Older adults at greater risk for Alzheimer's disease show stronger associations between sleep apnea severity in REM sleep and verbal memory.

BACKGROUND: Obstructive sleep apnea (OSA) increases risk for cognitive decline and Alzheimer's disease (AD). While the underlying mechanisms remain unclear, hypoxemia during OSA has been implicated in cognitive impairment. OSA during rapid eye movement (REM) sleep is usually more severe than in non-rapid eye movement (NREM) sleep, but the relative effect of oxyhemoglobin desaturation during REM versus NREM sleep on memory is not completely characterized. Here, we examined the impact of OSA, as well as the moderating effects of AD risk factors, on verbal memory in a sample of middle-aged and older adults with heightened AD risk. METHODS: Eighty-one adults (mean age:61.7 ± 6.0 years, 62% females, 32% apolipoprotein E ε4 allele (APOE4) carriers, and 70% with parental history of AD) underwent clinical polysomnography including assessment of OSA. OSA features were derived in total, NREM, and REM sleep. REM-NREM ratios of OSA features were also calculated. Verbal memory was assessed with the Rey Auditory Verbal Learning Test (RAVLT). Multiple regression models evaluated the relationships between OSA features and RAVLT scores while adjusting for sex, age, time between assessments, education years, body mass index (BMI), and APOE4 status or parental history of AD. The significant main effects of OSA features on RAVLT performance and the moderating effects of AD risk factors (i.e., sex, age, APOE4 status, and parental history of AD) were examined. RESULTS: Apnea-hypopnea index (AHI), respiratory disturbance index (RDI), and oxyhemoglobin desaturation index (ODI) during REM sleep were negatively associated with RAVLT total learning and long-delay recall. Further, greater REM-NREM ratios of AHI, RDI, and ODI (i.e., more events in REM than NREM) were related to worse total learning and recall. We found specifically that the negative association between REM ODI and total learning was driven by adults 60 + years old. In addition, the negative relationships between REM-NREM ODI ratio and total learning, and REM-NREM RDI ratio and long-delay recall were driven by APOE4 carriers. CONCLUSION: Greater OSA severity, particularly during REM sleep, negatively affects verbal memory, especially for people with greater AD risk. These findings underscore the potential importance of proactive screening and treatment of REM OSA even if overall AHI appears low.

Analysis of vitamin D3-sulfate and 25-hydroxyvitamin D3-sulfate in breastmilk by LC-MS/MS.

Sulfated metabolites of vitamin D have been suggested to be in breastmilk, although current methods to measure sulfated vitamin D compounds in breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have not adequately accounted for increased aqueous solubility of these sulfated metabolites. The purpose of this study was to generate a method of LC-MS/MS for measuring vitamin D3-3-sulfate (VitD3-S) and 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) specifically in human breastmilk. The resulting method uses methanol to precipitate protein and solid phase extraction to prepare the samples for LC-MS/MS. The limits of quantification for analytes in solvent were 0.23 ng/mL VitD3-S and 0.2 ng/mL 25OHD3-S. Various experiments observed concentrations ranging 0.53 to 1.7 ng/mL VitD3-S and ≤ 0.29 ng/mL 25OHD3-S. Both analytes were present in aqueous skim milk, demonstrating the enhanced aqueous solubility of these vitamin D sulfates. In conclusion, we describe an effective method for measuring VitD3-S and 25OHD3-S in breastmilk by LC-MS/MS.

M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation.

Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei (Mm) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation.

crosshap: R package for local haplotype visualization for trait association analysis.

