Customized versus Population-Based Birth Weight References for Predicting Fetal and Neonatal Undernutrition.

OBJECTIVES: The aim of our study was to construct a model of customized birth weight curves based on a Spanish population and to compare the ability of this customized model to our population-based chart to predict a neonatal ponderal index (PI) <10th percentile. METHODS: We developed a model that can predict the 10th percentile for a fetus according to gestational age and gender as well as maternal weight, height, and age. We compared the ability of this customized model to that of our own population-based model to predict a neonatal PI <10th percentile. Data from a large database were used (32,854 live newborns, from 1993 through 2012). Only singleton pregnancies with a gestational age at delivery of 32-42 weeks were included. RESULTS: In the entire pregnant population, the customized method was superior to the population-based method for detecting newborns with a PI <10th percentile (sensitivity: 55 vs. 40.96%; specificity: 99.6 vs. 91.23%; positive predictive value: 11.49 vs. 9.55%, and negative predictive value: 98.84 vs. 98.55%, respectively). In pregnant women with a BMI >90th percentile, the sensitivity was 75%, compared to 50% in the population-based method. In pregnant women with a height >90th percentile, the sensitivity was almost as high as in the population-based method (61.53 vs. 33.33%). CONCLUSION: The customized birth weight curve is superior to the population-based method for the detection of newborns with a PI <10th percentile. This is especially the case in women in the higher scales of height and weight as well as in preterm babies.

Normal Metabolic Levels in Prefrontal Cortex in Euthymic Bipolar I Patients with and without Suicide Attempts.

Introduction/Objective. Evidence suggests that the prefrontal cortex has been implicated in the pathophysiology of bipolar disorder (BD), but few neurochemical studies have evaluated this region in bipolar patients and there is no information from BD suicide attempters using Proton Magnetic Resonance Spectroscopy (H(+)MRS). The objective was to evaluate the metabolic function of the medial orbital frontal cortex in euthymic BD type I suicide and nonsuicide attempters compared to healthy subjects by H(+)MRS. Methods. 40 euthymic bipolar I outpatients, 19 without and 21 with history of suicide attempt, and 22 healthy subjects were interviewed using the Structured Clinical Interview with the DSM-IV axis I, the Hamilton Depression Rating Scale, the Young Mania Rating Scale, and the Barratt Impulsiveness Scale-11 and underwent H(+)MRS. Results. We did not find any metabolic abnormality in medial orbital frontal regions of suicide and nonsuicide BD patients and BD patients as a group compared to healthy subjects. Conclusions. The combined chronic use of psychotropic drugs with neuroprotective or neurotrophic effects leading to a euthymic state for longer periods of time may improve neurometabolic function, at least measured by H(+)MRS, even in suicide attempters. Besides, these results may implicate mood dependent alterations in brain metabolic activity. However, more studies with larger sample sizes of this heterogeneous disorder are warranted to clarify these data.

Prevalence and clinical correlates of alcohol use disorders among bipolar disorder patients: results from the Brazilian Bipolar Research Network.

OBJECTIVES: To investigate prevalence rates and clinical correlates of alcohol use disorders (AUD) among bipolar disorder (BD) patients in a large sample from the Brazilian Bipolar Research Network. METHODS: Four hundred and eighty-three DSM-IV BD patients, divided according to the presence or absence of a lifetime AUD diagnosis (BD-AUD vs. BD-nonAUD), were included. Demographic and clinical characteristics of these two groups were compared. Logistic regression was performed to identify which characteristics were most strongly associated with a lifetime AUD diagnosis. RESULTS: Nearly 23% presented a lifetime AUD diagnosis. BD-AUD patients were more likely to be male, to present rapid cycling, post-traumatic stress disorder (PTSD), anorexia, other substance use disorders (SUD), family history of SUD, any substance misuse during the first mood episode, history of psychosis, suicide attempts, and younger age at onset of illness than BD-nonAUD patients. Logistic regression showed that the variables most strongly associated with a lifetime AUD diagnosis were SUD (non-alcohol), any substance misuse during the first mood episode, PTSD, male gender, suicide attempt, family history of SUD, and younger age at onset of BD. CONCLUSIONS: BD-AUD patients begin their mood disorder earlier and present more suicidal behaviors than BD-nonAUD patients. Personal and family history of SUD may be good predictors of comorbid AUD among BD patients. These variables are easily assessed in the clinical setting and may help to identify a particularly severe subgroup of BD patients.

Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression.

