Systematic analysis of NDUFAF6 in complex I assembly and mitochondrial disease.

Isolated complex I (CI) deficiencies are a major cause of primary mitochondrial disease. A substantial proportion of CI deficiencies are believed to arise from defects in CI assembly factors (CIAFs) that are not part of the CI holoenzyme. The biochemistry of these CIAFs is poorly defined, making their role in CI assembly unclear, and confounding interpretation of potential disease-causing genetic variants. To address these challenges, we devised a deep mutational scanning approach to systematically assess the function of thousands of NDUFAF6 genetic variants. Guided by these data, biochemical analyses and cross-linking mass spectrometry, we discovered that the CIAF NDUFAF6 facilitates incorporation of NDUFS8 into CI and reveal that NDUFS8 overexpression rectifies NDUFAF6 deficiency. Our data further provide experimental support of pathogenicity for seven novel NDUFAF6 variants associated with human pathology and introduce functional evidence for over 5,000 additional variants. Overall, our work defines the molecular function of NDUFAF6 and provides a clinical resource for aiding diagnosis of NDUFAF6-related diseases.

Molecular insights into the prototypical single-stranded DNA-binding protein from E. coli.

The SSB protein of Escherichia coli functions to bind single-stranded DNA wherever it occurs during DNA metabolism. Depending upon conditions, SSB occurs in several different binding modes. In the course of its function, SSB diffuses on ssDNA and transfers rapidly between different segments of ssDNA. SSB interacts with many other proteins involved in DNA metabolism, with 22 such SSB-interacting proteins, or SIPs, defined to date. These interactions chiefly involve the disordered and conserved C-terminal residues of SSB. When not bound to ssDNA, SSB can aggregate to form a phase-separated biomolecular condensate. Current understanding of the properties of SSB and the functional significance of its many intermolecular interactions are summarized in this review.

COVID-19 infection and incident diabetes in American Indian and Alaska Native people: a retrospective cohort study.

Background: Evidence suggests an increased risk of new-onset diabetes following COVID-19 infection. American Indian/Alaska Native (AI/AN) people were disparately impacted by the COVID-19 pandemic and historically have had higher diabetes incidence than other racial/ethnic groups in the US. We measured the association between COVID-19 infection and incident diabetes in AI/AN people. Methods: We conducted a retrospective cohort study using de-identified patient data from the Indian Health Service's (IHS) National Patient Information Reporting System. We estimated age-adjusted diabetes incidence rates, incidence rate ratios, and adjusted hazard ratios among three cohorts spanning pre-pandemic (1/1/2018-2/28/2020) and pandemic (3/1/2020-12/31/2021) timeframes: 1) pre-pandemic cohort (1,503,085 individuals); 2) no-COVID-19 pandemic cohort (1,344,339 individuals); and 3) COVID-19 cohort (176,483 individuals). Findings: The COVID-19 cohort had an increased hazard of diabetes compared to the no-COVID-19 group (adjusted hazard ratio (aHR) = 1.56; 95% CI: 1.50-1.62) and the pre-pandemic group (aHR = 1.27; 95% CI: 1.22-1.32). The association between COVID-19 infection and new-onset diabetes was stronger in those with severe COVID-19 illness. A sensitivity analysis comparing the COVID-19 cohort to members of other cohorts that had acute upper respiratory infections showed an attenuated but higher risk of new-onset diabetes in those with COVID-19. Interpretation: AI/AN people diagnosed with COVID-19 had an elevated risk of a new diabetes diagnosis when compared to the no-COVID-19 group and the pre-pandemic group. The increased diabetes risk in the COVID-19 group remained in a sensitivity analysis that limited the comparator groups to individuals with an AURI diagnosis. Funding: US National Institute of Diabetes and Digestive and Kidney Diseases.

Biochemical characterization of Escherichia coli DnaC variants that alter DnaB helicase loading onto DNA.

DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6 complex with DnaB and opens a channel in the DnaB hexamer through which single-stranded DNA is thought to pass. During replication, replisomes frequently encounter DNA damage and nucleoprotein complexes that can lead to replication fork collapse. Such events require DnaB re-loading onto DNA to allow replication to continue. Replication restart proteins mediate this process by recruiting DnaB6/DnaC6 to abandoned DNA replication forks. Several dnaC mutations that bypass the requirement for replication restart proteins or that block replication restart have been identified in E. coli. To better understand how these DnaC variants function, we have purified and characterized the protein products of several such alleles. Unlike wild-type DnaC, three of the variants (DnaC 809, DnaC 809,820, and DnaC 811) can load DnaB onto replication forks bound by single-stranded DNA-binding protein. DnaC 809 can also load DnaB onto double-stranded DNA. These results suggest that structural changes in the variant DnaB6/DnaC6 complexes expand the range of DNA substrates that can be used for DnaB loading, obviating the need for the existing replication restart pathways. The protein product of dnaC1331, which phenocopies deletion of the priB replication restart gene, blocks loading through the major restart pathway in vitro. Overall, the results of our study highlight the utility of bacterial DnaC variants as tools for probing the regulatory mechanisms that govern replicative helicase loading.

The intrinsically disordered linker in the single-stranded DNA-binding protein influences DNA replication restart and recombination pathways in Escherichia coli K-12.

Tetrameric single-stranded (ss) DNA-binding proteins (SSBs) stabilize ssDNA intermediates formed during genome maintenance reactions in Bacteria. SSBs also recruit proteins important for these processes through direct SSB-protein interactions, including proteins involved in DNA replication restart and recombination processes. SSBs are composed of an N-terminal oligomerization and ssDNA-binding domain, a C-terminal acidic tip that mediates SSB-protein interactions, and an internal intrinsically disordered linker (IDL). Deletions and insertions into the IDL are well tolerated with few phenotypes, although the largest deletions and insertions exhibit some sensitivity to DNA-damaging agents. To define specific DNA metabolism processes dependent on IDL length, ssb mutants that lack 16, 26, 37, or 47 residues of the 57-residue IDL were tested for synthetic phenotypes with mutations in DNA replication restart or recombination genes. We also tested the impact of integrating a fluorescent domain within the SSB IDL using an ssb::mTur2 insertion mutation. Only the largest deletion tested or the insertion mutation causes sensitivity in any of the pathways. Mutations in two replication restart pathways (PriA-B1 and PriA-C) showed synthetic lethalities or small colony phenotypes with the largest deletion or insertion mutations. Recombination gene mutations del(recBCD) and del(ruvABC) show synthetic phenotypes only when combined with the largest ssb deletion. These results suggest that a minimum IDL length is important in some genome maintenance reactions in Escherichia coli. These include pathways involving PriA-PriB1, PriA-PriC, RecFOR, and RecG. The mTur2 insertion in the IDL may also affect SSB interactions in some processes, particularly the PriA-PriB1 and PriA-PriC replication restart pathways.IMPORTANCEssb is essential in Escherichia coli due to its roles in protecting ssDNA and coordinating genome maintenance events. While the DNA-binding core and acidic tip have well-characterized functions, the purpose of the intrinsically disordered linker (IDL) is poorly understood. In vitro studies have revealed that the IDL is important for cooperative ssDNA binding and phase separation. However, single-stranded (ss) DNA-binding protein (SSB) variants with large deletions and insertions in the IDL support normal cell growth. We find that the PriA-PriB1 and PriA-C replication restart, as well as the RecFOR- and RecG-dependent recombination, pathways are sensitive to IDL length. This suggests that cooperativity, phase separation, or a longer spacer between the core and acidic tip of SSB may be important for specific cellular functions.

"You can feel the fresh air " Rural Alaska Native household perceptions of home air purifiers and health.

Alaska Native and American Indian children experience frequent respiratory illness. Indoor air quality is associated with the severity and frequency of respiratory infections in children. High efficiency particulate air (HEPA) purifiers effectively improve indoor air quality and may protect respiratory health. In 2019, the Yukon-Kuskokwim Health Corporation implemented a pilot programme that provided education and HEPA purifiers to households of children with chronic lung conditions. The team evaluated HEPA purifier acceptability and use by interviewing representatives from 11 households that participated in the pilot programme. All interviewees reported improvement in their child's health, and some believed that the health of other household members was also improved because of the HEPA purifier. Interviewees reported that the HEPA purifiers were easy to use, quiet, and not expensive to run. Five of 11 households were still using the HEPA purifier at the time of the interview, which was about three years after receipt of the unit. The most common reasons for discontinuing use were equipment failure and lack of replacement filter, suggesting that programme support could increase sustainability. Our evaluation suggests that HEPA purifiers are acceptable and feasible for use in rural Alaska Native households.

