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PURPOSE: Mendelian disease genomic research has undergone a massive transformation over the past decade. With increasing availability of exome and genome sequencing, the role of Mendelian research has expanded beyond data collection, sequencing, and analysis to worldwide data sharing and collaboration. METHODS: Over the past 10 years, the National Institutes of Health–supported Centers for Mendelian Genomics (CMGs) have played a major role in this research and clinical evolution. RESULTS: We highlight the cumulative gene discoveries facilitated by the program, biomedical research leveraged by the approach, and the larger impact on the research community. Beyond generating a list of gene-phenotype relationships and participating in widespread data sharing, the CMGs have created resources, tools, and training for the larger community to foster understanding of genes and genome variation. The CMGs have participated in a wide range of data sharing activities, including deposition of all eligible CMG data into the Analysis, Visualization, and Informatics Lab-space (AnVIL), sharing candidate genes through the Matchmaker Exchange and the CMG website, and sharing variants in Genotypes to Mendelian Phenotypes (Geno2MP) and VariantMatcher. CONCLUSION: The work is far from complete; strengthening communication between research and clinical realms, continued development and sharing of knowledge and tools, and improving access to richly characterized data sets are all required to diagnose the remaining molecularly undiagnosed patients.

SUMMARYCopy number variants (CNVs) are major contributors to genetic diversity and disease. To date, exome sequencing (ES) has been generated for millions of individuals in international biobanks, human disease studies, and clinical diagnostic screening. While standardized methods exist for detecting short variants (single nucleotide and insertion/deletion variants) using tools such as the Genome Analysis ToolKit (GATK), technical challenges have confounded similarly uniform large-scale CNV analyses from ES data. Given the profound impact of rare and de novo coding CNVs on genome organization and human disease, the lack of widely-adopted and robustly benchmarked rare CNV discovery tools has presented a barrier to routine exome-wide assessment of this critical class of variation. Here, we introduce GATK-gCNV, a flexible algorithm to discover rare CNVs from genome sequencing read-depth information, which we distribute as an open-source tool packaged in GATK. GATK-gCNV uses a probabilistic model and inference framework that accounts for technical biases while simultaneously predicting CNVs, which enables self-consistency between technical read-depth normalization and variant calling. We benchmarked GATK-gCNV in 7,962 exomes from individuals in quartet families with matched genome sequencing and microarray data. These analyses demonstrated 97% recall of rare (≤1% site frequency) coding CNVs detected by microarrays and 95% recall of rare coding CNVs discovered by genome sequencing at a resolution of more than two exons. We applied GATK-gCNV to generate a reference catalog of rare coding CNVs in 197,306 individuals with ES from the UK Biobank. We observed strong correlations between CNV rates per gene and measures of mutational constraint, as well as rare CNV associations with multiple traits. In summary, GATK-gCNV is a tunable approach for sensitive and specific CNV discovery in ES, which can easily be applied across trait association and clinical screening.

ObjectiveBoth the gut microbiome and host genetics are known to play significant roles in the pathogenesis of IBD. However, the interaction between these two factors and its implications in the aetiology of IBD remain underexplored. Here, we report on the influence of host genetics on the gut microbiome in IBD.DesignTo evaluate the impact of host genetics on the gut microbiota of patients with IBD, we combined whole exome sequencing of the host genome and whole genome shotgun sequencing of 1464 faecal samples from 525 patients with IBD and 939 population-based controls. We followed a four-step analysis: (1) exome-wide microbial quantitative trait loci (mbQTL) analyses, (2) a targeted approach focusing on IBD-associated genomic regions and protein truncating variants (PTVs, minor allele frequency (MAF) >5%), (3) gene-based burden tests on PTVs with MAF ResultsWe identified 12 mbQTLs, including variants in the IBD-associated genes IL17REL, MYRF, SEC16A and WDR78. For example, the decrease of the pathway acetyl-coenzyme A biosynthesis, which is involved in short chain fatty acids production, was associated with variants in the gene MYRF (false discovery rate CYP2D6, GPR151 and CD160 genes. These genes are known for their function in the immune system. Moreover, interaction analyses confirmed previously known IBD disease-specific mbQTLs in TNFSF15.ConclusionThis study highlights that both common and rare genetic variants affecting the immune system are key factors in shaping the gut microbiota in the context of IBD and pinpoints towards potential mechanisms for disease treatment.

Context The genetic architecture of isolated hypogonadotropic hypogonadism (IHH) has not been completely defined. Objective To determine the role of copy number variants (CNVs) in IHH pathogenicity and define their phenotypic spectrum. Methods Exome sequencing (ES) data in IHH probands (n = 1394) (Kallmann syndrome [IHH with anosmia; KS], n = 706; normosmic IHH [nIHH], n = 688) and family members (n = 1092) at the Reproductive Endocrine Unit and the Center for Genomic Medicine of Massachusetts General Hospital were analyzed for CNVs and single nucleotide variants (SNVs)/indels in 62 known IHH genes. IHH subjects without SNVs/indels in known genes were considered “unsolved.” Phenotypes associated with CNVs were evaluated through review of patient medical records. A total of 29 CNVs in 13 genes were detected (overall IHH cohort prevalence: ~2%). Almost all (28/29) CNVs occurred in unsolved IHH cases. While some genes (eg, ANOS1 and FGFR1) frequently harbor both CNVs and SNVs/indels, the mutational spectrum of others (eg, CHD7) was restricted to SNVs/indels. Syndromic phenotypes were seen in 83% and 63% of IHH subjects with multigenic and single gene CNVs, respectively. Conclusion CNVs in known genes contribute to ~2% of IHH pathogenesis. Predictably, multigenic contiguous CNVs resulted in syndromic phenotypes. Syndromic phenotypes resulting from single gene CNVs validate pleiotropy of some IHH genes. Genome sequencing approaches are now needed to identify novel genes and/or other elusive variants (eg, noncoding/complex structural variants) that may explain the remaining missing etiology of IHH.

