Variant V6 Research Articles

Reactive transport model of the long-term geochemical evolution in a HLW repository in granite at the disposal cell scale: Variants, sensitivities, and model simplifications

The assessment of the long-term performance of the engineered barrier systems of high-level radioactive waste (HLW) repositories requires the use of reactive transport models. Montenegro et al. (2023) presented a non-isothermal reactive transport model of the long-term geochemical evolution of a HLW disposal cell in a granitic host rock corresponding to a generic reference concept. The model accounted for the vitrified waste, the carbon-steel canister, the bentonite buffer and the reference granitic rock. Here we extend their model by considering model variants (V), sensitivity cases (SC) and model abstractions (MA). Variants V1, V2 and V3 consist of considering MX-80 bentonite instead of FEBEX bentonite (V1), a larger groundwater flux through the granite (V2) and the Czech reference crystalline rock as a host rock (V3). Cases SC1 and SC2 consider a decrease of the silica concentration threshold value in the glass dissolution rate (SC1) and an earlier canister failure time (SC2), respectively. Runs MA1 to MA4 consider smectite as an unreactive mineral phase (MA1), the porosity feedback effect on chemical and transport parameters (MA2), a time-varying corrosion rate (MA3), and a coarser finite element grid (MA4), respectively. Model results of V1 show a larger pH, a smaller precipitation of magnetite, siderite and greenalite and a slightly smaller dissolution of ISG and smectite than the base run of Montenegro et al. (2023). Model predictions are very sensitive to the increase in the groundwater flow through the granitic host rock (V2). However, predictions are not sensitive to the chemical composition of the granite porewater (V3). The decrease in the silica saturation threshold from 1·10−3 to 5·10−4 mol/L in SC1 leads to a significant decrease in glass dissolution. Glass dissolution after 50,000 years in SC2 (earlier canister failure) is much larger than that of the base run. Model results are not sensitive to considering smectite as an unreactive mineral phase (MA1). However, model results are very sensitive to the porosity feedback effect (MA2). A 60% volume fraction of Fe(s) remains uncorroded after 50,000 years when a variable corrosion rate is considered in MA3. In this case the precipitation of corrosion products is much smaller than that of the base run. The general patterns of the numerical results in MA4 (coarser grid) are similar to those of the base case.

Delphi/AHP-Based Method for Biomass Sustainable Assessment in the Sugar Industry

Multi-criteria methods are highly attractive tools to address the inherent complexity of evaluating problems in various scientific areas. The combination of methods such as Delphi/AHP is emerging as a robust alternative to evaluate the sustainability of renewable energy sources. In this theoretical-descriptive research, the use of the Delphi method is proposed to select criteria and sub-criteria to obtain a high level of reliability, while the AHP method is used to establish an order of preference among the alternatives analyzed. This process requires the support of a committee of experts, whose role is to identify the various biomass alternatives that can be used in the sugar industry, considering aspects related to sustainability. The selected experts have identified energy, exergy, and emergetic indicators, in which economic, environmental, and social aspects are integrated. The multi-criteria analysis shows that the V1 variant was the most satisfactory in terms of biomass sustainability, representing 45% and 53% of the overall priorities in the evaluated case studies. In addition, the sensitivity analysis under an equal-weighted scenario for both study cases evidenced that variant V1 acquired the highest score (38.17%) among all alternatives. Variant V4 achieved the second highest score (31.79%), while alternative V2 achieved only 29.04%, respectively. The integration of Delphi/AHP methods emerges as a novel tool to assess sustainability in different industries of the energy sector.

Co-expression of CD44v6 and MMP2 predicts lung metastasis and unfavorable prognosis in osteosarcoma.

Aim: To evaluate the prognostic significance of CD44 variant v6 (CD44v6) and matrix metalloproteinases 2 (MMP2) expression in patients with surgically resected osteosarcoma.Methods: CD44v6 and MMP2 expression were immunohistochemically detected in 113 primary osteosarcoma patients at our institute between 2001 and 2019.Results: Both CD44v6 and MMP2 were independent predictors for metastasis-free and overall survival. An extended predictive range and improved sensitivity were observed when the combined effects of CD44v6 and MMP2 were considered. Specifically, patients with CD44v6+and MMP2+expression were more susceptible to lung metastasis and exhibited the poorest survival rates compared with the other groups.Conclusion: The combination of CD44v6 and MMP2 may serve as a precise prognostic indicator for predicting metastatic progression and survival outcomes in patients with osteosarcoma.

