Defects In Maintenance Research Articles

Human formin FHOD3-mediated actin elongation is required for sarcomere integrity in cardiomyocytes.

Contractility and cell motility depend on accurately controlled assembly of the actin cytoskeleton. Formins are a large group of actin assembly proteins that nucleate new actin filaments and act as elongation factors. Some formins may cap filaments, instead of elongating them, and others are known to sever or bundle filaments. The Formin HOmology Domain-containing protein (FHOD)-family of formins is critical to the formation of the fundamental contractile unit in muscle, the sarcomere. Specifically, mammalian FHOD3L plays an essential role in cardiomyocytes. Despite our knowledge of FHOD3L's importance in cardiomyocytes, its biochemical and cellular activities remain poorly understood. It has been proposed that FHOD-family formins act by capping and bundling, as opposed to assembling new filaments. Here, we demonstrate that FHOD3L nucleates actin and rapidly but briefly elongates filaments after temporarily pausing elongation, in vitro . We designed function-separating mutants that enabled us to distinguish which biochemical roles are req`uired in the cell. We found that human FHOD3L's elongation activity, but not its nucleation, capping, or bundling activity, is necessary for proper sarcomere formation and contractile function in neonatal rat ventricular myocytes. The results of this work provide new insight into the mechanisms by which formins build specific structures and will contribute to knowledge regarding how cardiomyopathies arise from defects in sarcomere formation and maintenance.

Guanylate Kinase 1 Deficiency: A Novel and Potentially Treatable Mitochondrial DNA Depletion/Deletions Disease.

Mitochondrial DNA (mtDNA) depletion/deletions syndrome (MDDS) comprises a group of diseases caused by primary autosomal defects of mtDNA maintenance. Our objective was to study the etiology of MDDS in 4 patients who lack pathogenic variants in known genetic causes. Whole exome sequencing of the probands was performed to identify pathogenic variants. We validated the mitochondrial defect by analyzing mtDNA, mitochondrial dNTP pools, respiratory chain activities, and GUK1 activity. To confirm pathogenicity of GUK1 deficiency, we expressed 2 GUK1 isoforms in patient cells. We identified biallelic GUK1 pathogenic variants in all 4 probands who presented with ptosis, ophthalmoparesis, and myopathic proximal limb weakness, as well as variable hepatopathy and altered T-lymphocyte profiles. Muscle biopsies from all probands showed mtDNA depletion, deletions, or both, as well as reduced activities of mitochondrial respiratory chain enzymes. GUK1 encodes guanylate kinase, originally identified as a cytosolic enzyme. Long and short isoforms of GUK1 exist. We observed that the long isoform is intramitochondrial and the short is cytosolic. In probands' fibroblasts, we noted decreased GUK1 activity causing unbalanced mitochondrial dNTP pools and mtDNA depletion in both replicating and quiescent fibroblasts indicating that GUK1 deficiency impairs de novo and salvage nucleotide pathways. Proband fibroblasts treated with deoxyguanosine and/or forodesine, a purine phosphatase inhibitor, ameliorated mtDNA depletion, indicating potential pharmacological therapies. Primary GUK1 deficiency is a new and potentially treatable cause of MDDS. The cytosolic isoform of GUK1 may contribute to the T-lymphocyte abnormality, which has not been observed in other MDDS disorders. ANN NEUROL 2024.

Phospholipase C zeta: a hidden face of sperm for oocyte activation and early embryonic development.

Oocyte activation is a fundamental event in mammalian fertilization and is initiated by a cascade of calcium signaling and oscillation pathways. Phospholipase C zeta (PLCζ) is involved in modulating cortical granule exocytosis, releasing oocyte meiotic arrest, regulating gene expression, and early embryogenesis. These processes are considered to be initiated and controlled by PLCζ activity via the inositol-1,4,5-triphosphate pathway. The decrease or absence of functional PLCζ due to mutational defects in protein expression or maintenance can impair male fertility. In this literature review, we highlight the significance of PLCζ as a sperm factor involved in oocyte activation, its mechanism of action, the signaling pathway involved, and its close association with oocyte activation. Finally, we discuss the relationship between male infertility and PLCζ deficiency.

Cell wall integrity modulates HOOKLESS1 and PHYTOCHROME INTERACTING FACTOR4 expression controlling apical hook formation.

