Development of a Tethered mRNA Amplifier to increase protein expression.

Herein, we present a novel method to specifically increase a messenger RNA's (mRNA) expression at the post-transcriptional level. This is accomplished using what we term a "Tethered mRNA Amplifier." The Tethered mRNA Amplifier specifically binds an mRNA's 3' untranslated region and enhances its stability/translation, often doubling protein output. We test this approach on several transcripts associated with haploinsufficiency disorders and increase their steady-state expression in cell culture. We suggest this approach may be a tenable therapeutic modality with precise activity and broad-spectrum application.

The lncRNA LOC102549805 (U1) modulates neurotoxicity of HIV-1 Tat protein.

HIV-1 Tat is a potent neurotoxic protein that is released by HIV-1 infected cells in the brain and perturbs neuronal homeostasis, causing a broad range of neurological disorders in people living with HIV-1. Furthermore, the effects of Tat have been addressed in numerous studies to investigate the molecular events associated with neuronal cells survival and death. Here, we discovered that exposure of rat primary neurons to Tat resulted in the up-regulation of an uncharacterized long non-coding RNA (lncRNA), LOC102549805 (lncRNA-U1). Our observations showed that increased expression of lncRNA-U1 in neurons disrupts bioenergetic pathways by dysregulating homeostasis of Ca2+, mitigating mitochondrial oxygen reduction, and decreasing ATP production, all of which point mitochondrial impairment in neurons via the Tat-mediated lncRNA-U1 induction. These changes were associated with imbalances in autophagy and apoptosis pathways. Additionally, this study showed the ability of Tat to modulate expression of the neuropeptide B/W receptor 1 (NPBWR1) gene via up-regulation of lncRNA-U1. Collectively, our results identified Tat-mediated lncRNA-U1 upregulation resulting in disruption of neuronal homeostasis.

Potential Role for Herpesviruses in Alzheimer's Disease.

Across the fields of virology and neuroscience, the role of neurotropic viruses in Alzheimer's disease (AD) has received renewed enthusiasm, with a particular focus on human herpesviruses (HHVs). Recent genomic analyses of brain tissue collections and investigations of the antimicrobial responses of amyloid-ß do not exclude a role of HHVs in contributing to or accelerating AD pathogenesis. Due to continued expansion in our aging cohort and the lack of effective treatments for AD, this composition examines a potential neuroviral theory of AD in light of these recent data. Consideration reveals a possible viral "Hit-and-Run" scenario of AD, as well as neurobiological mechanisms (i.e., neuroinflammation, protein quality control, oxidative stress) that may increase risk for AD following neurotropic infection. Although limitations exist, this theoretical framework reveals several novel therapeutic targets that may prove efficacious in AD.

Cross-talk between lipid homeostasis and endoplasmic reticulum stress in neurodegeneration: Insights for HIV-1 associated neurocognitive disorders (HAND).

The dysregulation of lipid homeostasis is emerging as a hallmark of many CNS diseases. As aberrant protein regulation is suggested to be a shared pathological feature amongst many neurodegenerative conditions, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), disruptions in neuronal lipid processing may contribute to disease progression in the CNS. Specifically, given the endoplasmic reticulum (ER) dual role in lipid homeostasis as well as protein quality control (PQC) via unfolded protein response (UPR), lipid dysregulation in the CNS may converge on ER functioning and constitute a crucial mechanism underlying aberrant protein aggregation. In the current review, we discuss the diverse roles of lipid species as essential components of the CNS. Moreover, given the importance of both lipid dysregulation and protein aggregation in pathology of CNS diseases, we attempt to assess the potential downstream cross-talk between lipid dysregulation and ER dependent PQC mechanisms, with special focus on HIV-associated neurodegenerative disorders (HAND).

Age-related neurodegenerative diseases.

Converging evidence indicates the dysregulation of unique cytosolic compartments called stress granules (SGs) might facilitate the accumulation of toxic protein aggregates that underlie many age-related neurodegenerative pathologies (ANPs). SG dynamics are particularly susceptible to the cellular conditions that are commonly induced by aging, including the elevation in reactive oxygen species and increased concentration of aggregate-prone proteins. In turn, the persistent formation of these compartments is hypothesized to serve as a seed for subsequent protein aggregation. Notably, the protein quality control (PQC) machinery responsible for inhibiting persistent SGs (e.g., Hsc70-BAG3) can become compromised with age, suggesting that the modulation of such PQC mechanisms could reliably inhibit pathological processes of ANPs. As exemplified in the context of accelerated aging syndromes (i.e., Hutchinson-Gilford progeria), PQC enhancement is emerging as a potential therapeutic strategy, indicating similar techniques might be applied to ANPs. Collectively, these recent findings advance our understanding of how the processes that might facilitate protein aggregation are particularly susceptible to aging conditions, and present investigators with an opportunity to develop novel targets for ANPs.

