Exploiting a rodent cell block for intrinsic resistance to HIV-1 gene expression in human T cells.

IMPORTANCE: Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells. CCNT1 is essential for efficient HIV-1 transcription, making it an intriguing target for gene-based inactivation of virus replication. We show that isogenic cell lines engineered to encode CCNT1 bearing a single mouse-informed amino acid change (tyrosine in place of cysteine at position 261) exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cell biology. These results provide proof of concept for targeting CCNT1 in the context of one or more functional HIV-1 cure strategies.

Factores asociados: características clínicas, microbiológicas y perfiles de resistencia en infecciones urinarias asociadas a catéter en dos hospitales de alta complejidad / Associated factors clinical and microbiological characteristics and resistance profiles in catheter-associated urinary tract infections in two highly complex hospitals

Resumen Objetivo: Determinar los factores asociados, las características clínicas, microbiológicas y perfiles de resistencia en las infecciones sintomáticas de tracto urinario asociado a catéter (ISTU-AC) en los dos Instituciones de alto nivel de complejidad. Materiales y métodos: Se llevó a cabo un estudio de casos y controles. Se incluyeron todos los pacientes con más de 48 horas de hospitalización con inserción de catéter urinario y se recolectaron todas las variables clínicas y microbiológicas de cada paciente. Se incluyeron 446 pacientes, 223 con infección sintomática del tracto urinario asociado a catéter. El análisis multivariado se realizó por medio de regresión logística. Resultados: Se evidenció una mayor proporción de hombres en los controles (60,5%) en comparación con los casos (51,1%), la mediana de la edad fue muy similar para los dos grupos de estudio. Se evidenció por el análisis de regresión logística multivariado que la estancia en UCI (OR 2,176; IC de 95% 1,332 - 3,555), más de 10 días de catéter urinario (OR 2,907; IC de 95% 1,744 - 4,846) y la terapia antibiótica previa (OR 0,060; IC de 95% 0,037 - 0,103) fueron los principales factores asociados con la ocurrencia de ISTU-AC. No se encontró asociación entre presentar el evento de interés y la edad, esta variable no está relacionada con la pre sencia de infecciones intrahospitalarias. Es probable que las comorbilidades, presentes con más frecuencia en el adulto mayor, hayan sobrepasado el efecto de la edad en esos estudios. La asociación con género no fue identificada como un factor de riesgo, lo cual podría esperarse, dado que el uso de la sonda vesical hace equiparable el riesgo entre los hombres y las mujeres. Conclusiones: Este estudio mostró que la infecciones sintomáticas de tracto urinario asociado a catéter sigue siendo la entidad más frecuente) en el ámbito hospi talario, poniendo en riesgo la seguridad de los pacientes y aumentando tanto las tasas de morbi-mortalidad

Summary Objective: To determine the associated factors, clinical and microbiological characteristics, and resistance profiles in symptomatic urinary tract infections associated with catheters in the two institutions with a high level of complexity. Materials and methods: A case-control study was carried out. All patients with more than 48 hours of hospitalization with urinary catheter insertion were included and all the clinical and microbiological variables of each patient were collected. 446 patients were included, 223 with symptomatic urinary tract infection associated with a catheter. The multivariate analysis was carried out by means of logistic regression. Results: A greater proportion of men was evidenced in the controls (60.5%) compared to the cases (51.1%), the median age was very similar for the two study groups. It was evidenced by the multivariate logistic regression analysis that the stay in the ICU (OR 2.176; 95% CI 1.322 - 3.555), more than 10 days of urinary catheter (OR 2.907; 95% CI 1.744 - 4.846) and antibiotic therapy previous (OR 0.060; 95% CI 0.037 - 0.103) were the main factors associated with the occurrence of CA-UTI. No association was found between presenting the event of interest and age, this variable is not related to the presence of intrahospital infections. Co morbidities, which are more frequently present in the elderly, are likely to have outweighed the effect of age in these studies. The association with gender was not identified as a risk factor, which could be expected, since the use of the urinary catheter makes the risk comparable between men and women. Conclusions: This study showed that symptomatic urinary tract infections associated with catheters are still a frequent entity in the hospital setting, putting the safety of patients at risk and increasing both morbidity and mortality rates

Broad and ultra-potent cross-clade neutralization of HIV-1 by a vaccine-induced CD4 binding site bovine antibody.