Modulation of PETase active site flexibility and activity on morphologically distinct polyethylene terephthalate substrates by surface charge engineering

Enzymatic hydrolysis of polyethylene terephthalate (PET) waste is a compelling strategy for environmentally friendly recycling of a major pollutant. Here, we investigate the effects of surface charge point mutations both proximal and distal to the active site of the mesophilic PET-degrading enzyme IsPETase and the thermostable V3 variant with superior activity. The vicinal K95A mutation significantly inhibited IsPETase activity on mechanically processed PET powder. Conversely, this mutation significantly increased hydrolysis of PET powder in the V3 PETase. Activity of both enzymes on PET film was inhibited by the K95A mutation, highlighting complex interplay between mutation context and substrate morphology. Further installing the distal R132N and R280A surface charge mutations potentiated activity of V3 on all substrates tested. This variant afforded 100 % degradation of pre-processed bottle-grade PET powder in 3 days at 40°C reaction temperature, a 3-fold improvement over IsPETase. Whilst reduction of positive charge on the PETase surface is known to reduce interaction with PET, molecular dynamics simulations suggest this can be offset by context-dependent modulation of active site flexibility, which differentially impacts both hydrolysis of morphologically distinct PET substrates and the concentration-dependent inhibition phenomenon observed for PETase.

Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality.

Signal recognition particles (SRPs) are essential for regulating intracellular protein transport and secretion. Patients with tumors with high SRP9 expression tend to have a poorer overall survival. However, to the best of our knowledge, no reports have described the relationship between SRP9 localization and prognosis in pancreatic cancer. Thus, the present study aimed to investigate this relationship. Immunohistochemical staining for SRP9 using excised specimens from pancreatic cancer surgery cases without preoperative chemotherapy or radiotherapy showed that SRP9 was preferentially expressed in the nucleus of the cancerous regions in some cases, which was hardly detected in other cases, indicating that SRP9 was transported to the nucleus in the former cases. To compare the prognosis of patients with SRP9 nuclear translocation, patients were divided into two groups: Those with a nuclear translocation rate of >50% and those with a nuclear translocation rate of ≤50%. The nuclear translocation rate of >50% group had a significantly better recurrence‑free survival than the nuclear translocation rate of ≤50% group (P=0.037). Subsequent in vitro experiments were conducted; notably, the nuclear translocation rate of SRP9 was reduced under amino acid‑deficient conditions, suggesting that multiple factors are involved in this phenomenon. To further study the function of SRP9 nuclear translocation, in vitro experiments were performed by introducing SRP9 splicing variants (v1 and v2) and their deletion mutants lacking C‑terminal regions into MiaPaCa pancreatic cancer cells. The results demonstrated that both splicing variants showed nuclear translocation regardless of the C‑terminal deletions, suggesting the role of the N‑terminal regions. Given that SRP9 is an RNA‑binding protein, the study of RNA immunoprecipitation revealed that signaling pathways involved in cancer progression and protein translation were downregulated in nuclear‑translocated v1 and v2. Undoubtedly, further studies of the nuclear translocation of SRP9 will open an avenue to optimize the precise evaluation and therapeutic control of pancreatic cancer.

Biological activities of gaba-producing probiotics from fermented panchagavya

Abstract Panchagavya, a venerable elixir from the Ayurvedic tradition, merits scholarly inspection. It denotes the sacred offerings of cow’s milk, urine, dung, ghee and curd, holding medicinal import within Ayurveda’s ancient practices. This study aims to illuminate differences between probiotic variants V2 and V7, through findings on their pharmacological applications and biochemical concentrations. It is proposed that fermented panchagavya’s psychobiotic strains may exhibit favorable reactions worth examining. Chiefly, the organic synthesis of neurotransmitters by beneficial bacteria is of interest. The regulatory neurotransmitter gamma-aminobutyric acid (GABA) could potentially be generated by a panchagavya-derived probiotic. Through serial dilution culture of bacteria obtained from fermented panchagavya, their growth and GABA formation, as evaluated by thin-layer chromatography and ultraviolet-visible spectrometric analysis, are observed. Strain V2 demonstrates promising pharmacological properties including antioxidant, anti-inflammatory, anti-diabetic and anti-obesity effects, as well as inhibition of albumin denaturation and heat-induced hemolysis. It presents a higher concentration of such activities than V7. Biochemical tests further indicate V2’s larger percentage of GABA synthesis, supporting prospective therapeutic applications. After sequencing selected strains to explore psychobiotic functioning, their isolation holds medical import as GABA-producing beneficial bacteria from fermented panchagavya.

