Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity

Abstract BACKGROUND Cell-free protein synthesis (CFPS) technology has emerged as a powerful tool for a variety of biotechnological applications, including the expression of different classes of biopharmaceutical products. L-Asparaginase (E.C. Number: 3.5.1.1, L-asparagine amidohydrolase) (L-ASNase) is an important biopharmaceutical used to treat leukemia, but expression of multiple proteoforms in CFPS systems and rapid characterization using standard colorimetric methods has not yet been fully exploited. Herein, recombinant expression and characterization of an L-ASNase from Erwinia chrysanthemi (Erwinase) using a new CFPS protocol is reported. RESULTS Expression and quantification of the enzymatic activity of a soluble his-tagged L-ASNase directly from a CFPS reaction was successfully achieved. Purification of the protein was not required in order to assess its biological activity. Activity of L-ASNase was significantly higher than the control reaction (7.07 ± 0.68 U mL–1 vs. 1.83 ± 0.14 U mL–1, respectively). Expression of a mutant Erwinase proteoform – V293M – was also achieved and it presented a similar enzymatic activity. No significant loss in L-ASNase enzymatic activity was noticed after removal of cyclic AMP, spermidine, transfer RNA, T7 RNA polymerase and, especially, ammonium acetate (a common interference in ASNase enzymatic assays) from the CFPS reaction. CONCLUSION The protocol developed in this work will facilitate the screening of novel clinically-relevant L-ASNase proteoforms. © 2021 Society of Chemical Industry (SCI).

Bites by Tomodon dorsatus (serpentes, dipsadidae): Clinical and epidemiological study of 86 cases.

A total of 86 proven cases of Tomodon dorsatus bites admitted to Hospital Vital Brazil (HVB) of Butantan Institute, in São Paulo, Brazil, between 1945 and 2018, were retrospectively analyzed. The cases included were those in which the snake was brought to HVB and was correctly identified. Of the 86 cases of snake bites, it was possible to describe the sex of the snake in 52 cases; 31 (59.6%) snakes were male. Only 52 snakes out of 86 could be studied because of their preservation status. The length of snakes (snout-vent length) ranged from 180 to 770 mm. Of the 86 snakes, 72 could be distinguished as adults (n = 63, 87.5%) or juveniles (n = 9, 12.5%). Most bites occurred in the spring and summer seasons (n = 57, 66.3%) and during warmer periods of the day (n = 61, 72.6%), between 9 a.m. and 3 p.m. The mean (±standard deviation) age of the victims was 26.9 ±â€¯17.2 years, and 60 (69.8%) were men. Approximately 90% of the patients sought medical care within 6 h after the bite. Both upper (n = 45, 52.3%) and lower (n = 37, 43.0%) limbs were the most frequently bitten, particularly the feet and hands (n = 54, 62.8%). The local clinical manifestations were pain (n = 55, 64.0%), transitory bleeding (n = 23, 26.7%), erythema (n = 22, 25.6%), edema (n = 14, 16.3%), paresthesia (n = 9, 10.5%), and ecchymosis (n = 3, 3.5%). Only 10 (11.6%) patients reported non-specific systemic symptoms characterized by transient dizziness or mild headache, and 21 (24.4%) patients showed no evidence of envenomation. A 20 min whole blood clotting test was performed in 31 (36.0%) patients on admission and all of them had coagulable blood. Supportive treatment was offered to 38 (44.2%) patients, namely, antiseptic (n = 20, 23.3%), antihistamines (n = 12, 14.0%), and analgesics (n = 9, 10.5%). Four (4.7%) patients were inappropriately treated with Bothrops antivenom before their admission to HVB. No sequelae or relevant complications were observed in patients, and the prognosis was benign. Therefore, although T. dorsatus bites can cause mild local symptomatology, it is important that health professionals know how to make the correct diagnosis to avoid unnecessary use of antivenom.

Acidentes por serpente falsa coral Oxyrhopus guibei (Hoge &Romano, 1978): estudo retrospectivo de 205 casos atendidos no Hospital Vital Brazil no período de 1945 a 2018

A total of 205 proven cases of Oxyrhopus guibei bites, a false coral snake, admitted to Hospital Vital Brazil (HVB) of Butantan Institute, in São Paulo, Brazil, between 1945 and 2018, were retrospectively analyzed. The cases included were those in which the snake was brought to HVB and was correctly identified. Of the 205 cases of snake bites, it was possible to describe the sex of the snake in 172 cases; 40 (65.5%) snakes were female. Only 83 snakes out of 205 could be studied because of their preservation status. The length of snakes (snout–vent length) ranged from 200 to 1250 mm. Of the 205 snakes, 172 could be distinguished as juveniles (n = 116, 68.4%) or adults (n = 56, 31.6%). Most bites occurred in the autumn and spring seasons (n = 127, 62.0%) and in the early evening (n = 84, 41.8%), between 6 PM and 12 PM. The mean (± standard deviation) age of the victims was 26.6 ± 15.9 years, and 130 (63.4%) were men. Approximately 75% of the patients sought medical care within 6 h after the bite. Both lower (n = 92, 49.8%) and upper (n = 86, 46.5%) limbs were the most frequently bitten, particularly the feet and hands (n = 135, 73.0%). The local clinical manifestations were pain (n = 101, 49.3%), transitory bleeding (n = 39, 19.0%), erythema (n = 31, 15.1%), edema (n = 21, 10.2%), paresthesia (n = 17, 8.3%), severe pain (n = 9, 4.4%), radiating pain (n = 8, 3.9%), and ecchymosis (n = 1, 0.5%). Only 38 (18.5%) patients reported nom-specific systemic symptoms characterized by transient dizziness or mild headache, and 69 (33.7%) patients showed no evidence of envenomation. A 20 min whole blood clotting test was performed in 36 (17.6%) patients on admission and all of them had coagulable blood. Supportive treatment was offered to 80 (39.0%) of patients, namely, antiseptic (n = 43, 21.0%), antihistamines (n = 25, 12.2%), and analgesics (n = 24, 11.7%). Fourteen (6.8%) patients were inappropriately treated with antivenom. No sequelae or relevant complications were observed in patients, and the prognosis was benign. Therefore, although O. guibei bites can cause mild local symptomatology, it can be confused with corals snakes (mimics the highly venomous coral snake Micrurus frontalis). Then, it very is important that health professionals know how to make the correct diagnosis to avoid unnecessary use of antivenom.

Foram retrospectivamente analisados 205 casos de picadas de Oxyrhopus guibei, uma falsa cobra coral, admitidos no Hospital Vital Brazil (HVB) do Instituto Butantan, em São Paulo, Brasil, entre 1945 e 2018. Os casos incluídos foram aqueles em que a cobra foi trazida para o HVB e corretamente identificada. Dos 205 casos, foi possível descrever o sexo da serpente em 172 casos: 40 (65,5%) serpentes eram fêmeas. Apenas 83 serpentes, de 205, puderam ser estudadas devido ao seu estado de preservação. O comprimento total das cobras variou de 200 a 1250 mm. Das 205 cobras, 172 puderam ser classificadas como filhotes (n = 116, 68,4%) ou adultas (n = 56, 31,6%). A maioria das picadas ocorreu no outono e na primavera (n = 127, 62,0%) e no final da tarde e início da noite (n = 84, 41,8%), entre 18 e 24 horas. A média das idades dos pacientes (± desvio padrão) foi de 26,6 ± 15,9 anos, e 130 (63,4%) pacientes eram homens. Aproximadamente 75% dos pacientes procuraram atendimento médico dentro das primeiras 6 horas após a picada. Os membros inferiores (n = 92, 49,8%) e superiores (n = 86, 46,5%) foram os locais mais frequentemente atingidos, particularmente os pés e as mãos (n = 135, 73,0%). As manifestações clínicas locais foram dor (n = 101, 49,3%), sangramento transitório (n = 39, 19,0%), eritema (n = 31, 15,1%), edema (n = 21, 10,2%), parestesia (n = 17, 8,3%), dor intensa (n = 9, 4,4%), dor irradiada (n = 8, 3,9%) e equimose (n = 1, 0,5%). Apenas 38 (18,5%) pacientes relataram sintomas sistêmicos não específicos, caracterizados por tontura transitória ou cefaleia leve, e 69 (33,7%) pacientes não apresentaram evidências de envenenamento. O teste do tempo de coagulação sanguínea foi realizado em 36 (17,6%) pacientes na admissão e em todos o sangue estava coagulável. Tratamento de suporte foi oferecido a 80 (39,0%) pacientes, notadamente o uso de antissépticos (n = 43, 21,0%), anti-histamínicos (n = 25, 12,2%) e analgésicos (n = 24, 11,7%). Quatorze (6,8%) pacientes foram tratados inadequadamente com o soro antiveneno. Nenhuma sequela ou complicações foram observadas nos pacientes e o prognóstico foi benigno. Portanto, embora as picadas de O. guibei possam causar leve sintomatologia local, ela pode ser confundida com serpentes corais verdadeiras (Micrurus frontalis). Portanto, é muito importante que os profissionais de saúde saibam como fazer o diagnóstico correto para evitar o uso desnecessário do soro antiveneno.

Bites by Tomodon dorsatus (serpentes, dipsadidae): clinical and epidemiological study of 86 cases

A total of 86 proven cases ofTomodon dorsatusbites admitted to Hospital Vital Brazil (HVB) of ButantanInstitute, in São Paulo, Brazil, between 1945 and 2018, were retrospectively analyzed. The cases included werethose in which the snake was brought to HVB and was correctly identified. Of the 86 cases of snake bites, it waspossible to describe the sex of the snake in 52 cases; 31 (59.6%) snakes were male. Only 52 snakes out of 86could be studied because of their preservation status. The length of snakes (snout–vent length) ranged from 180to 770 mm. Of the 86 snakes, 72 could be distinguished as adults (n = 63, 87.5%) or juveniles (n = 9, 12.5%).Most bites occurred in the spring and summer seasons (n = 57, 66.3%) and during warmer periods of the day(n = 61, 72.6%), between 9A.M. and 3P.M. The mean ( ± standard deviation) age of the victims was 26.9 ± 17.2 years, and 60 (69.8%) were men.Approximately 90% of the patients sought medical care within 6 h after the bite. Both upper (n = 45, 52.3%)and lower (n = 37, 43.0%) limbs were the most frequently bitten, particularly the feet and hands (n = 54,62.8%). The local clinical manifestations were pain (n = 55, 64.0%), transitory bleeding (n = 23, 26.7%), er-ythema (n = 22, 25.6%), edema (n = 14, 16.3%), paresthesia (n = 9, 10.5%), and ecchymosis (n = 3, 3.5%).Only 10 (11.6%) patients reported non-specific systemic symptoms characterized by transient dizziness or mildheadache, and 21 (24.4%) patients showed no evidence of envenomation. A 20 min whole blood clotting test wasperformed in 31 (36.0%) patients on admission and all of them had coagulable blood. Supportive treatment wasoffered to 38 (44.2%) patients, namely, antiseptic (n = 20, 23.3%), antihistamines (n = 12, 14.0%), and an-algesics (n = 9, 10.5%). Four (4.7%) patients were inappropriately treated withBothropsantivenom before theiradmission to HVB. No sequelae or relevant complications were observed in patients, and the prognosis wasbenign. Therefore, althoughT. dorsatusbites can cause mild local symptomatology, it is important that healthprofessionals know how to make the correct diagnosis to avoid unnecessary use of antivenom.

Bites by Tomodon dorsatus (serpentes, dipsadidae): clinical and epidemiological study of 86 cases

A total of 86 proven cases ofTomodon dorsatusbites admitted to Hospital Vital Brazil (HVB) of ButantanInstitute, in São Paulo, Brazil, between 1945 and 2018, were retrospectively analyzed. The cases included werethose in which the snake was brought to HVB and was correctly identified. Of the 86 cases of snake bites, it waspossible to describe the sex of the snake in 52 cases; 31 (59.6%) snakes were male. Only 52 snakes out of 86could be studied because of their preservation status. The length of snakes (snout–vent length) ranged from 180to 770 mm. Of the 86 snakes, 72 could be distinguished as adults (n = 63, 87.5%) or juveniles (n = 9, 12.5%).Most bites occurred in the spring and summer seasons (n = 57, 66.3%) and during warmer periods of the day(n = 61, 72.6%), between 9A.M. and 3P.M. The mean ( ± standard deviation) age of the victims was 26.9 ± 17.2 years, and 60 (69.8%) were men.Approximately 90% of the patients sought medical care within 6 h after the bite. Both upper (n = 45, 52.3%)and lower (n = 37, 43.0%) limbs were the most frequently bitten, particularly the feet and hands (n = 54,62.8%). The local clinical manifestations were pain (n = 55, 64.0%), transitory bleeding (n = 23, 26.7%), er-ythema (n = 22, 25.6%), edema (n = 14, 16.3%), paresthesia (n = 9, 10.5%), and ecchymosis (n = 3, 3.5%).Only 10 (11.6%) patients reported non-specific systemic symptoms characterized by transient dizziness or mildheadache, and 21 (24.4%) patients showed no evidence of envenomation. A 20 min whole blood clotting test wasperformed in 31 (36.0%) patients on admission and all of them had coagulable blood. Supportive treatment wasoffered to 38 (44.2%) patients, namely, antiseptic (n = 20, 23.3%), antihistamines (n = 12, 14.0%), and an-algesics (n = 9, 10.5%). Four (4.7%) patients were inappropriately treated withBothropsantivenom before theiradmission to HVB. No sequelae or relevant complications were observed in patients, and the prognosis wasbenign. Therefore, althoughT. dorsatusbites can cause mild local symptomatology, it is important that healthprofessionals know how to make the correct diagnosis to avoid unnecessary use of antivenom.