Evaluating Purifying Selection in the Mitochondrial DNA of Various Mammalian Species

Mitochondrial DNA (mtDNA), the circular DNA molecule inside the mitochondria of all eukaryotic cells, has been shown to be under the effect of purifying selection in several species. Traditional testing of purifying selection has been based simply on ratios of nonsynonymous to synonymous mutations, without considering the relative age of each mutation, which can be determined by phylogenetic analysis of this non-recombining molecule. The incorporation of a mutation time-ordering from phylogeny and of predicted pathogenicity scores for nonsynonymous mutations allow a quantitative evaluation of the effects of purifying selection in human mtDNA. Here, by using this additional information, we show that purifying selection undoubtedly acts upon the mtDNA of other mammalian species/genera, namely Bos sp., Canis lupus, Mus musculus, Orcinus orca, Pan sp. and Sus scrofa. The effects of purifying selection were comparable in all species, leading to a significant major proportion of nonsynonymous variants with higher pathogenicity scores in the younger branches of the tree. We also derive recalibrated mutation rates for age estimates of ancestors of these various species and proposed a correction curve in order to take into account the effects of selection. Understanding this selection is fundamental to evolutionary studies and to the identification of deleterious mutations.     

Polyanionic Candidate Microbicides Accelerate the Formation of Semen-Derived Amyloid Fibrils to Enhance HIV-1 Infection

Polyanionic candidate microbicides, including cellulose sulfate, carrageenan, PRO 2000, were proven ineffective in preventing HIV-1 transmission and even cellulose sulfate showed increased risk of HIV acquisition in the Phase III efficacy trials. Semen plays critical roles in HIV-1 sexual transmission. Specifically, amyloid fibrils formed by fragments of prostatic acidic phosphatase (PAP) in semen termed semen-derived enhancer of virus infection (SEVI) could drastically enhance HIV-1 infection. Here we investigated the interaction between polyanions and PAP248-286, a prototype peptide of SEVI, to understand the possible cause of polyanionic candidate microbicides to fail in clinical trials. We found anionic polymers could efficiently promote SEVI fibril formation, most likely mediated by the natural electrostatic interaction between polyanions and PAP248-286, as revealed by acid native PAGE and Western blot. The overall anti-HIV-1 activity of polyanions in the presence or absence of PAP248-286 or semen was evaluated. In the viral infection assay, the supernatants of polyanions/PAP248-286 or polyanions/semen mixtures containing the free, unbound polyanionic molecules showed a general reduction in antiviral efficacy, while the pellets containing amyloid fibrils formed by the polyanion-bound PAP248-286 showed aggravated enhancement of viral infection. Collectively, from the point of drug-host protein interaction, our study revealed that polyanions facilitate SEVI fibril formation to promote HIV-1 infection, thus highlighting a molecular mechanism underlying the failure of polyanions in clinical trials and the importance of drug-semen interaction in evaluating the anti-HIV-1 efficacy of candidate microbicides.     

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.     

In Vitro Activity of Sodium New Houttuyfonate Alone and in Combination with Oxacillin or Netilmicin against Methicillin-Resistant Staphylococcus aureus

Background

Staphylococcus aureus can cause severe infections, including bacteremia and sepsis. The spread of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for novel treatment options. Sodium new houttuyfonate (SNH) is an analogue of houttuynin, the main antibacterial ingredient of Houttuynia cordata Thunb. The aim of this study was to evaluate in vitro activity of SNH and its potential for synergy with antibiotics against hospital-associated MRSA.

Methodology

A total of 103 MRSA clinical isolates recovered in two hospitals in Beijing were evaluated for susceptibility to SNH, oxacillin, cephalothin, meropenem, vancomycin, levofloxacin, minocycline, netilmicin, and trimethoprim/sulfamethoxazole by broth microdilution. Ten isolates were evaluated for potential for synergy between SNH and the antibiotics above by checkerboard assay. Time-kill analysis was performed in three isolates to characterize the kill kinetics of SNH alone and in combination with the antibiotics that engendered synergy in checkerboard assays. Besides, two reference strains were included in all assays.

