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Orange peel powder was activated using different methods and was used to remove tartrazine (E102) from an aqueous solution. The following three adsorbents were synthethized: orange peel powder activated thermally (POAT), orange peel powder activated with sulfuric acid (POAA), orange peel powder activated with soda (POAS). These adsorbents were then characterized by Fourier Transform Infra-Red Spectrometry (FTIR), Raman spectroscopy, powder X-Ray Diffraction (XRD), and point-of-zero charge. The experimental parameters such as contact time, dose of adsorbent, initial concentration of tartrazine, pH, and temperature were studied. The adsorption capacities of tartrazine for the optimal POAT, POAA, and POAS were found to be 121.74, 122.25, and 116.35 mg/g, respectively. The experimental data were analyzed by Freundlich and Temkin isotherm models, as well as the pseudo-second-order kinetic model. HIGHLIGHTS Orange peel adsorbents were used for the removal of tartrazine from water.; The adsorption process followed a pseudo-second-order kinetic model (R2 = 0.999 and 1).; The adsorption capacity of tartrazine onto the adsorbent depends on the type of activation.; The adsorption capacity of tartrazine onto the adsorbent depends on parameter studies.; The effect of many factors on dye adsorption is negligible or little.;

This study aimed to evaluate the antibacterial, leishmanicidal, and cytotoxic potential of metabolites produced by bacteria isolated from rhizosphere soil samples. The bacterium was identified by genome sequencing as Streptomyces kronopolitis. A preliminary screening was carried out for the antimicrobial activity of S. kronopolitis, demonstrating activity against Staphylococcus aureus ATCC 6538, Corynebacterium diphtheriae ATCC 27010, C. diphtheriae ATCC 27012, and Mycobacterium abscessus, with inhibition halos of sizes 25, 36, 29, and 33 mm, respectively. To obtain secondary metabolites, the bacteria were subjected to submerged fermentation, and the metabolites were extracted using the liquid–liquid method with ethyl acetate. There was a similar MIC for M. abscessus and the two strains of C. diphtherium, reaching a concentration of 12.5 µg/mL, while that of S. aureus was 0.048 µg/mL. Assays for leishmanicidal activity and cytotoxicity against HEp-2 cells and red blood cells were performed. The metabolite showed an IC50 of 9.0 ± 0.9 µg/mL and CC50 of 221.2 ± 7.0 µg/mL. This metabolite does not have hemolytic activity and is more selective for parasites than for mammalian cells, with a selectivity index of 24.6. Thus, the studied metabolite may be a strong candidate for the development of less toxic drugs to treat diseases caused by pathogens.

Abstract Background Transcriptional profiling of prostate cancer (PC) has unveiled new markers of neoplasia and allowed insights into mechanisms underlying this disease. Genomewide analyses have also identified new chromosomal abnormalities associated with PC. The combination of both classes of data for the same sample cohort might provide better criteria for identifying relevant factors involved in neoplasia. Here we describe transcriptional signatures identifying distinct normal and tumoral prostate tissue compartments, and the inference and demonstration of a new, highly recurrent copy number gain on chromosome 17q25.3. Methods We have applied transcriptional profiling to tumoral and non-tumoral prostate samples with relatively homogeneous epithelial representations as well as pure stromal tissue from peripheral prostate and cultured cell lines, followed by quantitative RT-PCR validations and immunohistochemical analysis. In addition, we have performed in silico colocalization analysis of co-regulated genes and validation by fluorescent in situ hybridization (FISH). Results The transcriptomic analysis has allowed us to identify signatures corresponding to non-tumoral luminal and tumoral epithelium, basal epithelial cells, and prostate stromal tissue. In addition, in silico analysis of co-regulated expression of physically linked genes has allowed us to predict the occurrence of a copy number gain at chromosomal region 17q25.3. This computational inference was validated by fluorescent in situ hybridization, which showed gains in this region in over 65% of primary and metastatic tumoral samples. Conclusion Our approach permits to directly link gene copy number variations with transcript co-regulation in association with neoplastic states. Therefore, transcriptomic studies of carefully selected samples can unveil new diagnostic markers and transcriptional signatures highly specific of PC, and lead to the discovery of novel genomic abnormalities that may provide additional insights into the causes and mechanisms of prostate cancer.