Introduction:The middle-aged female population will increase in the coming years and will require more medical care to relieve climacteric symptomsObjective:To validate an instrument for evaluating climacteric and menopause in primary careMethods:Prospective, cross-sectional, instrument-validation study. The sample was composed of women attending primary care centers. Our research consisted of three stages: initial review (literature review, translation), validation (content validity, pilot study) and psychometric properties (internal consistency and confirmatory factorial analysis). We analyzed the data using SPSS version 20.Results:136 patients participated, with an average age of 48,6 years ± 5,3 and an average body mass index of 27,8 kg/m2 ± 4,4. In the first stage, we identified the most relevant symptoms: menstrual irregularities, hot flashes, vaginal problems, urinary incontinence, quality of sleep, and mood issues. The instrument was then translated by four professionals. For the second stage, we assessed content validity by expert judgment, obtaining an internal consistency of 0,77 and an initial reliability of 0,79. In the third stage, internal consistency was 0,69 and confirmatory factorial analysis was 0,7. The instrument consists of six questions; obtaining four or more positive answers warrants specialized care.Conclusion:We present a short instrument with adequate content validity and internal consistency, useful for managing climacteric symptoms at the primary care level. Introducción:La población de mujeres de mediana edad se incrementará en los próximos años y requerirá una mayor atención para aliviar los síntomas relacionados de atención con el climaterio.Objetivo:Validar un instrumento para la evaluación del climaterio y la menopausia en el primer nivel de atención.Métodos:Diseño de validación de instrumento, prospectivo y de corte transversal. Participaron mujeres que fueron atendidas en establecimientos de salud del primer nivel de atención. La investigación se realizó en tres fases, revisión inicial (revisión de literatura, traducción), ensayo (validez de contenido, prueba piloto) y propiedades psicométricas (análisis de consistencia interna, análisis factorial exploratorio). Para el análisis de datos se utilizó el programa SPSS versión 20.Resultados:Participaron 136 pacientes con una edad promedio de 48,6 años ± 5,3 y un promedio de índice de masa corporal 27,8 ± 4,4. En la primera fase se identificaron los síntomas más relevantes, irregularidades menstruales, sofocos, problemas vaginales, incontinencia urinaria, calidad de sueño y estado de ánimo. Se procedió a la traducción con la participación de cuatro profesionales. La segunda fase, de validez del contenido, fue mediante juicio de expertos, obteniendo una concordancia de 0,77 y una confiabilidad inicial 0,79. En la tercera fase, la consistencia interna fue 0,69 y 0,70 para análisis factorial exploratorio. El instrumento contiene seis preguntas, la obtención de ≥ 4 respuestas afirmativas justifican un manejo especializado.Conclusión:El instrumento es breve, tiene una adecuada validez de contenido y consistencia interna, útil para el manejo del climaterio en el primer nivel de atención.

Electric vehicle charging has shifted to higher voltages to achieve higher power for more rapid charging capabilities. This article provides a contactless magnetic plug solution that enables medium-voltage grid connections for electric vehicle charging to achieve 3.5- $\text{kV}_{\rm DC}$ -to-400- $\text{V}_{\rm DC}$ , 150-kW rapid charging capabilities. This novel magnetic plug improves upon existing electric vehicle charging solutions by guaranteeing safe operation and connection through galvanic and physical separation from the medium-voltage side. It achieves this with a gap and barrier in the transformer core. We introduce a unique asymmetry in the core to localize parasitic capacitance, fully separating the medium- and low-voltage regions. This approach eliminates arcing risk and allows rapid charging capabilities to be delivered to the general public. This gapped core constitutes the plug action of our proposed charging system. We present solutions for the unique challenges of this solution through a detailed analysis of the magnetic design. We confirm this analysis in finite-element analysis and experimentation. The solution is verified through a scaled laboratory prototype of 20 kW, 1 $\text{kV}_{\rm DC}$ to 50 $\text{V}_{\rm DC}$ that is representative of the proposed 150-kW design. We demonstrate safe, arc-free, disconnection in included active content, a new solution for high-power electric vehicle rapid charging.

Satellite DNAs (satDNAs) are among the most dynamically evolving components of eukaryotic genomes and play important roles in genome regulation, genome evolution, and speciation. Despite their abundance and functional impact, we know little about the evolutionary dynamics and molecular mechanisms that shape satDNA distributions in genomes. Here, we use high-quality genome assemblies to study the evolutionary dynamics of two complex satDNAs, Rsp-like and 1.688 g/cm3, in Drosophila melanogaster and its three nearest relatives in the simulans clade. We show that large blocks of these repeats are highly dynamic in the heterochromatin, where their genomic location varies across species. We discovered that small blocks of satDNA that are abundant in X chromosome euchromatin are similarly dynamic, with repeats changing in abundance, location, and composition among species. We detail the proliferation of a rare satellite (Rsp-like) across the X chromosome in D. simulans and D. mauritiana. Rsp-like spread by inserting into existing clusters of the older, more abundant 1.688 satellite, in events likely facilitated by microhomology-mediated repair pathways. We show that Rsp-like is abundant on extrachromosomal circular DNA in D. simulans, which may have contributed to its dynamic evolution. Intralocus satDNA expansions via unequal exchange and the movement of higher order repeats also contribute to the fluidity of the repeat landscape. We find evidence that euchromatic satDNA repeats experience cycles of proliferation and diversification somewhat analogous to bursts of transposable element proliferation. Our study lays a foundation for mechanistic studies of satDNA proliferation and the functional and evolutionary consequences of satDNA movement.

