IL-23 stabilizes an effector Treg cell program in the tumor microenvironment.

Interleukin-23 (IL-23) is a proinflammatory cytokine mainly produced by myeloid cells that promotes tumor growth in various preclinical cancer models and correlates with adverse outcomes. However, as to how IL-23 fuels tumor growth is unclear. Here, we found tumor-associated macrophages to be the main source of IL-23 in mouse and human tumor microenvironments. Among IL-23-sensing cells, we identified a subset of tumor-infiltrating regulatory T (Treg) cells that display a highly suppressive phenotype across mouse and human tumors. The use of three preclinical models of solid cancer in combination with genetic ablation of Il23r in Treg cells revealed that they are responsible for the tumor-promoting effect of IL-23. Mechanistically, we found that IL-23 sensing represents a crucial signal driving the maintenance and stabilization of effector Treg cells involving the transcription factor Foxp3. Our data support that targeting the IL-23/IL-23R axis in cancer may represent a means of eliciting antitumor immunity.

Morphological, cellular, and molecular basis of brain infection in COVID-19 patients.

Although increasing evidence confirms neuropsychiatric manifestations associated mainly with severe COVID-19 infection, long-term neuropsychiatric dysfunction (recently characterized as part of "long COVID-19" syndrome) has been frequently observed after mild infection. We show the spectrum of cerebral impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, ranging from long-term alterations in mildly infected individuals (orbitofrontal cortical atrophy, neurocognitive impairment, excessive fatigue and anxiety symptoms) to severe acute damage confirmed in brain tissue samples extracted from the orbitofrontal region (via endonasal transethmoidal access) from individuals who died of COVID-19. In an independent cohort of 26 individuals who died of COVID-19, we used histopathological signs of brain damage as a guide for possible SARS-CoV-2 brain infection and found that among the 5 individuals who exhibited those signs, all of them had genetic material of the virus in the brain. Brain tissue samples from these five patients also exhibited foci of SARS-CoV-2 infection and replication, particularly in astrocytes. Supporting the hypothesis of astrocyte infection, neural stem cell-derived human astrocytes in vitro are susceptible to SARS-CoV-2 infection through a noncanonical mechanism that involves spike-NRP1 interaction. SARS-CoV-2-infected astrocytes manifested changes in energy metabolism and in key proteins and metabolites used to fuel neurons, as well as in the biogenesis of neurotransmitters. Moreover, human astrocyte infection elicits a secretory phenotype that reduces neuronal viability. Our data support the model in which SARS-CoV-2 reaches the brain, infects astrocytes, and consequently, leads to neuronal death or dysfunction. These deregulated processes could contribute to the structural and functional alterations seen in the brains of COVID-19 patients.

The regeneration conferring transcription factor complex ERF115-PAT1 coordinates a wound-induced response in root-knot nematode induced galls.

The establishment of root-knot nematode (RKN; Meloidogyne spp.) induced galls in the plant host roots likely involves a wound-induced regeneration response. Confocal imaging demonstrates physical stress or injury caused by RKN infection during parasitism in the model host Arabidopsis thaliana. The ERF115-PAT1 heterodimeric transcription factor complex plays a recognized role in wound-induced regeneration. ERF115 and PAT1 expression flanks injured gall cells likely driving mechanisms of wound healing, implying a local reactivation of cell division which is also hypothetically involved in gall genesis. Herein, functional investigation revealed that ectopic ERF115 expression resulted in premature induction of galls, and callus formation adjacent to the expanding female RKN was seen upon PAT1 upregulation. Smaller galls and less reproduction were observed in ERF115 and PAT1 knockouts. Investigation of components in the ERF115 network upon overexpression and knockdown by qRT-PCR suggests it contributes to steer gall wound-sensing and subsequent competence for tissue regeneration. High expression of CYCD6;1 was detected in galls, and WIND1 overexpression resulted in similar ERF115OE gall phenotypes, also showing faster gall induction. Along these lines, we show that the ERF115-PAT1 complex likely coordinates stress signalling with tissue healing, keeping the gall functional until maturation and nematode reproduction.

