Interplay between protease and reverse transcriptase dimerization in a model HIV-1 polyprotein.

The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.

Preclinical assessment of a novel human antibody VH domain targeting mesothelin as an antibody-drug conjugate.

Mesothelin (MSLN) has been a validated tumor-associated antigen target for several solid tumors for over a decade, making it an attractive option for therapeutic interventions. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel heavy chain variable (VH) domain 3C9 from a large-size human immunoglobulin VH domain library. 3C9 exhibited high affinity (KD [dissociation constant] <3 nM) and binding specificity in a membrane proteome array (MPA). In a mouse xenograft model, 3C9 fused to human IgG1 Fc was detected at tumor sites as early as 8 h post-infusion and remained at the site for over 10 days. Furthermore, 3C9 fused to a human Fc domain drug conjugate effectively inhibited MSLN-positive tumor growth in a mouse xenograft model. The X-ray crystal structure of full-length MSLN in complex with 3C9 reveals interaction of the 3C9 domains with two distinctive residue patches on the MSLN surface. This newly discovered VH antibody domain has a high potential as a therapeutic candidate for MSLN-expressing cancers.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), 19 F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66L289K and p51 were predicted to form a heterodimer while p66 and p51L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

A synergy of activity, stability, and inhibitor-interaction of HIV-1 protease mutants evolved under drug-pressure.

A clinically-relevant, drug-resistant mutant of HIV-1 protease (PR), termed Flap+(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug-PR interactions, compared to wild-type PR. A similar mutant, Flap+(I54A) , which evolves from Flap+(I54V) and contains the single change at residue 54 relative to Flap+(I54V) , does not. Yet, how Flap+(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+(I54V) , Flap+(I54A) , and Flap+(I54) , a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.

Factores Pronóstico de Neumonía Grave Comunitaria en Unidad de Terapia Intensiva Pediátrica. Bayamo. 2016-2019 / Prognostic Factors for Severe Community Pneumonia in a Pediatric Intensive Care Unit. Bayamo. 2016-2019

Introducción: la neumonía adquirida en la comunidad (NAC) es una infección aguda del parénquima pulmonar que afecta a un paciente previamente sano o inmunocompetente expuesto a un microorganismo fuera del Hospital. Objetivo: determinar los principales factores pronósticos de mortalidad de la Neumonía grave adquirida en la comunidad, en pacientes ingresados con este diagnóstico en la UTIP de Bayamo, durante 2016-2019. Métodos: se realizó un estudio observacional analítico prospectivo, de cohorte transversal, para determinar los factores pronósticos asociados a la mortalidad por Neumonía grave comunitaria en la Unidad de Cuidados Intensivos (UTIP) en el Hospital Pediátrico Docente General Luís Ángel Milanés Tamayo de Bayamo, durante los años 2016-2019. Resultados: la presencia concomitante de Síndrome de Dificultad Respiratoria Aguda, sepsis y la edad menor de un año, resultaron factores favorecedores de muerte en pacientes con neumonía grave comunitaria. Conclusiones: la exposición de los pacientes a la asociación de los factores del modelo predictivo resultante, determinó una probabilidad de fallecer del 29 por ciento(AU)

Introduction: community acquired pneumonia (CAP) is an acute infection of the lung parenchyma that affects a previously healthy or immunocompetent patient exposed to a microorganism outside the Hospital. Objective: to determine the main prognostic factors for mortality of severe community-acquired pneumonia in patients admitted with this diagnosis to the Bayamo PICU, during 2016-2019. Methods: a prospective, analytical, observational, cross-sectional cohort study was carried out to determine the prognostic factors associated with mortality from severe community pneumonia in the Intensive Care Unit (PICU) at the General Luís Ángel Milanés Tamayo Pediatric Teaching Hospital in Bayamo, during the years 2016-2019. Results: the concomitant presence of Acute Respiratory Distress Syndrome, sepsis, and age less than one year, were factors favoring death in patients with severe community pneumonia. Conclusions: the exposure of the patients to the association of the factors of the resulting predictive model, determined a probability of death of 29 percent(EU)