Abstract Background The evolution of viral quasispecies can influence viral pathogenesis and the response to antiviral treatments. Mutant clouds in infected organisms represent the first stage in the genetic and antigenic diversification of RNA viruses, such as foot and mouth disease virus (FMDV), an important animal pathogen. Antigenic variants of FMDV have been classically diagnosed by immunological or RT-PCR-based methods. DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms (SNPs). Recently, a FMDV microarray was described to detect simultaneously the seven FMDV serotypes. These results encourage the development of new oligonucleotide microarrays to probe the fine genetic and antigenic composition of FMDV for diagnosis, vaccine design, and to gain insight into the molecular epidemiology of this pathogen. Results A FMDV microarray was designed and optimized to detect SNPs at a major antigenic site of the virus. A screening of point mutants of the genomic region encoding antigenic site A of FMDV C-S8c1 was achieved. The hybridization pattern of a mutant includes specific positive and negative signals as well as crosshybridization signals, which are of different intensity depending on the thermodynamic stability of each probe-target pair. Moreover, an array bioinformatic classification method was developed to evaluate the hybridization signals. This statistical analysis shows that the procedure allows a very accurate classification per variant genome. Conclusion A specific approach based on a microarray platform aimed at distinguishing point mutants within an important determinant of antigenicity and host cell tropism, namely the G-H loop of capsid protein VP1, was developed. The procedure is of general applicability as a test for specificity and discriminatory power of microarray-based diagnostic procedures using multiple oligonucleotide probes.

Abstract Background We use an approach based on Factor Analysis to analyze datasets generated for transcriptional profiling. The method groups samples into biologically relevant categories, and enables the identification of genes and pathways most significantly associated to each phenotypic group, while allowing for the participation of a given gene in more than one cluster. Genes assigned to each cluster are used for the detection of pathways predominantly activated in that cluster by finding statistically significant associated GO terms. We tested the approach with a published dataset of microarray experiments in yeast. Upon validation with the yeast dataset, we applied the technique to a prostate cancer dataset. Results Two major pathways are shown to be activated in organ-confined, non-metastatic prostate cancer: those regulated by the androgen receptor and by receptor tyrosine kinases. A number of gene markers (HER3, IQGAP2 and POR1) highlighted by the software and related to the later pathway have been validated experimentally a posteriori on independent samples. Conclusion Using a new microarray analysis tool followed by a posteriori experimental validation of the results, we have confirmed several putative markers of malignancy associated with peptide growth factor signalling in prostate cancer and revealed others, most notably ERRB3 (HER3). Our study suggest that, in primary prostate cancer, HER3, together or not with HER4, rather than in receptor complexes involving HER2, could play an important role in the biology of these tumors. These results provide new evidence for the role of receptor tyrosine kinases in the establishment and progression of prostate cancer.

The mechanisms that underlie the spontaneous and faithful assembly of virus particles are guiding the design of self-assembling protein-based nanostructures for biomedical or nanotechnological uses. In this study, the human immunodeficiency virus (HIV-1) capsid was used as a model to investigate what molecular feature(s) may determine whether a protein nanoparticle with the intended architecture, instead of an aberrant particle, will be self-assembled in vitro . Attempts of using the HIV-1 capsid protein CA for achieving in vitro the self-assembly of cone-shaped nanoparticles that contain CA hexamers and pentamers, similar to authentic viral capsids, had typically yielded hexamer-only tubular particles. We hypothesized that a reduction in the stability of a transient major assembly intermediate, a trimer of CA dimers (ToD), will increase the propensity of CA to assemble in vitro into cone-shaped particles instead of tubes. Certain amino acid substitutions at CA-CA interfaces strongly favored in vitro the assembly of cone-shaped nanoparticles that resembled authentic HIV-1 capsids. All-atom MD simulations indicated that ToDs formed by CA mutants with increased propensity for assembly into cone-shaped particles are destabilized relative to ToDs formed by wt CA or by another mutant that assembles into tubes. The results also indicated that ToD destabilization is mediated by conformational distortion of different CA-CA interfaces, which removes some interprotein interactions within the ToD. A model is proposed to rationalize the linkage between reduced ToD stability and increased propensity for the formation of CA pentamers during particle growth in vitro , favoring the assembly of cone-shaped HIV-1 capsid-like nanoparticles.

Motivation: Evolutionary inference depends crucially on the quality of multiple sequence alignments (MSA), which is problematic for distantly related proteins. Since protein structure is more conserved than sequence, it seems natural to use structure alignments for distant homologs. However, structure alignments may not be suitable for inferring evolutionary relationships., Results: Here we examined four protein similarity measures that depend on sequence and structure (fraction of aligned residues, sequence identity, fraction of superimposed residues, and contact overlap), finding that they are intimately correlated but none of them provides a complete and unbiased picture of conservation in proteins. Therefore, we propose the new hybrid protein sequence and structure similarity score PC_sim based on their main principal component. The corresponding divergence measure PC_div shows the strongest correlation with divergences obtained from individual similarities, suggesting that it infers accurate evolutionary divergences. We developed the program PC_ali that constructs protein MSAs either de novo or modifying an input MSA, using a similarity matrix based on PC_sim. The program constructs a starting MSA based on the maximal cliques of the graph of these PAs and it refines it through progressive alignments along the tree reconstructed with PC_div. Compared with eight state-of-the-art multiple structure or sequence alignment tools, PC_ali achieves higher or equal aligned fraction and structural scores, sequence identity higher than structure aligners although lower than sequence aligners, highest score PC_sim, and highest similarity with the MSAs produced by other tools and with the reference MSA Balibase., Availability and Implementation: https://github.com/ugobas/PC_ali., (© The Author(s) 2023. Published by Oxford University Press.)

Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Successful vaccines rely on activating a functional humoral immune response through the generation of class-switched high affinity immunoglobulins (Igs). The germinal center (GC) reaction is crucial for this process, in which B cells are selected in their search for antigen and T cell help. A major hurdle to understand the mechanisms of B cell:T cell cooperation has been the lack of an antigen-specific in vitro GC system. Here we report the generation of antigen-specific, high-affinity, class-switched Igs in simple 2-cell type cultures of naive B and T cells. B cell antigen uptake by phagocytosis is key to generate these Igs. We have used the method to interrogate if T cells confer directional help to cognate B cells that present antigen and to bystander B cells. We find that bystander B cells do not generate class-switched antibodies due to a defective formation of T-B conjugates and an early conversion into memory B cells., (© 2023. The Author(s).)

Background: Children are less susceptible than adults to symptomatic COVID-19 infection, but very few studies addressed their underlying cause. Moreover, very few studies analyzed why children highly exposed to the virus remain uninfected., Methods: We analyzed the serum levels of ACE2, angiotensin II, anti-spike and anti-N antibodies, cytokine profiles, and virus neutralization in a cohort of children at high risk of viral exposure, cohabiting with infected close relatives during the lockdown in Spain., Results: We analyzed 40 children who were highly exposed to the virus since they lived with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected relatives during the lockdown for several months without taking preventive measures. Of those, 26 reported mild or very mild symptoms. The induced immune response to the virus was analyzed 3 months after the household infection. Surprisingly, only 15 children had IgG anti-S (IgG + ) determined by a sensitive method indicative of a past infection. The rest, negative for IgG anti-N or S in various tests, could be further subdivided, according to IgM antibodies, into those having IgM anti-S and IgM anti-N (IgG - IgM high ) and those having only IgM anti-N (IgG - IgM low ). Interestingly, those two subgroups of children with IgM antibodies have strikingly different patterns of cytokines. The IgM high group had significantly higher IFN-α2 and IFN-γ levels as well as IL-10 and GM-CSF than the IgM low group. In contrast, the IgM low group had low levels of ACE2 in the serum. Both groups have a weaker but significant capacity to neutralize the virus in the serum than the IgG + group. Two children were negative in all immunological antibody tests., Conclusions: A significant proportion of children highly exposed to SARS-CoV-2 did not develop a classical adaptive immune response, defined by the production of IgG, despite being in close contact with infected relatives. A large proportion of those children show immunological signs compatible with innate immune responses (as secretion of natural antibodies and cytokines), and others displayed very low levels of the viral receptor ACE2 that may have protected them from the virus spreading in the body despite high and constant viral exposure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Úbeda, Maza, Delgado, Horndler, Abia, García-Bermejo, Serrano-Villar, Calvo, Bastolla, Sainz and Fresno.)