Principal Findings

SNH inhibited all test strains with minimum inhibitory concentrations (MICs) ranging from 16 to 64 µg/mL in susceptibility tests, and displayed inhibition to bacterial growth in concentration-dependent manner in time-kill analysis. In synergy studies, the combinations of SNH-oxacillin, SNH-cephalothin, SNH-meropenem and SNH-netilmicin showed synergistic effects against 12 MRSA strains with median fractional inhibitory concentration (FIC) indices of 0.38, 0.38, 0.25 and 0.38 in checkerboard assays. In time-kill analysis, SNH at 1/2 MIC in combination with oxacillin at 1/128 to 1/64 MIC or netilmicin at 1/8 to 1/2 MIC decreased the viable colonies by ≥2log₁₀ CFU/mL.

Conclusions/Significance

SNH demonstrated in vitro antibacterial activity against 103 hospital-associated MRSA isolates. Combinations of sub-MIC levels of SNH and oxacillin or netilmicin significantly improved the in vitro antibacterial activity against MRSA compared with either drug alone. The SNH-based combinations showed promise in combating MRSA.     

The Metastasis Suppressor,N-myc Downregulated Gene 1 (NDRG1), Is a Prognostic Biomarker for Human Colorectal Cancer

Metastasis remains to be one of the most prevalent causes leading to poor long-term survival of colorectal cancer (CRC) patients. The clinical significances of tumor metastatic suppressor, N-myc downregulated gene 1 (NDRG1), have been inconsistently reported in a variety of cancerous diseases. In this study with 240 CRC clinical specimens, we showed that NDRG1 expression was significantly decreased in most of CRC tissues compared to the paired non-tumor counterparts. Statistical analysis revealed a significant inverse correlation of NDRG1 expression with tumor stage, differentiation status and metastasis. Compared with NDRG1-negative group, NDRG1-positve group had better disease-free/overall survival (p = 0.000) over 5 years’ follow-up. Furthermore, NDRG1 was considered to be an independent prognostic factor for overall survival (p = 0.001) and recurrence (p = 0.003). Our study concludes that NDRG1 is a novel favorable predictor for the prognosis in CRC patients.     

Quantitative Analysis of Burden of Infectious Diarrhea Associated with Floods in Northwest of Anhui Province,China: A Mixed Method Evaluation

Background

Persistent and heavy rainfall in the upper and middle Huaihe River of China brought about severe floods during the end of June and July 2007. However, there has been no assessment on the association between the floods and infectious diarrhea. This study aimed to quantify the impact of the floods in 2007 on the burden of disease due to infectious diarrhea in northwest of Anhui Province.

Methods

A time-stratified case-crossover analysis was firstly conducted to examine the relationship between daily cases of infectious diarrhea and the 2007 floods in Fuyang and Bozhou of Anhui Province. Odds ratios (ORs) of the flood risk were quantified by conditional logistic regression. The years lived with disability (YLDs) of infectious diarrhea attributable to floods were then estimated based on the WHO framework of the calculating potential impact fraction in the Burden of Disease study.

Results

A total of 197 infectious diarrheas were notified during the exposure and control periods in the two study areas. The strongest effect was shown with a 2-day lag in Fuyang and a 5-day lag in Bozhou. Multivariable analysis showed that floods were significantly associated with an increased risk of the number cases of infectious diarrhea (OR = 3.175, 95%CI: 1.126–8.954 in Fuyang; OR = 6.754, 95%CI: 1.954–23.344 in Bozhou). Attributable YLD per 1000 of infectious diarrhea resulting from the floods was 0.0081 in Fuyang and 0.0209 in Bozhou.

Conclusions

Our findings confirm that floods have significantly increased the risks of infectious diarrhea in the study areas. In addition, prolonged moderate flood may cause more burdens of infectious diarrheas than severe flood with a shorter duration. More attention should be paid to particular vulnerable groups, including younger children and elderly, in developing public health preparation and intervention programs. Findings have significant implications for developing strategies to prevent and reduce health impact of floods.     

High Beta-Palmitate Fat Controls the Intestinal Inflammatory Response and Limits Intestinal Damage in Mucin Muc2 Deficient Mice

Background

Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha’-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha’-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2^−/−) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer.

Methods

Muc2^−/− mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed.

Results

Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2^−/− mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis.

Conclusions

This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2^−/− mice by inducing an immunosuppressive Treg cell response.     

