Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis

Signalling by the GTPase RhoA, a key regulator of epithelial cell behaviour, can stimulate opposing processes: RhoA can promote junction formation and apical constriction, and reduce adhesion and cell spreading. Molecular mechanisms are thus required that ensure spatially restricted and process-specific RhoA activation. For many fundamental processes, including assembly of the epithelial junctional complex, such mechanisms are still unknown. Here we show that p114RhoGEF is a junction-associated protein that drives RhoA signalling at the junctional complex and regulates tight-junction assembly and epithelial morphogenesis. p114RhoGEF is required for RhoA activation at cell-cell junctions, and its depletion stimulates non-junctional Rho signalling and induction of myosin phosphorylation along the basal domain. Depletion of GEF-H1, a RhoA activator inhibited by junctional recruitment, does not reduce junction-associated RhoA activation. p114RhoGEF associates with a complex containing myosin II, Rock II and the junctional adaptor cingulin, indicating that p114RhoGEF is a component of a junction-associated Rho signalling module that drives spatially restricted activation of RhoA to regulate junction formation and epithelial morphogenesis.     

Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.     

Nocturnal hypoxia and loss of kidney function

Background

Although obstructive sleep apnea (OSA) is more common in patients with kidney disease, whether nocturnal hypoxia affects kidney function is unknown.

Methods

We studied all adult subjects referred for diagnostic testing of sleep apnea between July 2005 and December 31 2007 who had serial measurement of their kidney function. Nocturnal hypoxia was defined as oxygen saturation (SaO2) below 90% for ≥12% of the nocturnal monitoring time. The primary outcome, accelerated loss of kidney function, was defined as a decline in estimated glomerular filtration rate (eGFR) ≥4 ml/min/1.73 m² per year.

Results

858 participants were included and followed for a mean study period of 2.1 years. Overall 374 (44%) had nocturnal hypoxia, and 49 (5.7%) had accelerated loss of kidney function. Compared to controls without hypoxia, patients with nocturnal hypoxia had a significant increase in the adjusted risk of accelerated kidney function loss (odds ratio (OR) 2.89, 95% confidence interval [CI] 1.25, 6.67).

Conclusion

Nocturnal hypoxia was independently associated with an increased risk of accelerated kidney function loss. Further studies are required to determine whether treatment and correction of nocturnal hypoxia reduces loss of kidney function.     

Influenza vaccination for immunocompromised patients: systematic review and meta-analysis from a public health policy perspective

Background

Immunocompromised patients are vulnerable to severe or complicated influenza infection. Vaccination is widely recommended for this group. This systematic review and meta-analysis assesses influenza vaccination for immunocompromised patients in terms of preventing influenza-like illness and laboratory confirmed influenza, serological response and adverse events.

Methodology/Principal Findings

Electronic databases and grey literature were searched and records were screened against eligibility criteria. Data extraction and risk of bias assessments were performed in duplicate. Results were synthesised narratively and meta-analyses were conducted where feasible. Heterogeneity was assessed using I² and publication bias was assessed using Begg''s funnel plot and Egger''s regression test. Many of the 209 eligible studies included an unclear or high risk of bias. Meta-analyses showed a significant effect of preventing influenza-like illness (odds ratio [OR] = 0.23; 95% confidence interval [CI] = 0.16–0.34; p<0.001) and laboratory confirmed influenza infection (OR = 0.15; 95% CI = 0.03–0.63; p = 0.01) through vaccinating immunocompromised patie nts compared to placebo or unvaccinated controls. We found no difference in the odds of influenza-like illness compared to vaccinated immunocompetent controls. The pooled odds of seroconversion were lower in vaccinated patients compared to immunocompetent controls for seasonal influenza A(H1N1), A(H3N2) and B. A similar trend was identified for seroprotection. Meta-analyses of seroconversion showed higher odds in vaccinated patients compared to placebo or unvaccinated controls, although this reached significance for influenza B only. Publication bias was not detected and narrative synthesis supported our findings. No consistent evidence of safety concerns was identified.

