Mutant Variants Research Articles

Insights into the physiological, molecular, and genetic regulators of albinism in Camellia sinensis leaves.

Introduction: Yanling Yinbiancha, a cultivar of Camellia sinensis (L.) O. Kuntze, is an evergreen woody perennial with characteristic albino leaves. A mutant variant with green leaves on branches has been recently identified. The molecular mechanisms underlying this color variation remain unknown. Methods: We aimed to utilize omics tools to decipher the molecular basis for this color variation, with the ultimate goal of enhancing existing germplasm and utilizing it in future breeding programs. Results and discussion: Albinotic leaves exhibited significant chloroplast degeneration and reduced carotenoid accumulation. Transcriptomic and metabolomic analysis of the two variants revealed 1,412 differentially expressed genes and 127 differentially accumulated metabolites (DAMs). Enrichment analysis for DEGs suggested significant enrichment of pathways involved in the biosynthesis of anthocyanins, porphyrin, chlorophyll, and carotenoids. To further narrow down the causal variation for albinotic leaves, we performed a conjoint analysis of metabolome and transcriptome and identified putative candidate genes responsible for albinism in C. sinensis leaves. 12, 7, and 28 DEGs were significantly associated with photosynthesis, porphyrin/chlorophyll metabolism, and flavonoid metabolism, respectively. Chlorophyllase 2, Chlorophyll a-Binding Protein 4A, Chlorophyll a-Binding Protein 24, Stay Green Regulator, Photosystem II Cytochrome b559 subunit beta along with transcription factors AP2, bZIP, MYB, and WRKY were identified as a potential regulator of albinism in Yanling Yinbiancha. Moreover, we identified Anthocyanidin reductase and Arabidopsis Response Regulator 1 as DEGs influencing flavonoid accumulation in albino leaves. Identification of genes related to albinism in C. sinensis may facilitate genetic modification or development of molecular markers, potentially enhancing cultivation efficiency and expanding the germplasm for utilization in breeding programs.

Rebound of multiple infections and prevalence of anti-malarial resistance associated markers following malaria upsurges in Dielmo village, Senegal, West Africa

BackgroundThanks to the scale up of malaria control interventions, the malaria burden in Senegal has decreased substantially to the point that the National Malaria Control Programme plans to achieve malaria elimination by 2030. To guide such efforts, measuring and monitoring parasite population evolution and anti-malarial drugs resistance is extremely important. Information on the prevalence of parasite mutations related to drug resistance can provide a first signal of emergence, introduction and selection that can help with refining drug interventions. The aim of this study was to analyse the prevalence of anti-malarial drug resistance-associated markers before and after the implementation of artemisinin-based combination therapy (ACT) from 2005 to 2014 in Dielmo, a model site for malaria intervention studies in Senegal.MethodsSamples from both malaria patients and Plasmodium falciparum asymptomatic carriers were analysed with high resolution melting (HRM) technique to genotype P. falciparum chloroquine resistance transporter (Pfcrt) gene haplotypes and multidrug-resistant protein 1 (Pfmdr1) gene at codons N86 and Y184.ResultsAmong the 539 samples analysed, 474, 486, and 511 were successfully genotyped for Pfmdr1 N86, Y184, and Pfcrt, respectively. The prevalence of drug resistance markers was high, particularly during the malaria upsurges. Following the scale-up in bed net distribution, only the mutant (86F-like) variant of Pfmdr1 86 was present while during the malaria upsurges the predominance of two types 86Y-86N (43%) and 86F-like (56%) were observed. Most infections (87%) carried the wild type Y-allele at Pfmdr1 184 during the period of nets scale-up while during the malaria upsurges only 16% of infections had wild type and 79% of infections had mixed (mutant/wild) type. The frequency of the mixed genotypes SVMNT-like_CVMNK and SVMNT-like_CVIET within Pfcrt gene was particularly low during bednet scale up. Their frequency increased significantly (P < 0.001) during the malaria upsurges.ConclusionThis data demonstrated the effect of multiple interventions on the dynamics of drug resistance-associated mutations in the main malaria parasite P. falciparum in an endemic village in Senegal. Monitoring drug resistance markers should be conducted periodically to detect threats of emergence or resurgence that could compromise the efficacy of anti-malarial drugs.

Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold

Cryoelectron microscopy (Cryo-EM) has enabled structural determination of proteins larger than about 50 kDa, including many intractable by any other method, but it has largely failed for smaller proteins. Here, we obtain structures of small proteins by binding them to a rigid molecular scaffold based on a designed protein cage, revealing atomic details at resolutions reaching 2.9 Å. We apply this system to the key cancer signaling protein KRAS (19 kDa in size), obtaining four structures of oncogenic mutational variants by cryo-EM. Importantly, a structure for the key G12C mutant bound to an inhibitor drug (AMG510) reveals significant conformational differences compared to prior data in the crystalline state. The findings highlight the promise of cryo-EM scaffolds for advancing the design of drug molecules against small therapeutic protein targets in cancer and other human diseases.

Efficient and accurate calculation of proline cis/trans isomerization free energies from Hamiltonian replica exchange molecular dynamics simulations

Proline cis/trans isomerization plays an important role in many biological processes but occurs on time scales not accessible to brute-force molecular dynamics (MD) simulations. We have designed a new Hamiltonian replica exchange scheme, ω-bias potential replica exchange molecular dynamics (ωBP-REMD), to efficiently and accurately calculate proline cis/trans isomerization free energies. ωBP-REMD is applied to various proline-containing tripeptides and a biologically important proline residue in the N2-domain of the gene-3-protein of phage fd in the wildtype and mutant variants of the protein. Excellent cis/trans transition rates are obtained. Reweighting of the sampled probability distribution along the peptide bond dihedral angle allows construction of the corresponding free-energy profile and calculation of the cis/trans isomerization free energy with high statistical precision. Very good agreement with experimental data is obtained. ωBP-REMD outperforms standard umbrella sampling in terms of convergence and agreement with experiment and strongly reduces perturbation of the local structure near the proline residue.

Evaluating the Cysteine-Rich and Catalytic Subdomains of Human Tyrosinase and OCA1-Related Mutants Using 1 μs Molecular Dynamics Simulation.

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 μs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a 'molten globule' state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain's rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism.

Structure-based virtual screening of natural compounds against wild and mutant (R1155K, A1156T and D1168A) NS3-4A protease of Hepatitis C virus

NS3-4A, a serine protease, is a primary target for drug development against Hepatitis C Virus (HCV). However, the effectiveness of potent next-generation protease inhibitors is limited by the emergence of mutations and resulting drug resistance. To address this, in this study a structure-based drug design approach is employed to screen a large library of 7320 natural compounds against both wild-type and mutant variants of NS3-4A protease. Telaprevir, a widely used protease inhibitor, was recruited as the control drug. The top 10 compounds with favorable binding affinities underwent drug-likeness evaluation. Based on ADMET studies, complexes of NP_024762 and NP_006776 were selected for molecular dynamic simulations. Principal component analysis (PCA) was employed to explore the conformational space and protein dynamics of the protein-ligand complex using a Free Energy Landscape (FEL) approach. The cosine values obtained from FEL analysis ranged from 0 to 1, and eigenvectors with cosine values below 0.2 were chosen for further analysis. To forecast binding free energies and evaluate energy contributions per residue, the MM-PBSA method was employed. The results highlighted the crucial role of amino acids in the catalytic domain for the binding of the protease with phytochemicals. Stable associations between the top compounds and the target protease were confirmed by the formation of hydrogen bonds in the binding pocket involving residues: His1057, Gly1137, Ser1139, and Ala1157. These findings suggest the potential of these compounds for further validation through biological evaluation. Communicated by Ramaswamy H. Sarma

Fighting Celiac Disease: Improvement of pH Stability of Cathepsin L In Vitro by Computational Design.

Roughly 1% of the global population is susceptible to celiac disease (CD)-inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect Tribolium castaneum-cathepsin L-hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2-4) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L-loss of the catalytically competent rotameric state of one of the active site residues, His 275. To "fix" the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people.

