Role of miRNA-671-5p in Mediating Wnt/β-Catenin-Triggered Podocyte Injury

Podocyte injury and proteinuria are the most common features of glomerular disease, which is the leading cause of end-stage renal failure. Hyperactivated Wnt/β-catenin signaling is closely associated with podocyte injury, but the underlying mechanisms are incompletely understood. Here we show that miRNA-671-5p (miR-671-5p) plays a crucial role in mediating β-catenin-triggered podocyte injury by targeting Wilms tumor 1 (WT1). Microarray-based expression profiling revealed that miR-671-5p was the most upregulated miRNA in podocytes after β-catenin activation. MiR-671-5p was colocalized with β-catenin in the glomeruli of proteinuric CKD in vivo. Bioinformatics analyses and luciferase reporter assays confirmed that miR-671-5p targeted WT1 mRNA. Overexpression of miR-671-5p mimics inhibited WT1 and impaired podocyte integrity, whereas miR-671-5p antagomir preserved the expression of WT1 and other podocyte-specific proteins under basal conditions or after β-catenin activation. In mouse remnant kidney model, overexpression of miR-671-5p aggravated podocyte injury, worsened kidney dysfunction and exacerbated renal fibrosis after 5/6 nephrectomy. In contrast, miR-671-5p antagomir alleviated podocyte injury and attenuated proteinuria and renal fibrotic lesions after glomerular injury in vivo. These studies underscore a pivotal role of miR-671-5p in mediating WT1 depletion and podocyte injury induced by β-catenin. Targeting miR-671-5p may serve as a new approach to prevent podocyte injury and proteinuria in proteinuric CKD.

How Molecular Topology Can Help in Amyotrophic Lateral Sclerosis (ALS) Drug Development: A Revolutionary Paradigm for a Merciless Disease

Even if amyotrophic lateral sclerosis is still considered an orphan disease to date, its prevalence among the population is growing fast. Despite the efforts made by researchers and pharmaceutical companies, the cryptic information related to the biological and physiological onset mechanisms, as well as the complexity in identifying specific pharmacological targets, make it almost impossible to find effective treatments. Furthermore, because of complex ethical and economic aspects, it is usually hard to find all the necessary resources when searching for drugs for new orphan diseases. In this context, computational methods, based either on receptors or ligands, share the capability to improve the success rate when searching and selecting potential candidates for further experimentation and, consequently, reduce the number of resources and time taken when delivering a new drug to the market. In the present work, a computational strategy based on Molecular Topology, a mathematical paradigm capable of relating the chemical structure of a molecule to a specific biological or pharmacological property by means of numbers, is presented. The result was the creation of a reliable and accessible tool to help during the early in silico stages in the identification and repositioning of potential hits for ALS treatment, which can also apply to other orphan diseases. Considering that further computational and experimental results will be required for the final identification of viable hits, three linear discriminant equations combined with molecular docking simulations on specific proteins involved in ALS are reported, along with virtual screening of the Drugbank database as a practical example. In this particular case, as reported, a clinical trial has been already started for one of the drugs proposed in the present study.

Efficient enrichment of synchronized mouse spermatocytes suitable for genome-wide analysis.

Chromatin-based mechanisms regulating developmental transitions during meiosis are fundamental but understudied aspects of male gametogenesis. Indeed, chromatin undergoes extensive remodeling dur-ing meiosis, leading to specific patterns of gene expression and chromosome organization, which ulti-mately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin binding sites by ChIP-seq. High quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin binding sites.

Durability and cross-reactivity of SARS-CoV-2 mRNA vaccine in adolescent children

