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We explored the hypothesis that progesterone direct effect on Trichinella spiralis might be mediated indeed by a new steroid-binding parasite protein. Our first results showed that Progesterone decreases the parasite molting rate. We amplify, isolated, cloned and sequenced the PGRMC2 sequence using specific primers from known species. Furthermore, we expressed the protein and developed an antibody to performance immunofluorescent confocal microscopy, where detected that parasite cells showed expression of a P4-binding protein exclusively located at the oocyte and the parasite´s cuticle. Presence of the PGRMC2 protein in these cells was also confirmed by western blot and flow cytometry. Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC2 is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone, and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that Trichinella spiralis PGRMC2 is related to a steroid-binding protein of another platyhelminths. Progesterone may probably act upon Trichinella spiralis oocytes probably by binding to PGRMC2. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.

BackgroundIn contrast to developed countries, in Mexico more than half of COVID-19 deaths have occurred in adults MethodsTo explore what contributes to the high COVID-19 mortality in Mexican middle-aged adults, we calculated age-stratified COVID-19 case fatality rates, YLL and relative risk (RR) of 9 comorbidities and 23 comorbidity combinations in a retrospective Mexican cohort with 905,579 PCR-confirmed COVID-19 cases/89,167 deaths, until Nov/2/2020.FindingsChronic kidney disease (CKD) had the highest RR for COVID-19 fatality, followed by diabetes and immunosuppression, that in turn had higher RR than obesity or hypertension as single comorbidities. The combination diabetes/hypertension with or without obesity had RR as high as CKD as a single comorbidity (>3 in ConclusionsThe Mexican COVID-19 outbreak illustrates that middle-aged adults 45-64-yo can have high COVID-19 mortality during large outbreaks, especially if they present chronic metabolic comorbidities, but also in their absence, making them an important group of concern after elders. COVID-19 mortality in middle-aged adults is likely proportional to the gradual decline in health that accompanies ageing, which presents earlier in poorer populations that also get more exposed to SARS-CoV-2 and have less access to specialized medical attention.

Toxoplasmosis is a zoonotic disease caused by the apicomplexa protozoan parasiteToxoplasma gondii. This disease is a health burden, mainly in pregnant women and immunocompromised individuals, in whom they can cause death. Despite advances in the medical area, nowadays there are no new drugs to treat toxoplasmosis. The standard therapy to toxoplasmosis has not had progress for last seven decades; it is a combination of sulfadiazine-pyrimethamine (S-P); which is co-administered with folic acid due to the adverse effects of the drug. Several studies have shown that the conventional treatment has limited effectiveness and severe adverse effects. Thus, the search of better treatments with greater efficacy and without the adverse effects becomes relevant. In the current work we demonstrate for the first time the parasiticidal effect of dehydroepiandrosterone (DHEA), a steroid hormone produced by many mammals, on extracellular tachyzoites (the infective stage ofT. gondii). In vitro treatment with DHEA reduces the viability of extracellular tachyzoites, and both the active and passive invasion processes. The ultrastructural analysis of treated parasites showed that DHEA alters the cytoskeleton structures, leading in the lost of the organelle structure and organization, as well as, the lost of the cellular shape. On a molecular level, we observed an important reduction of the expression of several proteins that are essential for the motility and virulence of parasites when they were exposed to DHEA. These results suggest that DHEA could be used as an alternative treatment against toxoplasmosis.