SUMMARY: Genome-wide association studies (GWAS) excels at harnessing dense genomic variant datasets to identify candidate regions responsible for producing a given phenotype. However, GWAS and traditional fine-mapping methods do not provide insight into the complex local landscape of linkage that contains and has been shaped by the causal variant(s). Here, we present crosshap, an R package that performs robust density-based clustering of variants based on their linkage profiles to capture haplotype structures in a local genomic region of interest. Following this, crosshap is equipped with visualization tools for choosing optimal clustering parameters (ɛ) before producing an intuitive figure that provides an overview of the complex relationships between linked variants, haplotype combinations, phenotype, and metadata traits. AVAILABILITY AND IMPLEMENTATION: The crosshap package is freely available under the MIT license and can be downloaded directly from CRAN with R >4.0.0. The development version is available on GitHub alongside issue support (https://github.com/jacobimarsh/crosshap). Tutorial vignettes and documentation are available (https://jacobimarsh.github.io/crosshap/).

A novel index for measuring the impact of devices on hypertension.

A key limitation in assessing the therapeutic impact of non-pharmacological approaches to treating hypertension is the method of reporting outcomes. Reducing the medications required to achieve the same blood pressure may be reported separately to a reduction in the blood pressure without change in medication, and thus lessen the reported beneficial impact of treatment. This study aims to derive a novel scoring system to gauge the therapeutic impact of non-drug treatment of hypertension by utilising a combination of excessive blood pressure and the number of anti-hypertensives into a combined score-the hypertensive index (HTi). The hypertensive index was empirically derived based on the systolic blood pressure and number of antihypertensive drugs, and applied retrospectively to a cohort undergoing intervention for renovascular hypertension. Subgroup and receiver operating characteristic analyses were used to compare the HTi to traditional methods of reporting outcomes. Following intervention (99 patients), 46% had improvement in both medication load and blood pressure, 29% had benefit in blood pressure without reduction in medication load, 15% had reduction in medication load without significant change in blood pressure and 9% showed no benefit in either parameter. The HTi was superior in detecting benefit from intervention compared with measuring blood pressure or medication load alone (AUC 0.94 vs 0.85;0.84). The hypertensive index may be a more sensitive marker of treatment effect than assessing blood pressure measurements alone. The use of such scoring systems in future trial design may allow more accurate reporting of the effects of interventions for hypertension.

Non-oral manifestations in adults with a clinical and molecularly confirmed diagnosis of periodontal Ehlers-Danlos syndrome.

Introduction: Periodontal Ehlers-Danlos Syndrome (pEDS) is a rare autosomal dominant type of EDS characterised by severe early-onset periodontitis, lack of attached gingiva, pretibial plaques, joint hypermobility and skin hyperextensibility as per the 2017 International EDS Classification. In 2016, deleterious pathogenic heterozygous variants were identified in C1R and C1S, which encode components of the complement system. Materials and Methods: Individuals with a clinical suspicion of pEDS were clinically and molecularly assessed through the National EDS Service in London and Sheffield and in genetic services in Austria, Sweden and Australia. Transmission electron microscopy and fibroblast studies were performed in a small subset of patients. Results: A total of 21 adults from 12 families were clinically and molecularly diagnosed with pEDS, with C1R variants in all families. The age at molecular diagnosis ranged from 21-73 years (mean 45 years), male: female ratio 5:16. Features of easy bruising (90%), pretibial plaques (81%), skin fragility (71%), joint hypermobility (24%) and vocal changes (38%) were identified as well as leukodystrophy in 89% of those imaged. Discussion: This cohort highlights the clinical features of pEDS in adults and contributes several important additional clinical features as well as novel deleterious variants to current knowledge. Hypothetical pathogenic mechanisms which may help to progress understanding and management of pEDS are also discussed.

Synthesis and Characterization of Multi-Reducing-End Polysaccharides.

Site-specific modification is a great challenge for polysaccharide scientists. Chemo- and regioselective modification of polysaccharide chains can provide many useful natural-based materials and help us illuminate fundamental structure-property relationships of polysaccharide derivatives. The hemiacetal reducing end of a polysaccharide is in equilibrium with its ring-opened aldehyde form, making it the most uniquely reactive site on the polysaccharide molecule, ideal for regioselective decoration such as imine formation. However, all natural polysaccharides, whether they are branched or not, have only one reducing end per chain, which means that only one aldehyde-reactive substituent can be added. We introduce a new approach to selective functionalization of polysaccharides as an entrée to useful materials, appending multiple reducing ends to each polysaccharide molecule. Herein, we reduce the approach to practice using amide formation. Amine groups on monosaccharides such as glucosamine or galactosamine can react with carboxyl groups of polysaccharides, whether natural uronic acids like alginates, or derivatives with carboxyl-containing substituents such as carboxymethyl cellulose (CMC) or carboxymethyl dextran (CMD). Amide formation is assisted using the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). By linking the C2 amines of monosaccharides to polysaccharides in this way, a new class of polysaccharide derivatives possessing many reducing ends can be obtained. We refer to this class of derivatives as multi-reducing-end polysaccharides (MREPs). This new family of derivatives creates the potential for designing polysaccharide-based materials with many potential applications, including in hydrogels, block copolymers, prodrugs, and as reactive intermediates for other derivatives.

Origin, synchronization, and propagation of sleep slow waves in children.

STUDY OBJECTIVES: Sleep slow wave activity, as measured using EEG delta power (<4 Hz), undergoes significant changes throughout development, mirroring changes in brain function and anatomy. Yet, age-dependent variations in the characteristics of individual slow waves have not been thoroughly investigated. Here we aimed at characterizing individual slow wave properties such as origin, synchronization, and cortical propagation at the transition between childhood and adulthood. METHODS: We analyzed overnight high-density (256 electrodes) EEG recordings of healthy typically developing children (N = 21, 10.3 ± 1.5 years old) and young healthy adults (N = 18, 31.1 ± 4.4 years old). All recordings were preprocessed to reduce artifacts, and NREM slow waves were detected and characterized using validated algorithms. The threshold for statistical significance was set at p = 0.05. RESULTS: The slow waves of children were larger and steeper, but less widespread than those of adults. Moreover, they tended to mainly originate from and spread over more posterior brain areas. Relative to those of adults, the slow waves of children also displayed a tendency to more strongly involve and originate from the right than the left hemisphere. The separate analysis of slow waves characterized by high and low synchronization efficiency showed that these waves undergo partially distinct maturation patterns, consistent with their possible dependence on different generation and synchronization mechanisms. CONCLUSIONS: Changes in slow wave origin, synchronization, and propagation at the transition between childhood and adulthood are consistent with known modifications in cortico-cortical and subcortico-cortical brain connectivity. In this light, changes in slow-wave properties may provide a valuable yardstick to assess, track, and interpret physiological and pathological development.

A Comparison of Microstructure and Microhardness Properties of IN718 Fabricated via Powder- and Wire-Fed Laser-Directed Energy Deposition.

The objective of this work is to compare the microstructure and microhardness properties of IN718 deposited by both powder- and wire-fed laser-directed energy deposition (L-DED) processes. The powder-fed L-DED is carried out on an Optomec LENS® system while the wire-fed L-DED is performed in an in-house custom-built system. Several single-layer single-track specimens are fabricated using different combinations of process parameters to down-select the optimal process parameters for both systems. The finalized parameters are, thereafter, used to build thin-wall specimens having identical designs. The specimens are characterized using optical and electron microscopy as well as microhardness measurements. The results demonstrate that the powder-fed specimen, built using optimal process parameters, does not exhibit any distortion. On the contrary, the wire-fed specimen, built with optimal process parameters, show lesser porosity. Differences in elemental segregation are also detected in the two specimens. For example, nitrides and carbides are observed in the wire-fed specimen but not in the powder-fed specimen. The microhardness measurements reveal the powder-fed specimen has higher microhardness values compared to the wire-fed specimen. These results can be used to fabricate parts with sequential powder and wire deposition to achieve biomimetic structures of varying microstructure and microhardness properties.

The Impact of Flame Burn Injuries on Patients Who Sustain Mild Injuries From Motor Vehicle Crashes.