Importance: Emergent SARS-CoV-2 variants and waning humoral immunity in vaccinated individuals are causing increased infections and hospitalizations. Children are not spared from infection nor complications of COVID-19, and the recent recommendation for boosters in individuals ages 12 years or older calls for broader understanding of the adolescent immune profile after mRNA vaccination. Objective: We sought to test the durability and cross-reactivity of anti-SARS-CoV-2 serologic responses over a six-month time course in vaccinated adolescents against the wildtype and Omicron antigens. Design, Setting and Participants: Adolescents who received a full (two-dose) series of the Pfizer-BioNTech mRNA vaccination participated in this longitudinal cohort study from May 2021 to January 2022. Blood samples were collected in clinical settings from thirty-one adolescents, nineteen of whom provided samples at four timepoints (prior to vaccination, two to three weeks after first dose, two to four weeks after second dose and six months after complete series). Sera were analyzed for antibody responses against wildtype and Omicron variant SARS-CoV-2-specific proteins. Main Outcomes and Measures: The main outcome was to analyze vaccine-induced immune responses over time by ELISA, as well as their cross-reactivity between antibody responses against wildtype SARS-CoV-2 and the Omicron variant of concern. Results: Thirty-one adolescents provided a blood sample for at least one timepoint. The median age of the cohort was 13.9 years. Half of the cohort was male, and one quarter of the population was Hispanic. Anti-Spike and anti-RBD antibodies waned after six months, nearing pre-vaccination levels. After the second dose of the vaccine, adolescent children displayed equal sensitivity for the Omicron-RBD and wildtype SARS-CoV-2-RBD, as well as an upward trend of Omicron-reactive antibodies six months after vaccination. Waning mRNA vaccine-induced immunity in adolescents highlights a vulnerability in pediatric protection against SARS-CoV-2 infection. Conclusions and Relevance: Vaccine-induced immunity wanes in adolescents over time to near pre-vaccinated levels. Cross-reactivity of antibodies generated by adolescents display efficacy against Omicron. These findings highlight the need for SARS-CoV-2 boosters to protect adolescents from highly infectious variants, illness and post-COVID-19 complications.

Definition of an Inflammatory Biomarker Signature in Plasma-Derived Extracellular Vesicles of Glioblastoma Patients

Glioblastoma (GB) is an aggressive type of tumour for which therapeutic options and biomarkers are limited. GB diagnosis mostly relies on symptomatic presentation of the tumour and, in turn, brain imaging and invasive biopsy that can delay its diagnosis. Description of easily accessible and effective biomarkers present in biofluids would thus prove invaluable in GB diagnosis. Extracellular vesicles (EVs) derived from both GB and stromal cells are essential to intercellular crosstalk in the tumour bulk, and circulating EVs have been described as a potential reservoir of GB biomarkers. Therefore, EV-based liquid biopsies have been suggested as a promising tool for GB diagnosis and follow up. To identify GB specific proteins, sEVs were isolated from plasma samples of GB patients as well as healthy volunteers using differential ultracentrifugation, and their content was characterised through mass spectrometry. Our data indicate the presence of an inflammatory biomarker signature comprising members of the complement and regulators of inflammation and coagulation including VWF, FCGBP, C3, PROS1, and SERPINA1. Overall, this study is a step forward in the development of a non-invasive liquid biopsy approach for the identification of valuable biomarkers that could significantly improve GB diagnosis and, consequently, patients’ prognosis and quality of life.

Comparative Secretome Analyses of Trichoderma/Arabidopsis Co-cultures Identify Proteins for Salt Stress, Plant Growth Promotion, and Root Colonization

Numerous Trichoderma strains are beneficial for plants, promote their growth, and confer stress tolerance. A recently described novel Trichoderma strain strongly promotes the growth of Arabidopsis thaliana seedlings on media with 50 mM NaCl, while 150 mM NaCl strongly stimulated root colonization and induced salt-stress tolerance in the host without growth promotion. To understand the dynamics of plant-fungus interaction, we examined the secretome from both sides and revealed a substantial change under different salt regimes, and during co-cultivation. Stress-related proteins, such as a fungal cysteine-rich Kp4 domain-containing protein which inhibits plant cell growth, fungal WSC- and CFEM-domain-containing proteins, the plant calreticulin, and cell-wall modifying enzymes, disappear when the two symbionts are co-cultured under high salt concentrations. In contrast, the number of lytic polysaccharide monooxygenases increases, which indicates that the fungus degrades more plant lignocellulose under salt stress and its lifestyle becomes more saprophytic. Several plant proteins involved in plant and fungal cell wall modifications and root colonization are only found in the co-cultures under salt stress, while the number of plant antioxidant proteins decreased. We identified symbiosis- and salt concentration-specific proteins for both partners. The Arabidopsis PYK10 and a fungal prenylcysteine lyase are only found in the co-culture which promoted plant growth. The comparative analysis of the secretomes supports antioxidant enzyme assays and suggests that both partners profit from the interaction under salt stress but have to invest more in balancing the symbiosis. We discuss the role of the identified stage- and symbiosis-specific fungal and plant proteins for salt stress, and conditions promoting root colonization and plant growth.

Tetraspanins as Potential Modulators of Glutamatergic Synaptic Function

Tetraspanins (Tspans) comprise a membrane protein family structurally defined by four transmembrane domains and intracellular N and C termini that is found in almost all cell types and tissues of eukaryotes. Moreover, they are involved in a bewildering multitude of diverse biological processes such as cell adhesion, motility, protein trafficking, signaling, proliferation, and regulation of the immune system. Beside their physiological roles, they are linked to many pathophysiological phenomena, including tumor progression regulation, HIV-1 replication, diabetes, and hepatitis. Tetraspanins are involved in the formation of extensive protein networks, through interactions not only with themselves but also with numerous other specific proteins, including regulatory proteins in the central nervous system (CNS). Interestingly, recent studies showed that Tspan7 impacts dendritic spine formation, glutamatergic synaptic transmission and plasticity, and that Tspan6 is correlated with epilepsy and intellectual disability (formerly known as mental retardation), highlighting the importance of particular tetraspanins and their involvement in critical processes in the CNS. In this review, we summarize the current knowledge of tetraspanin functions in the brain, with a particular focus on their impact on glutamatergic neurotransmission. In addition, we compare available resolved structures of tetraspanin family members to those of auxiliary proteins of glutamate receptors that are known for their modulatory effects.

Genome-Wide Identification and Comparative Analysis of the ASR Gene Family in the Rosaceae and Expression Analysis of PbrASRs During Fruit Development

The members of the Abscisic Acid (ABA) Stress and Ripening gene family (ASR) encode a class of plant-specific proteins with ABA/WDS domains that play important roles in fruit ripening, abiotic stress tolerance and biotic stress resistance in plants. The ASR gene family has been widely investigated in the monocotyledons and dicotyledons. Although the genome sequence is already available for eight fruit species of the Rosaceae, there is far less information about the evolutionary characteristics and the function of the ASR genes in the Rosaceae than in other plant families. Twenty-seven ASR genes were identified from species in the Rosaceae and divided into four subfamilies (I, II, III, and IV) on the basis of structural characteristics and phylogenetic analysis. Purifying selection was the primary force for ASR family gene evolution in eight Rosaceae species. qPCR experiments showed that the expression pattern of PbrASR genes from Pyrus bretschneideri was organ-specific, being mainly expressed in flower, fruit, leaf, and root. During fruit development, the mRNA abundance levels of different PbrASR genes were either down- or up-regulated, and were also induced by exogenous ABA. Furthermore, subcellular localization results showed that PbrASR proteins were mainly located in the nucleus and cytoplasm. These results provide a theoretical foundation for investigation of the evolution, expression, and functions of the ASR gene family in commercial fruit species of the Rosaceae family.

Optical Configuration Effect on the Structure and Reactivity of Diastereomers Revealed by Spin Effects and Molecular Dynamics Calculations

The peculiarities of spin effects in photoinduced electron transfer (ET) in diastereomers of donor-acceptor dyads are considered in order to study the influence of chirality on reactivity. Thus, the spin selectivity—the difference between the enhancement coefficients of chemically induced dynamic nuclear polarization (CIDNP)—of the dyad’s diastereomers reflects the difference in the spin density distribution in its paramagnetic precursors that appears upon UV irradiation. In addition, the CIDNP coefficient itself has demonstrated a high sensitivity to the change of chiral centers: when one center is changed, the hyperpolarization of all polarized nuclei of the molecule is affected. The article analyzes the experimental values of spin selectivity based on CIDNP calculations and molecular dynamic modeling data in order to reveal the effect of optical configuration on the structure and reactivity of diastereomers. In this way, we succeeded in tracing the differences in dyads with L- and D-tryptophan as an electron donor. Since the replacement of L-amino acid with D-analog in specific proteins is believed to be the cause of Alzheimer’s and Parkinson’s diseases, spin effects and molecular dynamic simulation in model dyads can be a useful tool for investigating the nature of this phenomenon.