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Cell Calcium Jun 2024Previous studies have identified RyR2 W4645R mutation, located in the caffeine-binding site, to associate with CPVT1 pathology. Caffeine binding to its site is thought...
AIMS
Previous studies have identified RyR2 W4645R mutation, located in the caffeine-binding site, to associate with CPVT1 pathology. Caffeine binding to its site is thought to displace the carboxyl-terminal domain to Ca-binding, allowing the tryptophan residue (W4645) to regulate Ca sensitivity of RyR2. To gain insights into regulation of RyR2 Ca-binding and its interaction with caffeine-binding site, we introduced W4645R-RyR2 point mutation via CRISPR/Cas9 gene-editing in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) and characterized their Ca-signaling phenotype compared to WT hiPSCCMs.
METHODS AND RESULTS
W4645R-RyR2 cardiomyocytes had: (1) no significant change in I magnitude or voltage-dependence; (2) slightly reduced CICR; (3) altered relaxation kinetics of Ca-transients with no change in isoproterenol sensitivity; (4) complete loss of caffeine-triggered Ca release; (5) larger SR Ca leak resulting in 40 % lower SR Ca content, as determined by myocytes' response to 4-CmC; (6) lower incidence of calcium sparks and asynchronous spontaneous SR Ca releases.
CONCLUSIONS
W4645R-RyR2 mutation induces loss of caffeine-triggered SR Ca release and enhances SR Ca leak that underlie asynchronous spontaneous Ca releases, triggering arrhythmia and impairing cardiac function.
PubMed: 38908063
DOI: 10.1016/j.ceca.2024.102925 -
Indian Journal of Medical Microbiology Jul 2024In India there is evidence of antimicrobial resistance in Helicobacter pylori, a definitive pathobiont whose only known niche is human gastric mucosa. This in turn can...
Antimicrobial resistance pattern of Helicobacter pylori in patients evaluated for dyspeptic symptoms in North-Eastern India with focus on detection of clarithromycin resistance conferring point mutations A2143G and A2142G within bacterial 23S rRNA gene.
PURPOSE
In India there is evidence of antimicrobial resistance in Helicobacter pylori, a definitive pathobiont whose only known niche is human gastric mucosa. This in turn can lead to failure of treatment, persistence or chronicity of infection. This hospital based, prospective, observational study investigates the presence of antimicrobial resistance in the organism with focus on detection of A2143G and A2142G major point mutations in domain V of H. pylori 23S rRNA gene as a molecular mechanism of conferring resistance.
METHODS
Endoscopic gastric biopsy samples from 52 patients presenting with dyspeptic symptoms from January 2016 to December 2016 were subjected to culture in a microaerophilic environment using Campylobacter agar with for 2-5 days. Isolates were identified using gram-staining, motility test and biochemical reactions. Modified Kirby-Bauer Disc diffusion method was used to determine antimicrobial susceptibility against Clarithromycin, Metronidazole, Amoxycillin, Levofloxacin, Tetracycline, Cotrimoxazole and Erythromycin. Additionally, detection of A2143G and A2142G point mutations conferring Clarithromycin resistance was carried out using real time PCR following extraction and quantification of bacterial DNA. Histopathological examination was carried out on all biopsy samples. Descriptive and inferential statistical analytical methods were used. Differences were considered significant for p < 0.05.
RESULTS
Culture positivity for H. pylori by phenotypic method was found to be 36.54%. Histopathologic Examination detected H. pylori in 55.7% and PCR detected 48.08% for either the wild type or one of two mutant strains A2143G and A2142G. No sample was found positive for both mutations. Metronidazole showed the highest resistance among antibiotics (78.9%) followed by Clarithromycin (47.3%).
CONCLUSION
Prevalence of antimicrobial resistance in H. pylori in North-Eastern India is substantially high with A2143G mutation being clinically most important in conferring Clarithromycin resistance. This resistance might be associated with low eradication rates despite initiation of therapy. ROC analysis of PCR proved it to be a good diagnostic tool.
PubMed: 38906329
DOI: 10.1016/j.ijmmb.2024.100652 -
PloS One 2024Syphilis, caused by Treponema pallidum, is resurging globally. Molecular typing allows for the investigation of its epidemiology. In Pakistan and other nations, T....
Syphilis, caused by Treponema pallidum, is resurging globally. Molecular typing allows for the investigation of its epidemiology. In Pakistan and other nations, T. pallidum subsp. pallidum has developed widespread macrolide resistance in the past decade. A study at the Peshawar Regional Blood Centre from June 2020-June 2021 analyzed serum samples from 32,812 blood donors in Khyber Pakhtunkhwa, Pakistan, to assess circulating T. pallidum strains and antibiotic resistance. Blood samples were initially screened for T. pallidum antibodies using a chemiluminescent microparticle immunoassay (CMIA). CMIA-reactive samples underwent polymerase chain reaction (PCR) targeted the polA, tpp47, bmp, and tp0319 genes. PCR-positive samples were further analyzed for molecular subtyping using a CDC-developed procedure and tp0548 gene examination. All PCR-positive samples were analyzed for the presence of point mutations A2058G and A2059G in 23S rRNA, as well as the G1058C mutation in 16S rRNA. These mutations are known to impart antimicrobial resistance to macrolides and doxycycline, respectively. Out of 32,812 serum samples, 272 (0.83%) were CMIA-reactive, with 46 being PCR-positive. Nine T. pallidum subtypes were identified, predominantly 14d/f. The A2058G mutation in 23S rRNA was found in 78% of cases, while G1058C in 16S rRNA and A2059G in 23S rRNA were absent. The research found donor blood useful for assessing T. pallidum molecular subtypes and antibiotic resistance, especially when chancres are not present. The prevalent subtype was 14d/f (51.85%), and the high macrolide resistance of 36 (78%) indicates caution in using macrolides for syphilis treatment in Khyber Pakhtunkhwa, Pakistan.
Topics: Treponema pallidum; Humans; Pakistan; Syphilis; Blood Donors; Anti-Bacterial Agents; Drug Resistance, Bacterial; Male; Female; Adult; Macrolides; RNA, Ribosomal, 23S; RNA, Ribosomal, 16S; Middle Aged; Doxycycline; Young Adult
PubMed: 38905249
DOI: 10.1371/journal.pone.0305720 -
Microbiology Spectrum Jun 2024To analyze the characteristics of as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. Thirteen clinical strains...
To analyze the characteristics of as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. Thirteen clinical strains isolated from 2003 to 2019 were selected, 10 of which were resistant to erythromycin (MIC >64 µg/mL), including 8 P1-type I and 2 P1-type II. Three were sensitive (<1 µg/mL) and P1-type II. One resistant strain had an A→G point mutation at position 2064 in region V of the 23S rRNA, the others had it at position 2063, while the three sensitive strains had no mutation here. Genome assembly and comparative genome analysis revealed a high level of genome consistency within the P1 type, and the primary differences in genome sequences concentrated in the region encoding the P1 protein. In P1-type II strains, three specific gene mutations were identified: C162A and A430G in L4 gene and T1112G mutation in the CARDS gene. Clinical information showed seven cases were diagnosed with severe pneumonia, all of which were infected with drug-resistant strains. Notably, BS610A4 and CYM219A1 exhibited a gene multi-copy phenomenon and shared a conserved functional domain with the DUF31 protein family. Clinically, the patients had severe refractory pneumonia, with pleural effusion, necessitating treatment with glucocorticoids and bronchoalveolar lavage. The primary variations between strains occur among different P1-types, while there is a high level of genomic consistency within P1-types. Three mutation loci associated with specific types were identified, and no specific genetic alterations directly related to clinical presentation were observed.IMPORTANCE is an important pathogen of community-acquired pneumonia, and macrolide resistance brings difficulties to clinical treatment. We analyzed the characteristics of as well as macrolide antibiotic resistance through whole-genome sequencing and comparative genomics. The work addressed primary variations between strains that occur among different P1-types, while there is a high level of genomic consistency within P1-types. In P1-type II strains, three specific gene mutations were identified: C162A and A430G in L4 gene and T1112G mutation in the CARDS gene. All the strains isolated from severe pneumonia cases were drug-resistant, two of which exhibited a gene multi-copy phenomenon, sharing a conserved functional domain with the DUF31 protein family. Three mutation loci associated with specific types were identified, and no specific genetic alterations directly related to clinical presentation were observed.
PubMed: 38904371
DOI: 10.1128/spectrum.03615-23 -
Frontiers in Genetics 2024Dysferlinopathy is an autosomal recessive disorder caused by mutations in the DYSF gene. This study reported two homozygous adjacent missense mutations in the DYSF...
Dysferlinopathy is an autosomal recessive disorder caused by mutations in the DYSF gene. This study reported two homozygous adjacent missense mutations in the DYSF gene, presenting clinically with bilateral lower limb weakness and calf swelling. Two homozygous adjacent missense mutations in the DYSF gene may be associated with the development of dysferlinopathy, but the exact mechanism needs further investigation. A retrospective analysis of clinical data from a dysferlinopathy-affected family was conducted. Peripheral blood samples were collected from members of this family for whole-exome sequencing (WES) and copy number variation analysis. Sanger sequencing was employed to confirm potential pathogenic variants. The Human Splicing Finder, SpliceAI, and varSEAK database were used to predict the effect of mutations on splicing function. The pathogenic mechanism of aberrant splicing in dysferlinopathy due to two homozygous adjacent missense mutations in the DYSF gene was determined by an splicing assay and an minigene assay. The proband was a 42-year-old woman who presented with weakness of the lower limbs for 2 years and edema of the lower leg. Two homozygous DYSF variants, c.5628C>A p. D1876E and c.5633A>T p. Y1878F, were identified in the proband. Bioinformatics databases suggested that the mutation c.5628C>A of DYSF had no significant impact on splicing signals. Human Splicing Finder Version 2.4.1 suggested that the c.5633A>T of DYSF mutation caused alteration of auxiliary sequences and significant alteration of the ESE/ESS motif ratio. VarSEAK and SpliceAI suggested that the c.5633A>T of DYSF mutation had no splicing effect. Both an splicing assay and an minigene assay showed two adjacent mutations: c.5628C>A p. D1876E and c.5633A>T p. Y1878F in the DYSF gene leading to an Exon50 jump that resulted in a 32-aa amino acid deletion within the protein. Point mutation c.5628C>A p. D1876E in the DYSF gene affected splicing , while point mutation c.5633A>T p. Y1878F in the DYSF gene did not affect splicing function. This study confirmed for the first time that two homozygous mutations of DYSF were associated with the occurrence of dysferlinopathy. The c.5628C>A p. D1876E mutation in DYSF affected the splicing function and may be one of the contributing factors to the pathogenicity.
PubMed: 38903757
DOI: 10.3389/fgene.2024.1404611 -
Neuro-oncology Advances 2024Fibroblast growth factor receptor 1 () mutations have been associated with poorer prognoses in pediatric central nervous system tumor patients. A recent study...
BACKGROUND
Fibroblast growth factor receptor 1 () mutations have been associated with poorer prognoses in pediatric central nervous system tumor patients. A recent study highlighted a link between mutations and spontaneous intracranial hemorrhage (ICH), demonstrating that all patients with an alteration experienced hemorrhage at some point during their course of treatment.
METHODS
The current study examined 50 out of 67 pediatric patients with low-grade gliomas (LGGs) who had genomic testing between 2011 and 2022 at our institution to determine whether a correlation exists between mutations and spontaneous ICH.
RESULTS
We found that of the 50 patients with genomic data, 7 (14%) experienced ICH, and an additional spontaneous hemorrhage was recorded; however, no genomic testing was performed for this case. Five of the seven patients (71.4%) had an modification. In our patient population, 6 expressed a detectable mutation (66.7% [4/6] had N546K alteration, 16.7% [1/6] exons duplication, and 16.7% [1/6] had a variant of unknown significance [VUS]). The patient with the VUS had no reported spontaneous hemorrhage. Statistical analysis found a significant association between and spontaneous intracranial hemorrhage (-value = < .0001). In the patient population, all cases of alterations ( = 3) co-occurred with mutations.
CONCLUSIONS
Our case series highlights this link between the FGFR1 mutation and spontaneous intracranial hemorrhage in pediatric LGGs.
PubMed: 38903142
DOI: 10.1093/noajnl/vdae074 -
Stem Cell Research & Therapy Jun 2024Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of...
Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34 HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45 cells in the peripheral blood, even when only 5,000-10,000 CD34 HSCs were transferred. Efficient engraftment of human CD45 cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34CD38 or CD34CD38 cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45 and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34 cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.
Topics: Animals; Humans; Proto-Oncogene Proteins c-kit; Hematopoiesis; Mice; Hematopoietic Stem Cells; Hematopoietic Stem Cell Transplantation; Mice, Transgenic; Cell Lineage; Antigens, CD34; Interleukin-3; Mutation
PubMed: 38902833
DOI: 10.1186/s13287-024-03799-w -
The Journal of Molecular Diagnostics :... Jul 2024The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity...
The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity with minimal equipment requirements are urgently needed to detect the many circulating species, subtypes, and variants of influenza at the point of need. Here, we introduce such a method using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a clustered regularly interspaced short palindromic repeats (CRISPR)-based RNA detection platform. Four SHINE assays were designed and validated for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously. This study demonstrates the utility of this duplex assay in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as in simultaneously detecting influenza A and human RNAse P in patient samples. These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance.
Topics: Humans; Influenza, Human; CRISPR-Cas Systems; Sensitivity and Specificity; RNA, Viral; Clustered Regularly Interspaced Short Palindromic Repeats; Molecular Diagnostic Techniques; Influenza A Virus, H3N2 Subtype; Influenza A Virus, H1N1 Subtype; Influenza A virus
PubMed: 38901927
DOI: 10.1016/j.jmoldx.2024.04.004 -
Surgical Case Reports Jun 2024No standard therapy for non-small lung cancer patients that have acquired resistance to tyrosine kinase inhibitor (TKI) therapy has been established. Some can be...
BACKGROUND
No standard therapy for non-small lung cancer patients that have acquired resistance to tyrosine kinase inhibitor (TKI) therapy has been established. Some can be effectively treated by salvage surgery, though indications for that procedure remain unclear. Reported here is the clinical course of a patient who experienced early post-operative distant metastases.
CASE PRESENTATION
A 48-year-old woman without symptoms was referred to another hospital for abnormal chest radiography findings and diagnosed with adenocarcinoma of the left lower lobe (cT2aN3M1b, stage IVB; TNM staging 7th edition). Gene mutation analysis revealed positive for epidermal growth factor receptor exon 19 deletion. Afatinib treatment was started, resulting in partial response, though regrowth of the main tumor was noted 1.5 years later. Bronchoscopic re-biopsy findings revealed a T790M point mutation and afatinib was switched to osimertinib. At 1.5 years following the start of osimertinib administration, the primary tumor was found to have regrown again and stereotactic radiation therapy was administered. Findings at 3.5 years after osimertinib administration indicated that all lymph nodes and distant metastases, excluding the primary tumor, were well controlled, and the patient was referred to our hospital for salvage surgery. Osimertinib was discontinued, and a left lower lobectomy with a left lingular segmentectomy and pleural biopsy were performed. The patient was discharged following an uneventful postoperative course. Three days after discharge, glossodynia developed and examination findings revealed tongue metastasis. The symptoms improved following re-administration of osimertinib, though right adrenal gland metastasis appeared 8 months after surgery. Radiation therapy was performed for tongue and right adrenal gland metastases, and the patient was alive 1 year after salvage surgery without out-of-control lesion appearing after the radiation therapy under the administration of osimertinib.
CONCLUSION
The present patient experienced multiple instances of systemic recurrence after undergoing salvage surgery. Experience with this case indicates that systemic therapy is essential for patients with distant metastatic lung cancer even following salvage surgery for the primary tumor.
PubMed: 38898314
DOI: 10.1186/s40792-024-01950-6 -
Nature Communications Jun 2024Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi...
Many bacterial pathogens, including the human exclusive pathogen Salmonella Typhi, express capsular polysaccharides as a crucial virulence factor. Here, through S. Typhi whole genome sequence analyses and functional studies, we found a list of single point mutations that make S. Typhi hypervirulent. We discovered a single point mutation in the Vi biosynthesis enzymes that control Vi polymerization or acetylation is enough to result in different capsule variants of S. Typhi. All variant strains are pathogenic, but the hyper Vi capsule variants are particularly hypervirulent, as demonstrated by the high morbidity and mortality rates observed in infected mice. The hypo Vi capsule variants have primarily been identified in Africa, whereas the hyper Vi capsule variants are distributed worldwide. Collectively, these studies increase awareness about the existence of different capsule variants of S. Typhi, establish a solid foundation for numerous future studies on S. Typhi capsule variants, and offer valuable insights into strategies to combat capsulated bacteria.
Topics: Salmonella typhi; Animals; Mice; Virulence; Polysaccharides, Bacterial; Mutation, Missense; Bacterial Capsules; Typhoid Fever; Humans; Bacterial Proteins; Virulence Factors; Female; Whole Genome Sequencing
PubMed: 38898034
DOI: 10.1038/s41467-024-49590-6