Factores Pronóstico de Neumonía Grave Comunitaria en Unidad de Terapia Intensiva Pediátrica. Bayamo. 2016-2019 / Prognostic Factors for Severe Community Pneumonia in a Pediatric Intensive Care Unit. Bayamo. 2016-2019 / Fatores prognósticos para Pneumonia Comunitária Grave em Unidade de Terapia Intensiva Pediátrica. Bayamo. 2016-2019

RESUMEN Introducción: la neumonía adquirida en la comunidad (NAC) es una infección aguda del parénquima pulmonar que afecta a un paciente previamente sano o inmunocompetente expuesto a un microorganismo fuera del Hospital. Objetivo: determinar los principales factores pronóstico de mortalidad de la Neumonía grave adquirida en la comunidad, en pacientes ingresados con este diagnóstico en la UTIP de Bayamo, durante 2016-2019. Métodos: se realizó un estudio observacional analítico prospectivo, de cohorte transversal, para determinar los factores pronóstico asociados a la mortalidad por Neumonía grave comunitaria en la Unidad de Cuidados Intensivos (UTIP) en el Hospital Pediátrico Docente General Luís Ángel Milanés Tamayo de Bayamo, durante los años 2016-2019. Resultados: la presencia concomitante de Síndrome de Dificultad Respiratoria Aguda, sepsis y la edad menor de un año, resultaron factores favorecedores de muerte en pacientes con neumonía grave comunitaria. Conclusiones: la exposición de los pacientes a la asociación de los factores del modelo predictivo resultante, determinó una probabilidad de fallecer del 29 %.

ABSTRACT Introduction: community acquired pneumonia (CAP) is an acute infection of the lung parenchyma that affects a previously healthy or immunocompetent patient exposed to a microorganism outside the Hospital. Objective: to determine the main prognostic factors for mortality of severe community-acquired pneumonia in patients admitted with this diagnosis to the Bayamo PICU, during 2016-2019. Methods: a prospective, analytical, observational, cross-sectional cohort study was carried out to determine the prognostic factors associated with mortality from severe community pneumonia in the Intensive Care Unit (PICU) at the General Luís Ángel Milanés Tamayo Pediatric Teaching Hospital in Bayamo, during the years 2016-2019. Results: the concomitant presence of Acute Respiratory Distress Syndrome, sepsis, and age less than one year, were factors favoring death in patients with severe community pneumonia. Conclusions: the exposure of the patients to the association of the factors of the resulting predictive model, determined a probability of death of 29%.

RESUMO Introdução: a pneumonia adquirida na comunidade (PAC) é uma infecção aguda do parênquima pulmonar que afeta um paciente previamente hígido ou imunocompetente exposto a um microrganismo fora do Hospital. Objetivo: determinar os principais fatores prognósticos de mortalidade por pneumonia grave adquirida na comunidade em pacientes admitidos com esse diagnóstico na UTIP Bayamo, durante o período de 2016-2019. Métodos: foi realizado um estudo de coorte prospectivo, analítico, observacional e transversal para determinar os fatores prognósticos associados à mortalidade por pneumonia comunitária grave na Unidade de Terapia Intensiva (UTIP) do Hospital Pediátrico General Luís Ángel Milanés Tamayo em Bayamo, durante os anos 2016-2019. Resultados: a presença concomitante de Síndrome do Desconforto Respiratório Agudo, sepse e idade inferior a um ano foram fatores que favoreceram o óbito em pacientes com pneumonia comunitária grave. Conclusões: a exposição dos pacientes à associação dos fatores do modelo preditivo resultante, determinou uma probabilidade de morte de 29%.

Effect of Lysyl-tRNA Synthetase on the Maturation of HIV-1 Reverse Transcriptase.

