Combination Of Mutations Research Articles

Specific sets of geranylgeranyl diphosphate synthases and phytoene synthases control the production of carotenoids and ABA in different tomato tissues.

Plant carotenoids are plastid-synthesized isoprenoids with roles as photoprotectants, pigments, and precursors of bioactive molecules such as the hormone abscisic acid (ABA). The first step of the carotenoid biosynthesis pathway is the production of phytoene from geranylgeranyl diphosphate (GGPP), catalyzed by phytoene synthase (PSY). GGPP produced by plastidial GGPP synthases (GGPPS) is channeled to the carotenoid pathway by direct interaction of GGPPS and PSY enzymes. Three plastid-localized GGPPS isoforms (referred to as SlG1-3) and three PSY enzymes (PSY1-3) are present in tomato (Solanum lycopersicum). Our previous work showed that SlG1 and PSY3 function together in the roots, whereas the rest of the isoforms are required in aerial tissues. Here we generated and analyzed combinations of double mutants lacking PSY1 or PSY2 and SlG2 or SlG3 to investigate the contribution of specific GGPPS and PSY pairs to the production of carotenoids and ABA in different tissues of the tomato plant. Despite that the loss of individual enzymes was found to trigger compensatory mechanisms that complicate interpretation of the results, the results confirm a major role for SlG3 in providing GGPP to PSY2 for housekeeping carotenoid biosynthesis in leaves, whereas SlG2 and PSY1 become most relevant when a more active production is required in flowers and breaker fruits, i.e., at the onset of ripening. We could also confirm that ABA production in the fruit pericarp is more dependent on PSY1 activity than on total carotenoid levels and that fruit size correlates with ABA levels accumulated in ripe rather than breaker fruits.

ATRX mutation modifies the DNA damage response in glioblastoma multiforme tumor cells and enhances patient prognosis.

The presence of specific genetic mutations in patients with glioblastoma multiforme (GBM) is associated with improved survival outcomes. Disruption of the DNA damage response (DDR) pathway in tumor cells enhances the effectiveness of radiotherapy drugs, while increased mutational burden following tumor cell damage also facilitates the efficacy of immunotherapy. The ATRX gene, located on chromosome X, plays a crucial role in DDR. The aim of this research is to elucidate the correlation between ATRX mutations and GBM. Dataset obtained from TCGA-GBM were conducted an analysis on the genomic features, biological characteristics, immunopathological markers, and clinical prognosis of patients carrying ATRX mutations. Our findings revealed a significantly elevated level of microsatellite instability in individuals with ATRX mutants, along with significant alterations in the receptor-tyrosine kinase (RTK)-ras pathway among patients exhibiting combined ATRX mutations. TCGA-GBM patients with concurrent ATRX mutations exhibited sensitivity to 26 chemotherapeutic and anticancer drugs, which exerted their effects by modulating the DDR of tumor cells through highly correlated mechanisms involving the RTK-ras pathway. Additionally, we observed an enrichment of ATRX mutations in specific pathways associated with DDR among TCGA-GBM patients. Our model also demonstrated prolonged overall survival in patients carrying ATRX mutations, particularly showing strong predictive value for 3- and 5-year survival rates. Furthermore, additional protective factors such as younger age, female gender, combined IDH mutations, and TP53 mutations were identified. The results underscore the protective role and prognostic significance of ATRX mutations in GBM as a potential therapeutic target and biomarker for patient survival.

Sterol-Targeted Laboratory Evolution Allows the Isolation of Thermotolerant and Respiratory-Competent Clones of the Industrial Yeast Saccharomyces cerevisiae.