Conclusions/Significance

Infection prevention and control strategies should recommend vaccinating immunocompromised patients. Potential for bias and confounding and the presence of heterogeneity mean the evidence reviewed is generally weak, although the directions of effects are consistent. Areas for further research are identified.     

Kinetic Changes of Ptdins (3,4,5) P3 within Fast and Slow Turnover Rates of Focal Adhesion

Background:The assembly and disassembly of the focal adhesions (FA) components occurs throughout life cycle of adhesion, with conservation of balance between removal and recruitment rate during temporal stages. Previous studies have demonstrated that phosphotidyilinositols play a role in regulating FA turnover. However, a little attention has been given to quantify the dynamics changes of Phosphatidylinositol 3,4,5-trisphosphate (PtdIns (3,4,5) P3) within and during fast and slow turnover rates of FA.Methods:In this study, we developed a protein purification MDA-MB-231 breast cancer cell line was used as a model in this study due to high metastatic and motile. These cells were co-transfected with GFP- paxillin/vinculin, as FA marker, and the GFP/mCherry-Btk-PH, as a biosensor to visualize PtdIns (3,4,5) P3. Confocal time-lapse images were used to monitor changes or differences in the local generation of PtdIns (3,4,5) P3 within and during assembly and disassembly of FA. Following transfection, immunostaining was used to examine the spatial co-localization between FA and PtdIns (3,4,5) P3.Results:Our data demonstrated that PtdIns (3,4,5) P3 co-localized with FAs and increase during assembly and decline during disassembly of FA which exhibits slow turnover rates and was in a constant level during assembly and disassembly of FA that displays fast turnover rates.DiscussionOur result suggested that the dynamic changes of PtdIns (3,4,5) P3, it may depend on components undergo turnover, such that early, nascent FA displays fast turnover rates and mature FA exhibits slow turnover rates. Thus, the local enrichment of PtdIns (3,4,5) P3 enhances FA assembly and disassembly activation.Key Words: Cancer cell migration, Focal adhesion turnover, MDA-MB-231 cell line, PtdIns (3,4,5) P3     

Virtual assessment,training, and evaluation of clinical laboratory technologists amidst peak Covid-19 pandemic: An observational study

Medical technologists are considered a neglected group when it comes to academic interventions. We developed and implemented an educational intervention and assessment for the technologists based on an online questionnaire as a pre-test consisting of questions related to knowledge (n=5), attitude (n=3), and practices (n=4) of daily internal quality control (QC) monitoring via Google Docs survey tool. This study served multiple purposes. It allowed keeping the technologists engaged during the peak of the COVID-19 pandemic while also improving the knowledge, attitude, and practices about the internal quality control using Bio-Rad Unity Real Time (URT) QC software. Subjects were graded based on the scores they received out of 100 (0-60 = poor; 61-79 = good; 80-100 = excellent). Training materials, i.e., a set of 5 videos every week via e-mail, were circulated. A voice-over PowerPoint presentation was also shared for easy comprehension. This activity was repeated after one month. A post-test was administered to assess the improvement. The study results show significant improvement in the technologists'' performance after the intervention.     

Influence of Androgenicity on Adipocytes and Precursor Cells in Female Rats

We examined the effects of overexposure of testosterone (T) on fat cell morphology and adipocyte precursor pools in inguinal and retroperitoneal fat depots of ovariectomized rats. In both tissues peripubertal T decreased weights without affecting adipocyte mean cell size or the size distribution profiles, but adipocyte number was decreased by 65% in the inguinal and by 38% in the retroperitoneal depots. Immunofluorescent flow cytometry utilizing a specific antibody to rat adipose tissue lipoprotein lipase was used to quantify regional precursor cell populations. T sharply reduced the percentages of differentiated and undifferentiated preadipocytes in the inguinal depot, from 43.2 ± 5.3 to 23.5 ± 2.1% and from 57.7 ± 4.0 to 43.6 ± 5.3%, respectively, with a concomitant increase in fibroblasts from 1.6 to 32.9%. On the other hand, T had no effect on retroperitoneal preadipocyte pools. Perinatal andro-genization exacerbated the decline in the inguinal weight (1.4 ± 0.1 vs. 2.2 ± 0.1g) but otherwise did not influence the actions of peripubertal T. Androgens may thus act in a tissue-specific manner to regulate fat cell growth potential in the femoral region in the female.     

Field evaluation of transgenic wheat plants stably expressing the HVA1 gene for drought tolerance

The barley HVA1 gene, encoding a member of the group 3 late embryogenesis abundant (LEA) proteins, has previously been introduced into spring wheat cv. Hi-Line to determine its effect on drought tolerance (Sivamani E, Bahieldin A, Wraith JM, Al-Niemi T, Dyer WE, Ho T-HD, Qu R (2000) Improved biomass productivity and water use efficiency under water deficit conditions in transgenic wheat constitutively expressing the barley HVA1 gene. Plant Sci 155, 1–9). T4 progeny from six independent transgenic events (lines 111/1, 1/1, 11/2, 84, 765 and 1201) were tested in nine field experiments over six cropping seasons. In the first two seasons, the total biomass per plot and the grain yield per plot of line 111/1 were higher than those of line 1/1, and higher than those of the wild-type control in the second season. The grain yield per plot of line 11/2 was significantly lower than that of the transgenic lines 111/1 and 1/1 in the third season, and this line was not tested further. In the fourth season, the plant height and grain yield per plot of line 111/1 were significantly higher than those of the wild-type control. Under dryland conditions in the fifth season, line 111/1 showed significantly greater plant height, total biomass per plot and grain yield per plot than the wild-type control in at least two of the four locations, as well as across locations. In the sixth season, newly developed transgenic lines 1201 and 765 significantly overyielded the two original transgenic lines 111/1 and 1/1, the non-expressing transgenic line 84 as well as the wild-type control in the three yield attributes and leaf water measurement, namely relative water content (RWC). This result coincided with the rate of HVA1 transgene expression of the different genotypes. Differences in total seed storage protein concentrations between the transgenic lines and the wild-type control within or across environmental conditions were insignificant. These field trials show that the HVA1 gene has the potential to confer drought stress protection in transgenic spring wheat.     

Female preponderance in congenital adrenal hyperplasia due to CYP21 deficiency in England: implications for neonatal screening

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase (CYP21) deficiency causes symptoms ranging from life-threatening neonatal adrenal crises to minimal virilization in adulthood. The relationship between CYP21 genotype and phenotypic markers in a non-screened population of 73 CAH children (44 female, 29 male; 54 white, 19 Asian) treated at the Royal Manchester Children's Hospital was investigated and ethnic and sex differences assessed. The patients were categorized according to the mutation on the mildest allele. The age at the time of diagnosis differed significantly between the groups (p = 0.02): all 25 Null and 25 of 26 of the I2 splice patients were diagnosed during the neonatal period, whereas 7 of 11 I172N patients were diagnosed late. Degree of female genital virilization, 17-hydroxyprogesterone level at diagnosis, and fludrocortisone requirement during the 1st year of treatment correlated with the genotype, although Asian Null patients required more fludrocortisone than their white counterparts (p = 0.055). There was an equal sex ratio in both the I2 splice (12 female/14 male) and I172N (5 female/6 male) groups. However, in the Null group, the ratio was 4.0 (20 female/5 male; p = 0.003), suggesting that some Null male infants perish before being clinically detected to have CYP21 deficiency. Our findings strongly support the need for implementation of a neonatal screening programme for CAH in the UK which may reduce the male infant mortality.     

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.