Structural and functional characterization of genes PYL-PP2C-SnRK2s in the ABA signalling pathway of Cucurbita pepo.

BACKGROUND: The core regulation of the abscisic acid (ABA) signalling pathway comprises the multigenic families PYL, PP2C, and SnRK2. In this work, we conducted a genome-wide study of the components of these families in Cucurbita pepo. RESULTS: The bioinformatic analysis of the C. pepo genome resulted in the identification of 19 CpPYL, 102 CpPP2C and 10 CpSnRK2 genes. The investigation of gene structure and protein motifs allowed to define 4 PYL, 13 PP2C and 3 SnRK2 subfamilies. RNA-seq analysis was used to determine the expression of these gene families in different plant organs, as well as to detect their differential gene expression during germination, and in response to ABA and cold stress in leaves. The specific tissue expression of some gene members indicated the relevant role of some ABA signalling genes in plant development. Moreover, their differential expression under ABA treatment or cold stress revealed those ABA signalling genes that responded to ABA, and those that were up- or down-regulated in response to cold stress. A reduced number of genes responded to both treatments. Specific PYL-PP2C-SnRK2 genes that had potential roles in germination were also detected, including those regulated early during the imbibition phase, those regulated later during the embryo extension and radicle emergence phase, and those induced or repressed during the whole germination process. CONCLUSIONS: The outcomes of this research open new research lines for agriculture and for assessing gene function in future studies.

An miR164-resistant mutation in the transcription factor gene CpCUC2B enhances carpel arrest and ectopic boundary specification in Cucurbita pepo flower development.

The sex determination process in cucurbits involves the control of stamen or carpel development during the specification of male or female flowers from a bisexual floral meristem, a function coordinated by ethylene. A gain-of-function mutation in the miR164-binding site of CpCUC2B, ortholog of the Arabidopsis transcription factor gene CUC2, not only produced ectopic floral meristems and organs, but also suppressed the development of carpels and promoted the development of stamens. The cuc2b mutation induced the transcription of CpCUC2B in the apical shoots of plants after female flowering but repressed other CUC genes regulated by miR164, suggesting a conserved functional redundancy of these genes in the development of squash flowers. The synergistic androecious phenotype of the double mutant between cuc2b and etr2b, an ethylene-insensitive mutation that enhances the production of male flowers, demonstrated that CpCUC2B arrests the development of carpels independently of ethylene and CpWIP1B. The transcriptional regulation of CpCUC1, CpCUC2, and ethylene genes in cuc2b and ethylene mutants also confirms this conclusion. However, the epistasis of cuc2b over aco1a, a mutation that suppresses stamen arrest in female flowers, and the down-regulation of CpACS27A in cuc2b female apical shoots, indicated that CpCUC2B promotes stamen development by suppressing the late ethylene production.

Production and characterization of the lipopeptide with anti-adhesion for oral biofilm on the surface of titanium for dental implants.

Titanium implants are subject to bacterial adhesion and peri-implantitis induction, and biosurfactants bring a new alternative to the fight against infections. This work aimed to produce and characterize the biosurfactant from Bacillus subtilis ATCC 19,659, its anti-adhesion and antimicrobial activity, and cell viability. Anti-adhesion studies were carried out against Streptococcus sanguinis, Staphylococcus aureus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Proteus mirabilis as the minimum inhibitory concentration and the minimum bactericidal concentration. Cell viability was measured against osteoblast and fibroblast cells. The biosurfactant was classified as lipopeptide, with critical micelle concentration at 40 µg mL- 1, and made the titanium surface less hydrophobic. The anti-adhesion effect was observed for Staphylococcus aureus and Streptococcus sanguinis with 54% growth inhibition and presented a minimum inhibitory concentration of 15.7 µg mL- 1 for Streptococcus sanguinis and Aggregatibacter actinomycetemcomitans. The lipopeptide had no cytotoxic effect and demonstrated high potential application against bacterial biofilms.

Increases in group A streptococcal infections in the pediatric population in Houston, TX, 2022.

Beginning in October 2022, we observed a substantial increase in the total number of cases of invasive GAS disease (iGAS) in the pediatric population in Houston, TX. Emm12 GAS strains were disproportionately represented but the overall proportion of iGAS infections observed during the current spike was similar to pre-pandemic years.

