Mutant Type Research Articles

Synthetically induced Arabidopsis thaliana autotetraploids provide insights into the analysis of meiotic mutants with altered crossover frequency.

Mutations affecting crossover (CO) frequency and distribution lead to the presence of univalents during meiosis, giving rise to aneuploid gametes and sterility. These mutations may have a different effect after chromosome doubling. The combination of altered ploidy and mutations could be potentially useful to gain new insights into the mechanisms and regulation of meiotic recombination; however, studies using autopolyploid meiotic mutants are scarce. Here, we have analyzed the cytogenetic consequences in colchicine-induced autotetraploids (colchiploids) from different Arabidopsis mutants with an altered CO frequency. We have found that there are three types of mutants: mutants in which chiasma frequency is doubled after chromosome duplication (zip4, mus81), as in the control; mutants in which polyploidy leads to a higher-than-expected increase in chiasma frequency (asy1, mer3, hei10, and mlh3); and mutants in which the rise in chiasma frequency produced by the presence of two extrachromosomal sets is less than doubled (msh5, fancm). In addition, the proportion of class I/class II COs varies after chromosome duplication in the control. The results obtained reveal the potential of colchiploid meiotic mutants for better understanding of the function of key proteins during plant meiosis. This is especially relevant considering that most crops are polyploids.

CRISPR-enabled investigation of fitness costs associated with the E198A mutation in β-tubulin of Colletotrichum siamense.

Understanding fitness costs associated with fungicide resistance is critical to improve resistance management strategies. E198A in b-tubulin confers resistance to the fungicide thiophanate-methyl and has been widely reported in several plant pathogens including Colletotrichum siamense. To better understand potential fitness costs associated with the resistance, a ribonucleoprotein (RNP) complex mediated CRISPR/Cas9 system was used to create a point mutation (E198A) through homology directed repair (HDR) in each of the sensitive (E198) C. siamense isolates collected from strawberries, raspberries, and peaches. The RNP complex was delivered into fungal protoplasts using polyethylene glycol-mediated (PEG) transfection. The transformation efficiency, the proportion of transformants of sensitive parental isolates containing the E198A mutation, averaged 72%. No off-target mutations were observed when sequences similar to the b-tubulin target region with a maximum of four mismatch sites were analyzed, suggesting that the CRISPR/Cas9 system used in this study was highly specific for genome editing in C. siamense. Of the 41 comparisons of fitness between mutant and wild type isolates through in vitro and detached fruit assays, mutant isolates appeared to be as fit (24 of 41 comparisons), if not more fit than wild-type isolates (10 of 41 comparisons). The use of CRISPR/Cas9 to evaluate fitness costs associated with point mutations in this study represents a novel and useful method, since wild-type and mutant isolates were genetically identical except for the target mutation.

Random X-Chromosome Inactivation in Adult Hemophilia B Carriers Does Not Attenuate Bleeding Risk