Synchronous rectification (SR) is widely adopted for low-voltage high-current applications to improve efficiency. However, most SR researches focus on low power levels at hundreds or several kilowatts, and there is yet any attempt to extend SR to tens of kilowatts. This paper presents the design and experimental results of a full-bridge SR using ON-Semi NCP4305 SR IC at 20kW for 48V data center application.

Contactless power transfer (CPT) methods have widely been explored for many applications. An isolated wireless power link enables a touch-safe interface without electrically energized contacts eliminating any arc flash or electrocution hazard. The concept presented here will enable safe medium voltage distribution inside data centers eliminating low voltage distribution with bulky cables bringing voltages larger than 1kV directly to the server rack. This paper proposes high-efficiency single-stage contactless power conversion from medium voltage to low voltage for data center applications. The proposed solution is evaluated using a 10kW design example and verified by experimental tests. A plug-in contactless transformer is developed with customized nanocrystalline cores and 3D printed bobbins to improve the DC-to-DC efficiency and manufacturability. A high efficiency converter is developed to maintain a 48V output voltage with a IOOOV input voltage over a wide load range. The DC-to-DC efficiency and frequency modulation performance are analyzed.

Wireless power transfer (WPT) techniques have been widely explored for various applications including for electric vehicles (EVs) and mobile phones. Less attention has been paid to the use of WPT in power distribution applications. An isolated wireless power link enables a touch safe interface without electrically energized contacts eliminating any arc flash or electrocution hazards. This concept will for example enable safe medium voltage distribution inside data centers eliminating low voltage distribution with its large bus bars and cables and bring voltages larger than 1kV directly to the server rack. It has been demonstrated that a converter using a planar contactless transformer can directly convert the 1kV DC input to 48V over a wide load range. In this paper, the planar contactless transformer is optimized with 3D printed bobbins to improve the manufacturability and technology readiness level. A new synchronous rectifier architecture is developed with MOSFET drain-to-source voltage-sensing for an H-bridge rectifier to improve the DC-to-DC efficiency. An adaptive dead time control method is used to compensate for layout parasitic inductances, to maximize the synchronous rectifier efficiency, and to avoid premature turn-off of MOSFETs. The DC-to-DC power transfer efficiency and loss distribution are analyzed.

Dubowitz syndrome (DubS) is considered a recognizable syndrome characterized by a distinctive facial appearance and deficits in growth and development. There have been over 200 individuals reported with Dubowitz or a "Dubowitz-like" condition, although no single gene has been implicated as responsible for its cause. We have performed exome (ES) or genome sequencing (GS) for 31 individuals clinically diagnosed with DubS. After genome-wide sequencing, rare variant filtering and computational and Mendelian genomic analyses, a presumptive molecular diagnosis was made in 13/27 (48%) families. The molecular diagnoses included biallelic variants in SKIV2L, SLC35C1, BRCA1, NSUN2; de novo variants in ARID1B, ARID1A, CREBBP, POGZ, TAF1, HDAC8, and copy-number variation at1p36.11(ARID1A), 8q22.2(VPS13B), Xp22, and Xq13(HDAC8). Variants of unknown significance in known disease genes, and also in genes of uncertain significance, were observed in 7/27 (26%) additional families. Only one gene, HDAC8, could explain the phenotype in more than one family (N = 2). All but two of the genomic diagnoses were for genes discovered, or for conditions recognized, since the introduction of next-generation sequencing. Overall, the DubS-like clinical phenotype is associated with extensive locus heterogeneity and the molecular diagnoses made are for emerging clinical conditions sharing characteristic features that overlap the DubS phenotype.

Generally, the data center server racks are supplied with power at or below 400V, primarily due to safety. However, due to the rapid increase in the size of data centers, the power demand is also rising sharply, and some of them consume power over a hundred MW. This results in bulky and inefficient low voltage distribution power cables to the server racks. In this paper, an attempt is made to directly feed medium voltage to data center servers using SiC converters, where the safety issues are addressed with a contactless power transfer technique. The selection of power converter circuit, steady-state operation, converter design, and contactless power transformer selection are reported. To verify the analysis and performance of the converter, a 20kW lab-prototype with input 1000V and output 48V is developed, and the details of building the system are discussed. The experimental results are included to verify the performances of the proposed system.

In this paper, a novel contactless electric vehicle charging architecture is proposed to achieve single-stage power conversion from the medium voltage (MV) DC grid to electric vehicle charging level by utilizing a detachable, galvanically isolated power transformer as the charging plug to provide a highly efficient, fast and safe charging experience to the end users. A scaled-down prototype of 20kW, 1000Vdc to 50Vdc solution is provided as a proof of concept of the full-scale 150kW, 8000Vdc to 400Vdc fast charging solution for electric vehicle.

Wireless power transfer (WPT) techniques have been widely explored for various applications including for electric vehicle (EV) and mobile phone charging. Less attention has been paid to the use of WPT for commercial or industrial applications and for power distribution. An isolated wireless power link can enable a touch safe interface without electrically energized contacts eliminating any arc flash or electrocution hazard. This concept can for example enable safe medium voltage distribution inside data centers eliminating low voltage distribution with its large bus bars and cables and bring voltages larger than 1kV directly to the rack. A design methodology is developed for a low loss, high voltage-transfer-ratio wireless power link and a high efficiency medium voltage power converter. The proposed design methodology is evaluated using a 10kW design example and verified by finite element analysis (FEA) and experimental tests. The WPT transformer is developed with a planar magnetics structure. A U-shape ferrite configuration is proposed to improve mutual coupling by providing a low magnetic reluctance coupling path for the magnetic flux. A high efficiency power converter is developed to maintain the output voltage (48V) with a 1kV input over a wide load range. The power transfer efficiency and loss distribution are analyzed.

Satellite DNAs (satDNAs) are among the most dynamically evolving components of eukaryotic genomes and play important roles in genome regulation, genome evolution, and speciation. Despite their abundance and functional impact, we know little about the evolutionary dynamics and molecular mechanisms that shape satDNA distributions in genomes. Here we use high-quality genome assemblies to study evolutionary dynamics of two complex satDNAs,Rsp-likeand1.688gm/cm3, inDrosophila melanogasterand its three nearest relatives in thesimulansclade. We show that large blocks of these repeats are highly dynamic in the heterochromatin, where their genomic location varies across species. We discovered that small blocks of satDNA that are abundant in X chromosome euchromatin are similarly dynamic, with repeats changing in abundance, location, and composition among species. We detail the proliferation of a rare satellite (Rsp-like) across the X chromosome inD. simulansandD. mauritiana. Rsp-likespreads by inserting into existing clusters of the older, more abundant1.688satellite, in events that were likely facilitated by microhomology-mediated repair pathways. We show thatRsp-likeis abundant on extrachromosomal circular DNA inD. simulans, which may have contributed to its dynamic evolution. Intralocus satDNA expansions via unequal exchange and the movement of higher-order repeats also contribute to the fluidity of the repeat landscape. We find evidence that euchromatic satDNA repeats experience cycles of proliferation and diversification somewhat analogous to bursts of transposable element proliferation. Our study lays a foundation for mechanistic studies of satDNA proliferation and the functional and evolutionary consequences of satDNA movement.

With the increase in size of data centers and cloud computing, its power demand is also rising sharply. Traditionally, this power distribution is achieved at about 400VDC which is inconvenient because it requires very bulky conductor to prevent high copper loss. In this paper, a new power distribution architecture for data centers is reported where direct medium voltage distribution to data-center racks are achieved with SiC based inverter. The safety issues raised due to bringing medium voltage to the racks are addressed with contactless power transfer technology. The rectifier circuit handles a high current at 48V, and synchronous rectification would be suitable to boost the efficiency. In this paper, a GaN based synchronous rectification is studied. This proposed converter circuit is analyzed and simulated in PowerSim 11. A 3kW experimental setup is developed in the lab to verify the analysis and simulation performances of the converter, where the input is 1000V and the output is 48V.

Introduction: While the role of single nucleotide variants (SNVs) in causal genes for Idiopathic Hypogonadotropic hypogonadism (IHH) is known, the contribution of copy number variants (CNVs) to IHH has not been systematically studied. Here, we examined the prevalence of CNVs in a large IHH cohort and their associated phenotypic spectrum. Methods: Exome sequencing (ES) from 1,441 IHH probands was analyzed using the GATK-gCNV pipeline to identify CNVs that spanned known IHH genes with a site frequency of Results: (i) CNV prevalence in IHH: Three percent of the IHH probands (46/1441 probands, 38 males and 8 females) harbored CNVs in 19/36 high confidence IHH genes (26 deletions 20 duplications). The vast majority of CNVs disrupted either the ANOS1 (26%) or FGFR1 (17%) genes. Intriguingly, CHD7 (a gene that carries SNVs in ~12% of our IHH subjects) did not harbor any CNVs. (ii) Phenotypic analysis: We found that CNVs were more common in IHH subjects with anosmia (Kallmann syndrome, N=33) compared to normosmic IHH (N=13). More than half of the subjects with CNVs affecting IHH genes carried at least 1 non-reproductive feature (26/46 IHH subjects, 56%). A syndromic presentation with multiple non-reproductive phenotypes was more common in IHH subjects harboring multigenic CNVs (IHH gene & additional genes) compared to IHH subjects with single IHH gene CNV or SNV in a single IHH gene (61% vs. 12% or 18%, respectively; p 0.0006). Conclusions: CNVs in known IHH genes contribute to 3% of the IHH genetic architecture in our cohort. IHH subjects with larger multigenic CNVs displayed phenotypes consistent with contiguous gene syndromes. The absence of genic CNVs in genes frequently found to carry SNVs, such as CHD7, requires additional analysis to establish the biologic or mechanistic reasons for their rarity.

