The ecto-enzyme CD38 modulates CD4 T cell immunometabolic responses and participates in HIV pathogenesis.

Despite abundant evidence correlating T cell CD38 expression and HIV infection pathogenesis, its role as a CD4 T cell immunometabolic regulator remains unclear. We find that CD38's extracellular glycohydrolase activity restricts metabolic reprogramming after TCR-engaging stimulation in Jurkat T CD4 cells, together with functional responses, while reducing intracellular NAD and NMN concentrations. Selective elimination of CD38's ectoenzyme function licenses them to decrease the OCR/ECAR ratio upon TCR signaling and to increase cycling, proliferation, survival, and CD40L induction. Pharmacological inhibition of ectoCD38 catalytic activity in memory CD4 T cells from chronic HIV-infected patients rescued TCR-triggered responses, including differentiation and effector functions, while reverting abnormally increased basal glycolysis, cycling, and spontaneous pro-inflammatory cytokine production. Additionally, ecto-CD38 blockage normalized basal and TCR-induced mitochondrial morpho-functionality, while increasing respiratory capacity in cells from HIV+ patients and healthy individuals. Ectoenzyme CD38's immunometabolic restriction of TCR-involving stimulation is relevant to CD4 T cell biology and to the deleterious effects of CD38 overexpression in HIV disease.

Exploration of low-frequency allelic variants of SARS-CoV-2 genomes reveals coinfections in Mexico occurred during periods of VOCs turnover.

A total of 14 973 alleles in 29 661 sequenced samples collected between March 2021 and January 2023 by the Mexican Consortium for Genomic Surveillance (CoViGen-Mex) and collaborators were used to construct a thorough map of mutations of the Mexican SARS-CoV-2 genomic landscape containing Intra-Patient Minor Allelic Variants (IPMAVs), which are low-frequency alleles not ordinarily present in a genomic consensus sequence. This additional information proved critical in identifying putative coinfecting variants included alongside the most common variants, B.1.1.222, B.1.1.519, and variants of concern (VOCs) Alpha, Gamma, Delta, and Omicron. A total of 379 coinfection events were recorded in the dataset (a rate of 1.28 %), resulting in the first such catalogue in Mexico. The most common putative coinfections occurred during the spread of Delta or after the introduction of Omicron BA.2 and its descendants. Coinfections occurred constantly during periods of variant turnover when more than one variant shared the same niche and high infection rate was observed, which was dependent on the local variants and time. Coinfections might occur at a higher frequency than customarily reported, but they are often ignored as only the consensus sequence is reported for lineage identification.

An amplicon-based protocol for whole-genome sequencing of human respiratory syncytial virus subgroup A.

It is convenient to study complete genome sequences of human respiratory syncytial virus (hRSV) for ongoing genomic characterization and identification of highly transmissible or pathogenic variants. Whole genome sequencing of hRSV has been challenging from respiratory tract specimens with low viral loads. Herein, we describe an amplicon-based protocol for whole genome sequencing of hRSV subgroup A validated with 24 isolates from nasopharyngeal swabs and infected cell cultures, which showed cycle threshold (Ct) values ranging from 10 to 31, as determined by quantitative reverse-transcription polymerase chain reaction. MinION nanopore generated 3200 to 5400 reads per sample to sequence over 93% of the hRSV-A genome. Coverage of each contig ranged from 130× to 200×. Samples with Ct values of 20.9, 25.2, 27.1, 27.7, 28.2, 28.8, and 29.6 led to the sequencing of over 99.0% of the virus genome, indicating high genome coverage even at high Ct values. This protocol enables the identification of hRSV subgroup A genotypes, as primers were designed to target highly conserved regions. Consequently, it holds potential for application in molecular epidemiology and surveillance of this hRSV subgroup.

Hypoxia Induces Alterations in the Circadian Rhythm in Patients with Chronic Respiratory Diseases.