BACKGROUND: The purpose of this study was to determine the differences in patient outcomes between motor vehicle crash (MVC) victims with an ISS < 15 and those with a similar ISS and a flame burn injury. METHODS: Data for patients involved in a MVC with a GCS ≥12 and an ISS < 15 with and without flame burn injury were reviewed from the American College of Surgeons National Trauma Data Bank between 2007 and 2017. International Classification of Diseases-9 and -10 revisions and External Injury Codes were used to identify patients who were divided into MVC only (Group 1) and MVC with additional flame burn injury (Group 2). In-hospital mortality was the primary outcome whereas secondary outcomes included ICU admission, ICU length of stay (LOS), hospital LOS, sepsis, deep vein thrombosis, acute respiratory distress syndrome, and pneumonia. Simple linear regression was used in the form of odds ratios to investigate risk factors for mortality and secondary outcomes. RESULTS: The mean LOS and ICU LOS were longer in Group 2 (5.9 vs 4.0 days, p-value <0.001, and 1.2 vs 0.6 days, p-value <0.001, respectively), with more patients being admitted to the ICU as well (22.9% vs 17.3%, p-value <0.001). Also, there were significantly higher rates of pneumonia (0.8% vs 0.5%, p-value 0.0014), deep vein thrombosis (0.6% vs 0.4%, p-value 0.028), and acute respiratory distress syndrome (0.5% vs 0.3%, p-value 0.004) in Group 2. Patients in Group 1 were older and more likely had hypertension, congestive heart failure, and COPD. There was no significant difference in mortality between Groups by odds ratios (OR 0.85, p-value 0.743) or raw percentages (0.3% vs 0.3%, p-value = 0.874). CONCLUSION: MVC victims with mild injuries who also sustain a burn injury are more likely to require admission to the ICU regardless of their comorbidities and more likely to develop respiratory complications, especially pneumonia and an increase in ICU and hospital LOS.

Postpartum Depression in a Community Sample of American Indian Mothers.

American Indian (AI) mothers experience high rates of postpartum depression (PPD). We evaluated the factor structure of the Edinburgh Postnatal Depression Scale (EPDS) among AI mothers from a rural AI serving health system. We also investigated potential associations between EPDS scores and selected psychosocial factors (n = 315). Exploratory Factor Analysis (n = 157) showed that a one-factor structure best fits the data. A Confirmatory Factor Analysis was then conducted to examine the fit of the one-factor model (n = 158). Goodness-of-fit statistics showed overall poor model fit (RMSEA = .13) which may be suggestive of an indicator of depression among Natives not detected by the EPDS. Results of the multiple regression analysis were non-significant. The findings demonstrated that while the EPDS measured aspects of PPD, there may be additional aspects of depression specific to the AI women in our sample not captured by the EPDS. Limitations and directions for future research are discussed.

Five recommendations for using large-scale publicly available data to advance health among American Indian peoples: the Adolescent Brain and Cognitive Development (ABCD) StudySM as an illustrative case.

American Indian and Alaska Native (AIAN) populations have suffered a history of exploitation and abuse within the context of mental health research and related fields. This history is rooted in assimilation policies, historical trauma, and cultural loss, and is promulgated through discrimination and disregard for traditional culture and community knowledge. In recognition of this history, it is imperative for researchers to utilize culturally sensitive approaches that consider the context of tribal communities to better address mental health issues for AIAN individuals. The public availability of data from large-scale studies creates both opportunities and challenges when studying mental health within AIAN populations. This manuscript has two goals; first, showcase an example of problematic use of Adolescent Brain Cognitive Development (ABCD) StudySM data to promulgate stereotypes about AIAN individuals and, second, in partnership with collaborators from Cherokee Nation, we provide five recommendations for utilizing data from publicly available datasets to advance health research in AIAN populations. Specifically, we argue for the consideration of (1) the heterogeneity of the communities represented, (2) the importance of focusing on AIAN health and well-being, (3) engagement of relevant communities and AIAN community leaders, (4) consideration of historical and ongoing injustices, and (5) engagement with AIAN regulatory agencies or review boards. These recommendations are founded on principles from broader indigenous research efforts emphasizing community-engaged research and principles of Indigenous Data Sovereignty and Governance.

Evaluation of consent forms for clinical practice in Spanish Public Hospitals.

OBJECTIVE: To evaluate the access, development, and quality of consents forms for clinical practice within the Spanish Public Hospitals. METHOD: A cross-sectional study was conducted in a two-stage process (January 2018-September 2021). In stage 1, A nationwide survey was undertaken across all public general hospitals (n=223) in the Spanish Healthcare System. In stage 2, Data was taken from the regional health services websites and Spanish regulations. Health Regional Departments were contacted to verify the accuracy of the findings. Data was analyzed using a descriptive and inferential statistics (frequencies, percentages, Chi-square & Fisher's exact tests). RESULTS: The response rate was 123 (55.16%) of Spanish Public Hospitals. The results revealed a range of hospital departments involved in the development of consent documents and the absence of a standardized approach to consent forms nationally. Consent audits are undertaken in 43.09% hospitals and translation of written consents into other languages is limited to a minority of hospitals (35.77%). The validation process of consent documentation is not in evidence in 13% of Spanish Hospitals. Regional Informed Consent Committees are not place in the majority (70.7%) of hospitals. Citizens can freely access to consent documents through the regional websites of Andalusia and Valencia only. CONCLUSION: Variability is found on access, development and quality of written consent across the Spanish Public Hospitals. This points to the need for a national informed consent strategy to establish policy, standards and an effective quality control system. National audits at regular intervals are necessary to improve the consistency and compliance of consent practice.

Ventilator Management in Extremely Preterm Infants.

Advances in ventilation strategies for infants in the NICU have led to increased survival of extremely preterm infants. More than 75% of infants born at less than or equal to 27 weeks' gestation require initial mechanical ventilation for survival due to developmental immaturity of their lungs and respiratory drive. Various ventilators using different technologies and involving multiple management strategies are available for use in this population. Centers across the world have successfully used conventional, high-frequency oscillatory and high-frequency jet ventilation to manage respiratory failure in extremely preterm infants. This review explores the existing evidence for each mode of ventilation and the importance of individualizing ventilator management strategies when caring for extremely preterm infants.

Two is better than one: Coupling DNA metabarcoding and stable isotope analysis improves dietary characterizations for a riparian-obligate, migratory songbird.

While an increasing number of studies are adopting molecular and chemical methods for dietary characterization, these studies often employ only one of these laboratory-based techniques; this approach may yield an incomplete, or even biased, understanding of diet due to each method's inherent limitations. To explore the utility of coupling molecular and chemical techniques for dietary characterizations, we applied DNA metabarcoding alongside stable isotope analysis to characterize the dietary niche of breeding Louisiana waterthrush (Parkesia motacilla), a migratory songbird hypothesized to preferentially provision its offspring with pollution-intolerant, aquatic arthropod prey. While DNA metabarcoding was unable to determine if waterthrush provision aquatic and terrestrial prey in different abundances, we found that specific aquatic taxa were more likely to be detected in successive seasons than their terrestrial counterparts, thus supporting the aquatic specialization hypothesis. Our isotopic analysis added greater context to this hypothesis by concluding that breeding waterthrush provisioned Ephemeroptera and Plecoptera, two pollution-intolerant, aquatic orders, in higher quantities than other prey groups, and expanded their functional trophic niche when such prey were not abundantly provisioned. Finally, we found that the dietary characterizations from each approach were often uncorrelated, indicating that the results gleaned from a diet study can be particularly sensitive to the applied methodologies. Our findings contribute to a growing body of work indicating the importance of high-quality, aquatic habitats for both consumers and their pollution-intolerant prey, while also demonstrating how the application of multiple, laboratory-based techniques can provide insights not offered by either technique alone.

Large-scale intramuscular electrode system for chronic electromyography and functional electrical stimulation.

To understand how the central nervous system (CNS) enacts movements, it seems important to monitor the activities of the many muscles involved. Likewise, to restore complex movements to paralyzed limbs with electrical stimulation requires access to most limb muscles. Intramuscular electrodes are needed to obtain isolated recordings or stimulation of individual muscles. As such, we developed and tested the stability of large arrays of implanted intramuscular electrodes. We implanted 58 electrodes in 29 upper limb muscles in each of three macaques. Electrode connectors were protected within a skull-mounted chamber. During surgery, wires were tunneled subcutaneously to target muscles, where gold anchors were crimped onto the leads. The anchors were then deployed with an insertion device. In two monkeys, the chamber was fixed to the skull with a titanium baseplate rather than acrylic cement. In multiple sessions up to 15 wk after surgery, electromyographic (EMG) signals were recorded while monkeys made the same reaching movement. EMG signals were stable, with an average (SD) coefficient of variation across sessions of 0.24 ± 0.15. In addition, at 4, 8, and 16 wk after surgery, forces to incrementing stimulus pulses were measured for each electrode. The threshold current needed to evoke a response at 16 wk was not different from that at 4 wk. Likewise, peak force evoked by 16 mA of current at 16 wk was not different from 4 wk. The stability of this system implies it could be effectively used to monitor and stimulate large numbers of muscles needed to understand the control of natural and evoked movements.NEW AND NOTEWORTHY A new method was developed to enable long-lasting recording and stimulation of large numbers of muscles with intramuscular electrodes. Electromyographic signals and evoked force responses in 29 upper limb muscles remained stable over several months when tested in nonhuman primates. This system could be used effectively to monitor and stimulate numerous muscles needed to more fully understand the control of natural and evoked movements.

Analysis of rare disruptive germline mutations in 2135 enriched BRCA-negative breast cancers excludes additional high-impact susceptibility genes.

BACKGROUND: Breast cancer has a significant heritable basis, of which ∼60% remains unexplained. Testing for BRCA1/BRCA2 offers useful discrimination of breast cancer risk within families, and identification of additional breast cancer susceptibility genes could offer clinical utility. PATIENTS AND METHODS: We included 2135 invasive breast cancer cases recruited via the Breast and Ovarian Cancer Susceptibility study, a retrospective UK study of familial breast cancer. ELIGIBILITY CRITERIA: female, BRCA-negative, white European ethnicity, and one of: (i) breast cancer family history, (ii) bilateral disease, (iii) young age of onset (<30 years), and (iv) concomitant ovarian cancer. We undertook exome sequencing of cases and carried out gene-level burden testing of rare damaging variants against those from 51 377 ethnicity-matched population controls from gnomAD. RESULTS: 159/2135 (7.4%) cases had a qualifying variant in an established breast cancer susceptibility gene, with minimal evidence of signal in other cancer susceptibility genes. Known breast cancer susceptibility genes PALB2, CHEK2, and ATM were the only genes to retain statistical significance after correcting for multiple testing. Due to the enrichment of hereditary cases in the series, we had good power (>80%) to detect a gene of BRCA1-like risk [odds ratio (OR) = 10.6] down to a population minor allele frequency of 4.6 × 10-5 (1 in 10 799, less than one-tenth that of BRCA1)and of PALB2-like risk (OR = 5.0) down to a population minor allele frequency of 2.8 × 10-4 (1 in 1779, less than half that of PALB2). Power was lower for identification of novel moderate penetrance genes (OR = 2-3) like CHEK2 and ATM. CONCLUSIONS: This is the largest case-control whole-exome analysis of enriched breast cancer published to date. Whilst additional breast cancer susceptibility genes likely exist, those of high penetrance are likely to be of very low mutational frequency. Contention exists regarding the clinical utility of such genes.

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.

How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids.

The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.