Sterol composition plays a crucial role in determining the ability of yeast cells to withstand high temperatures, an essential trait in biotechnology. Using a targeted evolution strategy involving fluconazole (FCNZ), an inhibitor of the sterol biosynthesis pathway, and the immunosuppressant FK506, we aimed to enhance thermotolerance in an industrial baker's yeast population by modifying their sterol composition. This approach yielded six isolates capable of proliferating in liquid YPD with μmax values ranging from 0.072 to 0.236 h-1 at 41.5°C, a temperature that completely inhibits the growth of the parental strain. The clones were categorised into two groups based on their respiratory competence or deficiency, the latter associated with mtDNA loss, an event seemingly linked to FCNZ and heat tolerance. Genome sequencing and ploidy-level analysis of all strains revealed aneuploidies, copy number variations (CNVs), and single nucleotide polymorphisms (SNPs). Notably, all evolved clones exhibited specific point mutations in MPM1 (P50S) and PDR1 (F749S). CRISPR-Cas9 experiments confirmed the role of the pdr1F749S mutation in the FCNZ-tolerance phenotype and demonstrated that Mpm1 is required for growth at high temperatures. However, no apparent heat tolerance benefit was observed from single or combined mutations in these genes, supporting the hypothesis that thermotolerance is mediated by multiple interacting mechanisms. In this context, all evolved clones exhibited altered sterol profiles, with differences observed between respiratory-competent and -deficient strains. In conclusion, our experimental evolution generated thermotolerant and fully competent strains and identified factors that could influence fluconazole and heat growth.

Biofoundry-Assisted Golden Gate Cloning with AssemblyTron.

Golden Gate assembly is a requisite method in synthetic biology that facilitates critical conventions such as genetic part abstraction and rapid prototyping. However, compared to robotic implementation, manual Golden Gate implementation is cumbersome, error-prone, and inconsistent for complex assembly designs. AssemblyTron is an open-source python package that provides an affordable automation solution using open-source OpenTrons OT-2 lab robots. Automating Golden Gate assembly with AssemblyTron can reduce failure-rate, resource consumption, and training requirements for building complex DNA constructs, as well as indexed and combinatorial libraries. Here, we dissect a panel of upgrades to AssemblyTron's Golden Gate assembly capabilities, which include Golden Gate assembly into modular cloning part vectors, error-prone polymerase chain reaction (PCR) combinatorial mutant library assembly, and modular cloning indexed plasmid library assembly. These upgrades enable a broad pool of users with varying levels of experience to readily implement advanced Golden Gate applications using low-cost, open-source lab robotics.

The scramblases VMP1 and TMEM41B are required for primitive endoderm specification by targeting WNT signaling.

The ER-resident proteins VMP1 and TMEM41B share a conserved DedA domain, which confers lipid scramblase activity. Loss of either gene results in embryonic lethality in mice and defects in autophagy and lipid droplet metabolism. To investigate their role in pluripotency and lineage specification, we generated Vmp1 and Tmem41b mutations in mouse embryonic stem cells (ESCs). We observed that ESCs carrying mutations in Vmp1 and Tmem41b show robust self-renewal and an unperturbed pluripotent expression profile but accumulate LC3-positive autophagosomes and lipid droplets consistent with defects in autophagy and lipid metabolism. ESCs carrying combined mutations in Vmp1 and Tmem41b can differentiate into a wide range of embryonic cell types. However, differentiation into primitive endoderm-like cells in culture is impaired, and the establishment of extra-embryonic endoderm stem (XEN) cells is delayed. Mechanistically, we show the deregulation of genes that are associated with WNT signaling. This is further confirmed by cell surface proteome profiling, which identified a significant reduction of the WNT-receptor FZD2 at the plasma membrane in Vmp1 and Tmem41b double mutant ESCs. Importantly, we show that transgenic expression of Fzd2 rescues XEN differentiation. Our findings identify the role of the lipid scramblases VMP1 and TMEM41B in WNT signaling during extra-embryonic endoderm development and characterize their distinct and overlapping functions.