Site-specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)-mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single-copy integration clones were then co-transfected with both Flp-containing plasmid and an EGFP-containing targeting cassette. Successful cassette exchange was suggested by emergence of G418-resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re-usable platform to produce multiple recombinant proteins for fundamental and applied research.

Sequential Social Exclusion in a Novel Cyberball Paradigm Leads to Reduced Behavioral Repair and Plasma Oxytocin in Borderline Personality Disorder.

Patients with borderline personality disorder (BPD) show interpersonal deficits, and altered emotional and oxytocin (OT) responses to social exclusion (Cyberball). In order to extend previous findings, this study applies a novel Cyberball variant. Nineteen BPD patients and 56 healthy controls (HC) played Cyberball for 2 minutes of inclusion, 5 minutes of partial exclusion by one of two co-players, and 2 minutes total exclusion by both. Plasma OT levels at baseline and after 7, 9, 15, and 40 minutes were measured with radioimmunoassay. BPD patients showed a greater aversive reaction and a trend for greater OT reduction after social exclusion than HC. BPD patients also tended to play less frequently with the excluder. Though limited by our sample size, we partially replicate previous findings. Our preliminary behavioral data support the notion of an altered OT regulation and reduced capacity for social cooperation in BPD.

Suicide in young Brazilian adults: 1997-2019 time series. / Suicídio em adultos jovens brasileiros: série temporal de 1997 a 2019.

This study aimed to analyze the time series of suicide mortality in Brazilian young adults between 1997 and 2019. This is an ecological study on time series with individuals aged 20 to 29 years. The Prais-Winsten regression was used for trend analysis. The mean suicide coefficient in young Brazilians was 6.36/100 thousand inhabitants in the period, showing increasing overall, females, and male trends. Regarding marital status, there was a higher proportion of suicide among single people (79.02%). As for the Brazilian regions, the South presented the highest mean mortality coefficient (9.18/100 thousand inhabitants), and the Northeast, North, and Southeast regions showed an increasing trend. Most deaths occured at home (54.93%), and there was an increasing trend both at home, at health establishments, and other places. We conclude that the suicide trend in Brazilian young adults was increasing in the period, with more significant proportions among single people. Brazilian regions showed different trends, and households, health facilities, and other locations followed an increasing trend toward suicide deaths in young adults.

Este estudo teve como objetivo analisar a série temporal da mortalidade por suicídio em adultos jovens brasileiros entre os anos de 1997 e 2019. Trata-se de um estudo ecológico, de séries temporais com indivíduos de 20 a 29 anos. Para a análise de tendência foi utilizada a regressão de Prais-Winsten. No período, o coeficiente médio de suicídio em jovens brasileiros foi de 6,36/100 mil habitantes, apresentando tendência crescente, tanto o geral, quanto no sexo feminino e masculino. Em relação ao estado civil, houve maior proporção de suicídio entre solteiros (79,02%). Quanto as regiões brasileiras, a Sul apresentou o maior coeficiente médio de mortalidade (9,18/100 mil habitantes), e as regiões Nordeste, Norte e Sudeste mostraram tendência crescente. A maioria dos óbitos ocorreram no domicílio (54,93%) e houve tendência crescente tanto no domicílio, como os estabelecimentos de saúde e outros locais. Conclui-se que a tendência do suicídio em adultos jovens brasileiros foi crescente no período, com maiores proporções entre os solteiros. As regiões brasileiras apresentaram diferentes tendências, e o domicílio, estabelecimentos de saúde e outros locais seguiram uma tendência crescente para as mortes por suicídio em adultos jovens.

Complications of laparoscopic inguinal herniorrhaphy including one case of atypical mycobacterial infection.

BACKGROUND: Although laparoscopic inguinal herniorrhaphy is considered safe, several complications may occur. This study aimed to evaluate the complications observed in 780 laparoscopic inguinal herniorrhaphies at the authors' hospital. METHODS: All the patients who underwent laparoscopic inguinal herniorrhaphy at the authors' hospital during a period of 11 years were enrolled retrospectively in this study. Patient demographics, operative data, and intra- and postoperative complications were evaluated. RESULTS: A total of 569 patients underwent 780 laparoscopic inguinal herniorrhaphies. The male-to-female ratio was 8.8 to 1, and the mean age was 54.8 ± 15.7 years. Hernia recurrence was recognized in 14 patients (2.5%). Intra- and postoperative complications were diagnosed in 28 (4.9%) and 35 (6.2%) patients respectively. There was no mortality. The most common intraoperative complication was extensive subcutaneous emphysema. Two patients with extensive subcutaneous emphysema had cardiac arrhythmia. Small bowel perforation and bladder perforation occurred in one patient each. One patient had extensive preperitoneal infection caused by Mycobacterium massiliense, which required mesh removal, tissue debridement, and prolonged antibiotic therapy. CONCLUSIONS: Although the mortality rate is low, potentially life-threatening complications such as small bowel and bladder perforation may be experienced by patients subjected to laparoscopic herniorrhaphy.