Formation of the apical hook in etiolated dicot seedlings results from differential growth in the hypocotyl apex and is tightly controlled by environmental cues and hormones, among which auxin and gibberellins (GAs) play an important role. Cell expansion is tightly regulated by the cell wall, but whether and how feedback from this structure contributes to hook development is still unclear. Here, we show that etiolated seedlings of the Arabidopsis (Arabidopsis thaliana) quasimodo2-1 (qua2) mutant, defective in pectin biosynthesis, display severe defects in apical hook formation and maintenance, accompanied by loss of asymmetric auxin maxima and of differential cell expansion. Moreover, qua2 seedlings show reduced expression of HOOKLESS 1 (HLS1) and PHYTOCHROME INTERACTING FACTOR 4 (PIF4), which are positive regulators of hook formation. Treatment of wild-type seedlings with the cellulose inhibitor isoxaben (isx) also prevents hook development and represses HLS1 and PIF4 expression. Exogenous GAs, loss of DELLA proteins or HLS1 overexpression partially restore hook development in qua2 and isx-treated seedlings. Interestingly, increased agar concentration in the medium restores, both in qua2 and isx-treated seedlings, hook formation, asymmetric auxin maxima and PIF4 and HLS1 expression. Analysis of plants expressing a FRET-based GA sensor indicate that isx reduces accumulation of GAs in the apical hook region in a turgor-dependent manner. Lack of the cell wall integrity sensor THESEUS 1, which modulates turgor loss point, restores hook formation in qua2 and isx-treated seedlings. We propose that turgor-dependent signals link changes in cell wall integrity to the PIF4-HLS1 signalling module to control differential cell elongation during hook formation.

Digital telomere measurement by long-read sequencing distinguishes healthy aging from disease

Telomere length is an important biomarker of organismal aging and cellular replicative potential, but existing measurement methods are limited in resolution and accuracy. Here, we deploy digital telomere measurement (DTM) by nanopore sequencing to understand how distributions of human telomere length change with age and disease. We measure telomere attrition and de novo elongation with up to 30 bp resolution in genetically defined populations of human cells, in blood cells from healthy donors and in blood cells from patients with genetic defects in telomere maintenance. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres. In patients with defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease.

Research on Surface Defect Detection Method of Photovoltaic Power Generation Panels——Comparative Analysis of Detecting Model Accuracy

INTRODUCTION: Research on intelligent defect detection technology using machine vision was conducted to address the challenging problem of detecting and localizing PV defects in photovoltaic power generation system operation and maintenance. OBJECTIVES: The aim is to improve the accuracy of PV defect detection and enhance the operation and maintenance efficiency of PV power plants. METHODS: In this paper, three detection methods such as image processing based detection, traditional machine learning based detection, and deep learning algorithm based detection are discussed and compared, and analyzed respectively. It is finally concluded that the deep learning based detection is more efficient in comparison. Then further analysis and simulation experiments are done through several detection algorithms based on deep learning. RESULTS: The experiment yields a high accuracy of the detection model based on the Faster-RCNN algorithm. Its mAP value reaches 92.6%. The detection model based on the YOLOv5 algorithm reaches a mAP value of 91.4%. But its speed is as much as 7 times faster than the model based on the Faster-RCNN algorithm. CONCLUSION: Comprehensive speed and accuracy index. Combining the needs of PV defect detection in the operation and maintenance of PV power generation systems with the results of simulation experiments. It is concluded that the detection model based on the YOLOv5 algorithm can provide better detection capability. Modeling with this algorithm is more suitable for PV defect detection.

Abstract LB185: High resolution telomere measurements in human cancer and aging using long-read nanopore sequencing