Targeted Directed Evolution of an α-L-Rhamnosidase on Hesperidin Through Six-Codon Combinatorial Mutagenesis

Targeted saturation mutagenesis at the residues located at the substrate-binding pocket for generating focused libraries has emerged as the technique of choice for enzyme engineering, but choosing the optimal residue number of the randomization site and the reduced amino acid alphabet to minimize the labor-determining screening effort remains a challenge. Herein, we propose the six-codon combinatorial mutagenesis (SCCM) strategy by using the BMT degeneracy codons encoding six amino acids with different chemical properties as the building blocks for the randomization of the amnio acid motif. SCCM requires only a small library of 646 clones for 95% coverage at the three-residue motif compared to conventional NNK degeneracy codons encoding all 20 canonical amino acids and requiring the screening of nearly 100,000 clones. SCCM generates a suitable number of mutant libraries, providing a new strategy for reducing the screening workload of saturated combination mutations in enzyme engineering. Using this approach, the α-L-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron had been successfully engineered for improving the hydrolytic activity on natural flavonoid diglycoside hesperidin via targeted directed evolution at the motifs positioning the entrance of the substrate-binding pocket. The results indicate that the conversion rates of the four mutants on hesperidin were increased by more than 30% compared with the wild type using whole-cell biotransformation. Moreover, the catalytic efficiency kcat/KM value of the mutant TM1-6-F5 was 1.4-fold higher than that of the wild type.

Gender and age characteristics of JAK2 V617F mutation allelic burden and CALR and MPL driver mutations detection during primary examination of patients with suspected myeloproliferative neoplasms

Background. Detection of a somatic mutation in the Janus kinase 2 (JAK2) gene, along with mutations in the CALR and MPL genes, is one of the main criteria for diagnosing Ph-negative myeloproliferative neoplasms (MN). At the same time, the JAK2 617F mutation is often associated with agedependent clonal hematopoiesis of undetermined potential (CHI). As a rule, the indicator reflecting the number of blood cells transformed by mutation – the mutant allelic burden (MA) of JAK2 617F in CHI does not exceed 1–2 %, however, there is no clear boundary between the level of this indicator separating CHI and MN. The previously described “JAK2 paradox”, according to which JAK2 617F positive CHI predominates in men, and for women this mutation is more associated with MN, suggests a different attitude to the diagnostic value of MA between men and women. A separate issue concerns the detection of low MA levels in combination with other MN driver mutations.Aim. To analyze the database of JAK2 617F mutation allelic burden quantitative assessment results and to identify CALR and MPL genes driver mutations in patients depending on their age and gender.Materials and methods. Data from records on the age and gender of 6210 patients (3061 men and 3149 women) were analyzed, in whom the JAK2 gene 617F somatic mutation in a quantitative format and MPL and CALR genes mutations were simultaneously determined. Of these, mutations were identified in 1226 women and 826 men with symptoms of MN. Statistical analysis was performed using Excel spreadsheets and Statistica 10 package.The results. The data obtained indicate that in women, compared to men, the frequency of the 617F mutation is higher in all age ranges. omen are also more likely to have mutations in the CALR and MPL genes. The gender and age dependence of identifying quantitative values of MA JAK2 617F differ significantly in the range of >1 and ˂ 1%. Also noteworthy is the fact of high detection (up to 17–27 %) of additional combined mutations in the CALR and MPL genes in patients with low MA JAK2 617F values.Conclusion. The data support the gender “JAK2 paradox”, which suggests differential interpretation of test results in men and women. To more clearly assess the characteristics of the diagnostic value of MAN JAK2 617F low levels and its dependence on the gender and age of patients, controlled multicenter clinical studies using standardized diagnostic test systems are needed.

The clinical features and prognostic implications of the co-mutated TP53 gene in advanced non-small cell lung cancer.