Introduction: The X-linked bleeding disorder Hemophilia B (HB) results from deficiency in coagulation Factor IX (FIX). In contrast to Hemophilia A, genotype-phenotype correlations in HB males are complex, as some F9 variants in males are associated with low plasma FIX activity (FIX:C) but milder than expected bleeding phenotypes. At least 75% of FIX is extravascular and not adequately measured from plasma. Hemostasis is likely influenced by this extravascular FIX which is bound reversibly to type IV collagen in the subendothelial basement membrane. We predict that genotype-phenotype correlations in female carriers of F9 mutations may have additional complexity as the mutant FIX can competitively interfere with wild type (WT) FIX for extravascular binding sites. F9 expression in female HB carriers is influenced by X-chromosome inactivation (XCI), a process that silences one X in XX females. XCI results in cells that randomly inactivate either the X carrying the mutant or wild type F9, although skewed XCI (>80:20) is found in ~25% of the general population. XCI skewing in HB carriers can lead to preferential expression of the X carrying the F9 mutation, which in turn influences plasma and extravascular levels of FIX and bleeding tendency. How frequently XCI is skewed in HB carriers, and the role of XCI in modulating FIX levels and bleeding phenotypes, remains poorly understood. We sought to determine whether XCI skewing may further differentiate bleeding risk in HB carriers. Methods: Subjects were enrolled in a prospective, observational cross-sectional study with informed consent. Both the Condensed MCMDM-1 bleeding assessment tool (BAT) and Pictorial Blood-loss Assessment Chart (PBAC) were completed at the time of the visit. F9 mutation analysis confirmed carrier status. FIX:C and FIX antigen ELISA assays were performed on all subjects. XCI skewing was assessed using established assays at the Androgen Receptor (AR) and Fragile X Mental Retardation (FMR1) loci. Results: A total of 15 subjects (11 adults) were confirmed F9 mutation carriers; six missense mutations and one frameshift mutation were identified. Abnormal bleeding is frequent in adult HB carriers, with 5/11 (45%) having BAT scores >4. Heavy menstrual bleeding (HMB) is particularly frequent, with all adult HB carriers reporting PBAC > 100, and 8/11 with BAT HMB subcategory score >1. FIX assessment by activity (mean 56% ± 28) and antigen (mean 0.55 IU/mL ± 0.2) assays are highly correlated (r 2= 0.83, p < 0.0001). Notably, only two adult HB carriers with significant bleeding have FIX levels < 50%, while two HB carriers with FIX levels < 50% have BAT scores < 4, indicating that neither FIX:C nor FIX antigen levels adequately predict bleeding risk in all HB carriers. Indeed, for all 11 adult HB carriers, no significant differences in either FIX:C or FIX antigen were observed between those with BAT scores ≥ 4 and < 4. We asked if XCI explained FIX levels or bleeding phenotypes. Nearly half (5/11) of the adult carriers have moderately skewed XCI (> 70:30); only one is extremely XCI skewed (> 80:20), and of the individuals with skewed XCI, 3/5 have BAT scores ≥ 4. Although random XCI is proposed to attenuate HB carrier bleeding phenotype, 2/6 HB carriers with random XCI also have BAT scores ≥ 4. XCI weakly correlated with FIX antigen (n = 15; r 2= 0.27, p < 0.05) but not FIX:C. When assessing this relationship for only the individuals with F9 c.1025C>T (n = 6), there was significant correlation with FIX:C (R 2= 0.73, p < 0.05) but not with FIX antigen or bleeding phenotype. The lack of correlation between XCI and FIX antigen in individuals with an identical F9 mutation argues for additional influences on intravascular, and likely extravascular, FIX levels and will require further investigation. Conclusions: Bleeding tendency in HB carriers is not rare and does not correlate with plasma FIX:C or FIX antigen. Random XCI is not fully protective against abnormal bleeding. Future studies are needed to better understand bleeding phenotypes in HB carriers that consider XCI, F9 mutation type and influences on circulating levels of WT and mutant FIX.

Structural basis of mannitol-induced activation of human taste receptor PKD2L1

Sour taste is generally considered as a non-preferred taste, but many organisms, including humans, evolved to consume sour-tasting foods. PKD2L1 is a proton-activated sour taste receptor. In this study, we found that mannitol, known to produce a sweet taste, acts on hPKD2L1 in a concentration-dependent and pH-sensitive manner, and in silico protein-ligand complex modeling indicated that D523 of PKD2L1 was the critical binding residue. When comparing the Vmax value, it was confirmed that the wild type has 55.7 ± 2.94 but changed to 0 ± 0 in the D523A mutant type. Fermented kimchi maintains an average pH of 3.5–4.5, and this low-pH condition supports the production of sweet and sour tastes by mannitol. A considerable quantity of mannitol is produced by heterofermentative lactic acid bacteria during fermentation of kimchi, and the acidity of kimchi increases after fermentation, which may impart a unique sweet and refreshing taste compared with fresh kimchi. These findings indicate that the differences in mannitol-induced taste perception, especially sour taste, depend on pH levels, which could help explain the conjugative taste perception associated with fermented kimchi.