Suicide trend among Brazilian adolescents between 1997 and 2016. / Tendência de suicídio em adolescentes brasileiros entre 1997 e 2016.

OBJECTIVE: To analyze the suicide mortality trend among Brazilian adolescents from 1997 to 2016. METHODS: This is an ecological time series study; Prais-Winsten regression was used. RESULTS: 14,852 suicide deaths were recorded, with mortality rates per 100,000 inhabitants of 1.95 in 1997, 2.65 in 2016 and average of 2.14 for the period 1997-2016; deaths predominated in males (67.59%), as well as deaths due to intentional self-harm (84.19%) and suicides at home (52.69%); the Midwest region had the highest rate in the period (3.71/100,000 inhabitants), in particular the state of Mato Grosso do Sul (8.3/100,000 inhabitants); the suicide trend rose 1.35% per annum in the general adolescent population (95%CI 0.56;2.15), 1.63% in males (95%CI 0.56;2.29), 3.11% in the North (95%CI 2.25;3.98) and 4.19% in the Northeast (95%CI 2.58;5.84). CONCLUSION: Suicide mortality in Brazilian adolescents showed an upward trend in the period studied.

Regular dental care in preschoolers in rural Southern Brazil.

OBJECTIVE: To evaluate if factors related to the mother's previous guidance on her children's dental health and the school attendance of children influence the regular dental care of preschoolers living in the rural area of a municipality in Southern Brazil. METHODS: A population-based study was conducted with 264 children under five years of age and their mothers. Socioeconomic and behavioral data were collected using a questionnaire, and the children were subjected to dental health tests. The outcome was the regular use of dental services. The main exposure variables were children's care in daycare centers or schools and maternal guidance on the child's dental health. Poisson regression analysis with robust variance adjustment was used to estimate prevalence ratios, considering a 95% confidence interval. RESULTS: The prevalence of regular use was 11.4% (95%CI 7.5-15.2). In the adjusted analysis, the regular use of services was associated with the child attending day care center/school (PR = 2.44; 95%CI 1.38-4.34), and the mother received dental health guidance (PR = 4.13; 95%CI 1.77-9.61), even with control for socioeconomic, maternal and child variables. CONCLUSION: When mothers receive previous information on child dental health care and children attend schools or daycare centers, the likelihood of regular dental appointments in preschoolers living in rural locations increases.

Oxytocin and bone quality in the femoral neck of rats in periestropause.

The objective of this study is to identify whether oxytocin (OT) contributes to the reduction of osteopenia in the femoral neck of rats in periestropause. Animals in irregular estrous cycles received two NaCl injections (0.15 mol/L) or OT (134 µg/kg) over a 12-h interval, and after thirty-five days without treatments, the biological sample collection was performed. The oxytocin group (Ot) demonstrated the highest enzymatic activity of alkaline phosphatase (p = 0.0138), lowest enzymatic activity of tartrate-resistant acid phosphatase (p = 0.0045), higher percentage of compact bone (p = 0.0359), cortical expression of runt-related transcription factor 2 (p = 0.0101), osterix (p = 0.0101), bone morphogenetic protein-2/4 (p = 0.0101) and periostin (p = 0.0455). Furthermore, the mineral-to-matrix ratio (ν1PO4/Proline) was higher and type-B carbonate substitution (CO3/ν1PO4) was lower (p = 0.0008 and 0.0303) in Ot group. The Ot showed higher areal bone mineral density (p = 0.0050), cortical bone area (p = 0.0416), polar moment of inertia, maximum, minimum (p = 0.0480, 0.0480, 0.0035), bone volume fraction (p = 0.0166), connectivity density (p < 0.0001), maximal load (p = 0.0003) and bone stiffness (p = 0.0145). In Ot percentage of cortical pores (p = 0.0102) and trabecular number (p = 0.0088) was lower. The results evidence action of OT in the reduction of osteopenia, suggesting that it is a promising anabolic strategy for the prevention of primary osteoporosis during the periestropause period.

Suicídio em adultos jovens brasileiros: série temporal de 1997 a 2019 / Suicide in young Brazilian adults: 1997-2019 time series

Resumo Este estudo teve como objetivo analisar a série temporal da mortalidade por suicídio em adultos jovens brasileiros entre os anos de 1997 e 2019. Trata-se de um estudo ecológico, de séries temporais com indivíduos de 20 a 29 anos. Para a análise de tendência foi utilizada a regressão de Prais-Winsten. No período, o coeficiente médio de suicídio em jovens brasileiros foi de 6,36/100 mil habitantes, apresentando tendência crescente, tanto o geral, quanto no sexo feminino e masculino. Em relação ao estado civil, houve maior proporção de suicídio entre solteiros (79,02%). Quanto as regiões brasileiras, a Sul apresentou o maior coeficiente médio de mortalidade (9,18/100 mil habitantes), e as regiões Nordeste, Norte e Sudeste mostraram tendência crescente. A maioria dos óbitos ocorreram no domicílio (54,93%) e houve tendência crescente tanto no domicílio, como os estabelecimentos de saúde e outros locais. Conclui-se que a tendência do suicídio em adultos jovens brasileiros foi crescente no período, com maiores proporções entre os solteiros. As regiões brasileiras apresentaram diferentes tendências, e o domicílio, estabelecimentos de saúde e outros locais seguiram uma tendência crescente para as mortes por suicídio em adultos jovens.

Abstract This study aimed to analyze the time series of suicide mortality in Brazilian young adults between 1997 and 2019. This is an ecological study on time series with individuals aged 20 to 29 years. The Prais-Winsten regression was used for trend analysis. The mean suicide coefficient in young Brazilians was 6.36/100 thousand inhabitants in the period, showing increasing overall, females, and male trends. Regarding marital status, there was a higher proportion of suicide among single people (79.02%). As for the Brazilian regions, the South presented the highest mean mortality coefficient (9.18/100 thousand inhabitants), and the Northeast, North, and Southeast regions showed an increasing trend. Most deaths occured at home (54.93%), and there was an increasing trend both at home, at health establishments, and other places. We conclude that the suicide trend in Brazilian young adults was increasing in the period, with more significant proportions among single people. Brazilian regions showed different trends, and households, health facilities, and other locations followed an increasing trend toward suicide deaths in young adults.

Regular dental care in preschoolers in rural Southern Brazil / Atendimento odontológico regular em pré-escolares da área rural do Sul do Brasil

ABSTRACT OBJECTIVE To evaluate if factors related to the mother's previous guidance on her children's dental health and the school attendance of children influence the regular dental care of preschoolers living in the rural area of a municipality in Southern Brazil. METHODS A population-based study was conducted with 264 children under five years of age and their mothers. Socioeconomic and behavioral data were collected using a questionnaire, and the children were subjected to dental health tests. The outcome was the regular use of dental services. The main exposure variables were children's care in daycare centers or schools and maternal guidance on the child's dental health. Poisson regression analysis with robust variance adjustment was used to estimate prevalence ratios, considering a 95% confidence interval. RESULTS The prevalence of regular use was 11.4% (95%CI 7.5-15.2). In the adjusted analysis, the regular use of services was associated with the child attending day care center/school (PR = 2.44; 95%CI 1.38-4.34), and the mother received dental health guidance (PR = 4.13; 95%CI 1.77-9.61), even with control for socioeconomic, maternal and child variables. CONCLUSION When mothers receive previous information on child dental health care and children attend schools or daycare centers, the likelihood of regular dental appointments in preschoolers living in rural locations increases.

RESUMO OBJETIVO Avaliar se fatores relacionados à orientação prévia da mãe sobre saúde bucal dos seus filhos e a frequência escolar das crianças influenciam o atendimento odontológico regular de pré-escolares moradores da área rural de um município do Sul do Brasil. MÉTODOS Um estudo de base populacional foi conduzido com 264 crianças menores de cinco anos e suas mães. Dados socioeconômicos e comportamentais foram coletados por meio de questionário e as crianças foram submetidas a exames de saúde bucal. O desfecho foi o uso regular de serviços odontológicos. As variáveis de exposição principais foram o atendimento infantil em creches ou escolas e orientação materna sobre a saúde bucal da criança. A análise de regressão de Poisson com ajuste robusto de variância foi utilizada para estimar as razões de prevalência e intervalos de confiança de 95%. RESULTADOS A prevalência de uso regular foi 11,4% (IC95% 7,5-15,2). Na análise ajustada o uso regular de serviços foi associado à criança frequentar creche/escola (RP = 2,44; IC95% 1,38-4,34) e a mãe ter recebido orientação de saúde bucal (RP = 4,13; IC95% 1,77-9,61), mesmo com controle para variáveis socioeconômicas, maternas e da criança. CONCLUSÃO Quando as mães recebem informações prévias sobre os cuidados com a saúde bucal infantil e as crianças frequentam escolas ou creches, aumenta a probabilidade de consultas odontológicas regulares em pré-escolares residentes em localidades rurais.

