MRP8/14 Is a Molecular Signature Triggered by Dopamine in HIV Latent Myeloid Targets That Increases HIV Transcription and Distinguishes HIV+ Methamphetamine Users with Detectable CSF Viral Load and Brain Pathology.

There is a significant overlap between HIV infection and substance-use disorders. Dopamine (DA) is the most abundantly upregulated neurotransmitter in methamphetamine abuse, with receptors (DRD1-5) that are expressed by neurons as well as by a large diversity of cell types, including innate immune cells that are the targets of HIV infection, making them responsive to the hyperdopaminergic environment that is characteristic of stimulant drugs. Therefore, the presence of high levels of dopamine may affect the pathogenesis of HIV, particularly in the brain. The stimulation of HIV latently infected U1 promonocytes with DA significantly increased viral p24 levels in the supernatant at 24 h, suggesting effects on activation and replication. Using selective agonists to different DRDs, we found that DRD1 played a major role in activating viral transcription, followed by DRD4, which increased p24 with a slower kinetic rate compared to DRD1. Transcriptome and systems biology analyses led to the identification of a cluster of genes responsive to DA, where S100A8 and S100A9 were most significantly correlated with the early increase in p24 levels following DA stimulation. Conversely, DA increased the expression of these genes' transcripts at the protein level, MRP8 and MRP14, respectively, which form a complex also known as calprotectin. Interestingly, MRP8/14 was able to stimulate HIV transcription in latent U1 cells, and this occurred via binding of the complex to the receptor for an advanced glycosylation end-product (RAGE). Using selective agonists, both DRD1 and DRD4 increased MRP8/14 on the surface, in the cytoplasm, as well as secreted in the supernatants. On the other hand, while DRD1/5 did not affect the expression of RAGE, DRD4 stimulation caused its downregulation, offering a mechanism for the delayed effect via DRD4 on the p24 increase. To cross-validate MRP8/14 as a DA signature with a biomarker value, we tested its expression in HIV+ Meth users' postmortem brain specimens and peripheral cells. MRP8/14+ cells were more frequently identified in mesolimbic areas such as the basal ganglia of HIV+ Meth+ cases compared to HIV+ non-Meth users or to controls. Likewise, MRP8/14+ CD11b+ monocytes were more frequent in HIV+ Meth users, particularly in specimens from participants with a detectable viral load in the CSF. Overall, our results suggest that the MRP8 and MRP14 complex may serve as a signature to distinguish subjects using addictive substances in the context of HIV, and that this may play a role in aggravating HIV pathology by promoting viral replication in people with HIV who use Meth.

Detection of H3K4me3 Identifies NeuroHIV Signatures, Genomic Effects of Methamphetamine and Addiction Pathways in Postmortem HIV+ Brain Specimens that Are Not Amenable to Transcriptome Analysis.

Human postmortem specimens are extremely valuable resources for investigating translational hypotheses. Tissue repositories collect clinically assessed specimens from people with and without HIV, including age, viral load, treatments, substance use patterns and cognitive functions. One challenge is the limited number of specimens suitable for transcriptional studies, mainly due to poor RNA quality resulting from long postmortem intervals. We hypothesized that epigenomic signatures would be more stable than RNA for assessing global changes associated with outcomes of interest. We found that H3K27Ac or RNA Polymerase (Pol) were not consistently detected by Chromatin Immunoprecipitation (ChIP), while the enhancer H3K4me3 histone modification was abundant and stable up to the 72 h postmortem. We tested our ability to use HeK4me3 in human prefrontal cortex from HIV+ individuals meeting criteria for methamphetamine use disorder or not (Meth +/-) which exhibited poor RNA quality and were not suitable for transcriptional profiling. Systems strategies that are typically used in transcriptional metadata were applied to H3K4me3 peaks revealing consistent genomic activity differences in regions where addiction and neuronal synapses pathway genes are represented, including genes of the dopaminergic system, as well as inflammatory pathways. The resulting comparisons mirrored previously observed effects of Meth on suppressing gene expression and provided insights on neurological processes affected by Meth. The results suggested that H3K4me3 detection in chromatin may reflect transcriptional patterns, thus providing opportunities for analysis of larger numbers of specimens from cases with substance use and neurological deficits. In conclusion, the detection of H3K4me3 in isolated chromatin can be an alternative to transcriptome strategies to increase the power of association using specimens with long postmortem intervals and low RNA quality.

The Diabits App for Smartphone-Assisted Predictive Monitoring of Glycemia in Patients With Diabetes: Retrospective Observational Study.

