Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing.

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.

Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Engineering Cancer Antigen-Specific T Cells to Overcome the Immunosuppressive Effects of TGF-ß.

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.

Ecto-5'-nucleotidase (Nt5e/CD73)-mediated adenosine signaling attenuates TGFß-2 induced elastin and cellular contraction.

Arterial calcification due to deficiency of CD73 (ACDC) is a rare genetic disease caused by a loss-of-function mutation in the NT5E gene encoding the ecto-5'-nucleotidase (cluster of differentiation 73, CD73) enzyme. Patients with ACDC develop vessel arteriomegaly, tortuosity, and vascular calcification in their lower extremity arteries. Histological analysis shows that patients with ACDC vessels exhibit fragmented elastin fibers similar to that seen in aneurysmal-like pathologies. It is known that alterations in transforming growth factor ß (TGFß) pathway signaling contribute to this elastin phenotype in several connective tissue diseases, as TGFß regulates extracellular matrix (ECM) remodeling. Our study investigates whether CD73-derived adenosine modifies TGFß signaling in vascular smooth muscle cells (SMCs). We show that Nt5e-/- SMCs have elevated contractile markers and elastin gene expression compared with Nt5e+/+ SMCs. Ecto-5'-nucleotidase (Nt5e)-deficient SMCs exhibit increased TGFß-2 and activation of small mothers against decapentaplegic (SMAD) signaling, elevated elastin transcript and protein, and potentiate SMC contraction. These effects were diminished when the A2b adenosine receptor was activated. Our results identify a novel link between adenosine and TGFß signaling, where adenosine signaling via the A2b adenosine receptor attenuates TGFß signaling to regulate SMC homeostasis. We discuss how disruption in adenosine signaling is implicated in ACDC vessel tortuosity and could potentially contribute to other aneurysmal pathogenesis.

The Clinical Utility of Two High-Throughput 16S rRNA Gene Sequencing Workflows for Taxonomic Assignment of Unidentifiable Bacterial Pathogens in Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming, and low throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests that are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by the respective built-in programs (MiSeq Reporter software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to that of Sanger 16S and better than that of Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78 h and 8.25 h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

Circular RNAs in organ injury: recent development.

Circular ribonucleic acids (circRNAs) are a class of long non-coding RNA that were once regarded as non-functional transcription byproducts. However, recent studies suggested that circRNAs may exhibit important regulatory roles in many critical biological pathways and disease pathologies. These studies have identified significantly differential expression profiles of circRNAs upon changes in physiological and pathological conditions of eukaryotic cells. Importantly, a substantial number of studies have suggested that circRNAs may play critical roles in organ injuries. This review aims to provide a summary of recent studies on circRNAs in organ injuries with respect to (1) changes in circRNAs expression patterns, (2) main mechanism axi(e)s, (3) therapeutic implications and (4) future study prospective. With the increasing attention to this research area and the advancement in high-throughput nucleic acid sequencing techniques, our knowledge of circRNAs may bring fruitful outcomes from basic and clinical research.

Chronic disease and elder mistreatment: A meta-analysis.

OBJECTIVE: This meta-analysis investigated the relationships between chronic diseases and different forms of elder mistreatment (physical, emotional, sexual, financial, neglect, or overall abuse). METHOD: Twelve different chronic disease risk markers linked to elder mistreatment were gathered from 48 studies (yielding 178 effect sizes (ESs) and a combined sample size of n = 390,785), then organized in to four broad chronic disease categories: endocrine disease, heart disease, neurological disease, and other chronic diseases. Data were analyzed with Comprehensive Meta-Analysis Software using a random effects approach. RESULTS: Neurological disease (odds ratio [OR] = 1.51), endocrine disease (OR = 1.38), heart disease (OR = 1.17), and other chronic diseases (OR = 1.26) were all significantly associated with elder mistreatments. Neurological disease (OR = 1.51) was found to have a significantly stronger association with elder mistreatment when compared to the heart disease category (OR = 1.17) and the other chronic disease category (OR = 1.26). When specifically investigating emotional abuse, there was a significantly stronger link with neurological disease (OR = 1.48) compared to other chronic diseases (OR = 1.21). CONCLUSIONS: This study provides the first meta-analytic benchmarks for understanding the links between chronic disease risk markers and different forms of elder mistreatment.

Young age is associated with decreased recurrence for renal cell carcinoma.