Genome-Wide Association Studies for Growth and Meat Production Traits in Sheep

Background

Growth and meat production traits are significant economic traits in sheep. The aim of the study is to identify candidate genes affecting growth and meat production traits at genome level with high throughput single nucleotide polymorphisms (SNP) genotyping technologies.

Methodology and Results

Using Illumina OvineSNP50 BeadChip, we performed a GWA study in 329 purebred sheep for 11 growth and meat production traits (birth weight, weaning weight, 6-month weight, eye muscle area, fat thickness, pre-weaning gain, post-weaning gain, daily weight gain, height at withers, chest girth, and shin circumference). After quality control, 319 sheep and 48,198 SNPs were analyzed by TASSEL program in a mixed linear model (MLM). 36 significant SNPs were identified for 7 traits, and 10 of them reached genome-wise significance level for post-weaning gain. Gene annotation was implemented with the latest sheep genome Ovis_aries_v3.1 (released October 2012). More than one-third SNPs (14 out of 36) were located within ovine genes, others were located close to ovine genes (878bp-398,165bp apart). The strongest new finding is 5 genes were thought to be the most crucial candidate genes associated with post-weaning gain: s58995.1 was located within the ovine genes MEF2B and RFXANK, OAR3_84073899.1, OAR3_115712045.1 and OAR9_91721507.1 were located within CAMKMT, TRHDE, and RIPK2 respectively. GRM1, POL, MBD5, UBR2, RPL7 and SMC2 were thought to be the important candidate genes affecting post-weaning gain too. Additionally, 25 genes at chromosome-wise significance level were also forecasted to be the promising genes that influencing sheep growth and meat production traits.

Conclusions

The results will contribute to the similar studies and facilitate the potential utilization of genes involved in growth and meat production traits in sheep in future.     

Sema6B,Sema6C,and Sema6D Expression and Function during Mammalian Retinal Development

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B^−/− or Sema6C^−/−; Sema6D^−/− retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization.     

Altered Pattern of Spontaneous Brain Activity in the Patients with End-Stage Renal Disease: A Resting-State Functional MRI Study with Regional Homogeneity Analysis

Purpose

To investigate the pattern of spontaneous neural activity in patients with end-stage renal disease (ESRD) with and without neurocognitive dysfunction using resting-state functional magnetic resonance imaging (rs-fMRI) with a regional homogeneity (ReHo) algorithm.

Materials and Methods

rs-fMRI data were acquired in 36 ESRD patients (minimal nephro-encephalopathy [MNE], n = 19, 13 male, 37±12.07 years; non-nephro-encephalopathy [non-NE], n = 17, 11 male, 38±12.13 years) and 20 healthy controls (13 male, 7 female, 36±10.27 years). Neuropsychological (number connection test type A [NCT-A], digit symbol test [DST]) and laboratory tests were performed in all patients. The Kendall''s coefficient of concordance (KCC) was used to measure the regional homogeneity for each subject. The regional homogeneity maps were compared using ANOVA tests among MNE, non-NE, and healthy control groups and post hoc t -tests between each pair in a voxel-wise way. A multiple regression analysis was performed to evaluate the relationships between ReHo index and NCT-A, DST scores, serum creatinine and urea levels, disease and dialysis duration.

Results

Compared with healthy controls, both MNE and non-NE patients showed decreased ReHo in the multiple areas of bilateral frontal, parietal and temporal lobes. Compared with the non-NE, MNE patients showed decreased ReHo in the right inferior parietal lobe (IPL), medial frontal cortex (MFC) and left precuneus (PCu). The NCT-A scores and serum urea levels of ESRD patients negatively correlated with ReHo values in the frontal and parietal lobes, while DST scores positively correlated with ReHo values in the bilateral PCC/precuneus, MFC and inferior parietal lobe (IPL) (all P<0.05, AlphaSim corrected). No significant correlations were found between any regional ReHo values and disease duration, dialysis duration and serum creatinine values in ESRD patients (all P>0.05, AlphaSim corrected).

Conclusion

Diffused decreased ReHo values were found in both MNE and non-NE patients. The progressively decreased ReHo in the default mode network (DMN), frontal and parietal lobes might be trait-related in MNE. The ReHo analysis may be potentially valuable for elucidating neurocognitive abnormalities of ESRD patients and detecting the development from non-NE to MNE.