EPHA2 polymorphisms and age-related cataract in India

Objective

We investigated whether previously reported single nucleotide polymorphisms (SNPs) of EPHA2 in European studies are associated with cataract in India.

Methods

We carried out a population-based genetic association study. We enumerated randomly sampled villages in two areas of north and south India to identify people aged 40 and over. Participants attended a clinical examination including lens photography and provided a blood sample for genotyping. Lens images were graded by the Lens Opacification Classification System (LOCS III). Cataract was defined as a LOCS III grade of nuclear ≥4, cortical ≥3, posterior sub-capsular (PSC) ≥2, or dense opacities or aphakia/pseudophakia in either eye. We genotyped SNPs rs3754334, rs7543472 and rs11260867 on genomic DNA extracted from peripheral blood leukocytes using TaqMan assays in an ABI 7900 real-time PCR. We used logistic regression with robust standard errors to examine the association between cataract and the EPHA2 SNPs, adjusting for age, sex and location.

Results

7418 participants had data on at least one of the SNPs investigated. Genotype frequencies of controls were in Hardy-Weinberg Equilibrium (p>0.05). There was no association of rs3754334 with cataract or type of cataract. Minor allele homozygous genotypes of rs7543472 and rs11260867 compared to the major homozygote genotype were associated with cortical cataract, Odds ratio (OR) = 1.8, 95% Confidence Interval (CI) (1.1, 3.1) p = 0.03 and 2.9 (1.2, 7.1) p = 0.01 respectively, and with PSC cataract, OR = 1.5 (1.1, 2.2) p = 0.02 and 1.8 (0.9, 3.6) p = 0.07 respectively. There was no consistent association of SNPs with nuclear cataract or a combined variable of any type of cataract including operated cataract.

Conclusions

Our results in the Indian population agree with previous studies of the association of EPHA2 variants with cortical cataracts. We report new findings for the association with PSC which is particularly prevalent in Indians.     

When maladaptive gene flow does not increase selection

Populations receiving high maladaptive gene flow are expected to experience strong directional selection—because gene flow pulls mean phenotypes away from local fitness peaks. We tested this prediction by means of a large and replicated mark‐recapture study of threespine stickleback (Gasterosteus aculeatus) in two stream populations. One of the populations (outlet) experiences high gene flow from the lake population and its morphology is correspondingly poorly adapted. The other population (inlet) experiences very low gene flow from the lake population and its morphology is correspondingly well adapted. Contrary to the above prediction, selection was not stronger in the outlet than in the inlet, a result that forced us to consider potential reasons for why maladaptive gene flow might not increase selection. Of particular interest, we show by means of a simple population genetic model that maladaptive gene flow can—under reasonable conditions—decrease the strength of directional selection. This outcome occurs when immigrants decrease mean fitness in the resident population, which decreases the strength of selection against maladapted phenotypes. We argue that this previously unrecognized effect of gene flow deserves further attention in theoretical and empirical studies.     

Optimization of a mouse recombinant antibody fragment for efficient production from Escherichia coli

A mutagenized mouse recombinant antibody fragment (rFab) that recognized HIV capsid protein was isolated from Escherichia coli at a level of 12 mg per liter of culture using standard shake flask methods. This is one of the highest yields of a modified antibody fragment obtained using non-fermentor-based methods. Recombinant Fab was isolated directly from the culture medium, which lacked complex materials such as tryptone and yeast extract. Fab isolated from the periplasm was not as homogeneous as that isolated directly from the culture medium. Optimization of the culture medium using recently developed media, the use of E. coli cell lines that contained rare tRNA codons, and mutagenesis of the Fab to improve the stability of the Fab were important factors in producing high-levels of the Fab. An isolation protocol easily adaptable to automation using a thiophilic-sepharose column followed by metal-chelate chromatography and the introduction of a non-traditional metal binding site for metal-chelate purification that bypasses the conventional hexahistidine tag cleavage step (to prevent the purification tag from interfering with crystallization) are additional features of this approach to produce a highly homogenous preparation of rFab. The resulting rFab binds to its antigen, p24, equivalent in character to the monoclonal from which the rFab was originally derived.     

Response surface optimization of the critical medium components for carbonyl reductase production by Candida viswanathii MTCC 5158

Culture conditions were optimized for the growth and carbonyl reductase production by a novel yeast strain Candida viswanathii. Response surface methodology was applied for the critical medium components (initial pH, mannitol, yeast extract and calcium chloride) identified earlier by one-factor-at-a-time approach. Central composite design was used for the optimization studies. Using this methodology, the optimal values for the concentration of mannitol, initial pH, yeast extract and calcium chloride were 1.9, 7.5, 1.6 and 4, respectively. This medium was projected to produce, theoretically, growth having an optical density of 1.1 (600 nm) and an enzyme activity of 81.5 U/ml. Using this optimized medium, an experimental growth of 1.1 OD (600 nm) and enzyme activity 80.9 U/ml verified the applied methodology. This approach for medium optimization led to an enhancement of the growth and enzyme activity by 1.3 and 2.3 times higher, respectively, as compared to the unoptimized media.     

The endogenous cellular protease inhibitor SPINT2 controls SARS-CoV-2 viral infection and is associated to disease severity

COVID-19 outbreak is the biggest threat to human health in recent history. Currently, there are over 1.5 million related deaths and 75 million people infected around the world (as of 22/12/2020). The identification of virulence factors which determine disease susceptibility and severity in different cell types remains an essential challenge. The serine protease TMPRSS2 has been shown to be important for S protein priming and viral entry, however, little is known about its regulation. SPINT2 is a member of the family of Kunitz type serine protease inhibitors and has been shown to inhibit TMPRSS2. Here, we explored the existence of a co-regulation between SPINT2/TMPRSS2 and found a tightly regulated protease/inhibitor expression balance across tissues. We found that SPINT2 negatively correlates with SARS-CoV-2 expression in Calu-3 and Caco-2 cell lines and was down-regulated in secretory cells from COVID-19 patients. We validated our findings using Calu-3 cell lines and observed a strong increase in viral load after SPINT2 knockdown, while overexpression lead to a drastic reduction of the viral load. Additionally, we evaluated the expression of SPINT2 in datasets from comorbid diseases using bulk and scRNA-seq data. We observed its down-regulation in colon, kidney and liver tumors as well as in alpha pancreatic islets cells from diabetes Type 2 patients, which could have implications for the observed comorbidities in COVID-19 patients suffering from chronic diseases.     

Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope–chromatin interactions

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin–nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.     

Heterologous expression in <Emphasis Type="Italic">Tritrichomonas foetus</Emphasis> of functional <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> AP65 adhesin

Background  

Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor.     

Targeting anti-aging protein sirtuin (Sirt) in the diagnosis of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a severe, incurable, age-associated respiratory disorder that has gained significance because of its unknown etiology and lack of therapeutic approaches. IPF causes maximum damage to the alveolar epithelial cells, thereby leading to lung remodeling and initiating epithelial to mesenchymal transition (EMT). The actual molecular mechanisms underlying IPF still remain unclear, and knowledge about these mechanisms would be helpful in its diagnosis. Sirtuins (Sirt) are class of NAD+-dependent proteins, widely known to exert positive and protective effects on age-related diseases such as diabetes, cancer, and so on, and are also involved in regulating IPF. The sirtuin family comprises of seven members (Sirt1 to Sirt7), out of which Sirt1, Sirt3, Sirt6, and Sirt7 exert positive effects on IPF. Sirt1 is associated with aging and inhibits cellular senescence and fibrosis. Sirt1 is well recognized in controlling pulmonary fibrosis and is also considered as a prime positive mediator of EMT. The expressions of Sirt3 protein tend to decline in IPF patients; hence it is known as an anti-fibrotic protein. Sirt6 indeed has been proven to reduce EMT during IPF. Decreased levels of Sirt7 during IPF regulate lung fibroblasts. Hence, active levels of Sirt1, Sirt3, Sirt6, and Sirt7 can be attractive target models to elucidate a novel potential therapeutic approach for IPF. In this prospect, we have discussed the role of Sirtuins in pulmonary fibrosis by exploring the recent research evidence that highlight the role of sirtuins and also describes their protective effects.