Selective elimination of mitochondrial mutations in the germline by genome editing.

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PAPERCLIP.

Impact of Early Antiretroviral Therapy Initiation on HIV-Specific CD4 and CD8 T Cell Function in Perinatally Infected Children.

Early initiation of antiretroviral therapy (ART) in vertically HIV-infected children limits the size of the virus reservoir, but whether the time of treatment initiation (TI) can durably impact host immune responses associated with HIV infection is still unknown. This study was conducted in PBMC of 20 HIV-infected virally suppressed children on ART (mean age 9.4 y), classified as early treated (ET; age at ART initiation ≤0.5 y, n = 14) or late treated (LT; age at ART initiation 1-10 y, n = 6). Frequencies and functions of Ag-specific CD4 (CD40L+) and CD8 (CD69+) T cells were evaluated by intracellular IL-2, IFN-γ, and TNF-α production with IL-21 in CD4 or CD107a, granzyme B and perforin in CD8 T cells following stimulation with HIV gp140 protein (ENV) or GAG peptides by multiparameter flow cytometry. ET showed a higher proportion of cytokine-producing ENV- and GAG-specific CD4 and CD8 T cells compared with LT. In particular, ET were enriched in polyfunctional T cells. RNA sequencing analysis showed upregulation of immune activation pathways in LT compared with ET. Our results suggest that timing of TI in HIV-infected children has a long-term and measurable impact on the quality of the HIV-specific T cell immune responses and transcriptional profiles of PBMC, reinforcing the importance of early TI.

Mitochondrial DNA sequence variation and risk of meningioma.

BACKGROUND: Risk factors for meningioma include female gender, African American race, high body mass index (BMI), and exposure to ionizing radiation. Although genome-wide association studies (GWAS) have identified two nuclear genome risk loci for meningioma (rs12770228 and rs2686876), the relation between mitochondrial DNA (mtDNA) sequence variants and meningioma is unknown. METHODS: We examined the association of 42 common germline mtDNA variants (minor allele frequency ≥ 5%), haplogroups, and genes with meningioma in 1080 controls and 478 meningioma cases from a case-control study conducted at medical centers in the southeastern United States. Associations were examined separately for meningioma overall and by WHO grade (n = 409 grade I and n = 69 grade II/III). RESULTS: Overall, meningioma was significantly associated with being female (OR 2.85; 95% CI 2.21-3.69), self-reported African American race (OR 2.38, 95% CI 1.41-3.99), and being overweight (OR 1.48; 95% CI 1.11-1.97) or obese (OR 1.70; 95% CI 1.25-2.31). The variant m.16362T > C (rs62581341) in the mitochondrial control region was positively associated with grade II/III meningiomas (OR 2.33; 95% CI 1.14-4.77), but not grade I tumors (OR 0.99; 95% CI 0.64-1.53). Haplogroup L, a marker for African ancestry, was associated with meningioma overall (OR 2.92; 95% CI 1.01-8.44). However, after stratifying by self-reported race, this association was only apparent among the few self-reported Caucasians with this haplogroup (OR 6.35; 95% CI 1.56-25.9). No other mtDNA variant, haplogroup, or gene was associated with meningioma. CONCLUSION: Common mtDNA variants and major mtDNA haplogroups do not appear to have associations with the odds of developing meningioma.

ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels.

Mutations in the mitochondrial inner membrane ATPase ATAD3A result in neurological syndromes in humans. In mice, the ubiquitous disruption of Atad3 (also known as Atad3a) was embryonic lethal, but a skeletal muscle-specific conditional knockout (KO) was viable. At birth, ATAD3 muscle KO mice had normal weight, but from 2 months onwards they showed progressive motor-impaired coordination and weakness. Loss of ATAD3 caused early and severe mitochondrial structural abnormalities, mitochondrial proliferation and muscle atrophy. There was dramatic reduction in mitochondrial cristae junctions and overall cristae morphology. The lack of mitochondrial cristae was accompanied by a reduction in high molecular weight mitochondrial contact site and cristae organizing system (MICOS) complexes, and to a lesser extent in OPA1. Moreover, muscles lacking ATAD3 showed altered cholesterol metabolism, accumulation of mitochondrial DNA (mtDNA) replication intermediates, progressive mtDNA depletion and deletions. Unexpectedly, decreases in the levels of some OXPHOS components occurred after cristae destabilization, indicating that ATAD3 is not crucial for mitochondrial translation, as previously suggested. Our results show a critical early role of ATAD3 in regulating mitochondrial inner membrane structure, leading to secondary defects in mtDNA replication and complex V and cholesterol levels in postmitotic tissue.This article has an associated First Person interview with the first author of the paper.

MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases.

We have designed mitochondrially targeted transcription activator-like effector nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach, we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNA(Lys) gene mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh syndrome. Transmitochondrial cybrid cells harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analyzed after different time periods. MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size will improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials.

Somatic mtDNA mutation spectra in the aging human putamen.

The accumulation of heteroplasmic mitochondrial DNA (mtDNA) deletions and single nucleotide variants (SNVs) is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq) to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the "common" deletion and other "major arc" deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m.3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ(-) mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states.

Overexpression of Twinkle-helicase protects cardiomyocytes from genotoxic stress caused by reactive oxygen species.

Mitochondrial DNA (mtDNA) in adult human heart is characterized by complex molecular forms held together by junctional molecules of unknown biological significance. These junctions are not present in mouse hearts and emerge in humans during postnatal development, concomitant with increased demand for oxidative metabolism. To analyze the role of mtDNA organization during oxidative stress in cardiomyocytes, we used a mouse model, which recapitulates the complex mtDNA organization of human hearts by overexpression of the mitochondrial helicase, TWINKLE. Overexpression of TWINKLE rescued the oxidative damage induced replication stalling of mtDNA, reduced mtDNA point mutation load, and modified mtDNA rearrangements in heterozygous mitochondrial superoxide dismutase knockout hearts, as well as ameliorated cardiomyopathy in mice superoxide dismutase knockout in a p21-dependent manner. We conclude that mtDNA integrity influences cell survival and reason that tissue specific modes of mtDNA maintenance represent an adaptation to oxidative stress.

Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse.

Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations.

Wastewater based surveillance can be used to reduce clinical testing intensity on a university campus.

Clinical testing has been a vital part of the response to and suppression of the COVID-19 pandemic; however, testing imposes significant burdens on a population. College students had to contend with clinical testing while simultaneously dealing with health risks and the academic pressures brought on by quarantines, changes to virtual platforms, and other disruptions to daily life. The objective of this study was to analyze whether wastewater surveillance can be used to decrease the intensity of clinical testing while maintaining reliable measurements of diseases incidence on campus. Twelve months of human health and wastewater surveillance data for eight residential buildings on a university campus were analyzed to establish how SARS-CoV-2 levels in the wastewater can be used to minimize clinical testing burden on students. Wastewater SARS-CoV-2 levels were used to create multiple scenarios, each with differing levels of testing intensity, which were compared to the actual testing volumes implemented by the university. We found that scenarios in which testing intensity fluctuations matched rise and falls in SARS-CoV-2 wastewater levels had stronger correlations between SARS-CoV-2 levels and recorded clinical positives. In addition to stronger correlations, most scenarios resulted in overall fewer weekly clinical tests performed. We suggest the use of wastewater surveillance to guide COVID-19 testing as it can significantly increase the efficacy of COVID-19 surveillance while reducing the burden placed on college students during a pandemic. Future efforts should be made to integrate wastewater surveillance into clinical testing strategies implemented on college campuses.

Expanding a Wastewater-Based Surveillance Methodology for DNA Isolation from a Workflow Optimized for SARS-CoV-2 RNA Quantification.