Human immunodeficiency virus type 1 (HIV-1) vaccination of cows has elicited broadly neutralizing antibodies (bNAbs). In this study, monoclonal antibodies (mAbs) are isolated from a clade A (KNH1144 and BG505) vaccinated cow using a heterologous clade B antigen (AD8). CD4 binding site (CD4bs) bNAb (MEL-1872) is more potent than a majority of CD4bs bNAbs isolated so far. MEL-1872 mAb with CDRH3 of 57 amino acids shows more potency (geometric mean half-maximal inhibitory concentration [IC50]: 0.009 µg/mL; breadth: 66%) than VRC01 against clade B viruses (29-fold) and than CHO1-31 against tested clade A viruses (21-fold). It also shows more breadth and potency than NC-Cow1, the only other reported anti-HIV-1 bovine bNAb, which has 60% breadth with geometric mean IC50 of 0.090 µg/mL in this study. Using successive different stable-structured SOSIP trimers in bovines can elicit bNAbs focusing on epitopes ubiquitous across subtypes. Furthermore, the cross-clade selection strategy also results in ultra-potent bNAbs.

Infrared Based Saliva Screening Test for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.

Enhancement of Antibody-Dependent Cellular Cytotoxicity and Phagocytosis in Anti-HIV-1 Human-Bovine Chimeric Broadly Neutralizing Antibodies.

No prophylactic vaccine has provided robust protection against human immunodeficiency virus type 1 (HIV-1). Vaccine-induced broadly neutralizing antibodies (bNAbs) have not been achieved in humans and most animals; however, cows vaccinated with HIV-1 envelope trimers produce bNAbs with unusually long third heavy complementarity-determining regions (CDRH3s). Alongside neutralization, Fc-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP), may be critical for in vivo bNAb antiviral activity. Here, we aimed to augment the Fc-dependent effector functions of a chimeric human-bovine bNAb, NC-Cow1, which binds the CD4 binding site (CD4bs) and exhibits broader and more potent neutralization than most human CD4bs bNAbs by using an exceptionally long 60-amino acid (aa) CDRH3. The bovine variable region of NC-Cow1 was paired with a human IgG1 Fc region mutated to create the following three variants: G236R/L328R (GRLR) that abrogates Fc-gamma receptor (FcγR) binding, and two variants that enhance binding, namely, G236A/S239D/I332E (GASDIE) and G236A/S239D/A330L/I332E (GASDALIE). Both GASDIE and GASDALIE improved binding to human FcγRIIA and FcγRIIIA, enhanced human natural killer (NK) cell activation, and mediated higher levels of ADCC and ADP activity than the wild-type human IgG1 Fc. GASDALIE mediated higher phagocytic activity than GASDIE. As expected, GRLR eliminated binding to FcγRs and did not mediate ADCC or ADP. We demonstrated that mutations in the human Fc region of bovine chimeric antibodies with ultralong CDRH3s could enhance antibody effector functions while maintaining envelope binding and neutralization. This study will have significant implications in the development of multifunctional anti-HIV antibodies, which may be important to prevent HIV-1 transmission in an antibody-based topical microbicide. IMPORTANCE Despite successful antiviral chemotherapy, human immunodeficiency virus (HIV) is still a lifelong persistent virus, and no vaccine yet prevents HIV transmission. Topical microbicides offer an important alternative method to prevent sexual transmission of HIV-1. With the production of highly potent anti-HIV-1 broadly neutralizing antibodies (bNAbs) and multifunctional antibodies, monoclonal antibodies are now important prophylactic agents. Recently discovered anti-HIV-1 bovine bNAbs (with higher potency and breadth than most human bNAbs) could be novel candidates as potent topical microbicides. Our study is significant as it demonstrates the compatibility of combining bovine-derived neutralization with human-derived antibody-effector functions. This study is a new approach to antibody engineering that strengthens the feasibility of using high-potency bovine variable region bNAbs with augmented Fc function and promotes them as a strong candidate for antibody-mediated therapies.

The "PalliActive Caregivers" Intervention for Caregivers of Patients With Cancer in Palliative Care: A Feasibility Pilot Study.

This pilot study aimed to assess the feasibility and possible effects of the "PalliActive Caregivers," nursing intervention, on the uncertainty in illness and quality of life of family caregivers of patients with cancer receiving palliative care. This pilot study used a randomized controlled design. The participants were 80 family caregivers. The experimental group received the novel "PalliActive Caregivers" intervention. Data were collected using a sociodemographic form, the Uncertainty in Illness Scale, the Quality of Life scale, and an Intervention satisfaction questionnaire. The caregivers who received the intervention "PalliActive Caregivers" reported a high degree of satisfaction (9.74 on a 10-point scale). The intervention showed a significant decrease in uncertainty regarding illness in the experimental group (P = .009), as well as a significant decrease in the psychological well-being of quality of life within the experimental and control groups, before and after the intervention (P = .013, P = .010). It is recommended that future studies using the "PalliActive Caregivers" intervention examine the effects on other variables such as the burden of patient's symptoms, caregiver burden and rewards, self-efficacy in symptom management, competence, unmet needs, and satisfaction with care.

Innovating a Nonconventional Yeast Platform for Producing Shikimate as the Building Block of High-Value Aromatics.

The shikimate pathway serves an essential role in many organisms. Not only are the three aromatic amino acids synthesized through this pathway, but many secondary metabolites also derive from it. Decades of effort have been invested into engineering Saccharomyces cerevisiae to produce shikimate and its derivatives. In addition to the ability to express cytochrome P450, S. cerevisiae is generally recognized as safe for producing compounds with nutraceutical and pharmaceutical applications. However, the intrinsically complicated regulations involved in central metabolism and the low precursor availability in S. cerevisiae has limited production levels. Here we report the development of a new platform based on Scheffersomyces stipitis, whose superior xylose utilization efficiency makes it particularly suited to produce the shikimate group of compounds. Shikimate was produced at 3.11 g/L, representing the highest level among shikimate pathway products in yeasts. Our work represents a new exploration toward expanding the current collection of microbial factories.

Combining Metabolic Engineering and Electrocatalysis: Application to the Production of Polyamides from Sugar.

Biorefineries aim to convert biomass into a spectrum of products ranging from biofuels to specialty chemicals. To achieve economically sustainable conversion, it is crucial to streamline the catalytic and downstream processing steps. In this work, a route that combines bio- and electrocatalysis to convert glucose into bio-based unsaturated nylon-6,6 is reported. An engineered strain of Saccharomyces cerevisiae was used as the initial biocatalyst for the conversion of glucose into muconic acid, with the highest reported muconic acid titer of 559.5 mg L(-1) in yeast. Without any separation, muconic acid was further electrocatalytically hydrogenated to 3-hexenedioic acid in 94 % yield despite the presence of biogenic impurities. Bio-based unsaturated nylon-6,6 (unsaturated polyamide-6,6) was finally obtained by polymerization of 3-hexenedioic acid with hexamethylenediamine.

Transcriptional activation of glutathione pathways and role of glucose homeostasis during copper imbalance.

Copper is an essential micronutrient for organism health. Dietary changes or pathologies linked to this metal induce changes in intracellular glutathione concentrations. Here, we studied the transcriptional activation of glutathione pathways in Jurkat cell lines, analyzing the effect of change in glucose homeostasis during a physiological and supra-physiological copper exposure. An immortalized line of human T lymphocyte cell line (Jurkat) was exposed to different copper and glucose conditions to mimic concentrations present in human blood. We applied treatments for 6 (acute) and 24 h (sustained) to 2 µM (physiological) or 20 µM (supra-physiological, Wilson disease scenario) of CuSO4 in combination with 25 mg/dL (hypoglycemia), 100 mg/dL (normal) and 200 mg/dL (hyperglycemia, diabetes scenario) of glucose. The results indicate that a physiological concentration of copper exposure does not induce transcriptional changes in the glutathione synthesis pathway after 6 or 24 h. The G6PDH gene (regeneration pathway), however, is induced during a supra-physiological copper condition. This data was correlated with the viability assays, where fluctuation in both glucose conditions (hypo and hyperglycemia scenario) affected Jurkat proliferation when 20 µM of CuSO4 was added to the culture media. Under a copper overload condition, the transcription of a component of glutathione regeneration pathway (G6PDH gene) is activated in cells chronically exposed to a hyperglycemia scenario, indicating that fluctuations in glucose concentration impact the resistance against the metal. Our findings illustrate the importance of glucose homeostasis during copper excess.

Hantavirus Gn and Gc envelope glycoproteins: key structural units for virus cell entry and virus assembly.

In recent years, ultrastructural studies of viral surface spikes from three different genera within the Bunyaviridae family have revealed a remarkable diversity in their spike organization. Despite this structural heterogeneity, in every case the spikes seem to be composed of heterodimers formed by Gn and Gc envelope glycoproteins. In this review, current knowledge of the Gn and Gc structures and their functions in virus cell entry and exit is summarized. During virus cell entry, the role of Gn and Gc in receptor binding has not yet been determined. Nevertheless, biochemical studies suggest that the subsequent virus-membrane fusion activity is accomplished by Gc. Further, a class II fusion protein conformation has been predicted for Gc of hantaviruses, and novel crystallographic data confirmed such a fold for the Rift Valley fever virus (RVFV) Gc protein. During virus cell exit, the assembly of different viral components seems to be established by interaction of Gn and Gc cytoplasmic tails (CT) with internal viral ribonucleocapsids. Moreover, recent findings show that hantavirus glycoproteins accomplish important roles during virus budding since they self-assemble into virus-like particles. Collectively, these novel insights provide essential information for gaining a more detailed understanding of Gn and Gc functions in the early and late steps of the hantavirus infection cycle.