Usually, the data center racks are supplied power at or below 400V, primarily due to safety. However, because of rapid increase in size of data centers, the power demand is also rising sharply, which results in bulky power cables and inefficient power distribution. In this paper, the idea of inductive coupling is used to increase the distribution voltage level to kVs, while maintaining the required safety aspects of the system. Based on the defined problem, a suitable converter topology is selected, where safety issue are addressed with a gapped-core (or split-core) transformer isolation. The performance of this converter circuit is analyzed, and component ratings are derived. Soft-switching performance of inverter devices are also reported. Due to low voltage and high current at the output, synchronous rectification technique promises significant improvement in efficiency. To verify the performances of the converter circuit, numerical simulation is performed in PowerSIM 11. A 2.5kW lab-prototype is developed, where the input/ output voltage is 1.1kV / 48V, and experimental results are included to justify the suitability of the selected converter.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle., eLife digest Glowing fireflies dancing in the dark are one of the most enchanting sights of a warm summer night. Their light signals are ‘love messages’ that help the insects find a mate – yet, they also warn a potential predator that these beetles have powerful chemical defenses. The light comes from a specialized organ of the firefly where a small molecule, luciferin, is broken down by the enzyme luciferase. Fireflies are an ancient group, with the common ancestor of the two main lineages originating over 100 million years ago. But fireflies are not the only insects that produce light: certain click beetles are also bioluminescent. Fireflies and click beetles are closely related, and they both use identical luciferin and similar luciferases to create light. This would suggest that bioluminescence was already present in the common ancestor of the two families. However, the specialized organs in which the chemical reactions take place are entirely different, which would indicate that the ability to produce light arose independently in each group. Here, Fallon, Lower et al. try to resolve this discrepancy and to find out how many times bioluminescence evolved in beetles. This required using cutting-edge DNA sequencing to carefully piece together the genomes of two species of fireflies (Photinus pyralis and Aquatica lateralis) and one species of click beetle (Ignelater luminosus). The genetic analysis revealed that, in all species, the genes for luciferases were very similar to the genetic sequences around them, which code for proteins that break down fat. This indicates that the ancestral luciferase arose from one of these metabolic genes getting duplicated, and then one of the copies evolving a new role. However, the genes for luciferase were very different between the fireflies and the click beetles. Further analyses suggested that bioluminescence evolved at least twice: once in an ancestor of fireflies, and once in the ancestor of the bioluminescent click beetles. More results came from the reconstituted genomes. For example, Fallon, Lower et al. identified the genes ‘turned on’ in the bioluminescent organ of the fireflies. This made it possible to list genes that may be involved in creating luciferin, and enable flies to grow brightly for long periods. In addition, the genetic information yielded sequences from bacteria that likely live inside firefly cells, and which may participate in the light-making process or the production of potent chemical defenses. Better genetic knowledge of beetle bioluminescence could bring new advances for both insects and humans. It may help researchers find and design better light-emitting molecules useful to track and quantify proteins of interest in a cell. Ultimately, it would allow a detailed understanding of firefly populations around the world, which could contribute to firefly ecotourism and help to protect these glowing insects from increasing environmental threats.

INTRODUCTION: Intubated patients with acute respiratory distress syndrome are thought to have a 5-12% incidence of barotrauma, even with protective ventilation However, little is known about the incidence of barotrauma in COVID-19 Due to high rates of observed barotrauma at this center, this retrospective cohort study aims to better characterize the incidence of barotrauma and identify predisposing factors such as inflammatory markers and disease severity indices for this high-mortality complication METHODS: Inclusion criteria were as follows: age over 18 years, positive RT-PCR for SARS-CoV2, admission to the ICU between 03/15/2020 and 06/15/2020, and a score of 5 or higher on the World Health Organization's Ordinal Scale or respiratory rate over 30 breaths per minute on admission Data were collected for the following categories developed by an internal committee of pulmonary/critical care faculty and housestaff based on similar studies: age, sex, body mass index, ferritin, d-dimer, APACHE II score, SOFA score, blood gas, ventilation mode and settings Patients with evidence of barotrauma (pneumothorax, pneumomediastinum, pneumopericardium, subcutaneous emphysema) on imaging had additional respiratory data points collected RESULTS: 78 patients met inclusion Among 38 patients who received invasive mechanical ventilation (IMV) 12 had barotrauma (32%) Of 40 patients who did not receive IMV 3 had barotrauma (8%) Of 15 cases of barotrauma, 8 had pneumothorax (2 bilateral, 6 unilateral), 9 had pneumomediastinum, 4 had pneumopericardium, 6 had subcutaneous emphysema 8 were found incidentally on imaging for non-respiratory indication Mortality in the barotrauma group was 72% for IMV & 50% for non-IMV (3 patients transferred to other hospital, 3 remain hospitalized) compared to 50% for IMV & 8% for non-IMV in patients without barotrauma Further analysis pending at submission, data to be finalized prior to presentation CONCLUSIONS: Barotrauma may be an underappreciated complication of COVID-19, perhaps serving as an independent predictor of disease severity or low lung compliance Many theories have been presented for the physiology of COVID-19 respiratory failure, but barotrauma could be evidence of or a herald sign for the low compliance phenotype