Our data on detailing metastasis localization and subtype characteristics in metastatic colorectal cancer patients treated with Bevacizumab

Aims: Our aim in this study was to determine the relationship between metastasis types and mutation subtypes in patients who were followed up in our center and received bevacizumab treatment, to determine the survival rates according to metastasis types, and to contribute to the literature on this subject.
 Methods: In our study, we retrospectively evaluated 42 consecutive metastatic colorectal cancer patients who were admitted to our hospital and diagnosed with colorectal cancer, thorax-abdominal CT scans were performed in our clinic to detect possible metastases, and the presence of metastases in one or more localizations was detected and treated with Bevacizumab.
 Results: The majority of colorectal cancers included in our study had the histopathological subtype of adenocarcinoma (90.5%). Genetic analyses revealed that 47.6% (20 patients) had mutant KRAS gene types, while 52.4% (22 patients) had wild type. The distribution of metastases was as follows; 31 (73.8%) cases with liver involvement, 12 (28.6%) with peritoneal involvement and 24 (57.1%) with lung involvement. In our study, median overall survival was 19 months and median disease-free survival was 7 months.
 Conclusion: The results of studies to date will be useful to help predict prognosis and to select appropriate regimens for treatment. We aimed to contribute to this process by presenting our own data in our own study. However, the true role of RAS genes as prognostic markers continues to be questioned, and multicenter studies are needed on the predictive and prognostic factors of colorectal cancers.

Effect Of Viable Mutants In M2 Generation Of Urdbean Vigna Mungo (L.) Hepper Through Induced Mutation In Variety ADT 3 and Co 6

Mutations were induced in Black gram (Vigna Mungo (L.) Hepper) varieties namely ADT 3 and Co- 6. Seeds treated with gamma rays doses at 200, 300 and 400 Gy and Electron beam viz., 200, 300 and 400 Gy and combined treatment (Electron beam + gamma rays) 200+200, 300+300 and 400 +400 Gy. Different types of Macro mutants and micro mutants were screened in M2 generation. The frequency and the spectrum of the viable mutants were estimated in M2 generation both in M1 plants basis and M2 seedling basis. The highest frequency of viable mutants was recorded in ADT 3.The highest number of mutants was recorded in electron beam followed by Gamma rays and combined treatment (Electron beam + gamma rays). The present study reveals good scope for isolation of suitable morphological traits mutants induced may be useful in breeding programmes for the improvement of blackgram with agronomically desirable character which force is utilized in future breeding programme.

The Breeding of Waxy Sorghum Using Traditional Three-Line Method and Marker-Assisted Selection

Sorghum (Sorghum bicolor) exhibits drought resistance and environmental adaptability, making it a crucial cereal crop for semi-arid regions. It has a wide range of uses, including as food, feed, brooms, alcohol production, and bioethanol. In particular, Taiwan imports nearly 50,000 tons of sorghum annually, primarily for the production of sorghum liquor. However, the ideal raw material for high-quality sorghum liquor is waxy sorghum, and not all sorghum varieties imported or promoted in Taiwan are of this waxy type. Consequently, there is a shortage of sufficient waxy sorghum raw materials to meet the demands of the Taiwan market. The occurrence of waxy sorghum (wx) is caused by the mutation of granule-bound starch synthase I (GBBS I), and there are currently several known types of mutants, carrying different wxa, wxb, and wxc waxy alleles. Among them, wxc is a novel mutation type, and in native sorghum in Taiwan, individuals with the waxy allele wxc have been found. The three-line method is a commonly used breeding strategy, which simplifies the process of emasculation to obtain hybrid F1 offspring. In this study, imported sorghum variety Liangnuo No.1 (with male sterility), native glutinous sorghum variety SB6 from Taiwan (carrying the wxc waxy allele), and sorghum reference genome variety BTx623 were used as research materials. The goal was to use the three-line method to produce waxy sorghums, including the male sterile line (A-line), male sterile maintenance line (B-line), and fertility-restoring line (R-line). The breeding results showed that by using backcross breeding, molecular-assisted selection, and traditional field selection methods, high-quality three-line materials (A-, B-, R-lines, named CNA1, CNB1 CNR1, respectively) and F1 hybrid (CNH1) with favorable agronomic traits and yield quality were successfully obtained.