BACKGROUND: Diabetes mellitus, which causes dysregulation of blood glucose in humans, is a major public health challenge. Patients with diabetes must monitor their glycemic levels to keep them in a healthy range. This task is made easier by using continuous glucose monitoring (CGM) devices and relaying their output to smartphone apps, thus providing users with real-time information on their glycemic fluctuations and possibly predicting future trends. OBJECTIVE: This study aims to discuss various challenges of predictive monitoring of glycemia and examines the accuracy and blood glucose control effects of Diabits, a smartphone app that helps patients with diabetes monitor and manage their blood glucose levels in real time. METHODS: Using data from CGM devices and user input, Diabits applies machine learning techniques to create personalized patient models and predict blood glucose fluctuations up to 60 min in advance. These predictions give patients an opportunity to take pre-emptive action to maintain their blood glucose values within the reference range. In this retrospective observational cohort study, the predictive accuracy of Diabits and the correlation between daily use of the app and blood glucose control metrics were examined based on real app users' data. Moreover, the accuracy of predictions on the 2018 Ohio T1DM (type 1 diabetes mellitus) data set was calculated and compared against other published results. RESULTS: On the basis of more than 6.8 million data points, 30-min Diabits predictions evaluated using Parkes Error Grid were found to be 86.89% (5,963,930/6,864,130) clinically accurate (zone A) and 99.56% (6,833,625/6,864,130) clinically acceptable (zones A and B), whereas 60-min predictions were 70.56% (4,843,605/6,864,130) clinically accurate and 97.49% (6,692,165/6,864,130) clinically acceptable. By analyzing daily use statistics and CGM data for the 280 most long-standing users of Diabits, it was established that under free-living conditions, many common blood glucose control metrics improved with increased frequency of app use. For instance, the average blood glucose for the days these users did not interact with the app was 154.0 (SD 47.2) mg/dL, with 67.52% of the time spent in the healthy 70 to 180 mg/dL range. For days with 10 or more Diabits sessions, the average blood glucose decreased to 141.6 (SD 42.0) mg/dL (P<.001), whereas the time in euglycemic range increased to 74.28% (P<.001). On the Ohio T1DM data set of 6 patients with type 1 diabetes, 30-min predictions of the base Diabits model had an average root mean square error of 18.68 (SD 2.19) mg/dL, which is an improvement over the published state-of-the-art results for this data set. CONCLUSIONS: Diabits accurately predicts future glycemic fluctuations, potentially making it easier for patients with diabetes to maintain their blood glucose in the reference range. Furthermore, an improvement in glucose control was observed on days with more frequent Diabits use.

Grain Yield Response of Corn (Zea mays L.) to Nitrogen Management Practices and Flooding.

Flooding can reduce corn growth and yield, but nitrogen (N) management practices may alter the degree to which plants are negatively impacted. Damage caused by flooded conditions may also affect the utilization of a post-flood N application to increase yield. The objectives of this study were to evaluate how pre-plant and pre-plant plus post-flood N applications contribute to corn growth and yield following flood conditions and to quantify the partial return of employing different N management strategies in the event of a flood. A field study was conducted in Ohio using four flood durations (FD; 0, 2, 4, or 6 days initiated at V4 to V5) and three N management practices (0 kg N ha-1, 134 kg N ha-1 applied pre-plant, and 134 pre-plant + 67 kg N ha-1 applied post-flooding). Application of 134 kg N ha-1 increased yield compared to 0 kg N ha-1 by 65%, 68%, 43% and 16% for 0 d, 2 d, 4 d, and 6 d FD, respectively; the application of 134 + 67 kg N ha-1 increased grain yield compared to 134 kg N ha-1 by 7%, 27%, 70%, or 55% for 0 d, 2 d, 4 d, or 6 d FD, respectively. Partial return analysis produced similar results to those for grain yield. Results suggest that in regions prone to early-season flooding, additional N applied post-flood can improve yield and partial return compared to the application of pre-plant alone at a lower rate or no N. Results indicate that total soil nitrate-N levels two weeks after flood initiation may serve as a good predictor of yield.

Dopamine and its receptors play a role in the modulation of CCR5 expression in innate immune cells following exposure to Methamphetamine: Implications to HIV infection.

