Stabilized Double-Stranded RNA Strategy Improves Cotton Resistance to CBW (Anthonomus grandis).

Cotton is the most important crop for fiber production worldwide. However, the cotton boll weevil (CBW) is an insect pest that causes significant economic losses in infested areas. Current control methods are costly, inefficient, and environmentally hazardous. Herein, we generated transgenic cotton lines expressing double-stranded RNA (dsRNA) molecules to trigger RNA interference-mediated gene silencing in CBW. Thus, we targeted three essential genes coding for chitin synthase 2, vitellogenin, and ecdysis-triggering hormone receptor. The stability of expressed dsRNAs was improved by designing a structured RNA based on a viroid genome architecture. We transformed cotton embryos by inserting a promoter-driven expression cassette that overexpressed the dsRNA into flower buds. The transgenic cotton plants were characterized, and positive PCR transformed events were detected with an average heritability of 80%. Expression of dsRNAs was confirmed in floral buds by RT-qPCR, and the T1 cotton plant generation was challenged with fertilized CBW females. After 30 days, data showed high mortality (around 70%) in oviposited yolks. In adult insects fed on transgenic lines, chitin synthase II and vitellogenin showed reduced expression in larvae and adults, respectively. Developmental delays and abnormalities were also observed in these individuals. Our data remark on the potential of transgenic cotton based on a viroid-structured dsRNA to control CBW.

Functional characterization of the pUceS8.3 promoter and its potential use for ectopic gene overexpression.

MAIN CONCLUSION: The pUceS8.3 is a constitutive gene promoter with potential for ectopic and strong genes overexpression or active biomolecules in plant tissues attacked by pests, including nematode-induced giant cells or galls. Soybean (Glycine max) is one of the most important agricultural commodities worldwide and a major protein and oil source. Herein, we identified the soybean ubiquitin-conjugating (E2) enzyme gene (GmUBC4; Glyma.18G216000), which is significantly upregulated in response to Anticarsia gemmatalis attack and Meloidogyne incognita-induced galls during plant parasitism by plant nematode. The GmUBC4 promoter sequence and its different modules were functionally characterized in silico and in planta using transgenic Arabidopsis thaliana and G. max lines. Its full-length transcriptional regulatory region (promoter and 5´-UTR sequences, named pUceS8.3 promoter) was able to drive higher levels of uidA (ß-glucuronidase) gene expression in different tissues of transgenic A. thaliana lines compared to its three shortened modules and the p35SdAMV promoter. Notably, higher ß-glucuronidase (GUS) enzymatic activity was shown in M. incognita-induced giant cells when the full pUceS8.3 promoter drove the expression of this reporter gene. Furthermore, nematode-specific dsRNA molecules were successfully overexpressed under the control of the pUceS8.3 promoter in transgenic soybean lines. The RNAi gene construct used here was designed to post-transcriptionally downregulate the previously characterized pre-mRNA splicing factor genes from Heterodera glycines and M. incognita. A total of six transgenic soybean lines containing RNAi gene construct were selected for molecular characterization after infection with M. incognita pre-parasitic second-stage (ppJ2) nematodes. A strong reduction in the egg number produced by M. incognita after parasitism was observed in those transgenic soybean lines, ranging from 71 to 92% compared to wild-type control plants. The present data demonstrated that pUceS8.3 is a gene promoter capable of effectively driving dsRNA overexpression in nematode-induced giant cells of transgenic soybean lines and can be successfully applied as an important biotechnological asset to generate transgenic crops with improved resistance to root-knot nematodes as well as other pests.

Case Report: Pott's Edematous Tumor: Complicated Frontal Sinusitis - An Unremembered Diagnosis.

Pott's Puffy tumor, also called Pott's edematous tumor (PET), is a subperiosteal abscess of the frontal bone, associated with osteomyelitis of the frontal bone. In this paper, we report the case of a 16-year-old patient who presented with headache associated with progressive forehead swelling and fever. Clinical and imaging exams pointed to the hypothesis of PET associated with brain abscess. Patient was submitted to surgical excision of the abscess and treatment of osteomyelitis, with intraoperative findings corroborating the condition. There was a good clinical-radiological recovery associated with prolonged antibiotic therapy and satisfactory follow-up after hospital. PET, which often results from an underdiagnosed or partially treated frontal sinusitis, is a condition that must be promptly recognized and directed to an adequate therapeutic approach due to the risk of serious complications that it entails.

Pyramiding dsRNAs increases phytonematode tolerance in cotton plants.

MAIN CONCLUSION: Host-derived suppression of nematode essential genes decreases reproduction of Meloidogyne incognita in cotton. Root-knot nematodes (RKN) represent one of the most damaging plant-parasitic nematode genera worldwide. RNAi-mediated suppression of essential nematode genes provides a novel biotechnological strategy for the development of sustainable pest-control methods. Here, we used a Host Induced Gene Silencing (HIGS) approach by stacking dsRNA sequences into a T-DNA construct to target three essential RKN genes: cysteine protease (Mi-cpl), isocitrate lyase (Mi-icl), and splicing factor (Mi-sf), called dsMinc1, driven by the pUceS8.3 constitutive soybean promoter. Transgenic dsMinc1-T4 plants infected with Meloidogyne incognita showed a significant reduction in gall formation (57-64%) and egg masses production (58-67%), as well as in the estimated reproduction factor (60-78%), compared with the susceptible non-transgenic cultivar. Galls of the RNAi lines are smaller than the wild-type (WT) plants, whose root systems exhibited multiple well-developed root swellings. Transcript levels of the three RKN-targeted genes decreased 13- to 40-fold in nematodes from transgenic cotton galls, compared with those from control WT galls. Finally, the development of non-feeding males in transgenic plants was 2-6 times higher than in WT plants, indicating a stressful environment for nematode development after RKN gene silencing. Data strongly support that HIGS of essential RKN genes is an effective strategy to improve cotton plant tolerance. This study presents the first application of dsRNA sequences to target multiple genes to promote M. incognita tolerance in cotton without phenotypic penalty in transgenic plants.

Improved cotton transformation protocol mediated by Agrobacterium and biolistic combined-methods.

MAIN CONCLUSION: The combined Agrobacterium- and biolistic-mediated methods of cotton transformation provide a straightforward and highly efficient protocol for obtaining transgenic cotton. Cotton (Gossypium spp.) is the most important crop for natural textile fiber production worldwide. Nonetheless, one of the main challenges in cotton production are the losses resulting from insect pests, pathogens, and abiotic stresses. One effective way to solve these issues is to use genetically modified (GM) varieties. Herein, we describe an improved protocol for straightforward and cost-effective genetic transformation of cotton embryo axes, merging biolistics and Agrobacterium. The experimental steps include (1) Agrobacterium preparation, (2) seed sterilization, (3) cotton embryo excision, (4) lesion of shoot-cells by tungsten bombardment, (5) Agrobacterium-mediated transformation, (6) embryo co-culture, (7) regeneration and selection of transgenic plants in vitro, and (8) molecular characterization of plants. Due to the high regenerative power of the embryonic axis and the exceptional ability of the meristem cells for plant regeneration through organogenesis in vitro, this protocol can be performed in approximately 4-10 weeks, with an average plant regeneration of about 5.5% (± 0.53) and final average transformation efficiency of 60% (± 0.55). The transgene was stably inherited, and most transgenic plants hold a single copy of the transgene, as desirable and expected in Agrobacterium-mediated transformation. Additionally, the transgene was stably expressed over generations, and transgenic proteins could be detected at high levels in the T2 generation of GM cotton plants. The T2 progeny showed no phenotypic or productivity disparity compared to wild-type plants. Collectively, the use of cotton embryo axes and the enhanced DNA-delivery system by combining particle bombardment and Agrobacterium infection enabled efficient transgenic plant recovery, overcoming usual limitations associated with the recalcitrance of several cotton genotypes subjected to somatic embryogenesis. The improved approach states this method's success for cotton genetic modification, allowing us to obtain GM cotton plants carrying traits, which are of fundamental relevance for the advancement of global agribusiness.