Ultrafast electron transfer of different vibronic states in flavodoxin

Understanding the ultrafast electron transfer (ET) processes involving various vibronic excitation in biological systems remains challenging and requires well-defined model proteins for in-depth studies. In this work, we selected three types of mutants with different ET lifetimes in flavodoxin to explore the excitation dependence of the photo-induced ET dynamics. For the ultrafast ET processes within the time window of intramolecular vibrational relaxation, with increasing excitation energies, we observed no change of ET lifetimes but with higher vibrational excitation in products, both of which can be attributed to the unique nature of ultrafast ET. For the slower ET, we did not find any dependence of product vibration excitation relative to the initial different excitation due to the complete vibrational relaxation. These results strongly indicate the vibrational energy can be propagated to the subsequent reactions during ultrafast ET processes.

ERK mediates interferon gamma-induced melanoma cell death

BackgroundInterferon-gamma (IFNγ) exerts potent growth inhibitory effects on a wide range of cancer cells through unknown signaling pathways. We pursued complementary screening approaches to characterize the growth inhibition pathway.MethodsWe performed chemical genomics and whole genome targeting CRISPR/Cas9 screens using patient-derived melanoma lines to uncover essential nodes in the IFNγ-mediated growth inhibition pathway. We used transcriptomic profiling to identify cell death pathways activated upon IFNγ exposure. Live imaging experiments coupled with apoptosis assays confirmed the involvement of these pathways in IFNγ-mediated cell death.ResultsWe show that IFNγ signaling activated ERK. Blocking ERK activation rescued IFNγ-mediated apoptosis in 17 of 23 (~ 74%) cell lines representing BRAF, NRAS, NF1 mutant, and triple wild type subtypes of cutaneous melanoma. ERK signaling induced a stress response, ultimately leading to apoptosis through the activity of DR5 and NOXA proteins.ConclusionsOur results provide a new understanding of the IFNγ growth inhibition pathway, which will be crucial in defining mechanisms of immunotherapy response and resistance.

Novel Fluoroquinolones with Possible Antibacterial Activity in Gram-Negative Resistant Pathogens: In Silico Drug Discovery.

Antibiotic resistance is a global threat to public health, and the search for new antibacterial therapies is a current research priority. The aim of this in silico study was to test nine new fluoroquinolones previously designed with potential leishmanicidal activity against Campylobacter jejuni, Escherichia coli, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Salmonella typhi, all of which are considered by the World Health Organization to resistant pathogens of global concern, through molecular docking and molecular dynamics (MD) simulations using wild-type (WT) and mutant-type (MT) DNA gyrases as biological targets. Our results showed that compound 9FQ had the best binding energy with the active site of E. coli in both molecular docking and molecular dynamics simulations. Compound 9FQ interacted with residues of quinolone resistance-determining region (QRDR) in GyrA and GyrB chains, which are important to enzyme activity and through which it could block DNA replication. In addition to compound 9FQ, compound 1FQ also showed a good affinity for DNA gyrase. Thus, these newly designed molecules could have antibacterial activity against Gram-negative microorganisms. These findings represent a promising starting point for further investigation through in vitro assays, which can validate the hypothesis and potentially facilitate the development of novel antibiotic drugs.

Vitamin C intake and colorectal cancer survival according to KRAS and BRAF mutation: a prospective study in two US cohorts.

The associations of vitamin C intake with colorectal cancer (CRC) survival according to tumour KRAS or BRAF mutation status remain unclear. We used the inverse probability weighted multivariable Cox proportional hazards regression model to calculate the hazard ratio (HR) of mortality, and spline analysis to evaluate the dose-response relationship in the Nurses' Health Study and Health Professionals Follow-up Study. We also assessed SLC2A1 mRNA expression according to KRAS or BRAF mutation in the TCGA database. During an average of 12.0 years of follow-up, we documented 2,096 CRC cases, of which 703 cases had KRAS and BRAF mutation data. The association between total vitamin C intake and CRC-specific mortality suggestively differed according to KRAS or BRAF mutation status (Pinteraction = 0.04), with the multivariable HR (95% CI) per 400 mg/day increase in vitamin C intake for CRC-specific mortality of 1.07 (0.87-1.32, Ptrend = 0.52) in cases with both wild type and 0.74 (0.55-1.00, Ptrend < 0.05) in cases with either KRAS or BRAF mutant type. TCGA analysis showed a higher mRNA SLC2A1 expression in KRAS or BRAF-mutated tumours than in wild-type tumours (P = 0.02). Our findings support the laboratory evidence for a potential benefit of vitamin C for CRC patients with KRAS or BRAF mutated tumours.