Jasmonate-deficient mutant lox3a reveals crosstalk between jasmonate and ethylene in the differential regulation of male and female flower opening and early fruit development in Cucurbita pepo.

Jasmonate (JA) has been found to be a relevant hormone in floral development in numerous species, but its function in cucurbit floral development and sex determination is unknown. Crosstalk between JA and ethylene (ET) in the differential regulation of male and female floral development was investigated by using the novel JA-deficient mutant lox3a, and the ET-deficient and -insensitive mutants, aco1a and etr2b, respectively, of Cucurbita pepo. The lox3a mutation suppresses male and female flower opening and induces the development of parthenocarpic fruit. A bulked-segregant analysis coupled with whole genome sequencing and fine mapping approach allowed the identification of lox3a mutation in CpLOX3A, a LIPOXYGENASE gene involved in JA biosynthesis. The reduced JA content and expression of JA-signalling genes in male and female flowers of lox3a, and the rescue of lox3a phenotype by external application of methyl jasmonate (MeJA), demonstrated that JA controls petal elongation and flower opening, as well as fruit abortion in the absence of fertilization. JA also rescued the phenotype of ET mutants aco1a and etr2b, which are both specifically defective in female flower opening and fruit abortion. ET, the sex determining hormone of cucurbits, is induced in female flowers towards anthesis, activating JA production and promoting the aperture of the female flower, and the abortion of the unfertilized ovary. Given the close association between flower closure and parthenocarpic fruit development, we propose that flower opening can act as a switch that triggers fruit set and development in fertilized ovaries, but may alternatively induce the abortion of the unfertilized ovary. Both ET and JA from mature and senescent petals can serve as remote signals that determine the alternative development of the ovary and fruit.

Intimate Partner Violence-Related Brain Injury Among Colombian Women.

OBJECTIVE: The aim of this study was to assess the prevalence of brain injury (BI) and its relationship to cognitive and psychological outcomes in women survivors of intimate partner violence (IPV) in Colombia, South America. SETTING: Women's shelters and organizations in Barranquilla, Colombia. PARTICIPANTS: Seventy women from the city of Barranquilla, Colombia, who experienced any form of IPV. DESIGN: Cross-sectional, retrospective. MAIN MEASURES: Participants were administered the computerized EMBRACED neuropsychological battery to assess learning, working and long-term memory, cognitive flexibility, and processing speed. Participants also completed measures of psychological symptoms. Partner violence severity was assessed with a semistructured interview for survivors of domestic violence. Presence and severity of IPV-related BI were assessed using the Brain Injury Severity Assessment (BISA). RESULTS: Thirty-one percent of women sustained at least one BI during an abusive relationship, and 10% sustained repetitive BIs. Furthermore, BI was negatively associated with measures of long-term and working memory, cognitive flexibility, as well as a trending ( P = .05) positive association with depression. With the exception of the relationship between BI and cognitive flexibility, which was substantially reduced and no longer significant, all of these relationships were nearly identical in strength when controlling for abuse severity, socioeconomic status, and educational level. CONCLUSION: These data are the first to specifically examine IPV-related BI in relation to cognitive and psychological functioning in a sample of Colombian women. These data add cross-cultural knowledge to the limited work in this area that has largely focused on women in North America.

The Alarm Fatigue Challenge in the Neonatal Intensive Care Unit: A "before" and "after" Study.

OBJECTIVE: Alarm fatigue (AF) happens when professionals are exposed to many alarms and they become desensitized to them. It is related to proliferation of devices, not standardized alarm limits, and high prevalence of "nonactionable alarms," i.e., false alarms (triggered by equipment issues) or nuisance alarms (physiological change not requiring clinical action). When AF happens, response time seems to be longer and important alarms could be dismissed. After evaluating the situation in our neonatal intensive care unit (NICU), an alarm management program (AMP) was developed to reduce AF. The objective of this study were to compare the proportion of true alarms, nonactionable alarms, and to measure response time to alarms in the NICU before and after implementing an AMP and also to determine variables associated with nonactionable alarms and response time. STUDY DESIGN: This was a cross-sectional study. A total of 100 observations were collected between December 2019 and January 2020. After an AMP was implemented, 100 new observations were collected between June 2021 and August 2021. We estimated the true and nonactionable alarms proportion. Univariate analyses were performed to determine variables associated with nonactionable alarms and response time. Logistic regression was performed to assess independent variables. RESULTS: The proportion of true alarms before and after AMP was 31 versus 57% (p = 0.001), whereas the proportion of nonactionable alarms was 69 versus 43% (p = 0.001). Median response time was significantly reduced (35 versus 12 seconds; p = 0.001). Before AMP, neonates with less intensive care needs had a higher proportion of nonactionable alarms and a longer response time. After AMP, response time was similar for true and nonactionable alarms. For both periods, the need of respiratory support was significantly associated with true alarms (p = 0.001). In the adjusted analysis, response time (p = 0.001) and respiratory support (p = 0.003) remained associated with nonactionable alarms. CONCLUSION: AF was highly prevalent in our NICU. This study shows that after the implementation of an AMP, response time to alarms and the proportion of nonactionable alarms can be significantly reduced. KEY POINTS: · AF happens when professionals are exposed to many alarms and they become desensitized to them.. · The presence of AF can compromise patients' safety.. · The implementation of an AMP can reduce AF..

Comparative RNA-Seq Analysis between Monoecious and Androecious Plants Reveals Regulatory Mechanisms Controlling Female Flowering in Cucurbita pepo.

In the monoecious Cucurbita pepo, the transition to female flowering is the time at which the plant starts the production of female flowers after an initial male phase of development. Ethylene plays an essential role in this process since some ethylene deficient and ethylene-insensitive mutants are androecious and only produce male flowers. To gain insight into the molecular mechanisms regulating the specification and early development of female flowers, we have compared the transcriptomic changes occurring in the shoot apices of WT and androecious ethylene-insensitive etr1b mutant plants upon female flowering transition. There were 1160 female flowering-specific DEGs identified in WT plants upon female flowering, and 284 of them were found to be modulated by the ethylene-insensitive etr1b mutation. The function of these DEGs indicated that female flower specification depends on the adoption of a transcriptional program that includes previously identified sex-determining genes in the ethylene pathway, but also genes controlling the biosynthesis and signaling pathways of other phytohormones, and those encoding for many different transcription factors. The transcriptomic changes suggested that gibberellins play a negative role in female flowering, while ethylene, auxins, ABA and cytokinins are positive regulators. Transcription factors from 34 families, including NAC, ERF, bHLH, bZIP, MYB and C2H2/CH3, were found to be regulating female flowering in an ethylene-dependent or -independent manner. Our data open a new perspective of the molecular mechanisms that control the specification and development of female flowers in C. pepo.

Advanced Sperm Selection Strategies as a Treatment for Infertile Couples: A Systematic Review.

Assisted reproductive technology (ART) is an essential tool to overcome infertility, and is a worldwide disease that affects millions of couples at reproductive age. Sperm selection is a crucial step in ART treatment, as it ensures the use of the highest quality sperm for fertilization, thus increasing the chances of a positive outcome. In recent years, advanced sperm selection strategies for ART have been developed with the aim of mimicking the physiological sperm selection that occurs in the female genital tract. This systematic review sought to evaluate whether advanced sperm selection techniques could improve ART outcomes and sperm quality/functionality parameters compared to traditional sperm selection methods (swim-up or density gradients) in infertile couples. According to preferred reporting items for systematic reviews and meta-analyses (PRISMA guidelines), the inclusion and exclusion criteria were defined in a PICOS (population, intervention, comparator, outcome, study) table. A systematic search of the available literature published in MEDLINE-PubMed until December 2021 was subsequently conducted. Although 4237 articles were recorded after an initial search, only 47 studies were finally included. Most reports (30/47; 63.8%) revealed an improvement in ART outcomes after conducting advanced vs. traditional sperm selection methods. Among those that also assessed sperm quality/functionality parameters (12/47), there was a consensus (10/12; 83.3%) about the beneficial effect of advanced sperm selection methods on these variables. In conclusion, the application of advanced sperm selection methods improves ART outcomes. In spite of this, as no differences in the reproductive efficiency between advanced methods has been reported, none can be pointed out as a gold standard to be conducted routinely. Further research addressing whether the efficiency of each method relies on the etiology of infertility is warranted.

Bempegaldesleukin (BEMPEG; NKTR-214) efficacy as a single agent and in combination with checkpoint-inhibitor therapy in mouse models of osteosarcoma.

Survival of patients with relapsed/refractory osteosarcoma has not improved in the last 30 years. Several immunotherapeutic approaches have shown benefit in murine osteosarcoma models, including the anti-programmed death-1 (anti-PD-1) and anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) immune checkpoint inhibitors. Treatment with the T-cell growth factor interleukin-2 (IL-2) has shown some clinical benefit but has limitations due to poor tolerability. Therefore, we evaluated the efficacy of bempegaldesleukin (BEMPEG; NKTR-214), a first-in-class CD122-preferential IL-2 pathway agonist, alone and in combination with anti-PD-1 or anti-CTLA-4 immune checkpoint inhibitors in metastatic and orthotopic murine models of osteosarcoma. Treatment with BEMPEG delayed tumor growth and increased overall survival of mice with K7M2-WT osteosarcoma pulmonary metastases. BEMPEG also inhibited primary tumor growth and metastatic relapse in lungs and bone in the K7M3 orthotopic osteosarcoma mouse model. In addition, it enhanced therapeutic activity of anti-CTLA-4 and anti-PD-1 checkpoint blockade in the DLM8 subcutaneous murine osteosarcoma model. Finally, BEMPEG strongly increased accumulation of intratumoral effector T cells and natural killer cells, but not T-regulatory cells, resulting in improved effector:inhibitory cell ratios. Collectively, these data in multiple murine models of osteosarcoma provide a path toward clinical evaluation of BEMPEG-based regimens in human osteosarcoma.

[Response to the COVID-19 Pandemic in Chile, Colombia, and Peru from a children's rights perspectiveResposta à pandemia de COVID-19 no Chile, Colômbia e Peru com foco nos direitos da criança]. / Respuesta con enfoque de derechos de la niñez frente a la pandemia por COVID-19 en Chile, Colombia y Perú.

OBJECTIVE: Describe the strategies established by Chile, Colombia, and Peru during the first year of the COVID-19 pandemic and compare them from a children's rights perspective. METHODS: A qualitative study with comparative analysis of public policies was conducted around seven cat-egories constructed by the Latin American Chapter of the International Society for Social Pediatrics and Child Health, based on the Convention on the Rights of the Child. Country documents were selected based on convenience sampling and were analyzed in deliberative dialogues. RESULTS: 173 documents from the three countries were reviewed. There was convergence around: prioritization of prevention of community transmission of the virus over promotion of the exercise of children's rights; lack of participation of children and adolescents in the process of developing public policies; and lack of progress in the recognition and protection of the exercise of children's rights overall. There were no major divergences beyond identified inequality gaps grounded in the reality of each country. CONCLUSION: The pandemic has affected the functioning of the economic, social, health, education, environmental, and governance systems in these three countries. While this study shows progress in the inclusion of the children's rights approach in formulated policies, the recognition of children and adolescents as holders of social and political rights could allow the construction of collective alternatives that guarantee health and well-being for all people throughout the life course.

OBJETIVO: Descrever as estratégias que foram utilizadas por Chile, Colômbia e Peru durante o primeiro ano da pandemia de COVID-19 e compará-las a partir da perspectiva dos direitos da criança. MÉTODOS: Realizou-se um estudo qualitativo de análise comparativa de políticas públicas, tomando como eixo sete categorias construídas pelo Capítulo Latino-americano da International Society for Social Pediatrics & Child Health a partir da Convenção sobre os Direitos da Criança (CDC). A seleção de documentos dos países foi feita por conveniência, e sua análise ocorreu mediante diálogos deliberativos. RESULTADOS: Foram revisados 173 documentos dos três países. A prioridade da prevenção da transmissão comunitária do vírus destaca-se por sua convergência, em detrimento da promoção do exercício dos direitos da criança, da falta de participação de crianças e adolescentes no processo de formulação de políticas públicas e da falta de progresso no reconhecimento e proteção da efetivação de todos os seus direitos. Não houve grandes divergências além das lacunas de desigualdade identificadas com base na realidade de cada país. CONCLUSÕES: A pandemia afetou o funcionamento dos sistemas econômico, social, de saúde, educação, meio ambiente e governança desses três países. Embora este estudo mostre avanços na inclusão da perspectiva dos direitos da criança e do adolescente nas políticas formuladas, a compreensão deles como sujeitos sociais e políticos titulares de direitos pode permitir a construção de alternativas coletivas que garantam saúde e bem-estar a todas as pessoas ao longo do curso da vida.