Estimates of potential harmful effects on ecosystems in the Canadian provinces of Alberta and Saskatchewan due to acidifying deposition were calculated, using a 1-year simulation of a high-resolution implementation of the Global Environmental Multiscale-Modelling Air-quality and Chemistry (GEM-MACH) model, and estimates of aquatic and terrestrial ecosystem critical loads. The model simulation was evaluated against two different sources of deposition data: total deposition in precipitation and total deposition to snowpack in the vicinity of the Athabasca oil sands. The model captured much of the variability of observed ions in wet deposition in precipitation (observed versus model sulfur, nitrogen and base cation R2 values of 0.90, 0.76 and 0.72, respectively), while being biased high for sulfur deposition, and low for nitrogen and base cations (slopes 2.2, 0.89 and 0.40, respectively). Aircraft-based estimates of fugitive dust emissions, shown to be a factor of 10 higher than reported to national emissions inventories (Zhang et al., 2018), were used to estimate the impact of increased levels of fugitive dust on model results. Model comparisons to open snowpack observations were shown to be biased high, but in reasonable agreement for sulfur deposition when observations were corrected to account for throughfall in needleleaf forests. The model–observation relationships for precipitation deposition data, along with the expected effects of increased (unreported) base cation emissions, were used to provide a simple observation-based correction to model deposition fields. Base cation deposition was estimated using published observations of base cation fractions in surface-collected particles (Wang et al., 2015).Both original and observation-corrected model estimates of sulfur, nitrogen, and base cation deposition were used in conjunction with critical load data created using the NEG-ECP (2001) and CLRTAP (2017) methods for calculating critical loads, using variations on the Simple Mass Balance model for terrestrial ecosystems, and the Steady State Water Chemistry and First-order Acidity Balance models for aquatic ecosystems. Potential ecosystem damage was predicted within each of the regions represented by the ecosystem critical load datasets used here, using a combination of 2011 and 2013 emissions inventories. The spatial extent of the regions in exceedance of critical loads varied between 1  ×  104 and 3.3  ×  105 km2, for the more conservative observation-corrected estimates of deposition, with the variation dependent on the ecosystem and critical load calculation methodology. The larger estimates (for aquatic ecosystems) represent a substantial fraction of the area of the provinces examined.Base cation deposition was shown to be sufficiently high in the region to have a neutralizing effect on acidifying deposition, and the use of the aircraft and precipitation observation-based corrections to base cation deposition resulted in reasonable agreement with snowpack data collected in the oil sands area. However, critical load exceedances calculated using both observations and observation-corrected deposition suggest that the neutralization effect is limited in spatial extent, decreasing rapidly with distance from emissions sources, due to the rapid deposition of emitted primary dust particles as a function of their size. We strongly recommend the use of observation-based correction of model-simulated deposition in estimating critical load exceedances, in future work.

This paper investigates the linkage between Canada's National Agri-Environmental Standards Initiative (NAESI) Biodiversity and Water themes by studying how patterns in terrestrial habitat, generated through land cover scenario modelling, influence water quality and quantity in the Raisin River watershed in southeastern Ontario. NAESI developed nonregulatory performance standards that define ideal and achievable levels of environmental quality. The indicators used to investigate the scenario risks included sediment and nutrient concentrations. The SWAT (Soil and Water Assessment Tool)-2005 model was calibrated and validated from 1985 to 2006 for current land cover and five other scenarios: potential natural vegetation (PNV); high biodiversity conservation (HBC); moderate biodiversity conservation; agricultural intensification with limited application of conservation direction; and agricultural intensification with no consideration of conservation direction (ANC). Scenario comparisons are provided for the average annual flow, and concentrations of total suspended sediment (TSS), total nitrogen, and total phosphorus for five watershed locations. The PNV scenario predicted the lowest total flows, and sediment and nutrient concentrations, and the ANC scenario predicted the highest sediment and nutrient concentrations. The SWAT median values for the HBC, "Current," and ANC scenarios at the outlet all exceeded the Ideal Performance Standards, except for the median TSS concentration of the HBC scenario.

Integrase (IN) catalyzes insertion of the retroviral genome into the host via two sequential reactions. The processing activity cleaves the 3'-dinucleotides from the two ends of the viral DNA which are then inserted into the host DNA. Tetramers are required for the joining step. While dimers have been shown to catalyze processing, they do so inefficiently, and the oligomeric requirement for processing is unknown. We have replaced loop202-208 at the putative dimer-dimer interface of the avian sarcoma virus IN with its analogue, loop188-194, from human immunodeficiency virus IN. The mutation abolished disintegration activity and a 2 x 10(-2) s-1 fast phase during single-turnover processing. A 3 x 10(-4) s-1 slow processing phase was unaffected. Preincubation with a DNA substrate known to promote tetramerization increased products formed during the fast phase by 2.5-fold only for wild-type IN, correlating the fast and slow phases with processing by tetramers and dimers, respectively. We propose a novel tetramer model for coupling processing and integration based on efficient processing by the tetramer. We provide for the first time direct evidence of the functional relevance of a structural element, loop202-208, which appears to be required for mediating the interaction between dimer halves of the active tetramer.

Background Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3°C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. Methodology/Principal Findings In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the “archaic” method of hand-transferring PCR tubes between water baths. Conclusions/Significance We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.

Lake Winnipeg eutrophication results from excess nutrient loading due to agricultural activities across the watershed. Estimating nonpoint-source pollution and the mitigation effects of beneficial management practices (BMPs) is an important step in protecting the water quality of streams and receiving waters. The use of computer models to systematically compare different landscapes and agricultural systems across the Red-Assiniboine basin has not been attempted at watersheds of this size in Manitoba. In this study, the Soil and Water Assessment Tool was applied and calibrated for three pilot watersheds of the Lake Winnipeg basin. Monthly flow calibration yielded overall satisfactory Nash-Sutcliffe efficiency (NSE), with values above 0.7 for all simulations. Total phosphorus (TP) calibration NSE ranged from 0.64 to 0.76, total N (TN) ranged from 0.22 to 0.75, and total suspended solids (TSS) ranged from 0.29 to 0.68. Based on the assessment of the TP exceedance levels from 1993 to 2007, annual loads were above proposed objectives for the three watersheds more than half of the time. Four BMP scenarios based on land use changes were studied in the watersheds: annual cropland to hay land (ACHL), wetland restoration (WR), marginal annual cropland conversion to hay land (MACHL), and wetland restoration on marginal cropland (WRMAC). Of these land use change scenarios, ACHL had the greatest impact: TSS loads were reduced by 33 to 65%, TN by 58 to 82%, and TP by 38 to 72% over the simulation period. By analyzing unit area and percentage of load reduction, the results indicate that the WR and WRMAC scenarios had a significant impact on water quality in high loading zones in the three watersheds. Such reductions of sediment, N, and P are possible through land use change scenarios, suggesting that land conservation should be a key component of any Lake Winnipeg restoration strategy.

