Sensitivities of Explicit Hail Predictions and Convective Scale Ensemble Forecasting to Microphysics Parameterizations and Ensemble Data Assimilation Configurations

The explicit prediction of deep, moist convection is challenging because small model and initial condition errors rapidly grow and degrade forecast skill. Microphysics schemes employed by convection-allowing models represent a substantial source of model error because microphysical processes are poorly understood and simplifying assumptions must be made to make simulations and forecasts computationally practical. Although data assimilation systems decrease initial condition errors, analysis and forecast skill is sensitive to the experiment design. This dissertation evaluates data assimilation and ensemble forecast system performances at convection-allowing/convection-resolving resolutions, when forecast models employ different multi-moment microphysics parameterization schemes, and the data assimilation configurations are varied. We address the related issues through detailed case studies that provide insights on optimizing the configuration of convection-allowing model forecasts. First, high-resolution hail size forecasts are made for a severe hail event on 19 May 2013 using the Advanced Regional Prediction System (ARPS). Forecasts using the National Severe Storms Laboratory (NSSL) variable density rimed ice double-moment microphysics scheme (referred to as NSSL) exhibit more skill than those using the Milbrandt and Yau double-moment (MY2) or triple-moment (MY3) schemes when verified against radar-derived hail size estimates. Although all three schemes predict severe surface hail coverage with moderate to high skill, MY2 and MY3 forecasts overpredict the maximum hail size. The NSSL scheme uses the two variable density rimed ice categories to generate large, dense hail through the wet growth of graupel. Both the MY2 and MY3 schemes predict hail to be smaller above the 0 °C isotherm because the category is primarily composed of small frozen raindrops; in the melting layer the hail quickly grows because the rimed ice accretes excessive water. MY2 and MY3 forecasts predict the largest hail sizes to be smaller when the accretion water is eliminated beneath the 0 °C isotherm. To improve hailstorm forecast initial conditions, CAPS Ensemble Kalman filter (EnKF) analyses are generated for the 8 May 2017 Colorado severe hail event using either the MY2 or the NSSL scheme in the forecast model. The results of the EnKF analyses are evaluated. With each microphysics scheme two experiments are conducted where reflectivity (Z) observations update either (1) only the hydrometeor mixing ratio or (2) all hydrometeor fields. Experiments that update only hydrometeor mixing ratios can create ensemble error covariances that are unreliable which increases analysis error. Despite improving initial condition estimates, experiments that update all hydrometeor fields underestimate surface hail size, which suggests additional constraint from observations is needed during data assimilation. Correlation patterns between observation prior estimates (e.g., Z) and model state variables are evaluated to determine the impact of hail growth assumptions in the MY and NSSL schemes on the forecast error covariances between microphysical and thermodynamic variables. For the MY2 scheme, Z is negatively correlated with updraft intensity because strong updrafts produce abundant, small hail aloft. The NSSL scheme predicts storm updrafts to produce fewer but larger hailstones aloft, which causes Z and updraft intensity to be positively correlated. Hail production processes also alter the background error covariances for in-cloud air temperature and hydrometeor species. This study documents strong sensitivity of ensemble data assimilation results of hailstorms to the parameterization of microphysical processes, and the need to reduce microphysics parameterization uncertainties. To improve data assimilation configurations for potential operational implementation, EnKF data assimilation experiments based on the operational GSI system employed by the Center for Analysis and Prediction of Storms (CAPS) realtime Spring Forecast Experiments are performed, followed by 6-hour forecasts for a mesoscale convective system (MCS) event on 28-29 May 2017. Experiments are run to evaluate the sensitivity of forecast skill to the configurations of the data assimilation system. Configurations examined include the ensemble initialization and covariance inflation as well as radar observation data thinning, covariance localization radii, observation error settings, and data assimilation frequency. Spin-up ensemble forecast surface temperatures are most skilled when the initial ensemble mean is centered upon the most recent NAM analysis, causing forecasts to predict a strong MCS. Experiments that assimilate radar observations every 5 minutes are better at the placement of high Z values near observed storms but exhibit a substantial decrease in forecast skill initially because of widespread spurious convection. Ensembles that assimilate more observations with less thinning of data or use a larger horizontal covariance localization radius for radar data overpredict the coverage of high Z values due to enhanced spurious convection. Both parameters have modestly positive impacts on forecast skill during the first forecast hour that are quickly lost due to the growth of forecast error. Forecast skill is less sensitive to the ensemble spread inflation factors and observation errors tested during this study. These results provide guidance towards optimizing the GSI EnKF system configuration, for this study the data assimilation configuration employed by the 2019 CAPS Spring Forecast Experiment produces the most skilled forecasts while remaining viable for realtime use

Kliničke praktične smjernice za perioperacijsku i poslijeoperacijsku skrb o arterijsko-venskim fistulama i umetcima za hemodijalizu u odraslih

Krvožilni pristup omogućuje hemodijalizu koja spašava život. Stoga je nužna dobra funkcija krvožilnog pristupa koja omogućuje prikladan krvni protok radi uklanjanja tvari koje se u uremiji zadržavaju u krvi bolesnika, uz istodobno sniženje rizika od sustavne infekcije na najmanju moguću mjeru. Godine 2007. Europske smjernice najbolje prakse (engl. European Best Practice Guidelines – EBPG), prethodnice trenutačne Europske najbolje bubrežne prakse (engl. European Renal Best Practice – ERBP), donijele su nacrt skupine preporuka – vodiča pri donošenju odluka o upućivanju na pregled radi krvožilnog pristupa, o procjeni i nadzoru izbora pristupa te o postupcima kod komplikacija. (1) Otad su se znatno razvili ne samo dokazi na kojima se temelje ove preporuke nego i procesi nastajanja smjernica. (2) Kao odgovor na to, ERBP je ažurirao prethodno djelo u suradnji s raznim stručnjacima iz tog područja uključujući i predstavnike Društva za krvožilni pristup (engl. Vascular Access Society – VAS), kirurge za krvožilni pristup, radiologe, medicinske sestre za dijalizu, znanstvenike, bolesnike i one koji se za njih brinu. Nastojanje da se pridržavaju sve strože metodike izrade smjernica nalagalo je određena odricanja u pogledu područja obuhvata ovih smjernica. Posljedično, one ne „pokrivaju” baš sve iste teme kao njihova prethodna verzija. Neka su područja zajednička, a neka su arhivirana da bi ustupila mjesto novim pitanjima kojima su prednost dali i pružatelji zdravstvene skrbi i oni za koje se skrbi. Odvojeno su objavljene pojedinosti postupka izbora djelokruga problematike koju su smjernice obuhvatile. (3) Nastajanje ovih smjernica slijedilo je strog proces pregleda i procjene dokaza koji se temeljio na sustavnim pregledima rezultata kliničkih istraživanja te opservacijskih podataka gdje je to bilo potrebno. Strukturirani pristup slijedio je model sustava GRADE (hrv. stupanj), koji svakoj preporuci pripisuje stupanj s obzirom na sigurnost sveukupnih dokaza te snagu. (4) Gdje je to bilo primjereno skupina za izradu smjernica unijela je nestupnjevan savjet za kliničku praksu, a koji nije proistekao iz pregleda sustavnih dokaza. Kliničke praktične smjernice iz 2019. godine specifično pokrivaju peritransplantacijske i poslijetransplantacijske aspekte arterijsko-venskih (AV) fistula i umetaka (graftova). Drugi dio, koji je bio u nastajanju kada su ove smjernice išle u tisak, pokrit će aspekte izbora krvožilnog pristupa, prijeoperacijske procjene krvnih žila i središnje venske katetere. Unatoč nedostatku dokaza velike sigurnosti za većinu područja krvožilnih pristupa, ERBP se posvetio izradi smjernica velike kakvoće, dajući smjernicu gdje god je moguće, a popis preporuka za istraživanje ondje gdje se nije moglo uputiti smjernicom. Nadamo se da će ove smjernice i one planirane pomoći stručnoj zajednici pri donošenju odluka o postupcima, postupnicima i skrbi vezanima s krvožilnim pristupima, pomoći bolesnicima i onima koji se za njih brinu da steknu uvid u problematiku te olakšati zajedničko donošenje odluka u tom području

Structure and mechanism of YopJ effector function and suppression of host immunity