Endoscopic submucosal dissection with an SB Knife® for the treatment of subcardial gastric leiomyoma.

Surgery has been considered the main treatment for submucosal tumors (SMTs). However, endoscopic resection is currently accepted for gastric SMTs smaller than 3 cm. Endoscopic submucosal dissection (ESD) is considered the technique of choice, and submucosal tunneling endoscopic resection has successful results with low complication rates according to the recent meta-analysis by Cao et al. The major limitation of these methods is the technical difficulty associated with certain anatomic locations.

Reliability and validity of a questionnaire of child development for national surveys. / Confiabilidad y validez de un cuestionario de desarrollo infantil en encuestas nacionales.

INTRODUCTION: The child development screening in national surveys supports the formulation of public policies that contribute to its diagnosis and early intervention, aimed at promoting comprehensive development and successful school performance until adolescence, however, few countries in Latin America have this information. OBJECTIVE: To assess the reliability and convergent validity of a cultural adaptation of the Screening Questionnaire of Child Development for Household Surveys (DIEH) and to des cribe the prevalence of developmental alterations in children aged between 2 months and 5 years in Bucaramanga-Colombia. SUBJECTS AND METHOD: Between July and December 2016, an evaluation of diagnostic tests was carried out in which participated 224 children aged between two months and five years and their parents or caregivers from Bucaramanga, Colombia. The Abbreviated Developmental Scale was applied by trained physiotherapists and the DIEH was answered by the parents or caregi vers. The reliability of the DIEH was established using the Cronbach's alpha, the Intraclass Correla tion Coefficient (ICC) and the Bland and Altman limits of agreement, and the convergent validity was established through the Spearman Correlation Coefficient. RESULTS: Based on the DIEH, the pre valence of lags and delays was 38.8% and 11.2% respectively, internal consistency ranged from 0.23 to 0.76; reproducibility showed an ICC between 0.60 and 0.92; and the convergent validity was almost perfect (p: 0.96). CONCLUSION: The cultural adaptation of the DIEH showed acceptable psychometric properties, which could be complemented with additional studies to recommend its use in national surveys in Latin America.

Age band 1 of the movement assessment battery for children -2. Reliability of the spanish version. / Batería para la evaluación del movimiento en niños -2- banda 1. Confiabilidad de la versión en español.

INTRODUCTION: The development of motor skills influences the capacity of the child to interact with the environment. Thus, several instruments have been created for their assessment. OBJECTIVE: To evaluate the internal consistency, reproducibility, and agreement level of age band 1 of the Movement Assessment Battery for Children - 2 in a preschool group. PATIENTS AND METHOD: Assessment study of diagnostic tests with 29 preschoolers, selected by convenience, enrollments in an educational ins titution in Bucaramanga city, Colombia. For the inter-evaluators reproducibility assessment, three evaluators watched each video independently. In the intra-evaluator reproducibility assessment, each evaluator watched the same video on two different occasions. The internal consistency, the intra- and inter-evaluator reproducibility, and the agreement level were determined using Cronbach's alpha co efficient, the Intraclass Correlation Coefficient (ICC), and the Bland and Altman limits of agreement method, respectively. RESULTS: Internal consistency of the total test for each of the three evaluators was higher than 0.60. Very good reliability was found for all items, domains, and total score of age band 1 of MABC-2 (ICC > 0.85), as well as good limits of agreement. CONCLUSIONS: age band 1 of MABC-2 Spanish version is an instrument with adequate reliability psychometric properties that can be used for the motor skills development evaluation in preschoolers.

In vitro cytotoxicity and genotoxicity of Furia®180 SC (zeta-cypermethrin) and Bulldock 125®SC (ß-cyfluthrin) pyrethroid insecticides in human peripheral blood lymphocytes.