The function of the circadian cycle is to determine the natural 24 h biological rhythm, which includes physiological, metabolic, and hormonal changes that occur daily in the body. This cycle is controlled by an internal biological clock that is present in the body's tissues and helps regulate various processes such as sleeping, eating, and others. Interestingly, animal models have provided enough evidence to assume that the alteration in the circadian system leads to the appearance of numerous diseases. Alterations in breathing patterns in lung diseases can modify oxygenation and the circadian cycles; however, the response mechanisms to hypoxia and their relationship with the clock genes are not fully understood. Hypoxia is a condition in which the lack of adequate oxygenation promotes adaptation mechanisms and is related to several genes that regulate the circadian cycles, the latter because hypoxia alters the production of melatonin and brain physiology. Additionally, the lack of oxygen alters the expression of clock genes, leading to an alteration in the regularity and precision of the circadian cycle. In this sense, hypoxia is a hallmark of a wide variety of lung diseases. In the present work, we intended to review the functional repercussions of hypoxia in the presence of asthma, chronic obstructive sleep apnea, lung cancer, idiopathic pulmonary fibrosis, obstructive sleep apnea, influenza, and COVID-19 and its repercussions on the circadian cycles.

Glial-derived mitochondrial signals affect neuronal proteostasis and aging.

The nervous system plays a critical role in maintaining whole-organism homeostasis; neurons experiencing mitochondrial stress can coordinate the induction of protective cellular pathways, such as the mitochondrial unfolded protein response (UPRMT), between tissues. However, these studies largely ignored nonneuronal cells of the nervous system. Here, we found that UPRMT activation in four astrocyte-like glial cells in the nematode, Caenorhabditis elegans, can promote protein homeostasis by alleviating protein aggregation in neurons. Unexpectedly, we find that glial cells use small clear vesicles (SCVs) to signal to neurons, which then relay the signal to the periphery using dense-core vesicles (DCVs). This work underlines the importance of glia in establishing and regulating protein homeostasis within the nervous system, which can then affect neuron-mediated effects in organismal homeostasis and longevity.

Glial-derived mitochondrial signals impact neuronal proteostasis and aging.

The nervous system plays a critical role in maintaining whole-organism homeostasis; neurons experiencing mitochondrial stress can coordinate the induction of protective cellular pathways, such as the mitochondrial unfolded protein response (UPRMT), between tissues. However, these studies largely ignored non-neuronal cells of the nervous system. Here, we found that UPRMT activation in four, astrocyte-like glial cells in the nematode, C. elegans, can promote protein homeostasis by alleviating protein aggregation in neurons. Surprisingly, we find that glial cells utilize small clear vesicles (SCVs) to signal to neurons, which then relay the signal to the periphery using dense-core vesicles (DCVs). This work underlines the importance of glia in establishing and regulating protein homeostasis within the nervous system, which can then impact neuron-mediated effects in organismal homeostasis and longevity.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.

Chirality control of a single carbene molecule by tip-induced van der Waals interactions.

Non-covalent interactions such as van der Waals interactions and hydrogen bonds are crucial for the chiral induction and control of molecules, but it remains difficult to study them at the single-molecule level. Here, we report a carbene molecule on a copper surface as a prototype of an anchored molecule with a facile chirality change. We examine the influence of the attractive van der Waals interactions on the chirality change by regulating the tip-molecule distance, resulting in an excess of a carbene enantiomer. Our model study provides insight into the change of molecular chirality controlled by van der Waals interactions, which is fundamental for understanding the mechanisms of chiral induction and amplification.

Monoclonal antibody generated against Nile tilapia (Oreochromis niloticus) IgT heavy chain using a peptide-based strategy.

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

C-C Coupling of Carbene Molecules on a Metal Surface in the Presence of Water.

