The splicing factor CCAR1 regulates the Fanconi anemia/BRCA pathway.

The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.

Fork restart: unloading FANCD2 to travel ahead.

In this issue of Molecular Cell, Brunner et al.1 reveal that eliminating FANCD2 from stalled forks via FBXL12-mediated degradation enables cells to tolerate oncogene-induced replication stress, making FBXL12 a promising target for cancer treatment.

CCAR2 functions downstream of the Shieldin complex to promote double-strand break end-joining.

The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.

TGFß pathway is required for viable gestation of Fanconi anemia embryos.

Overexpression of the TGFß pathway impairs the proliferation of the hematopoietic stem and progenitor cells (HSPCs) pool in Fanconi anemia (FA). TGFß promotes the expression of NHEJ genes, known to function in a low-fidelity DNA repair pathway, and pharmacological inhibition of TGFß signaling rescues FA HSPCs. Here, we demonstrate that genetic disruption of Smad3, a transducer of the canonical TGFß pathway, modifies the phenotype of FA mouse models deficient for Fancd2. We observed that the TGFß and NHEJ pathway genes are overexpressed during the embryogenesis of Fancd2-/- mice and that the Fancd2-/-Smad3-/- double knockout (DKO) mice undergo high levels of embryonic lethality due to loss of the TGFß-NHEJ axis. Fancd2-deficient embryos acquire extensive genomic instability during gestation which is not reversed by Smad3 inactivation. Strikingly, the few DKO survivors have activated the non-canonical TGFß-ERK pathway, ensuring expression of NHEJ genes during embryogenesis and improved survival. Activation of the TGFß-NHEJ axis was critical for the survival of the few Fancd2-/-Smad3-/- DKO newborn mice but had detrimental consequences for these surviving mice, such as enhanced genomic instability and ineffective hematopoiesis.

The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage.

Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin-binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C terminus of Ki-67 as an important module for genome stability.

In vitro analysis of monomer leaching in modern dental materials: CAD milled, printed, traditional heat-processed, and auto-polymerizing denture base resins.

PURPOSE: Denture fabrication has shifted from traditional heat-processed and auto-polymerizing materials to computer-aided design and computer-aided manufacturing (CAD-CAM) milled and printed materials. The monomer in traditional materials can induce an allergic reaction in some patients. With the rise in the edentulous population and increasing demand for the fabrication of dentures, these newer materials should be studied for monomer leaching. The purpose of this study was to evaluate the ratio of residual monomer in materials being used for denture bases: CAD-milled polymethylmethacrylate (PMMA), printed denture base resin, heat-processed PMMA, and auto-polymerizing PMMA comparatively. MATERIALS AND METHODS: Milled, printed, heat-activated, and auto-polymerizing denture base specimens (n = 3 for each group, each test run three times) were fabricated according to manufacturer recommendations. Specimens were first immersed in deuterated chloroform (CDCl3), a deuterated organic solvent, to evaluate monomer leaching and to observe physical properties of the materials. NMR spectroscopy was used to evaluate the dissolution of materials and residual monomer to crosslinked polymer ratios at 1, 4, and 9 days. A second group of specimens was then immersed in deuterium oxide (D2O) to evaluate if the residual monomers would leach out of the system. The solution was then analyzed using nuclear magnetic resonance (NMR) spectroscopy for 1 month. The deuterated forms of chloroform (CDCl3) and water (D2O) were used to enable sample characterization by NMR. RESULTS: While the heat-processed, auto-polymerizing, and milled specimens possessed residual monomers, no significant monomer leaching was noted in the printed specimen, while immersed in CDCl3. Similarly, the printed specimen was most resistant to dissolution, as compared to the rest; dissolution of the specimen is indicative of little to no cross-linking. No detectable dissolution of monomer was seen when all specimens were immersed in D2O for up to 1 month. CONCLUSIONS: Residual monomers were not found in the printed denture material in this study in either CDCl3 or D2O, whereas CAD-milled and traditionally processed denture bases still have residual monomers within their respective systems when immersed in organic solvent. None of the specimens tested leached monomers into D2O.

Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA.

In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents-MMS, 4NQO and bleomycin-that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.

High-Strength Organic-Inorganic Composites with Superior Thermal Insulation and Acoustic Attenuation.

We demonstrate facile fabrication of highly filled, lightweight organic-inorganic composites comprising polyurethanes covalently linked with naturally occurring clinoptilolite microparticles. These polyurethane/clinoptilolite (PUC) composites are shown to mitigate particle aggregation usually observed in composites with high particle loadings and possess enhanced thermal insulation and acoustic attenuation compared with conventionally employed materials (e.g., drywall and gypsum). In addition to these functional properties, the PUC composites also possess flexural strengths and strain capacities comparable to and higher than ordinary Portland cement (OPC), respectively, while being ∼1.5× lighter than OPC. The porosity, density, and mechanical and functional properties of these composites are tuned by systematically varying their composition (diisocyanate, polyurethane, and inorganic contents) and the nature of the organic (reactivity and source of polyol) components. The fabrication process involves mild curing conditions and uses commonly available reagents (naturally occurring aluminosilicate particles, polyols, and diisocyanate), thereby making the process scalable. Finally, the composite properties are shown to be independent of the polyol source (virgin or recycled), underlining the generality of this approach for the scalable utilization of recycled polyols.

Single-Stranded DNA Gap Accumulation Is a Functional Biomarker for USP1 Inhibitor Sensitivity.

Recent studies suggest that PARP and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA (ssDNA) gaps at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetically lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Herein, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells, and this phenotype correlates with drug sensitivity. USP1 inhibition increased monoubiquitinated proliferating cell nuclear antigen at replication forks, mediated by the ubiquitin ligase RAD18, and knockdown of RAD18 caused USP1 inhibitor resistance and suppression of ssDNA gaps. USP1 inhibition overcame PARP inhibitor resistance in a BRCA1-mutated xenograft model and induced ssDNA gaps. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. Finally, in patient-derived ovarian tumor organoids, sensitivity to USP1 inhibition alone or in combination correlated with the accumulation of ssDNA gaps. Assessment of ssDNA gaps in ovarian tumor organoids represents a rapid approach for predicting response to USP1 inhibition in ongoing clinical trials. Significance: USP1 inhibitors kill BRCA1-deficient cells and cause ssDNA gap accumulation, supporting the potential of using ssDNA gap detection as a functional biomarker for clinical trials on USP1 inhibitors.

Checkpoint regulation of replication forks: global or local?

Cell-cycle checkpoints are generally global in nature: one unattached kinetochore prevents the segregation of all chromosomes; stalled replication forks inhibit late origin firing throughout the genome. A potential exception to this rule is the regulation of replication fork progression by the S-phase DNA damage checkpoint. In this case, it is possible that the checkpoint is global, and it slows all replication forks in the genome. However, it is also possible that the checkpoint acts locally at sites of DNA damage, and only slows those forks that encounter DNA damage. Whether the checkpoint regulates forks globally or locally has important mechanistic implications for how replication forks deal with damaged DNA during S-phase.

High temporal resolution fluorescence measurements of a mitochondrial dye for detection of early stage apoptosis.

In the present study, early stage apoptosis is explored with high temporal resolution. In addition to monitoring early apoptosis induction in single cells by ultrasensitive confocal fluorescence microscopy (UCFM), the mitochondrial protein release kinetics was explored. The current study shows development and optimization of a novel, rapid apoptosis assay to explore the earliest changes in cells by the intrinsic apoptosis pathway. We show that early apoptotic changes in the mitochondria begin nearly simultaneously with the addition of an apoptosis-inducing drug, such as staurosporine. With a temporal resolution of five minutes, this non-invasive analytical technique can elucidate the earliest apoptotic events in living cells. Moreover, our results show that the mitochondrial inter-membrane proteins are not involved in the extrinsic pathway of Ramos cells mediated by an anti-CD95 antibody. Additional techniques such as light microscopy and flow cytometry were employed to confirm the results obtained by ultrasensitive confocal fluorescence microscopy. The results of this study help to understand the earliest mechanisms of apoptosis induction in cells, enabling new methods of drug testing and dose-response analyses.

Escherichia coli Adhesion and Biofilm Formation on Polymeric Nanostructured Surfaces.

Biofilm formation is a multistep process that requires initial contact between a bacterial cell and a surface substrate. Recent work has shown that nanoscale topologies impact bacterial cell viability; however, less is understood about how nanoscale surface properties impact other aspects of bacterial behavior. In this study, we examine the adhesive, viability, morphology, and colonization behavior of the bacterium Escherichia coli on 21 plasma-etched polymeric surfaces. Although we predicted that specific nanoscale surface structures of the surface would control specific aspects of bacterial behavior, we observed no correlation between any bacterial response or surface structures/properties. Instead, it appears that the surface composition of the polymer plays the most significant role in controlling and determining a bacterial response to a substrate, although changes to a polymeric surface via plasma etching alter initial bacteria colonization and morphology.

Short-term molecular consequences of chromosome mis-segregation for genome stability.

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.

CHAMP1 binds to REV7/FANCV and promotes homologous recombination repair.

A critical determinant of DNA repair pathway choice is REV7, an adaptor that binds to various DNA repair proteins through its C-terminal seatbelt domain. The REV7 seatbelt binds to either REV3, activating translesion synthesis, or to SHLD3, activating non-homologous end joining (NHEJ) repair. Recent studies have identified another REV7 seatbelt-binding protein, CHAMP1 (chromosome alignment-maintaining phosphoprotein 1), though its possible role in DNA repair is unknown. Here, we show that binding of CHAMP1 to REV7 activates homologous recombination (HR) repair. Mechanistically, CHAMP1 binds directly to REV7 and reduces the level of the Shieldin complex, causing an increase in double-strand break end resection. CHAMP1 also interacts with POGZ in a heterochromatin complex further promoting HR repair. Importantly, in human tumors, CHAMP1 overexpression promotes HR, confers poly (ADP-ribose) polymerase inhibitor resistance, and correlates with poor prognosis. Thus, by binding to either SHLD3 or CHAMP1 through its seatbelt, the REV7 protein can promote either NHEJ or HR repair, respectively.

Role of an Automated Deep Learning Algorithm for Reliable Screening of Abnormality in Chest Radiographs: A Prospective Multicenter Quality Improvement Study.

In medical practice, chest X-rays are the most ubiquitous diagnostic imaging tests. However, the current workload in extensive health care facilities and lack of well-trained radiologists is a significant challenge in the patient care pathway. Therefore, an accurate, reliable, and fast computer-aided diagnosis (CAD) system capable of detecting abnormalities in chest X-rays is crucial in improving the radiological workflow. In this prospective multicenter quality-improvement study, we have evaluated whether artificial intelligence (AI) can be used as a chest X-ray screening tool in real clinical settings. Methods: A team of radiologists used the AI-based chest X-ray screening tool (qXR) as a part of their daily reporting routine to report consecutive chest X-rays for this prospective multicentre study. This study took place in a large radiology network in India between June 2021 and March 2022. Results: A total of 65,604 chest X-rays were processed during the study period. The overall performance of AI achieved in detecting normal and abnormal chest X-rays was good. The high negatively predicted value (NPV) of 98.9% was achieved. The AI performance in terms of area under the curve (AUC), NPV for the corresponding subabnormalities obtained were blunted CP angle (0.97, 99.5%), hilar dysmorphism (0.86, 99.9%), cardiomegaly (0.96, 99.7%), reticulonodular pattern (0.91, 99.9%), rib fracture (0.98, 99.9%), scoliosis (0.98, 99.9%), atelectasis (0.96, 99.9%), calcification (0.96, 99.7%), consolidation (0.95, 99.6%), emphysema (0.96, 99.9%), fibrosis (0.95, 99.7%), nodule (0.91, 99.8%), opacity (0.92, 99.2%), pleural effusion (0.97, 99.7%), and pneumothorax (0.99, 99.9%). Additionally, the turnaround time (TAT) decreased by about 40.63% from pre-qXR period to post-qXR period. Conclusions: The AI-based chest X-ray solution (qXR) screened chest X-rays and assisted in ruling out normal patients with high confidence, thus allowing the radiologists to focus more on assessing pathology on abnormal chest X-rays and treatment pathways.

Ayurvedic Response to COVID-19 Pandemic in Kerala, India and Its Impact on Quarantined Individuals - A Community Case Study.

The SARS-CoV-2 infection has resulted in massive loss of valuable human lives, extensive destruction of livelihoods and financial crisis of unprecedented levels across the globe. Kerala, a province in India, like the rest of the country, launched preventive and control measures to mitigate the impact of COVID-19 early in 2020. The Government of Kerala started 1206 Ayur Raksha Clinics and associated Task Forces across the state in April 2020 to improve the reach and penetration of Ayurvedic preventive, therapeutic and convalescent care strategies for the COVID-19 pandemic. The implementation framework of the strategy was properly designed, and had a decentralized, people-centered, and participatory approach. Kerala has robust public health machinery with adequate human resource and infrastructure in the conventional medicine sector. This community case study examines how the decentralized organizational framework was effectively utilized for facilitating the delivery of Ayurvedic services in the COVID-19 situation. Key observations from the study are: Ayurvedic programs implemented systematically, under an organized framework with social participation enables wider utilization of the services. Such a framework is easily replicable even in resource-poor settings. Rather than a pluralistic approach, an integrative health system approach may be more viable in the Kerala scenario in public health emergencies.

AdpC is a Prevotella intermedia 17 leucine-rich repeat internalin-like protein.

The oral bacterium Prevotella intermedia attaches to and invades gingival epithelial cells, fibroblasts, and endothelial cells. Several genes encoding proteins that mediate both the adhesion and invasion processes are carried on the genome of this bacterium. Here, we characterized one such protein, AdpC, belonging to the leucine-rich repeat (LRR) protein family. Bioinformatics analysis revealed that this protein shares similarity with the Treponema pallidum LRR (LRR(TP)) family of proteins and contains six LRRs. Despite the absence of a signal peptide, this protein is localized on the bacterial outer membrane, indicating that it is transported through an atypical secretion mechanism. The recombinant form of this protein (rAdpC) was shown to bind fibrinogen. In addition, the heterologous host strain Escherichia coli BL21 expressing rAdpC (V2846) invaded fibroblast NIH 3T3 cells at a 40-fold-higher frequency than control E. coli BL21 cells expressing a sham P. intermedia 17 protein. Although similar results were obtained by using human umbilical vein endothelial cells (HUVECs), only a 3-fold-increased invasion of V2846 into oral epithelial HN4 cells was observed. Thus, AdpC-mediated invasion is cell specific. This work demonstrated that AdpC is an important invasin protein of P. intermedia 17.

Adaptation of Porphyromonas gingivalis to microaerophilic conditions involves increased consumption of formate and reduced utilization of lactate.

Porphyromonas gingivalis, previously classified as a strict anaerobe, can grow in the presence of low concentrations of oxygen. Microarray analysis revealed alteration in gene expression in the presence of 6 % oxygen. During the exponential growth phase, 96 genes were upregulated and 79 genes were downregulated 1.4-fold. Genes encoding proteins that play a role in oxidative stress protection were upregulated, including alkyl hydroperoxide reductase (ahpCF), superoxide dismutase (sod) and thiol peroxidase (tpx). Significant changes in gene expression of proteins that mediate oxidative metabolism, such as cytochrome d ubiquinol oxidase-encoding genes, cydA and cydB, were detected. The expression of genes encoding formate uptake transporter (PG0209) and formate tetrahydrofolate ligase (fhs) was drastically elevated, which indicates that formate metabolism plays a major role under aerobic conditions. The concomitant reduction of expression of a gene encoding the lactate transporter PG1340 suggests decreased utilization of this nutrient. The concentrations of both formate and lactate were assessed in culture supernatants and cells, and they were in agreement with the results obtained at the transcriptional level. Also, genes encoding gingipain protease secretion/maturation regulator (porR) and protease transporter (porT) had reduced expression in the presence of oxygen, which also correlated with reduced protease activities under aerobic conditions. In addition, metal transport was affected, and while iron-uptake genes such as the genes encoding the haemin uptake locus (hmu) were downregulated, expression of manganese transporter genes, such as feoB2, was elevated in the presence of oxygen. Finally, genes encoding putative regulatory proteins such as extracellular function (ECF) sigma factors as well as small proteins had elevated expression levels in the presence of oxygen. As P. gingivalis is distantly related to the well-studied model organism Escherichia coli, results from our work may provide further understanding of oxygen metabolism and protection in other related bacteria belonging to the phylum Bacteroidetes.

Behavior of E. coli with Variable Surface Morphology Changes on Charged Semiconductor Interfaces.

Bacterial behavior is often controlled by structural and composition elements of their cell wall. Using genetic mutant strains that change specific aspects of their surface structure, we modified bacterial behavior in response to semiconductor surfaces. We monitored the adhesion, membrane potential, and catalase activity of the Gram-negative bacterium Escherichia coli (E. coli) that were mutant for genes encoding components of their surface architecture, specifically flagella, fimbriae, curli, and components of the lipopolysaccharide membrane, while on gallium nitride (GaN) surfaces with different surface potentials. The bacteria and the semiconductor surface properties were recorded prior to the biofilm studies. The data from the materials and bioassays characterization supports the notion that alteration of the surface structure of the E. coli bacterium resulted in changes to bacterium behavior on the GaN medium. Loss of specific surface structure on the E. coli bacterium reduced its sensitivity to the semiconductor interfaces, while other mutations increase bacterial adhesion when compared to the wild-type control E. coli bacteria. These results demonstrate that bacterial behavior and responses to GaN semiconductor materials can be controlled genetically and can be utilized to tune the fate of living bacteria on GaN surfaces.

Years of Potential Life Lost Because of Cardiovascular Disease in Asian-American Subgroups, 2003-2012.

Background Asian-American subgroups (Asian-Indian, Chinese, Filipino, Korean, Japanese, and Vietnamese) display varied cardiovascular disease mortality patterns, especially at younger ages. This study aims to examine the years of potential life lost because of ischemic heart disease and cerebrovascular disease among the 6 largest Asian-American subgroups compared with non-Hispanic whites. Methods and Results We used National Center for Health Statistics Multiple Causes of Death mortality files from 2003 to 2012 to calculate race-specific life expectancy, mean years of potential life lost, and years of potential life lost per 100 000 population for each Asian subgroup and non-Hispanic whites. Asian-American subgroups display heterogeneity in cardiovascular disease burden. Asian-Indians had a high burden of ischemic heart disease; Asian-Indian men lost 724 years per 100 000 population in 2012 and a mean of 17 years to ischemic heart disease. Respectively, Vietnamese and Filipino men and women lost a mean of 17 and 16 years of life to cerebrovascular disease; Filipino men lost 352 years per 100 000 population in 2012. All Asian subgroups for both sexes had higher years of life lost to cerebrovascular disease compared with non-Hispanic whites. Conclusions Cardiovascular disease burden varies among Asian subgroups, and contributes to greater premature mortality in certain subgroups. Asian-Indian and Filipino populations have the highest years of life lost because of ischemic heart disease and Filipino and Vietnamese have the highest years of life lost because of cerebrovascular disease. Analysis of risk factors and development of subgroup-specific interventions are required to address these health disparities.