The Human Immunodeficiency Virus (HIV) infects cells in the Central Nervous System (CNS), where the access of antiretrovirals and antibodies that can kill the virus may be challenging. As a result of the early HIV entry in the brain, infected individuals develop inflammation and neurological deficits at various levels, which are aggravated by drugs of abuse. In the non-human primate model of HIV, we have previously shown that drugs of abuse such as Methamphetamine (Meth) increase brain viral load in correlation with a higher number of CCR5-expressing myeloid cells. CCR5 is a chemokine receptor that may be involved in increasing inflammation, but also, it is a co-receptor for viral entry into target cells. CCR5-expressing myeloid cells are the main targets of HIV in the CNS. Thus, the identification of factors and mechanisms that impact the expression of CCR5 in the brain is critical, as changes in CCR5 levels may affect the infection in the brain. Using a well-characterized in vitro system, with the THP1 human macrophage cell line, we have investigated the hypothesis that the expression of CCR5 is acutely affected by Meth, and examined pathways by which this effect could happen. We found that Meth plays a direct role by regulating the abundance and nuclear translocation of transcription factors with binding sites in the CCR5 promoter. However, we found that the main factor that modifies the CCR5 gene promoter at the epigenetic level towards transcription is Dopamine (DA), a neurotransmitter that is produced primarily in brain regions that are rich in dopaminergic neurons. In THP1 cells, the effect of DA on innate immune CCR5 transcription was mediated by DA receptors (DRDs), mainly DRD4. We also identified a role for DRD1 in suppressing CCR5 expression in this myeloid cell system, with potential implications for therapy. The effect of DA on innate immune CCR5 expression was also detectable on the cell surface during acute time-points, using low doses. In addition, HIV Tat acted by enhancing the surface expression of CCR5, in spite of its poor effect on transcription. Overall, our data suggests that the exposure of myeloid cells to Meth in the context of presence of HIV peptides such as Tat, may affect the number of HIV targets by modulating CCR5 expression, through a combination of DA-dependent and-independent mechanisms. Other drugs that increase DA may affect similar mechanisms. The implications of these epigenetic and translational mechanisms in enhancing HIV infection in the brain and elsewhere are demonstrated.

Modeling the Function of TATA Box Binding Protein in Transcriptional Changes Induced by HIV-1 Tat in Innate Immune Cells and the Effect of Methamphetamine Exposure.

Innate immune cells are targets of HIV-1 infection in the Central Nervous System (CNS), generating neurological deficits. Infected individuals with substance use disorders as co-morbidities, are more likely to have aggravated neurological disorders, higher CNS viral load and inflammation. Methamphetamine (Meth) is an addictive stimulant drug, commonly among HIV+ individuals. The molecular basis of HIV direct effects and its interactions with Meth in host response, at the gene promoter level, are not well understood. The main HIV-1 peptide acting on transcription is the transactivator of transcription (Tat), which promotes replication by recruiting a Tata-box binding protein (TBP) to the virus long-terminal repeat (LTR). We tested the hypothesis that Tat can stimulate host gene expression through its ability to increase TBP, and thus promoting its binding to promoters that bear Tata-box binding motifs. Genes with Tata-box domains are mainly inducible, early response, and involved in inflammation, regulation and metabolism, relevant in HIV pathogenesis. We also tested whether Tat and Meth interact to trigger the expression of Tata-box bearing genes. The THP1 macrophage cell line is a well characterized innate immune cell system for studying signal transduction in inflammation. These cells are responsive to Tat, as well as to Meth, by recruiting RNA Polymerase (RNA Pol) to inflammatory gene promoters, within 15 min of stimulation (1). THP-1 cells, including their genetically engineered derivatives, represent valuable tools for investigating monocyte structure and function in both health and disease, as a consistent system (2). When differentiated, they mimic several aspects of the response of macrophages, and innate immune cells that are the main HIV-1 targets within the Central Nervous System (CNS). THP1 cells have been used to characterize the impact of Meth and resulting neurotransmitters on HIV entry (1), mimicking the CNS micro-environment. Integrative consensus sequence analysis in genes with enriched RNA Pol, revealed that TBP was a major transcription factor in Tat stimulation, while the co-incubation with Meth shifted usage to a distinct and diversified pattern. For validating these findings, we engineered a THP1 clone to be deficient in the expression of all major TBP splice variants, and tested its response to Tat stimulation, in the presence or absence of Meth. Transcriptional patterns in TBP-sufficient and deficient clones confirmed TBP as a dominant transcription factor in Tat stimulation, capable of inducing genes with no constitutive expression. However, in the presence of Meth, TBP was no longer necessary to activate the same genes, suggesting promoter plasticity. These findings demonstrate TBP as mechanism of host-response activation by HIV-1 Tat, and suggest that promoter plasticity is a challenge imposed by co-morbid factors such as stimulant drug addiction. This may be one mechanism responsible for limited efficacy of therapeutic approaches in HIV+ Meth abusers.

Soil type affects Pinus ponderosa var. scopulorum (Pinaceae) seedling growth in simulated drought experiments.

PREMISE OF THE STUDY: Effects of drought stress and media type interactions on growth of Pinus ponderosa var. scopulorum germinants were investigated. • METHODS AND RESULTS: Soil properties and growth responses under drought were compared across four growth media types: two native soils (dolomitic limestone and granite), a soil-less industry standard conifer medium, and a custom-mixed conifer medium. After 35 d of growth, the seedlings under drought stress (reduced watering) produced less shoot and root biomass than watered control seedlings. Organic media led to decreased root biomass, but increased root length and shoot biomass relative to the mineral soils. • CONCLUSIONS: Media type affected root-to-shoot biomass partitioning of P. ponderosa var. scopulorum, which may influence net photosynthetic rates, growth, and long-term seedling survival. Further work should examine how specific soil properties like bulk density and organic matter influence biomass allocation in greenhouse studies.