A novel surface-confined C-C coupling reaction involving two carbene molecules and a water molecule was studied by scanning tunneling microscopy in real space. Carbene fluorenylidene was generated from diazofluorene in the presence of water on a silver surface. While in the absence of water, fluorenylidene covalently binds to the surface to form a surface metal carbene, and water can effectively compete with the silver surface in reacting with the carbene. Water molecules in direct contact with fluorenylidene protonate the carbene to form the fluorenyl cation before the carbene can bind to the surface. In contrast, the surface metal carbene does not react with water. The fluorenyl cation is highly electrophilic and draws electrons from the metal surface to generate the fluorenyl radical which is mobile on the surface at cryogenic temperatures. The final step in this reaction sequence is the reaction of the radical with a remaining fluorenylidene molecule or with diazofluorene to produce the C-C coupling product. Both a water molecule and the metal surface are essential for the consecutive proton and electron transfer followed by C-C coupling. This C-C coupling reaction is unprecedented in solution chemistry.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Microbiota composition in the lower respiratory tract is associated with severity in patients with acute respiratory distress by influenza.

Several factors are associated with the severity of the respiratory disease caused by the influenza virus. Although viral factors are one of the most studied, in recent years the role of the microbiota and co-infections in severe and fatal outcomes has been recognized. However, most of the work has focused on the microbiota of the upper respiratory tract (URT), hindering potential insights from the lower respiratory tract (LRT) that may help to understand the role of the microbiota in Influenza disease. In this work, we characterized the microbiota of the LRT of patients with Influenza A using 16S rRNA sequencing. We tested if patients with different outcomes (deceased/recovered) and use of antibiotics differ in their microbial community composition. We found important differences in the diversity and composition of the microbiota between deceased and recovered patients. In particular, we detected a high abundance of opportunistic pathogens such as Granulicatella, in patients either deceased or with antibiotic treatment. Also, we found antibiotic treatment correlated with lower diversity of microbial communities and with lower probability of survival in Influenza A patients. Altogether, the loss of microbial diversity could generate a disequilibrium in the community, potentially compromising the immune response increasing viral infectivity, promoting the growth of potentially pathogenic bacteria that, together with altered biochemical parameters, can be leading to severe forms of the disease. Overall, the present study gives one of the first characterizations of the diversity and composition of microbial communities in the LRT of Influenza patients and its relationship with clinical variables and disease severity.

Omicron-BA.1 Dispersion Rates in Mexico Varied According to the Regional Epidemic Patterns and the Diversity of Local Delta Subvariants.

PURPOSE: The Omicron subvariant BA.1 of SARS-CoV-2 was first detected in November 2021 and quickly spread worldwide, displacing the Delta variant. In this work, a characterization of the spread of this variant in Mexico is presented. METHODS: The time to fixation of BA.1, the diversity of Delta sublineages, the population density, and the level of virus circulation during the inter-wave interval were determined to analyze differences in BA.1 spread. RESULTS: BA.1 began spreading during the first week of December 2021 and became dominant in the next three weeks, causing the fourth COVID-19 epidemiological surge in Mexico. Unlike previous variants, BA.1 did not exhibit a geographically distinct circulation pattern. However, a regional difference in the speed of the replacement of the Delta variant was observed. CONCLUSIONS: Viral diversity and the relative abundance of the virus in a particular area around the time of the introduction of a new lineage seem to have influenced the spread dynamics, in addition to population density. Nonetheless, if there is a significant difference in the fitness of the variants, or if the time allowed for the competition is sufficiently long, it seems the fitter virus will eventually become dominant, as observed in the eventual dominance of the BA.1.x variant in Mexico.

Longitudinal Characterization of a Neutralizing and Total Antibody Response in Patients with Severe COVID-19 and Fatal Outcomes.

The host immune response to SARS-CoV-2 appears to play a critical role in disease pathogenesis and clinical manifestations in severe COVID-19 cases. Until now, the importance of developing a neutralizing antibody response in the acute phase and its relationship with progression to severe disease or fatal outcome among hospitalized patients remains unclear. In this study, we aim to characterize and compare longitudinally the primary humoral immune host response in the early stages of the disease, looking for an association between neutralization, antibody titers, infective viral lineage, and the clinical outcome in hospitalized and non-hospitalized patients. A total of 111 patients admitted at INER from November 2021 to June 2022 were included. We found that patients with negative or low neutralization showed a significant reduction in survival probability compared to patients with medium or high neutralization. We observed a significant decrease in the median of neutralization in patients infected with viral variants with changes in RBD of the spike protein. Our results suggest that developing an early and robust neutralizing response against SARS-CoV-2 may increase survival probability in critical patients.

