed that T allele homozygosity is significantly associated with MA (OR = 1.42; 95% CI, 1.10-1.82) and total migraine (OR = 1.37; 95% CI, 1.07-1.76), but not migraine without aura (OR = 1.16; 95% CI, 0.36-3.76). In studies of non-Caucasian population, the TT genotype was associated with total migraine (OR= 3.46; 95% CI, 1.22-9.82), whereas in studies of Caucasians this variant was associated with MA only (OR = 1.28; 95% CI, 1.002-1.63).
CONCLUSIONS: MTHFR C677T is associated with MA in individuals selected for depression study. A meta-analysis of 15 studies supports this association and demonstrated effects across ethnic groups.