Differential, Phosphorylation Dependent Trafficking of AQP2 in LLC-PK1 Cells

The kidney maintains water homeostasis by modulating aquaporin 2 (AQP2) on the plasma membrane of collecting duct principal cells in response to vasopressin (VP). VP mediated phosphorylation of AQP2 at serine 256 is critical for this effect. However, the role of phosphorylation of other serine residues in the AQP2 C-terminus is less well understood. Here, we examined the effect of phosphorylation of S256, S261 and S269 on AQP2 trafficking and association with recycling pathway markers. We used LLC-PK1 cells expressing AQP2(S-D) or (S-A) phospho mutants and a 20°C cold block, which allows endocytosis to continue, but prevents protein exit from the trans Golgi network (TGN), inducing formation of a perinuclear AQP2 patch. AQP2-S256D persists on the plasma membrane during cold block, while wild type AQP2, AQP2-S256A, S261A, S269A and S269D are internalized and accumulate in the patch. Development of this patch, a measure of AQP2 internalization, was most rapid with AQP2-S256A, and slowest with S261A and S269D. AQP2-S269D exhibited a biphasic internalization profile with a significant amount not internalized until 150 minutes of cold block. After rewarming to 37°C, wt AQP2, AQP2-S261A and AQP2-S269D rapidly redistributed throughout the cytoplasm within 20 minutes, whereas AQP2-S256A dissipated more slowly. Colocalization of AQP2 mutants with several key vesicular markers including clathrin, HSP70/HSC70, EEA, GM130 and Rab11 revealed no major differences. Overall, our data provide evidence supporting the role of S256 and S269 in the maintenance of AQP2 at the cell surface and reveal the dynamics of internalization and recycling of differentially phosphorylated AQP2 in cell culture

Genomic Characterization of Patient-Derived Xenograft Models Established from Fine Needle Aspirate Biopsies of a Primary Pancreatic Ductal Adenocarcinoma and from Patient-Matched Metastatic Sites

N-of-1 trials target actionable mutations, yet such approaches do not test genomically-informed therapies in patient tumor models prior to patient treatment. To address this, we developed patient-derived xenograft (PDX) models from fine needle aspiration (FNA) biopsies (FNA-PDX) obtained from primary pancreatic ductal adenocarcinoma (PDAC) at the time of diagnosis. Here, we characterize PDX models established from one primary and two metastatic sites of one patient. We identified an activating KRAS G12R mutation among other mutations in these models. In explant cells derived from these PDX tumor models with a KRAS G12R mutation, treatment with inhibitors of CDKs (including CDK9) reduced phosphorylation of a marker of CDK9 activity (phospho-RNAPII CTD Ser2/5) and reduced viability/growth of explant cells derived from PDAC PDX models. Similarly, a CDK inhibitor reduced phospho-RNAPII CTD Ser2/5, increased apoptosis, and inhibited tumor growth in FNA-PDX and patient-matched metastatic-PDX models. In summary, PDX models can be constructed from FNA biopsies of PDAC which in turn can enable genomic characterization and identification of potential therapies

Integrative structural biology of the penicillin-binding protein-1 from Staphylococcus aureus, an essential component of the divisome machinery

14 pags., 6 figs., 2 tabs.The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the “penicillin-binding protein and serine/threonine kinase associated” (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of saPBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.The work in the USA was supported by grants AI104987 (to SM)and AI116548 (to MC) from the NIH. The work in Spain was fundedby a grant from the Spanish Ministry of Science, Innovation and Competitiveness (BFU2017-90030-P and PID2020-115331GB-100 to JAH). We thank the staff from ALBA and Diamond Light Sourcesynchrotrons for help during X-ray and SAXS data collection,respectivel

Discovery of antibiotic (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3 H)-one

© 2015 American Chemical Society. In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.Peer Reviewe

Etiology of Severe Non-malaria Febrile Illness in Northern Tanzania: A Prospective Cohort Study.