INTRODUCTION: We aimed to examine stage-specific oncologic outcomes for young versus conventional-age patients with localized disease in a modern cohort. MATERIALS AND METHODS: The Surveillance, Epidemiology and End Results database was queried for patients with T1-T2N0M0 kidney cancer from 1975-2016, including clear cell, papillary, and chromophobe renal cell carcinoma. Patients were stratified into ≤ 40 years-old or > 40 years-old cohorts and underwent definitive treatment via percutaneous ablation, partial nephrectomy, or radical nephrectomy. Primary outcome was cancer-specific survival. Cox regression and Kaplan-Meier analysis were performed. RESULTS: A total of 44,673 patients were identified with 41,812 patients in the conventional-age and 2,861 patients in the young cohort with mean ages of 62.1 and 34.7 years old, respectively. The young cohort had a higher proportion of T1a disease compared to the conventional-age cohort (65.2% vs. 58.6%) and a lower proportion of the cT1b (24.4% vs. 29.3%), cT2a (6.8% vs. 8.4%), and cT2b (3.6% vs. 3.7%) disease. Chromophobe histology was more prevalent in the younger population (10.5% vs. 6.6%). Nuclear grade 3 or 4 were more prominent in the conventional-age population (24.8% vs. 19.1%). Cancer-specific death was significantly higher in the conventional-age cohort (2.4% vs. 0.7%). Cox regression analysis demonstrated patients > 40 years old, increasing stage, and higher grade were at independently increased risk of cancer-specific death. Kaplan-Meier analysis showed significantly improved 5-year cancer-specific survival for the young versus conventional-age cohorts when sub-stratified by stage. CONCLUSION: When stratified by stage, young patients with localized kidney cancer experience improved cancer-specific survival.

Increasing rate of pathologic upgrading in low risk prostate cancer patients in the active surveillance era.

INTRODUCTION: Management of prostate cancer has seen an increasing predilection for active surveillance in low risk (LR) patients. We aimed to evaluate the rate of pathologic upgrading in patients with very low (VLR) or LR prostate cancer after prostatectomy. MATERIALS AND METHODS: The National Cancer Database (NCDB) and the Surveillance, Epidemiology, and End Results (SEER) Database were queried for patients diagnosed with Gleason 6 prostate cancer and prostate specific antigen (PSA) < 10 ng/mL from 2010 to 2016. All patients underwent 12-core biopsy and a subsequent prostatectomy for final pathologic staging. Our primary outcome was rate of pathologic upgrading over the study period. RESULTS: A total of 35,332 patients from the NCDB and 7,186 patients from the SEER database were collected. Patient population had an average age of about 59 years old and was over 80% white. Mean pre-biopsy PSA was higher for the upgraded cohorts in the NCDB and SEER populations (5.3 versus 4.9 and 5.5 versus 5.1 respectively, p < 0.001). Upgraded cohorts were more likely to have a higher percentage of positive cores at biopsy (p < 0.001). Multivariable analysis demonstrated that increasing age, increasing PSA and year of diagnosis were all predictors of upgrading (p < 0.05) in both databases. African American race was significantly associated with upgrading in the NCDB database only (p = 0.001). Over the studied time period, the rate of upgrading at prostatectomy increased from 41.2% to 56.7% in the NCDB population and from 41.9% to 45.4% in the SEER population. CONCLUSIONS: The rate of pathologic upgrading of VLR and LR prostate cancer at prostatectomy has been increasing in recent years. Increasing age, pre-biopsy PSA and an increasing percentage of positive cores at biopsy are predictors of this outcome. This may relate to improved patient selection for active surveillance and definitive treatment.

Dysregulation of FOXO1 (Forkhead Box O1 Protein) Drives Calcification in Arterial Calcification due to Deficiency of CD73 and Is Present in Peripheral Artery Disease.

OBJECTIVE: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. CONCLUSIONS: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.

Risk factors of infective endocarditis in persons who inject drugs.

BACKGROUND: The rising incidence of infective endocarditis (IE) among people who inject drugs (PWID) has been a major concern across North America. The coincident rise in IE and change of drug preference to hydromorphone controlled-release (CR) among our PWID population in London, Ontario intrigued us to study the details of injection practices leading to IE, which have not been well characterized in literature. METHODS: A case-control study, using one-on-one interviews to understand risk factors and injection practices associated with IE among PWID was conducted. Eligible participants included those who had injected drugs within the last 3 months, were > 18 years old and either never had or were currently admitted for an IE episode. Cases were recruited from the tertiary care centers and controls without IE were recruited from outpatient clinics and addiction clinics in London, Ontario. RESULTS: Thirty three cases (PWID IE+) and 102 controls (PWID but IE-) were interviewed. Multivariable logistic regressions showed that the odds of having IE were 4.65 times higher among females (95% CI 1.85, 12.28; p = 0.001) and 5.76 times higher among PWID who did not use clean injection equipment from the provincial distribution networks (95% CI 2.37, 14.91; p < 0.001). Injecting into multiple sites and heating hydromorphone-CR prior to injection were not found to be significantly associated with IE. Hydromorphone-CR was the most commonly injected drug in both groups (90.9% cases; 81.4% controls; p = 0.197). DISCUSSION: Our study highlights the importance of distributing clean injection materials for IE prevention. Furthermore, our study showcases that females are at higher risk of IE, which is contrary to the reported literature. Gender differences in injection techniques, which may place women at higher risk of IE, require further study. We suspect that the very high prevalence of hydromorphone-CR use made our sample size too small to identify a significant association between its use and IE, which has been established in the literature.