COVID‐19 retreats and world recovers: A silver lining in the dark cloud

AbstractThe coronavirus disease (COVID‐19), which the World Health Organization classified as the Sixth Public Health Emergency Of International Concern (PHEIC) on January 30, 2020, is no longer a PHEIC. Millions were affected due to unawareness. The increase in fatalities and shortage of medicine was the first outrage of COVID‐19. As per the Johns Hopkins COVID‐19 resource center database, it was observed that the disease has spread dynamically across 200+ nations worldwide affecting more than 600 million people from 2019 to 2023, and over thousands of people were victimized regularly at a 2% mortality rate (approx.). In the midway, the mutant variants of concern like omicron, and delta have also created havoc and caused significant impact on public health, global economy, and lifestyle. Since 2019, 3 years now passed and the dynamic disease statistics seem decelerated; moreover, the prevalence of COVID‐19 is also fading. The Johns Hopkins resource center has also stopped recording the data of the global pandemic recently from March 10, 2023. Hence, based on the facts, we are presenting a concise report on the pandemic from 2019 to 2023, which includes a brief discussion of the global pandemic. We have highlighted global epidemiology, emphasizing the Indian COVID scenario, vaccination across the globe, and the psychosocial and geopolitical consequences of COVID‐19 with a brief background to pathology, clinical management, and the worldwide response against triage. A lot has changed and still needs to change after three tough years of COVID‐19. Even though science has progressed and advanced research in medicine is pointing toward future generations, there is no standard care supplied for COVID‐19‐like calamities. COVID‐19 cases might have declined but its influence on the society is still stagnant. This COVID experience has taught us that, despite our bleak beginnings, there is always hope for the future and that we must act with foresight to improve things for future generations.

Plant growth‐promoting rhizobacteria mediate soil hydro‐physical properties: An investigation with Bacillus subtilis and its mutants

AbstractPlant growth‐promoting rhizobacteria and other soil bacteria have the potential to improve soil hydro‐physical properties and processes through the production of extracellular polymeric substances (EPS). However, the mechanisms by which EPS mediates changes in soil properties and processes remain incompletely understood, partly due to variations in EPS composition produced under different environmental conditions. In this study, we investigated the influence of different bacterial traits on intrinsic soil properties and processes of evaporation and infiltration using sand treated with the wild‐type Bacillus subtilis variant (UD1022) and its two mutant variants, – and srf. The – mutant suppresses EPS production through alterations in the eps and tasA genes, while the srf mutant lacks the gene for surfactin production. Experimental results confirmed that the solution viscosity of the – mutant was the lowest and the solution surface tension of the srf mutant was the highest among the three tested bacteria strains. The distinct intrinsic properties of EPS produced by these bacterial strains resulted in varied hydro‐physical responses in the treated sand. Key influences included modifications in wettability, hydraulic decoupling (or mixed wettability), and aggregation, which collectively led to reduced evaporation rates and heterogeneous water distribution during infiltration in the bacteria‐treated sands. Our findings advance the understanding of the role bacterial EPS play in vadose zone hydrology and offer insights for the development of sustainable strategies for increasing water retention, supporting crop production in arid regions, and facilitating land restoration.

Untangling the Role of TREM2 in Conjugation with Microglia in Neuronal Dysfunction: A Hypothesis on a Novel Pathway in the Pathophysiology of Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder involving heterogenous pathophysiological characteristics, which has become a challenge to therapeutics. The major pathophysiology of AD comprises amyloid-β (Aβ), tau, oxidative stress, and apoptosis. Recent studies indicate the significance of Triggering receptor expressed on myeloid cells 2 (TREM2) and its mutant variants in AD. TREM2 are the transmembrane receptors of microglial cells that performs a broad range of physiological cell processes. Phagocytosis of Aβ is one of the physiological roles of TREM2, which plays a pivotal role in AD progression. R47H, a mutant variant of TREM2, increases the risk of AD by impairing TREM2-Aβ binding. Inconclusive evidence regarding the TREM2 signaling cascade mechanism of Aβ phagocytosis motivates the current review to propose a new hypothesis. The review systematically assesses the cross talk between TREM2 and other AD pathological domains and the influence of TREM2 on amyloid and tau seeding. Disease associated microglia (DAM), a novel state of microglia with unique transcriptional and functional signatures reported in neurodegenerative conditions, also depend on the TREM2 pathway for its differentiation. DAM is suggested to have a neuroprotective role. We hypothesize that TREM2, along with its signaling adaptors and endogenous proteins, play a key role in ameliorating Aβ clearance. We indicate that TREM2 has the potential to ameliorate the Aβ burden, though with differential clearance ability and may act as a potential therapeutic target.