This study analyzed the effects of ultra-low concentrations of multi-walled carbon nanotubes (MWNT) on the electromechanical properties of cement paste. Here, 0.025 and 0.050 wt% of MWNTs were dispersed using a poly(sodium 4-styrenesulfonate) aqueous solution, which was then used for casting specimens. Because these concentrations make large-scale applications much more feasible, sample consistency and effects of saturation were also studied. Their resistivity ranges and strain sensing properties (i.e., gage factors) were characterized.

Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic “push-pull” mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis. Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping. Parallel experiments detecting the protein fluorescence showed a slower Leu191 insertion step occurring after nucleotide flipping but before excision. The inserted Leu191 acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in “pushing” the uracil out of the DNA helix. The results define for the first time the proper sequence of events during a catalytic cycle and establish a “pull-push”, as opposed to a “push-pull”, mechanism for nucleotide flipping.

The integrase-catalyzed insertion of the retroviral genome into the host chromosome involves two reactionsin vivo: 1) the binding and endonucleolytic removal of the terminal dinucleotides of the viral DNA termini and 2) the recombination of the ends with the host DNA. Kukolj and Skalka (Kukolj, G., and Skalka, A. M. (1995) Genes Dev. 9, 2556–2567) have previously shown that tethering of the termini enhances the endonucleolytic activities of integrase. We have used 5′-5′ phosphoramidites to design reverse-polarity tethers that allowed us to examine the reactivity of two viral long terminal repeat-derived sequences when concurrently bound to integrase and, additionally, developed presteady-state assays to analyze the initial exponential phase of the reaction, which is a measure of the amount of productive nucleoprotein complexes formed during preincubation of integrase and DNA. Furthermore, the reverse-polarity tether circumvents the integrase-catalyzed splicing reaction (Bao, K., Skalka, A. M., and Wong, I. (2002) J. Biol. Chem. 277, 12089–12098) that obscures accurate analysis of the reactivities of synapsed DNA substrates. Consequently, we were able to establish a lower limit of 0.2 s−1 for the rate constant of the processing reaction. The analysis showed the physiologically relevant U3/U5 pair of viral ends to be the preferred substrate for integrase with the U3/U3 combination favored over the U5/U5 pair.

The Murray-Darling Basin in Australia is severely environmentally degraded due to anthropogenic changes. Problems include elevated river salinity levels, widespread blooms of toxic blue-green algae, decline of native fish and bird populations, and loss of floodplain wetlands. The community and the government are committed to improving the state of the environment in the Basin, both for its intrinsic ecological values, and to ensure the sustainability of production in Australia’s most economically important agricultural region. To facilitate the trade-off process between users of this resource, an environmental flows decision support system (EFDSS) is being developed to allow explicit prediction of the likely response of key features of the riverine environment to proposed flow management scenarios. The EFDSS is being developed using the RAISON shell (Lam et al. 1994), and will integrate a range of simple models of riverine ecology which are being developed. These models will include qualitative and quantitative models representing the response of different aspects of the instream and floodplain ecology dependent upon the river flow regime. The EFDSS will not include a hydrology model, but will use the outputs from the hydrology models currently in use in the Basin as inputs to the ecological models. The EFDSS will provide a range of tools to allow evaluation of scenarios, as well as explanations and supporting information to elucidate the ecological modelling.

The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reactionsthe asymmetric Rep dimer state, P2S, where ss DNA (dT(pT)15) is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition state analog ADP-AlF4. We found that when ADP-AlF4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s -1 per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP-AlF4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP a E-ATP a E'-ATP a E'- ADP-Pi a E-ADP-Pi a E-ADP + Pi a E + ADP + Pi, with K1 ) (2.0 ( 0.85) 10 5 M -1 , k2 ) 22 ( 3.5 s -1 , k-2 200 s -1 ), k4 ) 1.2 ( 0.14 s -1 , k-4 << 1.2 s -1 , K5 ) 1.0 ( 0.2 mM, and K6 ) 80 ( 8 IM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E '-ADP-Pi. In the context of our "subunit switching" model for Rep dimer translocation during processive DNA unwinding (Bjornson, K. B., Wong, I., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422), this state may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.

