A reference genome for the Andean cavefish Trichomycterus rosablanca (Siluriformes, Trichomycteridae): Building genomic resources to study evolution in cave environments.

Animals living in caves are of broad relevance to evolutionary biologists interested in understanding the mechanisms underpinning convergent evolution. In the Eastern Andes of Colombia, populations from at least two distinct clades of Trichomycterus catfishes (Siluriformes) independently colonized cave environments and converged in phenotype by losing their eyes and pigmentation. We are pursuing several research questions using genomics to understand the evolutionary forces and molecular mechanisms responsible for repeated morphological changes in this system. As a foundation for such studies, here we describe a diploid, chromosome-scale, long-read reference genome for Trichomycterus rosablanca, a blind, depigmented species endemic to the karstic system of the department of Santander. The nuclear genome comprises 1 Gb in 27 chromosomes, with a 40.0× HiFi long-read genome coverage having an N50 scaffold of 40.4 Mb and N50 contig of 13.1 Mb, with 96.9% (Eukaryota) and 95.4% (Actinopterygii) universal single-copy orthologs (BUSCO). This assembly provides the first reference genome for the speciose genus Trichomycterus, serving as a key resource for research on the genomics of phenotypic evolution.

Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes.

Merozoite surface antigen-1 is a glycoprotein expressed by Babesia bovis and is considered a vaccine candidate given that antibodies against it are able to partially block in vitro invasion of bovine erythrocytes. Despite this, no study to date has confirmed the target cell binding properties of the full MSA-1 or its fragments. This research has thus been focused on identifying protein regions playing a role in erythrocyte attachment, based on genetic diversity and natural selection analysis. Two regions under functional constraint (nucleotides 134-428 and 464-629) having a preponderance of negatively-selected signals were identified in silico. Three non-overlapping peptides derived from functionally constraint regions (42422 (39PEGSFYDDMSKFYGAVGSFD58), 42424 (91NALIKNNPMIRPDLFNATIV110) and 42426 (150TDIVEEDREKAVEYFKKHVY169)) were able to specifically bind to a sialoglycoprotein located on the bovine erythrocyte surface as confirmed by sensitive and specific peptide-cell interaction competition assays using both enzymatically treated and untreated red blood cells. Interestingly, it was predicted that peptides 42422 and 42426 have a helical structure and conserved motifs in all strain/isolates. These findings provide evidence, for the first time, related to B. bovis MSA-1 short regions used by the parasite in erythrocyte binding which could be predicted using natural selection analysis. Future work focused on evaluating these peptides' antigenic ability during natural infection, and their ability to induce protection in immunisation assays are needed to confirm their usefulness as synthetic vaccine candidates.

Phosphorous NMR Analysis and Activity of Chiral BINAP-Silver Complexes.

Chiral Ag-atropisomeric ligand species were studied in solution at different temperatures by 31 P-NMR spectroscopy. The analysis and understanding of key parameters in Ag-BINAP complexes were considered in the context of an enantioselective transformation. An efficient silver-catalyzed intramolecular [4+2] cycloaddition reaction of amide-1,6-enyne provided an enantiomerically enriched tricyclic compound using simple reagents and under mild reaction conditions.

SM-COLSARSPROT: Highly Immunogenic Supramutational Synthetic Peptides Covering the World's Population.

Fifty ~20-amino acid (aa)-long peptides were selected from functionally relevant SARS-CoV-2 S, M, and E proteins for trial B-21 and another 53 common ones, plus some new ones derived from the virus' main genetic variants for complementary trial C-21. Peptide selection was based on tremendous SARS-CoV-2 genetic variability for analysing them concerning vast human immunogenetic polymorphism for developing the first supramutational, Colombian SARS-protection (SM-COLSARSPROT), peptide mixture. Specific physicochemical rules were followed, i.e., aa predilection for polyproline type II left-handed (PPIIL) formation, replacing ß-branched, aromatic aa, short-chain backbone H-bond-forming residues, π-π interactions (n→π* and π-CH), aa interaction with π systems, and molecular fragments able to interact with them, disrupting PPIIL propensity formation. All these modified structures had PPIIL formation propensity to enable target peptide interaction with human leukocyte antigen-DRß1* (HLA-DRß1*) molecules to mediate antigen presentation and induce an appropriate immune response. Such modified peptides were designed for human use; however, they induced high antibody titres against S, M, and E parental mutant peptides and neutralising antibodies when suitably modified and chemically synthesised for immunising 61 major histocompatibility complex class II (MHCII) DNA genotyped Aotus monkeys (matched with their corresponding HLA-DRß1* molecules), predicted to cover 77.5% to 83.1% of the world's population. Such chemically synthesised peptide mixture represents an extremely pure, stable, reliable, and cheap vaccine for COVID-19 pandemic control, providing a new approach for a logical, rational, and soundly established methodology for other vaccine development.

The First Chemically-Synthesised, Highly Immunogenic Anti-SARS-CoV-2 Peptides in DNA Genotyped Aotus Monkeys for Human Use.

Thirty-five peptides selected from functionally-relevant SARS-CoV-2 spike (S), membrane (M), and envelope (E) proteins were suitably modified for immunising MHC class II (MHCII) DNA-genotyped Aotus monkeys and matched with HLA-DRß1* molecules for use in humans. This was aimed at producing the first minimal subunit-based, chemically-synthesised, immunogenic molecules (COLSARSPROT) covering several HLA alleles. They were predicted to cover 48.25% of the world's population for 6 weeks (short-term) and 33.65% for 15 weeks (long-lasting) as they induced very high immunofluorescent antibody (IFA) and ELISA titres against S, M and E parental native peptides, SARS-CoV-2 neutralising antibodies and host cell infection. The same immunological methods that led to identifying new peptides for inclusion in the COLSARSPROT mixture were used for antigenicity studies. Peptides were analysed with serum samples from patients suffering mild or severe SARS-CoV-2 infection, thereby increasing chemically-synthesised peptides' potential coverage for the world populations up to 62.9%. These peptides' 3D structural analysis (by 1H-NMR acquired at 600 to 900 MHz) suggested structural-functional immunological association. This first multi-protein, multi-epitope, minimal subunit-based, chemically-synthesised, highly immunogenic peptide mixture highlights such chemical synthesis methodology's potential for rapidly obtaining very pure, highly reproducible, stable, cheap, easily-modifiable peptides for inducing immune protection against COVID-19, covering a substantial percentage of the human population.

Babesia Bovis Ligand-Receptor Interaction: AMA-1 Contains Small Regions Governing Bovine Erythrocyte Binding.

Apical membrane antigen 1 is a microneme protein which plays an indispensable role during Apicomplexa parasite invasion. The detailed mechanism of AMA-1 molecular interaction with its receptor on bovine erythrocytes has not been completely defined in Babesia bovis. This study was focused on identifying the minimum B. bovis AMA-1-derived regions governing specific and high-affinity binding to its target cells. Different approaches were used for detecting ama-1 locus genetic variability and natural selection signatures. The binding properties of twelve highly conserved 20-residue-long peptides were evaluated using a sensitive and specific binding assay based on radio-iodination. B. bovis AMA-1 ectodomain structure was modelled and refined using molecular modelling software. NetMHCIIpan software was used for calculating B- and T-cell epitopes. The B. bovis ama-1 gene had regions under functional constraint, having the highest negative selective pressure intensity in the Domain I encoding region. Interestingly, B. bovis AMA-1-DI (100YMQKFDIPRNHGSGIYVDLG119 and 120GYESVGSKSYRMPVGKCPVV139) and DII (302CPMHPVRDAIFGKWSGGSCV321)-derived peptides had high specificity interaction with erythrocytes and bound to a chymotrypsin and neuraminidase-treatment sensitive receptor. DI-derived peptides appear to be exposed on the protein's surface and contain predicted B- and T-cell epitopes. These findings provide data (for the first-time) concerning B. bovis AMA-1 functional subunits which are important for establishing receptor-ligand interactions which could be used in synthetic vaccine development.

Malaria: Paving the way to developing peptide-based vaccines against invasion in infectious diseases.

Malaria remains a large-scale public health problem, killing more than 400,000 people and infecting up to 230 million worldwide, every year. Unfortunately, despite numerous efforts and research concerning vaccine development, results to date have been low and/or strain-specific. This work describes a strategy involving Plasmodium falciparum Duffy binding-like (DBL) and reticulocyte-binding protein homologue (RH) family-derived minimum functional peptides, netMHCIIpan3.2 parental and modified peptides' in silico binding prediction and modeling some Aotus major histocompatibility class II (MHCII) molecules based on known human molecules' structure to understand their differences. These are used to explain peptides' immunological behaviour when used as vaccine components in the Aotus model. Despite the great similarity between human and Aotus immune system molecules, around 50% of Aotus allele molecules lack a counterpart in the human immune system which could lead to an Aotus-specific vaccine. It was also confirmed that functional Plasmodium falciparum' conserved proteins are immunologically silent (in both the animal model and in-silico prediction); they must therefore be modified to elicit an appropriate immune response. Some peptides studied here had the desired behaviour and can thus be considered components of a fully-protective antimalarial vaccine.

Structural analysis of owl monkey MHC-DR shows that fully-protective malaria vaccine components can be readily used in humans.

More than 50 years ago the owl monkey (genus Aotus) was found to be highly susceptible to developing human malaria, making it an excellent experimental model for this disease. Microbes and parasites' (especially malaria) tremendous genetic variability became resolved during our malaria vaccine development, involving conserved peptides having high host cell binding activity (cHABPs); however, cHABPs are immunologically silent and must be specially modified (mHABPs) to induce a perfect fit into major histocompatibility complex (MHC) molecules (HLA in humans). Since malarial immunity is mainly antibody-mediated and controlled by the HLA-DRB genetic region, ∼1000 Aotus have been molecularly characterised for MHC-DRB, revealing striking similarity between human and Aotus MHC-DRB repertories. Such convergence suggested that a large group of immune protection-inducing protein structures (IMPIPS), highly immunogenic and protection inducers against malarial intravenous challenge in Aotus, could easily be used in humans for inducing full protection against malaria. We highlight the value of a logical and rational methodology for developing a vaccine in an appropriate animal model: Aotus monkeys.

Far from the Madding Crowd: the Molecular Basis for Immunological Escape of Plasmodium falciparum.

Like Thomas Hardy's famous novel Far from the Madding Crowd, Plasmodium falciparum parasites display their most relevant survival structures (proteins) involved in host cell invasion far away from the immune system's susceptible regions, displaying tremendous genetic variability, to attract the immune response and escape immune pressure. The 3D structure localisation of the conserved amino acid sequences of this deadly parasite's most relevant proteins involved in host cell invasion, as well as the location of the highly polymorphic, highly immunogenic regions, clearly demonstrates that such structures are far apart, sometimes 90° to 180° opposite, thereby rendering the immune response useless. It is also shown here that these conserved, functionally-relevant structures are immunologically silent, since no immune response has been induce.

Mycobacterium tuberculosis PE9 protein has high activity binding peptides which inhibit target cell invasion.

PE/PPE proteins are involved in several processes during Mycobacterium tuberculosis (Mtb) infection of target cells; studying them is extremely interesting as they are the only ones from the Mycobacterium genus, they abound in pathogenic species such as Mtb and their function remains yet unknown. The PE9 protein (Rv1088) was characterised, the rv1088 gene was identified by PCR in Mtb complex strains and its expression and localisation on mycobacterial surface was confirmed by Western blot and immunoelectron microscopy. Bioinformatics tools were used for predicting PE9 protein structural aspects and experimental study involved the circular dichroism of synthetic peptides. The peptides were tested in binding assays involving U937 and A549 cells; two high activity binding peptides (HABPs) were found for both cell lines (39226-(1)MSYMIATPAALTAAATDIDGI(21) and 39232-(125)YQRHFGTGGQPEFRQHSEHRR(144)), one for U937 (39231-(104)YAGAGRRQRRRRSGDGQWRLRQ(124)) and one for A549 (39230-(83)YGTGVFRRRRGRQTVTAAEHRA(103)). HABP 39232 inhibited mycobacterial entry to A549 cells (∼70%) and U937 cells (∼50%), peptides 39226 and 39231 inhibited entry to U937 cells (∼60% and 80%, respectively) and peptide 39230 inhibited entry to A549 cells (∼60%). This emphasised HABPs' functional importance in recognition between Mtb H37Rv and target cell receptors. These peptide sequences could be involved in invasion and were recognised by the host's immune system, thereby highlighting their use when designing an efficient anti-tuberculosis multiantigenic vaccine.

Immune protection-inducing protein structures (IMPIPS) against malaria: the weapons needed for beating Odysseus.

The review covers the functional and structural approach followed by our group for more than 34 years in the search for a methodology that allows the rational design of chemically synthesised vaccines. An analogy between Odysseus, the cunning hero of the epic poem Odyssey by Homer, and the elusive Plasmodium parasite has been made, to review our research group's main considerations when developing a rational methodology for designing second generation, modified peptide-based, minimal subunit, multi-antigen, multi-stage, chemically synthesised vaccines against Plasmodium falciparum malaria.