Abstract Telomere length is an important biomarker of cellular replicative potential and aging, but existing measurement methods are limited in resolution and accuracy. During human carcinogenesis, telomeres shorten profoundly in the earliest stages that precede tumor invasion. Telomere shortening can either halt cancer progression by inducing senescence and cell death or can promote cancer development by destabilizing chromosomes and generating chromosomal rearrangements. Stabilization of telomeres, typically by the upregulation of telomerase, is required for progression of nearly all human cancers. Here, we deploy digital telomere measurement by nanopore sequencing to understand how distributions of human telomere length change during aging and following carcinogenesis. We measure telomere attrition and de novo elongation with unprecedented resolution in genetically defined populations of human stem cells, in cells from the peripheral blood of healthy individuals and patients with genetic defects in telomere maintenance, cancer patients, and cancer cell lines. We find that human aging is accompanied by a progressive loss of long telomeres and an accumulation of shorter telomeres in white blood cells. In patients with inherited defects in telomere maintenance, the accumulation of short telomeres is more pronounced and correlates with phenotypic severity. We apply machine learning to train a binary classification model that distinguishes healthy individuals from those with telomere biology disorders. Next, we investigated the telomere lengths of a cohort of twenty colorectal tumors with patient-matched healthy colonic epithelia and found that two-thirds of colorectal tumors have telomere length distributions significantly shorter than the surrounding normal tissue. Finally, we explore the utility of telomere length distributions in cancer cell lines as clinically actionable disease biomarkers. This sequencing and bioinformatic pipeline will advance our understanding of telomere maintenance mechanisms and the use of telomere length as a clinical biomarker of aging and disease. Citation Format: Santiago Sanchez, Jessica Gu, Anudeep Golla, William Shomali, Annika Martin, Dirk Hockemeyer, Sharon Savage, Steven Artandi. High resolution telomere measurements in human cancer and aging using long-read nanopore sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB185.

Escargot a Snail superfamily member and its multiple roles in Drosophila melanogaster development.

The Snail superfamily of transcription factors plays a crucial role in metazoan development; one of the most important vertebrate members of this family is Snai1 which is orthologous to the Drosophila melanogaster esg gene. This review offers a comprehensive examination of the roles of the esg gene in Drosophila development, covering its expression pattern and downstream targets, and draws parallels between the vertebrate Snai1 family proteins on controlling the epithelial-to-mesenchymal transition and esg. This gene regulates stemness, ploidy, and pluripontency. esg is expressed in various tissues during development, including the gut, imaginal discs, and neuroblasts. The functions of the esg include the suppression of differentiation in intestinal stem cells and the preservation of diploidy in imaginal cells. In the nervous system development, esg expression also inhibits neuroblast differentiation, thus regulating the number of neurons and the moment in development of neuronal differentiation. Loss of esg function results in diverse developmental defects, including defects in intestinal stem cell maintenance and differentiation, and alters imaginal disc and nervous system development. Expression levels of esg also play a role in regulating longevity and metabolism in adult stages. This review provides an overview of the current understanding of esg's developmental role, emphasizing cellular and tissue effects that arise from its loss of function. The insights gained may contribute to a better understanding of evolutionary conserved developmental mechanisms and certain metabolic diseases.

Single-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival.

Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.

Abstract B025: Exceptional response to the ataxia telangiectasia and Rad3-related inhibitor (ATRi), camonsertib, in a patient with alternative lengthening of telomeres (ALT)-positive metastatic melanoma

Abstract One hallmark of cancer tumorigenesis is the maintenance of telomere length which canonically occurs through the reactivation of telomerase. Alternative lengthening of telomeres (ALT) is a minor telomere maintenance mechanism that uses homologous recombination (HR) to maintain telomere length and is associated with replication stress and defects in genome maintenance. In preclinical models, ALT positivity (ALT+) has been shown to sensitize tumor cells to ataxia telangiectasia and Rad3-related inhibitors (ATRi). Here we describe a confirmed clinical and molecular response to the ATRi, camonsertib, in a patient with ALT+ metastatic melanoma. To our knowledge, this is the first report of clinical experience with ATRi in ALT+ tumors. A 63-year-old male was diagnosed in July 2019 with stage IV (cTX, cNX, pM1c[1]) metastatic melanoma and treated with nivolumab (nivo) + ipilumumab (ipi) with initial partial response (PR) documented after 3 months which was confirmed on subsequent nivo maintenance. Ipi was resumed following suspected pseudoprogression in March 2020, and continued until confirmed progression in June 2020. Genomic profiling of a liver biopsy collected at the time of progression identified somatic alterations in TP53 (p.S127F, p.R65fs), NF1 (p.K1263*), BRCA2 (p.R2482C) and ATRX (p.R418*), a germline CHEK2 mutation (p.T376fs), and high tumor mutational burden. The patient received additional targeted regimens with olaparib (ola): nivo ± ola and later ola ± trametinib; no response was documented, and progression was noted in April 2021. In June 2021, the patient received camonsertib monotherapy at an initial dose of 120 mg QD 3 days on/4 days off in the phase I TRESR trial (NCT04497116). The patient tolerated treatment well and continued at the same dose until dose was reduced via a change in schedule (to 2 weeks on/1 week off) at 9 months due to anemia. The patient had an initial RECIST PR (-36%) at 3 months with circulating tumor DNA (ctDNA) clearance. The target lesions continued to decrease with a best decline of -74% after about 1 year of treatment. The cancer progressed at 19 months (+105% from nadir, -47% from baseline) though due to ongoing clinical benefit treatment was continued for a total duration of 22 months. Interrogation of ctDNA tumor fraction in longitudinal plasma samples is ongoing and will be reported. To understand the long and deep response to camonsertib treatment in this patient, we further characterized the screening biopsy by a custom targeted sequencing panel, SyNthetic lethal Interactions for Precision Diagnostics (SNiPDx), and whole genome sequencing (WGS). Both the BRCA2 and gCHEK2 mutations were monoallelic and genomic signature analysis defined the tumor as HR-proficient, consistent with the lack of benefit from ola. WGS confirmed a biallelic loss of ATRX with a genomic signature consistent with an ALT+ phenotype. The clinical experience of ATRi therapy in patients with ALT+ tumors is limited but further investigation of ATRi such as camonsertib is warranted in this patient population. Citation Format: Timothy A. Yap, Ian M. Silverman, Adrienne Johnson, Chenfeng Meng, Joseph D. Schonhoft, Michal Zimmermann, Danielle Ulanet, Victoria Rimkunas, Maria Koehler. Exceptional response to the ataxia telangiectasia and Rad3-related inhibitor (ATRi), camonsertib, in a patient with alternative lengthening of telomeres (ALT)-positive metastatic melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B025.

