Laminin alpha 5 regulates mammary gland remodeling through luminal cell differentiation and Wnt4-mediated epithelial crosstalk.

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.

MHCBI: a pipeline for calculating peptide-MHC binding energy using semi-empirical quantum mechanical methods with explicit/implicit solvent models.

Experimentally estimating peptide-major histocompatibility complex (pMHC) binding affinity has been quite challenging due to the many receptors and the many potential ligands implicated in it. We have thus proposed a straightforward computational methodology considering the different mechanisms involved in pMHC binding to facilitate studying such receptor-ligand interactions. We have developed a pipeline using semi-empirical quantum mechanical methods for calculating pMHC class I and II molecules' binding energy (BE). This pipeline can systematize the methodology for calculating pMHC system BE, enabling the rational design of T-cell epitopes to be used as pharmaceuticals and vaccines.

Plasmodium falciparum rhoptry neck protein 4 has conserved regions mediating interactions with receptors on human erythrocytes and hepatocyte membrane.

Plasmodium falciparum-related malaria represents a serious worldwide public health problem due to its high mortality rates. P. falciparum expresses rhoptry neck protein 4 (PfRON4) in merozoite and sporozoite rhoptries, it participates in tight junction-TJ formation via the AMA-1/RON complex and is refractory to complete genetic deletion. Despite this, which PfRON4 key regions interact with host cells remain unknown; such information would be useful for combating falciparum malaria. Thirty-two RON4 conserved region-derived peptides were chemically synthesised for determining and characterising PfRON4 regions having high host cell binding affinity (high activity binding peptides or HABPs). Receptor-ligand interaction/binding assays determined their specific binding capability, the nature of their receptors and their ability to inhibit in vitro parasite invasion. Peptides 42477, 42479, 42480, 42505 and 42513 had greater than 2% erythrocyte binding activity, whilst peptides 42477 and 42480 specifically bound to HepG2 membrane, both of them having micromolar and submicromolar range dissociation constants (Kd). Cell-peptide interaction was sensitive to treating erythrocytes with trypsin and/or chymotrypsin and HepG2 with heparinase I and chondroitinase ABC, suggesting protein-type (erythrocyte) and heparin and/or chondroitin sulphate proteoglycan receptors (HepG2) for PfRON4. Erythrocyte invasion inhibition assays confirmed HABPs' importance during merozoite invasion. PfRON4 800-819 (42477) and 860-879 (42480) regions specifically interacted with host cells, thereby supporting their inclusion in a subunit-based, multi-antigen, multistage anti-malarial vaccine.

PATTERNS OF SUBRETINAL AND/OR INTRARETINAL FLUID RECURRENCE IN PATIENTS WHO RECEIVED AS-NEEDED RANIBIZUMAB THERAPY IN THE HARBOR TRIAL.

PURPOSE: To evaluate subretinal fluid (SRF) and/or intraretinal fluid recurrence in patients with neovascular age-related macular degeneration who received as-needed (PRN) ranibizumab in a HARBOR (NCT00891735) post hoc analysis. METHODS: Analyses included patients with SRF and/or intraretinal fluid at baseline and fluid recurrence after a ≥3-month absence (N = 222). Baseline fluid location(s) were compared with location of recurrence after a ≥3-month absence. RESULTS: At baseline, fluid was equally distributed across all locations. On recurrence, the location was most frequently central (69%). Eyes with central fluid at baseline typically had recurrence in the same location (72% vs. 47%-53% with fluid in other locations). The type of recurrent fluid was typically the same as at baseline (SRF, 64%; intraretinal fluid, 75%). Overall, 37% (39/105) of eyes exhibited fluid recurrence in a new location, most frequently central (53%). There was a significant gain in best-corrected visual acuity (mean [95% confidence interval], +2.2 [0.4-4.0] letters) between the months of SRF resolution and recurrence. CONCLUSION: Although the location of SRF and/or intraretinal fluid was equally distributed at baseline, recurrent fluid was typically centrally located. The authors identified a subgroup of eyes exhibiting fluid recurrence in a different location than at baseline, potentially indicating new choroidal neovascularization.

A laminin 511 matrix is regulated by TAZ and functions as the ligand for the α6Bß1 integrin to sustain breast cancer stem cells.

Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 matrix that promotes self-renewal and tumor initiation by engaging the α6Bß1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the α5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.

Developing Anti-Babesia bovis Blood Stage Vaccines: A New Perspective Regarding Synthetic Vaccines.

Bovine babesiosis is caused by the Apicomplexa parasites from the genus Babesia. It is one of the most important tick-borne veterinary diseases worldwide; Babesia bovis being the species associated with the most severe clinical signs of the disease and causing the greatest economic losses. Many limitations related to chemoprophylaxis and the acaricides control of transmitting vectors have led to the adoption of live attenuated vaccine immunisation against B. bovis as an alternative control strategy. However, whilst this strategy has been effective, several drawbacks related to its production have prompted research into alternative methodologies for producing vaccines. Classical approaches for developing anti-B. bovis vaccines are thus discussed in this review and are compared to a recent functional approach to highlight the latter's advantages when designing an effective synthetic vaccine targeting this parasite.

Clinical manifestations and immune response to tuberculosis.

Tuberculosis is a far-reaching, high-impact disease. It is among the top ten causes of death worldwide caused by a single infectious agent; 1.6 million tuberculosis-related deaths were reported in 2021 and it has been estimated that a third of the world's population are carriers of the tuberculosis bacillus but do not develop active disease. Several authors have attributed this to hosts' differential immune response in which cellular and humoral components are involved, along with cytokines and chemokines. Ascertaining the relationship between TB development's clinical manifestations and an immune response should increase understanding of tuberculosis pathophysiological and immunological mechanisms and correlating such material with protection against Mycobacterium tuberculosis. Tuberculosis continues to be a major public health problem globally. Mortality rates have not decreased significantly; rather, they are increasing. This review has thus been aimed at deepening knowledge regarding tuberculosis by examining published material related to an immune response against Mycobacterium tuberculosis, mycobacterial evasion mechanisms regarding such response and the relationship between pulmonary and extrapulmonary clinical manifestations induced by this bacterium which are related to inflammation associated with tuberculosis dissemination through different routes.

The Black Box of Cellular and Molecular Events of Plasmodium vivax Merozoite Invasion into Reticulocytes.

Plasmodium vivax is the most widely distributed malaria parasite affecting humans worldwide, causing ~5 million cases yearly. Despite the disease's extensive burden, there are gaps in the knowledge of the pathophysiological mechanisms by which P. vivax invades reticulocytes. In contrast, this crucial step is better understood for P. falciparum, the less widely distributed but more often fatal malaria parasite. This discrepancy is due to the difficulty of studying P. vivax's exclusive invasion of reticulocytes, which represent 1-2% of circulating cells. Its accurate targeting mechanism has not yet been clarified, hindering the establishment of long-term continuous in vitro culture systems. So far, only three reticulocyte invasion pathways have been characterised based on parasite interactions with DARC, TfR1 and CD98 host proteins. However, exposing the parasite's alternative invasion mechanisms is currently being considered, opening up a large field for exploring the entry receptors used by P. vivax for invading host cells. New methods must be developed to ensure better understanding of the parasite to control malarial transmission and to eradicate the disease. Here, we review the current state of knowledge on cellular and molecular mechanisms of P. vivax's merozoite invasion to contribute to a better understanding of the parasite's biology, pathogenesis and epidemiology.

The molecular basis for peptide-based antimalarial vaccine development targeting erythrocyte invasion by P. falciparum.

This work describes a methodology for developing a minimal, subunit-based, multi-epitope, multi-stage, chemically-synthesised, anti-Plasmodium falciparum malaria vaccine. Some modified high activity binding peptides (mHABPs) derived from functionally relevant P. falciparum MSP, RH5 and AMA-1 conserved amino acid regions (cHABPs) for parasite binding to and invasion of red blood cells (RBC) were selected. They were highly immunogenic as assessed by indirect immunofluorescence (IFA) and Western blot (WB) assays and protective immune response-inducers against malarial challenge in the Aotus monkey experimental model. NetMHCIIpan 4.0 was used for predicting peptide-Aotus/human major histocompatibility class II (MHCII) binding affinity in silico due to the similarity between Aotus and human immune system molecules; ∼50% of Aotus MHCII allele molecules have a counterpart in the human immune system, being Aotus-specific, whilst others enabled recognition of their human counterparts. Some peptides' 1H-NMR-assessed structural conformation was determined to explain residue modifications in mHABPs inducing secondary structure changes. These directly influenced immunological behaviour, thereby highlighting the relationship with MHCII antigen presentation. The data obtained in such functional, immunological, structural and predictive approach suggested that some of these peptides could be excellent components of a fully-protective antimalarial vaccine.

Characterising four Sarconesiopsis magellanica (Diptera: Calliphoridae) larval fat body-derived antimicrobial peptides.

BACKGROUND: The inappropriate use of antibiotics has led to the accelerated growth of resistance to antibiotics. The search for new therapeutic strategies (i.e., antimicrobial peptides-AMPs) has thus become a pressing need. OBJECTIVE: Characterising and evaluating Sarconesiopsis magellanica larval fat body-derived AMPs. METHODS: Fat body extracts were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC); mass spectrometry was used for characterising the primary structure of the AMPs so found. ProtParam (Expasy) was used for analysing the AMPs' physico-chemical properties. Synthetic AMPs' antibacterial activity was evaluated. FINDINGS: Four new AMPs were obtained and called sarconesin III, IV, V and VI. Sarconesin III had an α-helix structure and sarconesins IV, V and VI had linear formations. Oligomer prediction highlighted peptide-peptide interactions, suggesting that sarconesins III, V and VI could form self-aggregations when in contact with the microbial membrane. AMPs synthesised from their native molecules' sequences had potent activity against Gram-positive bacteria and, to a lesser extent, against Gram-negative and drug-resistant bacteria. Sarconesin VI was the most efficient AMP. None of the four synthetic AMPs had a cytotoxic effect. MAIN CONCLUSIONS: S. magellanica larval fat body-derived antimicrobial peptides are an important source of AMPs and could be used in different antimicrobial therapies and overcoming bacterial resistance.

Structural Modelling of KCNQ1 and KCNH2 Double Mutant Proteins, Identified in Two Severe Long QT Syndrome Cases, Reveals New Insights into Cardiac Channelopathies.

Congenital long QT syndrome (LQTS) is a cardiac channelopathy characterized by a prolongation of the QT interval and T-wave abnormalities, caused, in most cases, by mutations in KCNQ1, KCNH2, and SCN5A. Although the predominant pattern of LQTS inheritance is autosomal dominant, compound heterozygous mutations in genes encoding potassium channels have been reported, often with early disease onset and more severe phenotypes. Since the molecular mechanisms underlying severe phenotypes in carriers of compound heterozygous mutations are unknown, it is possible that these compound mutations lead to synergistic or additive alterations to channel structure and function. In this study, all-atom molecular dynamic simulations of KCNQ1 and hERG channels were carried out, including wild-type and channels with compound mutations found in two patients with severe LQTS phenotypes and limited family history of the disease. Because channels can likely incorporate different subunit combinations from different alleles, there are multiple possible configurations of ion channels in LQTS patients. This analysis allowed us to establish the structural impact of different configurations of mutant channels in the activated/open state. Our data suggest that channels with these mutations show moderate changes in folding energy (in most cases of stabilizing character) and changes in channel mobility and volume, differentiating them from each other and from WT. This would indicate possible alterations in K+ ion flow. Hetero-tetrameric mutant channels showed intermediate structural and volume alterations vis-à-vis homo-tetrameric channels. These findings support the hypothesis that hetero-tetrameric channels in patients with compound heterozygous mutations do not necessarily lead to synergistic structural alterations.

Two 20-Residue-Long Peptides Derived from Plasmodium vivax Merozoite Surface Protein 10 EGF-Like Domains Are Involved in Binding to Human Reticulocytes.

Plasmodium parasites' invasion of their target cells is a complex, multi-step process involving many protein-protein interactions. Little is known about how complex the interaction with target cells is in Plasmodium vivax and few surface molecules related to reticulocytes' adhesion have been described to date. Natural selection, functional and structural analysis were carried out on the previously described vaccine candidate P. vivax merozoite surface protein 10 (PvMSP10) for evaluating its role during initial contact with target cells. It has been shown here that the recombinant carboxyl terminal region (rPvMSP10-C) bound to adult human reticulocytes but not to normocytes, as validated by two different protein-cell interaction assays. Particularly interesting was the fact that two 20-residue-long regions (388DKEECRCRANYMPDDSVDYF407 and 415KDCSKENGNCDVNAECSIDK434) were able to inhibit rPvMSP10-C binding to reticulocytes and rosette formation using enriched target cells. These peptides were derived from PvMSP10 epidermal growth factor (EGF)-like domains (precisely, from a well-defined electrostatic zone) and consisted of regions having the potential of being B- or T-cell epitopes. These findings provide evidence, for the first time, about the fragments governing PvMSP10 binding to its target cells, thus highlighting the importance of studying them for inclusion in a P. vivax antimalarial vaccine.

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.

NHP-immunome: A translational research-oriented database of non-human primate immune system proteins.

We are currently living the advent of a new age for medicine in which basic research is being quickly translated into marketable drugs, and the widespread access to genomics data is allowing the design and implementation of personalized solutions to medical conditions. Non-human primates (NHP) have gained an essential role in drug discovery and safety testing due to their close phylogenetic relationship to humans. In this study, a collection of well characterized genes of the human immune system was used to define the orthology-based immunome in four NHP species, with carefully curated annotations available based on multi-tissue RNA-seq datasets. A broad variation in the frequency of expressed protein isoforms was observed between species. Finally, this analysis also revealed the lack of expression of at least four different chemokines in new-world primates. In addition, transcripts corresponding to four genes including interleukin 12 subunit alpha were expressed in humans but no other primate species analyzed. Access to the non-human primate immunome is available in http://www.fidic.org.co:90/proyecto/.

Plasmodium falciparum pre-erythrocytic stage vaccine development.

Worldwide strategies between 2010 and 2017 aimed at controlling malarial parasites (mainly Plasmodium falciparum) led to a reduction of just 18% regarding disease incidence rates. Many biologically-derived anti-malarial vaccine candidates have been developed to date; this has involved using many experimental animals, an immense amount of work and the investment of millions of dollars. This review provides an overview of the current state and the main results of clinical trials for sporozoite-targeting vaccines (i.e. the parasite stage infecting the liver) carried out by research groups in areas having variable malaria transmission rates. However, none has led to promising results regarding the effective control of the disease, thereby making it necessary to complement such efforts at finding/introducing new vaccine candidates by adopting a multi-epitope, multi-stage approach, based on minimal subunits of the main sporozoite proteins involved in the invasion of the liver.

Sexual forms obtained in a continuous in vitro cultured Colombian strain of Plasmodium falciparum (FCB2).

BACKGROUND: The epidemiological control of malaria has been hampered by the appearance of parasite resistance to anti-malarial drugs and by the resistance of mosquito vectors to control measures. This has also been associated with weak transmission control, mostly due to poor control of asymptomatic patients associated with host-vector transmission. This highlights the importance of studying the parasite's sexual forms (gametocytes) which are involved in this phase of the parasite's life-cycle. Some African and Asian strains of Plasmodium falciparum have been fully characterized regarding sexual forms' production; however, few Latin-American strains have been so characterized. This study was aimed at characterizing the Colombian FCB2 strain as a gametocyte producer able to infect mosquitoes. METHODS: Gametocyte production was induced in in vitro cultured P. falciparum FCB2 and 3D7 strains. Pfap2g and Pfs25 gene expression was detected in FCB2 strain gametocyte culture by RT-PCR. Comparative analysis of gametocytes obtained from both strains was made (counts and morphological changes). In vitro zygote formation from FCB2 gametocytes was induced by incubating a gametocyte culture sample at 27 °C for 20 min. A controlled Anopheles albimanus infection was made using an artificial feed system with cultured FCB2 gametocytes (14-15 days old). Mosquito midgut dissection was then carried out for analyzing oocysts. RESULTS: The FCB2 strain expressed Pfap2g, Pfs16, Pfg27/25 and Pfs25 sexual differentiation-related genes after in vitro sexual differentiation induction, producing gametocytes that conserved the expected morphological features. The amount of FCB2 gametocytes produced was similar to that from the 3D7 strain. FCB2 gametocytes were differentiated into zygotes and ookinetes after an in vitro low-temperature stimulus and infected An. albimanus mosquitoes, developing to oocyst stage. CONCLUSIONS: Even with the history of long-term FCB2 strain in vitro culture maintenance, it has retained its sexual differentiation ability. The gametocytes produced here preserved these parasite forms' usual characteristics and An. albimanus infection capability, thus enabling its use as a tool for studying sexual form biology, An. albimanus infection comparative analysis and anti-malarial drug and vaccine development.

p-Methoxyphenol: A potent and effective scavenger for solid-phase peptide synthesis.

During the final step of t-Boc/Bzl, solid-phase peptide synthesis (SPPS)-protecting groups from amino acids (aa) side chains must be removed from the target peptides during cleavage from the solid support. These reaction steps involve hydrolysis with hydrogen fluoride (HF) in the presence of a nucleophile (scavenger), whose function is to trap the carbocations produced during SN 1-type reactions. Five peptide sequences were synthesised for evaluating p-methoxyphenol effectiveness as a potent scavenger. After the synthesis, the resin-peptide was then separated into two equal parts to be cleaved using two scavengers: conventional reactive p-cresol (reported in the literature as an effective acyl ion eliminator) and p-methoxyphenol (hypothesised as fulfilling the same functions as the routinely used scavenger). Detailed analysis of the electrostatic potential map (EPM) revealed similarities between these two nucleophiles, regarding net atomic charge, electron density distribution, and similar pKa values. Good scavenger efficacy was observed by chromatography and mass spectrometry results for the synthesised molecules, which revealed that p-methoxyphenol can be used as a potent scavenger during SPPS by t-Boc/Bzl strategy, as similar results were obtained using the conventional scavenger.

Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections.

The World Health Organisation (WHO) has placed twenty diseases into a group known as neglected tropical diseases (NTDs), twelve of them being parasitic diseases: Chagas' disease, cysticercosis/taeniasis, echinococcosis, food-borne trematodiasis, human African trypanosomiasis (sleeping sickness), leishmaniasis, lymphatic filariasis, onchocerciasis (river blindness), schistosomiasis, soil-transmitted helminthiasis (ascariasis, hookworm, trichuriasis), guinea-worm and scabies. Such diseases affect millions of people in developing countries where one of the main problems concerning the control of these diseases is diagnosis-based due to the most affected areas usually being far from laboratories having suitable infrastructure and/or being equipped with sophisticated equipment. Advances have been made during the last two decades regarding standardising and introducing techniques enabling diagnoses to be made in remote places, i.e., the loop-mediated isothermal amplification (LAMP) technique. This technique's advantages include being able to perform it using simple equipment, diagnosis made directly in the field, low cost of each test and the technique's high specificity. Using this technique could thus contribute toward neglected parasite infection (NPI) control and eradication programmes. This review describes the advances made to date regarding LAMP tests, as it has been found that even though several studies have been conducted concerning most NPI, information is scarce for others.

Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion.

Protein-protein interactions (IPP) play an essential role in practically all biological processes, including those related to microorganism invasion of their host cells. It has been found that a broad repertoire of receptor-ligand interactions takes place in the binding interphase with host cells in malaria, these being vital interactions for successful parasite invasion. Several trials have been conducted for elucidating the molecular interface of interactions between some Plasmodium falciparum and Plasmodium vivax antigens with receptors on erythrocytes and/or reticulocytes. Structural information concerning these complexes is available; however, deeper analysis is required for correlating structural, functional (binding, invasion, and inhibition), and polymorphism data for elucidating new interaction hotspots to which malaria control methods can be directed. This review describes and discusses recent structural and functional details regarding three relevant interactions during erythrocyte invasion: Duffy-binding protein 1 (DBP1)-Duffy antigen receptor for chemokines (DARC); reticulocyte-binding protein homolog 5 (PfRh5)-basigin, and erythrocyte binding antigen 175 (EBA175)-glycophorin A (GPA).