Effect of Hypoxia in the Transcriptomic Profile of Lung Fibroblasts from Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by exacerbated extracellular matrix deposition that disrupts oxygen exchange. Hypoxia and its transcription factors (HIF-1α and 2α) influence numerous circuits that could perpetuate fibrosis by increasing myofibroblasts differentiation and by promoting extracellular matrix accumulation. Therefore, this work aimed to elucidate the signature of hypoxia in the transcriptomic circuitry of IPF-derived fibroblasts. To determine this transcriptomic signature, a gene expression analysis with six lines of lung fibroblasts under normoxia or hypoxia was performed: three cell lines were derived from patients with IPF, and three were from healthy donors, a total of 36 replicates. We used the Clariom D platform, which allows us to evaluate a huge number of transcripts, to analyze the response to hypoxia in both controls and IPF. The control's response is greater by the number of genes and complexity. In the search for specific genes responsible for the IPF fibroblast phenotype, nineteen dysregulated genes were found in lung fibroblasts from IPF patients in hypoxia (nine upregulated and ten downregulated). In this sense, the signaling pathways revealed to be affected in the pulmonary fibroblasts of patients with IPF may represent an adaptation to chronic hypoxia.

Metagenomic analysis reveals differences in the co-occurrence and abundance of viral species in SARS-CoV-2 patients with different severity of disease.

BACKGROUND: SARS-CoV-2 infections have a wide spectrum of clinical manifestations whose causes are not completely understood. Some human conditions predispose to severe outcome, like old age or the presence of comorbidities, but many other facets, including coinfections with other viruses, remain poorly characterized. METHODS: In this study, the eukaryotic fraction of the respiratory virome of 120 COVID-19 patients was characterized through whole metagenomic sequencing. RESULTS: Genetic material from respiratory viruses was detected in 25% of all samples, whereas human viruses other than SARS-CoV-2 were found in 80% of them. Samples from hospitalized and deceased patients presented a higher prevalence of different viruses when compared to ambulatory individuals. Small circular DNA viruses from the Anneloviridae (Torque teno midi virus 8, TTV-like mini virus 19 and 26) and Cycloviridae families (Human associated cyclovirus 10), Human betaherpesvirus 6, were found to be significantly more abundant in samples from deceased and hospitalized patients compared to samples from ambulatory individuals. Similarly, Rotavirus A, Measles morbillivirus and Alphapapilomavirus 10 were significantly more prevalent in deceased patients compared to hospitalized and ambulatory individuals. CONCLUSIONS: Results show the suitability of using metagenomics to characterize a broader peripheric virological landscape of the eukaryotic virome in SARS-CoV-2 infected patients with distinct disease outcomes. Identified prevalent viruses in hospitalized and deceased patients may prove important for the targeted exploration of coinfections that may impact prognosis.

Advanced Molecular Tweezers with Lipid Anchors against SARS-CoV-2 and Other Respiratory Viruses.

The COVID-19 pandemic caused by SARS-CoV-2 presents a global health emergency. Therapeutic options against SARS-CoV-2 are still very limited but urgently required. Molecular tweezers are supramolecular agents that destabilize the envelope of viruses resulting in a loss of viral infectivity. Here, we show that first-generation tweezers, CLR01 and CLR05, disrupt the SARS-CoV-2 envelope and abrogate viral infectivity. To increase the antiviral activity, a series of 34 advanced molecular tweezers were synthesized by insertion of aliphatic or aromatic ester groups on the phosphate moieties of the parent molecule CLR01. A structure-activity relationship study enabled the identification of tweezers with a markedly enhanced ability to destroy lipid bilayers and to suppress SARS-CoV-2 infection. Selected tweezer derivatives retain activity in airway mucus and inactivate the SARS-CoV-2 wildtype and variants of concern as well as respiratory syncytial, influenza, and measles viruses. Moreover, inhibitory activity of advanced tweezers against respiratory syncytial virus and SARS-CoV-2 was confirmed in mice. Thus, potentiated tweezers are broad-spectrum antiviral agents with great prospects for clinical development to combat highly pathogenic viruses.