Tendência de suicídio em adolescentes brasileiros entre 1997 e 2016 / Tendencia al suicidio en adolescentes brasileños entre 1997 y 2016 / Suicide trend among Brazilian adolescents between 1997 and 2016

Objetivo: Analisar a tendência da mortalidade por suicídio em adolescentes brasileiros no período de 1997 a 2016. Métodos: Estudo ecológico de séries temporais; empregou-se regressão de Prais-Winsten. Resultados: Foram registrados 14.852 óbitos por suicídio, com taxas de mortalidade por 100 mil habitantes de 1,95 em 1997, 2,65 em 2016, e média de 2,14 no período 1997-2016; predominaram óbitos no sexo masculino (67,59%), por lesões autoprovocadas intencionalmente (84,19%), ocorridos no domicílio (52,69%); a região Centro-Oeste apresentou a maior taxa no período (3,71/100 mil hab.), com destaque para Mato Grosso do Sul (8,3/100 mil hab.); observou-se tendência de aumento de 1,35% ao ano na mortalidade por suicídio na população geral de adolescentes (IC95% 0,56;2,15), 1,63% no sexo masculino (IC95% 0,56;2,29), 3,11% na região Norte (IC95% 2,25;3,98) e 4,19% no Nordeste (IC95% 2,58;5,84). Conclusão: A mortalidade por suicídio em adolescentes brasileiros mostrou tendência ascendente no período estudado.

Objetivo: analizar la tendencia al suicidio en adolescentes brasileños de 1997 a 2016. Método: estudio ecológico de series temporales; se utilizó la regresión de Prais-Winsten. Resultados: se registraron 14.852 suicidios, con tasas de mortalidad de 1,95 por 100.000 habitantes en 1997, 2,65 en 2016 y promedio de 2,14 en el período 1997-2016; las muertes predominaron en el sexo masculino (67,59%), por autolesión intencional (84,19%) y ocurridas en casa (52,69%); la región Centro-Oeste tuvo la tasa más alta en el período (3,71/100,000 habitantes), con énfasis en Mato Grosso do Sul (8,3/100,000 habitantes); hubo una tendencia de aumento del 1,35% al año de la mortalidad por suicidio en la población adolescente general (IC95% 0,56; 2,15), 1,63% en varones (IC95% 0,56; 2,29), 3,11% en la región Norte (IC95% 2,25; 3,98) y 4.19% en el Nordeste (IC95% 2,58; 5;84). Conclusión: la mortalidad por suicidio en adolescentes brasileños mostró una tendencia al alza en el período estudiado.

Objective: To analyze the suicide mortality trend among Brazilian adolescents from 1997 to 2016. Methods: This is an ecological time series study; Prais-Winsten regression was used. Results: 14,852 suicide deaths were recorded, with mortality rates per 100,000 inhabitants of 1.95 in 1997, 2.65 in 2016 and average of 2.14 for the period 1997-2016; deaths predominated in males (67.59%), as well as deaths due to intentional self-harm (84.19%) and suicides at home (52.69%); the Midwest region had the highest rate in the period (3.71/100,000 inhabitants), in particular the state of Mato Grosso do Sul (8.3/100,000 inhabitants); the suicide trend rose 1.35% per annum in the general adolescent population (95%CI 0.56;2.15), 1.63% in males (95%CI 0.56;2.29), 3.11% in the North (95%CI 2.25;3.98) and 4.19% in the Northeast (95%CI 2.58;5.84). Conclusion: Suicide mortality in Brazilian adolescents showed an upward trend in the period studied.

Prefrontal gray matter volume in adults bipolar I outpatients is associated with history of suicide attempts?

ABSTRACT Background: Patients with Bipolar Disorder (BD) have the highest lifetime risk for suicidal behavior (SB) compared to other psychiatric disorders. Neuroimaging research provides evidence of some structural and functional abnormalities in the brain of BD suicide attempters (SA), but interpretation of these findings may represent a number of features. Objective: The purpose of this study was to evaluate the volume of the prefrontal cortex in euthymic BD type I outpatients, with and without history of SA. Methods: 36 euthymic BD I outpatients (18 with and 18 without suicide attempt history) were underwent structural MRI and total and regional gray matter volumes were assessed and compared with 22 healthy controls (HC). Results: We did not found any differences in all areas between suicidal and non-suicidal BD I patients and BD patients as a group compared to HC as well. Discussion: our findings suggest that can be a different subgroups of patients in relation to prefrontal cortex volumes according to some clinical and socio-demographic caractheristics, such as number of previous episodes and continuous use of medical psychotropic drugs that may induce neuroplasticity phenomena, which restore cerebral volume and possibly can lead to long-term euthymia state.

Insect cell line development using FLP-mediated cassette exchange technology.

Traditional cell line development is quite laborious and time-consuming as it is based on the random integration of the gene of interest which leads to unpredictable expression behavior. In opposition, recombinase-mediated cassette exchange systems represent a powerful genetic engineering approach, allowing site-specific insertion of recombinant genes into pre-tagged genomic loci with superior expression characteristics, thus bypassing the need for extensive clone screening and shortening the development timelines. Such systems have not been widely implemented in insect cell lines used for the production of recombinant proteins most commonly through the baculovirus expression vector system. Herein, it is provided the protocol for the implementation of a FLP-mediated cassette exchange system in Spodoptera frugiperda Sf 9 cells, in order to grant a flexible cell line for the stable production of recombinant proteins.

Método do índice de qualidade (MIQ) na estimativa da vida de prateleira de peixes de espécies nativas brasileiras / Method of quality index method (QIM) for shelf life estimation of the Brazilian native species fish

O presente estudo teve como objetivo desenvolver um Método do Índice de Qualidade específico para três espécies de peixes nativos do Brasil, Hoplias malabaricus (Traíra), Hoplias lacerdae (Traírão) e Genidens barbus (Bagre branco) para estimar a vida de prateleira destas espécies através de uma avaliação espécie-específica sensorial. Na realização do estudo foram utilizados 20 peixes frescos de cada espécie, coletados no dia da captura, que foram avaliados sensorialmente até os 14 dias pós captura a cada dois dias. Para determinar a vida útil, também foi feito o monitoramento do pH. Os resultados obtidos mostram que o método do índice de qualidade pode ser utilizado como ferramenta para estimar a vida de prateleira das espécies analisadas. Acredita-se que mais estudos são necessários para validar o método proposto em espécies brasileiras.

Production of rotavirus core-like particles in Sf9 cells using recombinase-mediated cassette exchange.

A flexible Sf9 insect cell line was recently developed leveraging the recombinase-mediated cassette exchange (RMCE) technology, which competes with the popular baculovirus expression vector system (BEVS) in terms of speed to produce new proteins. Herein, the ability of this cell platform to produce complex proteins, such as rotavirus core-like particles, was evaluated. A gene construct coding for a VP2-GFP fusion protein was targeted to a pre-characterized high recombination efficiency locus flanked by flipase (Flp) recognition target sites and, after three weeks in selection, an isogenic cell population was obtained. Despite the lower cell specific productivities with respect to those obtained by baculovirus infection, the titers of VP2-GFP reached in shake flask batch cultures were comparable as a result of higher cell densities. To further improve the VP2-GFP levels from stable expression, analysis of exhausted medium was undertaken to design feeding strategies enabling higher cell densities as well as increased culture duration. The implementation of the best strategy allowed reaching 20 million cells per ml in bioreactor cultures; the integrity of the rotavirus core-like particles could be confirmed by electron microscopy. Overall, we show that this Sf9-Flp cell platform represents a valuable alternative to the BEVS for producing complex recombinant proteins, such as rotavirus core-like particles.

Insect cells as a production platform of complex virus-like particles.

Virus-like particles (VLPs) are multiprotein structures that resemble the conformation of native viruses but lack a viral genome, potentiating their application as safer and cheaper vaccines. The production of VLPs has been strongly linked with the use of insect cells and the baculovirus expression vector system, especially those particles composed of two or more structural viral proteins. In fact, this expression platform has been extensively improved over the years to address the challenges of coexpression of multiple proteins and their proper assembly into complexes in the same cell. In this article, the role of insect cell technology in the development and production of complex VLPs is overviewed; recent achievements, current bottlenecks and future trends are also highlighted.

A cell sorting protocol for selecting high-producing sub-populations of Sf9 and High Five™ cells.

Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells.