RESUMO
MOTIVATION: The next-generation sequencing technologies have transformed our understanding of immunoglobulin (Ig) profiles in various immune states. Clonotyping, which groups Ig sequences into B cell clones, is crucial in investigating the diversity of repertoires and changes in antigen exposure. Despite its importance, there is no widely accepted method for clonotyping, and existing methods are computationally intensive for large sequencing datasets. RESULTS: To address this challenge, we introduce YClon, a fast and efficient approach for clonotyping Ig repertoire data. YClon uses a hierarchical clustering approach, similar to other methods, to group Ig sequences into B cell clones in a highly sensitive and specific manner. Notably, our approach outperforms other methods by being more than 30 to 5000 times faster in processing the repertoires analyzed. Astonishingly, YClon can effortlessly handle up to 2 million Ig sequences on a standard laptop computer. This enables in-depth analysis of large and numerous antibody repertoires. AVAILABILITY AND IMPLEMENTATION: YClon was implemented in Python3 and is freely available on GitHub.
Assuntos
Linfócitos B , Imunoglobulinas , Células Clonais , Imunoglobulinas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise por ConglomeradosRESUMO
Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and exudate samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. Envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.
Assuntos
Venenos de Aranha , Aranhas , Animais , Colorimetria , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Diester Fosfórico Hidrolases/genética , Coelhos , Sensibilidade e Especificidade , Venenos de Aranha/genética , Venenos de Aranha/toxicidade , Aranhas/genéticaRESUMO
Learning synthetic biology is often seen as a far distant possibility, restricted to those who have the privilege of an academic career. We propose a student-centered discussion group around synthetic biology, aimed at people from high school onwards with different backgrounds to interact and learn about synthetic biology. We developed a 14-week long program with three modules: "Leveling," "Introducing," and "Discussion." By completing the first two modules, the members should be more comfortable with biological names, structures, concepts, and techniques. The modules developed are available in Portuguese, Spanish, and English via the Open Lab Idea Real website (https://ideareal.org/clube-de-biologia-sintetica/) and can be used to implement the Club either in place or virtually around the world. We put it to practice at Universidade Federal de Minas Gerais (UFMG) creating the Club named BioSin. There are programs such as the International Genetically Engineered Machine (iGEM) competition focused on disseminating synthetic biology. Although iGEM is one fantastic way of learning about synthetic biology, there is a high cost. Because of that, a study and discussion Club is a tool to spread knowledge and engage with the study area.
Assuntos
Engenharia Genética , Biologia Sintética , Humanos , Estudos Interdisciplinares , Instituições Acadêmicas , Estudantes , Biologia Sintética/educaçãoRESUMO
In patients with severe forms of COVID-19, thromboelastometry has been reported to display a hypercoagulant pattern. However, an algorithm to differentiate severe COVID-19 patients from nonsevere patients and healthy controls based on thromboelastometry parameters has not been developed. Forty-one patients over 18 years of age with positive qRT-PCR for SARS-CoV-2 were classified according to the severity of the disease: nonsevere (NS, n = 20) or severe (S, n = 21). A healthy control (HC, n = 9) group was also examined. Blood samples from all participants were tested by extrinsic (EXTEM), intrinsic (INTEM), non-activated (NATEM) and functional assessment of fibrinogen (FIBTEM) assays of thromboelastometry. The thrombodynamic potential index (TPI) was also calculated. Severe COVID-19 patients exhibited a thromboelastometry profile with clear hypercoagulability, which was significantly different from the NS and HC groups. Nonsevere COVID-19 cases showed a trend to thrombotic pole. The NATEM test suggested that nonsevere and severe COVID-19 patients presented endogenous coagulation activation (reduced clotting time and clot formation time). TPI data were significantly different between the NS and S groups. The maximum clot firmness profile obtained by FIBTEM showed moderate/elevated accuracy to differentiate severe patients from NS and HC. A decision tree algorithm based on the FIBTEM-MCF profile was proposed to differentiate S from HC and NS. Thromboelastometric parameters are a useful tool to differentiate the coagulation profile of nonsevere and severe COVID-19 patients for therapeutic intervention purposes.
Assuntos
Coagulação Sanguínea , COVID-19/sangue , Tromboelastografia , Trombofilia/sangue , Adulto , Idoso , Algoritmos , COVID-19/complicações , COVID-19/diagnóstico , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Trombofilia/diagnóstico , Trombofilia/etiologia , Adulto JovemRESUMO
As antibodies are a very important tool for diagnosis, therapy, and experimental biology, a large number of antibody structures and sequences have become available in recent years. Therefore, tools that allow the analysis, comparison, and visualization of this large amount of antibody data are crucially needed. We developed the antibody high-density alignment visualization and analysis (Yvis) platform to provide an innovative, robust and high-density data visualization of antibody sequence alignments, called Collier de Diamants. The Yvis platform also provides an integrated structural database, which is updated weekly, and many different search and filter options. This platform can help to formulate hypotheses concerning the key residues in antibody structures or interactions to improve the understanding of antibody properties. The Yvis platform is available at http://bioinfo.icb.ufmg.br/yvis/.
Assuntos
Anticorpos/química , Alinhamento de Sequência , Software , Gráficos por Computador , Bases de Dados de Proteínas , Anticorpos Anti-HIV/química , Humanos , Região Variável de Imunoglobulina/química , Análise de Sequência de ProteínaRESUMO
Horse serum antibodies have been used for greater than a century for the treatment and prophylaxis of infectious diseases and envenomations. Little is known, however, about the immunogenetic diversity that produces horse serum antibodies. Here, we employed next-generation sequencing for a first-in-kind comprehensive analysis of the equine B-cell repertoire. Nearly 45,000 and 30,000 clonotypes were obtained for the heavy-chain (IGH) and lambda light-chain (IGL) loci, respectively. We observed skewed use of the common subgroups IGHV2 (92.49%) and IGLV8 (82.50%), consistent with previous reports, but also novel use of the rare genes IGHV6S1 and IGLV4S2. CDR-H3 amino acid composition revealed different amino acid patterns at positions 106 and 116 compared to human, rabbit, and mouse, suggesting that an extended conformation predominates among horse CDR-H3 loops. Our analysis provides new insights regarding the mechanisms employed to generate antibody diversity in the horse, and could be applicable to the optimized design of synthetic antibodies intended for future therapeutic use.
Assuntos
Regiões Determinantes de Complementaridade/genética , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Cavalos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias lambda de Imunoglobulina/genética , Animais , Regiões Determinantes de Complementaridade/imunologia , Cavalos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias lambda de Imunoglobulina/imunologia , Camundongos , CoelhosRESUMO
Accidents with venomous animals pose a health issue in Brazil, and those involving brown spiders (Loxosceles sp.) figure between the most frequent ones. The accidental envenomation by brown spiders causes a strong local dermonecrotic effect, which can be followed by systemic manifestations that in some cases lead to death. The production of antivenoms for the treatments of such accidents relies on a variety of animal experiments, from the spider venom extraction to the production of antivenom in horses. In the present work, there is an attempt to reduce and optimize animal experiments with the construction and production of a chimeric protein, named Lil, containing immunodominant epitopes previously mapped from the main proteins of the Loxosceles venom, the Sphingomyelinases D. The Lil protein contains epitopes from Sphinomyelinases D of the three-main species found in Brazil and this chimeric protein was found capable of inducing antibodies with the potential to partially neutralize the toxic effects of Loxosceles intermedia venom in an animal model. Therefore, in order to reduce spider usage and to improve the lifespan of the horses used for immunization we suggest the Lil protein as a potential candidate to replace the venom usage in the antivenom production protocols.
Assuntos
Aranha Marrom Reclusa/enzimologia , Epitopos de Linfócito B/imunologia , Epitopos Imunodominantes/imunologia , Diester Fosfórico Hidrolases/imunologia , Proteínas Recombinantes de Fusão/imunologia , Venenos de Aranha/imunologia , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização , Testes de Neutralização , Diester Fosfórico Hidrolases/genética , Coelhos , Venenos de Aranha/genéticaRESUMO
In the present investigation we used a recombinant LiD1 toxin, named rLiD1his, from Loxosceles intermedia brown spider to elicit specific antibodies in mice carrying different Human Leukocyte Antigens class II (HLAII) {DRB1.0401 (DR4), DQB1.0601 (DQ6) and DQB1.0302 (DQ8)} as well as in BALB/C and C57BL/6 control mice. All mice strains produced high antibody titers against rLiD1his but DR4 mice antibodies (the lower responder mice) were not able to recognize L. intermedia crude venom. The anti-rLiD1his sera, except from DR4 mice, were able to neutralize dermonecrotic, hemorrhagic and edematogenic effects of rLiD1his in naïve rabbits. Overlapping peptides from the amino acid sequence of LiD1 toxin were prepared by SPOT method and differences in LiD1 epitope recognition were observed using different mice anti-rLiD1his sera. The region (160)DKVGHDFSGNDDISDVGK(177) was recognized by transgenic DQ8 and DQ6 mice sera. Other epitopes were recognized by at least two different animals' sera including (10)MGHMVNAIGQIDEFVNLG(27), (37)FDDNANPEYTYHGIP(51), (70)GLRSATTPGNSKYQEKLV(87) and (259)AAYKKKFRVATYDDN(273). Among these epitopes, the epitopes 37-51 and 160-177 have already been shown in previously studies as good candidates to be used alone or combined with other peptides to induce protective immune response against Loxosceles venoms. The results presented here highlight the importance of HLAII in antibody response and recognition of specific B-cell epitopes of rLiD1his spider toxin according to HLAII type and impact in the epitopic vaccine development against this spider.
Assuntos
Anticorpos/imunologia , Epitopos de Linfócito B/imunologia , Diester Fosfórico Hidrolases/imunologia , Venenos de Aranha/enzimologia , Sequência de Aminoácidos , Animais , Patrimônio Genético , Soros Imunes/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Testes de Neutralização , CoelhosRESUMO
The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for approximately 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver-Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.
Assuntos
Peptídeo Hidrolases/metabolismo , Ativadores de Plasminogênio/metabolismo , Venenos de Víboras/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Ensaio de Imunoadsorção Enzimática , Glicosilação , Cinética , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Ativadores de Plasminogênio/química , Ativadores de Plasminogênio/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Sandwich-type enzyme linked immunosorbent assays (ELISA) were developed to detect Lachesis muta muta (bushmaster) snake venom using antibodies against the plasminogen activator enzyme (LV-PA). Antibodies to LV-PA were obtained by immunization of one rabbit with the purified enzyme. The IgG fraction was purified from rabbit blood in a single step on a column of Sepharose-L. m. muta venom and used to coat the microtiter plates. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with L. m. muta venom from those in mice inoculated with venoms from Bothrops atrox, B. brazili, B. castelnaudi, Bothriopsis taeniata, B. bilineata, Crotalus durissus ruruima and the antigenic Bothrops (AgB) and Crotalus (AgC) pools venoms used to produce Bothropic and Crotalic antivenoms at Fundacao Ezequiel Dias (FUNED). Measurable absorbance signals were obtained with 1.5 ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice.
Assuntos
Especificidade de Anticorpos/imunologia , Venenos de Crotalídeos/isolamento & purificação , Viperidae , Animais , Anticorpos/imunologia , Brasil , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Camundongos , Ativadores de Plasminogênio/imunologia , Especificidade da EspécieRESUMO
A serine proteinase with kallikrein-like activity (LV-Ka) has been purified to homogeneity from bushmaster snake (Lachesis muta muta) venom. Physicochemical studies indicated that LV-Ka is a single chain glycoprotein with a molecular mass (Mr) of 33 kDa under reducing conditions which was reduced to 28 kDa after treatment with N-Glycosidase F (PNGase F). LV-Ka can be bounded and neutralized by serum alpha2-macroglobulin (alpha2-M), a prevalent mammalian protease inhibitor that is capable of forming a macromolecular complex with LV-Ka (Mr >180 kDa). Cleavage of alpha2-M by the enzyme resulted in the formation of 90-kDa fragments. The proteolytic activity of LV-Ka against dimethylcasein could be inhibited by alpha2-M, and the binding ratio of the inhibitor:enzyme complex was found to be 1:1. The Michaelis constant, Km, and catalytic rate constant, kcat, of LV-Ka on four selective chromogenic substrates were obtained from Lineweaver-Burk plots. LV-Ka exhibits substrate specificities not only for the glandular kallikrein H-D-Val-Leu-Arg-pNA (S-2266) but also for the plasmin substrates S-2251 and Tos-Gly-Pro-Lys-pNA. Bovine kininogen incubated with LV-Ka generated a polypeptide that dose dependently contracted mesenteric arterial rings from spontaneously hypertensive rats (SHR) in a similar way as bradykinin (BK) does. As it happens with BK, LV-Ka generated polypeptide was inhibited by HOE-140, a bradykinin B2-receptor antagonist and by indomethacin, a cyclo-oxygenase inhibitor. These results strongly suggest that the polypeptide generated by LV-Ka by cleavage of bovine kininogen is bradykinin. In addition, our studies may help to understand the mechanism of action involved in hypotension produced by envenomation of bushmaster snake.
Assuntos
Cininas/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Serina Endopeptidases/química , Venenos de Víboras/química , Viperidae , Animais , Bovinos , Humanos , Técnicas In Vitro , Masculino , Artérias Mesentéricas/metabolismo , Ratos , Ratos Endogâmicos SHR , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/toxicidade , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Venenos de Víboras/isolamento & purificação , Venenos de Víboras/toxicidadeRESUMO
A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (0.8 microg/g) was shown to lower blood pressure in experimental rats. In vitro, the isolated proteinase was shown to have neither fibrin(ogeno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity=13.0 and 31.5 U/mg, respectively; crude venom=0.25 and 6.0 U/mg) but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB and PMSF, indicating that the enzyme is a serine proteinase. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen was one of the activities exhibited by LV-Ka.