Double Variant Research Articles

HFE gene mutation and oxidative damage biomarkers in patients with myelodysplastic syndromes and its relation to transfusional iron overload: an observational cross-sectional study

ObjectiveA relation between transfusional IOL (iron overload), HFE status and oxidative damage was evaluated.Design, setting and participantsAn observational cross-sectional study involving 87 healthy individuals and 78 patients with myelodysplastic syndromes...

Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2.

Facioscapulohumeral muscular dystrophy (FSHD) predominantly affects the muscles in the face, trunk and upper extremities and is marked by large clinical variability in disease onset and progression. FSHD is associated with partial chromatin relaxation of the D4Z4 repeat array on chromosome 4 and the somatic expression of the D4Z4 encoded DUX4 gene. The most common form, FSHD1, is caused by a contraction of the D4Z4 repeat array on chromosome 4 to a size of 1-10 units. FSHD2, the less common form of FSHD, is most often caused by heterozygous variants in the chromatin modifier SMCHD1, which is involved in the maintenance of D4Z4 methylation. We identified three families in which the proband carries two potentially damaging SMCHD1 variants. We investigated whether these variants were located in cis or in trans and determined their functional consequences by detailed clinical information and D4Z4 methylation studies. In the first family, both variants in trans were shown to act synergistically on D4Z4 hypomethylation and disease penetrance, in the second family both SMCHD1 function-affecting variants were located in cis while in the third family one of the two variants did not affect function. This study demonstrates that having two SMCHD1 missense variants that affect function is compatible with life in males and females, which is remarkable considering its role in X inactivation in mice. The study also highlights the variability in SMCHD1 variants underlying FSHD2 and the predictive value of D4Z4 methylation analysis in determining the functional consequences of SMCHD1 variants of unknown significance.

Concurrent Nucleotide Substitution Mutations in the Human Genome Are Characterized by a Significantly Decreased Transition/Transversion Ratio

There is accumulating evidence that the number of multiple-nucleotide substitutions (MNS) occurring in closely spaced sites in eukaryotic genomes is significantly higher than would be predicted from the random accumulation of independently generated single-nucleotide substitutions (SNS). Although this excess can in principle be accounted for by the concept of transient hypermutability, a general mutational signature of concurrent MNS mutations has not so far been evident. Employing a dataset (N = 449) of "concurrent" double MNS mutations causing human inherited disease, we have identified just such a mutational signature: concurrently generated double MNS mutations exhibit a >twofold lower transition/transversion ratio (termed RTs/Tv ) than independently generated de novo SNS mutations (<0.80 vs. 2.10; P = 2.69 × 10(-14) ). We replicated this novel finding through a similar analysis employing two double MNS variant datasets with differing abundances of concurrent events (150,521 variants with both substitutions on the same haplotypic lineage vs. 94,875 variants whose component substitutions were on different haplotypic lineages) plus 5,430,874 SNS variants, all being derived from the whole-genome sequencing of seven Chinese individuals. Evaluation of the newly observed mutational signature in diverse contexts provides solid support for the postulated role of translesion synthesis DNA polymerases in transient hypermutability.

HCV RNA measurement in samples with diverse genotypes using versions 1 and 2 of the Roche COBAS® AmpliPrep/COBAS® TaqMan® HCV test

BackgroundAccurate HCV RNA measurement is required for monitoring treatment. Underquantification has been reported with some genotypes, particularly genotype 4, using version 1 of the Roche COBAS® AmpliPrep/COBAS® TaqMan® HCV Test. ObjectivesCompare the viral loads of clinical specimens representing diverse genotypes from across the United States using versions 1 (V1) and 2 (V2) of the Roche COBAS® AmpliPrep/COBAS® TaqMan® HCV Test. Assess the frequency of nt145 and nt165 variants in the 5′ UTR associated with detection failures and underquantification in a large clinical sample database. Study designThree hundred archived clinical samples were measured using V1 and V2. Bland–Altman analysis was performed on log-transformed results and compared by genotype. The frequencies of nt145 and nt165 variants from 15,817 sequences were calculated. ResultsOn average, V2 results were 0.16logIU/mL lower than V1 results. The average genotype 4 sample was 0.08logIU/mL higher in V1 than V2. The largest individual sample differences were −2.10 (genotype 2b) and 1.57 (genotype 2a) logIU/mL. For genotype 4 samples, the greatest underquantification by V1 was 1.46logIU/mL. There were 13 (0.082%) variants at nt145 and 24 variants at nt165 (0.152%), including one sequence with variants at both positions (0.0063%). ConclusionsGenotype 4 samples from the U.S. are rarely underquantified and not disproportionately so compared to other genotypes using the COBAS® AmpliPrep/COBAS® TaqMan® HCV Tests. Variants at the nt145 and nt165 positions are uncommon in the U.S. and double variants are exceedingly rare. Underquantification of HCV samples with the V1 assay is likely a very rare occurrence in U.S. patients.

Noncanonical amino acids to improve the pH response of pHLIP insertion at tumor acidity.

The pH low insertion peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membranes with a pH50 of 6.1, while most solid tumors have extracellular pH (pH(e)) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH response. Replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH50 to 6.74, matching average tumor acidity, and replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH response (transition over 0.5 instead of 1 pH unit). These effects are additive: the Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79, with sharper transition than Asp25Aad. Furthermore, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.

Enhancing allosteric inhibition in Thermus thermophilus Phosphofructokinase.

The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol–1 (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol–1 increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild type TtPFK. These results support previous suggestions that heterotropic inhibition and activation occur by different pathways prokaryotic PFK.

Engineering of Artificial pH Switch Proteins using Internal Ionizable Residues with Anomalous PKA Values

Many proteins act as pH sensors. They can respond to small changes in pH with a large conformational transition of functional significance. The thermodynamic basis of pH-driven conformational changes in proteins is well understood: a change in pH can drive a conformational transition when final and initial states have different affinity for H+. This requires that some ionizable groups titrate with different pKa values in the two states. Here we demonstrate how buried Lys or Glu residues with highly anomalous pKa values can be used for rational engineering of artificial pH switch protein that undergo global, cooperative unfolding in response to small changes in pH in the physiological range. This study was enabled by our previous demonstration that most Lys and Glu residues buried in the interior of staphylococcal nuclease titrate with highly anomalous pKa values as a consequence of being buried in the hydrophobic interior of the protein. The ionization of a single buried ionizable group is not sufficient to drive the unfolding of the protein, thus we engineered variants with two buried ionizable groups. The V66E/A109E and T62K/L125K variants of SNase were selected because, based on the pKa values measured for these buried residues, the protein was expected to act as a switch near pH 7. This was confirmed with equilibrium thermodynamic experiments. The double Glu variant was stable at low pH and unfolded at high pH and the double Lys variant was stable at high pH and unfolded at low pH, as expected from the nature of the shifts in pKa values. Crystallography and NMR spectroscopy were used to characterize the folded and unfolded states. This strategy for the design of artificial pH sensing proteins is completely general and transferable to other proteins.

Interactions between Pairs of Charges Buried in the Hydrophobic Interior of a Protein are Unexpectedly Weak

Internal ion pairs buried in the hydrophobic protein interior are essential for many important biochemical processes, including H+ transport, e- transfer, ion homeostasis, and catalysis. Despite the importance of these pairs, their properties remain poorly understood. It has been suggested that, for some systems, medium or long-range Coulomb interactions between buried groups could play a role in determining biological function. In principle, this should only be possible if the protein interior behaves like a medium of low dielectric constant, as assumed in most electrostatics models. These motifs are then of special interest not only for their functional roles, but also as probes to examine the balance between Coulomb and hydration energies experienced by buried charges. As the distance between the internal charges increases, the balance of these energies will disfavor burying the groups in the charged state, eliminating any Coulomb interaction between them. To examine the balance between Coulomb and dehydration energies in the protein interior we engineered a series of double variants in a highly stable variant of staphylococcal nuclease. Each variant included an internal histidine paired with either an aspartate or glutamate buried at various internal positions throughout the protein interior. This set of proteins used to probe the distance dependence of potential Coulomb interactions. No significant electrostatic coupling was observed for pairs that interacted through medium- or long-range Coulomb interactions. When the distance between the groups was short, favorable coupling energies were only observed when the side chains of the pair could achieve a geometry favorable for a H-bond; spatial proximity alone was insufficient to create favorable Coulomb interactions. Simple electrostatics models that describe ionized states as point charges interacting through space are unlikely to be able to reproduce these data.

Combinatorial active-site variants confer sustained clavulanate resistance in BlaC β-lactamase from Mycobacterium tuberculosis.

Bacterial resistance to β-lactam antibiotics is a global issue threatening the success of infectious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilient to β-lactam treatment, primarily due to the chromosomally encoded BlaC β-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant β-lactam compounds currently in use. Recent laboratory and clinical studies have nevertheless shown that specific β-lactam-BlaC inhibitor combinations can be used to inhibit the growth of extensively drug-resistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimens against resistant strains. In the present work, we performed combinatorial active-site replacements in BlaC to demonstrate that specific inhibitor-resistant (IRT) substitutions at positions 69, 130, 220, and/or 234 can act synergistically to yield active-site variants with several thousand fold greater in vitro resistance to clavulanate, the most common clinical β-lactamase inhibitor. While most single and double variants remain sensitive to clavulanate, double mutants R220S-K234R and S130G-K234R are substantially less affected by time-dependent clavulanate inactivation, showing residual β-lactam hydrolytic activities of 46% and 83% after 24 h incubation with a clinically relevant inhibitor concentration (5 μg/ml, 25 µM). These results demonstrate that active-site alterations in BlaC yield resistant variants that remain active and stable over prolonged bacterial generation times compatible with mycobacterial proliferation. These results also emphasize the formidable adaptive potential of inhibitor-resistant substitutions in β-lactamases, potentially casting a shadow on specific β-lactam-BlaC inhibitor combination treatments against M. tuberculosis.

Permissive changes in the neuraminidase play a dominant role in improving the viral fitness of oseltamivir-resistant seasonal influenza A(H1N1) strains

Permissive neuraminidase (NA) substitutions such as R222Q, V234M and D344N have facilitated the emergence and worldwide spread of oseltamivir-resistant influenza A/Brisbane/59/2007 (H1N1)-H275Y viruses. However, the potential contribution of genetic changes in other viral segments on viral fitness remains poorly investigated. A series of recombinant A(H1N1)pdm09 and A/WSN/33 7:1 reassortants containing the wild-type (WT) A/Brisbane/59/2007 NA gene or its single (H275Y) and double (H275Y/Q222R, H275Y/M234V and H275Y/N344D) variants were generated and their replicative properties were assessed in vitro. The Q222R reversion substitution significantly reduced viral titers when evaluated in both A(H1N1)pdm09 and A/WSN/33 backgrounds. The permissive role of the R222Q was further confirmed using A/WSN/33 7:1 reassortants containing the NA gene of the oseltamivir-susceptible or oseltamivir-resistant influenza A/Mississippi/03/2001 strains. Therefore, NA permissive substitutions play a dominant role for improving viral replication of oseltamivir-resistant A (H1N1)-H275Y viruses in vitro.

G-protein receptor kinase 4 polymorphism and response to antihypertensive therapy.

G-protein receptor kinase 4 polymorphism influences blood pressure regulation via modulation of dopamine receptor D1 in renal proximal tubular cells. We investigated the role of G-protein receptor kinase 4 polymorphism in the response to hypertensive therapy in patients with essential hypertension. In a prospective study, we assessed the G-protein receptor kinase 4 polymorphisms R65L, A142V, and A486V in 100 hypertensive patients. We analyzed the association of the 3 gene variants on blood pressure control and response to antihypertensive therapy with single-locus analysis, haplotype analysis, and regression analysis. Hypertensive individuals with a homozygous double variant of 65 L and 142 V needed significantly more antihypertensive treatment (number of antihypertensives 2.59 vs 1.95, P = 0.043) and especially diuretic therapy (0.82 vs 0.49, P = 0.029) to reach the same mean arterial blood pressure than did homozygous carriers of only 1 variant or heterozygous/wild-type carriers of R65L, A142V, and A486V alleles. G-protein receptor kinase 4 polymorphism is associated with antihypertensive treatment response in patients with essential hypertension. Determination of G-protein receptor kinase 4 polymorphism may improve individual antihypertensive blood pressure control in patients with essential hypertension.

Structure and stability of an unusual zinc-binding protein from Bacteroides thetaiotaomicron

The crystal structure of a putative protease from Bacteroides thetaiotaomicron (ppBat) suggested the presence of a zinc ion in each protomer of the dimer as well as a flavin in the dimer interface. Since the chemical identity of the flavin and the exact mode of binding remained unclear, we have determined the crystal structure of ppBat in complex with riboflavin. The obtained structure revealed that the isoalloxazine ring is sandwiched between two tryptophan residues (Trp164) from both chains and adopts two alternate orientations with the N(10)-ribityl side chain protruding from the binding site in opposite directions. In order to characterize the zinc-binding site, we generated two single variants and one double variant in which the two coordinating cysteine residues (Cys74 and Cys111) were replaced by alanine. All three variants were unable to bind zinc demonstrating that both cysteine residues are essential for binding. Moreover, the lack of zinc binding also resulted in drastically reduced thermal stability (11–15°C). A similar effect was obtained when wild-type protein was incubated with EDTA supporting the conclusion that the zinc-binding site plays an important structural role in ppBat. On the other hand, attempts to identify proteolytic activity failed suggesting that the zinc may not act as a catalytic center in ppBat. Structurally similar zinc binding motives in other proteins were also found to play a structural rather than catalytic role and hence it appears that neither the flavin nor the zinc binding sites possess a catalytic function in ppBat.

A type system for Continuation Calculus

Continuation Calculus (CC), introduced by Geron and Geuvers, is a simple foundational model for functional computation. It is closely related to lambda calculus and term rewriting, but it has no variable binding and no pattern matching. It is Turing complete and evaluation is deterministic. Notions like "call-by-value" and "call-by-name" computation are available by choosing appropriate function definitions: e.g. there is a call-by-value and a call-by-name addition function. In the present paper we extend CC with types, to be able to define data types in a canonical way, and functions over these data types, defined by iteration. Data type definitions follow the so-called "Scott encoding" of data, as opposed to the more familiar "Church encoding". The iteration scheme comes in two flavors: a call-by-value and a call-by-name iteration scheme. The call-by-value variant is a double negation variant of call-by-name iteration. The double negation translation allows to move between call-by-name and call-by-value.

An evolutionarily conserved alternate metal ligand is important for activity in α-isopropylmalate synthase from Mycobacterium tuberculosis

Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon–carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to α-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of α-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for α-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes.

Molecular epidemiology and population structure of the honey bee brood pathogen Melissococcus plutonius.

Melissococcus plutonius is the causative agent of European foulbrood (EFB), which is a serious brood disease of the European honey bee (Apis mellifera). EFB remains a threat because of a poor understanding of disease epidemiology. We used a recently published multi-locus sequence typing method to characterise 206 M. plutonius isolates recovered from outbreaks in England and Wales over the course of 2 years. We detected 15 different sequence types (STs), which were resolved by eBURST and phylogenetic analysis into three clonal complexes (CCs) 3, 12 and 13. Single and double locus variants within CC3 were the most abundant and widespread genotypes, accounting for 85% of the cases. In contrast, CCs 12 and 13 were rarer and predominantly found in geographical regions of high sampling intensity, consistent with a more recent introduction and localised spread. K-function analysis and interpoint distance tests revealed significant geographical clustering in five common STs, but pointed to different dispersal patterns between STs. We noted that CCs appeared to vary in pathogenicity and that infection caused by the more pathogenic variants is more likely to lead to honey bee colony destruction, as opposed to treatment. The importance of these findings for improving our understanding of disease aetiology and control are discussed.

Two BRM promoter insertion polymorphisms increase the risk of early‐stage upper aerodigestive tract cancers

Brahma (BRM) has a key function in chromatin remodeling. Two germline BRM promoter insertion–deletion polymorphisms, BRM-741 and BRM-1321, have been previously associated with an increased risk of lung cancer in smokers and head and neck cancer. To further evaluate their role in cancer susceptibility particularly in early disease, we conducted a preplanned case–control study to investigate the association between the BRM promoter variants and stage I/II upper aerodigestive tract (UADT) cancers (i.e., lung, esophageal, head and neck), a group of early-stage malignancies in which molecular and genetic etiologic factors are poorly understood. The effects of various clinical factors on this association were also studied. We analyzed 562 cases of early-stage UADT cancers and 993 matched healthy controls. The double homozygous BRM promoter variants were associated with a significantly increased risk of early stage UADT cancers (adjusted odds ratio [aOR], 2.46; 95% confidence interval [CI], 1.7–3.8). This association was observed in lung (aOR, 2.61; 95% CI, 1.5–4.9) and head and neck (aOR, 2.75; 95% CI, 1.4–5.6) cancers, but not significantly in esophageal cancer (aOR, 1.66; 95% CI, 0.7–5.8). There was a nonsignificant trend for increased risk in the heterozygotes or single homozygotes. The relationship between the BRM polymorphisms and early-stage UADT cancers was independent of age, sex, smoking status, histology, and clinical stage. These findings suggest that the BRM promoter double insertion homozygotes may be associated with an increased risk of early-stage UADT cancers independent of smoking status and histology, which must be further validated in other populations.

Age-related prevalence of dermatoscopic patterns of acral melanocytic nevi

The objective of this study was to evaluate the relation between age and dermatoscopic features of acral nevi. We evaluated 159 dermatoscopic images of melanocytic nevi from 146 individuals filed at the Dermatoscopy Outpatient Clinic of Tokyo Women’s Medical University Medical Center East between April 2006 and March 2009. All images of melanocytic lesions on acral volar skin that showed a clear-cut dermatoscopic pattern of an acral nevus at the time of initial observation were included. The dermatoscopic patterns of all images were retrospectively examined in a blinded fashion according to the standard dermatoscopic classification criteria for acral melanocytic nevi. Images were classified using 15 structural variants of the parallel furrow pattern. These variants were then re-classified into two groups; the “single” line group and “double” line group. Patients of the double line group (age, 25.5 years) were significantly younger than those of the single line group (32.4 years). There was no significant difference in the age-related predominance between the solid line patterns and dotted line patterns. There was a significant age difference between patients with nevi showing the crista dotted pattern (mean age 24.9 years) and patients with nevi without the crista dotted pattern (mean age 34.6 years). We conclude that the double line variant of the parallel furrow pattern and crista dotted pattern, which probably correspond to the congenital type acral nevus, tend to be more common in young patients.

Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O2 Activation

Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes.

Genetic variation in the two-pore domain potassium channel, TASK-1, may contribute to an atrial substrate for arrhythmogenesis

The two-pore domain potassium channel, K2P3.1 (TASK-1) modulates background conductance in isolated human atrial cardiomyocytes and has been proposed as a potential drug target for atrial fibrillation (AF). TASK-1 knockout mice have a predominantly ventricular phenotype however, and effects of TASK-1 inactivation on atrial structure and function have yet to be demonstrated in vivo. The extent to which genetic variation in KCNK3, that encodes TASK-1, might be a determinant of susceptibility to AF is also unknown. To address these questions, we first evaluated the effects of transient knockdown of the zebrafish kcnk3a and kcnk3b genes and cardiac phenotypes were evaluated using videomicroscopy. Combined kcnk3a and kcnk3b knockdown in 72 hour post fertilization embryos resulted in lower heart rate (p<0.001), marked increase in atrial diameter (p<0.001), and mild increase in end-diastolic ventricular diameter (p=0.01) when compared with control-injected embryos. We next performed genetic screening of KCNK3 in two independent AF cohorts (373 subjects) and identified three novel KCNK3 variants. Two of these variants, present in one proband with familial AF, were located at adjacent nucleotides in the Kozak sequence and reduced expression of an engineered reporter. A third missense variant, V123L, in a patient with lone AF, reduced resting membrane potential and altered pH sensitivity in patch-clamp experiments, with structural modeling predicting instability in the vicinity of the TASK-1 pore. These in vitro data suggest that the double Kozak variants and V123L will have loss-of-function effects on ITASK. Cardiac action potential modeling predicted that reduced ITASK prolongs atrial action potential duration, and that this is potentiated by reciprocal changes in activity of other ion channel currents. Our findings demonstrate the functional importance of ITASK in the atrium and suggest that inactivation of TASK-1 may have diverse effects on atrial size and electrophysiological properties that can contribute to an arrhythmogenic substrate.

Understanding the Psychological Issues in The Geriatric Population in The Times of COVID-19- A Critical Review from The Lens of a Psychologist

The first wave of the Covid-19 pandemic hit India in January 2020 when the first confirmed case was reported at a general hospital of Kerala. Since then, the number of reported cases kept on increasing at a steady pace, we saw deaths, rise, fall and stagnancy in the number of cases in India while the figures of deaths reported remained worrisome. While India is currently battling with the deadly double mutant variant of the virus, the present critical review is an attempt to study, analyse and understand the need of catering to the needs of geriatric population, especially in a collectivistic culture like India, where elderly issues, psychological difficulties, and coping mechanism takes a back seat while there is constant fear of leading a healthy life especially in the testing times of a national pandemic, COVID-19. This critical review is an attempt to highlight the various issues dealt by the average Indian elderly, their lifestyle after retirement, sustaining means of livelihood in times of uncertainty and keeping a positive mindset for a better quality of life. This paper also attempts to enumerate few strategies required to inculcate a sense of life satisfaction and improved quality of life among the elderly.