An acute bout of resistance exercise increases BDNF in hippocampus and restores the long-term memory of insulin-resistant rats.

A sedentary lifestyle, inadequate diet, and obesity are substantial risk factors for Type 2 diabetes mellitus (T2DM) development. A major picture of T2DM is insulin resistance (IR), which causes many impairments in brain physiology, such as increased proinflammatory state and decreased brain-derived neurotrophic factor (BDNF) concentration, hence reducing cognitive function. Physical exercise is a non-pharmacological tool for managing T2DM/IR and its complications. Thus, this study investigated the effects of IR induction and the acute effects of resistance exercise (RE) on memory, neurotrophic, and inflammatory responses in the hippocampus and prefrontal cortex of insulin-resistant rats. IR was induced by a high-fat diet and fructose-rich beverage. Insulin-resistant rats performed acute resistance exercise (IR.RE; vertical ladder climb at 50-100% of the maximum load) or rest (IR.REST; 20 min). Cognitive parameters were assessed by novel object recognition (NOR) tasks, and biochemical analyses were performed to assess BDNF concentrations and inflammatory profile in the hippocampus and prefrontal cortex. Insulin-resistant rats had 20% worse long-term memory (LTM) (p < 0.01) and lower BDNF concentration in the hippocampus (-14.6%; p < 0.05) when compared to non-insulin-resistant rats (CON). An acute bout of RE restored LTM (-9.7% pre vs. post; p > 0.05) and increased BDNF concentration in the hippocampus (9.1%; p < 0.05) of insulin-resistant rats compared to REST. Thus, an acute bout of RE can attenuate the adverse effects of IR on memory and neurotrophic factors in rats, representing a therapeutic tool to alleviate the IR impact on the brain.

Carbamoylase-based impedimetric electronic tongue for rapid detection of paralytic shellfish toxins.

Phytotoxins produced by marine microalgae, such as paralytic shellfish toxins (PSTs), can accumulate in bivalve molluscs, representing a human health concern due to the life-threatening symptoms they cause. To avoid the commercialization of contaminated bivalves, monitoring programs were established in the EU. The purpose of this work is the implementation of a PST transforming enzyme-carbamoylase-in an impedimetric test for rapid simultaneous detection of several carbamate and N-sulfocarbamoyl PSTs. Carbamoylase hydrolyses carbamate and sulfocarbamoyl toxins, which may account for up to 90% of bivalve toxicity related to PSTs. Conformational changes of carbamoylase accompanying enzymatic reactions were probed by Fourier transform mid-infrared spectroscopy (FT-MIR) and electrochemical impedance spectroscopy (EIS). Furthermore, a combination of EIS with a metal electrode and a carbamoylase-based assay was employed to harness changes in the enzyme conformation and adsorption on the electrode surface during the enzymatic reaction as an analytical signal. After optimization of the working conditions, the developed impedimetric e-tongue could quantify N-sulfocarbamoyl toxins with a detection limit of 0.1 µM. The developed e-tongue allows the detection of these toxins at concentration levels observed in bivalves with PST toxicity close to the regulatory limit. The quantification of a sum of N-sulfocarbamoyl PSTs in naturally contaminated mussel extracts using the developed impedimetric e-tongue has been demonstrated.

Phylogeographic patterns of Cyphocharax from trans-Andean rivers and northward expansion to lower Central America (Teleostei, Curimatidae).

Phylogenetic analyses of mitochondrial and nuclear data of 31 specimens of Cyphocharax from trans-Andean rivers support the presence of one lineage of Cyphocharax aspilos in Lago Maracaibo and three cryptic lineages of Cyphocharax magdalenae: (1) Cauca-Magdalena and Ranchería, (2) León and Atrato, and (3) Chucunaque-Tuira, Santa María, and Chiriquí basins of Central America. Results suggest that the Serranía del Perijá facilitated Late Miocene cladogenetic events, whereas post-Isthmian C. magdalenae expansion was enabled by gene flow across the lower Magdalena valley and Central American lowlands. Time-calibrated phylogenetics indicate that the C. magdalenae colonized lower Central America in the Pliocene (3.7 MYA; Ma), the divergence Atrato-Magdalena occurred in Late Pliocene (3.0 Ma) and the split Ranchería-Magdalena during the Middle Pleistocene (1.3 Ma). Updated geographic distribution data support the hypothesis that the Cordillera de Talamanca functions as a barrier to northward expansion of C. magdalenae in Central America.