Background We have previously reported that progesterone (P4) has a direct in vitro effect on the scolex evagination and growth of Taenia solium cysticerci. Here, we explored the hypothesis that the P4 direct effect on T. solium might be mediated by a novel steroid-binding parasite protein. Methods By way of using immunofluorescent confocal microscopy, flow cytometry analysis, double-dimension electrophoresis analysis, and sequencing the corresponding protein spot, we detected a novel PGRMC in T. solium. Molecular modeling studies accompanied by computer docking using the sequenced protein, together with phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is from parasite origin. Results Our results show that P4 in vitro increases parasite evagination and scolex size. Using immunofluorescent confocal microscopy, we detected that parasite cells showed expression of a P4-binding like protein exclusively located at the cysticercus subtegumental tissue. Presence of the P4-binding protein in cyst cells was also confirmed by flow cytometry. Double-dimension electrophoresis analysis, followed by sequencing the corresponding protein spot, revealed a protein that was previously reported in the T. solium genome belonging to a membrane-associated progesterone receptor component (PGRMC). Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus, both of them being nested within a cluster including similar proteins present in platyhelminths such as Schistocephalus solidus and Schistosoma haematobium. Conclusion Progesterone may directly act upon T. solium cysticerci probably by binding to PGRMC. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions. Electronic supplementary material The online version of this article (10.1186/s13071-018-2703-1) contains supplementary material, which is available to authorized users.

Toxoplasmosis is a zoonotic disease caused by the apicomplexa protozoan parasite Toxoplasma gondii. This disease is a health burden, mainly in pregnant women and immunocompromised individuals. Dehydroepiandrosterone (DHEA) has proved to be an important molecule that could drive resistance against a variety of infections, including intracellular parasites such as Plasmodium falciparum and Trypanozoma cruzi, among others. However, to date, the role of DHEA on T. gondii has not been explored. Here, we demonstrated for the first time the toxoplasmicidal effect of DHEA on extracellular tachyzoites. Ultrastructural analysis of treated parasites showed that DHEA alters the cytoskeleton structures, leading to the loss of the organelle structure and organization as well as the loss of the cellular shape. In vitro treatment with DHEA reduces the viability of extracellular tachyzoites and the passive invasion process. Two-dimensional (2D) SDS-PAGE analysis revealed that in the presence of the hormone, a progesterone receptor membrane component (PGRMC) with a cytochrome b5 family heme/steroid binding domain-containing protein was expressed, while the expression of proteins that are essential for motility and virulence was highly reduced. Finally, in vivo DHEA treatment induced a reduction of parasitic load in male, but not in female mice.

Pesticides are used extensively in agriculture, and their residues in food must be monitored to prevent toxicity. The most abundant protein in cow&rsquo, s milk, &beta, lactoglobulin (BLG), shows high affinity for diverse hydrophobic ligands in its central binding pocket, called the calyx. Several of the most frequently used pesticides are hydrophobic. To predict if BLG may be an unintended carrier for pesticides, we tested its ability to bind 555 pesticides and their isomers, for a total of 889 compounds, in a rigid docking screen. We focused on the analysis of 60 unique molecules belonging to the five pesticide classes defined by the World Health Organization, that docked into BLG&rsquo, s calyx with &Delta, Gs ranging from &minus, 8.2 to &minus, 12 kcal mol&minus, 1, chosen by statistical criteria. These &ldquo, potential ligands&rdquo, were further analyzed using molecular dynamic simulations, and the binding energies were explored with Molecular Mechanics/Generalized Born/Surface Area (MMGBSA). Hydrophobic pyrethroid insecticides, like cypermethrin, were found to bind as deeply and tightly into the calyx as BLG&rsquo, s natural ligand, palmitate, while polar compounds, like paraquat, were expelled. Our results suggest that BLG could be a carrier for pesticides, in particular for pyrethroid insecticides, allowing for their accumulation in cow&rsquo, s milk beyond their solubility restrictions. This analysis opens possibilities for pesticide biosensor design based on BLG.