Essential functions of RNA helicase Vasa in maintaining germline stem cells and piRNA-guided Stellate silencing in Drosophila spermatogenesis.

DEAD-box RNA helicase Vasa is required for gonad development and fertility in multiple animals. Vasa is implicated in many crucial aspects of Drosophila oogenesis, including translation regulation, primordial germ cell specification, piRNA silencing of transposable elements, and maintenance of germline stem cells (GSCs). However, data about Vasa functions in Drosophila spermatogenesis remain controversial. Here we showed that loss-of-function vasa mutations led to failures of GSC maintenance in the testes, a severe loss of total germ cell content, and a cessation of male fertility over time. Defects in GSC maintenance in vasa mutant testes were not associated with an increasing frequency of programmed cell death, indicating that a premature loss of GSCs occurred via entering differentiation. We found that Vasa is implicated in the positive regulation of rhino expression both in the testes and ovaries. The introduction of a transgene copy of rhino, encoding a nuclear component of piRNA pathway machinery, in vasa mutant background allowed us to restore premeiotic stages of spermatogenesis, including the maintenance of GSCs and the development of spermatogonia and spermatocytes. However, piRNA-guided repression of Stellate genes in spermatocytes of vasa mutant testes with additional rhino copy was not restored, and male fertility was disrupted. Our study uncovered a novel mechanistic link involving Vasa and Rhino in a regulatory network that mediates GSC maintenance but is dispensable for the perfect biogenesis of Su(Ste) piRNAs in testes. Thus, we have shown that Vasa functions in spermatogenesis are essential at two distinct developmental stages: in GSCs for their maintenance and in spermatocytes for piRNA-mediated silencing of Stellate genes.

DNA Damage Response Constrains Cell Growth and Drives Clonal Hematopoiesis in Telomere Biology Disorders

