Repurposing AZD5438 and Dabrafenib for Cisplatin-Induced AKI.

SIGNIFICANCE STATEMENT: To combat both untoward effects of nephrotoxicity and ototoxicity in cisplatin-treated patients, two potential therapeutic oral anticancer drugs AZD5438 and dabrafenib, a phase-2 clinical trial protein kinase CDK2 inhibitor and an US Food and Drug Administration-approved drug BRAF inhibitor, respectively, were tested in an established mouse AKI model. Both drugs have previously been shown to protect significantly against cisplatin-induced hearing loss in mice. Each drug ameliorated cisplatin-induced increases in the serum biomarkers BUN, creatinine, and neutrophil gelatinase-associated lipocalin. Drugs also improved renal histopathology and inflammation, mitigated cell death by pyroptosis and necroptosis, and significantly enhanced overall survival of cisplatin-treated mice. BACKGROUND: Cisplatin is an effective chemotherapy agent for a wide variety of solid tumors, but its use is dose-limited by serious side effects, including AKI and hearing loss. There are no US Food and Drug Administration-approved drugs to treat both side effects. Recently, two anticancer oral drugs, AZD5438 and dabrafenib, were identified as protective against cisplatin-induced hearing loss in mice. We hypothesize that similar cell stress and death pathways are activated in kidney and inner ear cells when exposed to cisplatin and tested whether these drugs alleviate cisplatin-induced AKI. METHODS: The HK-2 cell line and adult FVB mice were used to measure the protection from cisplatin-induced cell death and AKI by these drugs. Serum markers of kidney injury, BUN, creatinine, and neutrophil gelatinase-associated lipocalin as well as histology of kidneys were analyzed. The levels of markers of kidney cell death, including necroptosis and pyroptosis, pERK, and proliferating cell nuclear antigen, were also examined by Western blotting and immunofluorescence. In addition, CDK2 knockout (KO) mice were used to confirm AZD5438 protective effect is through CDK2 inhibition. RESULTS: The drugs reduced cisplatin-induced cell death in the HK-2 cell line and attenuated cisplatin-induced AKI in mice. The drugs reduced serum kidney injury markers, inhibited cell death, and reduced the levels of pERK and proliferating cell nuclear antigen, all of which correlated with prolonged animal survival. CDK2 KO mice were resistant to cisplatin-induced AKI, and AZD5438 conferred no additional protection in the KO mice. CONCLUSIONS: Cisplatin-induced damage to the inner ear and kidneys shares similar cellular beneficial responses to AZD5438 and dabrafenib, highlighting the potential therapeutic use of these agents to treat both cisplatin-mediated kidney damage and hearing loss.

Computational Prediction of Structure, Function, and Interaction of Myzus persicae (Green Peach Aphid) Salivary Effector Proteins.

Similar to plant pathogens, phloem-feeding insects such as aphids deliver effector proteins inside their hosts that act to promote host susceptibility and enable feeding and infestation. Despite exciting progress toward identifying and characterizing effector proteins from these insects, their functions remain largely unknown. The recent groundbreaking development in protein structure prediction algorithms, combined with the availability of proteomics and transcriptomic datasets for agriculturally important pests, provides new opportunities to explore the structural and functional diversity of effector repertoires. In this study, we sought to gain insight into the infection strategy used by the Myzus persicae (green peach aphid) by predicting and analyzing the structures of a set of 71 effector candidate proteins. We used two protein structure prediction methods, AlphaFold and OmegaFold, that produced mutually consistent results. We observed a wide continuous spectrum of structures among the effector candidates, from disordered proteins to globular enzymes. We made use of the structural information and state-of-the-art computational methods to predict M. persicae effector protein properties, including function and interaction with host plant proteins. Overall, our investigation provides novel insights into prediction of structure, function, and interaction of M. persicae effector proteins and will guide the necessary experimental characterization to address new hypotheses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Synthetic and biological surfactant effects on freshwater biofilm community composition and metabolic activity.

Surfactants are used to control microbial biofilms in industrial and medical settings. Their known toxicity on aquatic biota, and their longevity in the environment, has encouraged research on biodegradable alternatives such as rhamnolipids. While previous research has investigated the effects of biological surfactants on single species biofilms, there remains a lack of information regarding the effects of synthetic and biological surfactants in freshwater ecosystems. We conducted a mesocosm experiment to test how the surfactant sodium dodecyl sulfate (SDS) and the biological surfactant rhamnolipid altered community composition and metabolic activity of freshwater biofilms. Biofilms were cultured in the flumes using lake water from Lake Lunz in Austria, under high (300 ppm) and low (150 ppm) concentrations of either surfactant over a four-week period. Our results show that both surfactants significantly affected microbial diversity. Up to 36% of microbial operational taxonomic units were lost after surfactant exposure. Rhamnolipid exposure also increased the production of the extracellular enzymes, leucine aminopeptidase, and glucosidase, while SDS exposure reduced leucine aminopeptidase and glucosidase. This study demonstrates that exposure of freshwater biofilms to chemical and biological surfactants caused a reduction of microbial diversity and changes in biofilm metabolism, exemplified by shifts in extracellular enzyme activities. KEY POINTS: • Microbial biofilm diversity decreased significantly after surfactant exposure. • Exposure to either surfactant altered extracellular enzyme activity. • Overall metabolic activity was not altered, suggesting functional redundancy.

Is Preoperative Serum Albumin Predictive of Adverse Surgical Outcomes in Maxillofacial Fracture Repair?

PURPOSE: Malnutrition has been recognized as a predictor of postoperative adverse outcomes across many surgical subspecialties. The purpose of this study was to evaluate the relationship between serum albumin and adverse outcomes in patients undergoing operative repair of maxillofacial fractures. METHODS: The authors utilized the 2011 to 2018 American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) databases to identify patients with facial fractures undergoing operative repair. The primary predictor variable was preoperative serum albumin level. Outcome variables included complications and other adverse outcomes occurring within 30 days of the index operation. Descriptive, bivariate, and multiple logistic regression statistics were utilized to evaluate the relationship between serum albumin and adverse outcomes. RESULTS: During the study period 1211 subjects underwent operative repair of a facial fracture and had a documented serum albumin level. Of these subjects, 1037 (85.6%) had normal albumin levels and 174 (14.4%) had hypoalbuminemia. A total of 90 subjects experienced a complication (7.43%), although albumin level was not associated with surgical complications or any complication. In bivariate analysis, subjects with hypoalbuminemia were significantly more likely to have an extended length of stay (P ≤ .001), adverse discharge disposition (P ≤ .001), and be readmitted (P = .002). In multivariate analysis, hypoalbuminemia was an independent predictor of an extended length of stay (P ≤ .001, 95% CI 2.50 to 7.62), adverse discharge disposition (P = .048, 95% CI 1.01 to 3.75), and readmission (P = .041, 95% CI 1.03 to 3.47). CONCLUSIONS: Serum albumin was not an independent predictor of complications after maxillofacial trauma repair. However, it was an independent predictor of other adverse outcomes including extended length of stay, adverse discharge disposition, and readmission. Targeted nutritional optimization may represent an opportunity to improve outcomes in this demographic.

Does the Modified Frailty Index (mFI-5) Predict Adverse Outcomes in Maxillofacial Fracture Repair?

PURPOSE: Frailty has been recognized as a predictor of postoperative adverse outcomes in many surgical subspecialties. The purpose of this study was to evaluate the relationship between frailty and complications in patients undergoing operative repair of facial fractures. METHODS: The authors utilized the 2011 to 2018 American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) databases to identify patients with facial fractures undergoing operative repair. The primary predictor variable was frailty as measured by the 5-Factor Modified Frailty Index (mFI-5). The primary outcome variable was the postoperative complication rate. Descriptive, bivariate, and multiple logistic regression statistics were utilized to evaluate the relationship between frailty and complications. RESULTS: During the study period, 4,290 subjects underwent operative repair of a facial fracture. Of these subjects, 4,086 (83.0%) were classified as nonfrail, 626 (12.7%) as moderately frail, and 208 (4.20%) as severely frail. A total of 237 subjects experienced a complication (4.82%), and the incidence of complications increased in a stepwise manner with increasing frailty (P ≤ .001). In multivariate regression, age (P = .050, 95% CI = 1.00 to 1.02), Native Hawaiian/Pacific Islander race (P = .018, 95% CI = 1.23 to 8.63), classification as moderately frail (P = .010, 95% CI = 1.15 to 2.66), classification as severely frail (P = .032, 95% CI = 1.06 to 3.70), mandibular fractures (P = .004, 95% CI = 1.24 to 2.98), and wound classification as contaminated (P ≤ .001, 95% CI = 1.53 to 4.57) or dirty/infected (P = .020, 95% CI = 1.16 to 5.55) were independent predictors of complications. Severely frail subjects also had greater length of hospital admission (P ≤ .001) and higher 30-day readmission rates (P ≤ .001). CONCLUSIONS: Frailty is an independent predictor of complications following facial fracture repair and is associated with greater length of hospital admission and 30-day readmission rates.

Females Have Worse Overall and Disease-Specific Survival in Human Papillomavirus-Negative Oropharyngeal Squamous Cell Carcinoma.

PURPOSE: Although sex (male vs female) has been identified as an independent prognostic factor in human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC), the role of sex in HPV-negative OPSCC is less understood. The purpose of this study is to measure the association between sex and HPV-negative OPSCC disease-specific survival (DSS) and overall survival (OS). METHODS: This longitudinal, retrospective study examined cases of HPV-negative OPSCC diagnosed in the United States between 2013 and 2016 from the Surveillance, Epidemiology, and End Results database. Patients with primary OPSCC and known HPV-negative status were included. Those with HPV-positive or unknown status and primary lesions located outside the oropharynx were excluded. The primary predictor variable was patient sex (male vs female). Primary outcome variables of interest included DSS and OS. The following patient-level covariates were also assessed: age, race, insurance status, primary anatomical site and histological type of lesion, histologic grade and stage, and disease outcome. A survival analysis was conducted using univariate and multivariate analyses via a cox proportional hazard regression model. An α value less than 0.05 was considered statistically significant. RESULTS: The study sample consisted of 2,565 cases (25.1% female) of HPV-negative OPSCC. Females presented with lower histologic grade (P = .015) and earlier stage (P = .003). Females demonstrated worse DSS (P < .001) and OS (P < .001). After multivariate adjustment, female sex (hazard ratio [HR] = 1.38; 95% confidence interval [CI], 1.13 to 1.67; P = .002), advanced age (HR = 1.672; 95% CI, 1.07 to 2.60; P = .023), advanced overall stage (HR = 4.69; 95% CI, 1.54 to 14.267; P = .006), TNM stage (T4: HR = 5.74; 95% CI, 3.86 to 8.55, P < .001, N3: HR = 3.48; 95% CI, 2.17 to 5.58; P < .001, and M1: HR = 2.80; 95% CI, 2.09 to 3.74; P < .001), subjects residing in counties with the highest rates of smoking (HR = 1.29; 95% CI, 1.01 to 1.65; P = .044), and the lack of surgical treatment in patients treated with radiation and/or chemotherapy (HR = 1.44; 95% CI, 1.08 to 1.91; P = .012) were correlated with poorer DSS and OS. CONCLUSION: Females with HPV-negative OPSCC demonstrated worse DSS and OS despite better typical prognostic signs such as histologic grade and clinical stage.

A Cadaveric Study Addressing the Feasibility of Remote Patient Monitoring Prosthesis for Total Knee Arthroplasty.

BACKGROUND: Since the COVID-19 pandemic of 2020, there has been a marked rise in the use of telemedicine to evaluate patients after total knee arthroplasty (TKA). The purpose of our study was to assess a novel stem with an embedded sensor that can remotely and objectively monitor a patient's mobility after TKA. METHODS: A single anatomically designed knee system was implanted in concert with an interconnected tibial stem extension containing 3D accelerometers, 3D gyroscopes, a power source, and a telemetry transmission capability in 3 cadaveric pelvis to toe specimens. The legs were moved by hand to preset tibial positions at full knee extension, midflexion, flexion, and back to midflexion and extension for a total of 16 trials across 6 knees. RESULTS: Sensor data were successfully transmitted with good quality of signal to an external base station. Good correlation to the range of motion of the tibia was found (mean error 0.1 degrees; root mean square error 3.8 degrees). The signal from the heel drop tests suggests the sensor could detect heel strike during activities of daily living in vivo and the potential for additional signal processing to analyze vibratory and motion patterns detected by the sensors. A frequency domain analysis of a properly cemented and poorly cemented implant during the heel drop test suggests a difference in accelerometer signal in these implant states. CONCLUSION: The results confirm signals generated from an embedded TKA sensor can transmit through bone and cement, providing accurate range of motion data and may be capable of detecting changes in prosthesis fixation remotely.