Wastewater-based surveillance (WBS) is a noninvasive, epidemiological strategy for assessing the spread of COVID-19 in communities. This strategy was based upon wastewater RNA measurements of the viral target, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The utility of WBS for assessing the spread of COVID-19 has motivated research to measure targets beyond SARS-CoV-2, including pathogens containing DNA. The objective of this study was to establish the necessary steps for isolating DNA from wastewater by modifying a long-standing RNA-specific extraction workflow optimized for SARS-CoV-2 detection. Modifications were made to the sample concentration process and included an evaluation of bead bashing prior to the extraction of either DNA or RNA. Results showed that bead bashing reduced detection of RNA from wastewater but improved recovery of DNA as assessed by quantitative polymerase chain reaction (qPCR). Bead bashing is therefore not recommended for the quantification of RNA viruses using qPCR. Whereas for Mycobacterium bacterial DNA isolation, bead bashing was necessary for improving qPCR quantification. Overall, we recommend 2 separate workflows, one for RNA viruses that does not include bead bashing and one for other microbes that use bead bashing for DNA isolation. The experimentation done here shows that current-standing WBS program methodologies optimized for SARS-CoV-2 need to be modified and reoptimized to allow for alternative pathogens to be readily detected and monitored, expanding its utility as a tool for public health assessment.

Predicting COVID-19 cases using SARS-CoV-2 RNA in air, surface swab and wastewater samples.

Genomic footprints of pathogens shed by infected individuals can be traced in environmental samples, which can serve as a noninvasive method of infectious disease surveillance. The research evaluates the efficacy of environmental monitoring of SARS-CoV-2 RNA in air, surface swabs and wastewater to predict COVID-19 cases. Using a prospective experimental design, air, surface swabs, and wastewater samples were collected from a college dormitory housing roughly 500 students from March to May 2021 at the University of Miami, Coral Gables, FL. Students were randomly screened for COVID-19 during the study period. SARS-CoV-2 concentration in environmental samples was quantified using Volcano 2nd Generation-qPCR. Descriptive analyses were conducted to examine the associations between time-lagged SARS-CoV-2 in environmental samples and COVID-19 cases. SARS-CoV-2 was detected in air, surface swab and wastewater samples on 52 (63.4 %), 40 (50.0 %) and 57 (68.6 %) days, respectively. On 19 (24 %) of 78 days SARS-CoV-2 was detected in all three sample types. COVID-19 cases were reported on 11 days during the study period and SARS-CoV-2 was also detected two days before the case diagnosis on all 11 (100 %), 9 (81.8 %) and 8 (72.7 %) days in air, surface swab and wastewater samples, respectively. SARS-CoV-2 detection in environmental samples was an indicator of the presence of local COVID-19 cases and a 3-day lead indicator for a potential outbreak at the dormitory building scale. Proactive environmental surveillance of SARS-CoV-2 or other pathogens in multiple environmental media has potential to guide targeted measures to contain and/or mitigate infectious disease outbreaks within communities.

Geospatially-resolved public-health surveillance via wastewater sequencing.

Wastewater, which contains everything from pathogens to pollutants, is a geospatially-and temporally-linked microbial fingerprint of a given population. As a result, it can be leveraged for monitoring multiple dimensions of public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (n=1,419 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County from 2020-2022. First, we used targeted amplicon sequencing (n=966) to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with clinical caseloads from University students (N = 1,503) and Miami-Dade County hospital patients (N = 3,939 patients), as well as an 8-day earlier detection of the Delta variant in wastewater vs. in patients. Additionally, in 453 metatranscriptomic samples, we demonstrate that different wastewater sampling locations have clinically and public-health-relevant microbiota that vary as a function of the size of the human population they represent. Through assembly, alignment-based, and phylogenetic approaches, we also detect multiple clinically important viruses (e.g., norovirus ) and describe geospatial and temporal variation in microbial functional genes that indicate the presence of pollutants. Moreover, we found distinct profiles of antimicrobial resistance (AMR) genes and virulence factors across campus buildings, dorms, and hospitals, with hospital wastewater containing a significant increase in AMR abundance. Overall, this effort lays the groundwork for systematic characterization of wastewater to improve public health decision making and a broad platform to detect emerging pathogens.

