A novel matrix derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed immobilized-metal affinity chromatography

Agarose coated gigaporous polystyrene microspheres were evaluated as a novel matrix for immobilized-metal affinity chromatography (IMAC). With four steps, nickel ions were successfully immobilized on the microspheres. The gigaporous structure and chromatographic properties of IMAC medium were characterized. A column packed with the matrix showed low column backpressure and high column efficiency at high flow velocity. Furthermore, this matrix was used for purifying superoxide dismutase (SOD), which was expressed in Escherichia coli (E. coli) in submerged fermentation, on an Äkta purifier 100 system under different flow velocities. The purity of the SOD from this one-step purification was 79% and the recovery yield was about 89.6% under the superficial flow velocity of 3251 cm/h. In conclusion, all the results suggested that the gigaporous matrix has considerable advantages for high-speed immobilized-metal affinity chromatography.     

Comparative Effects of Hispidulin,Genistein, and Icariin with Estrogen on Bone Tissue in Ovariectomized Rats

Icariin, Genistein, and Hispidulin have been proven to have estrogen-like and antiosteoporotic activity and can be potentially used for the treatment of osteoporosis. The present study found that Icariin, Genistein, and Hispidulin treatments, emulating estrogen, significantly contributed to bone density. Comparative effects of Icariin, Genistein, and Hispidulin with estrogen on in ovariectomized rats were investigated. Our results showed that genistein was found to have superior bone protective effects against osteoporosis among genistein, Icariin, and Hispidulin.     

The molecular basis of spinocerebellar ataxia type 48 caused by a de novo mutation in the ubiquitin ligase CHIP

The spinocerebellar ataxias (SCAs) are a class of incurable diseases characterized by degeneration of the cerebellum that results in movement disorder. Recently, a new heritable form of SCA, spinocerebellar ataxia type 48 (SCA48), was attributed to dominant mutations in STIP1 homology and U box-containing 1 (STUB1); however, little is known about how these mutations cause SCA48. STUB1 encodes for the protein C terminus of Hsc70 interacting protein (CHIP), an E3 ubiquitin ligase. CHIP is known to regulate proteostasis by recruiting chaperones via a N-terminal tetratricopeptide repeat domain and recruiting E2 ubiquitin-conjugating enzymes via a C-terminal U-box domain. These interactions allow CHIP to mediate the ubiquitination of chaperone-bound, misfolded proteins to promote their degradation via the proteasome. Here we have identified a novel, de novo mutation in STUB1 in a patient with SCA48 encoding for an A52G point mutation in the tetratricopeptide repeat domain of CHIP. Utilizing an array of biophysical, biochemical, and cellular assays, we demonstrate that the CHIP^A52G point mutant retains E3-ligase activity but has decreased affinity for chaperones. We further show that this mutant decreases cellular fitness in response to certain cellular stressors and induces neurodegeneration in a transgenic Caenorhabditis elegans model of SCA48. Together, our data identify the A52G mutant as a cause of SCA48 and provide molecular insight into how mutations in STUB1 cause SCA48.     

Ion-current-based Proteomic Profiling of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome

Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ∼40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS.Smith-Lemli-Opitz syndrome (SLOS)¹ is an autosomal recessive disorder associated with subnormal growth and failure to thrive, mental retardation and neurosensory deficits, and multiple congenital anomalies, including dysmorphologies (1, 2). Early epidemiological studies estimated the incidence of SLOS as 1 in 20,000 to 1 in 60,000 live births, primarily among Caucasians (1, 2). However, more recent studies suggest that the SLOS carrier frequency is ∼1 in 30 to 1 in 50; this predicts a much higher actual disease frequency, ranging from 1 in 1,590 to 1 in 17,000 (3, 4), making SLOS the fourth most common autosomal recessive human disease (after cystic fibrosis, phenylketonuria, and hemochromatosis). Mutation of the DHCR7 gene is the intrinsic cause of SLOS; this gene encodes the enzyme DHCR7 (3β-hydroxysterol-Δ7-reductase, a.k.a. 7-dehydrocholesterol reductase; EC1.3.1.21), which catalyzes the final step in the cholesterol biosynthetic pathway, reducing the Δ7 double bond and thus converting 7-dehydrocholesterol (7DHC) to cholesterol (4, 5). As a consequence, markedly reduced levels of cholesterol and aberrantly elevated levels of the cholesterol precursor 7DHC (and its epimer, 8DHC) are observed in the majority of affected SLOS patients (6, 7). Therefore, the clinical suspicion of SLOS is confirmed by elevated 7DHC in plasma or tissues, typically demonstrated via chromatographic methods (e.g. HPLC or GC/MS) (8, 9).Visual capacity may become compromised in SLOS patients because of a variety of congenital or postnatal pathologies, such as cataracts, aniridia, corneal endothelium defects, sclerocornea, electrophysiological defects in the retina, optic nerve abnormalities, or other ophthalmologic problems (10, 11). We currently lack full knowledge of the exact pathobiological mechanism underlying SLOS, but additional insights may be afforded by studies employing a rodent model of the disease in which rats are treated with AY9944 (trans-1,4-bis[2-chlorobenzylaminomethyl] cyclohexane dihydrochloride), a relatively selective inhibitor of DHCR7 (12–14). We previously described progressive retinal degeneration in this rat model of SLOS, which is characterized by the shortening of retinal rod outer segments, pyknosis and thinning of the outer nuclear layer (ONL) of the retina (which contains the photoreceptor nuclei), and accumulation of membranous/lipid inclusions in the retinal pigment epithelium (RPE) (12, 13). Reduced rod outer segment membrane fluidity, primarily caused by a dramatic (30 to 40 mol%) decline in docosahexaenoic acid (22:6, n3) levels relative to age-matched controls, also was observed in the SLOS rat model by three postnatal months (15, 16). Retinal function and sterol steady-state in the same rat model of SLOS can be partially rescued using a high-cholesterol diet (2% by weight), although histological degeneration of the retina still occurs (17). However, the molecular mechanisms that underlie the observed electrophysiological defects in the retina, the accumulation of membranous/lipid inclusions in the RPE, the shortening of retinal rod outer segments, and the initiation of ONL pyknosis in the SLOS rat model remain poorly understood. Therefore, a comprehensive profiling of the retinal proteomes of AY9944-treated versus age-matched untreated control rats may contribute to further understanding of the underlying mechanisms responsible for the retinopathy associated with the SLOS model and, by extension, the human disease.Nevertheless, extensive and reliable expression profiling of the retinal proteome remains a prominent challenge, owing to the need to quantify data from multiple animals and a high percentage of integral membrane and membrane-associated proteins (18, 19). Label-free approaches can compare multiple replicates (20–22) with quantitative accuracy comparable to that attained with stable isotope-labeling methods (23–25). However, in order to achieve reliable relative quantification, highly quantitative and reproducible sample preparation and LC/MS analysis are required for relatively large-scale sample cohorts.In the present study, we performed a reproducible, well-controlled, ion-current-based comparative proteomic analysis of the retinas from AY9944-treated versus age/sex-matched control rats (n = 5 animals per group). A high-concentration detergent mixture was used for the efficient extraction of proteins from retinas, and samples then underwent a reproducible precipitation/on-pellet-digestion procedure and long-column, 7-h nano-LC-MS analysis. These approaches ensured extensive comparative analysis of retina samples with 10 animals. The preparative and analytical procedures were carefully optimized and controlled to ensure optimal reproducibility. Two label-free approaches, the ion-current-based method and a spectral counting method, were compared in parallel. The altered proteins were subjected to functional annotation, and selected groups of proteins of interest were further validated by means of Western blot and correlative immunohistochemical analysis.     