An ex vitro hairy root system from petioles of detached soybean leaves for in planta screening of target genes and CRISPR strategies associated with nematode bioassays.

MAIN CONCLUSION: The ex vitro hairy root system from petioles of detached soybean leaves allows the functional validation of genes using classical transgenesis and CRISPR strategies (e.g., sgRNA validation, gene activation) associated with nematode bioassays. Agrobacterium rhizogenes-mediated root transformation has been widely used in soybean for the functional validation of target genes in classical transgenesis and single-guide RNA (sgRNA) in CRISPR-based technologies. Initial data showed that in vitro hairy root induction from soybean cotyledons and hypocotyls were not the most suitable strategies for simultaneous performing genetic studies and nematode bioassays. Therefore, an ex vitro hairy root system was developed for in planta screening of target molecules during soybean parasitism by root-knot nematodes (RKNs). Applying this method, hairy roots were successfully induced by A. rhizogenes from petioles of detached soybean leaves. The soybean GmPR10 and GmGST genes were then constitutively overexpressed in both soybean hairy roots and tobacco plants, showing a reduction in the number of Meloidogyne incognita-induced galls of up to 41% and 39%, respectively. In addition, this system was evaluated for upregulation of the endogenous GmExpA and GmExpLB genes by CRISPR/dCas9, showing high levels of gene activation and reductions in gall number of up to 58.7% and 67.4%, respectively. Furthermore, morphological and histological analyses of the galls were successfully performed. These collective data validate the ex vitro hairy root system for screening target genes, using classical overexpression and CRISPR approaches, directly in soybean in a simple manner and associated with nematode bioassays. This system can also be used in other root pathosystems for analyses of gene function and studies of parasite interactions with plants, as well as for other purposes such as studies of root biology and promoter characterization.

Minc03328 effector gene downregulation severely affects Meloidogyne incognita parasitism in transgenic Arabidopsis thaliana.

MAIN CONCLUSION: Minc03328 effector gene downregulation triggered by in planta RNAi strategy strongly reduced plant susceptibility to Meloidogyne incognita and suggests that Minc03328 gene is a promising target for the development of genetically engineered crops to improve plant tolerance to M. incognita. Meloidogyne incognita is the most economically important species of root-knot nematodes (RKN) and causes severe damage to crops worldwide. M. incognita secretes several effector proteins to suppress the host plant defense response, and manipulate the plant cell cycle and other plant processes facilitating its parasitism. Different secreted effector proteins have already been identified in M. incognita, but not all have been characterized or have had the confirmation of their involvement in nematode parasitism in their host plants. Herein, we characterized the Minc03328 (Minc3s00020g01299) effector gene, confirmed its higher expression in the early stages of M. incognita parasitism in plants, as well as the accumulation of the Minc03328 effector protein in subventral glands and its secretion. We also discuss the potential for simultaneous downregulation of its paralogue Minc3s00083g03984 gene. Using the in planta RNA interference strategy, Arabidopsis thaliana plants overexpressing double-stranded RNA (dsRNA) were generated to specifically targeting and downregulating the Minc03328 gene during nematode parasitism. Transgenic Minc03328-dsRNA lines that significantly downregulated Minc03328 gene expression during M. incognita parasitism were significantly less susceptible. The number of galls, egg masses, and [galls/egg masses] ratio were reduced in these transgenic lines by up to 85%, 90%, and 87%, respectively. Transgenic Minc03328-dsRNA lines showed the presence of fewer and smaller galls, indicating that parasitism was hindered. Overall, data herein strongly suggest that Minc03328 effector protein is important for M. incognita parasitism establishment. As well, the in planta Minc03328-dsRNA strategy demonstrated high biotechnological potential for developing crop species that could efficiently control RKN in the field.

Overexpression of a soybean Globin (GmGlb1-1) gene reduces plant susceptibility to Meloidogyne incognita.

MAIN CONCLUSION: The overexpression of the GmGlb1-1 gene reduces plant susceptibility to Meloidogyne incognita. Non-symbiotic globin class #1 (Glb1) genes are expressed in different plant organs, have a high affinity for oxygen, and are related to nitric oxide (NO) turnover. Previous studies showed that soybean Glb1 genes are upregulated in soybean plants under flooding conditions. Herein, the GmGlb1-1 gene was identified in soybean as being upregulated in the nematode-resistant genotype PI595099 compared to the nematode-susceptible cultivar BRS133 during plant parasitism by Meloidogyne incognita. The Arabidopsis thaliana and Nicotiana tabacum transgenic lines overexpressing the GmGlb1-1 gene showed reduced susceptibility to M. incognita. Consistently, gall morphology data indicated that pJ2 nematodes that infected the transgenic lines showed developmental alterations and delayed parasitism progress. Although no significant changes in biomass and seed yield were detected, the transgenic lines showed an elongated, etiolation-like growth under well-irrigation, and also developed more axillary roots under flooding conditions. In addition, transgenic lines showed upregulation of some important genes involved in plant defense response to oxidative stress. In agreement, higher hydrogen peroxide accumulation and reduced activity of reactive oxygen species (ROS) detoxification enzymes were also observed in these transgenic lines. Thus, based on our data and previous studies, it was hypothesized that constitutive overexpression of the GmGlb1-1 gene can interfere in the dynamics of ROS production and NO scavenging, enhancing the acquired systemic acclimation to biotic and abiotic stresses, and improving the cellular homeostasis. Therefore, these collective data suggest that ectopic or nematode-induced overexpression, or enhanced expression of the GmGlb1-1 gene using CRISPR/dCas9 offers great potential for application in commercial soybean cultivars aiming to reduce plant susceptibility to M. incognita.

Phylogenomic analysis of the Neotropical fish subfamily Characinae using ultraconserved elements (Teleostei: Characidae).

Characinae is one of the most species-rich subfamilies of Characidae and holds special taxonomic importance because it includes Charax, type-genus of Characidae and Characiformes. Currently, the monophyly and the hypotheses of intergeneric and interspecific relationships of Characinae are based on a few morphological and molecular studies but all with low species coverage. Given their diversity, taxonomic importance, and the lack of a taxon-dense phylogeny, we sought to buttress the systematic understanding of Characinae collecting DNA sequence data from ultraconserved elements (UCEs) of the genome from 98 specimens covering 57 species (61%) plus 17 characiforms as outgroups. We used maximum likelihood, Bayesian inference, and coalescent-based species tree approaches and the resulting phylogeny with 1,300 UCE loci (586,785 characters) reinforced the monophyly of the subfamily as well as of six genera: Acestrocephalus, Charax, Cynopotamus, Galeocharax, Phenacogaster, and Roeboides. The phylogeny provides a hypothesis of intergeneric and interspecific relationships for the subfamily with Phenacogaster sister to all genera, and Acanthocharax sister to Cynopotamini (Cynopotamus (Acestrocephalus Galeocharax)) and Characini (Charax Roeboides). We propose a new tribe Acanthocharacini to allocate Acanthocharax, two subclades for Phenacogaster, two for Cynopotamus, three for Charax, and reinforced the four subclades for Roeboides previously identified by morphological studies. Additionally, we generated a time-calibrated phylogeny for Characinae that suggested an initial diversification during the Miocene at around 19 million years ago and discussed historical biogeographic events for major subclades. The results obtained here will contribute to the development of further research on the evolutionary processes modulating species diversification in Characinae.

Accelerated Diversification Explains the Exceptional Species Richness of Tropical Characoid Fishes.

