Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons.

Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both "classical" and "modulatory" signals that could produce diverse and/or complementary effects in associated circuits. Here, we establish that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. Our findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network.

Accounting for misclassified and unknown cause of death data in vital registration systems for estimating trends in HIV mortality.

INTRODUCTION: Misclassification of HIV deaths can substantially diminish the usefulness of cause of death data for decision-making. In this study, we describe the methods developed by the Global Burden of Disease Study to account for the misclassified cause of death data from vital registration systems for estimating HIV mortality in 132 countries and territories. METHODS: The cause of death data were obtained from the World Health Organization Mortality Database and official country-specific mortality databases. We implemented two steps to adjust the raw cause of death data: (1) redistributing garbage codes to underlying causes of death, including HIV/AIDS by applying methods, such as analysis of multiple cause data and proportional redistribution, and (2) reassigning HIV deaths misclassified as other causes to HIV/AIDS by examining the age patterns of underlying causes in location and years with and without HIV epidemics. RESULTS: In 132 countries, during the period from 1990 to 2018, 1,848,761 deaths were reported as caused by HIV/AIDS. After garbage code redistribution in these 132 countries, this number increased to 4,165,015 deaths. An additional 1,944,291 deaths were added through correction of HIV deaths misclassified as other causes in 44 countries. The proportion of HIV deaths derived from garbage code redistribution decreased over time, from 0.4 in 1990 to 0.1 in 2018. The proportion of deaths derived from HIV misclassification correction peaked at 0.4 in 2006 and declined afterwards to 0.08 in 2018. The greatest contributors to garbage code redistribution were "immunodeficiency antibody" (ICD 9: 279-279.1; ICD 10: D80-D80.9) and "immunodeficiency other" (ICD 9: 279, 279.5-279.9; ICD 10: D83-D84.9, D89, D89.8-D89.9), which together contributed 77% of all redistributed deaths at their peak in 1995. Respiratory tuberculosis (ICD 9: 010-012.9; ICD 10: A10-A14, A15-A16.9) contributed the greatest proportion of all HIV misclassified deaths (25-62% per year) over the most years. CONCLUSIONS: Correcting for miscoding and misclassification of cause of death data can enhance the utility of the data for analyzing trends in HIV mortality and tracking progress toward the Sustainable Development Goal targets.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

Methyl-CpG binding domain proteins inhibit interspecies courtship and promote aggression in Drosophila.

Reproductive isolation and speciation are driven by the convergence of environmental and genetic variation. The integration of these variation sources is thought to occur through epigenetic marks including DNA methylation. Proteins containing a methyl-CpG-binding domain (MBD) bind methylated DNA and interpret epigenetic marks, providing a dynamic yet evolutionarily adapted cellular output. Here, we report the Drosophila MBD-containing proteins, dMBD-R2 and dMBD2/3, contribute to reproductive isolation and survival behavioral strategies. Drosophila melanogaster males with a reduction in dMBD-R2 specifically in octopamine (OA) neurons exhibit courtship toward divergent interspecies D. virilis and D. yakuba females and a decrease in conspecific mating success. Conspecific male-male courtship is increased between dMBD-R2-deficient males while aggression is reduced. These changes in adaptive behavior are separable as males with a hypermethylated OA neuronal genome exhibited a decrease in aggression without altering male-male courtship. These results suggest Drosophila MBD-containing proteins are required within the OA neural circuitry to inhibit interspecies and conspecific male-male courtship and indicate that the genetically hard-wired neural mechanisms enforcing behavioral reproductive isolation include the interpretation of the epigenome.