An Omics Approach to Extracellular Vesicles from HIV-1 Infected Cells.

Human Immunodeficiency Virus-1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infecting nearly 37 million people worldwide. Currently, there is no definitive cure, mainly due to HIV-1's ability to enact latency. Our previous work has shown that exosomes, a small extracellular vesicle, from uninfected cells can activate HIV-1 in latent cells, leading to increased mostly short and some long HIV-1 RNA transcripts. This is consistent with the notion that none of the FDA-approved antiretroviral drugs used today in the clinic are transcription inhibitors. Furthermore, these HIV-1 transcripts can be packaged into exosomes and released from the infected cell. Here, we examined the differences in protein and nucleic acid content between exosomes from uninfected and HIV-1-infected cells. We found increased cyclin-dependent kinases, among other kinases, in exosomes from infected T-cells while other kinases were present in exosomes from infected monocytes. Additionally, we found a series of short antisense HIV-1 RNA from the 3' LTR that appears heavily mutated in exosomes from HIV-1-infected cells along with the presence of cellular noncoding RNAs and cellular miRNAs. Both physical and functional validations were performed on some of the key findings. Collectively, our data indicate distinct differences in protein and RNA content between exosomes from uninfected and HIV-1-infected cells, which can lead to different functional outcomes in recipient cells.

Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells.

HIV-1 Tat is known to be released by HIV infected non-neuronal cells in the brain, and after entering neurons, compromises brain homeostasis by impairing pro-survival pathways, thus contributing to the development of HIV-associated CNS disorders commonly observed in individuals living with HIV. Here, we demonstrate that synapsins, phosphoproteins that are predominantly expressed in neuronal cells and play a vital role in modulating neurotransmitter release at the pre-synaptic terminal, and neuronal differentiation become targets for Tat through autophagy and protein quality control pathways. We demonstrate that the presence of Tat in neurons results in downregulation of BAG3, a co-chaperone for heat shock proteins (Hsp70/Hsc70) that is implicated in protein quality control (PQC) processes by eliminating mis-folded and damaged proteins, and selective macroautophagy. Our results show that treatment of cells with Tat or suppression of BAG3 expression by siRNA in neuronal cells disturbs subcellular distribution of synapsins and synaptotagmin 1 (Syt1) leading to their accumulation in the neuronal soma and along axons in a punctate pattern, rather than being properly distributed at axon-terminals. Further, our results revealed that synapsins partially lost their stability and their removal via lysosomal autophagy was noticeably impaired in cells with low levels of BAG3. The observed impairment of lysosomal autophagy, under this condition, is likely caused by cells losing their ability to process LC3-I to LC3-II, in part due to a decrease in the ATG5 levels upon BAG3 knockdown. These observations ascribe a new function for BAG3 in controlling synaptic communications and illuminate a new downstream target for Tat to elicit its pathogenic effect in impacting neuronal cell function and behavior.

Dysregulation of Neuronal Cholesterol Homeostasis upon Exposure to HIV-1 Tat and Cocaine Revealed by RNA-Sequencing.

HIV-1 Tat protein is released from HIV-1-infected cells and can enter non-permissive cells including neurons. Tat disrupts neuronal homeostasis and may contribute to the neuropathogenesis in people living with HIV (PLWH). The use of cocaine by PLWH exacerbates neuronal dysfunction. Here, we examined the mechanisms by which Tat and cocaine facilitate alterations in neuronal homeostatic processes. Bioinformatic interrogation of the results from RNA deep sequencing of rat hippocampal neurons exposed to Tat alone indicated the dysregulation of several genes involved in lipid and cholesterol metabolism. Following exposure to Tat and cocaine, the activation of cholesterol biosynthesis genes led to increased levels of free cholesterol and cholesteryl esters in rat neurons. Results from lipid metabolism arrays validated upregulation of several processes implicated in the biogenesis of ß-amyloid and Alzheimer's disease (AD), including sterol o-acyltransferase 1/acetyl-coenzyme A acyltransferase 1 (SOAT1/ACAT1), sortilin-related receptor L1 (SORL1) and low-density lipoprotein receptor-related protein 12 (LRP12). Further studies in Tat-treated primary neuronal cultures and brain tissues from HIV-1 transgenic mice as well as SIV-infected macaques confirmed elevated levels of SOAT1/ACAT 1 proteins. Our results offer novel insights into the molecular events involved in HIV and cocaine-mediated neuronal dysfunction that may also contribute to neuropathogenic events associated with the development of AD.