The Neotropics harbor the most species-rich freshwater fish fauna on the planet, but the timing of that exceptional diversification remains unclear. Did the Neotropics accumulate species steadily throughout their long history, or attain their remarkable diversity recently? Biologists have long debated the relative support for these museum and cradle hypotheses, but few phylogenies of megadiverse tropical clades have included sufficient taxa to distinguish between them. We used 1288 ultraconserved element loci spanning 293 species, 211 genera, and 21 families of characoid fishes to reconstruct a new, fossil-calibrated phylogeny and infer the most likely diversification scenario for a clade that includes a third of Neotropical fish diversity. This phylogeny implies paraphyly of the traditional delimitation of Characiformes because it resolves the largely Neotropical Characoidei as the sister lineage of Siluriformes (catfishes), rather than the African Citharinodei. Time-calibrated phylogenies indicate an ancient origin of major characoid lineages and reveal a much more recent emergence of most characoid species. Diversification rate analyses infer increased speciation and decreased extinction rates during the Oligocene at around 30 Ma during a period of mega-wetland formation in the proto-Orinoco-Amazonas. Three species-rich and ecomorphologically diverse lineages (Anostomidae, Serrasalmidae, and Characidae) that originated more than 60 Ma in the Paleocene experienced particularly notable bursts of Oligocene diversification and now account collectively for 68% of the approximately 2150 species of Characoidei. In addition to paleogeographic changes, we discuss potential accelerants of diversification in these three lineages. While the Neotropics accumulated a museum of ecomorphologically diverse characoid lineages long ago, this geologically dynamic region also cradled a much more recent birth of remarkable species-level diversity. [Biodiversity; Characiformes; macroevolution; Neotropics; phylogenomics; ultraconserved elements.].

Discovery and functional characterization of novel cotton promoters with potential application to pest control.

KEY MESSAGE: pGhERF105 and pGhNc-HARBI1 promoters are highly responsive to CBW infestation and exhibit strong activity in vegetative and reproductive tissues, increasing their potential application in GM crop plants for pest control. The main challenge to cotton (Gossypium hirsutum) crop productivity is the constant attack of several pests, including the cotton boll weevil (CBW, Anthonomus grandis), which uses cotton floral buds for feeding and egg-laying. The endophytic nature of the early developmental stages of CBW makes conventional pesticide-based control poorly efficient. Most biotechnological assets used for pest control are based on Bacillus thurigiensis insecticidal Cry toxins or the silencing of insect-pest essential genes using RNA-interference technology. However, suitable plant promoter sequences are required to efficiently drive insecticidal molecules to the target plant tissue. This study selected the Ethylene Responsive Factor 105 (GhERF105) and Harbinger transposase-derived nuclease (GhNc-HARBI1) genes based on available transcriptome-wide data from cotton plants infested by CBW larvae. The GhERF105 and GhNc-HARBI1 genes showed induction kinetics from 2 to 96 h under CBW's infestation in cotton floral buds, uncovering the potential application of their promoters. Therefore, the promoter regions (1,500 base pairs) were assessed and characterized using Arabidopsis thaliana transgenic plants. The pGhERF105 and pGhNc-HARBI1 promoters showed strong activity in plant vegetative (leaves and roots) and reproductive (flowers and fruits) tissues, encompassing higher GUS transcriptional activity than the viral-constitutive Cauliflower Mosaic Virus 35S promoter (pCaMV35S). Notably, pGhERF105 and pGhNc-HARBI1 promoters demonstrated more efficiency in driving reporter genes in flowers than other previously characterized cotton flower-specific promoters. Overall, the present study provides a new set of cotton promoters suitable for biotechnological application in cotton plants for pest resistance.

Late Outcomes of Elective Endovascular Abdominal Aortic Aneurysm Repair in a Tertiary Low-Volume Hospital in Brazil.