A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma.

BACKGROUND: Circulating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Reliable detection of global expression profiles is required to maximise miRNA biomarker discovery. Although developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. In this study we compare commercially available RNA extraction methods using MagnaZol (Bioo Scientific) or miRNeasy (QIAGEN) and three library preparation methods - CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (QIAGEN) - which aim to address one or both of these issues. RESULTS: Different RNA extractions and library preparation protocols result in differential detection of miRNAs. A greater proportion of reads mapped to miRNAs in libraries prepared with MagnaZol RNA than with miRNeasy RNA. Libraries prepared using QIAseq demonstrated the greatest miRNA diversity with many more very low abundance miRNAs detected (~ 2-3 fold more with < 10 reads), whilst CleanTag detected the fewest individual miRNAs and considerably over-represented miR-486-5p. Libraries prepared with QIAseq had the strongest correlation with RT-qPCR quantification. Analysis of unique molecular indices (UMIs) incorporated in the QIAseq protocol indicate that little PCR bias is introduced during small RNA library preparation. CONCLUSIONS: Small RNAs were consistently detected using all RNA extraction and library preparation protocols tested, but with some miRNAs at significantly different levels. Choice of the most suitable protocol should be informed by the relative importance of minimising the total sequencing required, detection of rare miRNAs or absolute quantification.

Influence of lifestyle choices on risks of CYP1B1 polymorphisms for prostate cancer.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5' untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G-C-T-G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non-smokers and non-drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G-C-T-G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease.

Efficient Electrosteric Assembly of Nanoparticle Heterodimers and Linear Heteroassemblies.

Bottom-up approaches to the synthesis of nanostructures are of particular interest because they offer several advantages over the traditional top-down approaches. In this work, we present a new method to self-assemble nanoparticles into controlled heteroaggregates. The technique relies on carefully balancing attractive electrostatic forces with repulsive steric hindrance that is provided by surface-grafted polyethylene glycol (PEG). Two different-sized gold nanoparticles (GNPs) were used as a model system: 13 nm GNPs were functionalized with PEG-thiol and mercapto dodecanoic acid, while 7 nm GNPs were functionalized with PEG-thiol and (11- Mercaptoundecyl)trimethylammonium bromide. When mixed together, these oppositely charged particles self-assemble into stable colloidal structures (i.e., nanoclusters) whose structure depends strongly on the surface concentration of PEG. Smaller structures are obtained as the PEG surface concentration increases because steric hindrance dominates and prevents uncontrolled aggregation. In particular, under the right conditions, we were able to selectively synthesize heterodimers (which are effectively Janus particles) and linear heteroassemblies. This method is scalable, and it provides a step forward in bottom-up synthesis of nanomaterials.

Ligature-induced peri-implantitis and periodontitis in mice.

AIM: Peri-implantitis (PI), inflammation around dental implants, shares characteristics with periodontitis (PD). However, PI is more difficult to control and treat, and detailed pathophysiology is unclear. We aimed to compare PI and PD progression utilizing a murine model. MATERIALS AND METHODS: Four-week-old male C57BL/6J mice had their left maxillary molars extracted. Implants were placed in healed extraction sockets and osseointegrated. Ligatures were tied around the implants and second molars. Controls did not receive ligatures. Mice were sacrificed 1 week, 1 and 3 months (n ≥ 5/group/time point) post-ligature placement. Bone loss analysis was performed. Histology was performed for: haematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-8 (MMP-8), nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), toluidine blue and calcein. RESULTS: PI showed statistically greater bone loss compared to PD at 1 and 3 months. At 3 months, 20% of implants in PI exfoliated; no natural teeth exfoliated in PD. H&E revealed that alveolar bone surrounding implants in PI appeared less dense compared to PD. PI presented with increased osteoclasts, MMP-8 and NF-κB, compared to PD. CONCLUSION: PI exhibited greater tissue and bone destruction compared to PD. Future studies will characterize the pathophysiological differences between the two conditions.

Downregulation of MicroRNA-126 Contributes to the Failing Right Ventricle in Pulmonary Arterial Hypertension.