Wastewater Surveillance for Identifying SARS-CoV-2 Infections in Long-Term Care Facilities, Kentucky, USA, 2021-2022.

Persons living in long-term care facilities (LTCFs) were disproportionately affected by COVID-19. We used wastewater surveillance to detect SARS-CoV-2 infection in this setting by collecting and testing 24-hour composite wastewater samples 2-4 times weekly at 6 LTCFs in Kentucky, USA, during March 2021-February 2022. The LTCFs routinely tested staff and symptomatic and exposed residents for SARS-CoV-2 using rapid antigen tests. Of 780 wastewater samples analyzed, 22% (n = 173) had detectable SARS-CoV-2 RNA. The LTCFs reported 161 positive (of 16,905) SARS-CoV-2 clinical tests. The wastewater SARS-CoV-2 signal showed variable correlation with clinical test data; we observed the strongest correlations in the LTCFs with the most positive clinical tests (n = 45 and n = 58). Wastewater surveillance was 48% sensitive and 80% specific in identifying SARS-CoV-2 infections found on clinical testing, which was limited by frequency, coverage, and rapid antigen test performance.

Simple SARS-CoV-2 concentration methods for wastewater surveillance in low resource settings.

Wastewater-based epidemiology (WBE) measures pathogens in wastewater to monitor infectious disease prevalence in communities. Due to the high dilution of pathogens in sewage, a concentration method is often required to achieve reliable biomarker signals. However, most of the current concentration methods rely on expensive equipment and labor-intensive processes, which limits the application of WBE in low-resource settings. Here, we compared the performance of four inexpensive and simple concentration methods to detect SARS-CoV-2 in wastewater samples: Solid Fraction, Porcine Gastric Mucin-conjugated Magnetic Beads, Calcium Flocculation-Citrate Dissolution (CFCD), and Nanotrap® Magnetic Beads (NMBs). The NMBs and CFCD methods yielded the highest concentration performance for SARS-CoV-2 (∼16-fold concentration and âˆ¼ 41 % recovery) and require <45 min processing time. CFCD has a relatively low consumable cost (<$2 per four sample replicates). All methods can be performed with basic laboratory equipment and minimal electricity usage which enables further application of WBE in remote areas and low resource settings.

Identification of recG genetic interactions in Escherichia coli by transposon sequencing.

IMPORTANCE: DNA damage and subsequent DNA repair processes are mutagenic in nature and an important driver of evolution in prokaryotes, including antibiotic resistance development. Genetic screening approaches, such as transposon sequencing (Tn-seq), have provided important new insights into gene function and genetic relationships. Here, we employed Tn-seq to gain insight into the function of the recG gene, which renders Escherichia coli cells moderately sensitive to a variety of DNA-damaging agents when they are absent. The reported recG genetic interactions can be used in combination with future screens to aid in a more complete reconstruction of DNA repair pathways in bacteria.

Evaluating Scalable Supervised Learning for Synthesize-on-Demand Chemical Libraries.

Traditional small-molecule drug discovery is a time-consuming and costly endeavor. High-throughput chemical screening can only assess a tiny fraction of drug-like chemical space. The strong predictive power of modern machine-learning methods for virtual chemical screening enables training models on known active and inactive compounds and extrapolating to much larger chemical libraries. However, there has been limited experimental validation of these methods in practical applications on large commercially available or synthesize-on-demand chemical libraries. Through a prospective evaluation with the bacterial protein-protein interaction PriA-SSB, we demonstrate that ligand-based virtual screening can identify many active compounds in large commercial libraries. We use cross-validation to compare different types of supervised learning models and select a random forest (RF) classifier as the best model for this target. When predicting the activity of more than 8 million compounds from Aldrich Market Select, the RF substantially outperforms a naïve baseline based on chemical structure similarity. 48% of the RF's 701 selected compounds are active. The RF model easily scales to score one billion compounds from the synthesize-on-demand Enamine REAL database. We tested 68 chemically diverse top predictions from Enamine REAL and observed 31 hits (46%), including one with an IC50 value of 1.3 µM.

Wastewater Surveillance for SARS-CoV-2 at Long-Term Care Facilities: Mixed Methods Evaluation.

BACKGROUND: Wastewater surveillance provided early indication of COVID-19 in US municipalities. Residents of long-term care facilities (LTCFs) experienced disproportionate morbidity and mortality early in the COVID-19 pandemic. We implemented LTCF building-level wastewater surveillance for SARS-CoV-2 at 6 facilities in Kentucky to provide early warning of SARS-CoV-2 in populations considered vulnerable. OBJECTIVE: This study aims to evaluate the performance of wastewater surveillance for SARS-CoV-2 at LTCFs in Kentucky. METHODS: We conducted a mixed methods evaluation of wastewater surveillance following Centers for Disease Control and Prevention (CDC) guidelines for evaluating public health surveillance systems. Evaluation steps in the CDC guidelines were engaging stakeholders, describing the surveillance system, focusing the evaluation design, gathering credible evidence, and generating conclusions and recommendations. We purposively recruited stakeholders for semistructured interviews and undertook thematic content analysis of interview data. We integrated wastewater, clinical testing, and process data to characterize or calculate 7 surveillance system performance attributes (simplicity, flexibility, data quality, sensitivity and positive predictive value [PPV], timeliness, representativeness, and stability). RESULTS: We conducted 8 stakeholder interviews. The surveillance system collected wastewater samples (N=811) 2 to 4 times weekly at 6 LTCFs in Kentucky from March 2021 to February 2022. Synthesis of credible evidence indicated variable surveillance performance. Regarding simplicity, surveillance implementation required moderate human resource and technical capacity. Regarding flexibility, the system efficiently adjusted surveillance frequency and demonstrated the ability to detect additional pathogens of interest. Regarding data quality, software identified errors in wastewater sample metadata entry (110/3120, 3.53% of fields), technicians identified polymerase chain reaction data issues (140/7734, 1.81% of reactions), and staff entered all data corrections into a log. Regarding sensitivity and PPV, using routine LTCF SARS-CoV-2 clinical testing results as the gold standard, a wastewater SARS-CoV-2 signal of >0 RNA copies/mL was 30.6% (95% CI 24.4%-36.8%) sensitive and 79.7% (95% CI 76.4%-82.9%) specific for a positive clinical test at the LTCF. The PPV of the wastewater signal was 34.8% (95% CI 27.9%-41.7%) at >0 RNA copies/mL and increased to 75% (95% CI 60%-90%) at >250 copies/mL. Regarding timeliness, stakeholders received surveillance data 24 to 72 hours after sample collection, with delayed reporting because of the lack of weekend laboratory staff. Regarding representativeness, stakeholders identified challenges delineating the population contributing to LTCF wastewater because of visitors, unknown staff toileting habits, and the use of adult briefs by some residents preventing their waste from entering the sewer system. Regarding stability, the reoccurring cost to conduct 1 day of wastewater surveillance at 1 facility was approximately US $144.50, which included transportation, labor, and materials expenses. CONCLUSIONS: The LTCF wastewater surveillance system demonstrated mixed performance per CDC criteria. Stakeholders found surveillance feasible and expressed optimism regarding its potential while also recognizing challenges in interpreting and acting on surveillance data.

Generation of the NeoThy mouse model for human immune system studies.

Humanized mouse models, created via transplantation of human hematopoietic tissues into immune-deficient mice, support a number of research applications, including transplantation immunology, virology and oncology studies. As an alternative to the bone marrow, liver, thymus humanized mouse, which uses fetal tissues for generating a chimeric human immune system, the NeoThy humanized mouse uses nonfetal tissue sources. Specifically, the NeoThy model incorporates hematopoietic stem and progenitor cells from umbilical cord blood (UCB) as well as thymus tissue that is typically discarded as medical waste during neonatal cardiac surgeries. Compared with fetal thymus tissue, the abundant quantity of neonatal thymus tissue offers the opportunity to prepare over 1,000 NeoThy mice from an individual thymus donor. Here we describe a protocol for processing of the neonatal tissues (thymus and UCB) and hematopoietic stem and progenitor cell separation, human leukocyte antigen typing and matching of allogenic thymus and UCB tissues, creation of NeoThy mice, assessment of human immune cell reconstitution and all experimental steps from planning and design to data analysis. This entire protocol takes a total of ~19 h to complete, with steps broken up into multiple sessions of 4 h or less that can be paused and completed over multiple days. The protocol can be completed, after practice, by individuals with intermediate laboratory and animal handling skills, enabling researchers to make effective use of this promising in vivo model of human immune function.

Biochemical properties of naturally occurring human bloom helicase variants.

Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo, there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro, we purified the BLM catalytic core (BLMcore, residues 636-1298) with either the P868L or G1120R substitution. We also purified a BLMcore K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLMcore P868L and G1120R proteins were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of one lobe of the ATPase domain. Because BLMcore P868L and G1120R retain helicase function in vitro, it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo. Interestingly, we found that BLMcore K869A K870A has diminished ATPase activity, weakened binding to duplex DNA structures, and less robust helicase activity compared to wild-type BLMcore. Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM.

Simultaneous evaluation of treatment efficacy and toxicity for bispecific T-cell engager therapeutics in a humanized mouse model.

Immuno-oncology (IO)-based therapies such as checkpoint inhibitors, bi-specific antibodies, and CAR-T-cell therapies have shown significant success in the treatment of several cancer indications. However, these therapies can result in the development of severe adverse events, including cytokine release syndrome (CRS). Currently, there is a paucity of in vivo models that can evaluate dose-response relationships for both tumor control and CRS-related safety issues. We tested an in vivo PBMC humanized mouse model to assess both treatment efficacy against specific tumors and the concurrent cytokine release profiles for individual human donors after treatment with a CD19xCD3 bispecific T-cell engager (BiTE). Using this model, we evaluated tumor burden, T-cell activation, and cytokine release in response to bispecific T-cell-engaging antibody in humanized mice generated with different PBMC donors. The results show that PBMC engrafted NOD-scid Il2rgnull mice lacking expression of mouse MHC class I and II (NSG-MHC-DKO mice) and implanted with a tumor xenograft predict both efficacy for tumor control by CD19xCD3 BiTE and stimulated cytokine release. Moreover, our findings indicate that this PBMC-engrafted model captures variability among donors for tumor control and cytokine release following treatment. Tumor control and cytokine release were reproducible for the same PBMC donor in separate experiments. The PBMC humanized mouse model described here is a sensitive and reproducible platform that identifies specific patient/cancer/therapy combinations for treatment efficacy and development of complications.

Replication fork binding triggers structural changes in the PriA helicase that govern DNA replication restart in E. coli.

Bacterial replisomes often dissociate from replication forks before chromosomal replication is complete. To avoid the lethal consequences of such situations, bacteria have evolved replication restart pathways that reload replisomes onto prematurely terminated replication forks. To understand how the primary replication restart pathway in E. coli (PriA-PriB) selectively acts on replication forks, we determined the cryogenic-electron microscopy structure of a PriA/PriB/replication fork complex. Replication fork specificity arises from extensive PriA interactions with each arm of the branched DNA. These interactions reshape the PriA protein to create a pore encircling single-stranded lagging-strand DNA while also exposing a surface of PriA onto which PriB docks. Together with supporting biochemical and genetic studies, the structure reveals a switch-like mechanism for replication restart initiation in which restructuring of PriA directly couples replication fork recognition to PriA/PriB complex formation to ensure robust and high-fidelity replication re-initiation.

Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities.

The bacterial RadD enzyme is important for multiple genome maintenance pathways, including RecA DNA strand exchange and RecA-independent suppression of DNA crossover template switching. However, much remains unknown about the precise roles of RadD. One potential clue into RadD mechanisms is its direct interaction with the single-stranded DNA binding protein (SSB), which coats single-stranded DNA exposed during genome maintenance reactions in cells. Interaction with SSB stimulates the ATPase activity of RadD. To probe the mechanism and importance of RadD-SSB complex formation, we identified a pocket on RadD that is essential for binding SSB. In a mechanism shared with many other SSB-interacting proteins, RadD uses a hydrophobic pocket framed by basic residues to bind the C-terminal end of SSB. We found that RadD variants that substitute acidic residues for basic residues in the SSB binding site impair RadD:SSB complex formation and eliminate SSB stimulation of RadD ATPase activity in vitro. Additionally, mutant Escherichia coli strains carrying charge reversal radD changes display increased sensitivity to DNA damaging agents synergistically with deletions of radA and recG, although the phenotypes of the SSB-binding radD mutants are not as severe as a full radD deletion. This suggests that cellular RadD requires an intact interaction with SSB for full RadD function.