Periplasmic Binding Proteins (PBPs) trap nutrients for their internalization into bacteria by ABC transporters. Ligand binding triggers PBP closure by bringing its two domains together like a Venus flytrap. The atomic determinants that control PBP opening and closure for nutrient capture and release are not known, although it is proposed that opening and ligand release occur while in contact with the ABC transporter for concurrent substrate translocation. In this paper we evaluated the effect of the isomerization of a conserved proline, located near the binding site, on the propensity of PBPs to open and close. ArgT/LAO from Salmonella typhimurium and HisJ from Escherichia coli were studied through molecular mechanics at two different temperatures: 300 and 323 K. Eight microseconds were simulated per protein to analyze protein opening and closure in the absence of the ABC transporter. We show that when the studied proline is in trans, closed empty LAO and HisJ can open. In contrast, with the proline in cis, opening transitions were much less frequent and characterized by smaller changes. The proline in trans also renders the open trap prone to close over a ligand. Our data suggest that the isomerization of this conserved proline modulates the PBP mechanism: the proline in trans allows the exploration of conformational space to produce trap opening and closure, while in cis it restricts PBP movement and could limit ligand release until in productive contact with the ABC transporter. This is the first time that a proline isomerization has been related to the control of a large conformational change like the PBP flytrap mechanism.

Summary Protein–DNA recognition is fundamental to transcriptional regulation. Transcription factors must be capable of locating their specific sites situated throughout the genome and distinguishing them from related sites. Mlc and NagC control uptake and use of the sugars, glucose and N-acetylglucosamine. Both their helix–turn–helix motifs and their consensus binding sites on DNA are very similar. One distinguishing feature is that most NagC sites have a C/G bp at positions −11 and +11 from the centre of symmetry of the operator, while all Mlc sites have A/T. By constructing Mlc and NagC chimeras, we show that the helix–turn–helix motif per se is not responsible for specific recognition of Mlc or NagC sites, but that a linker, joining the DNA-binding domain to the rest of the protein, is the major determinant. We show that a change of just two amino acids in the NagC linker is sufficient to allow NagC to recognize an A/T bp at positions +/−11 and repress Mlc targets. Modelling of the NagC linker suggests that it forms an extended structure containing two arginines and we suggest that these arginines interact differently with the minor groove at positions +/−11 depending upon the presence of a C/G or A/T bp.

The effects of testosterone (T4) and dihydrotestosterone (DHT) on the survival of the helminth cestode parasite Taenia crassiceps, as well as their effects on actin, tubulin and myosin expression and their assembly into the excretory system of flame cells are described in this paper. In vitro evaluations on parasite viability, flow cytometry, confocal microscopy, video-microscopy of live flame cells, and docking experiments of androgens interacting with actin, tubulin, and myosin were conducted. Our results show that T4 and DHT reduce T. crassiceps viability in a dose- and time-dependent fashion, reaching 90% of mortality at the highest dose used (40 ng/ml) and time exposed (10 days) in culture. Androgen treatment does not induce differences in the specific expression pattern of actin, tubulin, and myosin isoforms as compared with control parasites. Confocal microscopy demonstrated a strong disruption of the parasite tegument, with reduced assembly, shape, and motion of flame cells. Docking experiments show that androgens are capable of affecting parasite survival and flame cell morphology by directly interacting with actin, tubulin and myosin without altering their protein expression pattern. We show that both T4 and DHT are able to bind actin, tubulin, and myosin affecting their assembly and causing parasite intoxication due to impairment of flame cell function. Live flame cell video microscopy showing a reduced motion as well changes in the shape of flame cells are also shown. In summary, T4 and DHT directly act on T. crassiceps cysticerci through altering parasite survival as well as the assembly and function of flame cells.

NagC and Mlc, paralogous members of the ROK family of proteins with almost identical helix-turn-helix DNA binding motifs, specifically regulate genes for transport and utilization of N-acetylglucosamine and glucose. We previously showed that two amino acids in a linker region outside the canonical helix-turn-helix motif are responsible for Mlc site specificity. In this work we identify four amino acids in the linker, which are required for recognition of NagC targets. These amino acids allow Mlc and NagC to distinguish between a C/G and an A/T bp at positions ±11 of the operators. One linker position, glycine in NagC and arginine in Mlc, corresponds to the major specificity determinant for the two proteins. In certain contexts it is possible to switch repression from Mlc-style to NagC-style, by interchanging this glycine and arginine. Secondary determinants are supplied by other linker positions or the helix-turn-helix motif. A wide genomic survey of unique ROK proteins shows that glycine- and arginine-rich sequences are present in the linkers of nearly all ROK family repressors. Conserved short sequence motifs, within the branches of the ROK evolutionary tree, suggest that these sequences could also be involved in operator recognition in other ROK family members.

Mg-ATP particles from bovine heart mitochondria have more than 95% of their F1 in complex with the inhibitor protein (IF1). The F1–IF1 complex was solubilized and purified. The question addressed was if this naturally occurring complex existed as monomers or dimers. Size exclusion chromatography and electron microscopy showed that most of the purified F1–IF1 complex was a dimer of two F1–IF1. As determined by the former method, the relative concentrations of dimeric and monomeric F1–IF1 depended on the concentration of protein that was applied to the column. Apparently, there is an equilibrium between the two forms of F1–IF1.

Mitochondrial F1FO-ATP synthase of chlorophycean algae is a complex partially embedded in the inner mitochondrial membrane that is isolated as a highly stable dimer of 1600kDa. It comprises 17 polypeptides, nine of which (subunits Asa1 to 9) are not present in classical mitochondrial ATP synthases and appear to be exclusive of the chlorophycean lineage. In particular, subunits Asa2, Asa4 and Asa7 seem to constitute a section of the peripheral stalk of the enzyme. Here, we over-expressed and purified subunits Asa2, Asa4 and Asa7 and the corresponding amino-terminal and carboxy-terminal halves of Asa4 and Asa7 in order to explore their interactions in vitro, using immunochemical techniques, blue native electrophoresis and affinity chromatography. Asa4 and Asa7 interact strongly, mainly through their carboxy-terminal halves. Asa2 interacts with both Asa7 and Asa4, and also with subunit α in the F1 sector. The three Asa proteins form an Asa2/Asa4/Asa7 subcomplex. The entire Asa7 and the carboxy-terminal half of Asa4 seem to be instrumental in the interaction with Asa2. Based on these results and on computer-generated structural models of the three subunits, we propose a model for the Asa2/Asa4/Asa7 subcomplex and for its disposition in the peripheral stalk of the algal ATP synthase.

Protein-DNA recognition is fundamental to transcriptional regulation. Transcription factors must be capable of locating their specific sites situated throughout the genome and distinguishing them from related sites. Mlc and NagC control uptake and use of the sugars, glucose and N-acetylglucosamine. Both their helix-turn-helix motifs and their consensus binding sites on DNA are very similar. One distinguishing feature is that most NagC sites have a C/G bp at positions -11 and +11 from the centre of symmetry of the operator, while all Mlc sites have A/T. By constructing Mlc and NagC chimeras, we show that the helix-turn-helix motif per se is not responsible for specific recognition of Mlc or NagC sites, but that a linker, joining the DNA-binding domain to the rest of the protein, is the major determinant. We show that a change of just two amino acids in the NagC linker is sufficient to allow NagC to recognize an A/T bp at positions +/-11 and repress Mlc targets. Modelling of the NagC linker suggests that it forms an extended structure containing two arginines and we suggest that these arginines interact differently with the minor groove at positions +/-11 depending upon the presence of a C/G or A/T bp.

The Gγ subunit of the heterotrimeric G protein is not required to trigger the mating pathway in K. lactis. When the Gγ subunit is missing, the Gα subunit carry Gβ to the plasma membrane and the pheromone pathway is then activated by the Gα/Gβ heterodimer., The Kluyveromyces lactis heterotrimeric G protein is a canonical Gαβγ complex; however, in contrast to Saccharomyces cerevisiae, where the Gγ subunit is essential for mating, disruption of the KlGγ gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Gγ, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and ΔGγ cells of K. lactis. In contrast to the moderate sterility shown by the single ΔKlGα, the double ΔKlGα ΔKlGγ mutant displayed full sterility. A partial sterile phenotype of the ΔKlGγ mutant was obtained in conditions where the KlGβ subunit interacted defectively with the Gα subunit. The addition of a CCAAX motif to the C-end of KlGβ, partially suppressed the lack of both KlGα and KlGγ subunits. In cells lacking KlGγ, the KlGβ subunit cofractionated with KlGα in the plasma membrane, but in the ΔKlGα ΔKlGγ strain was located in the cytosol. When the KlGβ-KlGα interaction was affected in the ΔKlGγ mutant, most KlGβ fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gβ subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Gγ subunit.

The soluble and membrane-anchored isoforms of Mgm1 are only active when they work together (in trans)., Two dynamin-related protein (DRP) families are essential for fusion of the outer and inner mitochondrial membranes, Fzo1 (yeast)/Mfn1/Mfn2 (mammals) and Mgm1 (yeast)/Opa1 (mammals), respectively. Fzo1/Mfns possess two medial transmembrane domains, which place their critical GTPase and coiled-coil domains in the cytosol. In contrast, Mgm1/Opa1 are present in cells as long (l) isoforms that are anchored via the N terminus to the inner membrane, and short (s) isoforms were predicted to be soluble in the intermembrane space. We addressed the roles of Mgm1 isoforms and how DRPs function in membrane fusion. Our analysis indicates that in the absence of a membrane, l- and s-Mgm1 both exist as inactive GTPase monomers, but that together in trans they form a functional dimer in a cardiolipin-dependent manner that is the building block for higher-order assemblies.

Summary Mlc and NagC are two homologous transcription factors which bind to similar DNA targets but for which the inducing signals and mechanisms of activation are very different. Displacing Mlc from its DNA binding sites necessitates its sequestration to the inner membrane via an interaction with PtsG (EIICBGlc), while NagC is displaced from its DNA targets by interacting with GlcNAc6P. We have isolated mutations in both proteins which prevent the inactivation of the repressors by growth on glucose or GlcNAc. These mutations are located in different and specific regions of each protein. For Mlc changes at the C-terminal make it a constitutive repressor and also prevent it from binding to EIIBGlc. Mutations in NagC, at positions which form a structural motif resembling a glucose binding site in Mlc, produce permanently repressing forms of NagC, suggesting that this motif forms a GlcNAc6P binding site in NagC. The pattern of repression by chimeric proteins of NagC and Mlc confirms the importance of the C-terminal region of Mlc for both repression and inducer binding and demonstrate that the helix–turn–helix DNA-binding motif is not sufficient to determine the specificity of interaction of the repressor with DNA.

β-lactoglobulin (BLG) is an abundant milk protein relevant for industry and biotechnology, due significantly to its ability to bind a wide range of polar and apolar ligands. While hydrophobic ligand sites are known, sites for hydrophilic ligands such as the prevalent milk sugar, lactose, remain undetermined. Through the use of molecular docking we first, analyzed the known fatty acid binding sites in order to dissect their atomistic determinants and second, predicted the interaction sites for lactose with monomeric and dimeric BLG. We validated our approach against BLG structures co-crystallized with ligands and report a computational setup with a reduced number of flexible residues that is able to reproduce experimental results with high precision. Blind dockings with and without flexible side chains on BLG showed that: i) 13 experimentally-determined ligands fit the calyx requiring minimal movement of up to 7 residues out of the 23 that constitute this binding site. ii) Lactose does not bind the calyx despite conformational flexibility, but binds the dimer interface and an alternate Site C. iii) Results point to a probable lactolation site in the BLG dimer interface, at K141, consistent with previous biochemical findings. In contrast, no accessible lysines are found near Site C. iv) lactose forms hydrogen bonds with residues from both monomers stabilizing the dimer through a claw-like structure. Overall, these results improve our understanding of BLG's binding sites, importantly narrowing down the calyx residues that control ligand binding. Moreover, our results emphasize the importance of the dimer interface as an insufficiently explored, biologically relevant binding site of particular importance for hydrophilic ligands. Furthermore our analyses suggest that BLG is a robust scaffold for multiple ligand-binding, suitable for protein design, and advance our molecular understanding of its ligand sites to a point that allows manipulation to control binding.