Aphid BCR4 Structure and Activity Uncover a New Defensin Peptide Superfamily

Aphids (Hemiptera: Aphidoidea) are among the most detrimental insects for agricultural plants, and their management is a great challenge in agronomical research. A new class of proteins, called Bacteriocyte-specific Cysteine-Rich (BCR) peptides, provides an alternative to chemical insecticides for pest control. BCRs were initially identified in the pea aphid Acyrthosiphon pisum. They are small disulfide bond-rich proteins expressed exclusively in aphid bacteriocytes, the insect cells that host intracellular symbiotic bacteria. Here, we show that one of the A. pisum BCRs, BCR4, displays prominent insecticidal activity against the pea aphid, impairing insect survival and nymphal growth, providing evidence for its potential use as a new biopesticide. Our comparative genomics and phylogenetic analyses indicate that BCRs are restricted to the aphid lineage. The 3D structure of BCR4 reveals that this peptide belongs to an as-yet-unknown structural class of peptides and defines a new superfamily of defensins.     

Alu Deletions in LAMA2 and CDH4 Genes Are Key Components of Polygenic Predictors of Longevity

Longevity is a unique human phenomenon and a highly stable trait, characterized by polygenicity. The longevity phenotype occurs due to the ability to successfully withstand the age-related genomic instability triggered by Alu elements. The purpose of our cross-sectional study was to evaluate the combined contribution of ACE*Ya5ACE, CDH4*Yb8NBC516, COL13A1*Ya5ac1986, HECW1*Ya5NBC182, LAMA2*Ya5-MLS19, PLAT*TPA25, PKHD1L1*Yb8AC702, SEMA6A*Yb8NBC597, STK38L*Ya5ac2145 and TEAD1*Ya5ac2013 Alu elements to longevity. The study group included 2054 unrelated individuals aged from 18 to 113 years who are ethnic Tatars from Russia. We analyzed the dynamics of the allele and genotype frequencies of the studied Alu polymorphic loci in the age groups of young (18–44 years old), middle-aged (45–59 years old), elderly (60–74 years old), old seniors (75–89 years old) and long-livers (90–113 years old). Most significant changes in allele and genotype frequencies were observed between the long-livers and other groups. The search for polygenic predictors of longevity was performed using the APSampler program. Attaining longevity was associated with the combinations LAMA2*ID + CDH4*D (OR = 2.23, P_Bonf = 1.90 × 10⁻²) and CDH4*DD + LAMA2*ID + HECW1*D (OR = 4.58, P_Bonf = 9.00 × 10⁻³) among persons aged between 18 and 89 years, LAMA2*ID + CDH4*D + SEMA6A*I for individuals below 75 years of age (OR = 3.13, P_Bonf = 2.00 × 10⁻²), LAMA2*ID + HECW1*I for elderly people aged 60 and older (OR = 3.13, P_Bonf = 2.00 × 10⁻²) and CDH4*DD + LAMA2*D + HECW1*D (OR = 4.21, P_Bonf = 2.60 × 10⁻²) and CDH4*DD + LAMA2*D + ACE*I (OR = 3.68, P_Bonf = 1.90 × 10⁻²) among old seniors (75–89 years old). The key elements of combinations associated with longevity were the deletion alleles of CDH4 and LAMA2 genes. Our results point to the significance for human longevity of the Alu polymorphic loci in CDH4, LAMA2, HECW1, SEMA6A and ACE genes, involved in the integration systems.     

Towards an In Vitro 3D Model for Photosynthetic Cancer Treatment: A Study of Microalgae and Tumor Cell Interactions

As hypoxic tumors show resistance to several clinical treatments, photosynthetic microorganisms have been recently suggested as a promising safe alternative for oxygenating the tumor microenvironment. The relationship between organisms and the effect microalgae have on tumors is still largely unknown, evidencing the need for a simple yet representative model for studying photosynthetic tumor oxygenation in a reproducible manner. Here, we present a 3D photosynthetic tumor model composed of human melanoma cells and the microalgae Chlamydomonas reinhardtii, both seeded into a collagen scaffold, which allows for the simultaneous study of both cell types. This work focuses on the biocompatibility and cellular interactions of the two cell types, as well as the study of photosynthetic oxygenation of the tumor cells. It is shown that both cell types are biocompatible with one another at cell culture conditions and that a 10:1 ratio of microalgae to cells meets the metabolic requirement of the tumor cells, producing over twice the required amount of oxygen. This 3D tumor model provides an easy-to-use in vitro resource for analyzing the effects of photosynthetically produced oxygen on a tumor microenvironment, thus opening various potential research avenues.     

Reaction of Corroles with Sarcosine and Paraformaldehyde: A New Facet of Corrole Chemistry

Details on the unexpected formation of two new (dimethylamino)methyl corrole isomers from the reaction of 5,10,15-tris(pentafluorophenyl)corrolatogallium(III) with sarcosine and paraformaldehyde are presented. Semi-empirical calculations on possible mechanism pathways seem to indicate that the new compounds are probably formed through a Mannich-type reaction. The extension of the protocol to the free-base 5,10,15-tris(pentafluorophenyl)corrole afforded an unexpected new seven-membered ring corrole derivative, confirming the peculiar behavior of corroles towards known reactions when compared to the well-behaved porphyrin counterparts.     

CS12192, a Novel JAK3/JAK1/TBK1 Inhibitor,Synergistically Enhances the Anti-Inflammation Effect of Methotrexate in a Rat Model of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a common disease worldwide and is treated commonly with methotrexate (MTX). CS12192 is a novel JAK3 inhibitor discovered by Chipscreen Biosciences for the treatment of autoimmune diseases. In the present study, we examined the therapeutic effect of CS12192 against RA and explored if the combinational therapy of CS12192 and MTX produced a synergistic effect against RA in rat collagen-induced arthritis (CIA). Arthritis was induced in male Sprague-Dawley rats by two intradermal injections of bovine type II collagen (CII) and treated with MTX, CS12192, or the combination of CS12192 and MTX daily for two weeks. Effects of different treatments on arthritis score, X-ray score, pathology, and expression of inflammatory cytokines and biomarkers were examined. We found that treatment with either CS12192 or MTX produced a comparable therapeutic effect on CIA including: (1) significantly lowering the arthritis score, X-ray score, serum levels of rheumatic factor (RF), C-reactive protein (CRP), and anti-nuclear antibodies (ANA); (2) largely alleviating histopathological damage, reducing infiltration of Th17 cells while promoting Treg cells; (3) inhibiting the expression of inflammatory cytokines and chemokines such as IL-1β, TNF-α, IL-6, CCL2, and CXCL1. All these inhibitory effects were further improved by the combinational therapy with MTX and CS12192. Of importance, the combinational treatment also resulted in a marked switching of the Th17 to Treg and the M1 to M2 immune responses in synovial tissues of CIA. Thus, when compared to the monotherapy, the combination treatment with CS12192 and MTX produces a better therapeutic effect against CIA with a greater suppressive effect on T cells and macrophage-mediated joint inflammation.     

The Future of Point-of-Care Nucleic Acid Amplification Diagnostics after COVID-19: Time to Walk the Walk

Since the onset of the COVID-19 pandemic, over 610 million cases have been diagnosed and it has caused over 6.5 million deaths worldwide. The crisis has forced the scientific community to develop tools for disease control and management at a pace never seen before. The control of the pandemic heavily relies in the use of fast and accurate diagnostics, that allow testing at a large scale. The gold standard diagnosis of viral infections is the RT-qPCR. Although it provides consistent and reliable results, it is hampered by its limited throughput and technical requirements. Here, we discuss the main approaches to rapid and point-of-care diagnostics based on RT-qPCR and isothermal amplification diagnostics. We describe the main COVID-19 molecular diagnostic tests approved for self-testing at home or for point-of-care testing and compare the available options. We define the influence of specimen selection and processing, the clinical validation, result readout improvement strategies, the combination with CRISPR-based detection and the diagnostic challenge posed by SARS-CoV-2 variants for different isothermal amplification techniques, with a particular focus on LAMP and recombinase polymerase amplification (RPA). Finally, we try to shed light on the effect the improvement in molecular diagnostics during the COVID-19 pandemic could have in the future of other infectious diseases.     

Whole Exome Sequencing Identifies a Heterozygous Variant in the Cav1.3 Gene CACNA1D Associated with Familial Sinus Node Dysfunction and Focal Idiopathic Epilepsy

Cav1.3 voltage-gated L-type calcium channels (LTCCs) are involved in cardiac pacemaking, hearing and hormone secretion, but are also expressed postsynaptically in neurons. So far, homozygous loss of function mutations in CACNA1D encoding the Cav1.3 α₁-subunit are described in congenital sinus node dysfunction and deafness. In addition, germline mutations in CACNA1D have been linked to neurodevelopmental syndromes including epileptic seizures, autism, intellectual disability and primary hyperaldosteronism. Here, a three-generation family with a syndromal phenotype of sinus node dysfunction, idiopathic epilepsy and attention deficit hyperactivity disorder (ADHD) is investigated. Whole genome sequencing and functional heterologous expression studies were used to identify the disease-causing mechanisms in this novel syndromal disorder. We identified a heterozygous non-synonymous variant (p.Arg930His) in the CACNA1D gene that cosegregated with the combined clinical phenotype in an autosomal dominant manner. Functional heterologous expression studies showed that the CACNA1D variant induces isoform-specific alterations of Cav1.3 channel gating: a gain of ion channel function was observed in the brain-specific short CACNA1D isoform (Cav1.3_S), whereas a loss of ion channel function was seen in the long (Cav1.3_L) isoform. The combined gain-of-function (GOF) and loss-of-function (LOF) induced by the R930H variant are likely to be associated with the rare combined clinical and syndromal phenotypes in the family. The GOF in the Cav1.3_S variant with high neuronal expression is likely to result in epilepsy, whereas the LOF in the long Cav1.3_L variant results in sinus node dysfunction.     

Altered Cortical Palmitoylation Induces Widespread Molecular Disturbances in Parkinson’s Disease

The relationship between Parkinson’s disease (PD), the second-most common neurodegenerative disease after Alzheimer’s disease, and palmitoylation, a post-translational lipid modification, is not well understood. In this study, to better understand the role of protein palmitoylation in PD and the pathways altered in this disease, we analyzed the differential palmitoyl proteome (palmitome) in the cerebral cortex of PD patients compared to controls (n = 4 per group). Data-mining of the cortical palmitome from PD patients and controls allowed us to: (i) detect a set of 150 proteins with altered palmitoylation in PD subjects in comparison with controls; (ii) describe the biological pathways and targets predicted to be altered by these palmitoylation changes; and (iii) depict the overlap between the differential palmitome identified in our study with protein interactomes of the PD-linked proteins α-synuclein, LRRK2, DJ-1, PINK1, GBA and UCHL1. In summary, we partially characterized the altered palmitome in the cortex of PD patients, which is predicted to impact cytoskeleton, mitochondrial and fibrinogen functions, as well as cell survival. Our study suggests that protein palmitoylation could have a role in the pathophysiology of PD, and that comprehensive palmitoyl-proteomics offers a powerful approach for elucidating novel cellular pathways modulated in this neurodegenerative disease.     

Crystal Structure of CYP3A4 Complexed with Fluorol Identifies the Substrate Access Channel as a High-Affinity Ligand Binding Site

Cytochrome P450 3A4 (CYP3A4) is a major human drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity, allosteric behavior, and implications in drug–drug interactions. Despite extensive investigations, the mechanism of ligand binding to CYP3A4 is not fully understood. We determined the crystal structure of CYP3A4 complexed with fluorol, a small fluorescent dye that can undergo hydroxylation. In the structure, fluorol associates to the substrate channel, well suited for the binding of planar polyaromatic molecules bearing polar groups, through which stabilizing H-bonds with the polar channel residues, such as Thr224 and Arg372, can be established. Mutagenesis, spectral, kinetic, and functional data confirmed the involvement but not strict requirement of Thr224 for the association of fluorol. Collectively, our data identify the substrate channel as a high-affinity ligand binding site and support the notion that hydrophobic ligands first dock to the nearby peripheral surface, before migrating to the channel and, subsequently, into the active site.