Multimodal single-cell analysis of non-random heteroplasmy distribution in human retinal mitochondrial disease.

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non-random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non-random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

Association, Conformational Rearrangements and the Reverse Process of Aggregates Dissociation during Apomyoglobin Amyloid Formation

Amyloid formation is linked with serious human diseases that are currently incurable. Usually, in the study of amyloid aggregation, the description of the protein’s association is in focus. Whereas the mechanism of the cross-β-structure formation, and the presence of aggregation reversibility, remain insufficiently explored. In this work, the kinetics of amyloid aggregation of apomyoglobin (ApoMb) have been studied using thioflavin fluorescence, electron microscopy, and non-denaturing electrophoresis. An analysis of the concentration dependence of the aggregation rates allows the conclusion that ApoMb amyloid formation includes the stages of conformational rearrangements in the aggregates, followed by their association and the fibril formation. The study of the mutant variants aggregation kinetics showed that the association rate is determined by the amino acids’ hydrophobicity, while the rate of conformational rearrangements is affected by the localization of the substitution. An unexpected result was the discovery that ApoMb amyloid formation is reversible, and under native-like conditions, the amyloid can dissociate, producing monomers. A consequence of the reversibility of amyloid aggregation is the presence of the monomer after aggregation completion. Since the aggregation reversibility indicates the possibility of dissociation of already formed fibrils, presented data and approaches can be useful in finding ways for amyloid diseases treatment.

Effects of Ataxia-Telangiectasia Mutated Variants on Radionecrosis and Local Control After Stereotactic Radiation Surgery for Non-Small Cell Lung Cancer Brain Metastases

Genetic variants affecting the radiation response protein ataxia-telangiectasia mutated (ATM) have been associated with increased adverse effects of radiation but also with improved local control after conventional radiation therapy. However, it is unknown whether ATM variants affect rates of radionecrosis (RN) and local intracranial progression (LIP) after stereotactic radiosurgery (SRS) for brain metastases. Patients undergoing an initial course of SRS for non-small cell lung cancer (NSCLC) brain metastases at a single institution were retrospectively identified. Kaplan-Meier estimates were calculated and Cox proportional hazards testing was performed based on ATM variant status. A total of 541 patients completed SRS for brain metastasis secondary to NSCLC, of whom 260 completed molecular profiling. Variants of ATM were identified in 36 cases (13.8%). Among patients who completed molecular profiling, RN incidence was 4.9% (95% CI, 1.6%-8.2%) at 6 months and 9.9% (95% CI, 4.8%-15.0%) at 12 months. Incidence of RN was not significantly increased among patients with ATM variants, with an RN incidence of 5.3% (95% CI, 0.0%-15.3%) at both 6 and 12 months (P=.46). For all patients who completed genomic profiling, LIP was 5.4% (95% CI, 2.4%-8.4%) at 6 months and 9.8% (5.5%-14.1%) at 12 months. A significant improvement in LIP was not detected among patients with ATM variants, with an LIP incidence of 3.1% (0.0%-9.1%) at both 6 and 12 months (P=.26). Although differences according to ATM variant type (pathologic variant or variant of unknown significance) did not reach significance, no patients with ATM pathologic variants experienced LIP. We did not detect significant associations between ATM variant status and RN or LIP after SRS for NSCLC brain metastases. The current data set allows estimation of patient cohort sizes needed to power future investigations to identify genetic variants that associate with significant differences in outcomes after SRS.

Identification of key amino acid residues in AtUMAMIT29 for transport of glucosinolates.

Glucosinolates are key defense compounds of plants in Brassicales order, and their accumulation in seeds is essential for the protection of the next generation. Recently, members of the Usually Multiple Amino acids Move In and Out Transporter (UMAMIT) family were shown to be essential for facilitating transport of seed-bound glucosinolates from site of synthesis within the reproductive organ to seeds. Here, we set out to identify amino acid residues responsible for glucosinolate transport activity of the main seed glucosinolate exporter UMAMIT29 in Arabidopsis thaliana. Based on a predicted model of UMAMIT29, we propose that the substrate transporting cavity consists of 51 residues, of which four are highly conserved residues across all the analyzed homologs of UMAMIT29. A comparison of the putative substrate binding site of homologs within the brassicaceous-specific, glucosinolate-transporting clade with the non-brassicaceous-specific, non-glucosinolate-transporting UMAMIT32 clade identified 11 differentially conserved sites. When each of the 11 residues of UMAMIT29 was individually mutated into the corresponding residue in UMAMIT32, five mutant variants (UMAMIT29#V27F, UMAMIT29#M86V, UMAMIT29#L109V, UMAMIT29#Q263S, and UMAMIT29#T267Y) reduced glucosinolate transport activity over 75% compared to wild-type UMAMIT29. This suggests that these residues are key for UMAMIT29-mediated glucosinolate transport activity and thus potential targets for blocking the transport of glucosinolates to the seeds.

Impact of E484Q and L452R Mutations on Structure and Binding Behavior of SARS-CoV-2 B.1.617.1 Using Deep Learning AlphaFold2, Molecular Docking and Dynamics Simulation.

During the outbreak of COVID-19, many SARS-CoV-2 variants presented key amino acid mutations that influenced their binding abilities with angiotensin-converting enzyme 2 (hACE2) and neutralizing antibodies. For the B.1.617 lineage, there had been fears that two key mutations, i.e., L452R and E484Q, would have additive effects on the evasion of neutralizing antibodies. In this paper, we systematically investigated the impact of the L452R and E484Q mutations on the structure and binding behavior of B.1.617.1 using deep learning AlphaFold2, molecular docking and dynamics simulation. We firstly predicted and verified the structure of the S protein containing L452R and E484Q mutations via the AlphaFold2-calculated pLDDT value and compared it with the experimental structure. Next, a molecular simulation was performed to reveal the structural and interaction stabilities of the S protein of the double mutant variant with hACE2. We found that the double mutations, L452R and E484Q, could lead to a decrease in hydrogen bonds and higher interaction energy between the S protein and hACE2, demonstrating the lower structural stability and the worse binding affinity in the long dynamic evolutional process, even though the molecular docking showed the lower binding energy score of the S1 RBD of the double mutant variant with hACE2 than that of the wild type (WT) with hACE2. In addition, docking to three approved neutralizing monoclonal antibodies (mAbs) showed a reduced binding affinity of the double mutant variant, suggesting a lower neutralization ability of the mAbs against the double mutant variant. Our study helps lay the foundation for further SARS-CoV-2 studies and provides bioinformatics and computational insights into how the double mutations lead to immune evasion, which could offer guidance for subsequent biomedical studies.

Vaccine equity and access: A comparative assessment of Covaxin, Covishield, and Sputnik V.

The RNA virus known as SARS-CoV-2, which causes severe acute respiratory syndrome, was discovered for the first time in Wuhan, China, in December 2019. The World Health Organization (WHO) declared the outbreak a global pandemic on March 11, 2020, as a result of the virus's subsequent spread throughout the planet. The urgent creation of safe and effective vaccines has elevated to a top priority in the global healthcare industry because of the terrible effects of the COVID-19 outbreak. The Covaxin and Covishield vaccines were administered as part of the start of the SARS-CoV-2 vaccination campaign in India on January 13, 2021. Covaxin is made up of adjuvant-inactivated viral particles, whereas Covishield is an adenovirus vector-based vaccine. The utility and effectiveness of each vaccine are significantly influenced by its formulation, adjuvants, and mode of action. Vaccine efficacy depends on various factors, including the creation of memory cells, cell-mediated immunity, and antibodies. Results from third-phase trials have shown that Covishield exhibits an effectiveness of approximately 90%, while Covaxin demonstrates an effectiveness of around 80%. Both vaccines have demonstrated satisfactory efficacy against several mutant variants of SARS-CoV-2. The effectiveness of Covishield, however, should be noted as compromised if there are significant changes in the spike (S) protein structure in future variants. In contrast, Covaxin may remain effective against such variants due to its ability to elicit multiple antibodies targeting different epitopes. The objective of this study is to evaluate and contrast Covaxin, Covishield &amp; Sputnik v immunogenic and therapeutic efficacy. Additionally, potential vaccination challenges in the coming days will be discussed. Understanding the relative strengths and limitations of these vaccines can inform decision-making and strategies related to vaccine deployment, public health interventions, and future vaccine development efforts."

Five novel dysfunctional variants in the TSPAN12 gene in familial exudative vitreoretinopathy

Familial exudative vitreoretinopathy (FEVR) is an inheritable vitreoretinal disease characterized by incomplete retinal vascular development, which often leads to multiple retinal complications and causes severe vision loss in children. We reported the TSPAN12 variants' frequency in a cohort of FEVR and five novel TSPAN12 variants and related clinical features in six Chinese families. Seven hundred thirty-four families’ genetic in-house data were reviewed. Whole-exome sequencing (WES) was performed in all probands; Sanger sequencing was conducted in the family members. Five novel variants from six families were noted, and clinical data were collected. Luciferase assays were applied to test the activity of the Norrin/β-catenin signal caused by the mutant TSPAN12 genes. The frequency of TSPAN12 variants in FEVR is 8.79% (50/569). Five novel variants in TSPAN12 were identified in six families, including two missense variants, c.476G > A(p.Cys159Tyr) and c.81T > G(p.Ser27Arg), two frameshift variants, c.628_629insA(p.Met210Asnfs*42) and c.251delG(p.Gly84Glufs*3) and one nonsense, c.352G > T(p.Glu118*). Low vision, high myopia, nystagmus, and leukocoria are the common symptom at the first presentation. All variants were also predicted as pathogenic in silico. Moreover, the luciferase assay demonstrated that all variants caused severely compromised Norrin/β-catenin signaling activity. In conclusion, the frequency of TSPAN12 variants in FEVR was 8.79% in our cohort. Five novel variants of TSPAN12 were identified. Moreover, we demonstrated the dysfunction of mutant variants via the downregulation of Norrin/β-catenin signaling. These findings expanded the genetic and clinical spectrum of FEVR with TSPAN12 variants.

Clinical significance of mutational variants in beta and alpha genes in patients with hemoglobinopathies from two large Greek centers: a complex interplay between genotype and phenotype.

Hemoglobinopathies affect patients in the wider Mediterranean area consisting of 4 distinct subgroups: beta thalassemia major (TM), beta thalassemia intermedia (TI), sickle cell disease (SCD) and hemoglobin H disease (alpha thalassemia). The clinical spectrum varies from mild to severe. Complex interactions between genes and environmental factors form the clinical manifestations. There is an unmet need to clarify these multifactorial mechanisms.This is the first Greek study describing mutational alleles (HBB and HBA1/HBA2 gene variants) in 217 patients with hemoglobinopathies of two large centers in Greece (Larissa and Athens) and associating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). Thus, the complex interplay between corresponding genotypes and phenotypes was investigated.Our results are in accordance with previous national studies with limited variations, due to regional prevalence of specific gene variants, as expected. It is also a description of the prevalence of hemoglobinopathies in the Greek population. The type and prevalence of beta and alpha globin gene variants differ significantly among countries. We also confirm the well-known observation of many studies that in our beta thalassemic or SCD patients, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course, whereas the inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype.In cases in whom the genotype and phenotype did not correlate, factors like the function or modification of possible regulatory genes or additional nutritional-environmental effects should be investigated. KEY MESSAGES: • This is the first Greek study, fully molecularly defining the beta and alpha mutational alleles in 217 patients with hemoglobinopathies of two large centers in Greece and correlating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). • In the beta thalassemic or SCD patients of our cohort, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course (confirmation of a well-known previous observation). • The inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype (confirmation of a well known previous observation). • The function or modification of possible regulatory genes should be investigated in cases in whom the genotype and phenotype did not correlate.