TP53 is a frequently mutated oncogene within non-small cell lung cancer (NSCLC). However, the clinical and prognostic significance of co-mutations in TP53 in patients with advanced NSCLC has not been fully elucidated. A total of 174 patients with advanced NSCLC were enrolled in this study. All patients were subjected to sequencing analysis of tumor-related genes and information such as PD-L1 expression, TMB, and co-mutation changes were collected. Patients were categorized into TP53 mutant and TP53 wild-type groups according to their TP53 mutation status and then statistically analyzed. TP53 mutations were the most common among all patients, accounting for 56.32%, followed by epidermal growth factor receptor mutations at 48.27%. The most common mutation sites in the TP53 mutation group were exons 5-8.TP53 mutations were significantly associated with PD-L1 and TMB levels. Univariate Cox analysis showed that gender and EGFR mutation affected the prognosis of TP53-mutated NSCLC patients, and multivariate Cox regression analysis identified EGFR mutation as an independent risk factor. The OS of NSCLC patients in the TP53 mutation group was significantly shorter than that of the TP53wt group. Survival curves in the TP53/EGFR combined mutation group showed that patients with combined EGFR mutation had a lower survival rate. TP53 mutations are associated with different clinical indicators and have important implications in clinical treatment. TP53 is a poor prognostic factor for NSCLC patients, and TP53/EGFR co-mutation will affect the survival time of patients. TP53/EGFR co-mutation may be a new prognostic marker for NSCLC.

NS2B-D55E and NS2B-E65D Variations Are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model.

Japanese encephalitis virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and an NS2B co-factor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at the internal C, NS2A/NS2B, NS2B/NS3, and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging the NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in the regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residues 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in the hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. However, it will be crucial to explore these mutations in other in vivo and/or in vitro models. Collectively, these observations will be useful for understanding the replication of JEV GI and GIII viruses.

Enhanced thermostability and catalytic activity for arginine deiminase from Enterobacter faecalis SK32.001 via combinatorial mutagenesis

Arginine deiminase (ADI) exhibits potential for clinical and industrial applications, yet its low thermostability and catalytic efficiency under physiological conditions limit its utility. In this work, the ADI of Enterococcus faecalis SK32.001 was rationally designed. A total of 120 combinatorial mutants, ranging from two-point to six-point mutations, were constructed by sequentially stacking single-point positive mutants (F44W, N163P, E220L, N318E, A336G, T340I). Among them, the mutants S604, S700, S601, and S606 exhibited higher Tm values, while the mutants S605, S547, S602, S607, S517, and S557 demonstrated enhanced enzymatic activity. Notably, the five-point mutant S547 (F44W/N163P/E220I/A336G/T340I) exhibited remarkable pH tolerance (pH 4.5–9.5, with over 80 % residual enzyme activity). Its specific enzyme activity reached 131.60 U/mg, which was 2-fold higher than that of wild enzyme. The Tm value of this enzyme increases to 64.04 °C, 11.62 °C higher than that of the wild-type enzyme. The structure predicted by AlphaFold 2 revealed that the increased rigidity, formation of new hydrogen bonds, and an increase in hydrophobic residues may account for the enhanced enzyme activity and thermostability. This research demonstrates that rational design strategies can effectively optimize enzyme properties, providing insights for the development of microbial enzymes with industrial relevance.

Features of Highly Homologous T-Cell Receptor Repertoire in the Immune Response to Mutations in Immunogenic Epitopes.

CD8+ T-cell immunity, mediated through interactions between human leukocyte antigen (HLA) and the T-cell receptor (TCR), plays a pivotal role in conferring immune memory and protection against viral infections. The emergence of SARS-CoV-2 variants presents a significant challenge to the existing population immunity. While numerous SARS-CoV-2 mutations have been associated with immune evasion from CD8+ T cells, the molecular effects of most mutations on epitope-specific TCR recognition remain largely unexplored, particularly for epitope-specific repertoires characterized by common TCRs. In this study, we investigated an HLA-A*24-restricted NYN epitope (Spike448-456) that elicits broad and highly homologous CD8+ T cell responses in COVID-19 patients. Eleven naturally occurring mutations in the NYN epitope, all of which retained cell surface presentation by HLA, were tested against four transgenic Jurkat reporter cell lines. Our findings demonstrate that, with the exception of L452R and the combined mutation L452Q + Y453F, these mutations have minimal impact on the avidity of recognition by NYN peptide-specific TCRs. Additionally, we observed that a similar TCR responded differently to mutant epitopes and demonstrated cross-reactivity to the unrelated VYF epitope (ORF3a112-120). The results contradict the idea that immune responses with limited receptor diversity are insufficient to provide protection against emerging variants.

Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of berberine bridge enzyme-like mutants with reduced nicotine content

Tobacco (Nicotiana tabacum) plants synthesize the psychoactive pyridine alkaloid nicotine, which has sparked growing interest in reducing nicotine levels through genome editing aiming at inactivating key biosynthetic genes. Although stable transformation-mediated genome editing is effective in tobacco, its polyploid nature complicates the complete knockout of genes and the segregation of transgenes from edited plants. In this study, we developed a non-transgenic genome editing method in tobacco by delivering the CRISPR/Cas machinery via an engineered negative-strand RNA rhabdovirus vector, followed by the regeneration of mutant plants through tissue culture. Using this method, we targeted six berberine bridge enzyme-like protein (BBL) family genes for mutagenesis, which are implicated in the last steps of pyridine alkaloid biosynthesis, in the commercial tobacco cultivar Hongda. We generated a panel of 16 mutant lines that were homozygous for mutations in various combinations of BBL genes. Alkaloid profiling revealed that lines homozygous for BBLa and BBLb mutations exhibited drastically reduced nicotine levels, while other BBL members played a minor role in nicotine synthesis. The decline of nicotine content in these lines was accompanied by reductions in anatabine and cotinine levels but increases in nornicotine and its derivative myosmine. Preliminary agronomic evaluation identified two low-nicotine lines with growth phenotypes comparable to those of wild-type plants under greenhouse and field conditions. Our work provides potentially valuable genetic materials for breeding low-nicotine tobacco and enhances our understanding of alkaloid biosynthesis.

Integrating neural mutation into mutation-based fault localization: A hybrid approach

Fault localization is an important part of software testing and debugging, helping improve the process of fixing faults. Mutation-Based Fault Localization (MBFL) is widely used, but the reliance of Traditional-MBFL on syntactical mutants often limits its accuracy. To address this, we propose Neural-MBFL, which introduces neural mutation to generate semantically richer mutants using deep learning to better mimic real faults. Additionally, we present NeuraIntegra-MBFL, which combines neural and traditional mutation strategies through mutant combination and suspiciousness aggregation. Experiments on 835 faulty programs from the Defects4J benchmark show that Neural-MBFL improves fault localization compared to Traditional-MBFL, with a 35.50% relative improvement in MAP and 127 more faults localized at TOP-5, while maintaining acceptable computational cost. Compared to Neural-MBFL, NeuraIntegra-MBFL further enhances performance, particularly with suspiciousness aggregation, achieving an additional 11.96% MAP improvement and localizes 45 more faults at TOP-5, demonstrating the effectiveness of integrating suspiciousness scores. Using overlap and correlation analyses, we confirmed the complementarity between Neural-MBFL and Traditional-MBFL. Neural-MBFL is more effective at localizing faults that require understanding deep code semantics, while Traditional-MBFL performs better at handling rule-based modifications. NeuraIntegra-MBFL successfully integrates the strengths of both methods, offering better performance than either approach alone.

Analysis of molecular mechanisms of delafloxacin resistance in Escherichia coli

In this study delafloxacin resistance mechanisms in Escherichia coli strains were analyzed. Delafloxacin is a new fluoroquinolone, that is approved for clinical application however, resistance against this agent is scarcely reported. In our study 37 E. coli strains were included and antimicrobial susceptibility testing was performed for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, imipenem. Six delafloxacin resistant E. coli strains were selected for whole-genome sequencing and all of them exhibited resistance to other fluoroquinonlones and showed an extended-spectrum beta-lactamase phenotype. The six delafloxacin resistant E. coli strains belonged to different sequence types (STs) namely, ST131 (2 strains), ST57 (2 strains), ST162 and ST15840. Each delafloxacin resistant strain possessed multiple mutations in quinolone resistance-determining regions (QRDRs). Notably, three mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were in strains of ST162, ST57 and ST15840. However, the two strains of ST131 carried five combined mutations namely, gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, parE Ile549Leu. Association of delafloxacin resistance and production of CTX-M-15 in ST131, CMY-2 in ST162 and ST15840 was detected. In this study a new ST, ST15840 of clonal complex 69 was identified. Our results demonstrate, that at least three mutations in QRDRs are required for delafloxacin resistance in E. coli.

The Association Between Janus Kinase 2 and Factor V Leiden Mutations and Thrombotic Complications in Patients With Myeloproliferative Disorders: A Study From Saudi Arabia.

Background The Janus kinase 2 (JAK2) V617F mutations are related to increased thrombotic risk in patients with myeloproliferative disorders (MPDs). However, little is known aboutwhether inherited thrombophilia represents an additive risk factor in mutated subjects. We addressed the association between combined mutations of JAK2 and factor V Leiden (FVL) and thrombotic complications in Saudi Arabian patients with MPDs. Methods We studied 60 patients with MPDs, 32 with polycythemia vera (PV), 24 with essential thrombocythemia (ET), and four with primary myelofibrosis (PMF). All patients were examined for JAK2 V617F and FVL mutations. Results The study included 50 (83.3%) males and 10 (16.7%) females, with a mean age of 44.23 ± 11.32 years. JAK2 was found positive among all (100%) ofthe studied patients. Thirty-eight patients out of 60 (63.3%) had thrombotic events.FVL was found positive in 12 (20%) patients. The patients with JAK2 and FVL mutations had a higher incidence of thrombotic events (11/38, 28.9%) than those with JAK2 but without FVL mutations (1/22, 4.5%). Therelative risk ratios for increased risk for having thrombotic events were 2.1 (95% confidence interval (95% CI): 1.2-3.8, p=0.03) and 4.3 (95% CI: 2.1-9.5, p<0.001) for patients with JAK2 mutations alone, and those with both JAK2 and FVL mutations, respectively. Conclusions In the present study of patients with MPDs from Saudi Arabia, JAK2 mutations were found among all the studied patients, and FVL mutations were encountered in 20% of patients. The patients with both JAK2 and FVL mutations had a higher incidence of thrombotic events than those with JAK2 but without FVL mutations. The relative risk ratios for increased risk for thrombotic events among patients with MPDs were 2.1 and 4.3 for patients with JAK2 mutations alone and those with JAK2 and FVL mutations, respectively.Further larger prospective studies are warranted.

Cdk8 and Hira mutations trigger X chromosome elimination in naive female hybrid mouse embryonic stem cells

Mouse embryonic stem cells (ESCs) possess a pluripotent developmental potential and a stable karyotype. An exception is the frequent loss of one X chromosome in female ESCs derived from inbred mice. In contrast, female ESCs from crosses between different Mus musculus subspecies often maintain two X chromosomes and can model X chromosome inactivation. Here we report that combined mutations of Hira and Cdk8 induce rapid loss of one X chromosome in a Mus musculus castaneus hybrid female ESC line that originally maintains two X chromosomes. We show that MEK1 inhibition, which is used for culturing naive pluripotent ESCs is sufficient to induce X chromosome loss. In conventional ESC media, Hira and Cdk8 mutant ESCs maintain both X chromosomes. Induction of X chromosome loss by switching to naive culture media allows us to perform kinetic measurements for calculating the chromosome loss rate. Our analysis shows that X chromosome loss is not explained by selection of XO cells, but likely driven by a process of chromosome elimination. We show that elimination of the X chromosome occurs with a rate of 0.3% per cell per division, which exceeds reported autosomal loss rates by 3 orders of magnitude. We show that chromosomes 8 and 11 are stably maintained. Notably, Xist expression from one of the two X chromosomes rescues X chromosomal instability in ΔHiraΔCdk8 ESCs. Our study defines mutations of Hira and Cdk8 as molecular drivers for X chromosome elimination in naive female ESCs and describes a cell system for elucidating the underlying mechanism.

ARR1 and ARR12 modulate arsenite toxicity responses in Arabidopsis roots by transcriptionally controlling the actions of NIP1;1 and NIP6;1.

Cytokinin is central to coordinating plant adaptation to environmental stresses. Here, we first demonstrated the involvement of cytokinin in Arabidopsis responses to arsenite [As(III)] stress. As(III) treatment reduced cytokinin contents, while cytokinin treatment repressed further primary root growth in Arabidopsis plants under As(III) stress. Subsequently, we revealed that the cytokinin signaling members ARR1 and ARR12, the type-B ARABIDOPSIS RESPONSE REGULATORs, participate in cytokinin signaling-mediated As(III) responses in plants as negative regulators. A comprehensive transcriptome analysis of the arr1 and arr12 single and arr1,12 double mutants was then performed to decipher the cytokinin signaling-mediated mechanisms underlying plant As(III) stress adaptation. Results revealed important roles for ARR1 and ARR12 in ion transport, nutrient responses, and secondary metabolite accumulation. Furthermore, using hierarchical clustering and regulatory network analyses, we identified two NODULIN 26-LIKE INTRINSIC PROTEIN (NIP)-encoding genes, NIP1;1 and NIP6;1, potentially involved in ARR1/12-mediated As(III) uptake and transport in Arabidopsis. By analyzing various combinations of arr and nip mutants, including high-order triple and quadruple mutants, we demonstrated that ARR1 and ARR12 redundantly function as negative regulators of As(III) tolerance by acting upstream of NIP1;1 and NIP6;1 to modulate their function in arsenic accumulation. ChIP-qPCR, EMSA, and transient dual-LUC reporter assays revealed that ARR1 and ARR12 transcriptionally activate the expression of NIP1;1 and NIP6;1 by directly binding to their promoters and upregulating their expression, leading to increased arsenic accumulation under As(III) stress. These findings collectively provide insights into cytokinin signaling-mediated plant adaptation to excessive As(III), contributing to the development of crops with low arsenic accumulation.

Regulate catalytic performance by engineering non-regular structure of extradiol dioxygenase: An entrance to bottom strategy

Extradiol dioxygenase Tcu3516 is a home-sourced enzyme demonstrating potent aromatic phenol degradation capacity. To add to the advantageous modifications inside active cavity, this work reported a novel strategy to engineer rarely concerned non-regular structures around the entrance towards the active site at the bottom of cavity. Three structures, Loop region 1 (Loop1: Met173-Arg185), Loop region 2 (Loop2: Ala201-Val212) and C-terminal (C-tail: His290-Lys306) were therefore identified through structural flexibility analysis. Highly rigid prolines within the structures were mutated into smaller alanine, glycine, or serine to improve structural flexibilities; while only P183S on Loop1 showed 3-fold activity enhancement vs the WT when subjected to cleavage of mono-cyclic catechol analogues. The analysis of Root Mean Square Fluctuation showed that P183S presents certain enhancement on Loop1 flexibility without dramatic changes of other domains. Furthermore, the synergetic effects from mutation P183S and cavity-based mutations V186L, V212N and D285A were evaluated by characterizing combinatorial mutants. Temperature dependence and thermostability of the combined mutants showed a more flexible catalytic domain without sacrificing structural integrity and stability. kcat value of P183S/V186L (SL) towards monocyclic catechols significantly surpasses any other combinatorial mutants around Tcu3516 active sites. Moreover, the synergetic effects on conformational plasticity were analyzed by molecular dynamic simulations to shed light into the interplay between structural changes and catalytic performance.

Exploring the Prevalence and Prognostic Impact of Wilms Tumor 1 Exon 7 Mutation Status with Combinations of FLT3-ITD and NPM1 Mutations as Potential Molecular Biomarkers in Acute Myeloid Leukemia Patients with Normal Cytogenetics.

This study explores the prognostic impact of FLT3-ITD, NPM1, and WT1 mutations both independently and in combination in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) patients as they exhibit varying clinical outcomes. 150 CN-AML patients were selected to assess the prevalence and prognostic significance of WT1 mutations in combination with FLT3-ITD and NPM1 status using polymerase chain reaction (PCR) followed by Sanger sequencing. WT1 exon 7 mutations were present in 12.6% of patients. Elderly individuals, with a mean age of 49.4 years, were more prone to NPM1 mutations, though the association was not statistically significant (p=0.094). Significant associations were observed between lactate dehydrogenase (LDH) and hemoglobin (Hb) levels with FLT3-ITD and NPM1 mutations (p=0.003 and p=0.04, respectively). The M4 subtype exhibited the highest prevalence of WT1 mutations (p=0.0036). Patients with NPM1 mutations had a higher overall survival rate compared to NPM1 wild-type cases (p=0.057). There was no significant correlation between overall survival and WT1 and FLT3-ITD mutations. Regarding relapse-free survival, NPM1 mutation cases exhibited a higher survival probability compared to NPM1 wild-type cases. Similarly, WT1 mutated cases had a higher survival probability compared to WT1 wild-type cases, although these differences were not statistically significant. The combined mutation statuses of NPM1/FLT3-ITD with WT1 did not yield significant outcomes. The study suggests that larger cohort studies may reveal more relevant associations, given the relatively small cohort in this study. This study found a significant association between patient survival outcomes and NPM1 mutation status, as well as the combined FLT3-ITD and NPM1 status. Profiling both NPM1 and FLT3-ITD mutations at the time of diagnosis serves as a robust prognostic marker in AML treatment. WT1 mutation status did not show a significant association with patient outcomes. Larger population studies may provide more relevant insights.

The bifunctional transcription factor NAC32 modulates nickel toxicity responses through repression of root-nickel compartmentalization and activation of auxin biosynthesis

Nickel (Ni) is an important micronutrient, but excess Ni is toxic to many plant species. Currently, relatively little is known about the genetic basis of the plant responses to Ni toxicity. Here, we demonstrate that NAC32 transcription factor functions as a core genetic hub to regulate the Ni toxicity responses in Arabidopsis. NAC32 negatively regulates root-Ni concentration through the IREG2 (IRON REGULATED2) encoding a transporter. NAC32 also induces local auxin biosynthesis in the root-apex transition zone by upregulating YUCCA 7 (YUC7)/8/9 expression, which results in a local enhancement of auxin signaling in root tips, especially under Ni toxicity, thereby impaired primary root growth. By analyses of various combinations of nac32 and ireg2 mutants, as well as nac32 and yuc7/8/9 triple mutants, including high-order quadruple mutant, we demonstrated that NAC32 negatively regulates Ni stress tolerance by acting upstream of IREG2 and YUC7/8/9 to modulate their function in Ni toxicity responses. ChIPqPCR, EMSA (electrophoretic mobility shift assay) and transient dual-LUC reporter assays showed that NAC32 transcriptionally represses IREG2 expression but activates YUC7/8/9 expression by directly binding to their promoters. Our work demonstrates that NAC32 coordinates Ni compartmentation and developmental plasticity in roots, providing a conceptual framework for understanding Ni toxicity responses in plants.