YopJs are a family of bacterial effector proteins involved in regulating host immune response upon infection. This family of effectors is found in a wide variety of plant and animal pathogens, and targets a variety of substrates. YopJs inactivate their targets through acetylation, using acetyl coenzyme A (AcCoA) as the source of the acetyl group and inositol hexakisphospate (IP6) as a cofactor. Interestingly, this family of acetyltransferases is most closely related to cysteine proteases. The biochemical consequences of acetylation vary depending on the function of the target. Currently, there are three well characterized YopJs from animal pathogens: YopJ from Yersinia pestis, VopA from Vibrio parahaemolyticus, and AvrA from Salmonella enterica, and two from plant pathogens: HopZ1 from Pseudomonas syringae, and PopP2 from Ralstonia solanacearum. There are other YopJs whose functions are less well characterized. PopP2 acetylates plant transcription factors involved in plant defense gene expression, resulting in destabilization of their interaction with DNA and suppression of these genes. HopZ1 targets a variety of factors involved in plant secretion pathways and metabolism, all implicated in defense against infection. To date, all known substrates of YopJs from animal infecting pathogens are kinases involved in innate immune signaling. However, these effectors do not target the same kinases, despite the fact that these innate immune signaling kinases are structurally related. Consequently, these effectors have different effects on host immune response.Crystal structures of PopP2 and HopZ1 have been solved. These structures reveal the overall fold and mode of IP6 and AcCoA binding. Moreover, a crystal structure of PopP2 in complex with its transcription factor target was solved, which reveal how this particular YopJiinteracts with its target. Despite these advances, there is less structural information regarding kinase acetylating YopJs from animal pathogens. In the first part of this dissertation, I present a crystal structure of a variant of the kinase acetylating YopJ, AvrA. This structure reveals that the fold, and mode of IP6 and AcCoA binding, is conserved between YopJs from plant and animal infecting pathogens. I also delineate regions on AvrA, which are involved in kinase binding.In the second part of the dissertation, I address the mechanism of the acetyltransferase reaction and address how different YopJs target different kinases. Since YopJs are structurally more closely related to proteases compared to other known acetyltransferases, their mechanism of function is unique. I present evidence that AvrA acts though a ping-pong mechanism. This mechanism has been previously proposed, and evidence in support of it in the context of PopP2 presented. My work on AvrA adds to this body of evidence. With respect to differential kinase targeting, I present evidence that the structural features of different kinase acetylating YopJs regulate the extent of kinase acetylation and this how efficiently different kinases are suppressed.La famille de protéines effectrices bactériennes YopJ participe à la régulation de la réponse immunitaire de l'hôte lors d'une infection. Ces effecteurs sont présents dans une vaste gamme de pathogènes infectant les plantes et les animaux, et ciblent divers substrats. Les YopJs inactivent leurs cibles par l'acétylation, en utilisant l'acétyl-coenzyme A (AcCoA) comme source de groupe acétyle ainsi que l'acide myo-inositol hexaphosphorique (IP6) en tant que cofacteur. Fait intéressant, cette famille d'acétyltransférases est apparentée aux protéases à cystéine, et non point aux autres familles d'acétyltransférases connues. Les effets biochimiques de l'acétylation varient dépendamment de la fonction de la cible. A présent, trois YopJs provenant de pathogènes animaux (YopJ de Yersinia pestis, VopA de Vibrio parahaemolyticus, et AvrA de Salmonella enterica) ainsi que deux provenant de phytopathogènes (HopZ1 de Pseudomonas syringae et PopP2 de Ralstonia solanacearum) sont bien caractérisés. D'autres YopJs sont connues, mais leurs fonctions sont mal caractériséesPopP2 acétyle des facteurs de transcription participant à l'expression de gènes de défense chez les plantes, causant la déstabilisation de leur interaction avec l'ADN et la suppression de ces gènes. HopZ1 cible diverses protéines impliquées dans les voies métaboliques de sécrétion, toutes étant reliées à la défense des plantes contre l'infection. Toutes les YopJs des pathogènes animaux – YopJ, VopA et AvrA – ciblent des kinases participant à la signalisation dans le système immunitaire inné. Toutefois, ces effecteurs ne ciblent pas tous les mêmes kinases, en dépit de la similarité structurelle de ces cibles. En conséquence, ces YopJs exercent des effets variés sur la physiologie de l'hôte.Les structures cristallines de PopP2 et HopZ1 ont été déterminées, et ont révélé leur repliement et leur mode de liaison à l'IP6 et AcCoA. De plus, la structure cristalline de PopP2iiiassociée a son facteur de transcription cible a été déterminée, illustrant comment ce membre des YopJs interagit avec sa cible. En dépit de toutes ces informations, peu est connu concernant la structures des YopJs des pathogènes animaux, qui ont des kinases pour cibles. Dans la première partie de cette thèse, je présente la structure cristalline d'une de ces YopJs – AvrA. Ces résultats révèlent que le repliement ainsi que le mode de liaison à l'IP6 AcCoA sont conservés entre AvrA, PopP2 et HopZ1. Je souligne aussi les parties d'AvrA impliquées dans la liaison à ses kinases cibles.Dans la deuxième partie de la thèse, j'aborde le mécanisme de l'acétylation et explore la manière par laquelle les YopJs ciblent des kinases différentes. Puisque les YopJs ont évolué à partir des protéases, leur mode d'action est unique parmi les acétyltransférases. Je présente des preuves d'un mécanisme catalytique de type ping-pong pour AvrA. Un tel mécanisme a précédemment été proposé pour PopP2; mes recherches sur AvrA suggèrent que toutes les YopJs emploient un tel mécanisme. En ce qui concerne la spécificité du ciblage de kinases, des caractéristiques structurales chez les YopJs régulent l'efficacité de ce processus

Structural Sensitivity of a Prokaryotic Pentameric Ligand-gated Ion Channel to Its Membrane Environment

International audienceAlthough the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state

Peptide-Antibody Fusions Engineered by Phage Display Exhibit an Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants

The spread of COVID-19 has been exacerbated by the emergence of variants of concern (VoC). Many VoC contain mutations in the spike protein (S-protein) and are implicated in infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but they are limited by low potency and vulnerability to RBD mutations in VoC. To address these issues, we used naiv e phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the Nterminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate-affinity nAb to generate tetravalent peptide-IgG fusions, and we showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent K-D < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrated that an RBDbinding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format, including the dominant B.1.1.529 (Omicron) variant that is resistant to most clinically approved therapeutic nAbs. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug and showed that it enabled the neutralization of a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2