Premature aging syndrome in ataxia telangiectasia patients

Ataxia telangiectasia (AT) is a genetic disorder caused by the mutation of the atm gene. It is characterized by progressive neurological abnormalities in combination with oculocutaneous telangiectasias, immunodeficiency, and increased frequency of malignancy. Cells of AT patients display increased radiosensitivity and premature aging markers, including shortened telomer length beginning at birth and limited proliferation potential. We studied radiosensitivity (at a dose 2 Gy) and the manifestation of premature aging markers in cultured skin fibroblasts derived from two unrelated AT patients and their heterozygous parents. We have shown that all the markers studied, i.e., HP1-γ, histone H2AX phosphorylated for serine-139 (γ-H2AX) and foci of 53BP1 protein, indicate the premature aging of the cells of both patients and their blood relatives. However, cells of heterozygous carriers express premature aging to a lesser extent. A study of the repair process (the amount of γ-H2AX and the number of cells with 53BP1 foci in their nuclei) after X-ray irradiation showed that patients’ cells only halfway completed repairs, even 24 h after irradiation, while the healthy donor cells completed repairs in 24 h. In cells from atm heterozygous donors, DNA repair was also slower. Heterozygous cells also differ reliably from healthy donor cells. Only amounts of p21^Waf1/Cip1 protein, an inhibitor of cyclin-dependent kinases, in heterozygous cells do not differ from normal cells. However, the patients’ cells differ significantly. It was found that the mutation of the atm gene was related to the suppression of the reparation of DNA double-strand breaks (DSBs), which is in good agreement with increased radiosensitivity and premature aging in AT families at the cellular level.     

Fitness Impact and Stability of a Transgene Conferring Resistance to Dengue-2 Virus following Introgression into a Genetically Diverse Aedes aegypti Strain

In 2006, we reported a mariner (Mos1)-transformed Aedes aegypti line, Carb77, which was highly resistant to dengue-2 virus (DENV2). Carb77 mosquitoes expressed a DENV2-specific inverted-repeat (IR) RNA in midgut epithelial cells after ingesting an infectious bloodmeal. The IR-RNA formed double-stranded DENV2-derived RNA, initiating an intracellular antiviral RNA interference (RNAi) response. However, Carb77 mosquitoes stopped expressing the IR-RNA after 17 generations in culture and lost their DENV2-refractory phenotype. In the current study, we generated new transgenic lines having the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 for >33 generations. Southern blot analysis identified two transgene integration sites in Carb109M. Northern blot analysis detected abundant, transient expression of the IR-RNA 24 h after a bloodmeal. Carb109M mosquitoes were refractory to different DENV2 genotypes but not to other DENV serotypes. To further test fitness and stability, we introgressed the Carb109M transgene into a genetically diverse laboratory strain (GDLS) by backcrossing for five generations and selecting individuals expressing the transgene''s EGFP marker in each generation. Comparison of transgene stability in replicate backcross 5 (BC₅) lines versus BC₁ control lines demonstrated that backcrossing dramatically increased transgene stability. We subjected six BC₅ lines to five generations of selection based on EGFP marker expression to increase the frequency of the transgene prior to final family selection. Comparison of the observed transgene frequencies in the six replicate lines relative to expectations from Fisher''s selection model demonstrated lingering fitness costs associated with either the transgene or linked deleterious genes. Although minimal fitness loss (relative to GDLS) was manifest in the final family selection stage, we were able to select homozygotes for the transgene in one family, Carb109M/GDLS.BC₅.HZ. This family has been genetically stable and DENV2 refractory for multiple generations. Carb109M/GDLS.BC₅.HZ represents an important line for testing proof-of-principle vector population replacement.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Specific features of the apoptotic response of urinary bladder cancer cells to neoadjuvant chemotherapy

The TUNEL reaction was used to study induced apoptosis in the tumor cells of urinary bladder cancer (UBC) patients. It has been shown that an increase in the values of induced apoptosis correlates well with the indicators showing a tumor’s corresponding response to the application of neoadjuvant chemotherapy, using the gemcitabine-cisplatin regimen. It has been concluded that assessing the level of induced apoptosis in UBC tumor cells using the TUNEL assay enables making a prognosis for the efficacy of chemotherapy at the single-cell level in patients with this type of cancer.     

The central role of protein S12 in organizing the structure of the decoding site of the ribosome

The ribosome decodes mRNA by monitoring the geometry of codon–anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EF-Tu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.