In the present study, human peripheral blood lymphocytes were exposed in vitro to 0, 6, 12, 18, 24, and 30 µg/mL Furia®180 SC (zeta-cypermethrin) and 0, 6.3, 12.5, 18.8, 25, and 31.3 µg/mL Bulldock®125 SC (ß-cyfluthrin). Exposure to 32 µg/mL bleomycin for 24 h served as a positive control. The cytotoxic and genotoxic effects of each insecticide were analyzed using alkaline comet and trypan blue dye exclusion assays. DNA damage was evaluated through three genotoxicity parameters: tail length (TL), tail moment (TM) and tail intensity (TI). Furia®180 SC and Bulldock®125 SC pyrethroid insecticides and bleomycin significantly increased DNA damage in a concentration-dependent manner. Bulldock®125 SC induced more DNA damage than Furia. Lymphocyte viability did not change after exposure to different concentrations of the two pyrethroid insecticides and bleomycin. Moreover, genotoxic results demonstrated that Furia®180 SC and Bulldock®125 SC insecticides caused in vitro DNA damage in human peripheral lymphocytes.

Intracellular characterization of Gag VLP production by transient transfection of HEK 293 cells.

Transient transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in transient transfection, little is known regarding the transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes' internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag-GFP fusion construct that forms fluorescent Gag-VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 min of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 1010 VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 h post-transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet (total Gag-GFP), indicating that there is a fraction of Gag that remains inside the cells. The maximum VLP concentration in the cell culture supernatant with cell viability >89% was observed at 72 hpt, which was determined to be the optimal harvest time. Biotechnol. Bioeng. 2017;114: 2507-2517. © 2017 Wiley Periodicals, Inc.

Abnormality of adipokines and endothelial dysfunction in Mexican obese adolescents with insulin resistance.

PURPOSE: The aim of this study was to investigate the possible relationship among insulin resistance (IR), endothelial dysfunction, and alteration of adipokines in Mexican obese adolescents and their association with metabolic syndrome (MetS). MATERIALS AND METHODS: Two hundred and twenty-seven adolescents were classified according to the body mass index (BMI) (control: N=104; obese: N=123) and homeostasis model of the assessment-insulin resistance index (HOMA-IR) (obese with IR: N=65). The circulating concentrations of leptin, adiponectin, soluble intercellular adhesion molecule-1 (sICAM-1), and IR were determined by standard methods. RESULTS: The obese adolescents with IR presented increased presence of MetS and higher circulating concentrations in sICAM-1 in comparison with the obese subjects without IR. The lowest concentrations of adiponectin were observed in the obese with IR. In multivariate linear regression models, sICAM-1 along with triglycerides, total cholesterol, and waist circumference was strongly associated with HOMA-IR (R2=0.457, P=0.008). Similarly, after adjustment for age, BMI-SDS, lipids, and adipokines, HOMA-IR remained associated with sICAM-1 (R2=0.372, P=0.008). BMI-SDS was mildly associated with leptin (R2=0.176, P=0.002) and the waist circumference was mild and independent determinant of adiponectin (R2=0.136, P=0.007). CONCLUSIONS: Our findings demonstrated that the obese adolescents, particularly the obese subjects with IR exhibited increased presence of MetS, abnormality of adipokines, and endothelial dysfunction. The significant interaction between IR and endothelial dysfunction may suggest a novel therapeutic approach to prevent or delay systemic IR and the genesis of cardiovascular diseases in obese patients.

Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells.

HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated transient transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 10(10) VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics.

Involvement of chlororespiration in chilling stress in the tropical species Spathiphyllum wallisii.

Spathiphyllum wallisii plants were used to study the effect of chilling stress under high illumination on photosynthesis and chlororespiration. Leaves showed different responses that depended on root temperature. When stem, but not root, was chilled, photosystem II (PSII) was strongly photoinhibited. However, when the whole plant was chilled, the maximal quantum yield of PSII decreased only slightly below the normal values and cyclic electron transport was stimulated. Changes were also observed in the chlororespiration enzymes and PGR5. In whole plants chilled under high illumination, the amounts of NADH dehydrogenase (NDH) complex and plastid terminal oxidase (PTOX) remained similar to control and increased when only stem was chilled. In contrast, the amount of PGR5 polypeptide was higher in plants when both root and stem were chilled than in plants in which only stem was chilled. The results indicated that the contribution of chlororespiration to regulating photosynthetic electron flow is not relevant when the whole plant is chilled under high light, and that another pathway, such as cyclic electron flow involving PGR5 polypeptide, may be more important. However, when PSII activity is strongly photoinhibited in plants in which only stem is chilled, chlororespiration, together with other routes of electron input to the electron transfer chain, is probably essential.

Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered.