Subtractive hybridization used to identify mRNA associated with the maturation of bovine oocytes

The main objective of this project is to identify mRNA associated with oocyte maturation and embryonic developmental competency. The knowledge of genes and their accumulated mRNA is essential to better understand the mechanisms involved in the oocyte maturation and the survival of the in vitro produced embryo. We used bovine slaughterhouse-recovered ovaries and collected the oocytes from two follicle size categories: <2 mm and 3-5 mm. The mRNA content of oocytes from follicles 3-5 mm where considered to be more competent when compared to the content of oocytes from follicles <2 mm. In this report we compare two different technical approaches both involving PCR to compare the mRNA pools of the oocytes. In the first approach we performed the differential display (DDRT) technique to amplify and display side by side the cDNAs of groups of 10 denuded oocytes. From this approach, we isolated 28 different bands. After analysis, three of those bands had strong homology with known genes. In the second approach pools of 50 denuded oocytes were submitted to suppressive subtraction hybridization (SSH). We identified several known genes like cyclin B1, splicing factor ccl.4, cytochrome c oxidase, and mineralocorticoid receptor while numerous other clones remain unidentified. The cyclin B1 clone was used as a probe to evaluate its follicular size specificity on virtual Northern blot. The PCR basis of these techniques allows comparison of mRNA from tissues of low abundance such as oocytes. In this study the SSH resulted in longer clones than DDRT and showed high specificity.     

Polymorphic analysis of CYP2C9 gene in Vietnamese population

Molecular Biology Reports - Genetic variations in CYP2C9 are associated to inter-individual variability of drugs metabolism and response. The only report has been done previously mainly focusing on...     

Inside Front Cover: Near‐infrared bone densitometry: A feasibility study on distal radius measurement (J. Biophotonics 7/2018)

This study provides a simple method to detect human distal radius bone density based on near infrared (NIR) imaging. The information of bone mineral density can be measured by transluminational optical bone densitometric system. Compared to dual‐energy x‐ray absorptiometry (DXA) results in clinical trial, NIR images show a strong correlation to DXA. Further details can be found in the article by Chun Chung, Yu‐Pin Chen, Tsai‐Hsueh Leu, and Chia‐Wei Sun ( e201700342 ).

     

Near‐infrared bone densitometry: A feasibility study on distal radius measurement

Osteoporosis, defined as decreased bone mineral density (BMD), poses patients in dangers for fracture risk and has become a major public health problem worldwide because of is associated morbidity, mortality and costs. Without doubt, early detection and timely intervention are important to successfully manage osteoporosis and its associated complications. The dual‐energy x‐ray absorptiometry (DXA) is the most popular and standard method to measure BMD. However, limitations including radiation exposure and availability restrict its application for osteoporosis screening among general population. In this study, we developed a simple method to detect human distal radius bone density based on near infrared (NIR) image system. Among 10 volunteers (including 5 young and 5 elderly participants) receiving bone density measurement using our NIR image system at the ultradistal part of bilateral distal radius, we demonstrated a strong correlation between the optical attenuation and BMD measured with DXA, which may facilitate predicting bone density status. We hope our potential NIR image system may open a new avenue for development of osteoporosis screening facilities and help in prevention of osteoporosis related fracture and its associated complications in the near future. Pearson's correlations between BMD values from the DXA and light intensity of NIR system.      

Cell type‐specific regulation of ciliary transition zone assembly in vertebrates

Ciliopathies are life‐threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans. By performing quantitative immunofluorescence studies in RPGRIP1L^?/? mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type‐specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate‐specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.     

Rapid and dynamic arginylation of the leading edge β‐actin is required for cell migration

β‐actin plays key roles in cell migration. Our previous work demonstrated that β‐actin in migratory non‐muscle cells is N‐terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of β‐actin arginylation in cell migration. We found that arginylated β‐actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact‐inhibited cells in confluent cultures, suggesting that arginylated β‐actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact‐inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of β‐actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.      

Sildenafil improves long-term effect of endothelial progenitor cell-based treatment for monocrotaline-induced rat pulmonary arterial hypertension

Background aimsWe hypothesized that the long-term therapeutic effect of combined sildenafil and bone marrow-derived endothelial progenitor cells (BMDEPCs) on monocrotaline (MCT)-induced rat pulmonary arterial hypertension (PAH) is superior to either treatment alone.MethodsMale Sprague-Dawley rats (n = 40) were equally divided into normal controls, MCT (65 mg/kg, subcutaneously) only, MCT + sildenafil (25 mg/kg/day, orally), MCT + BMDEPCs (2.0 × 10⁶ autologous cells, intravenously) and MCT + sildenafil+ BMDEPCs. BMDEPCs and sildenafil were given on day 21 after MCT administration. Animals were sacrificed by day 90 after MCT administration.ResultsThe apoptotic (caspase 3, Bax) and inflammatory (tumor necrosis factor-α, matrix metalloproteinase-9) biomarkers in right ventricle and lung and pulmonary expressions of fibrotic biomarkers (transforming growth factor-β, p-Smad3) and connexin 43 protein were lower in monotherapy groups (i.e., MCT + sildenafil and MCT + BMDEPCs) and further decreased in normal controls and combined treatment groups (i.e., MCT + sildenafil + BMDEPCs) compared with untreated animals (i.e., MCT only) (all P < 0.01). Expressions of anti-fibrotic biomarkers (bone morphogenetic protein-2, p-Smad1/5) and numbers of alveolar sacs and arterioles in lung were higher in monotherapy groups and further increased in normal controls and combined treatment groups compared with untreated animals (all P < 0.005). In right ventricle, connexin 43 and α-myosin heavy chain (MHC) expressions were higher in the monotherapy groups and further elevated in normal controls and combined treatment groups compared with untreated animals, whereas β-MHC exhibited the opposite pattern (all P < 0.01). Right ventricular systolic pressure and weight were lower in the monotherapy animals and further reduced in normal controls and combined treatment groups compared with untreated animals (all P < 0.0001).ConclusionsCombined therapy with BMDEPCs and sildenafil was superior to either treatment alone in attenuating rodent MCT-induced PAH.     

Differential regulation of eEF2 and p70S6K by AMPKalpha2 in heart

Eukaryotic elongation factor 2 (eEF-2) and mammalian target of rapamycin (mTOR)–p70 ribosomal protein S6 kinase (p70S6K) signaling pathways control protein synthesis and are inhibited during myocardial ischemia. Intracellular acidosis and AMP-activated protein kinase (AMPK) activation, both occurring during ischemia, have been proposed to participate in this inhibition. We evaluated the contribution of AMPKα2, the main cardiac AMPK catalytic subunit isoform, in eEF2 and mTOR–p70S6K regulation using AMPKα2 KO mice. Hearts were perfused ex vivo with or without insulin, and then submitted or not to ischemia. Insulin pre-incubation was necessary to activate mTOR–p70S6K and evaluate their subsequent inhibition by ischemia. Ischemia decreased insulin-induced mTOR–p70S6K phosphorylation in WT and AMPKα2 KO mice to a similar extent. This AMPKα2-independent p70S6K inhibition correlated well with the inhibition of PKB/Akt, located upstream of mTOR–p70S6K and can be mimicked in cardiomyocytes by decreasing pH. By contrast, ischemia-induced inhibitory phosphorylation of eEF-2 was drastically reduced in AMPKα2 KO mice. Interestingly, AMPKα2 also played a role under normoxia. Its deletion increased the insulin-induced p70S6K stimulation. This p70S6K over-stimulation was associated with a decrease in inhibitory phosphorylation of Raptor, an mTOR partner identified as an AMPK target. In conclusion, AMPKα2 controls cardiac p70S6K under normoxia and regulates eEF-2 but not the mTOR–p70S6K pathway during ischemia. This challenges the accepted notion that mTOR–p70S6K is inhibited by myocardial ischemia mainly via an AMPK-dependent mechanism.     

Increased Circulating Endothelial Apoptotic Microparticle to Endothelial Progenitor Cell Ratio Is Associated with Subsequent Decline in Glomerular Filtration Rate in Hypertensive Patients

Background

Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs) and increased levels of endothelial apoptotic microparticles (EMP). However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR) remains unclear.

Methods and Results

We enrolled totally 100 hypertensive out-patients with eGFR ≥30 mL/min/1.73 m². The mean annual rate of GFR decline (△GFR/y) was −1.49±3.26 mL/min/1.73 m² per year during the follow-up period (34±6 months). Flow cytometry was used to assess circulating EPC (CD34⁺/KDR⁺) and EMP levels (CD31⁺/annexin V⁺) in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r = −0.465, p<0.001), microalbuminuria (r = −0.329, p = 0.001), and the Framingham risk score (r = −0.245, p = 0.013). When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08±3.04 vs. −0.50±2.84 vs. −1.25±2.49 vs. −4.42±2.82, p<0.001). Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y.

Conclusions

An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.