The eastern part of the Fertile Crescent concealed an unexpected route of olive (Olea europaea L.) differentiation.

Background and Aims: Olive is considered a native plant of the eastern side of the Mediterranean basin, from where it should have spread westward along the Mediterranean shores, while little is known about its diffusion in the eastern direction. Methods: Genetic diversity levels and population genetic structure of a wide set of olive ecotypes and varieties collected from several provinces of Iran, representing a high percentage of the entire olive resources present in the area, was screened with 49 chloroplast and ten nuclear simple sequence repeat markers, and coupled with archaeo-botanical and historical data on Mediterranean olive varieties. Approximate Bayesian Computation was applied to define the demographic history of olives including Iranian germplasm, and species distribution modelling was performed to understand the impact of the Late Quaternary on olive distribution. Key Results: The results of the present study demonstrated that: (1) the climatic conditions of the last glacial maximum had an important role on the actual olive distribution, (2) all Iranian olive samples had the same maternal inheritance as Mediterranean cultivars, and (3) the nuclear gene flow from the Mediterranean basin to the Iranian plateau was almost absent, as well as the contribution of subspecies cuspidata to the diversity of Iranian olives. Conclusions: Based on this evidence, a new scenario for the origin and distribution of this important fruit crop has been traced. The evaluation of olive trees growing in the eastern part of the Levant highlighted a new perspective on the spread and distribution of olive, suggesting two routes of olive differentiation, one westward, spreading along the Mediterranean basin, and another moving towards the east and reaching the Iranian plateau before its domestication.

Machine Learning Based Classification of Microsatellite Variation: An Effective Approach for Phylogeographic Characterization of Olive Populations.

Finding efficient analytical techniques is overwhelmingly turning into a bottleneck for the effectiveness of large biological data. Machine learning offers a novel and powerful tool to advance classification and modeling solutions in molecular biology. However, these methods have been less frequently used with empirical population genetics data. In this study, we developed a new combined approach of data analysis using microsatellite marker data from our previous studies of olive populations using machine learning algorithms. Herein, 267 olive accessions of various origins including 21 reference cultivars, 132 local ecotypes, and 37 wild olive specimens from the Iranian plateau, together with 77 of the most represented Mediterranean varieties were investigated using a finely selected panel of 11 microsatellite markers. We organized data in two '4-targeted' and '16-targeted' experiments. A strategy of assaying different machine based analyses (i.e. data cleaning, feature selection, and machine learning classification) was devised to identify the most informative loci and the most diagnostic alleles to represent the population and the geography of each olive accession. These analyses revealed microsatellite markers with the highest differentiating capacity and proved efficiency for our method of clustering olive accessions to reflect upon their regions of origin. A distinguished highlight of this study was the discovery of the best combination of markers for better differentiating of populations via machine learning models, which can be exploited to distinguish among other biological populations.

High genetic diversity detected in olives beyond the boundaries of the Mediterranean Sea.

BACKGROUND: Olive trees (Olea europaea subsp. europaea var. europaea) naturally grow in areas spanning the Mediterranean basin and towards the East, including the Middle East. In the Iranian plateau, the presence of olives has been documented since very ancient times, though the early history of the crop in this area is shrouded in uncertainty. METHODS: The varieties presently cultivated in Iran and trees of an unknown cultivation status, surviving under extreme climate and soil conditions, were sampled from different provinces and compared with a set of Mediterranean cultivars. All samples were analyzed using SSR and chloroplast markers to establish the relationships between Iranian olives and Mediterranean varieties, to shed light on the origins of Iranian olives and to verify their contribution to the development of the current global olive variation. RESULTS: Iranian cultivars and ecotypes, when analyzed using SSR markers, clustered separately from Mediterranean cultivars and showed a high number of private alleles, on the contrary, they shared the same single chlorotype with the most widespread varieties cultivated in the Mediterranean. CONCLUSION: We hypothesized that Iranian and Mediterranean olive trees may have had a common origin from a unique center in the Near East region, possibly including the western Iranian area. The present pattern of variation may have derived from different environmental conditions, distinct levels and selection criteria, and divergent breeding opportunities found by Mediterranean and Iranian olives.These unexpected findings emphasize the importance of studying the Iranian olive germplasm as a promising but endangered source of variation.