The effect of amino acid deletions and substitutions in the longest loop of GFP

<p>Abstract</p> <p>Background</p> <p>The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from <it>Aequorea victoria </it>has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region.</p> <p>Results</p> <p>In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Δ I129 and sgGFP-Δ D130. Interestingly, both mutants were thermosensitive and at 30°C sgGFP-Δ D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein.</p> <p>Conclusion</p> <p>The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.</p

Combining metagenomics, metatranscriptomics and viromics to explore novel microbial interactions: towards a systems-level understanding of human microbiome

AbstractThe advances in experimental methods and the development of high performance bioinformatic tools have substantially improved our understanding of microbial communities associated with human niches. Many studies have documented that changes in microbial abundance and composition of the human microbiome is associated with human health and diseased state. The majority of research on human microbiome is typically focused in the analysis of one level of biological information, i.e., metagenomics or metatranscriptomics. In this review, we describe some of the different experimental and bioinformatic strategies applied to analyze the 16S rRNA gene profiling and shotgun sequencing data of the human microbiome. We also discuss how some of the recent insights in the combination of metagenomics, metatranscriptomics and viromics can provide more detailed description on the interactions between microorganisms and viruses in oral and gut microbiomes. Recent studies on viromics have begun to gain importance due to the potential involvement of viruses in microbial dysbiosis. In addition, metatranscriptomic combined with metagenomic analysis have shown that a substantial fraction of microbial transcripts can be differentially regulated relative to their microbial genomic abundances. Thus, understanding the molecular interactions in the microbiome using the combination of metagenomics, metatranscriptomics and viromics is one of the main challenges towards a system level understanding of human microbiome

The microbiome and the gut-lung axis in tuberculosis: interplay in the course of disease and treatment

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB) that remains a significant global health challenge. The extensive use of antibiotics in tuberculosis treatment, disrupts the delicate balance of the microbiota in various organs, including the gastrointestinal and respiratory systems. This gut-lung axis involves dynamic interactions among immune cells, microbiota, and signaling molecules from both organs. The alterations of the microbiome resulting from anti-TB treatment can significantly influence the course of tuberculosis, impacting aspects such as complete healing, reinfection, and relapse. This review aims to provide a comprehensive understanding of the gut-lung axis in the context of tuberculosis, with a specific focus on the impact of anti-TB treatment on the microbiome

Type 2 Diabetes Variants Disrupt Function of SLC16A11 through Two Distinct Mechanisms

Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains ∼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. Video Abstract [Figure presented] Keywords: type 2 diabetes (T2D); genetics; disease mechanism; SLC16A11; MCT11; solute carrier (SLC); monocarboxylates; fatty acid metabolism; lipid metabolism; precision medicin

Protein evolution by codon-based random deletions

A method to delete in-phase codons throughout a defined target region of a gene has been developed. This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode. Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis. The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 β-lactamase. Functional ceftazidime-resistant β-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found. The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space

La agenda de investigación científica en México, ¿hay espacio para los medios?: Entrevista a Xavier Soberón Mainero

Tema del mesEl Sistema Nacional de Investigadores (SNI) es un programa del Consejo Nacional de Ciencia y Tecnología (CONACYT), creado en 1984, con el fin de responder a la crisis económica de 1982 y evitar así la fuga de cerebros. Funciona a través de estímulos salariales individuales que se otorgan con base a una evaluación por pares del desempeño académico. En la actualidad, agrupa a cerca de 20 mil científicos y tecnólogos de todo el país que laboran en diversas instituciones donde se realiza investigación científica, conjuntados en siete grandes áreas del conocimiento. Puede decirse que el SNI representa a una buena parte de la comunidad científica nacional, entre la cual se encuentran un gran número de mexicanos con el más alto grado de preparación en el país. A poco más de un cuarto de siglo de su creación, y tras la reciente experiencia de la emergencia sanitaria vivida en el país en 2009, diversas voces cuestionan si el SNI no debería tener un papel más preponderante en coordinar a la comunidad científica para vincularla con los problemas nacionales y con quienes toman las decisiones que afectan al país. Xavier Soberón Mainero es especialista en biotecnología e investigador de la UNAM desde 1981. Fue director del Instituto de Biotecnología entre 1997 y 2005 y actualmente es Director General del Instituto Nacional de Medicina Genómica. Soberón fungió como Director del SNI en 2008 y 2009, cuando la pandemia de influenza A (H1N1) puso a prueba la capacidad de organización de los investigadores nacionales para responder ante la emergencia sanitaria. Nos reunimos con él para hablar sobre la agenda de investigación en México, la vinculación de dicha agenda con los problemas nacionales, sobre cuál puede ser el papel de los medios en esta ecuación y sobre su experiencia personal durante la emergencia sanitaria al frente del Sistema Nacional de Investigadore