BACKGROUND: Right ventricular (RV) failure is the most important factor of both morbidity and mortality in pulmonary arterial hypertension (PAH). However, the underlying mechanisms resulting in the failed RV in PAH remain unknown. There is growing evidence that angiogenesis and microRNAs are involved in PAH-associated RV failure. We hypothesized that microRNA-126 (miR-126) downregulation decreases microvessel density and promotes the transition from a compensated to a decompensated RV in PAH. METHODS AND RESULTS: We studied RV free wall tissues from humans with normal RV (n=17), those with compensated RV hypertrophy (n=8), and patients with PAH with decompensated RV failure (n=14). Compared with RV tissues from patients with compensated RV hypertrophy, patients with decompensated RV failure had decreased miR-126 expression (quantitative reverse transcription-polymerase chain reaction; P<0.01) and capillary density (CD31(+) immunofluorescence; P<0.001), whereas left ventricular tissues were not affected. miR-126 downregulation was associated with increased Sprouty-related EVH1 domain-containing protein 1 (SPRED-1), leading to decreased activation of RAF (phosphorylated RAF/RAF) and mitogen-activated protein kinase (MAPK); (phosphorylated MAPK/MAPK), thus inhibiting the vascular endothelial growth factor pathway. In vitro, Matrigel assay showed that miR-126 upregulation increased angiogenesis of primary cultured endothelial cells from patients with decompensated RV failure. Furthermore, in vivo miR-126 upregulation (mimic intravenous injection) improved cardiac vascular density and function of monocrotaline-induced PAH animals. CONCLUSIONS: RV failure in PAH is associated with a specific molecular signature within the RV, contributing to a decrease in RV vascular density and promoting the progression to RV failure. More importantly, miR-126 upregulation in the RV improves microvessel density and RV function in experimental PAH.

Assessing the Effects of Acute Amyloid ß Oligomer Exposure in the Rat.

Alzheimer's disease (AD) is the most common form of dementia, yet there are no therapeutic treatments that can either cure or delay its onset. Currently, the pathogenesis of AD is still uncertain, especially with respect to how the disease develops from a normal healthy brain. Amyloid ß oligomers (AßO) are highly neurotoxic proteins and are considered potential initiators to the pathogenesis of AD. Rat brains were exposed to AßO via bilateral intracerebroventricular injections. Rats were then euthanized at either 1, 3, 7 or 21-days post surgery. Rat behavioural testing was performed using the Morris water maze and open field tests. Post-mortem brain tissue was immunolabelled for Aß, microglia, and cholinergic neurons. Rats exposed to AßO showed deficits in spatial learning and anxiety-like behaviour. Acute positive staining for Aß was only observed in the corpus callosum surrounding the lateral ventricles. AßO exposed rat brains also showed a delayed increase in activated microglia within the corpus callosum and a decreased number of cholinergic neurons within the basal forebrain. Acute exposure to AßO resulted in mild learning and memory impairments with co-concomitant white matter pathology within the corpus callosum and cholinergic cell loss within the basal forebrain. Results suggest that acute exposure to AßO in the rat may be a useful tool in assessing the early phases for the pathogenesis of AD.

Characterizing the neurotranscriptomic states in alternative stress coping styles.

BACKGROUND: Animals experience stress in many contexts and often successfully cope. Individuals exhibiting the proactive versus reactive stress coping styles display qualitatively different behavioral and neuroendocrine responses to stressors. The predisposition to exhibiting a particular coping style is due to genetic and environmental factors. In this study we explore the neurotranscriptomic and gene network biases that are associated with differences between zebrafish (Danio rerio) lines selected for proactive and reactive coping styles and reared in a common garden environment. RESULTS: Using RNA-sequencing we quantified the basal transcriptomes from the brains of wild-derived zebrafish lines selectively bred to exhibit the proactive or reactive stress coping style. We identified 1953 genes that differed in baseline gene expression levels. Weighted gene coexpression network analyses identified one gene module associated with line differences. Together with our previous pharmacological experiment, we identified a core set of 62 genes associated with line differences. Gene ontology analyses reveal that many of these core genes are implicated in neurometabolism (e.g. organic acid biosynthetic and fatty acid metabolic processes). CONCLUSIONS: Our results show that proactive and reactive stress coping individuals display distinct basal neurotranscriptomic states. Differences in baseline expression of select genes or regulation of specific gene modules are linked to the magnitude of the behavioral response and the display of a coping style, respectively. Our results expand the molecular mechanisms of stress coping from one focused on the neurotransmitter systems to a more complex system that involves an organism's capability to handle neurometabolic loads and allows for comparisons with other animal taxa to uncover potential conserved mechanisms.