Electronically Temperature-Dependent Interplay between He and Trivacancy in Tungsten Plasma-Facing Materials.

Both microvoids and helium (He) impurities are widely present in tungsten (W) plasma-facing materials (PFMs), where the interaction between microvoids and He atoms has led to the intriguing development of microvoids. In this paper, we comprehensively investigated the interaction between He atoms and trivacancy (V3), a fundamental microvoid in W-PFMs, at the level of tight-binding theory. Our study showed that He atoms can catalyze the decomposition of the original V3 or facilitate its transformation into another V3 variant. We propose that a He atom near the V3 defect induces significant changes in the distribution of d-electron charges within the W atoms lining the inner wall of the V3 defect, making the W atom nearest to this He atom cationic and the other W atoms anionic. The attractive interaction between them promotes the decomposition and deformation of V3. As electronic excitation increases, the ionization of W atoms on the V3 wall gradually intensifies, thereby enhancing the cationic characteristics of the W atoms closest to the He atom. This process also prompts other W atoms to shift from anions to cations, leading to a transition in the electrostatic interactions between them from attraction to repulsion. This transformation, driven by electronic excitation, plays a significant inhibitory role in the decomposition and deformation of V3.

Reversible reorientation of martensite variants in stress-induced martensite aged Co35Ni35Al30 single crystals

The paper reports the temperature dependence of the reversible reorientation of martensite variants during loading/unloading along the [001]B2||[001]L10-direction in compression in stress-induced martensite (SIM) aged at 423 K for 0.5 h under a compressive stress of 500 MPa applied along the [110]B2||[100]L10-direction Co35Ni35Al30 single crystals. A wide temperature range of reversible strain from 203 K to 413 K (210 K) is observed due to the stabilisation of the tetragonal L10-martensite variant V1 ([001]B2||[001]L10) during SIM-aging in these single crystals. The reversible strain in the range of 157 K (203 K<Tt<As=Af=360 K) is because of the reorientation of the L10-martensite variants V1-V2/V3. The superelasticity at Tt>Af=360 K occurred due to the stress-induced B2-L10(V2/V3) martensitic transformation (MT). Rubber-like behaviour develops in the range of 52 K (203 K<Tt<Mf*=255 K) and is characterised by a narrow stress hysteresis of 13 MPa and a reversible strain of −12.0 % due to the movement of the twinning boundaries (reversible twinning) in L10-martensite. The reversible strain reaches −10.2 % in the temperature range from Mf*=255 K to As=Af=360 K. This strain is associated with a new mechanism of L10-martensite variant reorientation, which occurs through a sequence of reverse and forward stress-induced L10(V1)-B2-L10(V2/V3) MTs and leads to the stages on the stress–strain curves. The reverse L10(V1)-B2 MT at loading and forward B2-L10(V1) MT at unloading on the first stage of the stress–strain curves are characterised by low critical stresses of 8–23 MPa, a narrow stress hysteresis of 3–19 MPa and a completely reversible strain of −6.0 %, which is stable for 100 loading/unloading cycles.

Clinical difference on the variants and co-mutation in a Chinese cohort with ALK-positive advanced non-small cell lung cancer.