Surface Diffusion Aided by a Chirality Change of Self-Assembled Oligomers under 2D Confinement.

Chirality switching of self-assembled molecular structures is of potential interest for designing functional materials but is restricted by the strong interaction between the embedded molecules. Here, we report on an unusual approach based on reversible chirality changes of self-assembled oligomers using variable-temperature scanning tunneling microscopy supported by quantum mechanical calculations. Six functionalized diazomethanes each self-assemble into chiral wheel-shaped oligomers on Ag(111). At 130 K, a temperature far lower than expected, the oligomers change their chirality even though the molecules reside in an embedded self-assembled structure. Each chirality change is accompanied by a slight center-of-mass shift. We show how the identical activation energies of the two processes result from the interplay of the chirality change with surface diffusion, findings that open the possibility of implementing various functional materials from self-assembled supramolecular structures.

Clinical and Virological Features of Patients Hospitalized with Different Types of COVID-19 Vaccination in Mexico City.

Coronavirus disease 2019 (COVID-19) vaccines effectively protect against severe disease and death. However, the impact of the vaccine used, viral variants, and host factors on disease severity remain poorly understood. This work aimed to compare COVID-19 clinical presentations and outcomes in vaccinated and unvaccinated patients in Mexico City. From March to September 2021, clinical, demographic characteristics, and viral variants were obtained from 1014 individuals with a documented SARS-CoV-2 infection. We compared unvaccinated, partially vaccinated, and fully vaccinated patients, stratifying by age groups. We also fitted multivariate statistical models to evaluate the impact of vaccination status, SARS-CoV-2 lineages, vaccine types, and clinical parameters. Most hospitalized patients were unvaccinated. In patients over 61 years old, mortality was significantly higher in unvaccinated compared to fully vaccinated individuals. In patients aged 31 to 60 years, vaccinated patients were more likely to be outpatients (46%) than unvaccinated individuals (6.1%). We found immune disease and age above 61 years old to be risk factors, while full vaccination was found to be the most protective factor against in-hospital death. This study suggests that vaccination is essential to reduce mortality in a comorbid population such as that of Mexico.

PPI-Affinity: A Web Tool for the Prediction and Optimization of Protein-Peptide and Protein-Protein Binding Affinity.

Virtual screening of protein-protein and protein-peptide interactions is a challenging task that directly impacts the processes of hit identification and hit-to-lead optimization in drug design projects involving peptide-based pharmaceuticals. Although several screening tools designed to predict the binding affinity of protein-protein complexes have been proposed, methods specifically developed to predict protein-peptide binding affinity are comparatively scarce. Frequently, predictors trained to score the affinity of small molecules are used for peptides indistinctively, despite the larger complexity and heterogeneity of interactions rendered by peptide binders. To address this issue, we introduce PPI-Affinity, a tool that leverages support vector machine (SVM) predictors of binding affinity to screen datasets of protein-protein and protein-peptide complexes, as well as to generate and rank mutants of a given structure. The performance of the SVM models was assessed on four benchmark datasets, which include protein-protein and protein-peptide binding affinity data. In addition, we evaluated our model on a set of mutants of EPI-X4, an endogenous peptide inhibitor of the chemokine receptor CXCR4, and on complexes of the serine proteases HTRA1 and HTRA3 with peptides. PPI-Affinity is freely accessible at https://protdcal.zmb.uni-due.de/PPIAffinity.