BACKGROUND: The aim of this study is to report early and long-term results of elective endovascular aneurysm repair (EVAR) in a tertiary low-volume hospital in Brazil. METHODS: Between October 2006 and May 2017, 120 patients underwent elective EVAR for infrarenal aortic aneurysm. The interventions were reviewed retrospectively, focusing on 30-day mortality, long-term survival, and freedom from reintervention. Late outcomes were assessed by the Kaplan-Meier method and Cox regression. RESULTS: The follow-up's median and interquartile range was 3 (1-5) years. Overall, most patients were males (75%) and the median age was 74 years. Mostly patients were at a high risk for intervention (79.1%) and the majority was classified as American Society of Anesthesiologists III (53.3%). Preoperative aneurysm diameter median was 60 mm, interquartile range was 52.7-69. As per the postoperative aneurysm sac evolution, the number of patients with a reduction, stabilization, or an increase was 93 (77.5%), 18 (15%), and 9 (7.5%), respectively. The 30-day mortality was 6.6% and no late aneurysm-related deaths were identified. The overall incidence of late endoleaks was 24.1%, with the predominance of type II (23.3%), followed by type IA (0.8 %). Secondary interventions were necessary for 9 patients (7.5%). The 6-year analyses revealed freedom from reintervention and overall survival of 87.9% and 57.7%, respectively. The Cox regression analyses identified age > 75 years as an adverse factor for overall survival (hazard ratio = 2.5; P = 0.021). CONCLUSIONS: In the present study, EVAR in a low-volume center was associated with high 30-day mortality, but satisfactory long-term results were identified.

In planta RNAi approach targeting three M. incognita effector genes disturbed the process of infection and reduced plant susceptibility.

Meloidogyne incognita is the most economically important species of the root-knot nematode complex causing damage to several crops worldwide. During parasitism in host plants, M. incognita secretes several effector proteins to suppress the plant immune system, manipulate the plant cell cycle, and promote parasitism. Several effector proteins have been identified, but their relationship with plant parasitism by M. incognita has not been fully confirmed. Herein, the Minc01696, Minc00344, and Minc00801 putative effector genes were evaluated to assess their importance during soybean and Nicotiana tabacum parasitism by M. incognita. For this study, we used in planta RNAi technology to overexpress dsRNA molecules capable of producing siRNAs that target and downregulate these nematode effector genes. Soybean composite roots and N. tabacum lines were successfully generated, and susceptibility level to M. incognita was evaluated. Consistently, both transgenic soybean roots and transgenic N. tabacum lines carrying the RNAi strategy showed reduced susceptibility to M. incognita. The number of galls per plant and the number of egg masses per plant were reduced by up to 85% in transgenic soybean roots, supported by the downregulation of effector genes in M. incognita during parasitism. Similarly, the number of galls per plant, the number of egg masses per plant, and the nematode reproduction factor were reduced by up to 83% in transgenic N. tabacum lines, which was also supported by the downregulation of the Minc00801 effector gene during parasitism. Therefore, our data indicate that all three effector genes can be a target in the development of new biotechnological tools based on the RNAi strategy in economically important crops for M. incognita control.

The multigenerational effects of clothianidin on Chironomus xanthus: Larvae exposed to this acetylcholine super agonist show no clear resistance.

Clothianidin (CLO) is an insecticide belonging to the second-generation class of neonicotinoids. In this study, we evaluated how CLO affects the survival and the complete life cycle of the tropical insect Chironomus xanthus, a non-target species, considering the Parental (P) and Filial (F1) generations. We found a 48 h-lethal concentration (LC50) of CLO of 3.78 µg/L. The lowest observed effect concentrations (LOECs) were: i) for body growth and head capsule width in P generation = 47.3 ng/L CLO; ii) for body growth and head capsule width in F1 generation larvae = 80 and 36.4 ng/L CLO, respectively; iii) for cumulative emergence it was 80 ng/L CLO in the P generation, while there was no significant difference in the F1 generation; iv) for total developmental time for males and females = 61.53 ng/L in P generation; v) in the F1 generation, the LOEC was determined to be 36.4 ng/L for males and 80 ng/L for females; vi) The number of total hatched eggs and total hatched eggs/female had LOECs of 36.4 ng/L CLO for both generations. Our study reveals that environmentally relevant concentrations of the CLO-based insecticide are highly toxic to C. xanthus. It also shows that the F1 generation, resulting from parents exposed to CLO was not clearly resistant to the insecticide. This fact might be explained by the different effects observed for males and females of F1 generation. Understanding the sub-types of acetylcholine receptors present on target and non-target insect species and toxicological effects of neonicotinoids seems to be desirable for the insecticide industry to deal with insect pests and the environmental protection of non-target organisms.