Effect of ethyl methanesulfonate mediated mutation for enhancing morpho-physio-biochemical and yield contributing traits of fragrant rice.

Chemical mutagenesis has been successfully used for increasing genetic diversity in crop plants. More than 800 novel mutant types of rice (Oryza sativa L.) have been developed through the successful application of numerous mutagenic agents. Among a wide variety of chemical mutagens, ethyl-methane-sulfonate (EMS) is the alkylating agent that is most commonly employed in crop plants because it frequently induces nucleotide substitutions as detected in numerous genomes. In this study, seeds of the widely consumed Basmati rice variety (Super Basmati, Oryza sativa L.) were treated with EMS at concentrations of 0.25%, 0.50%, 0.75%, 1.0%, and 1.25% to broaden its narrow genetic base. Sensitivity to a chemical mutagen such as ethyl methanesulfonate (EMS) was determined in the M1 generation. Results in M1 generation revealed that as the levels of applied EMS increased, there was a significant reduction in the germination percent, root length, shoot length, plant height, productive tillers, panicle length, sterile spikelet, total spikelet, and fertility percent as compared to the control under field conditions. All the aforementioned parameters decreased but there was an increase in EMS mutagens in an approximately linear fashion. Furthermore, there was no germination at 1.25% of EMS treatment for seed germination. A 50% germination was recorded between 0.50% and 0.75% EMS treatments. After germination, the subsequent parameters, viz. root length and shoot length had LD50 between 05.0% and 0.75% EMS dose levels. Significant variation was noticed in the photosynthetic and water related attributes of fragrant rice. The linear increase in the enzymatic attributes was noticed by the EMS mediated treatments. After the establishment of the plants in the M1 generation in the field, it was observed that LD50 for fertility percentage was at EMS 1.0% level, for the rice variety. Hence, it is concluded that for creating genetic variability in the rice variety (Super Basmati), EMS doses from 0.5% to 0.75% are the most efficient, and effective.

Loss of HES1 expression is associated with extracellular matrix remodeling and tumor immune suppression in KRAS mutant colon adenocarcinomas

The loss of HES1, a canonical Notch signaling target, may cooperate with KRAS mutations to remodel the extracellular matrix and to suppress the anti-tumor immune response. While HES1 expression is normal in benign hyperplastic polyps and normal colon tissue, HES1 expression is often lost in sessile serrated adenomas/polyps (SSAs/SSPs) and colorectal cancers (CRCs) such as those right-sided CRCs that commonly harbor BRAF or KRAS mutations. To develop a deeper understanding of interaction between KRAS and HES1 in colorectal carcinogenesis, we selected microsatellite stable (MSS) and KRAS mutant or KRAS wild type CRCs that show aberrant expression of HES1 by immunohistochemistry. By comparing the transcriptional landscapes of microsatellite stable (MSS) CRCs with or without nuclear HES1 expression, we investigated differentially expressed genes and activated pathways. We identified pathways and markers in the extracellular matrix and immune microenvironment that are associated with mutations in KRAS. We found that loss of HES1 expression positively correlated with matrix remodeling and epithelial-mesenchymal transition but negatively correlated with tumor cell proliferation. Furthermore, loss of HES1 expression in KRAS mutant CRCs correlates with a higher M2 macrophage polarization and activation of IL6 and IL10 immunosuppressive signature. Identifying these HES1-related markers may be useful for prognosis stratification and developing treatment for KRAS-mutant CRCs.

Preclinical characterization of tunlametinib, a novel, potent, and selective MEK inhibitor.

Background: Aberrant activation of RAS-RAF-MEK-ERK signaling pathway has been implicated in more than one-third of all malignancies. MEK inhibitors are promising therapeutic approaches to target this signaling pathway. Though four MEK inhibitors have been approved by FDA, these compounds possess either limited efficacy or unfavorable PK profiles with toxicity issues, hindering their broadly application in clinic. Our efforts were focused on the design and development of a novel MEK inhibitor, which subsequently led to the discovery of tunlametinib. Methods: This study verified the superiority of tunlametinib over the current MEK inhibitors in preclinical studies. The protein kinase selectivity activity of tunlametinib was evaluated against 77 kinases. Anti-proliferation activity was analyzed using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. ERK and phospho-ERK levels were evaluated by Western blot analysis. Flow cytometry analysis was employed to investigate cell cycle and arrest. Cell-derived xenograft (CDX) and Patient-derived xenograft (PDX) models were used to evaluate the tumor growth inhibition. The efficacy of tunlametinib as monotherapy treatment was evaluated in KRAS/BRAF mutant or wild type xenograft model. Furthermore, the combination studies of tunlametinib with BRAF/KRASG12C/SHP2 inhibitors or chemotherapeutic agent were conducted by using the cell proliferation assay in vitro and xenograft models in vivo. Results: In vitro, tunlametinib demonstrated high selectivity with approximately 19-fold greater potency against MEK kinase than MEK162, and nearly 10-100-fold greater potency against RAS/RAF mutant cell lines than AZD6244. In vivo, tunlametinib resulted in dramatic tumor suppression and profound inhibition of ERK phosphorylation in tumor tissue. Mechanistic study revealed that tunlametinib induced cell cycle arrest at G0/G1 phase and apoptosis of cells in a dose-proportional manner. In addition, tunlametinib demonstrated a favorable pharmacokinetic profile with dose-proportionality and good oral bioavailability, with minimal drug exposure accumulation. Furthermore, tunlametinib combined with BRAF/KRASG12C/SHP2 inhibitors or docetaxel showed synergistically enhanced response and marked tumor inhibition. Conclusion: Tunlametinib exhibited a promising approach for treating RAS/RAF mutant cancers alone or as combination therapies, supporting the evaluation in clinical trials. Currently, the first-in-human phase 1 study and pivotal clinical trial of tunlametinib as monotherapy have been completed and pivotal trials as combination therapy are ongoing.

Lipopolysaccharide triggers exacerbated microglial activation, excessive cytokine release and behavioural disturbances in mice with truncated Fused-in-Sarcoma Protein (FUS)

CNS inflammation, including microglial activation, in response to peripheral infections are known to contribute to the pathology of both familial and sporadic neurodegenerative disease. The relationship between Fused-in-Sarcoma Protein (FUS)-mediated disease in the transgenic FUS[1–359] animals and the systemic inflammatory response have not been explored. Here, we investigated microglial activation, inflammatory gene expression and the behavioural responses to lipopolysaccharide-induced (LPS; 0.1 mg/kg) systemic inflammation in the FUS[1–359] transgenic mice. The pathology of these mice recapitulates the key features of mutant FUS-associated familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Here, pre-symptomatic 8-week-old mutant or wild type controls were challenged with LPS or with saline and sucrose intake, novel cage exploration, marble burying and swimming behaviours were analyzed. The level of pro-inflammatory gene expression was also determined, and microglial activation was evaluated. In chronic experiments, to discover whether the LPS challenge would affect the onset of ALS-like paralysis, animals were evaluated for clinical signs from 5 to 7 weeks post-injection. Compared to controls, acutely challenged FUS[1–359]-tg mice exhibited decreased sucrose intake and increased floating behaviours. The FUS[1–359]-tg mice exhibited an increase in immunoreactivity for Iba1-positive cells in the prefrontal cortex and ventral horn of the spinal cord, which was accompanied by increased expression of interleukin-1β, tumour necrosis factor, cyclooxygenase-(COX)-1 and COX-2. However, the single LPS challenge did not alter the time to development of paralysis in the FUS[1–359]-tg mice. Thus, while the acute inflammatory response was enhanced in the FUS mutant animals, it did not have a lasting impact on disease progression.

The clinical impact of chemokine receptor CCR5 Δ32 mutation in SARS-CoV-2 infected patients

Since the first outbreak of coronavirus disease (COVID-19), many infected people have developed a severe infection, which is usually a sequel of cytokine overproduction. The chemokine receptor, such as chemokine receptor 5, also denoted as (CCR5) has a role in the pathogenicity of COVID-19 disease. The ongoing research paper tried to assess the impact role of CCR5Δ32 mutation in a group of Iraq SARS-CoV-2 infected patients. A total of 180 samples were enrolled in this study; 120 were patients infected with COVID-19 and verified by reverse transcriptase polymerase chain reaction (RT-PCR) in nasopharyngeal swabs. Those patients were categorized into two groups based on the severity of the disease: severe COVID-19, which included 60 patients and mild/moderate COVID-19 with 60 patients. Furthermore, 60 subjects confirmed to be COVID-19-negative were enrolled in this study as a control group. The nucleic DNA was obtained from whole blood, and the CCR5Δ32 mutation was genotyped and detected by polymerase chain reaction using specific primer sequences. Results of the current study mentioned that out of the 180 samples in this study, 100 (100%) were wild type for the CCR5 gene (CCR5-wt), while none (0%) were mutant type for the CCR5-Δ32. This research has demonstrated that none of the study patients have the mutant CCR5 gene type (CCR5-32), assuming a lack of the role of CCR5Δ32 in the prognosis of COVID-19 infection. Keywords: Chemokine receptor 5 (CCR5), CCR5Δ32 mutation, COVID-19, SARS-CoV-2.

BAP1 mutations inhibit the NF-κB signaling pathway to induce an immunosuppressive microenvironment in uveal melanoma

BackgroundTumor immune microenvironment regulates the growth and metastasis of uveal melanoma (UM). This study aims to reveal the possible molecular mechanism of BRCA1-associated protein 1 (BAP1) mutations in affecting the tumor immune microenvironment in UM through mediating the nuclear factor-κB (NF-κB) signaling pathway.MethodsTCGA and cBioPortal databases jointly analyzed the genes with high mutation frequency in UM samples. Following survival analysis of UM patients, UM samples with BAP1 mutations were subjected to immune cell infiltration analysis. The signaling pathways associated with the mutated genes were screened by GSEA. Subsequently, the differential BAP1 expression was analyzed in the selected UM cell lines with wild type (WT) or mutant type (MUT) BAP1.ResultsBioinformatics analysis identified 12 genes mutated in the UM samples, while only BAP1 mutations were related to the prognosis of UM patients. UM patients with BAP1 mutations had higher immune cell infiltration. BAP1 mutations inhibited the NF-κB signaling pathway, suppressing the cytokine secretion and antigen presentation by macrophages. Rescue experiments confirmed that overexpressed NF-κB could reverse the effect of BAP1 mutations on the immunosuppressive microenvironment, thus suppressing the malignant phenotypes of UM cells.ConclusionBAP1 mutations may inhibit the NF-κB signaling pathway, repressing the cytokine secretion and antigen presentation by macrophages, which induces the immunosuppressive microenvironment, enhances the malignant phenotypes of UM cells and ultimately promotes the growth and metastasis of UM.

RatioCRISPR: A ratiometric biochip based on CRISPR/Cas12a for automated and multiplexed detection of heteroplasmic SNPs in mitochondrial DNA

Mitochondrial genetic diseases are often characterized by heteroplasmic single nucleotide polymorphisms (SNPs) where both wild-type (WT) and mutant-type (MT) coexist, making detection of accurate SNP abundance critical for diagnosis. Here, we present RatioCRISPR, an automated ratiometric biochip sensor based on the CRISPR/Cas12a system for detecting multiple heteroplasmic SNPs in mitochondrial DNA (mtDNA). The ratiometric sensor output is only influenced by the relative abundance of WT and MT, with minimal impact from sample concentration. Biochips allow the simultaneous detection of multiple SNP sites for more accurate disease diagnosis. RatioCRISPR can accurately detect 8 samples simultaneously within 25 min with a limit of detection (LOD) of 15.7 aM. We successfully detected 13 simulated samples of three mtDNA point mutations (m.3460G>A, m.11778G>A, and m.14484T>C), which lead to Leber's hereditary optic neuropathy (LHON) and set a threshold (60%) of heteroplasmy to evaluate disease risk. This automated and accurate biosensor has broad applications in diagnosing multiple SNPs, especially those with heteroplasmic variations, making it an advanced and convenient tool for mtDNA disease diagnosis.

High frequency of WNT-activated medulloblastomas with CTNNB1 wild type suggests a higher proportion of hereditary cases in a Latin-Iberian population.

Medulloblastomas are the most common primary malignant brain tumors in children. They are divided into molecular subgroups: WNT-activated, SHH-Activated, TP53 mutant or wild type, and non-WNT/non-SHH (Groups 3 and 4). WNT-activated medulloblastomas are usually caused by mutations in the CTNNB1 gene (85%-90%), and most remaining cases of CTNNB1 wild type are thought to be caused by germline mutations in APC. So far, the frequencies of CTNNB1 have been reported mainly in North American and European populations. The aim of this study was to report the frequency of CTNNB1 mutations in WNT-activated medulloblastomas in a Latin-Iberian population and correlate with their clinicopathological characteristics. A total of 266 medulloblastomas from seven different institutions from Brazil (n=211), Portugal (n=38), and Argentina (n=17) were evaluated. Following RNA and DNA isolation from formalin-fixed, paraffin-embedded (FFPE) tumor tissues, the molecular classification and CTNNB1 mutation analysis were performed by nCounter and Sanger sequencing, respectively. WNT-activated medulloblastomas accounted for 15% (40/266) of the series. We observed that 73% of WNT-activated medulloblastomas harbored CTNNB1 mutations. CTNNB1 wild-type cases (27%) were more prevalent in female individuals and suggested to be associated with a worse outcome. Among the CTNNB1 wild-type cases, the available analysis of family history revealed two cases with familiar adenomatous polyposis, harboring APC germline variants. We observed a lower incidence of CTNNB1 mutations in WNT-activated medulloblastomas in our Latin-Iberian cohort compared to frequencies previously described in other populations. Considering that CTNNB1 wild-type cases may exhibit APC germline mutations, our study suggests a higher incidence (~30%) of hereditary WNT-activated medulloblastomas in the Latin-Iberian population.

Development of a high-resolution melt-based assay to rapidly detect the azole-resistant Candida auris isolates.

Candida auris is a multidrug-resistant yeast that rapidly spreads, making it the leading Candidate for the next pandemic. One main leading cause of emerging resistant C. auris isolates is nonsynonymous mutations. This study aimed to detect the Y132F mutation, one of the most important azole resistance-associated mutations in the ERG-11 gene of C. auris, by developing a reliable high-resolution melt (HRM)-based method. Five C. auris isolates from Iran, plus three control isolates from other Clades were used in the study. The antifungal susceptibility testing through micro broth dilution was performed to recheck their susceptibility to three azole antifungals, including fluconazole, itraconazole, and voriconazole. Moreover, the polymerase chain reaction (PCR) sequencing of the ERG-11 gene was performed. Following the bioinformatic analysis and HRM-specific primer design, an HRM-based assay was developed and evaluated to detect ERG-11 mutations. The minimum inhibitory concentrations of fluconazole among Iranian C. auris isolates ranged from 8 to 64 μg/mL. The PCR-sequencing of the ERG-11 gene and bioinformatic analyses revealed the mutation of Y132F, a substitution consequence of A to T on codon 395 in one fluconazole-resistant isolate (IFRC4050). The developed HRM assay successfully differentiated the targeted single nucleotide polymorphism between mutant and wild types (temperature [Tm]: 81.79 ℃ - cycle threshold [CT]: 20.06 for suspected isolate). For both mutant and non-mutant isolates, the mean Tm range was 81.79-82.39 °C and the mean CT value was 20.06-22.93. These results were completely in accordance with the findings of DNA sequencing. The fast-track HRM-based method successfully detected one of the most common mechanisms of resistance in the ERG-11 gene of C. auris within 3 h. Finally, the development of more panels of HRM assays for the detection of all azole resistance mutations in C. auris ERG-11 is recommended to expand the scope of the field and facilitate the elaboration of rapid and accurate methods of antifungal resistance assessment.