In human immunodeficiency virus-1 (HIV-1), reverse transcriptase (RT) is encoded as a 66 kDa protein, p66, in the Gag-Pol polyprotein. This protein is proteolytically cleaved by HIV-1 protease (PR) to finally generate a mature RT that is a heterodimer, composed of a p66 subunit and a p66-derived 51 kDa subunit, p51. In our prior work, we demonstrated that tRNALys3 binding to p66/p66 facilitates efficient cleavage of p66 to p51 by PR. However, tRNALys3 is known to be recruited to the virus by forming a complex with lysyl-tRNA synthetase (LysRS). Herein, we tested whether LysRS can have an effect on RT maturation in vitro. Importantly, our data show no significant differences in RT maturation in the presence of LysRS. Furthermore, no apparent p66/66 interaction with LysRS was observed. Although PR cleaved LysRS, it did not immediately release tRNALys3 from LysRS. Thus, we conclude that a free fraction of tRNALys3, which is in equilibrium with a LysRS-bound form, interacts with p66/p66 without any additional mechanism involving release of tRNALys3 from LysRS. Given that only transient tRNALys3-p66/p66 interaction is needed for efficient RT maturation, a small amount of free tRNA may be sufficient for this process. These studies reveal molecular level insights into RT maturation and will be useful for the design of cellular/viral experiments to better understand the role of tRNA in HIV-1 replication.

Probing Structural Changes among Analogous Inhibitor-Bound Forms of HIV-1 Protease and a Drug-Resistant Mutant in Solution by Nuclear Magnetic Resonance.

In the era of state-of-the-art inhibitor design and high-resolution structural studies, detection of significant but small protein structural differences in the inhibitor-bound forms is critical to further developing the inhibitor. Here, we probed differences in HIV-1 protease (PR) conformation among darunavir and four analogous inhibitor-bound forms and compared them with a drug-resistant mutant using nuclear magnetic resonance chemical shifts. Changes in amide chemical shifts of wild-type (WT) PR among these inhibitor-bound forms, ΔCSP, were subtle but detectable and extended >10 Å from the inhibitor-binding site, asymmetrically between the two subunits of PR. Molecular dynamics simulations revealed differential local hydrogen bonding as the molecular basis of this remote asymmetric change. Inhibitor-bound forms of the drug-resistant mutant also showed a similar long-range ΔCSP pattern. Differences in ΔCSP values of the WT and the mutant (ΔΔCSPs) were observed at the inhibitor-binding site and in the surrounding region. Comparing chemical shift changes among highly analogous inhibitors and ΔΔCSPs effectively eliminated local environmental effects stemming from different chemical groups and enabled exploitation of these sensitive parameters to detect subtle protein conformational changes and to elucidate asymmetric and remote conformational effects upon inhibitor interaction.

Novel signature fatty acid profile of the giant manta ray suggests reliance on an uncharacterised mesopelagic food source low in polyunsaturated fatty acids.

Traditionally, large planktivorous elasmobranchs have been thought to predominantly feed on surface zooplankton during daytime hours. However, the recent application of molecular methods to examine long-term assimilated diets, has revealed that these species likely gain the majority from deeper or demersal sources. Signature fatty acid analysis (FA) of muscle tissue was used to examine the assimilated diet of the giant manta ray Mobula birostris, and then compared with surface zooplankton that was collected during feeding and non-feeding events at two aggregation sites off mainland Ecuador. The FA profiles of M. birostris and surface zooplankton were markedly different apart from similar proportions of arachidonic acid, which suggests daytime surface zooplankton may comprise a small amount of dietary intake for M. birostris. The FA profile of M. birostris muscle was found to be depleted in polyunsaturated fatty acids, and instead comprised high proportions of 18:1ω9 isomers. While 18:1ω9 isomers are not explicitly considered dietary FAs, they are commonly found in high proportions in deep-sea organisms, including elasmobranch species. Overall, the FA profile of M. birostris suggests a diet that is mesopelagic in origin, but many mesopelagic zooplankton species also vertically migrate, staying deep during the day and moving to shallower waters at night. Here, signature FA analysis is unable to resolve the depth at which these putative dietary items were consumed and how availability of this prey may drive distribution and movements of this large filter-feeder.