Nucleoid-associated proteins (NAPs) play a central role in chromosome organization and environment-responsive transcription regulation. The Bacillus subtilis-encoded NAP Rok binds preferentially AT-rich regions of the genome, which often contain genes of foreign origin that are silenced by Rok binding. Additionally, Rok plays a role in chromosome architecture by binding in genomic clusters and promoting chromosomal loop formation. Based on this, Rok was proposed to be a functional homolog of E. coli H-NS. However, it is largely unclear how Rok binds DNA, how it represses transcription and whether Rok mediates environment-responsive gene regulation. Here, we investigated Rok's DNA binding properties and the effects of physico-chemical conditions thereon. We demonstrate that Rok is a DNA bridging protein similar to prototypical H-NS-like proteins. However, unlike these proteins, the DNA bridging ability of Rok is not affected by changes in physico-chemical conditions. The DNA binding properties of the Rok interaction partner sRok are affected by salt concentration. This suggests that in a minority of Bacillus strains Rok activity can be modulated by sRok, and thus respond indirectly to environmental stimuli. Despite several functional similarities, the absence of a direct response to physico-chemical changes establishes Rok as disparate member of the H-NS family., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Signaling via the T cell receptor (TCR) is critical during the development, maintenance, and activation of T cells. Quantitative aspects of TCR signaling have an important role during positive and negative selection, lineage choice, and ability to respond to small amounts of antigen. By using a mutant mouse line expressing a hypomorphic allele of the CD3ζ chain, we show here that the strength of pre-TCR–mediated signaling during T cell development determines the diversity of the TCRβ repertoire available for positive and negative selection, and hence of the final αβTCR repertoire. This finding uncovers an unexpected, pre-TCR signaling–dependent and repertoire–shaping role for β-selection beyond selection of in-frame rearranged TCRβ chains. Our data furthermore support a model of pre-TCR signaling in which the arrangement of this receptor in stable nanoclusters determines its quantitative signaling capacity.

Background: COVID-19 can generate a broad spectrum of severity and symptoms. Many studies analysed the determinants of severity but not among some types of symptoms. More importantly, very few studies analysed patients highly exposed to the virus that nonetheless remain uninfected., Methods: We analysed serum levels of ACE2, Angiotensin II and anti-Spike antibodies in 2 different cohorts at high risk of viral exposure, highly exposed but uninfected subjects, either high risk health care workers or persons cohabiting with infected close relatives and seropositive patients with symptoms. We tested the ability of the sera of these subjects to neutralize lentivirus pseudotyped with the Spike-protein., Results: We found that the serum levels of ACE2 are significantly higher in highly exposed but uninfected subjects. Moreover, sera from this seronegative persons can neutralize SARS-CoV-2 infection in cellular assays more strongly that sera from non-exposed negative controls eventhough they do not have anti-CoV-2 IgG antibodies suggesting that high levels of ACE2 in serum may somewhat protect against an active infection without generating a conventional antibody response. Finally, we show that among patients with symptoms, ACE2 levels were significantly higher in infected patients who developed cutaneous as compared with respiratory symptoms and ACE2 was also higher in those with milder symptoms., Conclusions: These findings suggest that soluble ACE2 could be used as a potential biomarker to predict SARS-CoV-2 infection risk and to discriminate COVID-19 disease subtypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Maza, Úbeda, Delgado, Horndler, Llamas, van Santen, Alarcón, Abia, García-Bermejo, Serrano-Villar, Bastolla and Fresno.)

The emergence of COVID-19 has led to a worldwide challenge for the rapid development of vaccines. Several types of safe and effective vaccines have been available in a time frame never seen before. Now that several hundred million people have been vaccinated there is an opportunity to compare vaccines in terms of protection and immune response. Here, we have applied a highly sensitive multiplexed flow cytometry method to measure simultaneously IgM, IgG1 and IgA anti-spike protein antibodies generated in response to three vaccines: ChAdOx1 (Oxford-AstraZeneca), mRNA-1273 (Moderna), and BNT162b2 (Pfizer-BioNTech). We have found that mRNA vaccines (mRNA-1273 and BNT162b2) induce a stronger humoral response, both after the first and the second dose, than the adenovirus-based ChAdOx1 vaccine. We also found that, in the elderly, antibody titers negatively correlate with the age of the donor but, also, that antibody titers remain stable for at least 6 months after complete vaccination. Finally, we found that one dose of BNT162b2 is sufficient to induce the highest antibody titers in seropositive pre-vaccination donors. We hope these data will help to guide future decisions on vaccination strategies., Competing Interests: The authors have issued a patent application owned by CSIC. Authors SR-P, MQ, SÁ-F, and AO are employed by VITRO S.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Romero-Pinedo, Quesada, Horndler, Álvarez-Fernández, Olmo, Abia, Alarcón and Delgado.)

Background: Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRAS, HRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL., Methods: Conditional knock-in mice were generated to overexpress wild type RRAS2 and prove its driver role. RT-qPCR analysis of a human CLL sample cohort was carried out to measure RRAS2 transcriptional expression. Sanger DNA sequencing was used to identify a SNP in the 3'UTR region of RRAS2 in human CLL samples. RNAseq of murine CLL was carried out to identify activated pathways, molecular mechanisms and to pinpoint somatic mutations accompanying RRAS2 overexpression. Flow cytometry was used for phenotypic characterization and shRNA techniques to knockdown RRAS2 expression in human CLL., Results: RRAS2 mRNA is found overexpressed in its wild type form in 82% of the human CLL samples analyzed (n = 178, mean and median = 5-fold) as well as in the explored metadata. A single nucleotide polymorphism (rs8570) in the 3'UTR of the RRAS2 mRNA has been identified in CLL patients, linking higher expression of RRAS2 with more aggressive disease. Deliberate overexpression of wild type RRAS2 in mice, but not an oncogenic Q72L mutation in the coding sequence, provokes the development of CLL. Overexpression of wild type RRAS2 in mice is accompanied by a strong convergent selection of somatic mutations in genes that have been identified in human CLL. R-RAS2 protein is physically bound to the BCR and mediates BCR signals in CLL., Conclusions: The results indicate that overexpression of wild type RRAS2 is behind the development of CLL., (© 2022. The Author(s).)

During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts' defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and-importantly-transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes.

Conjugation plays important roles in genome plasticity, adaptation and evolution but is also the major horizontal gene-transfer route responsible for spreading toxin, virulence and antibiotic resistance genes. A better understanding of the conjugation process is required for developing drugs and strategies to impede the conjugation-mediated spread of these genes. So far, only a limited number of conjugative elements have been studied. For most of them, it is not known whether they represent a group of conjugative elements, nor about their distribution patterns. Here we show that pLS20 from the Gram-positive bacterium Bacillus subtilis is the prototype conjugative plasmid of a family of at least 35 members that can be divided into four clades, and which are harboured by different Bacillus species found in different global locations and environmental niches. Analyses of their phylogenetic relationship and their conjugation operons have expanded our understanding of a family of conjugative plasmids of Gram-positive origin., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Transcriptional regulation allows adaptive and coordinated gene expression, and is essential for life. Processive antitermination systems alter the transcription elongation complex to allow the RNA polymerase to read through multiple terminators in an operon. Here, we describe the discovery of a novel bipartite antitermination system that is widespread among conjugative elements from Gram-positive bacteria, which we named conAn. This system is composed of a large RNA element that exerts antitermination, and a protein that functions as a processivity factor. Besides allowing coordinated expression of very long operons, we show that these systems allow differential expression of genes within an operon, and probably contribute to strict regulation of the conjugation genes by minimizing the effects of spurious transcription. Mechanistic features of the conAn system are likely to decisively influence its host range, with important implications for the spread of antibiotic resistance and virulence genes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Conjugation, the process by which a DNA element is transferred from a donor to a recipient cell, is the main horizontal gene transfer route responsible for the spread of antibiotic resistance and virulence genes. Contact between a donor and a recipient cell is a prerequisite for conjugation, because conjugative DNA is transferred into the recipient via a channel connecting the two cells. Conjugative elements encode proteins dedicated to facilitating the recognition and attachment to recipient cells, also known as mating pair formation. A subgroup of the conjugative elements is able to mediate efficient conjugation during planktonic growth, and mechanisms facilitating mating pair formation will be particularly important in these cases. Conjugative elements of Gram-negative bacteria encode conjugative pili, also known as sex pili, some of which are retractile. Far less is known about mechanisms that promote mating pair formation in Gram-positive bacteria. The conjugative plasmid pLS20 of the Gram-positive bacterium Bacillus subtilis allows efficient conjugation in liquid medium. Here, we report the identification of an adhesin gene in the pLS20 conjugation operon. The N-terminal region of the adhesin contains a class II type thioester domain (TED) that is essential for efficient conjugation, particularly in liquid medium. We show that TED-containing adhesins are widely conserved in Gram-positive bacteria, including pathogens where they often play crucial roles in pathogenesis. Our study is the first to demonstrate the involvement of a class II type TED-containing adhesin in conjugation. IMPORTANCE Bacterial resistance to antibiotics has become a serious health care problem. The spread of antibiotic resistance genes between bacteria of the same or different species is often mediated by a process named conjugation, where a donor cell transfers DNA to a recipient cell through a connecting channel. The first step in conjugation is recognition and attachment of the donor to a recipient cell. Little is known about this first step, particularly in Gram-positive bacteria. Here, we show that the conjugative plasmid pLS20 of Bacillus subtilis encodes an adhesin protein that is essential for effective conjugation. This adhesin protein has a structural organization similar to adhesins produced by other Gram-positive bacteria, including major pathogens, where the adhesins serve in attachment to host tissues during colonization and infection. Our findings may thus also open novel avenues to design drugs that inhibit the spread of antibiotic resistance by blocking the first recipient-attachment step in conjugation., (Copyright © 2021 Gago-Córdoba et al.)