Contrasting roles of GmNAC065 and GmNAC085 in natural senescence, plant development, multiple stresses and cell death responses.

NACs are plant-specific transcription factors involved in controlling plant development, stress responses, and senescence. As senescence-associated genes (SAGs), NACs integrate age- and stress-dependent pathways that converge to programmed cell death (PCD). In Arabidopsis, NAC-SAGs belong to well-characterized regulatory networks, poorly understood in soybean. Here, we interrogated the soybean genome and provided a comprehensive analysis of senescence-associated Glycine max (Gm) NACs. To functionally examine GmNAC-SAGs, we selected GmNAC065, a putative ortholog of Arabidopsis ANAC083/VNI2 SAG, and the cell death-promoting GmNAC085, an ANAC072 SAG putative ortholog, for analyses. Expression analysis of GmNAC065 and GmNAC085 in soybean demonstrated (i) these cell death-promoting GmNACs display contrasting expression changes during age- and stress-induced senescence; (ii) they are co-expressed with functionally different gene sets involved in stress and PCD, and (iii) are differentially induced by PCD inducers. Furthermore, we demonstrated GmNAC065 expression delays senescence in Arabidopsis, a phenotype associated with enhanced oxidative performance under multiple stresses, higher chlorophyll, carotenoid and sugar contents, and lower stress-induced PCD compared to wild-type. In contrast, GmNAC085 accelerated stress-induced senescence, causing enhanced chlorophyll loss, ROS accumulation and cell death, decreased antioxidative system expression and activity. Accordingly, GmNAC065 and GmNAC085 targeted functionally contrasting sets of downstream AtSAGs, further indicating that GmNAC85 and GmNAC065 regulators function inversely in developmental and environmental PCD.

Fatal Outcome of Chikungunya Virus Infection in Brazil.

BACKGROUND: Chikungunya virus (CHIKV) emerged in the Americas in 2013 and has caused approximately 2.1 million cases and >600 deaths. A retrospective investigation was undertaken to describe clinical, epidemiological, and viral genomic features associated with deaths caused by CHIKV in Ceará state, northeast Brazil. METHODS: Sera, cerebrospinal fluid (CSF), and tissue samples from 100 fatal cases with suspected arbovirus infection were tested for CHIKV, dengue virus (DENV), and Zika virus (ZIKV). Clinical, epidemiological, and death reports were obtained for patients with confirmed CHIKV infection. Logistic regression analysis was undertaken to identify independent factors associated with risk of death during CHIKV infection. Phylogenetic analysis was conducted using whole genomes from a subset of cases. RESULTS: Sixty-eight fatal cases had CHIKV infection confirmed by reverse-transcription quantitative polymerase chain reaction (52.9%), viral antigen (41.1%), and/or specific immunoglobulin M (63.2%). Co-detection of CHIKV with DENV was found in 22% of fatal cases, ZIKV in 2.9%, and DENV and ZIKV in 1.5%. A total of 39 CHIKV deaths presented with neurological signs and symptoms, and CHIKV-RNA was found in the CSF of 92.3% of these patients. Fatal outcomes were associated with irreversible multiple organ dysfunction syndrome. Patients with diabetes appear to die at a higher frequency during the subacute phase. Genetic analysis showed circulation of 2 CHIKV East-Central-South African (ECSA) lineages in Ceará and revealed no unique virus genomic mutation associated with fatal outcome. CONCLUSIONS: The investigation of the largest cross-sectional cohort of CHIKV deaths to date reveals that CHIKV-ECSA strains can cause death in individuals from both risk and nonrisk groups, including young adults.