Telomere biology disorders (TBD) are inherited diseases caused by genetic defects in telomere maintenance. Patients with TBD have critically shortened telomeres, leading to bone marrow failure (BMF) and an increased risk of myeloid neoplasms (MN). We conducted a study to understand the genetic changes driving clonal evolution in TBD patients and their distinct mechanisms compared to normal aging and sporadic myelodysplastic syndrome (MDS). We analyzed acquired genetic alterations in hematopoietic cells from 105 TBD patients. The diagnosis of TBD was made using a combination of clinical history, germline mutation status, and telomere length. Germline mutations were identified in 84% of patients, a family history consistent with TBD was seen in 46%, and 76% of patients had lymphocyte telomere lengths (TL) <1st %ile for their age, while 22% fell between the 1st and 10th %ile. Complications observed in our cohort included BMF (42%), myeloid neoplasms (17%), solid tumors (17%), cirrhosis (14%), and interstitial lung disease (34%). Clonal hematopoiesis was detected in 44 of 105 patients (42%), including 8 of 27 (30%) patients 18 years or younger. Somatic genetic alterations primarily involved the DNA damage response (DDR) pathway, with ATM and TP53 being the most frequently mutated genes (11 ATM mutations in 5 of 80 patients; 5 TP53 mutations in 4 of 84 patients; Fig. 1). Mutations typical of age-related clonal hematopoiesis were seen in 4 patients (3 ASXL1, 1 TET2). Somatic reversion of TBD germline lesions was found in 7 patients. Additionally, 11 somatic mutations were detected in genes involved in DDR, telomere maintenance, and cell senescence, including KAT6B, PALB2, TELO2, TOP1, and RTEL1. MDS-associated gene mutations were found in 15 patients (18%), 8 in the setting of MN. The most common mutations affected spliceosome genes with U2AF1 p.S34F and SF3B1 being most notable, including 1 U2AF1 S34F identified in a 15-year-old. Mutations in NPM1, RUNX1, WT1, SF3B1, and FLT3 were identified only after malignant transformation. Acquired cytogenetic changes were observed in 16 of 87 evaluable patients (18%), 8 in the setting of MN, and included gain of 1q in 5 patients (5.7%). Two patients with TP53 mutations developed a complex karyotype (Fig 1). To understand selective pressures contributing to clonal hematopoiesis development in TBD, we analyzed transcriptional changes in bone marrow (n=2) and peripheral blood (n=1) mononuclear cells (MNCs) of 3 TBD patients with different germline lesions using single cell RNA sequencing (scRNASeq). MNCs from TBD patients exhibited significant differences compared to healthy controls, showing downregulation of MYC targets, reduced translation, cell cycle arrest, and upregulation of ATM-dependent DDR pathway and FAS pathway (Fig 2). We hypothesized that mutations in DDR pathway genes, particularly somatic ATM loss, enable TBD hematopoietic stem and progenitor cells to overcome telomere-induced DDR induction and premature senescence. Consistent with our hypothesis, low dose ATM inhibition in TBD patients' fibroblasts significantly enhanced growth rate of TERC-mutated and DKC1-mutated cells, whereas control fibroblasts were unaffected. Somatic mutations in ATM in our cohort were typically observed in patients with TERC-mutated TBD (3 of 5, 3 of 34 with TERC-mutated TBD), very low TL, and extra-hematopoietic manifestations. Of the 11 ATM mutations, 3 were predicted loss of function and 4 were missense kinase domain mutations, known or predicted to result in loss of kinase activity. Somatic ATM loss occurred both as heterozygous or compound heterozygous mutations. The development of ATM mutations alone was not causative of malignancy; however, 2 patients with somatic ATM mutations developed myeloid neoplasms after acquisition of additional mutations (subclonal mutation in NPM1 in one patient and clonal replacement with a TP53-mutant clone in another patient). In conclusion, our findings highlight the role of telomere dysfunction-induced DDR and cellular senescence in bone marrow failure and clonal selection in TBD. ATM loss selectively enhances growth of TBD cells by inhibiting DDR activation which may ameliorate marrow failure but has the potential to increase the risk of myeloid neoplasms after acquisition of additional genetic events. Future studies should explore the possibility of a therapeutic window involving very low doses of ATM inhibition in TBD.

Large-Scale Characterization and Functional Assessment of Adaptive Immunity in Patients with MGUS and SMM Reveals Significant Defects