Correlative analysis of wastewater trends with clinical cases and hospitalizations through five dominant variant waves of COVID-19.

Wastewater-based epidemiology (WBE) has been utilized to track community infections of Coronavirus Disease 2019 (COVID-19) by detecting RNA of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), within samples collected from wastewater. The correlations between community infections and wastewater measurements of the RNA can potentially change as SARS-CoV-2 evolves into new variations by mutating. This study analyzed SARS-CoV-2 RNA, and indicators of human waste in wastewater from two sewersheds of different scales (University of Miami (UM) campus and Miami-Dade County Central District wastewater treatment plant (CDWWTP)) during five internally defined COVID-19 variant dominant periods (Initial, Pre-Delta, Delta, Omicron and Post-Omicron wave). SARS-CoV-2 RNA quantities were compared against COVID-19 clinical cases and hospitalizations to evaluate correlations with wastewater SARS-CoV-2 RNA. Although correlations between documented clinical cases and hospitalizations were high, prevalence for a given wastewater SARS-CoV-2 level varied depending upon the variant analyzed. The correlative relationship was significantly steeper (more cases per level found in wastewater) for the Omicron-dominated period. For hospitalization, the relationships were steepest for the Initial wave, followed by the Delta wave with flatter slopes during all other waves. Overall results were interpreted in the context of SARS-CoV-2 virulence and vaccination rates among the community.

Mitochondrial DNA sequence variation and risk of glioma.

BACKGROUND: Malignant gliomas are the most common primary adult brain tumors, with a poor prognosis and ill-defined etiology. Mitochondrial DNA (mtDNA) sequence variation has been linked with certain cancers; however, research on glioma is lacking. METHODS: We examined the association of common (minor allele frequency ≥ 5%) germline mtDNA variants and haplogroups with glioma risk in 1,566 glioma cases and 1,017 controls from a US case-control study, and 425 glioma cases and 1,534 matched controls from the UK Biobank cohort (UKB). DNA samples were genotyped using the UK Biobank array that included a set of common and rare mtDNA variants. Risk associations were examined separately for glioblastoma (GBM) and lower grade tumors (non-GBM). RESULTS: In the US study, haplogroup W was inversely associated with glioma when compared with haplogroup H (OR = 0.43, 95%CI: 0.23-0.79); this association was not demonstrated in the UKB (OR = 1.07, 95%CI: 0.47-2.43). In the UKB, the variant m.3010G > A was significantly associated with GBM (OR = 1.32; 95%CI: 1.01-1.73; p = 0.04), but not non-GBM (1.23; 95%CI: 0.78-1.95; p = 0.38); no similar association was observed in the US study. In the US study, the variant m.14798 T > C, was significantly associated with non-GBM (OR = 0.72; 95%CI: 0.53-0.99), but not GBM (OR = 0.86; 95%CI: 0.66-1.11), whereas in the UKB, a positive association was observed between this variant and GBM (OR = 1.46; 95%CI: 1.06-2.02) but not non-GBM (OR = 0.92; 95%CI: 0.52-1.63). None of these associations were significant after adjustment for multiple testing. CONCLUSION: The association of inherited mtDNA variation, including rare and singleton variants, with glioma risk merits further study.

A pilot study to troubleshoot quality control metrics when assessing circulating miRNA expression data reproducibility across study sites.

BACKGROUND: Given the growing interest in using microRNAs (miRNAs) as biomarkers of early disease, establishment of robust protocols and platforms for miRNA quantification in biological fluids is critical. OBJECTIVE: The goal of this multi-center pilot study was to evaluate the reproducibility of NanoString nCounter™ technology when analyzing the abundance of miRNAs in plasma and cystic fluid from patients with pancreatic lesions. METHODS: Using sample triplicates analyzed across three study sites, we assessed potential sources of variability (RNA isolation, sample processing/ligation, hybridization, and lot-to-lot variability) that may contribute to suboptimal reproducibility of miRNA abundance when using nCounter™, and evaluated expression of positive and negative controls, housekeeping genes, spike-in genes, and miRNAs. RESULTS: Positive controls showed a high correlation across samples from each site (median correlation coefficient, r> 0.9). Most negative control probes had expression levels below background. Housekeeping and spike-in genes each showed a similar distribution of expression and comparable pairwise correlation coefficients of replicate samples across sites. A total of 804 miRNAs showed a similar distribution of pairwise correlation coefficients between replicate samples (p= 0.93). After normalization and selecting miRNAs with expression levels above zero in 80% of samples, 55 miRNAs were identified; heatmap and principal component analysis revealed similar expression patterns and clustering in replicate samples. CONCLUSIONS: Findings from this pilot investigation suggest the nCounter platform can yield reproducible results across study sites. This study underscores the importance of implementing quality control procedures when designing multi-center evaluations of miRNA abundance.

Comparison of Electronegative Filtration to Magnetic Bead-Based Concentration and V2G-qPCR to RT-qPCR for Quantifying Viral SARS-CoV-2 RNA from Wastewater.

Methods of wastewater concentration (electronegative filtration (ENF) versus magnetic bead-based concentration (MBC)) were compared for the analysis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), beta-2 microglobulin, and human-coronavirus OC43. Using ENF as the concentration method, two quantitative Polymerase Chain Reaction (qPCR) analytical methods were also compared: Volcano 2nd Generation (V2G)-qPCR and reverse transcriptase (RT)-qPCR measuring three different targets of the virus responsible for the COVID-19 illness (N1, modified N3, and ORF1ab). Correlations between concentration methods were strong and statistically significant for SARS-CoV-2 (r=0.77, p<0.001) and B2M (r=0.77, p<0.001). Comparison of qPCR analytical methods indicate that, on average, each method provided equivalent results with average ratios of 0.96, 0.96 and 1.02 for N3 to N1, N3 to ORF1ab, and N1 to ORF1ab and were supported by significant (p<0.001) correlation coefficients (r =0.67 for V2G (N3) to RT (N1), r =0.74 for V2G (N3) to RT (ORF1ab), r = 0.81 for RT (N1) to RT (ORF1ab)). Overall results suggest that the two concentration methods and qPCR methods provide equivalent results, although variability is observed for individual measurements. Given the equivalency of results, additional advantages and disadvantages, as described in the discussion, are to be considered when choosing an appropriate method.

COVID-19 Prediction using Genomic Footprint of SARS-CoV-2 in Air, Surface Swab and Wastewater Samples.

Importance: Genomic footprints of pathogens shed by infected individuals can be traced in environmental samples. Analysis of these samples can be employed for noninvasive surveillance of infectious diseases. Objective: To evaluate the efficacy of environmental surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for predicting COVID-19 cases in a college dormitory. Design: Using a prospective experimental design, air, surface swabs, and wastewater samples were collected from a college dormitory from March to May 2021. Students were randomly screened for COVID-19 during the study period. SARS-CoV-2 in environmental samples was concentrated with electronegative filtration and quantified using Volcano 2 nd Generation-qPCR. Descriptive analyses were conducted to examine the associations between time-lagged SARS-CoV-2 in environmental samples and clinically diagnosed COVID-19 cases. Setting: This study was conducted in a residential dormitory at the University of Miami, Coral Gables campus, FL, USA. The dormitory housed about 500 students. Participants: Students from the dormitory were randomly screened, for COVID-19 for 2-3 days / week while entering or exiting the dormitory. Main Outcome: Clinically diagnosed COVID-19 cases were of our main interest. We hypothesized that SARS-CoV-2 detection in environmental samples was an indicator of the presence of local COVID-19 cases in the dormitory, and SARS-CoV-2 can be detected in the environmental samples several days prior to the clinical diagnosis of COVID-19 cases. Results: SARS-CoV-2 genomic footprints were detected in air, surface swab and wastewater samples on 52 (63.4%), 40 (50.0%) and 57 (68.6%) days, respectively, during the study period. On 19 (24%) of 78 days SARS-CoV-2 was detected in all three sample types. Clinically diagnosed COVID-19 cases were reported on 11 days during the study period and SARS-CoV-2 was also detected two days before the case diagnosis on all 11 (100%), 9 (81.8%) and 8 (72.7%) days in air, surface swab and wastewater samples, respectively. Conclusion: Proactive environmental surveillance of SARS-CoV-2 or other pathogens in a community/public setting has potential to guide targeted measures to contain and/or mitigate infectious disease outbreaks. Key Points: Question: How effective is environmental surveillance of SARS-CoV-2 in public places for early detection of COVID-19 cases in a community?Findings: All clinically confirmed COVID-19 cases were predicted with the aid of 2 day lagged SARS-CoV-2 in environmental samples in a college dormitory. However, the prediction efficiency varied by sample type: best prediction by air samples, followed by wastewater and surface swab samples. SARS-CoV-2 was also detected in these samples even on days without any reported cases of COVID-19, suggesting underreporting of COVID-19 cases.Meaning: SARS-CoV-2 can be detected in environmental samples several days prior to clinical reporting of COVID-19 cases. Thus, proactive environmental surveillance of microbiome in public places can serve as a mean for early detection of location-time specific outbreaks of infectious diseases. It can also be used for underreporting of infectious diseases.