Residential Exposure to 50 Hz Magnetic Fields and the Association with Miscarriage Risk: A 2-Year Prospective Cohort Study

Objective

The hypothesis of whether exposure to extremely low-frequency magnetic fields (ELF-MF) may increase miscarriage risk is controversial. A 2-year prospective cohort study was designed to study the association between exposure to 50 Hz magnetic fields (MF) and the miscarriage risk for women residing in the area of the Pearl-River Delta of China.

Method

Two towns with densely distributed power supply constructions were selected as the study sites. From 2010 to 2012, 552 women in the region who were at approximately 8 weeks of gestation or who planned to have a baby within 1 year were selected as candidate subjects. Exposure to MF was estimated by measurements at their front doors and in the alley in front of the subjects’ houses. The average exposure level was used as a cutoff point to define the exposed group. Clinical miscarriage was diagnosed by local obstetricians. Staffs from the local population and family planning service stations were responsible for the follow-up interviews every 2 months.

Results

Four hundred and thirteen pregnant women were selected for the cohort study. The average residential exposure to MF was 0.099 µT. No significantly increased risk of miscarriage was found to be associated with the average front-door exposure (p>0.05). However, miscarriage risk was found to be significantly associated with maximum alley exposure (p=0.001). The relative risk (RR) of miscarriage from maximum alley exposure was 2.35 (95% C.I.: 1.18-4.71). In addition, Cox regression analysis showed that the adjusted hazard ratio of maximum alley exposure for miscarriage was 1.72 (95% C.I.:1.10-2.69).

Conclusion

Although the miscarriage incidence was shown to be positively associated with the maximum alley MF exposure, the association between miscarriage risk and the exposure to MF was not confirmed in the study. The results of this study are of interest concerning MF exposure assessment and pregnancy outcomes.     

Life history of the plateau pika (Ochotona curzoniae) in alpine meadows of the Tibetan Plateau

The life history of a species is a result of natural selection and reflects how the species is adapted to its environment. Knowledge of life history is crucial for further ecological studies and conservation management. This paper presents aspects of the life history of the plateau pika (Ochotona curzoniae), a small mammal native to alpine meadows of the Tibetan Plateau. The mean lifespan of juveniles from first litters was longer than the mean lifespan of juveniles from second litters. The population consisted of more juveniles than adults (over-wintered animals) in August and these juveniles came primarily from the first litter of the year. The sex ratio of juveniles was female-biased even though the sex ratio of adults did not differ from 1:1. The mortality rate of juveniles and adults during the warm season (May–August) was greater than the mortality rate of these groups during the cold season (September–April). Mean juvenile growth rate during the warm season was 1.4 g/d and the growth rate of the first litters was remarkably slower than that of the second litters.     

RNA-Seq analysis of compatible and incompatible styles of Pyrus species at the beginning of pollination

Plant Molecular Biology - At the early stage of pollination, the difference in gene expression between compatibility and incompatibility is highly significant about the pollen-specific expression...     

Exome sequencing reveals CCDC111 mutation associated with high myopia

Myopia is a refractive error of the eye that is prevalent worldwide. The most extreme form, high myopia, is usually associated with other ocular disorders such as retinal detachment, macular degeneration, cataract, and glaucoma, and is one of leading causes of blindness. The etiology is complex and has not been fully elucidated. In this study, we identified a novel missense variant of the CCDC111 gene (NM_152683.2: c.265T > G; p.Y89D) in a high myopia family by exome sequencing. The variant was identified in 4 patients from an additional 270 sporadic high myopia patients, but not found in 270 controls. The amino acid is highly conserved across species, and variants giving rise to amino acid substitutions are predicted to be functionally damaging. The CCDC111 gene was ubiquitously expressed in primary cell cultures from human eye tissue, including corneal epithelial cells, choroidal melanoma cells, scleral fibroblasts, retinal epithelial cells, retinal Müller cells, and lens capsule epithelial cells. In summary, our results suggested that the CCDC111 may be a susceptibility gene for high myopia.