Improving species status assessments under the U.S. Endangered Species Act and implications for multispecies conservation challenges worldwide.

Despite its successes, the U.S. Endangered Species Act (ESA) has proven challenging to implement due to funding limitations, workload backlog, and other problems. As threats to species survival intensify and as more species come under threat, the need for the ESA and similar conservation laws and policies in other countries to function efficiently has grown. Attempts by the U.S. Fish and Wildlife Service (USFWS) to streamline ESA decisions include multispecies recovery plans and habitat conservation plans. We address species status assessment (SSA), a USFWS process to inform ESA decisions from listing to recovery, within the context of multispecies and ecosystem planning. Although existing SSAs have a single-species focus, ecosystem-based research can efficiently inform multiple SSAs within a region and provide a foundation for transition to multispecies SSAs in the future. We considered at-risk grassland species and ecosystems within the southeastern United States, where a disproportionate number of rare and endemic species are associated with grasslands. To initiate our ecosystem-based approach, we used a combined literature-based and structured World Café workshop format to identify science needs for SSAs. Discussions concentrated on 5 categories of threats to grassland species and ecosystems, consistent with recommendations to make shared threats a focus of planning under the ESA: (1) habitat loss, fragmentation, and disruption of functional connectivity; (2) climate change; (3) altered disturbance regimes; (4) invasive species; and (5) localized impacts. For each threat, workshop participants identified science and information needs, including database availability, research priorities, and modeling and mapping needs. Grouping species by habitat and shared threats can make the SSA process and other planning processes for conservation of at-risk species worldwide more efficient and useful. We found a combination of literature review and structured discussion effective for identifying the scientific information and analysis needed to support the development of multiple SSAs. Article impact statement: Species status assessments can be improved by an ecosystem-based approach that groups imperiled species by shared habitats and threats.

Mejoramiento de la Evaluación del Estado de una Especie bajo el Acta de Especies en Peligro de los Estados Unidos y Sus Consecuencias para los Retos de la Conservación Multiespecie a Nivel Mundial Resumen A pesar de su éxito, el Acta de Especies en Peligro de los E.U.A. (AEP) ha sido un reto de implementación por las limitaciones en su financiamiento, el retraso en la carga de trabajo y otros problemas. Conforme se intensifican las amenazas a la supervivencia de las especies y más especies resultan amenazadas, aumenta la necesidad de que la AEP y las políticas similares de otros países funcionen efectivamente. Los intentos por parte del Servicio Estadounidense de Pesca y Fauna (SEPF) para optimizar las decisiones de la AEP incluyen planes multiespecie de recuperación y planes de conservación de hábitat (PRH). Abordamos la evaluación del estado de las especies (EEE), un proceso del SEPF para orientar las decisiones del AEP desde el listado hasta la recuperación, dentro del contexto de la planeación multiespecie y de ecosistemas. Aunque las EEE existentes tienen un enfoque sobre una única especie, la investigación basada en el ecosistema puede orientar eficientemente a múltiples EEE dentro de una región y proporcionar una base para la transición a las EEE multiespecie en el futuro. Consideramos a las especies y los ecosistemas en riesgo de los pastizales del sureste de los Estados Unidos, en donde un número desproporcionado de especies raras y endémicas está asociado con los pastizales. Para iniciar nuestra estrategia basada en el ecosistema, usamos un formato de taller de World Café estructurado y basado en la literatura para identificar la necesidad de tener EEE. Las discusiones se centraron en cinco categorías de amenazas para las especies y ecosistemas de los pastizales, consistentes con las recomendaciones para volver a las amenazas compartidas un foco de la planeación bajo la AEP: (1) pérdida del hábitat, fragmentación y disrupción de la conectividad funcional; (2) cambio climático; (3) regímenes alterados de perturbación; (4) especies invasoras; y (5) impactos localizados. Para cada amenaza, los participantes del taller identificaron las necesidades científicas y de información, incluyendo la disponibilidad de bases de datos, prioridades de la investigación y necesidades de modelado y mapeado. La agrupación de las especies por hábitat y amenaza compartida puede hacer más eficientes y útiles el proceso de EEE y otros procesos de planeación de la conservación de especies en riesgo a nivel mundial. Encontramos una combinación de revisiones bibliográficas y discusiones estructuradas para identificar la información y el análisis necesarios para respaldar el desarrollo de múltiples EEE.

Using the Tea Bag Index to determine how two human pharmaceuticals affect litter decomposition by aquatic microorganisms.

This study demonstrates that independent additive effects of two human pharmaceuticals, the antibiotic trimethoprim and the artificial estrogen 17a-Ethinylestradiol (EE2), inhibit plant litter decomposition by aquatic microorganisms. The constant release of pharmaceuticals, such as these, has the potential to affect aquatic microbial metabolism and alter biogeochemical cycling of carbon and nutrients. Here we advance the Tea Bag Index (TBI) for decomposition by using it in a series of contaminant exposure experiments testing how interactions between trimethoprim and EE2 affect aquatic microbial activity. The TBI is a citizen science tool used to test microbial activity by measuring the differential degradation of green and rooibos tea as proxies for respectively labile and recalcitrant litter decomposition. Exposure to either trimethoprim or EE2 decreased decomposition of green tea, suggesting additive effects upon microbial activity. Exposure to EE2 alone decreased rooibos tea decomposition. Consequently, trimethoprim and EE2 stabilized labile organic matter against microbial degradation and restricted decomposition. We propose that the method outlined could provide a powerful tool for testing the impacts of multiple interacting pollutants upon microbial activity, at a range of scales, across aquatic systems and over ecologically relevant time scales.

Amino acid substitutions in the human homomeric ß3 GABAA receptor that enable activation by GABA.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α1ß2γ2 receptor is the major subtype in the brain; GABA binds at the ß2(+)α1(-) interface. The structure of the homomeric ß3 GABAAR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ß3(+)α1(-) heteromeric interface in the homomeric human ß3 GABAAR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ß3 GABAAR variants containing Y87F, Q89R, and G152T. Reversion of Phe87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABAARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ß3 GABAAR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr87 prevents inhibitory effects of GABA.

A Structural Rationale for N-Methylbicuculline Acting as a Promiscuous Competitive Antagonist of Inhibitory Pentameric Ligand-Gated Ion Channels.

Bicuculline, a valued chemical tool in neurosciences research, is a competitive antagonist of specific GABAA receptors and affects other pentameric ligand-gated ion channels including the glycine, nicotinic acetylcholine and 5-hydroxytryptamine type 3 receptors. We used a fluorescence-quenching assay and isothermal titration calorimetry to record low-micromolar dissociation constants for N-methylbicuculline interacting with acetylcholine-binding protein and an engineered version called glycine-binding protein (GBP), which provides a surrogate for the heteromeric interface of the extracellular domain of the glycine receptor (GlyR). The 2.4 Šresolution crystal structure of the GBP:N-methylbicuculline complex, sequence and structural alignments reveal similarities and differences between GlyR and the GABAA receptor-bicuculline interactions. N-methylbicuculline displays a similar conformation in different structures, but adopts distinct orientations enforced by interactions and steric blocks with key residues and plasticity in the binding sites. These features explain the promiscuous activity of bicuculline against the principal inhibitory pentameric ligand-gated ion channels in the CNS.

Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens.

The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high-resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a zinc-containing metalloenzyme, and in vitro assays demonstrate that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp-120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.

Open and compressed conformations of Francisella tularensis ClpP.

Caseinolytic proteases are large oligomeric assemblies responsible for maintaining protein homeostasis in bacteria and in so doing influence a wide range of biological processes. The functional assembly involves three chaperones together with the oligomeric caseinolytic protease catalytic subunit P (ClpP). This protease represents a potential target for therapeutic intervention in pathogenic bacteria. Here, we detail an efficient protocol for production of recombinant ClpP from Francisella tularensis, and the structural characterization of three crystal forms which grow under similar conditions. One crystal form reveals a compressed state of the ClpP tetradecamer and two forms an open state. A comparison of the two types of structure infers that differences at the enzyme active site result from a conformational change involving a highly localized disorder-order transition of a ß-strand α-helix combination. This transition occurs at a subunit-subunit interface. Our study may now underpin future efforts in a structure-based approach to target ClpP for inhibitor or activator development. Proteins 2016; 85:188-194. © 2016 Wiley Periodicals, Inc.

Can Religion Protect Against Suicide?

The vast majority of the world's population is affiliated with a religious belief structure, and each of the major faith traditions (in its true form) is strongly opposed to suicide. Ample literature supports the protective effect of religious affiliation on suicide rates. Proposed mechanisms for this protective effect include enhanced social network and social integration, the degree of religious commitment, and the degree to which a particular religion disapproves of suicide. We review the sociological data for these effects and the general objections to suicide held by the faith traditions. We explore how clinicians may use such knowledge with individual patients, including routinely taking a religious/spiritual history. The clinician who is aware of the common themes among the faith traditions in opposition to suicide is better prepared to address religious/spiritual matters, as appropriate, in crisis situations. The clinician who understands the patient's belief system is also better prepared to request consultation with religious professionals when indicated.

EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system.

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1-D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein.

Depth of Interaction Calibration and Capabilities in 2×2 Discrete Crystal Arrays and Digital Silicon Photomultipliers.

Digital silicon photomultiplers (dSiPMs) have potential in the advancement of PET detectors. Their advantages include decreased dark counts through selective microcell activation, fast timing, and flexibility configuring event triggering and collection. Further improvements in PET image resolution are possible when photon depth of interaction (DOI) is available, as this reduces parallax error caused by mispositioning events at the peripheral field of view. These improvements are desirable in smaller ring diameter PET systems, such as whole body PET/MRI. In this study we quantify the DOI capabilities of a unique crystal array design (termed dual light sharing arrays or DLSA) that takes advantage of the 2-by-2-pixel die readout logic of a PDPC dSiPM (Philips Digital Photon Counting 3200) device by Philips Medical Systems. The DLSA is comprised of a 2×2 array of 4×4×22 mm3 LYSO crystals; inter-crystal surfaces were optically coupled in part with high-index optical adhesive and optically isolated in complimentary parts with mirror-film reflector such that light sharing was depth-dependent and different along two axes. The DLSA was mounted to one die of a PDPC and its depth-dependent response to 511-keV gamma rays was calibrated using a coincidence-collimated beam from both side and entrance surfaces. Entrance surface DOI calibration was performed through an iterative application of maximum likelihood calculations based on the signal ratio in crystals adjacent to the crystal of interaction. Results showed timing resolutions of 350-370 ps and energy resolutions of 10-12% while achieving a DOI position estimation of 6-7 mm FWHM. Significant improvements in depth estimation error were found when using maximum likelihood estimation and 3-4 depth bins. Furthermore, similar calibration results were obtained for both side-surface and entrance-surface illumination methods, which suggest that PET system calibrations may be easily performed using a monoenergetic flood source with entrance surface illumination.

Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile.

Tryptophan is an important precursor for chemical entities that ultimately support the biosynthesis of key metabolites. The second stage of tryptophan catabolism is catalysed by kynurenine formamidase, an enzyme that is different between eukaryotes and prokaryotes. In the present study, we characterize the catalytic properties and present the crystal structures of three bacterial kynurenine formamidases. The structures reveal a new amidase protein fold, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site. The structure of a complex with 2-aminoacetophenone delineates aspects of molecular recognition extending to the observation that the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing. The cations occupy a crowded environment, and, unlike most Zn2+-dependent enzymes, there is little scope to increase co-ordination number during catalysis. We propose that the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism.

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase.

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function.