Virus-Mediated shRNA Knockdown of Prodynorphin in the Rat Nucleus Accumbens Attenuates Depression-Like Behavior and Cocaine Locomotor Sensitization

Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition.     

Linking Student Performance in Massachusetts Elementary Schools with the “Greenness” of School Surroundings Using Remote Sensing

Various studies have reported the physical and mental health benefits from exposure to “green” neighborhoods, such as proximity to neighborhoods with trees and vegetation. However, no studies have explicitly assessed the association between exposure to “green” surroundings and cognitive function in terms of student academic performance. This study investigated the association between the “greenness” of the area surrounding a Massachusetts public elementary school and the academic achievement of the school’s student body based on standardized tests with an ecological setting. Researchers used the composite school-based performance scores generated by the Massachusetts Comprehensive Assessment System (MCAS) to measure the percentage of 3^rd-grade students (the first year of standardized testing for 8–9 years-old children in public school), who scored “Above Proficient” (AP) in English and Mathematics tests (Note: Individual student scores are not publically available). The MCAS results are comparable year to year thanks to an equating process. Researchers included test results from 2006 through 2012 in 905 public schools and adjusted for differences between schools in the final analysis according to race, gender, English as a second language (proxy for ethnicity and language facility), parent income, student-teacher ratio, and school attendance. Surrounding greenness of each school was measured using satellite images converted into the Normalized Difference Vegetation Index (NDVI) in March, July and October of each year according to a 250-meter, 500-meter, 1,000-meter, and 2000-meter circular buffer around each school. Spatial Generalized Linear Mixed Models (GLMMs) estimated the impacts of surrounding greenness on school-based performance. Overall the study results supported a relationship between the “greenness” of the school area and the school-wide academic performance. Interestingly, the results showed a consistently positive significant association between the greenness of the school in the Spring (when most Massachusetts students take the MCAS tests) and school-wide performance on both English and Math tests, even after adjustment for socio-economic factors and urban residency.     

Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

The non-visual arrestins, arrestin-2 and arrestin-3, belong to a small family of multifunctional cytosolic proteins. Non-visual arrestins interact with hundreds of G protein-coupled receptors (GPCRs) and regulate GPCR desensitization by binding active phosphorylated GPCRs and uncoupling them from heterotrimeric G proteins. Recently, non-visual arrestins have been shown to mediate G protein-independent signaling by serving as adaptors and scaffolds that assemble multiprotein complexes. By recruiting various partners, including trafficking and signaling proteins, directly to GPCRs, non-visual arrestins connect activated receptors to diverse signaling pathways. To investigate arrestin-mediated signaling, a structural understanding of arrestin activation and interaction with GPCRs is essential. Here we identified global and local conformational changes in the non-visual arrestins upon binding to the model GPCR rhodopsin. To detect conformational changes, pairs of spin labels were introduced into arrestin-2 and arrestin-3, and the interspin distances in the absence and presence of the receptor were measured by double electron electron resonance spectroscopy. Our data indicate that both non-visual arrestins undergo several conformational changes similar to arrestin-1, including the finger loop moving toward the predicted location of the receptor in the complex as well as the C-tail release upon receptor binding. The arrestin-2 results also suggest that there is no clam shell-like closure of the N- and C-domains and that the loop containing residue 136 (homolog of 139 in arrestin-1) has high flexibility in both free and receptor-bound states.     

Herpes Virus MicroRNA Expression and Significance in Serous Ovarian Cancer

Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality.     

The intrinsically disordered CARDs‐Helicase linker in RIG‐I is a molecular gate for RNA proofreading

The innate immune receptor RIG‐I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG‐I''s C‐terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non‐specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG‐I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs‐Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD‐chosen RNAs to bind the helicase domain, while at the same time blocking non‐specific RNAs. These findings also indicate that CHL could represent a novel target for RIG‐I‐based therapeutics.     

QTL mapping and phenotypic variation for root architectural traits in maize (Zea mays L.)

Key message

QTL were identified for root architectural traits in maize.

Abstract

Root architectural traits, including the number, length, orientation, and branching of the principal root classes, influence plant function by determining the spatial and temporal domains of soil exploration. To characterize phenotypic patterns and their genetic control, three recombinant inbred populations of maize were grown for 28 days in solid media in a greenhouse and evaluated for 21 root architectural traits, including length, number, diameter, and branching of seminal, primary and nodal roots, dry weight of embryonic and nodal systems, and diameter of the nodal root system. Significant phenotypic variation was observed for all traits. Strong correlations were observed among traits in the same root class, particularly for the length of the main root axis and the length of lateral roots. In a principal component analysis, relationships among traits differed slightly for the three families, though vectors grouped together for traits within a given root class, indicating opportunities for more efficient phenotyping. Allometric analysis showed that trajectories of growth for specific traits differ in the three populations. In total, 15 quantitative trait loci (QTL) were identified. QTL are reported for length in multiple root classes, diameter and number of seminal roots, and dry weight of the embryonic and nodal root systems. Phenotypic variation explained by individual QTL ranged from 0.44 % (number of seminal roots, NyH population) to 13.5 % (shoot dry weight, OhW population). Identification of QTL for root architectural traits may be useful for developing genotypes that are better suited to specific soil environments.     

Synthesis and biological evaluation of aryl-oxadiazoles as inhibitors of Mycobacterium tuberculosis

Despite increased research efforts to find new treatments for tuberculosis in recent decades, compounds with novel mechanisms of action are still required. We previously identified a series of novel aryl-oxadiazoles with anti-tubercular activity specific for bacteria using butyrate as a carbon source. We explored the structure activity relationship of this series. Structural modifications were performed in all domains to improve potency and physico-chemical properties. A number of compounds displayed sub-micromolar activity against M. tuberculosis utilizing butyrate, but not glucose as the carbon source. Compounds showed no or low cytotoxicity against eukaryotic cells. Three compounds were profiled in mouse pharmacokinetic studies. Plasma clearance was low to moderate but oral exposure suggested solubility-limited drug absorption in addition to first pass metabolism. The presence of a basic nitrogen in the linker slightly increased solubility, and salt formation optimized aqueous solubility. Our findings suggest that the 1,3,4-oxadiazoles are useful tools and warrant further investigation.     

Placeholder Nucleosomes Underlie Germline-to-Embryo DNA Methylation Reprogramming

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