Combination of Aerobic Training and Cocoa Flavanols as Effective Therapies to Reduce Metabolic and Inflammatory Disruptions in Insulin-Resistant Rats: The Exercise, Cocoa, and Diabetes Study.

We aimed to investigate the combined effects of aerobic exercise (EXE) and cocoa flavanol (COCOA) supplementation on performance, metabolic parameters, and inflammatory and lipid profiles in obese insulin-resistant rats. Therefore, 32 male Wistar rats (230-250 g) were fed a high-fat diet and a fructose-rich beverage for 30 days to induce insulin resistance. Next, the rats were randomized into four groups, orally administered placebo solution or COCOA supplementation (45 mg·kg-1), and either remained sedentary or were subjected to EXE on a treadmill at 60% peak velocity for 30 min, for 8 weeks. Blood samples and peripheral tissues were collected and processed to analyze metabolic and inflammatory parameters, lipid profiles, and morphological parameters. Supplementation with COCOA and EXE improved physical performance and attenuated body mass gain, adipose index, and adipocyte area. When analyzed as individual interventions, supplementation with COCOA and EXE improved glucose intolerance and the lipid profile reduced the concentrations of leptin, glucose, and insulin, and reduced homeostasis assessment index (all effects were p < .001 for both interventions), while ameliorated some inflammatory mediators in examined tissues. In skeletal muscles, both COCOA supplementation and EXE increased the expression of glucose transporter (p < .001 and p < .001), and combined intervention showed additive effects (p < .001 vs. COCOA alone or EXE alone). Thus, combining COCOA with EXE represents an effective nonpharmacological strategy to treat insulin resistance; it could prevent Type 2 diabetes mellitus by improving physical performance, glucose metabolism, neuroendocrine control, and lipid and inflammatory mediators in the liver, pancreas, adipose tissue, and skeletal muscle in obese male insulin-resistant rats.

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.

Striatal and septo-hypothalamic responses to anticipation and outcome of affiliative rewards.

Humans are intrinsically motivated to bond with others. The ability to experience affiliative emotions (such as affection/tenderness, sexual attraction, and admiration/awe) may incentivize and promote these affiliative bonds. Here, we interrogate the role of the critical reward circuitry, especially the Nucleus Accumbens (NAcc) and the septo-hypothalamic region, in the anticipation of and response to affiliative rewards using a novel incentive delay task. During Functional Magnetic Resonance Imaging (FMRI), participants (n = 23 healthy humans; 14 female) anticipated and watched videos involving affiliative (tenderness, erotic desire, and awe) and nonaffiliative (i.e., food) rewards, as well as neutral scenes. On the one hand, anticipation of both affiliative and nonaffiliative rewards increased activity in the NAcc, anterior insula, and supplementary motor cortex, but activity in the amygdala and the ventromedial prefrontal cortex (vmPFC) increased in response to reward outcomes. On the other hand, affiliative rewards more specifically increased activity in the septo-hypothalamic area. Moreover, NAcc activity during anticipation correlated with positive arousal for all rewards, whereas septo-hypothalamic activity during the outcome correlated with positive arousal and motivation for subsequent re-exposure only for affiliative rewards. Together, these findings implicate a general appetitive response in the NAcc to different types of rewards but suggests a more specific response in the septo-hypothalamic region in response to affiliative rewards outcomes. This work also presents a new task for distinguishing between neural responses to affiliative and non-affiliative rewards.

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.

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.