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike "S" protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti-S antibodies with protective neutralizing activity are long-lasting and can be detected in sera 8 months after infection., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Signal strength controls the outcome of αβ T cell selection in the thymus, resulting in death if the affinity of the rearranged TCR is below the threshold for positive selection, or if the affinity of the TCR is above the threshold for negative selection. Here we show that deletion of the GTPase RRAS2 results in exacerbated negative selection and above-normal expression of positive selection markers. Furthermore, Rras2 -/- mice are resistant to autoimmunity both in a model of inflammatory bowel disease (IBD) and in a model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). We show that MOG-specific T cells in Rras2 -/- mice have reduced affinity for MOG/I-A b tetramers, suggesting that enhanced negative selection leads to selection of TCRs with lower affinity for the self-MOG peptide. An analysis of the TCR repertoire shows alterations that mostly affect the TCRα variable (TRAV) locus with specific VJ combinations and CDR3α sequences that are absent in Rras2 -/- mice, suggesting their involvement in autoimmunity., (© 2019 Martínez-Riaño et al.)

During conjugation a genetic element is transferred from a bacterial donor to a recipient cell via a connecting channel. It is the major route responsible for the spread of antibiotic resistance. Conjugative elements can contain exclusion system(s) that inhibit its transfer to a cell already harboring the element. Our limited knowledge on exclusion systems is mainly based on plasmids of Gram-negative bacteria. Here we studied the conjugative plasmid pLS20 of the Gram-positive Bacillus subtilis . We demonstrate that pLS20 contains an exclusion system and identified the single gene responsible for exclusion, named ses pLS20 , which is embedded in the conjugation operon. Ses pLS20 is the founding member of a novel family of surface exclusion proteins encoded by conjugative elements of Gram-positive origin. We show that the extent of surface exclusion correlates with the level of ses pLS20 expression, and that ses pLS20 is expressed at basal low-levels in all donor cells but becomes highly expressed in conjugating cells. Accordingly, the transfer of pLS20 from a conjugation-primed donor cell to an un-primed or conjugation-primed donor is inhibited moderately and very efficiently, respectively. The consequences of this differential regulation, which appears to be a conserved feature of surface exclusion systems of Gram-positive and Gram-negative origin, are discussed.

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition.

Neurotransmitter removal from glycine-mediated synapses relies on two sodium-driven high-affinity plasma membrane GlyTs that control neurotransmitter availability. Mostly glial GlyT1 is the main regulator of glycine synaptic levels, whereas neuronal GlyT2 promotes the recycling of synaptic glycine and supplies neurotransmitter for presynaptic vesicle refilling. The GlyTs differ in sodium:glycine symport stoichiometry, showing GlyT1 a 2:1 and GlyT2 a 3:1 sodium:glycine coupling. Sodium binds to the GlyTs at two conserved Na + sites: Na1 and Na2. The location of GlyT2 Na3 site remains unknown, although Glu650 has been involved in the coordination. Here, we have used comparative MD simulations of a GlyT2 model constructed by homology to the crystalized DAT from Drosophila melanogaster by placing the Na3 ion at two different locations. By combination of in silico and experimental data obtained by biochemical and electrophysiological analysis of GlyTs mutants, we provide evidences suggesting the GlyT2 third sodium ion is held by Glu-250 and Glu-650, within a region with robust allosteric properties involved in cation-specific sensitivity. Substitution of Glu650 in GlyT2 by the corresponding methionine in GlyT1 reduced the charge-to-flux ratio to the level of GlyT1 without producing transport uncoupling. Chloride dependence of glycine transport was almost abolished in this GlyT2 mutant but simultaneous substitution of Glu250 and Glu650 by neutral amino acids rescued chloride sensitivity, suggesting that protonation/deprotonation of Glu250 substitutes chloride function. The differential behavior of equivalent GlyT1 mutations sustains a GlyT2-specific allosteric coupling between the putative Na3 site and the chloride site.

The stability, binding, and tissue penetration of variable new-antigen receptor (VNAR) single-domain antibodies have been tested as part of an investigation into their ability to serve as novel therapeutics. V13 is a VNAR that recognizes vascular endothelial growth factor 165 (VEGF 165 ). In the present study V13 was used as a parental molecule into which we introduced mutations designed in silico . Two of the designed VNAR mutants were expressed, and their ability to recognize VEGF 165 was assessed in vitro and in vivo . One mutation (Pro98Tyr) was designed to increase VEGF 165 recognition, while the other (Arg97Ala) was designed to inhibit VEGF 165 binding. Compared to parental V13, the Pro98Tyr mutant showed enhanced VEGF 165 recognition and neutralization, as indicated by inhibition of angiogenesis and tumor growth. This molecule thus appears to have therapeutic potential for neutralizing VEGF 165 in cancer treatment., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

Bacterial conjugation is the process by which a conjugative element (CE) is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer ( oriT ), where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis . We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1 LS20 and aux2 LS20 , and which we show are essential for conjugation. Both Aux1 LS20 and Aux2 LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1 LS20 and Aux2 LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriT LS20 , although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1 LS20 and/or Aux2 LS20 are located upstream of almost 400 relaxase genes of the Rel LS20 family (MOB L ) of relaxases. Thus, Aux1 LS20 and Aux2 LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

Spliceosome activity is tightly regulated to ensure adequate splicing in response to internal and external cues. It has been suggested that core components of the spliceosome, such as the snRNPs, would participate in the control of its activity. The experimental indications supporting this proposition, however, remain scarce, and the operating mechanisms poorly understood. Here, we present genetic and molecular evidence demonstrating that the LSM2-8 complex, the protein moiety of the U6 snRNP, regulates the spliceosome activity in Arabidopsis, and that this regulation is controlled by the environmental conditions. Our results show that the complex ensures the efficiency and accuracy of constitutive and alternative splicing of selected pre-mRNAs, depending on the conditions. Moreover, miss-splicing of most targeted pre-mRNAs leads to the generation of nonsense mediated decay signatures, indicating that the LSM2-8 complex also guarantees adequate levels of the corresponding functional transcripts. Interestingly, the selective role of the complex has relevant physiological implications since it is required for adequate plant adaptation to abiotic stresses. These findings unveil an unanticipated function for the LSM2-8 complex that represents a new layer of posttranscriptional regulation in response to external stimuli in eukaryotes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC 50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases., (Copyright © 2016, American Association for the Advancement of Science.)

The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice., Competing Interests: Competing Interests: We have the following interests: This study was supported by Biotherapix, S.L.U. Patricia Garrido, María Pernas and José Luis Torán were employed by Biotherapix, S.L.U. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Approximately seven hundred 45S rRNA genes (rDNA) in the Arabidopsis thaliana genome are organised in two 4 Mbp-long arrays of tandem repeats arranged in head-to-tail fashion separated by an intergenic spacer (IGS). These arrays make up 5 % of the A. thaliana genome. IGS are rapidly evolving sequences and frequent rearrangements inside the rDNA loci have generated considerable interspecific and even intra-individual variability which allows to distinguish among otherwise highly conserved rRNA genes. The IGS has not been comprehensively described despite its potential importance in regulation of rDNA transcription and replication. Here we describe the detailed sequence variation in the complete IGS of A. thaliana WT plants and provide the reference/consensus IGS sequence, as well as genomic DNA analysis. We further investigate mutants dysfunctional in chromatin assembly factor-1 (CAF-1) (fas1 and fas2 mutants), which are known to have a reduced number of rDNA copies, and plant lines with restored CAF-1 function (segregated from a fas1xfas2 genetic background) showing major rDNA rearrangements. The systematic rDNA loss in CAF-1 mutants leads to the decreased variability of the IGS and to the occurrence of distinct IGS variants. We present for the first time a comprehensive and representative set of complete IGS sequences, obtained by conventional cloning and by Pacific Biosciences sequencing. Our data expands the knowledge of the A. thaliana IGS sequence arrangement and variability, which has not been available in full and in detail until now. This is also the first study combining IGS sequencing data with RFLP analysis of genomic DNA.

Although the T cell antigen receptor (TCR) is long known to contain multiple signaling subunits (CD3γ, CD3δ, CD3ɛ and CD3ζ), their role in signal transduction is still not well understood. The presence of at least one immunoreceptor tyrosine-based activation motif (ITAM) in each CD3 subunit has led to the idea that the multiplication of such elements essentially serves to amplify signals. However, the evolutionary conservation of non-ITAM sequences suggests that each CD3 subunit is likely to have specific non-redundant roles at some stage of development or in mature T cell function. The CD3ɛ subunit is paradigmatic because in a relatively short cytoplasmic sequence (∼55 amino acids) it contains several docking sites for proteins involved in intracellular trafficking and signaling, proteins whose relevance in T cell activation is slowly starting to be revealed. In this review we will summarize our current knowledge on the signaling effectors that bind directly to the TCR and we will propose a hierarchy in their response to TCR triggering., (Copyright © 2014 Elsevier B.V. All rights reserved.)

At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed., Competing Interests: The authors have declared that no competing interests exist.

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. PA is caused by mutations in either the PCCA or PCCB genes encoding the α- and β-subunits of the PCC enzyme which are assembled as an α6β6 dodecamer. In this study we have investigated the molecular basis of the defect in ten fibroblast samples from PA patients. Using homology modeling with the recently solved crystal structure of the PCC holoenzyme and a eukaryotic expression system we have analyzed the structural and functional effect of novel point mutations, also revealing a novel splice defect by minigene analysis. In addition, we have investigated the contribution of oxidative stress to cellular damage measuring reactive oxygen species (ROS) levels and apoptosis parameters in patient fibroblasts, as recent studies point to a secondary mitochondrial dysfunction as pathophysiological mechanism in this disorder. The results show an increase in intracellular ROS content compared to controls, correlating with the activation of the JNK and p38 signaling pathways. Highest ROS levels were present in cells harboring functionally null mutations, including one severe missense mutation. This work provides molecular insight into the pathogenicity of PA variants and indicates that oxidative stress may be a major contributing factor to the cellular damage, supporting the proposal of antioxidant strategies as novel supplementary therapy in this rare disease.

Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php.

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r(2) ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations.

Deficiency of phosphomannomutase (PMM2, MIM#601785) is the most common congenital disorder of glycosylation. Herein we report the genetic analysis of 22 Spanish PMM2 deficient patients and the functional analysis of 14 nucleotide changes in a prokaryotic expression system in order to elucidate their molecular pathogenesis. PMM2 activity assay revealed the presence of six protein changes with no enzymatic activities (p.R123Q, p.R141H, p.F157S, p.P184T, p.F207S and p.D209G) and seven mild protein changes with residual activities ranging from 16 to 54% (p.L32R, p.V44A p.D65Y, p.P113L p.T118S, p.T237M and p.C241S) and also one variant change with normal activity (p.E197A). The results obtained from Western blot analysis, degradation time courses of 11 protein changes and structural analysis of the PMM2 protein, suggest that the loss-of-function of most mutant proteins is based on their increased susceptibility to degradation or aggregation compared to the wild type protein, considering PMM2 deficiency as a conformational disease. We have identified exclusively catalytic protein change (p.D209G), catalytic protein changes affecting protein stability (p.R123Q and p.R141H), two protein changes disrupting the dimer interface (p.P113L and p.T118S) and several misfolding changes (p.L32R, p.V44A, p.D65Y, p.F157S, p.P184T, p.F207S, p.T237M and p.C241S). Our current work opens a promising therapeutic option using pharmacological chaperones to revert the effect of the characterized misfolding mutations identified in a wide range of PMM2 deficient patients.