Fluorescence Spectroscopy of the Tryptophan Microenvironment in Carcinus aestuarii Hemocyanin

The steady-state and time-resolved fluorescence properties of the multitryptophan minimal subunit CaeSS2 from Carcinus aestuarii hemocyanin have been studied with the aim of probing the environment of the fluorophores within the protein matrix. Subunit a of Panulirus interruptus hemocyanin, whose X-ray structure is known, has been also studied. The results are compared with those collected with other two monomeric fractions (CaeSS1, CaeSS3) produced by dissociation of the native, oligomeric protein as well as with those of the hexameric aggregate. Three classes of tryptophan residues can be singled out by a combination of fluorescence quenching and lifetime measurements on the holo-Hc (the copper containing, oxygen binding form) and the apo-Hc (the copper-free derivative). One class of tryptophans is exposed to the protein surface. Some of these residues are proposed to be involved in the intersubunit interactions in CaeSS1 and CaeSS3 fractions whereas in CaeSS2 the protein matrix masks them. This suggests the occurrence of conformational rearrangements after detachment of the subunit from the native aggregate, which could explain the inability of CaeSS2 to reassociate. A second class of tryptophan has been correlatively assigned, by comparison with the results obtained with Panulirus interruptus hemocyanin, to residues in close proximity to the active site. The third class includes buried, active site-distant, residues

Selected Policy Measures Against the Debt Distress in Mongolia

The objective of this report is to examine the public external debt sustainability of Mongolia, and to propose appropriate regulatory actions for ongoing debates about economic reform. Following sharp external shocks that include a drop in foreign direct investment and a depreciation of the national currency, the country is at a critical moment of determining whether to default on its external debts or correct structural policy failures. Therefore, it is important that Mongolia identify its level of debt distress and determine which structural reforms should take place

Development of an amplicon-based sequencing approach in response to the global emergence of mpox

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.This publication was made possible by CTSA Grant Number UL1 TR001863 from the National Center for Advancing Translational Science (NCATS), a component of the National Institutes of Health (NIH) awarded to CBFV. INSA was partially funded by the HERA project (Grant/ 2021/PHF/23776) supported by the European Commission through the European Centre for Disease Control (to VB).info:eu-repo/semantics/publishedVersio

Incipient parallel evolution of SARS-CoV-2 Deltacron variant in South Brazil

With the coexistence of multiple lineages and increased international travel, recombination and gene flow are likely to become increasingly important in the adaptive evolution of SARS-CoV-2. These processes could result in genetic introgression and the incipient parallel evolution of multiple recombinant lineages. However, identifying recombinant lineages is challenging, and the true extent of recombinant evolution in SARS-CoV-2 may be underestimated. This study describes the first SARS-CoV-2 Deltacron recombinant case identified in Brazil. We demonstrate that the recombination breakpoint is at the beginning of the Spike gene. The 5′ genome portion (circa 22 kb) resembles the AY.101 (Delta), and the 3′ genome portion (circa 8 kb nucleotides) is most similar to the BA.1.1 (Omicron). Furthermore, evolutionary genomic analyses indicate that the new strain emerged after a single recombination event between lineages of diverse geographical locations in December 2021 in South Brazil. This Deltacron, AYBA-RS, is one of the dozens of recombinants described in 2022. The submission of only four sequences in the GISAID database suggests that this lineage had a minor epidemiological impact. However, the recent emergence of this and other Deltacron recombinant lineages (XD, XF, and XS) suggests that gene flow and recombination may play an increasingly important role in the COVID-19 pandemic. We explain the evolutionary and population genetic theory that supports this assertion, concluding that this stresses the need for continued genomic surveillance. This monitoring is vital for countries where multiple variants are present, as well as for countries that receive significant inbound international travel

Differentially expressed proteins identified inthe cambial region of Eucalyptus grandis during reaction wood formation

A madeira é um tecido complexo e variável que se encontra sob o controle dos diversos estádios de desenvolvimento e do ambiente. Um modelo experimental freqüentemente utilizado para o estudo do processo da formação da madeira tem sido a indução da formação da madeira de reação. A madeira de reação é formada em resposta a um estímulo gravitacional e está associada com um aumento na taxa de crescimento, modificação química e estrutural na parede celular das fibras. A madeira de reação formada no lado superior do tronco inclinado é chamada de madeira de tração, enquanto que a madeira formada no lado inferior do tronco inclinado é chamada de madeira oposta. Uma das principais características da madeira de tração é a deposição de uma camada gelatinosa na parede celular secundária, que é rica em celulose altamente cristalina. Com o objetivo de caracterizar as alterações na expressão gênica e no perfil de proteínas durante a formação da madeira de reação em Eucalyptus grandis, árvores de 1,8 anos de idade foram inclinadas sob um ângulo fixo (45º) e amostras da região cambial foram coletadas após 15, 30 e 60 dias da região do tronco com a curvatura mais acentuada. Proteínas e RNA foram extraídos da região do xilema em diferenciação da madeira de tração e da madeira oposta, e foram comparadas às da madeira normal (controle) utilizando uma abordagem proteômica (géis 2D e LC-MS/MS) e PCRsq. As análises foram focadas em genes e proteínas relacionadas à modificação e formação da parede celular. 480 spots de proteínas foram identificados e classificados em categorias funcionais, com particular atenção dada às proteínas relacionadas ao metabolismo de UDP-açúcares (incluindo a biossíntese de celulose), biossíntese de lignina e citoesqueleto. Além disso, a expressão relativa dos transcritos relacionados à biossíntese de celulose e lignina, ao metabolismo de pectinas e ao citoesqueleto foram analisadas através de PCRsq na madeira controle, de tração e oposta. Diversas diferenças no padrão de expressão de genes e proteínas envolvidas nas mudanças estruturais e da composição da parede celular secundária em relação à madeira normal foram observadas ao longo do período de indução. A deposição da camada G foi observada após 60 dias de indução da madeira de tração, através de secções transversais coradas com safranina e azul de astra. Ao longo do tempo de indução, os resultados sugerem um aumento no fluxo do carbono para a biossíntese de lignina na madeira oposta e para a biossíntese de celulose na madeira de tração. Assim, este trabalho resultou na caracterização dos perfis de expressão de proteínas e genes envolvidos nos mecanismos moleculares da formação da madeira sob estresse gravitacional e mais precisamente no remodelamento da parede celular secundária durante a formação da camada G.Wood is a complex and highly variable tissue under developmental and environmental control. Usually, an experimental model used to study wood formation is the induction of reaction wood formation. Reaction wood is formed to response a gravitational stimuli and is associated with an increased growth rate, modified fiber cell walls chemistry and structure. The reaction wood formed in the upper side of a bent trunk is called tension wood, and that one formed in the lower side is called opposite wood. One of the main characteristics of tension wood is the deposition of gelatinous layer (G layer) on the secondary cell wall, which is rich in highly crystalline cellulose. With the aim to characterize the changes in gene expression and protein profile during tenson wood formation in Eucalyptus grandis, 1,8 year-old trees were bent at a fixed angle (45º) and samples from the cambial region taken after 15, 30 and 60 days in the region of the trunk with the most accentuated curve. Proteins and RNA were extracted from the differentiating xylem region of the tension wood and opposite wood and were compared to those of normal wood (control) using 2D electrophoresis (LC-MS/MS) and sqPCR. The analysis was focused on genes and proteins related to the modification and formation of the cell wall. 480 spots of proteins were extracted, identified and classified into functional categories, with particular attention to those related to nucleotide sugar metabolism (including cellulose), lignin biosynthesis and the cytoskeleton. In addition, the relative expression of transcripts related to cellulose and lignin biosynthesis pectin metabolism and the cytoskeleton were analyzed by sqPCR in control, tension and opposite wood. Several differences in the expression pattern of genes and proteins involved in structural and secondary cell wall composition changes were observed when compared to normal wood during the induction period. The deposition of G layer was observed after 60 days of tension wood induction using transversal sections stained with safranin-astra blue. Throughout the induction period, the results suggest an increase in the flow carbon for lignin biosynthesis in the opposite wood and for cellulose biosynthesis in the tension wood. Therefore, the results of this work characterize the protein and gene expression profiles involved in the molecular mechanisms underlying wood formation under gravitational stress and, more specifically, in secondary cell wall remodeling during G layer formation

Differentially expressed proteins identified inthe cambial region of Eucalyptus grandis during reaction wood formation

A madeira é um tecido complexo e variável que se encontra sob o controle dos diversos estádios de desenvolvimento e do ambiente. Um modelo experimental freqüentemente utilizado para o estudo do processo da formação da madeira tem sido a indução da formação da madeira de reação. A madeira de reação é formada em resposta a um estímulo gravitacional e está associada com um aumento na taxa de crescimento, modificação química e estrutural na parede celular das fibras. A madeira de reação formada no lado superior do tronco inclinado é chamada de madeira de tração, enquanto que a madeira formada no lado inferior do tronco inclinado é chamada de madeira oposta. Uma das principais características da madeira de tração é a deposição de uma camada gelatinosa na parede celular secundária, que é rica em celulose altamente cristalina. Com o objetivo de caracterizar as alterações na expressão gênica e no perfil de proteínas durante a formação da madeira de reação em Eucalyptus grandis, árvores de 1,8 anos de idade foram inclinadas sob um ângulo fixo (45º) e amostras da região cambial foram coletadas após 15, 30 e 60 dias da região do tronco com a curvatura mais acentuada. Proteínas e RNA foram extraídos da região do xilema em diferenciação da madeira de tração e da madeira oposta, e foram comparadas às da madeira normal (controle) utilizando uma abordagem proteômica (géis 2D e LC-MS/MS) e PCRsq. As análises foram focadas em genes e proteínas relacionadas à modificação e formação da parede celular. 480 spots de proteínas foram identificados e classificados em categorias funcionais, com particular atenção dada às proteínas relacionadas ao metabolismo de UDP-açúcares (incluindo a biossíntese de celulose), biossíntese de lignina e citoesqueleto. Além disso, a expressão relativa dos transcritos relacionados à biossíntese de celulose e lignina, ao metabolismo de pectinas e ao citoesqueleto foram analisadas através de PCRsq na madeira controle, de tração e oposta. Diversas diferenças no padrão de expressão de genes e proteínas envolvidas nas mudanças estruturais e da composição da parede celular secundária em relação à madeira normal foram observadas ao longo do período de indução. A deposição da camada G foi observada após 60 dias de indução da madeira de tração, através de secções transversais coradas com safranina e azul de astra. Ao longo do tempo de indução, os resultados sugerem um aumento no fluxo do carbono para a biossíntese de lignina na madeira oposta e para a biossíntese de celulose na madeira de tração. Assim, este trabalho resultou na caracterização dos perfis de expressão de proteínas e genes envolvidos nos mecanismos moleculares da formação da madeira sob estresse gravitacional e mais precisamente no remodelamento da parede celular secundária durante a formação da camada G.Wood is a complex and highly variable tissue under developmental and environmental control. Usually, an experimental model used to study wood formation is the induction of reaction wood formation. Reaction wood is formed to response a gravitational stimuli and is associated with an increased growth rate, modified fiber cell walls chemistry and structure. The reaction wood formed in the upper side of a bent trunk is called tension wood, and that one formed in the lower side is called opposite wood. One of the main characteristics of tension wood is the deposition of gelatinous layer (G layer) on the secondary cell wall, which is rich in highly crystalline cellulose. With the aim to characterize the changes in gene expression and protein profile during tenson wood formation in Eucalyptus grandis, 1,8 year-old trees were bent at a fixed angle (45º) and samples from the cambial region taken after 15, 30 and 60 days in the region of the trunk with the most accentuated curve. Proteins and RNA were extracted from the differentiating xylem region of the tension wood and opposite wood and were compared to those of normal wood (control) using 2D electrophoresis (LC-MS/MS) and sqPCR. The analysis was focused on genes and proteins related to the modification and formation of the cell wall. 480 spots of proteins were extracted, identified and classified into functional categories, with particular attention to those related to nucleotide sugar metabolism (including cellulose), lignin biosynthesis and the cytoskeleton. In addition, the relative expression of transcripts related to cellulose and lignin biosynthesis pectin metabolism and the cytoskeleton were analyzed by sqPCR in control, tension and opposite wood. Several differences in the expression pattern of genes and proteins involved in structural and secondary cell wall composition changes were observed when compared to normal wood during the induction period. The deposition of G layer was observed after 60 days of tension wood induction using transversal sections stained with safranin-astra blue. Throughout the induction period, the results suggest an increase in the flow carbon for lignin biosynthesis in the opposite wood and for cellulose biosynthesis in the tension wood. Therefore, the results of this work characterize the protein and gene expression profiles involved in the molecular mechanisms underlying wood formation under gravitational stress and, more specifically, in secondary cell wall remodeling during G layer formation

Métodos e estratégias em proteômica e suas aplicações na área vegetal Methods and strategies in proteomics and their applications in plants

A implementação da espectrometria de massa (MS) para as análises de peptídeos (MS) e de aminoácidos (MS em tandem ou MS/MS) tornou possível a identificação de centenas de proteínas em experimentos únicos. Uma grande variedade de estratégias está disponível atualmente para o fracionamento e a purificação de amostras, a identificação de proteínas, a quantificação, a análise de modificações pós-traducionais (MPT's) e os estudos de interação. Dessa forma, a proteômica abre novas perspectivas na biologia de plantas com ênfase nos estudos de variabilidade genética, estresses fisiológicos e desenvolvimento de plantas.<br>The implementation of mass spectrometry (MS) for peptides (MS) and amino acids (tandem MS or MS/MS) analysis allowed the identification of hundreds of proteins in single experiments. A number of different strategies are current available for sample fractioning and purification, proteins identification, quantification, post-translational modifications (PTM) and interaction analyses. In this way, the proteomics open up new perspectives in plant biology with emphasis on studies of genetic variability, physiological stresses and plant development