Relationships between SARS-CoV-2 in Wastewater and COVID-19 Clinical Cases and Hospitalizations, with and without Normalization against Indicators of Human Waste.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in wastewater has been used to track community infections of coronavirus disease-2019 (COVID-19), providing critical information for public health interventions. Since levels in wastewater are dependent upon human inputs, we hypothesize that tracking infections can be improved by normalizing wastewater concentrations against indicators of human waste [Pepper Mild Mottle Virus (PMMoV), ß-2 Microglobulin (B2M), and fecal coliform]. In this study, we analyzed SARS-CoV-2 and indicators of human waste in wastewater from two sewersheds of different scales: a University campus and a wastewater treatment plant. Wastewater data were combined with complementary COVID-19 case tracking to evaluate the efficiency of wastewater surveillance for forecasting new COVID-19 cases and, for the larger scale, hospitalizations. Results show that the normalization of SARS-CoV-2 levels by PMMoV and B2M resulted in improved correlations with COVID-19 cases for campus data using volcano second generation (V2G)-qPCR chemistry (r s = 0.69 without normalization, r s = 0.73 with normalization). Mixed results were obtained for normalization by PMMoV for samples collected at the community scale. Overall benefits from normalizing with measures of human waste depend upon qPCR chemistry and improves with smaller sewershed scale. We recommend further studies that evaluate the efficacy of additional normalization targets.

Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks.

To investigate mtDNA recombination induced by multiple double strand breaks (DSBs) we used a mitochondria-targeted form of the ScaI restriction endonuclease to introduce DSBs in heteroplasmic mice and cells in which we were able to utilize haplotype differences to trace the origin of recombined molecules. ScaI cleaves multiple sites in each haplotype of the heteroplasmic mice (five in NZB and three in BALB mtDNA) and prolonged expression causes severe mtDNA depletion. After a short pulse of restriction enzyme expression followed by a long period of recovery, mitochondrial genomes with large deletions were detected by PCR. Curiously, we found that some ScaI sites were more commonly involved in recombined molecules than others. In intra-molecular recombination events, deletion breakpoints were close to or upstream of ScaI cleavage sites, confirming the recombinogenic character of DSBs in mtDNA. A region adjacent to the D-loop was preferentially involved in recombination of all molecules. Sequencing through NZB and BALB haplotype markers in recombined molecules enabled us to show that in addition to intra-molecular mtDNA recombination, rare inter-molecular mtDNA recombination events can also occur. This study underscores the role of DSBs in the generation of mtDNA rearrangements and supports the existence of recombination hotspots.

In vivo methylation of mtDNA reveals the dynamics of protein-mtDNA interactions.

To characterize the organization of mtDNA-protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in transcription and replication. The transcription termination region (TERM) within the tRNA(Leu(UUR)) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL). Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that nucleoids are dynamically associated with proteins.