Hormone profiling and the root proteome analysis of itpk1 mutant seedlings of barley (Hordeum vulgare) during the red-light induced photomorphogenesis

Inositol phosphates represent crucial primary metabolites, known as phosphate storage molecules or molecules enabling protein interactions. Inositol phosphates and their pyrophosphate forms direct the assembly of the COP9 signalosome and Cullin Ring Ligase complexes. Thus, inositol phosphates participate on protein degradation, which is a means of signal transduction for several plant hormone classes and light signaling cascade. In plants, production of inositol phosphates is maintained by several inositol phosphate kinases including the group of inositol-1,3,4-trisphosphate 5/6 kinases (ITPKs). The members of ITPKs connect plant phosphate status with light perception and hormonal balance. Photomorphogenesis in red light was investigated using allelic variants of barley Hvitpk1 mutants produced by gene editing. Barley ITPK promoter regions, conserved protein motifs, and expression profiles were investigated to characterize their evolutionary diversification and transcriptional regulation. We focused on ITPK1 relation to auxins, cytokinins, gibberellins, jasmonates, abscisic acid, and salicylic acid in red light-induced photomorphogenesis. Global proteome analyses was conducted in the mutant and wild-type (WT) seedlings grown under continuous red light. Our results demonstrate that different allelic variants of itpk1 mutants manifested defects in producing important phytohormones with consequences for root growth. Mutations in itpk1 affected hormone biosynthesis and metabolite, while proteome analysis revealed significant differences in seedling root proteomes between mutant lines. Based on our results and published data, a model of ITPK activity is proposed depicting them as regulators of hormone signal transduction at the level of protein degradation.

Interaction of Wild Type and V804l and V804m-Mutated Ret Protein Kinase with Emodin: In Silico Approach

Medullary thyroid carcinoma (MTC) is a malignant endocrine tumor originating from parafollicular calcitonin-producing C cells. The proto-oncogene RET (REarranged during Transfection) is known as the main actor in the development and outbreak of MTC. Gain of function mutations of RET constitutively activate the receptor which are found to be responsible for the high percentage of MTC cases. The two FDA-approved drugs used for MTC, vandetanib and cabozantinib, are resistant to two mutant variants of RET which are V804L and V804M. In this study, the interactions of emodin, a natural molecule found in plants, with wild-type as well as V804L and V804M-mutated RET kinase were investigated via molecular docking. Pymol was used to create point mutations on wild type RET. Vandetanib and cabozantinib were used as the reference drugs. The binding free energy of vandetanib with wild-type RET, V804L and V804M variants were found to be -9.3, -9.1 and -8.6 kcal/mol. Similarly, the binding free energy of cabozantinib with wild-type RET, V804L and V804M variants were found to be -10.6, -10.4, and -9.5 kcal/mol, respectively. Clearly, the binding affinity of vandetanib and cabozantinib to RET kinase was found to be reduced in mutated variants as compared to wild type. In the meantime, the binding energy between emodin and wild-type RET was shown to be -9.3 kcal/mol. Interestingly, the binding affinity of emodin to V804L and V804M variants was determined to be increased (-9.9 and -9.8 kcal/mol, respectively) compared to wild type. Furthermore, many H-bonds and hydrophobic interactions between emodin and mutated RET variants were shown. Therefore, strong binding affinity of emodin to wild-type and the mutated variants of RET was suggested in this study. In conclusion, emodin was found to be a potential molecule to inhibit RET kinase activity and could be used as a therapeutical agent against medullary thyroid carcinoma.