DNA helicases are motor proteins that unwind duplex DNA during DNA replication, recombination and repair in reactions that are coupled to ATP binding and hydrolysis. In the process of unwinding duplex DNA processively, DNA helicases must also translocate along the DNA filament. To probe the mechanism of ATP-driven translocation by the dimeric E. coli Rep helicase along single stranded (ss) DNA, we examined the effects of ATP on the dissociation kinetics of ssDNA from the Rep dimer. Stopped-flow experiments show that the dissociation rate of a fluorescent ss oligodeoxynucleotide bound to one subunit of the dimeric Rep helicase is stimulated by ssDNA binding to the other subunit, and that the rate of this ssDNA exchange reaction is further stimulated ∼60-fold upon ATP hydrolysis. This ssDNA exchange process occurs via an intermediate in which ssDNA is transiently bound to both subunits of the Rep dimer. These results suggest a rolling or subunit switching mechanism for processive ATP-driven translocation of the dimeric Rep helicase along ssDNA. Such a mechanism requires the extreme negative cooperativity for DNA binding to the second subunit of the Rep dimer, which insures that the doubly DNA-ligated Rep (P 2 S 2 ) dimer is formed only transiently and relaxes back to the singly ligated Rep (P 2 S) dimer. The fact that other oligomeric DNA helicases share many functional features with the dimeric Rep helicase suggests that similar mechanisms for translocation and DNA unwinding may apply to other dimeric as well as hexameric DNA helicases.

To examine the coupling of ATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a single-stranded hexadecameric oligodeoxynucleotide (S). Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized. The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-crosslinked Rep complexes formed with dT16. Therefore, ATP hydrolysis by both PS and P2S complexes are not coupled to DNA dissociation. This also rules out a strictly unidirectional sliding mechanism for ATP-driven translocation along single-stranded DNA by either PS or the P2S dimer. However, ATP hydrolysis by the doubly ligated P2S2 Rep dimer is coupled to single-stranded DNA dissociation from one subunit of the dimer, although loosely (low efficiency). These results suggest that ATP hydrolysis can drive translocation of the dimeric Rep helicase along DNA by a "rolling" mechanism where the two DNA binding sites of the dimer alternately bind and release DNA. Such a mechanism is biologically important when one subunit binds duplex DNA, followed by subsequent unwinding.

The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis. Rep functions as a dimer but assembles to its active dimeric form only on binding DNA. Each promoter of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA. The dimer can bind up to two oligodeoxynucleotides in five DNA-ligation states: two half-ligated states, P2S and P2D, and three fully-ligated states, P2S2, P2D2, and P2SD. We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an "active rolling" model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T.M., (1992), Science 256, 350-355]. THe basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization. We have measured the steady-state ATPase activities of Rep bound to dT(pT)15 in each distinct ss DNA ligation state (PS, P2S, and P2S2) to compare with our previous measurements with unligated Rep monomer (P) [Moore, K.J.M., & Lohman, T.M. (1994) Biochemistry 33, 14550]. We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following kcat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 x 10(-3) s(-1) for P, 2.17 +/- 0.04 s(-1) for PS, 16.5 +/- 0.2 s(-1) for P2S, and 71 +/- 2.5 s(-1) for P2S2 (20 mM Tris-HCl, pH 7.5, 6mM NaCl, 5 mM MgCl2, 10% glycerol, 4 degrees C). The apparent KM's for ATP hydrolysis are 2.05 +/- 0.1 microM for PS and 2.7 +/- 0.2 microM for P2S. These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA site of both subunits of the Rep dimer. These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements.

The Lake Winnipeg Basin Initiative was created as part of Canada's Action Plan on clean water. Its focus is to deal with excessive lake inputs of nu- trients from surface runoff and municipal wastewater. Understanding the dy- namics of nutrient loading, the associated algal blooms and resulting changes in fish populations, beach closures, and ecosystem imbalance requires access to various sources of data, information, knowledge, expertise and tools. Such criti- cal components are delivered through the Lake Winnipeg Basin Initiative In- formation Portal, which integrates multiple geospatial and non-geospatial data- sets of information pertaining to the basin and serves as a data, information and modelling portal. With data coming from many disparate sources, the Canadian Geospatial Data Infrastructure standards are applied to ensure interoperability. The "Community of Models" allows the modellers to post their model and re- sults, and also allows the portal users to comment on the results to ensure a healthy dialogue.

Nitrocellulose-filter binding is a powerful technique commonly used to study protein-nucleic acid interactions; however, its utility in quantitative studies is often compromised by its lack of precision. To improve precision and accuracy, we have introduced two modifications to the traditional technique: the use of a 96-well dot-blot apparatus and the addition of a DEAE membrane beneath the nitrocellulose membrane. Using the dot-blot apparatus, an entire triplicate set of data spanning 20-24 titrant concentrations can be collected on a single 4.5 x 5 inch sheet of nitrocellulose, obviating the need to manipulate separate filters for each titration point. The entire titration can then be quantitated simultaneously with direct two-dimensional beta-emission imaging technology. The DEAE second membrane traps all DNA that does not bind to the nitrocellulose, enabling a direct determination of the total amount of DNA filtered. This measurement improves precision by allowing the amount of DNA retained by the nitrocellulose to be normalized against the total amount of DNA filtered. The DEAE membrane also permits a more accurate quantitation of filter-retention efficiency and nonspecific background retention based on free DNA rather than total DNA filtered. The general approach and methods of analysis to obtain equilibrium binding isotherms are discussed, using as examples our studies of the Escherichia coli Rep protein, a helicase, and its interactions with short oligodeoxynucleotides.

Environmental decision support systems have been implemented in Environmental Canada. They are proved to be practical. Not only the modellers increase their productivity with the assist of the EDSSs, government officials and policy makers also gain valuable insights by using these EDSSs to set policy guidelines and objectives. The EEM-SAT DSS has been tested by the EEM National office as well as by external users and has proven to substantially improve the ability of the user to generate accurate and consistent analyses that are required under the Canadian EEM program. The responses from external users have been positive. A significant proportion of the time and effort spent on

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)