Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.     

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class “S1.2 Other Anabolic Agents” of the list of prohibited substances of the World Anti-Doping Agency.     

In silico characterization of a novel pathogenic deletion mutation identified in XPA gene in a Pakistani family with severe xeroderma pigmentosum

Background

Xeroderma Pigmentosum (XP) is a rare skin disorder characterized by skin hypersensitivity to sunlight and abnormal pigmentation. The aim of this study was to investigate the genetic cause of a severe XP phenotype in a consanguineous Pakistani family and in silico characterization of any identified disease-associated mutation.

Results

The XP complementation group was assigned by genotyping of family for known XP loci. Genotyping data mapped the family to complementation group A locus, involving XPA gene. Mutation analysis of the candidate XP gene by DNA sequencing revealed a novel deletion mutation (c.654del A) in exon 5 of XPA gene. The c.654del A, causes frameshift, which pre-maturely terminates protein and result into a truncated product of 222 amino acid (aa) residues instead of 273 (p.Lys218AsnfsX5). In silico tools were applied to study the likelihood of changes in structural motifs and thus interaction of mutated protein with binding partners. In silico analysis of mutant protein sequence, predicted to affect the aa residue which attains coiled coil structure. The coiled coil structure has an important role in key cellular interactions, especially with DNA damage-binding protein 2 (DDB2), which has important role in DDB-mediated nucleotide excision repair (NER) system.

Conclusions

Our findings support the fact of genetic and clinical heterogeneity in XP. The study also predicts the critical role of DDB2 binding region of XPA protein in NER pathway and opens an avenue for further research to study the functional role of the mutated protein domain.     

Quantitative,high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans

Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypomethylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period.Key words: cord blood, birth weight, folic acid, homocysteine, BeadArray, hierarchical clustering, Illumina     

CORE: a phylogenetically-curated 16S rDNA database of the core oral microbiome

Comparing bacterial 16S rDNA sequences to GenBank and other large public databases via BLAST often provides results of little use for identification and taxonomic assignment of the organisms of interest. The human microbiome, and in particular the oral microbiome, includes many taxa, and accurate identification of sequence data is essential for studies of these communities. For this purpose, a phylogenetically curated 16S rDNA database of the core oral microbiome, CORE, was developed. The goal was to include a comprehensive and minimally redundant representation of the bacteria that regularly reside in the human oral cavity with computationally robust classification at the level of species and genus. Clades of cultivated and uncultivated taxa were formed based on sequence analyses using multiple criteria, including maximum-likelihood-based topology and bootstrap support, genetic distance, and previous naming. A number of classification inconsistencies for previously named species, especially at the level of genus, were resolved. The performance of the CORE database for identifying clinical sequences was compared to that of three publicly available databases, GenBank nr/nt, RDP and HOMD, using a set of sequencing reads that had not been used in creation of the database. CORE offered improved performance compared to other public databases for identification of human oral bacterial 16S sequences by a number of criteria. In addition, the CORE database and phylogenetic tree provide a framework for measures of community divergence, and the focused size of the database offers advantages of efficiency for BLAST searching of large datasets. The CORE database is available as a searchable interface and for download at http://microbiome.osu.edu.     

Cigarette Smoking and Risk of Breast Cancer in a New Zealand Multi-Ethnic Case-Control Study

Background

The association between breast cancer and tobacco smoke is currently unclear. The aim of this study was to assess the effect of smoking behaviours on the risk of breast cancer among three ethnic groups of New Zealand women.

Methods

A population-based case-control study was conducted including breast cancer cases registered on the New Zealand Cancer Registry between 2005 and 2007. Controls were matched by ethnicity and 5-year age-group. Logistic regression was used to estimate the association between breast cancer and smoking at different time points across the lifecourse, for each ethnic group. Estimated odds ratios (OR) were adjusted for established risk factors.

Results

The study comprised 1,799 cases (302 Māori, 70 Pacific, 1,427 non-Māori/non-Pacific) and 2,540 controls (746 Māori, 191 Pacific, 1,603 non-Māori/non-Pacific). There was no clear association between smoking and breast cancer for non-Māori/non-Pacific women, although non-Māori/non-Pacific ex-smokers had statistically significant increased risk of breast cancer when smoking duration was 20 years or more, and this remained significant in the fully adjusted model (OR 1.31, 95% CI 1.03 to 1.66). Māori showed more consistent increased risk of breast cancer with increasing duration among current smokers (<20 years OR 1.61, 95% CI 0.55 to 4.74; 20+ years OR 2.03, 95% CI 1.29 to 3.22). There was a clear pattern of shorter duration since smoking cessation being associated with increased likelihood of breast cancer, and this was apparent for all ethnic groups.

Conclusion

There was no clear pattern for cigarette smoking and breast cancer incidence in non-Māori/non-Pacific women, but increased risks were observed for Māori and Pacific women. These findings suggest that lowering the prevalence of smoking, especially among Māori and Pacific women, could be important for reducing breast cancer incidence.     

Effects of morning vs. evening combined strength and endurance training on physical performance,sleep and well-being

The aim of the present study was to examine how combined strength and endurance training in the morning and evening influences the adaptations in strength and endurance performance, perception of time management, psychological well-being and sleep. The combined training period lasted for 24 weeks and the participants were divided into the morning training (MG, n = 18), evening training (EG, n = 24) and control groups (CG, n = 10). Isometric leg press force (iLP), maximal oxygen consumption (VO₂max), sleep behavior, fatigue, time management, motivation, self-esteem and health-related quality of life (HRQoL) were assessed. Morning to evening difference in iLP was observed in both MG and EG at Pre and Post, with higher force values in the evening, but not for VO₂max. iLP force increased significantly in EG in the morning (p < 0.001) and evening (p = 0.010). VO₂max increased in MG and EG both in the morning (both p < 0.001) and in the evening (MG: p < 0.001; EG: p = 0.003). Participants of the present study slept 7–8 h per night and the self-reported sleep duration, get-up time and the average time to go to bed were similar between the groups and did not change from Pre to Post. From HRQoL dimensions, the score for bodily pain decreased in MG (p = 0.029) and significant between-group differences were observed for Pre-Post changes in MG and EG (p = 0.001) as well as between MG and CG (p < 0.001). In vitality, a significant between-group difference was observed for Pre to Post changes in MG and EG (p = 0.014). Perception of time management decreased in EG (p = 0.042) but stayed unchanged for MG and CG. For the intrinsic motivation to participate, significant between-group differences were observed for MG and EG (p = 0.033) and between MG and CG (p = 0.032) for Pre to Post changes. Self-esteem improved in MG (p = 0.029) and EG (p = 0.024). The present combined strength and endurance training program performed in the morning and in the evening led to similar improvements in strength and endurance performance. Training in the morning or in the evening did not disrupt the already good sleep behavior and it was able to further increase the self-esteem. Although training in the morning hours may leave more time for free time activities or social life (i.e. family and friends) compared to the evening training, it might be more challenging to stay motivated to participate in prolonged training programs in the morning hours.