Sepse neonatal: manejo hospitalar / Neonatal sepsis: hospital management

A sepse neonatal é definida como uma síndrome clínica que se manifesta com sinais de infecção e resposta inflamatória sistêmica nos primeiros 28 dias de vida. Sua etiologia pode ser bacteriana, viral ou fúngica. Sepse neonatal precoce é uma infecção adquirida por via vertical, na qual um patógeno é transmitido da mãe para o recém-nascido por via ascendente ou transplacentária. Já a sepse neonatal tardia é tipicamente adquirida horizontalmente através de patógenos provenientes do ambiente do neonato. O diagnóstico baseia-se em: fatores de risco maternos e neonatais, manifestações clínicas e exames laboratoriais. A presença de três ou mais sinais clínicos no RN ou no mínimo dois sinais associados a fatores de risco maternos autoriza o diagnóstico de sepse clínica ou síndrome séptica, justificando-se o início da antibioticoterapia sem o auxílio de exames laboratoriais. A infecção por estreptococo do grupo B (EGB) é responsável por aproximadamente 45% dos casos de sepse neonatal precoce confirmada por cultura entre os recém-nascidos a termo. Atualmente, recomenda a realização de cultura retal-vaginal para colonização por EGB no pré-natal de todas as gestantes e a profilaxia antibiótica intraparto é indicada para todas as mulheres com colonização por EGB identificada por cultura retal-vaginal, para mulheres com bacteriúria por EGB identificada em qualquer ponto durante a gravidez, para mulheres com uma história de um bebê anterior com infecção por EGB, e para mulheres em trabalho de parto prematuro e/ou com ruptura prematura de membranas ovulares com menos de 37 semanas de gestação. O tratamento antibiótico inicial da sepse neonatal precoce é empírico, sendo o mesmo para todas as causas, até que os resultados das culturas estejam disponíveis. A terapia de primeira linha recomendada é Ampicilina associada a um Aminoglicosídeo para recém-nascidos de até 7 dias de idade. Nos pacientes com sepse e neutropenia acentuada (< 700 neutrófilos) está indicado o uso de fator estimulador humano de colônias de granulócitos. Palavras-chave: Sepse Neonatal, Neonatologia, Antibioticoterapia, Protocolo de sepse neonatal, Fatores de Risco, Estreptococo do grupo B.

Soybean Embryonic Axis Transformation: Combining Biolistic and Agrobacterium-Mediated Protocols to Overcome Typical Complications of In Vitro Plant Regeneration.

The first successful attempt to generate genetically modified plants expressing a transgene was preformed via T-DNA-based gene transfer employing Agrobacterium tumefaciens-mediated genetic transformation. Limitations over infectivity and in vitro tissue culture led to the development of other DNA delivery systems, such as the biolistic method. Herein, we developed a new one-step protocol for transgenic soybean recovery by combining the two different transformation methods. This protocol comprises the following steps: agrobacterial preparation, seed sterilization, soybean embryo excision, shoot-cell injury by tungsten-microparticle bombardment, A. tumefaciens-mediated transformation, embryo co-cultivation in vitro, and selection of transgenic plants. This protocol can be completed in approximately 30-40 weeks. The average efficiency of producing transgenic soybean germlines using this protocol was 9.84%, similar to other previously described protocols. However, we introduced a more cost-effective, more straightforward and shorter methodology for transgenic plant recovery, which allows co-cultivation and plant regeneration in a single step, decreasing the chances of contamination and making the manipulation easier. Finally, as a hallmark, our protocol does not generate plant chimeras, in contrast to traditional plant regeneration protocols applied in other Agrobacterium-mediated transformation methods. Therefore, this new approach of plant transformation is applicable for studies of gene function and the production of transgenic cultivars carrying different traits for precision-breeding programs.

A major chikungunya epidemic with high mortality in northeastern Brazil.

INTRODUCTION: Chikungunya causes fever and severe and persistent joint pain. METHODS: We reported a chikungunya outbreak that occurred in Ceará State, Brazil between 2016 and 2017 with emphasis on epidemiological characterization of cases, high number of deaths, mortality-associated factors, and spatial and temporal spread of the epidemic among municipalities. RESULTS: In November 2015, the first autochthonous cases of chikungunya were confirmed in Ceará, Brazil. In 2016-2017, 195,993 cases were reported, with an incidence of 2,186.5/100,000 inhabitants and 244 confirmed deaths. CONCLUSIONS: Rapid transmission and high mortality rate are serious problems, especially in regions with co-circulating arboviruses.

A major chikungunya epidemic with high mortality in northeastern Brazil

Abstract INTRODUCTION Chikungunya causes fever and severe and persistent joint pain. METHODS We reported a chikungunya outbreak that occurred in Ceará State, Brazil between 2016 and 2017 with emphasis on epidemiological characterization of cases, high number of deaths, mortality-associated factors, and spatial and temporal spread of the epidemic among municipalities. RESULTS: In November 2015, the first autochthonous cases of chikungunya were confirmed in Ceará, Brazil. In 2016-2017, 195,993 cases were reported, with an incidence of 2,186.5/100,000 inhabitants and 244 confirmed deaths. CONCLUSIONS: Rapid transmission and high mortality rate are serious problems, especially in regions with co-circulating arboviruses.

Lupus cystitis presenting with hidronephrosis and gastrointestinal involvement.

INTRODUCTION: Lupus cystitis is a rare manifestation of systemic lupus erythematosus, characterized by thickening of the bladder wall, associated with ureterohydronephrosis. In several cases gastrointestinal symptoms are the main manifestation. The optimal immunosuppressive regimen is still unknown. METHODS: 16-year-old girl with systemic lupus erythematosus was admitted with dysuria, renal impairment, diarrhea, abdominal pain, nausea and vomiting. An abdominal CT scan demonstrated moderate bilateral dilation from the pelvis to the ureterovesical junction, and the bladder exhibited reduced capacity and diffusely thickened walls. A diagnosis of chronic interstitial cystitis was performed and treatment with high dose methylprednisolone resulted in a significant relief of gastrointestinal and urinary symptoms and a reduction of the bladder thickness with improvement of their capacity. CONCLUSION: Lupus cystitis is a rare and underdiagnosed urinary manifestation of LES and the presence of mild urinary symptoms and abdominal pain may alert about this possibility.

Lupus cystitis presenting with hidronephrosis and gastrointestinal involvement / Cistite lúpica apresentando hidronefrose e envolvimento gastrointestinal

Abstract Introduction: Lupus cystitis is a rare manifestation of systemic lupus erythematosus, characterized by thickening of the bladder wall, associated with ureterohydronephrosis. In several cases gastrointestinal symptoms are the main manifestation. The optimal immunosuppressive regimen is still unknown. Methods: 16-year-old girl with systemic lupus erythematosus was admitted with dysuria, renal impairment, diarrhea, abdominal pain, nausea and vomiting. An abdominal CT scan demonstrated moderate bilateral dilation from the pelvis to the ureterovesical junction, and the bladder exhibited reduced capacity and diffusely thickened walls. A diagnosis of chronic interstitial cystitis was performed and treatment with high dose methylprednisolone resulted in a significant relief of gastrointestinal and urinary symptoms and a reduction of the bladder thickness with improvement of their capacity. Conclusion: Lupus cystitis is a rare and underdiagnosed urinary manifestation of LES and the presence of mild urinary symptoms and abdominal pain may alert about this possibility.

Resumo Introdução: A Cistite Lúpica é uma manifestação rara do Lúpus Eritematoso Sistémico, caracterizada pelo espessamento da parede da bexiga, podendo estar associada com ureterohidronefrose. Em vários casos, os sintomas gastrointestinais são a principal manifestação e ainda não se sabe qual o regime imunossupressor mais adequado nesta situação. Método: Uma jovem de 16 anos de idade com lúpus eritematoso sistémico foi admitida com disúria, insuficiência renal, diarreia, dor abdominal, náuseas e vómitos. A tomografia computadorizada de abdome demonstrou dilatação moderada de ureter e pelve renal bilateralmente e bexiga exibindo capacidade reduzida e paredes difusamente espessadas. Foi feito o diagnóstico de cistite intersticial crónica e o tratamento com dose elevada de metilprednisolona resultou em um alívio significativo dos sintomas urinários e gastrointestinais e uma redução da espessura da bexiga, com melhora da sua capacidade. Conclusão: A Cistite Lúpica é uma manifestação urinária rara e subdiagnosticada do LES e a presença de sintomas urinários leves e dor abdominal devem alertar para essa possibilidade diganóstica.

Societal Preferences for EQ-5D Health States from a Brazilian Population Survey.

OBJECTIVE: To elicit preference weights for a subset of EuroQol five-dimensional (EQ-5D) questionnaire health states from a representative sample for the state of Minas Gerais, Brazil, using a time trade-off (TTO) method and to analyze these data so as to estimate social preference weights for the complete set of 243 states. METHODS: Data came from a valuation study with 3362 literate individuals aged between 18 and 64 years living in urban areas. The present study was based on quota sampling by age and sex. Face-to-face interviews were conducted in participants' own homes. A total of 99 EQ-5D questionnaire health states were selected, presorted into 26 blocks of six unique health states. Each participant valued one block together with the full health, worst health, and dead states. Each health state was evaluated by more than 100 individuals. TTO data were modeled at both individual and aggregate levels by using ordinary least squares and random effects methods. RESULTS: Values estimated by different models yielded very similar results with satisfactory goodness-of-fit statistics: the mean absolute error was around 0.03 and fewer than 25% of the states had a mean absolute error greater than 0.05. Dummies coefficients for each level within the EQ-5D questionnaire dimensions of health displayed an internally consistent ordering, with the mobility dimension demonstrating the largest value decrement. The values of mean observed transformed TTO values range from 0.869 to-0.235. CONCLUSIONS: The study demonstrates the feasibility of conducting face-to-face interviews using TTO in a Brazilian population setting. The estimated values for EQ-5D questionnaire health states based on this Minas Gerais survey represent an important first step in establishing national Brazilian social preference weights for the EQ-5D questionnaire.

Restless legs syndrome and quality of sleep in type 2 diabetes.

OBJECTIVE: To investigate the presence of restless legs syndrome (RLS) and the quality of sleep in a population of type 2 diabetic patients. RESEARCH DESIGN AND METHODS: The study population was composed of 100 consecutive patients regularly attending a diabetes clinic at the University Hospital of the Federal University of Ceará. The subjects' quality of sleep was assessed by the Pittsburgh Sleep Quality Index, and excessive daytime sleepiness (EDS) was measured by the Epworth Sleepiness Scale. The RLS was diagnosed using the four minimum criteria defined by the International Restless Legs Syndrome Study Group. Other relevant clinical and laboratory parameters were obtained by interview and chart review. RESULTS: RLS was found in 27% of patients. Poor sleep quality was present in 45% of cases and was associated with age (P = 0.04), peripheral neuropathy (P = 0.001), and RLS (P = 0.000). EDS was found in 26% of patients. Logistic regression analysis revealed an association between RLS and peripheral neuropathy (odds ratio 12.85 [95% CI 2.83-58.40], P = 0.001). CONCLUSIONS: RLS is common in type 2 diabetic patients and can be a major cause of sleep disruption in these patients.