Introduction Patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) or Smoldering Multiple Myeloma (SMM) exhibit signs of immune dysregulation despite their early stage of oncogenesis. The SARS-CoV-2 pandemic and subsequent broad vaccination of the population presented an opportunity to compare the real-life immune response of patients and healthy individuals following exposure to a common antigen. Here, we have used SARS-CoV-2 vaccination as a model to study the adaptive immune response and have thus comprehensively assessed the adaptive immune response of patients with MGUS/SMM to SARS-CoV-2 vaccination compared to healthy donors (HD). Methods We performed (i) paired 5' single-cell RNA and T cell receptor (TCR) sequencing on 224 peripheral blood mononuclear cell (PBMC) samples drawn serially before and after two doses of SARS-CoV-2 vaccination (HD, n=26; MGUS, n=20; SMM, n=48; Multiple Myeloma (MM), n=24), (ii) ELISA for anti-Spike IgG antibodies post-vaccination (post-vx), (HD, n=237; MGUS, n=550; SMM, n=947) and MMR antibodies at baseline (HD, n=20; SMM, n=20), (iii) bulk TCR sequencing pre- and post-vx (HD, n=2; SMM, n=2) (Adaptive Biotechnologies), and (iv) IFN gamma ELISpot assay following in vitro stimulation with Spike peptide (HD, n=20; SMM, n=20). All post-vx samples were drawn after 2 doses of vaccination. Results Patients and HD showed comparable anti-Spike antibody titers within 14-50 days post-vx, however, we observed faster waning in patients with MGUS/SMM, who had significantly lower titers 50-100 days post-vx (p=0.014 & p=0.007, respectively), suggesting a potential defect in long-lived plasma cell maintenance. Indeed, patients with SMM showed significantly lower titers of antibodies against Mumps and Rubella, antigens encountered in childhood, compared to age-matched HD (p=0.002 & p=0.03, respectively), showing that this defect may also lead to loss of plasma cell memory. Next, we performed single-cell RNA/TCR-seq to assess the patients' T cell response to vaccination. Patients with SMM showed a significant increase in TCR repertoire diversity post-vx (paired t-test, p=0.046), suggesting at least partial preservation of T cell responses with recruitment of rare clones. Indeed, using bulk TCR-seq, we observed Spike-specific T cells in patients with MGUS/SMM. Interestingly, however, a significant enrichment in Spike-specific clones by single-cell TCR-seq was only observed in HD post-vx (paired t-test, p=0.01), suggesting patient T cell responses may be suboptimal. Functional assessment of T cell activation following stimulation with the Spike peptide showed significantly lower activation in patients with SMM compared to HD (t-test, p=0.002), confirming the suspected defect. Notably, a larger proportion of Spike-specific clones in patients was of the Treg phenotype compared to HD (empirical p=0.002), which may partly explain the observed defect. Conclusions This study demonstrates that despite the absence of symptoms, patients with MGUS/SMM may demonstrate defective humoral and cellular immune responses, including loss of plasma cell memory. Specific interventions, such as changes in the vaccination schedule, may be warranted for this population, and the morbidity associated with these defects should be studied further.

Saccharomyces cerevisiae survival against heat stress entails a communication between CCT and cell wall integrity pathway.

The chaperonin TRiC/CCT is cytosolic cylindrical complex of 16 subunits encoded by eight essential genes CCT1-8. It contributes to folding 10% of cellular polypeptides in yeast. The strain carrying substitution point mutation G412E in the equatorial domain of Cct7p resulted in the improper folding of substrates. In this study, the Cct7p mutant exhibited sensitivity to non-optimal growth temperatures and cell wall stressors. Heat shock is known to disrupt cell wall and protein stability in budding yeast. Mitogen-activated protein kinase-mediated cell wall integrity pathway gets activated to compensate the perturbed cell wall. Overexpression of the PKC1 and SLT2 genes of MAPK signaling pathway in mutant rescued the growth and cell division defects. Additionally, the genes of the CWI pathway such as SED1, GFA1, PIR1, and RIM21 are down-regulated. The Cct7p mutant strain (G412E) is unable to withstand the heat stress due to the underlying defects in protein folding and cell wall maintenance. Taken together, our results strongly indicate the interaction between CCT and cell wall integrity pathway.

Two mitochondrial HMG-box proteins, Cim1 and Abf2, antagonistically regulate mtDNA copy number in Saccharomyces cerevisiae.

The mitochondrial genome, mtDNA, is present in multiple copies in cells and encodes essential subunits of oxidative phosphorylation complexes. mtDNA levels have to change in response to metabolic demands and copy number alterations are implicated in various diseases. The mitochondrial HMG-box proteins Abf2 in yeast and TFAM in mammals are critical for mtDNA maintenance and packaging and have been linked to mtDNA copy number control. Here, we discover the previously unrecognized mitochondrial HMG-box protein Cim1 (copy number influence on mtDNA) in Saccharomyces cerevisiae, which exhibits metabolic state dependent mtDNA association. Surprisingly, in contrast to Abf2's supportive role in mtDNA maintenance, Cim1 negatively regulates mtDNA copy number. Cells lacking Cim1 display increased mtDNA levels and enhanced mitochondrial function, while Cim1 overexpression results in mtDNA loss. Intriguingly, Cim1 deletion alleviates mtDNA maintenance defects associated with loss of Abf2, while defects caused by Cim1 overexpression are mitigated by simultaneous overexpression of Abf2. Moreover, we find that the conserved LON protease Pim1 is essential to maintain low Cim1 levels, thereby preventing its accumulation and concomitant repressive effects on mtDNA. We propose a model in which the protein ratio of antagonistically acting Cim1 and Abf2 determines mtDNA copy number.

Enterovirus D68 capsid formation and stability requires acidic compartments.

The respiratory picornavirus enterovirus D68 is a causative agent of acute flaccid myelitis, a childhood paralysis disease identified in the last decade. Poliovirus, another picornavirus associated with paralytic disease, is a fecal-oral virus that survives acidic environments when passing from host to host. Here, we follow up on our previous work showing a requirement for acidic intracellular compartments for maturation cleavage of poliovirus particles. Enterovirus D68 requires acidic vesicles for an earlier step, assembly, and maintenance of viral particles themselves. These data have strong implications for the use of acidification blocking treatments to combat enterovirus diseases.

Genetic Counseling and Family Screening Recommendations in Patients with Telomere Biology Disorders.

Telomere biology disorders (TBDs) encompass a spectrum of genetic diseases with a common pathogenesis of defects in telomerase function and telomere maintenance causing extremely short telomere lengths. Here, we review the current literature surrounding genetic testing strategies, cascade testing, reproductive implications, and the role of genetic counseling. The understanding of the genetic causes and clinical symptoms of TBDs continues to expand while genetic testing and telomere length testing are nuanced tools utilized in the diagnosis of this condition. Access to genetic counseling is becoming more abundant and is valuable in supporting patients and their families in making informed decisions. Patient resources and support groups are valuable to this community. Defining which populations should be offered genetic counseling and testing is imperative to provide proper diagnoses and medical management for not only the primary patient, but also their biological relatives.

Mitochondrial DNA Maintenance Disorders: Impact of Impaired Deoxynucleoside Triphosphates Metabolism

The maintenance of mitochondrial DNA (mtDNA) is dependent upon several nuclear gene-encoded proteins including enzymes forming the replisome needed to synthesise mtDNA. These enzymes need to be present in balanced quantities to function properly. In addition, mtDNA synthesis requires a balanced supply of nucleotides that is achieved by nucleoside recycling inside the mitochondria, and nucleotide import from the cytosol. Mitochondrial DNA maintenance defects are a group of diseases caused by pathogenic variants in the nuclear genes involved in mtDNA maintenance, and result from impaired mtDNA replication. Pathogenic nuclear gene variants identified to date include genes that encode enzymes of mtDNA replication machinery (such as POLG), genes that encode proteins that help to maintain a balanced mitochondrial nucleotide pool (such as TK2), and genes that encode proteins involved in mitochondrial fusion. Here, the presentation provided by Ramon Martí, Research Group on Neuromuscular and Mitochondrial Diseases, Vall d’Hebron Institut de Recerca, Barcelona, Spain, and CIBERER, Madrid, Spain, is summarised. A leading expert on mitochondrial pathology, Martí presented at the Euromit 2023 International Conference on Mitochondrial Disease, which took place in Bologna, Italy, in May 2023.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

Haploinsufficiency of the essential gene Rps12 causes defects in erythropoiesis and hematopoietic stem cell maintenance.

Ribosomal protein (Rp) gene haploinsufficiency can result in Diamond-Blackfan Anemia (DBA), characterized by defective erythropoiesis and skeletal defects. Some mouse Rp mutations recapitulate DBA phenotypes, although others lack erythropoietic or skeletal defects. We generated a conditional knockout mouse to partially delete Rps12. Homozygous Rps12 deletion resulted in embryonic lethality. Mice inheriting the Rps12KO/+ genotype had growth and morphological defects, pancytopenia, and impaired erythropoiesis. A striking reduction in hematopoietic stem cells (HSCs) and progenitors in the bone marrow (BM) was associated with decreased ability to repopulate the blood system after competitive and non-competitive BM transplantation. Rps12KO/+ lost HSC quiescence, experienced ERK and MTOR activation, and increased global translation in HSC and progenitors. Post-natal heterozygous deletion of Rps12 in hematopoietic cells using Tal1-Cre-ERT also resulted in pancytopenia with decreased HSC numbers. However, post-natal Cre-ERT induction led to reduced translation in HSCs and progenitors, suggesting that this is the most direct consequence of Rps12 haploinsufficiency in hematopoietic cells. Thus, RpS12 has a strong requirement in HSC function, in addition to erythropoiesis.