Despite the generally favourable prognoses observed in patients with ALK-positive non-small cell lung cancer (NSCLC), there remains significant variability in clinical outcomes. The objective of this study is to enhance patient stratification by examining both the specific sites of gene fusion and the presence of co-occurring mutations. We collected retrospective clinical and pathological data on ALK-positive patients with locally advanced or metastatic disease. ALK fusion variants and concomitant mutations were identified through next-generation sequencing technology. We then assessed treatment efficacy via tumor response and survival metrics. This study included a total of 59 patients, with 49 harboring echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions and 10 presenting with rare fusions. The median follow-up period was 33 months. Clinical outcomes between non-EML4-ALK and EML4-ALK patients were comparable. Among the EML4-ALK cohort, patients with longer variants (v1, v2, v8) demonstrated superior progression-free survival (PFS) (median PFS: 34 months vs. 11 months; hazard ratio [HR]: 2.28; P = 0.05) compared to those with shorter variants (v3, v5). Furthermore, patients treated with second-generation ALK inhibitors (ALKi) displayed a progression-free survival advantage (median PFS: not reached [NR] vs. 9 months; HR: 5.37; P = 0.013). Baseline TP53 co-mutation were linked with a substantially shorter OS (median OS,37 months vs. NR; HR 2.74; P = 0.047). In ALK+ NSCLC, longer EML4-ALK variants correlate with improved prognosis and enhanced response to second-generation ALKi, while TP53 co-mutations indicate a negative prognosis.

Abstract 5163: Determining the role of septin expression in response of breast cancer patients to microtubule targeted chemotherapy

Abstract Drugs that disrupt the microtubule cytoskeleton, including both microtubule stabilizing taxanes and destabilizers like eribulin, are regularly used in the treatment of metastatic breast cancer. However, there are currently no molecular biomarkers to guide their use. Septins, a class of small GTPases, interact with both microtubules and actin to functionally link these cytoskeletal components and regulate cell biological processes shown to be critical for the invasion and migration of cancer cells. In particular, septin 9 (SEPT9) directly interacts directly with microtubules through an N-terminal domain on the longest isoform, SEPT9i1, which is sufficient to promote migration and invasion of breast cancer cells in vitro. We hypothesized that the relative expression of septin9 isoforms in breast tumors could play a role in their response to treatment with microtubule-targeted chemotherapy, particularly in the metastatic setting. A retrospective investigation of the relationship between septin expression and response to taxane chemotherapy was conducted utilizing full transcriptome sequencing data from biopsies of 75 breast cancer patients treated at our NCI-Designated Mays Cancer Center who received molecular profiling through Caris Life Sciences. Levels of total SEPT9 expression as well as the expression of specific variants that encode mechanistically distinct septin9 isoforms, SEPT9v1, v2, and v3, were determined for each sample and correlated with demographic, treatment, and outcome data for these patients. The oncogenic sept9_v1 variant (which produces the septin9_i1 isoform) was detected in over half of tumors and those with the highest total septin9 expression also expressed significantly higher levels of this isoform. Strikingly, the expression of total septin9, as well as the v3 variant, was significantly increased in taxane-treated patients who survived over a year after diagnosis with metastatic breast cancer, suggesting that septin9 expression could be a molecular correlate of response to taxane chemotherapy. We found that the expression of total septin9 and its variants was not significantly correlated with race, ethnicity, or cancer type suggesting that our findings would apply to our patient population as a whole, which is over 50% Hispanic. These data were used to support an ongoing prospective study to determine effect of septin expression on response to taxane chemotherapy in the neoadjuvant setting and determine the effect of taxane treatment on septin expression by comparing samples pre and post treatment. Together this work supports our overall goal of identifying predictive biomarkers to facilitate the use of microtubule targeted chemotherapy in a more personalized manner. Citation Format: Tamarah Aldawoodi, Jacob Nathaniel Essif, Amna Naqvi, Lauren D. Boyle, Daniela Urueta Portillo, Kate Lathrop, April L. Risinger. Determining the role of septin expression in response of breast cancer patients to microtubule targeted chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5163.

NanoLuc Binary Technology as a methodological approach: an important new tool for studying the localization of androgen receptor and androgen receptor splice variant V7 homo and heterodimers

BackgroundThe androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT). The clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. Clinical failure of ADT is often characterized by the synthesis of a constitutively active AR splice variant, termed AR-V7. AR-V7 mRNA expression is considered as a resistance mechanism following ADT. AR-V7 no longer needs androgenic stimuli for nuclear entry and/or dimerization.MethodsOur goal was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology under androgen stimulation and deprivation conditions.ResultsOur data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 homodimers localize in the nucleus. However, after androgen stimulation, all the AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers were localized in the nucleus.ConclusionsWe showed that AR-FL and AR-V7 form heterodimers that localize to the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus.

Energy Sustainability Indicators for the Use of Biomass as Fuel for the Sugar Industry

There are numerous analytical and/or computational tools for evaluating the energetic sustainability of biomass in the sugar industry. However, the simultaneous integration of the energetic–exergetic and emergetic criteria for such evaluation is still insufficient. The objective of the present work is to propose a range of indicators to evaluate the sustainability of the use of biomass as fuel in the sugar industry. For this purpose, energy, exergy, and emergy evaluation tools were theoretically used as sustainability indicators. They were validated in five variants of different biomass and their mixtures in two studies of technologies used in Cuba for the sugar industry. As a result, the energy method showed, for all variants, an increase in efficiency of about 5% in the VU-40 technology compared to the Retal technology. There is an increase in energy efficiency when considering AHRs of 2.8% or Marabu (Dichrostachys cinerea) (5.3%) compared to the V1 variant. Through the study of the exergetic efficiency, an increase of 2% was determined in both technologies for the case of the V1 variant, and an increase in efficiency is observed in the V2 variant of 5% and the V3 variant (5.6%) over the V1 variant. The emergetic method showed superior results for the VU-40 technology over the Retal technology due to higher fuel utilization. In the case of the V1 variant, there was a 7% increase in the renewability ratio and an 11.07% increase in the sustainability index. This is because more energy is produced per unit of environmental load.

Extensive Genetic Diversity and Epidemiological Patterns of Factor H-Binding Protein Variants among Neisseria meningitidis in China.

Factor H-binding protein (fHbp) is a virulence factor expressed by Neisseria meningitidis (N. meningitidis), the primary causative agent of invasive meningococcal disease (IMD) in humans. fHbp is utilized as the main component in vaccines to provide protection against IMD caused by serogroup B N. meningitidis. In order to comprehensively investigate the genetic diversity and epidemiological patterns of fHbp variants within isolates of Chinese N. meningitidis, we utilized the NEIS0349 locus, which encompasses the complete coding sequences of fHbp. This enabled us to identify allelic variants of fHbp with enhanced resolution. A total of 109 fHbp variants were identified in 1013 Chinese N. meningitidis isolates. We reconstructed a phylogenetic tree and analyzed the epidemiological characteristics of each variant. Considering both temporal and geographical distribution patterns, only four fHbp variants (v2.16, v2.18, v2.404, and v2.21) exhibited persistent nationwide prevalence during the previous decade (2011-2021). These variants were highly prevalent in both serogroup B strains from patients and healthy individuals, suggesting their potential as suitable vaccine candidates for nationwide implementation against IMD caused by serogroup B strains. Our study emphasizes the significance of conducting continuous surveillance of meningococcal strains to monitor the genetic diversity of fHbp for the purpose of vaccine development.

GRAINS MOISTURE VARIATION IN FIELD PEAS IN RELATION TO FERTILIZATION

The study evaluated the moisture content of field pea’s grains at the time of harvest, in relation to mineral fertilization. The field researches took place within ARDS Lovrin. The field pea, 'Boxer' variety, was cultivated in non-irrigated conditions, on a chernozem type soil. Mineral fertilization was done with nitrogen (0, 25, 75 kg ha-1 a.s.; a.s. – active substance), phosphorus (0, 80 kg ha-1 a.s.) and potassium (0, 40, 80, 120 kg ha-1 a.s.). The combination of the three fertilizers resulted in 16 fertilization options (V1 to V16). At the moment of physiological maturity, BBCH code 9, the plants on the experimental variants were harvested. On pea grains samples, on the experimental variants, the humidity (M, %) was determined with the Wile 200 – Granit device. The moisture values varied between 13.70% (V3) and 17.90% (V13). The grain moisture values showed a normal distribution (r = 0.975). Compared to the average value (MAvg = 16.36%), higher values were recorded for variants V1, V4, V7, V9, V11, V12, V13, V14. The cluster analysis facilitated the generation of a dendrogram in which the variants were grouped based on Euclidean distances (Coph. corr. = 0.739). A cluster C1 included the variants with low moisture content, within which a high level of similarity was recorded between V5 and V8 (SDI = 0.00). Within cluster C2, the variants with higher moisture content were grouped, and a high level of similarity was recorded between variants V4 and V11 (SDI = 0.00). The model in the form of equations and graphic models resulted from the regression analysis, which described the variation of grain moisture depending on the applied fertilizers. Divergent action was recorded between nitrogen and phosphorus, respectively potassium in relation to grain moisture values.

Evaluation of different lines of vegetable soybean Glycine max L. Merr. under conditions of the Moscow region

Edamame is a special soybean ( Glycine max (L.) Merr .) harvested as a vegetable when the seeds are immature (R6 and R7 stage) and have expanded to fill 80 to 90 percent of the pod width. The study was conducted in the experimental field of Federal Scientific Vegetable Center in the Moscow Region in 2020-2022 and Agro-biotechnological department, Agrarian and Technological Institute, RUDN University. The object of research was 3 accessions of soybean ( Glycine max L.): accession A, accession F, Hidaka and Lira. Before sowing, soybean seeds were inoculated with Optimays 400 ( Bradyrhizobium japonicum ) and Biobesta ( Sinorhizobium fredii ). In the field experiment, 10 soybean samples were taken from each plot at the stage of biological ripeness to determine plant height (cm), number of pods per plant, number of stems per plant, stem width (mm), average number of seeds per plant, weight of 1000 seeds (g) and yield (t/ha). The experiment was designed in a complete random block with a factorial arrangement. Thus, we had 4 accessions and 3 treatments (C - control, V1 - Bradyrhizobium japonicum and V2 - Sinorhizobium fredii ) with 3 replacations. The study showed that accession A had the highest average yield per hectare (2.70 t/ha) and weight of 1000 seeds (159 g). Under conditions of the Moscow region, variant V2 ( Sinorhizobium fredii ) had the highest average yield (2.57 t/ha) and weight of 1000 seeds (100.25 g). Therefore, more than 90 % of the results obtained for these two indicators are associated with the use of V2 treatment. For the rest of the studied indicators, no significant differences were found, and they did not affect the efficiency indicators. The combination of accession A and treatment V2 led to the highest average yield (2.99 t/ha). The product based on Sinorhizobium fredii showed the best indicators for all the studied samples.

An Atypical Somatic Calr Variant Acquired in Cis to a Germline Calr variant Induces Constitutive Signaling of the Thrombopoietin Receptor and Results in Essential Thrombocythemia

Acquired mutations in the gene encoding the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are responsible for ~ 30% of essential thrombocythemia (ET) and myelofibrosis (MF) cases. Two CALR mutations, a deletion of 52 base pairs (del52) and an insertion of 5 base pairs (ins5) are the most frequent in ET and MF patients. All CALR mutations result in a +1-frameshift leading to 1) the replacement of the negatively-charged wild-type (WT) C-terminus that binds calcium in the ER by a positively-charged mutant tail including 44 residues that are common to all CALR mutant proteins and 2) the loss of the ER retention KDEL; together leading to the deregulation of calcium homeostasis. The CALR mutants are also secreted and bind to MPL in the ER but induce its constitutive dimerization and downstream signaling pathways at the cell surface. A 16-year-old girl presented with a platelet count of 797 x 10 3/mL rising to 1,228 x 10 3/mL within 2 years. After Informed Consent, we detected two atypical CALR variants in her blood cells by NGS analysis: V1 (c.1122-1125del) at 47% variant allele frequency (VAF) and V2 (c.1227-1231del) in cis of the V1 leading to V1-V2 (c.1122-1125del ; c.1227-1231del) at 5% VAF. The presence of V1 in blood and nails from the propositus and her healthy mother confirmed its germline origin. The V1-V2 was only found in the propositus at 5% in isolated monocytes and at very low levels in NK and B cells, confirming its somatic acquisition. The germline V1 induces a frameshift resulting in the replacement of the CALR WT C-terminus by the positively charged 44 residue-long C-terminal mutant tail present in del52 and ins5 and the loss of KDEL together with an insertion of 10 amino acids not present in CALR del52 nor ins5. In contrast, the V1-V2 generates a similar but shortened C-terminus containing the Arg-rich MPL binding site and KDEL (Fig. 1A). To investigate the functional impact of these CALR variants, we cloned V1 and V1-V2 with a N-terminal hemagglutinin (HA) tag as well as CALR WT and del52 as controls in a retroviral plasmid. Viral particles were produced to transduce Ba/F3 cell lines expressing MPL. Structural modeling using AlphaFold was used to investigate the role of KDEL restoration in V1-V2 in producing an oncogenic protein leading to ET. Both CALR WT and V1-V2 variants that contain the KDEL motif were detected to a similar extent in Ba/F3-MPL cell lysates. In contrast, del52 and V1, that lost the ER-retention KDEL motif, were barely detected in cell lysates. Unexpectedly, despite the presence of the KDEL motif, V1-V2 was found at a similar level than del52 at the Ba/F3-MPL cell surface. In contrast, V1 was barely detectable at the cell surface, like CALR WT. In order to investigate the role of the KDEL in V1-V2 cell surface expression, we generated a CALR WT lacking the KDEL and added the KDEL motif to del52. The presence of the KDEL motif stabilized the intracellular expression of del52 without affecting cell surface localization while the removal of the KDEL motif in CALR WT appeared to destabilize the protein and induce a slight leak to the cell surface. Structural modeling using AlphaFold predicted that the KDEL in V1-V2 structure is accessible to KDEL receptors, suggesting that the motif may be masked by the interaction of V1-V2 with MPL while trafficking to the cell surface. Indeed, cell-surface expression of V1-V2 was decreased in Ba/F3 that do not express MPL. We show that V1-V2, but not V1, constitutively activates the phosphorylation of STATs, ERK and AKT in Ba/F3-MPL. TPO further increases activation of STATs but not of AKT and ERK by V1-V2 and del52. Coherently, V1-V2, but not V1, sustained oncogenic MPL stimulation able to sustain cell autonomous Ba/F3-MPL cell proliferation (Fig. 1B). Both del52 and V1-V2 up-regulated BCL-2 mRNA. Dose-response cytotoxicity assays with the BCL-2 inhibitor venetoclax showed that these two mutants are sensitive to lower doses compared to CALR WT- or V1-expressing Ba/F3-MPL cells. This is the first report of a germline loss-of-function CALR variant (V1) expressed as an unstable protein and that might predispose to the acquisition of a somatic gain-of-function CALR variant (V1-V2) responsible for ET. The presence of KDEL in V1-V2 leads to stabilized CALR expression that does not prevent cell surface localization. Moreover, it leads to induction of MPL signaling and cytokine-independent cell growth.

Single nucleotide polymorphisms and their association with QTc and left ventricular function in peripartum cardiomyopathy

Abstract Background Peripartum cardiomyopathy (PPCM) is a form of pregnancy-associated heart failure that affects previously healthy women toward the end of pregnancy or in the months following delivery. PPCM is associated with left ventricular (LV) non-recovery in about half of patients, with 10% dying within the first year after diagnosis. Recent clinical investigations have also shown that about 50% of patients with PPCM have a prolonged QTc at presentation, which was a predictor of poor outcome (death, readmission, persistent poor LV function) by 6 months. The mechanism of QT prolongation in PPCM, however, remains poorly understood. In this study, we tested the hypothesis that SNPs, known to be associated with long QT syndrome (LQTS), could be used to predict the QT intervals in patients with PPCM. Methods We recruited forty-three patients with PPCM and genotyped them for 12 single nucleotide polymorphisms (SNPs) known to have an association with the QT interval: AGT (rs699); NOS1AP (rs16847548); CYP11B2 (rs1799998); NOS1AP (rs4657139); AKAP9 (rs7808587); AKAP9 (rs2961024); AKAP9 (rs11772585); AGTR1 (rs5186); AKAP9 (rs2282972); KCNQ1 (rs2074238); KCNH2 (rs1805123); ACE (rs4646994). These variants were correlated with clinical and echocardiographic parameters, as well as the QT interval, as corrected by the Bazett’s formula (QTcB). Results The median age of this cohort was 30.4 years (IQR 25.3-34.9). More than half (74%) of patients presented with a New York Heart Association (NYHA) functional class III or IV. Overall, the median LVEDD at presentation was 58mm (IQR 53-63), LVEF 31% (IQR 24-36) and QTcB (in lead V3) 450ms (IQR 429-476). SNP analysis showed that 40% of the PPCM cohort that had the NOS1AP (rs16847548) variant were associated with significantly longer QTc in V3 (471ms [443-480] vs 441ms [426-458], p=0.028; the NOS1AP (rs16847548) variant was also associated with both prolonged QTc and LVEF &amp;lt; 35% (47.1 vs 15.4%, p=0.024). Our results also showed that PPCM patients with the AKAP9 (rs2282972) variant had a shorter QTc in lead II (468ms [425-490] vs 431ms [415.0-459.5], p=0.012), a smaller LVEDD (63.0 [57.0-67.5] vs 57.0 [53.0-59.5]; p= 0.022) and were likely to have both a shorter QTc and LVEF &amp;lt; 35% (57.1 vs 24.1, p=0.033). None of the other SNPs showed any significance. Conclusion Our study found that, in a cohort of PPCM patients, the NOS1AP (rs16847548) variant is associated with a prolonged QTc and the AKAP9 (rs2282972) variant is associated with a shorter QTc interval. As prolonged QTc and severely impaired LV function are well-known predictors of poor outcome, we postulate that NOS1AP could be used a marker of outcome in PPCM patients. We also postulate that the AKAP9 variant, known to be associated with a decreased risk for a cardiac event, might also indicate a decreased the risk in a certain subset of our PPCM patients. However, a larger study is needed to determine whether these SNPs may have a prognostic value in PPCM.

ВЛИЯНИЕ ВЫСОКОШТАМБОВЫХ ФОРМИРОВОК НА ПЛОДОНОСНОСТЬ, УРОЖАЙ И КАЧЕСТВО ВИНОГРАДА СОРТА БОЛГАР

The effect of different high-stem formations and winter bud load during pruning on the fertility, yield and quality of grapes of the Bolgar variety was studied. It was found that the formations, the fruiting unit type and winter bud load had a significant effect on the studied indicators of actual fertility, mass and structure of the cluster, mass per 100 grains, sugar content, titratable acids and yield. With comparatively higher values of most of these indicators, had the variants V4 – Geneva double curtain with planting distances of 4.0×1.0 m and pruning loads of 4 plugs of 2 winter buds and 1 fruit rod of 8 winter buds and V3 – Single curtain with planting distances of 3.2×1.2 m and pruning loads of 6 plugs of 2 winter buds and 2 fruit rods of 9 winter buds.

A Competitive High-Throughput Screening Platform for Designing Polylactic Acid-Specific Binding Peptides.

Among biobased polymers, polylactic acid (PLA) is recognized as one of the most promising bioplastics to replace petrochemical-based polymers. PLA is typically blended with other polymers such as polypropylene (PP) for improved melt processability, thermal stability, and stiffness. A technical challenge in recycling of PLA/PP blends is the sorting/separation of PLA from PP. Material binding peptides (MBPs) can bind to various materials. Engineered MBPs that can bind in a material-specific manner have a high potential for material-specific detection or enhanced degradation of PLA in mixed PLA/PP plastics. To obtain a material-specific MBP for PLA binding (termed PLAbodies ), protein engineering of MBP Cg-Def for improved PLA binding specificity is reported in this work. In detail, a 96-well microtiter plate based high-throughput screening system for PLA specific binding (PLABS) was developed and validated in a protein engineering (KnowVolution) campaign. Finally, the Cg-Def variant V2 (Cg-Def S19K/K10L/N13H) with a 2.3-fold improved PLA binding specificity compared to PP was obtained. Contact angle and surface plasmon resonance measurements confirmed improved material-specific binding of V2 to PLA (1.30-fold improved PLA surface coverage). The established PLABS screening platform represents a general methodology for designing PLAbodies for applications in detection, sorting, and material-specific degradation of PLA in mixed plastics.

Novel Inhibitors of androgen receptor's DNA binding domain identified using an ultra-large virtual screening.

Androgen receptor (AR) inhibition remains the primary strategy to combat the progression of prostate cancer (PC). However, all clinically used AR inhibitors target the ligand-binding domain (LBD), which is highly susceptible to truncations through splicing or mutations that confer drug resistance. Thus, there exists an urgent need for AR inhibitors with novel modes of action. We thus launched a virtual screening of an ultra-large chemical library to find novel inhibitors of the AR DNA-binding domain (DBD) at two sites: protein-DNA interface (P-box) and dimerization site (D-box). The compounds selected through vigorous computational filtering were then experimentally validated. We identified several novel chemotypes that effectively suppress transcriptional activity of AR and its splice variant V7. The identified compounds represent previously unexplored chemical scaffolds with a mechanism of action that evades the conventional drug resistance manifested through LBD mutations. Additionally, we describe the binding features required to inhibit AR DBD at both P-box and D-box target sites.