The syndrome of fever is a commonly presenting complaint among persons seeking healthcare in low-resource areas, yet the public health community has not approached fever in a comprehensive manner. In many areas, malaria is over-diagnosed, and patients without malaria have poor outcomes. We prospectively studied a cohort of 870 pediatric and adult febrile admissions to two hospitals in northern Tanzania over the period of one year using conventional standard diagnostic tests to establish fever etiology. Malaria was the clinical diagnosis for 528 (60.7%), but was the actual cause of fever in only 14 (1.6%). By contrast, bacterial, mycobacterial, and fungal bloodstream infections accounted for 85 (9.8%), 14 (1.6%), and 25 (2.9%) febrile admissions, respectively. Acute bacterial zoonoses were identified among 118 (26.2%) of febrile admissions; 16 (13.6%) had brucellosis, 40 (33.9%) leptospirosis, 24 (20.3%) had Q fever, 36 (30.5%) had spotted fever group rickettsioses, and 2 (1.8%) had typhus group rickettsioses. In addition, 55 (7.9%) participants had a confirmed acute arbovirus infection, all due to chikungunya. No patient had a bacterial zoonosis or an arbovirus infection included in the admission differential diagnosis. Malaria was uncommon and over-diagnosed, whereas invasive infections were underappreciated. Bacterial zoonoses and arbovirus infections were highly prevalent yet overlooked. An integrated approach to the syndrome of fever in resource-limited areas is needed to improve patient outcomes and to rationally target disease control efforts

Malaria is an uncommon cause of adult sepsis in south-western Uganda

Malaria is often considered a cause of adult sepsis in malaria endemic areas. However, diagnostic limitations can make distinction between malaria and other infections challenging. Therefore, the objective of this study was to determine the relative contribution of malaria to adult sepsis in south-western Uganda

Refined physical parameters for Chariklo's body and rings from stellar occultations observed between 2013 and 2020

Context. The Centaur (10199) Chariklo has the first ring system discovered around a small object. It was first observed using stellar occultation in 2013. Stellar occultations allow sizes and shapes to be determined with kilometre accuracy, and provide the characteristics of the occulting object and its vicinity. Aims. Using stellar occultations observed between 2017 and 2020, our aim is to constrain the physical parameters of Chariklo and its rings. We also determine the structure of the rings, and obtain precise astrometrical positions of Chariklo. Methods. We predicted and organised several observational campaigns of stellar occultations by Chariklo. Occultation light curves were measured from the datasets, from which ingress and egress times, and the ring widths and opacity values were obtained. These measurements, combined with results from previous works, allow us to obtain significant constraints on Chariklo's shape and ring structure. Results. We characterise Chariklo's ring system (C1R and C2R), and obtain radii and pole orientations that are consistent with, but more accurate than, results from previous occultations. We confirm the detection of W-shaped structures within C1R and an evident variation in radial width. The observed width ranges between 4.8 and 9.1 km with a mean value of 6.5 km. One dual observation (visible and red) does not reveal any differences in the C1R opacity profiles, indicating a ring particle size larger than a few microns. The C1R ring eccentricity is found to be smaller than 0.022 (3σ), and its width variations may indicate an eccentricity higher than ~0.005. We fit a tri-axial shape to Chariklo's detections over 11 occultations, and determine that Chariklo is consistent with an ellipsoid with semi-axes of 143.8-1.5+1.4, 135.2-2.8+1.4, and 99.1-2.7+5.4 km. Ultimately, we provided seven astrometric positions at a milliarcsecond accuracy level, based on Gaia EDR3, and use it to improve Chariklo's ephemeris.Fil: Morgado, B.E.. Centre National de la Recherche Scientifique. Observatoire de Paris; Francia. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; BrasilFil: Sicardy, Bruno. Centre National de la Recherche Scientifique. Observatoire de Paris; FranciaFil: Braga Ribas, Felipe. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; Brasil. Centre National de la Recherche Scientifique. Observatoire de Paris; Francia. Universidade Tecnologia Federal do Parana; BrasilFil: Desmars, Josselin. Centre National de la Recherche Scientifique. Observatoire de Paris; FranciaFil: Gomes Júnior, Altair Ramos. Universidade de Sao Paulo; BrasilFil: Bérard, D.. Centre National de la Recherche Scientifique. Observatoire de Paris; FranciaFil: Leiva, Rodrigo. Universidad de Chile; Chile. Centre National de la Recherche Scientifique. Observatoire de Paris; FranciaFil: Vieira Martins, Roberto. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; Brasil. Universidade Federal do Rio de Janeiro; BrasilFil: Benedetti Rossi, G.. Centre National de la Recherche Scientifique. Observatoire de Paris; Francia. Universidade Federal de Sao Paulo; BrasilFil: Santos Sanz, Pablo. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; BrasilFil: Camargo, Julio Ignacio Bueno. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; BrasilFil: Duffard, R.. Universidade Federal do Rio de Janeiro; BrasilFil: Rommel, F.L.. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; BrasilFil: Assafin, M.. Centre National de la Recherche Scientifique. Observatoire de Paris; FranciaFil: Boufleur, R.C.. Universidad Nacional de Córdoba; ArgentinaFil: Colas, F.. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; BrasilFil: Kretlow, Mike. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; BrasilFil: Beisker, W.. University of North Carolina; Estados UnidosFil: Sfair, Rafael. Centre National de la Recherche Scientifique. Observatoire de Paris; FranciaFil: Snodgrass, Colin. University of Edinburgh; Reino UnidoFil: Morales, N.. Pontificia Universidad Católica de Chile; Chile. Universidad Católica de Chile; ChileFil: Fernández Valenzuela, E.. Pontificia Universidad Católica de Chile; Chile. Universidad Católica de Chile; ChileFil: Amaral, L.S.. Massachusetts Institute of Technology; Estados UnidosFil: Amarante, A.. Ministério de Ciencia, Tecnologia e Innovacao. Observatorio Nacional; BrasilFil: Artola, R.A.. Centre National de la Recherche Scientifique. Observatoire de Paris; FranciaFil: Backes, M.. Universidad Nacional de Córdoba; ArgentinaFil: Bath, K. L.. University of North Carolina; Estados UnidosFil: Bouley, S.. University of St. Andrews; Reino UnidoFil: Garcia Lambas, Diego Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; ArgentinaFil: Schneiter, Ernesto Matías. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Ingeniería Económica y Legal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Astronomía Teórica y Experimental. Universidad Nacional de Córdoba. Observatorio Astronómico de Córdoba. Instituto de Astronomía Teórica y Experimental; Argentin

Geomorphology of the Imhotep region on comet 67P/Churyumov-Gerasimenko from OSIRIS observations

Context. Since August 2014, the OSIRIS Narrow Angle Camera (NAC) onboard the Rosetta spacecraft has acquired high spatial resolution images of the nucleus of comet 67P/Churyumov-Gerasimenko, down to the decimeter scale. This paper focuses on the Imhotep region, located on the largest lobe of the nucleus, near the equator. Aims. We map, inventory, and describe the geomorphology of the Imhotep region. We propose and discuss some processes to explain the formation and ongoing evolution of this region. Methods. We used OSIRIS NAC images, gravitational heights and slopes, and digital terrain models to map and measure the morphologies of Imhotep. Results. The Imhotep region presents a wide variety of terrains and morphologies: smooth and rocky terrains, bright areas, linear features, roundish features, and boulders. Gravity processes such as mass wasting and collapse play a significant role in the geomorphological evolution of this region. Cometary processes initiate erosion and are responsible for the formation of degassing conduits that are revealed by elevated roundish features on the surface. We also propose a scenario for the formation and evolution of the Imhotep region; this implies the presence of large primordial voids inside the nucleus, resulting from its formation process

(704) Interamnia: a transitional object between a dwarf planet and a typical irregular-shaped minor body

Context. With an estimated diameter in the 320–350 km range, (704) Interamnia is the fifth largest main belt asteroid and one of the few bodies that fills the gap in size between the four largest bodies with D > 400 km (Ceres, Vesta, Pallas and Hygiea) and the numerous smaller bodies with diameter ≤200 km. However, despite its large size, little is known about the shape and spin state of Interamnia and, therefore, about its bulk composition and past collisional evolution. Aims. We aimed to test at what size and mass the shape of a small body departs from a nearly ellipsoidal equilibrium shape (as observed in the case of the four largest asteroids) to an irregular shape as routinely observed in the case of smaller (D ≤ 200 km) bodies. Methods. We observed Interamnia as part of our ESO VLT/SPHERE large program (ID: 199.C-0074) at thirteen different epochs. In addition, several new optical lightcurves were recorded. These data, along with stellar occultation data from the literature, were fed to the All-Data Asteroid Modeling algorithm to reconstruct the 3D-shape model of Interamnia and to determine its spin state. Results. Interamnia’s volume-equivalent diameter of 332 ± 6 km implies a bulk density of ρ = 1.98 ± 0.68 g cm−3, which suggests that Interamnia – like Ceres and Hygiea – contains a high fraction of water ice, consistent with the paucity of apparent craters. Our observations reveal a shape that can be well approximated by an ellipsoid, and that is compatible with a fluid hydrostatic equilibrium at the 2σ level. Conclusions. The rather regular shape of Interamnia implies that the size and mass limit, under which the shapes of minor bodies with a high amount of water ice in the subsurface become irregular, has to be searched among smaller (D ≤ 300 km) less massive (m ≤ 3 × 1019 kg) bodies

Genome-Wide Identification of HrpL-Regulated Genes in the Necrotrophic Phytopathogen Dickeya dadantii 3937

BACKGROUND: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. METHODOLOGY/PRINCIPAL FINDINGS: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens