The LcrG tip chaperone protein of the Yersinia pestis type III secretion system is partially folded

The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG-LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded alpha helices spanning residues 7-38, 41-46, and 58-73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7-73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52-73) shows tight-binding interaction with LcrV while the N-terminal region (residues 7-51) shows weaker interaction with LcrV. This suggests there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52-73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV

Cellulose-derived oligomers act as damage-associated molecular patterns and trigger defense-like responses

The plant cell wall, often the site of initial encounters between plants and their microbial pathogens, is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides as well as proteins. The concept of damage-associated molecular patterns (DAMPs) was proposed to describe plant elicitors like oligogalacturonides (OGs), which can be derived by the breakdown of the pectin homogalacturon by pectinases. OGs act via many of the same signaling steps as pathogen- or microbe-associated molecular patterns (PAMPs) to elicit defenses and provide protection against pathogens. Given both the complexity of the plant cell wall and the fact that many pathogens secrete a wide range of cell wall-degrading enzymes, we reasoned that the breakdown products of other cell wall polymers may be similarly biologically active as elicitors and may help to reinforce the perception of danger by plant cells. Our results indicate that oligomers derived from cellulose are perceived as signal molecules in Arabidopsis (Arabidopsis thaliana), triggering a signaling cascade that shares some similarities to responses to well-known elicitors such as chitooligomers and OGs. However, in contrast to other known PAMPs/DAMPs, cellobiose stimulates neither detectable reactive oxygen species production nor callose deposition. Confirming our idea that both PAMPs and DAMPs are likely to cooccur at infection sites, cotreatments of cellobiose with flg22 or chitooligomers led to synergistic increases in gene expression. Thus, the perception of cellulose-derived oligomers may participate in cell wall integrity surveillance and represents an additional layer of signaling following plant cell wall breakdown during cell wall remodeling or pathogen attack

Equipe de teleatendimento da terapia ocupacional (E-TO): reflexões sobre a prática remota na infância e adolescência/ Occupational therapy tele-service team (E-TO): Reflections about remote practice in childhood and adolescence

Contextualização: Diante da pandemia COVID-19, declarada em março de 2020, a Universidade Federal de Minas Gerais se adaptou aos desafios da telessaúde para o estágio supervisionado em Terapia Ocupacional, na saúde da criança e adolescente, respaldada pela resolução nº 516 do Conselho Federal de Fisioterapia e Terapia Ocupacional. Processo de Intervenção/acompanhamento: Este trabalho relata a criação do serviço remoto de Terapia Ocupacional, descrevendo seu processo organizacional coordenado para assistir 16 famílias de crianças com transtornos do desenvolvimento. Análise crítica da prática: As metas funcionais das crianças de nove das famílias foram alcançadas totalmente e 85,7% responderam positivamente, evidenciando a eficácia do modelo remoto. No entanto, observou-se dilatação da carga horária do estágio, devido à intensificação das atividades assíncronas para elaboração de materiais personalizados, aumentando a percepção de sobrecarga da equipe.  Síntese das considerações: Para manter resultados funcionais positivos, evitando a sobrecarga, faz-se necessário reavaliar a carga horária destinada às atividades assíncronas.Palavras-chave: Telemonitoramento. Telessaúde. Autismo. Crianças. Terapia Ocupacional. Contextualization: : Faced with the COVID-19 pandemic declared in March 2020, the Federal University of Minas Gerais adapted to the challenges of telehealth for the supervised internship in Occupational Therapy, in child and adolescent health, supported by Resolution No. 516 of the Federal Council of Physiotherapy and Occupational Therapy. Intervention process/accompaniment: The present work reports the creation of the remote Occupational Therapy service describing its coordinated organizational process to assist 16 families of children with developmental disorders. Critical analysis of the practice: The functional goals of the children of nine of the families were fully achieved and 85.7% responded positively, evidencing the effectiveness of the remote model. However, there was an increase in the workload of the internship, due to the intensification of asynchronous activities for the development of personalized materials, increasing the perception of the team's overload.  Summary of considerations: To maintain positive functional results, avoiding overload, it is necessary to reassess the workload allocated to asynchronous activities.Keywords: Telemonitoring. Telehealth. Autism. Kids. Occupational Therapy.Resumen Contextualización Ante la pandemia COVID-19 declarada en marzo de 2020, la Universidad Federal de Minas Gerais se adaptó a los desafíos de la telesalud para la pasantía supervisada en Terapia Ocupacional, en salud infantil y adolescente, respaldada por la Resolución No. 516 del Consejo Federal. de Fisioterapia y Terapia Ocupacional. Proceso de intervención/seguimento: El presente trabajo reporta la creación del servicio de Terapia Ocupacional a distancia describiendo su proceso organizativo coordinado para asistir a 16 familias de niños con trastornos del desarrollo. Análisis crítico de la práctica: : Las metas funcionales de los hijos de nueve de las familias se cumplieron en su totalidad y el 85,7% respondió positivamente, evidenciando la efectividad del modelo remoto. Sin embargo, hubo un aumento en la carga de trabajo de la pasantía, debido a la intensificación de actividades asincrónicas para el desarrollo de materiales personalizados, aumentando la percepción de sobrecarga del equipo.  Resumen de consideraciones: Para mantener resultados funcionales positivos, evitando la sobrecarga, es necesario reevaluar la carga de trabajo asignada a las actividades asincrónicas.Palabras clave: Telemonitorización. Telesalud. Autismo. Niños. Terapia Ocupaciona

Palladium(II) complexes with thiosemicarbazones: syntheses, characterization and cytotoxicity against breast cancer cells and Anti-Mycobacterium tuberculosis activity

Três complexos de PdII com tiossemicarbazonas N(4)-substituídas foram preparados: [Pd(aptsc)(PPh3)](NO3) H2O, 1, [Pd(apmtsc)(PPh3)](NO3), 2, e [Pd(apptsc)(PPh3)](NO3) H2O, 3, sendo PPh3 = trifenilfosfina; Haptsc = 2-acetilpyridina-tiossemicarbazona; Hapmtsc = 2-acetilpiridina-N(4)-metil-tiossemicarbazona e Happtsc = 2-acetilpiridina-N(4)-fenil-tiossemicarbazona. Os complexos foram caracterizados por análise elementar, IR, UV-Vis, ¹H e 31P{¹H} NMR e tiveram suas estruturas cristalinas determinadas por difratometria de raios X em monocristal. Os ligantes tiossemicarbazonatos monoaniônicos atuam de modo tridentado, ligando-se ao metal pelos átomos de nitrogênio piridínico, nitrogênio azometínico e enxofre. A atividade citotóxica frente à linhagem de células tumorais MDA-MB231 (tumor de mama) e a atividade anti-Mycobacterium tuberculosis H37Rv ATCC 27294 dos compostos foram investigadas. Os complexos de PdII mostraram-se altamente ativos contra as células tumorais, com valores de IC50 em torno de 5 µmol L-1, enquanto o agente antitumoral em uso clínico cisplatina mostrou-se inativo. Os compostos apresentaram atividade anti-M. tuberculosis significante, com valores de CIM comparáveis ou melhores que aqueles referentes a alguns fármacos usados clinicamente contra tuberculose.Three PdII complexes were prepared from N(4)-substituted thiosemicarbazones: [Pd(aptsc)(PPh3)](NO3) H2O, 1, [Pd(apmtsc)(PPh3)](NO3), 2, and [Pd(apptsc)(PPh3)](NO3) H2O, 3, where PPh3 = triphenylphosphine; Haptsc = 2-acetylpyridine-thiosemicarbazone; Hapmtsc = 2-acetylpyridine-N(4)-methyl-thiosemicarbazone and Happtsc = 2-acetylpyridine-N(4)-phenyl-thiosemicarbazone. All complexes were characterized by elemental analysis, IR, UV-Vis, ¹H and 31P{¹H} NMR spectroscopies, and had their crystalline structures determined by X-ray diffractometry from single crystals. The monoanionic thiosemicarbazonate ligands act in a tridentate mode, binding to the metal through the pyridine nitrogen, the azomethine nitrogen and the sulfur atoms. The cytotoxic activity against the breast cancer cell line MDA-MB231 and the anti-Mycobacterium tuberculosis H37Rv ATCC 27294 activity were evaluated for the compounds. All PdII complexes were highly active against the studied cell line, presenting similar values of IC50, around 5 µmol L-1, while the clinically applied antitumor agent cisplatin was inactive. The compounds show remarkable anti-M. tuberculosis activities, presenting MIC values comparable or better than some commercial anti-M tuberculosis drugs

The Acyl-CoA ligase-like (ACLL) gene family in Arabidopsis and poplar

Many genes of unknown function have been annotated in plant genome projects, and many of these may encode undiscovered enzymes. For example, completion of the Arabidopsis thaliana genome sequence revealed large families of phenylpropanoid-like enzymes of unknown functions. Using an in silico similarity search based on the amino-acid sequences of known Arabidopsis genes encoding 4-coumarate:CoA ligase (4CL), I identified nine putative genes as members of the Arabidopsis acyl-CoA ligase-like (ACLL) gene superfamily which encode a plant-specific clade of enzymes closely related to true 4CLs. I also identified all ACLLs in the fully sequenced poplar and rice genomes. Phylogenetic analysis of amino-acid sequences revealed five ACLL clades, each containing at least one ACLL member from each species, suggesting conserved biochemical functions for ACLL enzymes. In four of five clades, most of the ACLL representatives have the PTS1 peroxisomal target sequence, indicating a likely function in that organelle. I established tissue expression profiles and the wound and herbivory responsiveness of Arabidopsis and poplar ACLL genes, and this revealed similar expression patterns for potentially orthologous genes. Finally, I mined publicly available microarray databases for co-expressed Arabidopsis genes, and this data provides clues for potential ACLL biochemical functions. The only non-peroxisomal clade is the one most closely related to true 4CLs and contains a single copy gene in Arabidopsis (ACLL5) and poplar (ACLL13). These genes are flower and anther-preferred in expression, and because of the apparent conservation in sequence and in expression, were chosen for functional analysis. ACLL5 is transiently expressed in tapetum cells just prior to release of microspores from tetrads, suggesting a role in pollen wall and/or sporopollenenin formation. In support of this, an acll5 transposon insertion mutant is male sterile and fails to produce pollen grains. These data suggest that ACLL5 and similar enzymes from other species, produce CoA ester intermediates used in an unknown pathway required for pollen wall formation. In silico co-expression analysis in Arabidopsis has revealed potential other members of this pathway, also conserved across angiosperms. This work highlights the utility of the Arabidopsis model system in the discovery of genes in other plant species with genome sequence information.Science, Faculty ofBotany, Department ofGraduat

The YscE/YscG Chaperone and YscF N-Terminal sequences Target YscF to the Yersinia pestis Type III Secretion Apparatus

The needle structures of type III secretion (T3S) systems are formed by the secretion and polymerization of a needle subunit protein, YscF in Yersinia pestis. A subset of T3S systems employ unique heterodimeric chaperones, YscE and YscG in Y. pestis, to prevent the polymerization of needle subunits within the bacterial cell. We demonstrate that the YscE/YscG chaperone is also required for stable YscF expression and for secretion of YscF. Overexpression of a functional maltose-binding protein (MBP)–YscG hybrid protein stabilized cytoplasmic YscF but YscF was not secreted in the absence of YscE. Furthermore, a YscE mutant protein was identified that functioned with YscG to stabilize cytosolic YscF; however, YscF was not secreted. These findings confirm a role for the YscE/YscG chaperone in YscF secretion and suggest that YscE may have a specific role in this process. Recent studies have shown that YscF deleted of its N-terminal 15 residues is still secreted and functional, suggesting that YscF may not require an N-terminal secretion signal. However, we demonstrate that YscF contains an N-terminal secretion signal and that a functional N-terminal signal is required for YscF secretion

Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis[C][W]

Although it is one of central importance to plant reproductive success, the structure of sporopollenin, the main constituent of exine in pollen, is still poorly understood. This work shows that two Arabidopsis oxidoreductases are involved in a biosynthetic pathway leading to sporopollenin that may have been a key determinant in the evolution of land plants

A novel fatty Acyl-CoA Synthetase is required for pollen development and sporopollenin biosynthesis in Arabidopsis

Acyl-CoA Synthetase (ACOS) genes are related to 4-coumarate:CoA ligase (4CL) but have distinct functions. The Arabidopsis thaliana ACOS5 protein is in clade A of Arabidopsis ACOS proteins, the clade most closely related to 4CL proteins. This clade contains putative nonperoxisomal ACOS enzymes conserved in several angiosperm lineages and in the moss Physcomitrella patens. Although its function is unknown, ACOS5 is preferentially expressed in the flowers of all angiosperms examined. Here, we show that an acos5 mutant produced no pollen in mature anthers and no seeds by self-fertilization and was severely compromised in pollen wall formation apparently lacking sporopollenin or exine. The phenotype was first evident at stage 8 of anther development and correlated with maximum ACOS5 mRNA accumulation in tapetal cells at stages 7 to 8. Green fluorescent protein–ACOS5 fusions showed that ACOS5 is located in the cytoplasm. Recombinant ACOS5 enzyme was active against oleic acid, allowing kinetic constants for ACOS5 substrates to be established. Substrate competition assays indicated broad in vitro preference of the enzyme for medium-chain fatty acids. We propose that ACOS5 encodes an enzyme that participates in a conserved and ancient biochemical pathway required for sporopollenin monomer biosynthesis that may also include the Arabidopsis CYP703A2 and MS2 enzymes