Doctoral Degree Granting Institutions: Recent submissions
Now showing items 21-40 of 1908
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Participation in a US community-based cardiovascular health study: investigating nonrandom selection effects related to employment, perceived stress, work-related stress, and family caregivingPurpose: Participation in health studies may be inversely associated with employment and stress. We investigated whether employment, perceived stress, work-related stress, and family caregiving were related to participation in a longitudinal US community-based health study of black and white men and women aged ≥45 years. Methods: Prevalence ratios and confidence intervals were estimated for completion of the second stage (S2) of a two-stage enrollment process by employment (status, type), and stress (perceived stress, work-related stress, caregiving), adjusting for age, sex, race, region, income, and education. Eligibility and consent for a follow-up occupational survey were similarly evaluated. Results: Wage- but not self-employed participants were less likely than the unemployed to complete S2. Among the employed, S2 completion did not vary by stress; however, family caregivers with a short time burden of care (<2 hour/d) were more likely to complete S2, compared to noncaregivers. Eligibility and participation in the follow-up occupational survey were higher among those employed (vs. unemployed) at enrollment but were not associated with enrollment stress levels. Conclusions: Limited evidence of selection bias was seen by employment and stress within a large US community-based cohort, but findings suggest the need for enrollment procedures to consider possible barriers to participation among wage-employed individuals.
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WHO/ILO work-related burden of disease and injury: Protocol for systematic reviews of exposure to long working hours and of the effect of exposure to long working hours on ischaemic heart diseaseBackground: The World Health Organization (WHO) and the International Labour Organization (ILO) are developing a joint methodology for estimating the national and global work-related burden of disease and injury (WHO/ILO joint methodology), with contributions from a large network of experts. In this paper, we present the protocol for two systematic reviews of parameters for estimating the number of deaths and disability-adjusted life years of ischaemic heart disease from exposure to long working hours, to inform the development of the WHO/ILO joint methodology. Objectives: We aim to systematically review studies on occupational exposure to long working hours (Systematic Review 1) and systematically review and meta-analyse estimates of the effect of long working hours on ischaemic heart disease (Systematic Review 2), applying the Navigation Guide systematic review methodology as an organizing framework. The selection of both, the exposure and the health outcome is justified by substantial scientific evidence on adverse effects of long working hours on ischaemic heart disease risk. Data sources: Separately for Systematic Reviews 1 and 2, we will search electronic academic databases for potentially relevant records from published and unpublished studies, Medline, EMBASE, Web of Science, CISDOC and PsychINFO. We will also search electronic grey literature databases, Internet search engines and organizational websites; hand-search reference list of previous systematic reviews and included study records; and consult additional experts. Study eligibility and criteria: We will include working-age (≥15 years) workers in the formal and informal economy in any WHO and/or ILO Member State, but exclude children (<15 years) and unpaid domestic workers. For Systematic Review 1, we will include quantitative prevalence studies of relevant levels of exposure to long working hours (i.e. 35-40, 41-48, 49-54 and ≥55 h/week) stratified by country, sex, age and industrial sector or occupation. For Systematic Review 2, we will include randomized controlled trials, cohort studies, case-control studies and other non-randomized intervention studies with an estimate of the relative effect of relevant level(s) of long working hours on the prevalence of, incidence of or mortality from ischaemic heart disease, compared with the theoretical minimum risk exposure level (i.e. 35-40 h/week). Study appraisal and synthesis methods: At least two review authors will independently screen titles and abstracts against the eligibility criteria at a first stage and full texts of potentially eligible records at a second stage, followed by extraction of data from qualifying studies. At least two review authors will assess risk of bias and the quality of evidence, using the most suited tools currently available. For Systematic Review 2, if feasible, we will combine relative risks using meta-analysis. We will report results using the guidelines for accurate and transparent health estimates reporting (GATHER) for Systematic Review 1 and the preferred reporting items for systematic reviews and meta-analyses guidelines (PRISMA) for Systematic Review 2. PROSPERO registration number: CRD42017084243.
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Prevention of hypertension due to long working hours and other work hazards is needed to reduce the risk of cardiovascular diseaseHypertension is the foremost risk factor for cardiovascular disease (CVD), which is the leading cause of death globally. In some countries, such as the US, the prevalence of hypertension and working-age CVD mortality are increasing. CVD is also the most common work-related disease worldwide. Long working hours and other psychosocial stressors at work are important modifiable risk factors for hypertension and CVD. However, there has been inadequate attention paid to the primary prevention of work-related hypertension and CVD. The state-of-the art method for blood pressure (BP) measurement is 24-hour ambulatory BP (ABP), necessary for accurate clinical decision making and to assess risk factors for BP elevation. Thus, ABP should be used in workplace screening and surveillance programs (along with surveys) to identify occupational risk factors, high-risk job titles, worksites and shifts, and evaluate programs designed to improve work organization. For example, after 30 months of an organizational intervention designed to lower psychosocial stressors at work among >2000 public sector white-collar workers in Quebec, Canada, BP and prevalence of hypertension significantly decreased in the intervention group, with no change in the control group, and a significant difference between the intervention and control groups. Further research is also needed on mechanisms linking work-related factors to hypertension and CVD, the cardiovascular effects of understudied work stressors, high-CVD risk worker groups, potential "upstream" intervention points, and country differences in working conditions, hypertension and CVD. Important organizational interventions, such as collective bargaining, worker cooperatives, or legislative and regulatory-level interventions, need to be evaluated.
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The Use of Three-Dimensionally Printed β-Tricalcium Phosphate/Hydroxyapatite to Understand the Regulation of Adenosine Receptors in Osteoclast Formation and Promotion in Bone Regeneration.Introduction: Bone defects resulting from trauma or infection need timely and effective treatments to replace damaged bone. Using specialized three-dimensional (3-D) printing technology, combined with bioactive molecules, we can design custom 3-D scaffolds for bone repair. The Hydroxyapatite (HA)/Beta-Tri-Calcium Phosphate (β-TCP) scaffold components provide mechanical strength, conduct bone throughout the scaffold and remodel over time. Adenosine, acting via adenosine receptors (A1, A2A, A2B and A3), plays a critical role in regulating bone metabolism. Dipyridamole (DIPY) increases local adenosine levels by blocking cellular uptake of adenosine and stimulates bone regeneration. We tested the capacity of DIPY, hypothesize that with a bioactive filler, such as DIPY, these scaffolds may successfully regenerate bone over critical sized bone defects in an in vivo model Methods: 15% HA:85% β-TCP scaffolds were designed using Robocad software, fabricated using a 3-D Robot, and sintered at 1100°C for 4h. Scanning electron microscopy (SEM) and micro-computed tomography (micro-CT) were used to examine structural aspects on pre/post-sintering, while x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and inductive coupled plasma (ICP) were used to evaluate porosity, crystalline phase quantification, and Ca:P ratio, respectively. Vehicle, BMP-2 and combination drug scaffolds (scaffold + PBS, scaffold + drug, scaffold+ collagen + drug) were implanted in C57B6 mice with 3mm critical size defect for 2, 4 and 8 weeks. DIPY release from scaffold was assayed in vitro spectrophotometrically over time. MicroCT and histological analysis were conducted to determine the degree of new bone formation and remodeling. Results: Qualitative microstructural evaluation using SEM showed a broader pore/particle size distribution for materials sintered. XRD, FT-IR and ICP results showed substantial deviations in the original 15/85% HA/β-TCP formulation with the detection of ~10%calcium pyrophosphate. As sintering temperature was increased, lower amounts of the HA (5% HA: ~95% β-TCP) phase was observed. DIPY release assays showed a constant release of the compound in collagen for a period of 10 days. Quantitative and qualitative results from microCT showed similar and significant bone formation and remodeling in HA/β-TCP- DIPY and HA/β-TCP-BMP-2 scaffolds when compared to vehicle at 2, 4 and 8 weeks (P≤ 0.05, P≤ 0.05 and P≤ 0.01, respectively). Histological analysis showed increased bone formation and remodeling in HA/β-TCP- DIPY and HA/β-TCP-BMP-2 scaffolds. Conclusion: Targeting osteoblasts and osteoclasts via appropriate adenosine receptor blockade or stimulation leads to increased bone regeneration in a murine model. Micro-CT and histology results show that the delivery of DIPY in the 3-D ceramic scaffolds promotes bone formation as effectively as BMP-2 in vivo. In the future, different inks can be used to fabricate different regions of the scaffold, depending on anticipated mechanical and remodeling requirements and scaffolds may be filled with different component materials that can be released at different times.
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MicroRNA-30c Mitigates Hypercholesterolemia and Atherosclerosis in Mice.High plasma cholesterol levels are found in several metabolic disorders and their reductions are advocated to reduce risk of atherosclerosis. A way to lower plasma lipids is to curtail lipoprotein assembly and secretion; however, this is associated with steatosis. Previously, we showed that lentiviral mediated hepatic expression of miR-30 reduces plasma cholesterol and atherosclerosis in mice. Since lentiviral delivery of miR-30c in humans for therapeutic purposes may pose several challenges and given the rapid progress of miRs from discovery to their potential development as a novel class of therapeutics, we hypothesized that a stabilized analogue of miR-30c (“mimic”) can replace viral delivery and be a suitable agent to lower hypercholesterolemia and atherosclerosis in male and female mice. To test this hypothesis, we complexed the synthetic miR-30c mimic with lipid emulsions and injected intravenously into mice placed on a Western diet to induce hyperlipidemia. Here, we show for the first time that weekly injections of a miR-30c mimic elevates hepatic miR-30c levels. This treatment results in sustained reductions in plasma cholesterol levels in Western diet-fed WT and Apoe−/− mice. The effect of miR-30c was dose dependent and the maximum effect on plasma cholesterol was observed 6 days after each injection. Further, we show that weekly injections of a miR-30c mimic also reduces atherosclerosis in Western diet-fed Apoe−/− mice. These reductions in plasma cholesterol and atherosclerosis were not accompanied with hepatic lipid accumulation or increases in plasma ALT, AST and CK, indicating that the miR-30c mimic was not causing hepatic steatosis or any obvious liver and muscle injury. To investigate more comprehensive role of miR-30c, we tested whether it can reduce plasma cholesterol and atherosclerosis in additional mouse models besides Western diet-induced hyperlipidemic mice. We found that miR-30c mimic significantly and safely lowers plasma cholesterol and atherosclerosis in Ldlr-deficient mice (Ldlr−/−), suggesting it a potential therapeutic modality for homozygous familial hypercholesterolemia (HoFH). Moreover, it dampens hypercholesterolemia in chow-fed type 2 diabetic and hypercholesterolemic ob/ob and db/db mice. However, it had no effect on plasma cholesterol in chow-fed STZ-induced diabetic and WT mice. Thus, these studies provide further proof of concept that miR-30c mimic could be developed into a safe, long-lasting and effective therapeutic agent for mitigating hypercholesterolemia in HoFH patients and in other hypercholesterolemic patients in general independent of the origin of hypercholesterolemia.
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Examining the effects of PKMzeta Inhibition on Primary Sensory (S1) cortex in Rat and Non-Human Primate.The primary somatosensory cortex (S1) is organized in a somatotopic manner. It has previously been demonstrated that responses to localized stimuli, termed a cortical response field (CRF) are characterized by a central excitatory response surrounded by a border of local inhibition. The size and shape of these CRFs can change, sometimes rapidly, as a result of behavioral training or chronic changes in sensory input. It has been hypothesized that mechanisms of synaptic plasticity, such as long-term potentiation (LTP) may play an important role in developing and maintaining the structure of a CRF. In this study we injected Zeta Inhibitory Peptide (ZIP), an inhibitor of the protein kinase PKMz known to be involved in the maintenance of LTP, into the primary sensory cortex (S1) of both rodents and non-human primates. We found that administration of ZIP caused a simultaneous disruption of CRF boundaries as well as a net decrease in response to tactile stimulation in both species. The evidence from these studies supports a model of sensory cortex that includes ongoing maintenance of CRF borders via PKMz dependent LTP.
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Sleep disturbances and racial-ethnic disparities in 10-year dementia risk among a national sample of older adults in the USARace/ethnicity and sleep disturbances are associated with dementia risk.
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Inequities in palliative care delivery to patients with HIV and Stage IV cancers in the US (2004-2020)Background: People with HIV (PWH) diagnosed with stage-IV cancer are less likely to receive palliative care (PC) compared to those without HIV. Our objective was to evaluate inequities in PC receipt among PWH with stage IV cancer in the US. Methods: We used the National Cancer Database (2004-2020), including adult (18-89 years) PWH with the 14 most common cancers that occur among PWH. PC was defined as treatment provided with non-curative intent. Our main exposures included % quartiles of adults without a high school degree (educational attainment) and median income quartiles within the patient's zip code. We used hierarchical multivariable Poisson regression to estimate adjusted prevalence ratios(aPR) with 95% confidence intervals (95% CI), adjusting for age, sex, year of diagnosis, race/ethnicity, and cancer type. Results: Among the included 10,120 PWH with stage IV cancer, 72% were men, 51% were either non-Hispanic(NH)-Black or Hispanic/Latinx, 38% were aged ≥60 years, and 97% resided in urban areas. Fourteen percent received PC. NH-Black PWH living in zip-codes with lower quartiles of educational attainment were more likely to receive PC compared to those in the highest quartile (Q1vs.Q4: aPR:1.93;95% CI:1.29-2,86) For income overall, compared to those in the highest quartile (Q4) of income, those in the lowest quartile had 26% higher likelihood of receiving PC (Q1vs.Q4: aPR:1.26;95% CI:1.05-1.52), particularly among NH-Black adults (Q1vs.Q4: aPR:1.67;95% CI:1.25-2.22; Q2 vs.Q4; aPR:1.48;95% CI:1.09-2.01). Conclusions: PC use among PWH with stage-IV cancer is low. Contextual poverty plays a role in PC delivery to PWH and cancer, particularly among NH-Black PWH.
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Spatially resolved transcriptomic signatures of hippocampal subregions and Arc-expressing ensembles in active place avoidance memoryThe rodent hippocampus is a spatially organized neuronal network that supports the formation of spatial and episodic memories. We conducted bulk RNA sequencing and spatial transcriptomics experiments to measure gene expression changes in the dorsal hippocampus following the recall of active place avoidance (APA) memory. Through bulk RNA sequencing, we examined the gene expression changes following memory recall across the functionally distinct subregions of the dorsal hippocampus. We found that recall induced differentially expressed genes (DEGs) in the CA1 and CA3 hippocampal subregions were enriched with genes involved in synaptic transmission and synaptic plasticity, while DEGs in the dentate gyrus (DG) were enriched with genes involved in energy balance and ribosomal function. Through spatial transcriptomics, we examined gene expression changes following memory recall across an array of spots encompassing putative memory-associated neuronal ensembles marked by the expression of the IEGs Arc, Egr1, and c-Jun. Within samples from both trained and untrained mice, the subpopulations of spatial transcriptomic spots marked by these IEGs were transcriptomically and spatially distinct from one another. DEGs detected between Arc + and Arc- spots exclusively in the trained mouse were enriched in several memory-related gene ontology terms, including "regulation of synaptic plasticity" and "memory." Our results suggest that APA memory recall is supported by regionalized transcriptomic profiles separating the CA1 and CA3 from the DG, transcriptionally and spatially distinct IEG expressing spatial transcriptomic spots, and biological processes related to synaptic plasticity as a defining the difference between Arc + and Arc- spatial transcriptomic spots.
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Dopamine release and dopamine-related gene expression in the amygdala are modulated by the gastrin-releasing peptide in opposite directions during stress-enhanced fear learning and extinctionFear extinction leads to a decrease of originally acquired fear responses after the threat is no longer present. Fear extinction is adaptive and critical for organism's survival, but deficits in extinction may lead to exaggerated fear in animals or post-traumatic stress disorder (PTSD) in humans. Dopamine has recently emerged as essential for fear extinction and PTSD, however the neural circuits serving this dopamine function are only beginning to be investigated, and the dopamine intracellular signaling pathways are unknown. We generated gastrin-releasing peptide gene knockout (Grp-/-) mice and found that they exhibit enhanced fear memory in a stress-enhanced fear learning (SEFL) paradigm, which combines stress exposure and fear extinction, two features critical for developing PTSD. Using in vivo fiber photometry to record dopamine signals, we found that the susceptibility of Grp-/- mice to SEFL is paralleled by an increase in basolateral amygdala (BLA) dopaminergic binding during fear conditioning and early extinction. Combined optogenetics and ex vivo electrophysiology showed an increase in presynaptic ventral tegmental area (VTA)-BLA connectivity in Grp-/- mice, demonstrating a role of dysregulated input from the VTA on BLA function in the absence of the GRP. When examining gene transcription using RNA-seq and qPCR, we discovered concerted down-regulation in dopamine-related genes in the BLA of Grp-/- mice following long-term SEFL memory recall that was not observed in naïve conditions. These experiments demonstrate that the GRP regulates dopamine function in stress-enhanced fear processing and identify the Grp as the first gene known to regulate dopaminergic control of fear extinction.
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Comparative effectiveness of anterior and posterior approaches for interscalene brachial plexus block: A systematic review and meta-analysisIntroduction: Interscalene Brachial Plexus Blocks (ISBPB) are highly effective forms of anesthesia for surgeries involving the upper arm, shoulder, and neck. Recently, there has been a growing interest in comparing the advantages and limitations of the anterior and posterior approaches. Methods: This systematic review and meta-analysis aimed to determine whether the anterior or posterior approach to ISBPB offers a clinical advantage regarding complete block rates and time to block completion. We included randomized controlled trials comparing the anterior and posterior techniques for ISBPB while excluding studies with overlapping populations, comparisons of blocks other than interscalene, and articles written in a non-English language. Results: The search strategy identified 2229 articles, of which six Randomized Controlled Trials (RCTs) met the inclusion criteria for the meta-analysis. A total of 414 patients were included, with 210 patients in the anterior group and 204 in the posterior group. The Odds Ratio (OR) for a complete sensory block between the two techniques did not reach statistical significance (OR = 0.56 [0.20, 1.58], 95% CI, p = 0.27). Similarly, the Standardized Mean Difference (SMD) for the time to complete the block also did not reach statistical significance (SMD: -0.77 [-2.12, 0.59], 95% CI, p = 0.27). Heterogeneity for complete block was not significant (I2 = 0%), while procedure time showed high heterogeneity (I2 = 97%). Conclusion: Both techniques have shown effectiveness in providing surgical analgesia. The choice of technique should be determined by the provider's comfort and proficiency, as well as ensuring the highest level of safety for the patient.
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Optimal timing of oral anticoagulation initiation in patients with acute ischaemic stroke and atrial fibrillation: a comprehensive meta-analysis and systematic reviewThe optimal timing for initiating direct oral anticoagulants (DOACs) for secondary stroke prevention in patients with atrial fibrillation and acute ischaemic stroke remains controversial due to concerns about haemorrhagic transformation. This study aimed to analyse the efficacy and safety of early versus late DOAC initiation. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a systematic review was conducted, searching major databases (PubMed, Embase, Cochrane Library and ClinicalTrials.gov) up to May 2024. A total of 11 studies were identified, comprising nine cohort studies (75.5% weight) and two randomised controlled trials (RCTs) (24.5% weight), involving 13 020 participants. The early DOAC group (mean initiation 3.5±1.29 days) included 6250 participants, while the late group (5.7±1.25 days) had 6770 participants. Outcome measures included recurrent ischaemic stroke (RIS), intracranial haemorrhage (ICH), systemic embolism, major haemorrhage (MH), non-major haemorrhage (NMH) and all-cause mortality. Statistical analysis using the Cochrane Review Manager calculated ORs and 95% CIs via the Mantel-Haenszel random effects model. This pooled meta-analysis revealed that the early DOAC group had lower rates of RIS (2.2% vs 2.9%, OR 0.72, 95% CI 0.52 to 0.98, p=0.04, I2=40%) and ICH (0.51% vs 0.93%, OR 0.45, 95% CI 0.29 to 0.70, p<0.05, I2=0%) compared with the late DOAC group. Subgroup analysis of RCTs and cohort studies showed reduced RIS and ICH risks in the early DOAC group, with moderate heterogeneity. In the sensitivity analysis, the early group (<4 days) had a lower risk of RIS compared with the late group (>4 days) without a statistically significant impact on ICH. No significant differences in MH, NMH, systemic embolism or all-cause mortality were observed between either group; however, a limited number of RCTs and moderate heterogeneity weakened the conclusions. Additional RCTs are needed to provide more definitive insights.
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The Role of Cardiac Magnetic Resonance to Predict Response to Cardiac Resynchronization Therapy: A Systematic Review and Meta-analysis.Cardiac resynchronization therapy (CRT) has revolutionized heart failure (HF) management, offering benefits in morbidity, mortality, and symptom alleviation. However, optimal response rates are not universally achieved, necessitating enhanced patient-selection strategies. Myocardial scar patterns, quantified by delayed-enhancement cardiac magnetic resonance (DE-CMR), have been implicated in CRT outcomes. We conducted a meta-analysis of observational studies assessing CRT responses by performing a systematic literature search using PubMed, Embase, Ovid MEDLINE, Scopus, the Cochrane Library, ScienceDirect, and the Web of Science. Scar burden, left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) were evaluated. CRT response rates among ischemic and non-ischemic cardiomyopathy patients were also explored. This meta-analysis incorporated eight studies meeting the eligibility criteria. CRT responders exhibited a significantly lower scar burden (-11.7%; 95% confidence interval, 6.6%-16.8%) compared to non-responders, supporting the predictive value of scar quantification ( = 95.25%; < .001). Responders demonstrated an increased mean LVEF (from 25.2% to 31.9%), while non-responders showed modest changes (from 23.3% to 24.4%). Responders experienced a decrease in mean LVESV from 158.8 to 132.8 mL, contrasting with a more stable mean LVESV value in non-responders (reduction from 160.9 to 157.6 mL). Responders experienced a reduced mean LVEDV from 219.4 to 196.7 mL, while non-responders showed more minimal changes (from 213.4 to 210.6 mL). Limited data suggested a CRT response rate of 34.7% in ischemic cardiomyopathy; non-ischemic data were insufficient. In conclusion, DE-CMR, assessing the scar burden, emerges as a valuable tool for predicting the CRT response. A lower scar burden correlates with improved responses, supporting the role of DE-CMR in refining patient selection for CRT. This meta-analysis contributes insights into personalized CRT strategies, emphasizing the potential of imaging modalities to enhance therapeutic outcomes in HF patients. Further research is warranted to solidify these findings and refine clinical applications.
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TFE3 and TFEB are E-cadherin and CD40 ligand transcription activators.TFE3 and TFEB are two closely related transcription factors belonging to the family of basic helix-loop-helix zipper (bHLH-Zip) transcription factors. TFE3 and TFEB regulate gene expression by forming dimers that bind to E3 sites in target gene promoters and/or enhancers. Some evidence suggests that TFE3 and TFEB have multiple common target genes in various types of cells and tissues, which reflects their sequence and distribution similarity. Moreover, translocations of TFE3 and TFEB genes that lead to over-expression are associated with rare types of renal cell carcinoma. However, the physiological target genes of TFE3 and TFEB in vivo, and how mutations of TFE3 and TFEB genes cause cancers are largely unknown. In the first set of my thesis studies, I found that TFE3 and TFEB might be functionally important for kidney development by regulating the transcription of Ecadherin, an epithelial marker, in response to the signals of leukemia inhibitory factor (LIF), an established mesenchymal to epithelial transition (MET) inducer in kidney development. This conclusion was based on following experiment results: over expression of TFE3 or TFEB induced E-cadherin and WT1 expression in transformed mesenchymal cells, and inhibiting both endogenous TFE3 and TFEB with RNA interference (RNAi) or a trans-dominant negative (TDN) protein inhibited E-cadherin expression in primary mesenchymal cells. In addition, TFE3 and TFEB were LIF responsive transcription activators of E-cadherin via the MAPK pathway in primary mesenchymal cells. Surprisingly, our data also showed that over- expressed TFE3 and TFEB partially inhibited E-cadherin expression in renal epithelial cells. Given the knowledge that down regulation of E-cadherin is associated with cancer progression and metastasis, this result implies that over expressed TFE3 and TFEB may contribute to renal malignancy by dysregulating tumor repressor genes’ expression, and gives new insight into etiology of the renal cancer. In the remainder of the thesis studies, I also found an important in vivo function of TFE3 and TFEB in the immune system via regulating the expression of CD40 ligand (CD40L), a critical mediator of thymus (T)-dependent immune responses that is mainly expressed by CD4 T cells, based on following experimental results: chromatin immunoprecipitation (ChIP) and reporter gene assays showed that both TFE3 and TFEB bound to the CD40L promoter and activated CD40L transcription in CD4 T cells, whereas knocking down TFEB in TFE3-deficient T cells via RNAi impaired CD40L expression. Moreover, inhibition of TFE3 and TFEB simultaneously in T cells with the TDN protein via transgenesis resulted in a phenotype similar to X-linked hyper-IgM syndrome (HIGM), a human disease caused by loss-of-function mutations of the CD40L gene. Given the knowledge that abnormal CD40L expression in CD4 T cells is associated with a wide variety of autoimmune and inflammatory diseases, and the mechanism of the abnormal CD40L expression in CD4 T cells is still unknown, we provide a new direction to study and treat these diseases by identifying TFE3 and TFEB as CD40L physiological transcriptional regulators.
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The Role of de novo Synthesis of PKMζ in Dorsal Hippocampus during Long-term Potentiation and Spatial Memory Storage.Protein kinase Mζ (PKMζ) is an autonomously active PKC isoform that plays a key role in long-term memory (LTM) storage. Postsynaptic perfusion of PKMζ causes an increase in the amplitude of EPSCs recorded from CA1 pyramidal cells (Ling, Benardo, & Sacktor, 2006), and overexpression of PKMζ in the neocortex enhances the LTM of conditioned taste aversion (Shema et al., 2011). In addition, inhibition of PKMζ by selective ζ-pseudosubstrate inhibitory peptide (ZIP) reverses the late phase of long-term potentiation (late-LTP) (Serrano, Yao, & Sacktor, 2005; Pastalkova et al., 2006), and disrupts the maintenance of spatial LTM (Pastalkova, et al., 2006; Serrano et al., 2008). However, little is known about the localization of the changes in PKMζ expression after spatial training. Moreover, because ZIP inhibits the enzymatic activity of PKMζ including that of both newly synthesized and pre-existing pools of the kinase, it is not clear which of the two pools mediates the effects of PKMζ on LTM and late-LTP. To address these questions, the localization of the change of PKMζ distribution 24 hr after both massed and spaced training of active place avoidance by immunohistochemistry was first investigated, and the results showed that the increased PKMζ mainly located in stratum pyramidale, radiatum and lacunosum-moleculare in hippocampal CA1 region. This pattern of increased PKMζ persisted 1 month after spaced training. The intracranial injection of PKMζ antisense oligodeoxynucleotides to this area to selectively inhibit de novo PKMζ synthesis was then conducted. The results showed that the antisense injection did not affect the basal level of PKMζ but blocked the increased PKMζ after LTP-inducing tetani or behavioral training. Furthermore, the antisense also disrupted the late-LTP and spatial LTM without affecting early-LTP and short-term memory. The results suggest that de novo synthesis of PKMζ is required for the newly formed LTP and LTM.
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Haplodeficiency of Hexim1 Enhances Skeletal Muscle Regeneration by Promoting Satellite Cell Post-Injury ExpansionSkeletal muscle constitutes the most abundant tissue of the human body and possesses remarkable regeneration capacity for the repair of muscle damage in injury or muscular disorders. However, the regeneration capacity of skeletal muscle varies depending on a number of factors, including age, anatomic location and the local tissue environment. Loss of muscle regeneration capacity, such as that seen in dystrophic muscle diseases characterized by progressive muscle degeneration and weakness, leads to reduced mobility and pre-mature death. Hexamethylene bis-acetamide inducible 1 (HEXIM1, aka cardiac lineage protein 1, CLP-1) is a well-established inhibitory component of the positive transcription elongation factor (P-TEFb) complex. Our lab has previously reported that down-regulation of HEXIM1 in mouse myoblast cell line C2C12 inhibits myogenic differentiation. Since myogenic differentiation of muscle progenitor cells is a critical event of skeletal muscle regeneration, I hypothesize that HEXIM1 can regulate the myogenic differentiation of muscle progenitor cells, the satellite cells, via the P-TEFb-dependent transcription elongation process and thus influence the regeneration of skeletal muscle. By application of an established skeletal muscle acute injury and regeneration model, I observed enhanced regeneration of muscle fibers and improved recovery of muscle contractile function following acute injury in Hexim1 haplodeficient mice in comparison with wild type controls. Immunofluorescence-based cell counting and flow cytometry analysis both showed that Hexim1 haplodeficient muscles harbored more satellite cells than wild type controls during regeneration. Hexim1 haplodeficient satellite cells proliferate faster than wild type satellite cells in vitro and are resistant to low-serum induced myogenic differentiation. Transplantation of Hexim1 haplodeficient satellite cells into injured wild type muscles improved regeneration more effectively than transplantation of wild type satellite cells. Conversely, HMBA induced HEXIM1 overexpression repressed satellite cell proliferation, induced myogenic differentiation and restrained muscle regeneration. This study would establish HEXIM1/P-TEFb pathway as a regulator of skeletal muscle regeneration and satellite cell maintenance, and may offer a valuable approach of modulating HEXIM1 to enhance cell-based therapies for muscular disorders.
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The Role of a Novel Calpain Inhibitor for the Treatment of Experimental Autoimmune Encephalomyelitis (EAE).Aberrant activation of calpain plays a key role in the pathophysiology of neurodegenerative disorders including multiple sclerosis (MS). Calpain is increasingly expressed in inflammatory cells in experimental autoimmune encephalomyelitis (EAE) and is significantly elevated in the white matter of patients with multiple sclerosis, thus calpain inhibition could be a target for therapeutic intervention. A novel drug cysteic-leucyl-argininal (CYLA) was designed by attaching a cysteic acid to the protease inhibiting part of leupeptin. Cysteic acid has similar functional group as in taurine, an amino acid that is being transported across the blood-brain barrier (BBB) using two taurine transporters. The experiments of this thesis addressed the effect of CYLA, targeted to the central nervous system (CNS), in a myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57Bl/6 mice. Daily intraperitoneal application of 0.5 to 2mg of this novel calpain inhibitor or oral administration of 2mg of the prodrug was found to significantly reduce the clinical signs, demyelination, and inflammatory infiltration in a dose-and time-dependant manner. Measurement of calpain content by a fluorogenic method and calpain activity by Western blot analysis of substrate degradation (alpha-fodrin) revealed that this inhibitor could cross the blood-brain barrier and inhibit calpain. In addition, the effect of CYLA in suppressing axonal injury was examined in chronic EAE. Markers for axonal injury were barely detectable in the CYLA treated mice groups. Both the free aldehyde form of CYLA, which was administered by injection, and the diacetal prodrug which was given orally, were able to ameliorate the disease course and its progression. We concluded that CYLA has a potential for the treatment of MS and other neurodegenerative disease in which elevated calpain activity is suspected.
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Spontaneous Withdrawal from THC during Adolescence: Sex Differences in Locomotor Activity and Anxiety.Research suggests that the use and abuse of marijuana can be especially harmful if it occurs during adolescence, a period of vast developmental changes throughout the brain. Because of the localization of cannabinoid receptors within the limbic system (Rossi et al., 2009; Katona et al., 2001), and the established effects of cannabinoids on emotional states/anxiety levels of rats and humans, we wanted to study the sex- and dose-related effects of Δ9-tetrahydrocannabinol (THC, the main psychoactive component in marijuana) on behavior and anxiety in a spontaneous withdrawal paradigm. Male and female Sprague Dawley rats were administered 2, 7.5 or 15 mg/kg THC or vehicle from postnatal day 35-41, a period approximating mid-adolescence in humans. Locomotor activity, anxiety-related behaviors, corticosterone and neurosteroid levels were measured during drug administration and subsequent drug abstinence. THC caused significant dose-dependent locomotor depression during drug administration. Locomotor depression initially abated upon drug cessation, but then re-emerged by the end of the 2-week drug abstinence period and was greater in female rats than male rats. We also found the development of sensitization to the locomotor-depressing effects of THC in middle- and high-dose rats and the subsequent development of tolerance in high-dose rats. The high dose of THC increased anxiety-like behaviors while the low dose decreased anxiety-like behaviors during the drug administration period, with females more sensitive to the anxiogenic effects of THC. During drug abstinence, females were again especially sensitive to the anxiogenic effects of THC, while THC had an anxiolytic-like effect on males. Measurements of plasma corticosterone and neurosteroid levels during the drug abstinence period may help to explain sex differences in these results. In conclusion, this study demonstrates sexually-dimorphic effects of THC on anxiety-related behaviors and locomotor activity after cessation of THC administration, information that may be useful in the development of therapeutic remedies for clinical marijuana withdrawal.
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PaCa-Ag1: A Candidate Pancreatic Cancer-specific Tumor MarkerThere is an urgent need to identify novel pancreatic carcinoma-specific markers to improve the early diagnosis and treatment of this lethal disease. The present investigation describes the purification of a new mouse monoclonal antibody, mAb3C4, its use in the characterization of the pancreatic carcinoma–specific PaCa-Ag1 and the preclinical exploration of mAb3C4 and PaCa-Ag1 in the context of pancreatic carcinoma. MAb3C4 (an IgG1) was generated by Immuno-substractive Hyperimmunization (ISHIP) in the context of a model of in vitro pancreatic carcinogenesis. Fluorescence staining and ultrastructural analyses of intact rat pancreatic carcinoma cells showed the expression of PaCa-Ag1 on the cells’ plasma membrane. Most importantly, mAb3C4 showed strong cross reactivity with the cells of six human pancreatic carcinoma cell lines examined. PaCa-Ag1 was absent from untransformed, normal BMRPA1 cells, normal rodent and human pancreatic and other normal tissues. Traces of PaCa-Ag1 were seen in lysates of renal, colorectal and prostate adenocarcinomas. By immunoblotting with mAb3C4, PaCa-Ag1 was identified as a poorly glycosylated polypeptide of Mr ~ 43.5kD with a pI of 3.67 for both the human and rat species. Subcellular fractionation and membrane extraction from pancreatic carcinoma cells confirmed PaCa-Ag1 as an integral membrane protein. Cytofluorimetric quantitation showed the presence of up to 14.8x104 PaCa-Ag1 sites on rodent and up to 10.5 times (up to 8.14x105) PaCa-Ag1 sites on human pancreatic carcinoma cells. A fast ka of 7.26x106M-1s-1 and a slow Kd of 1.9x10-6s-1 indicated a strong affinity km of 3.89x1011M-1 of mAb3C4 for PaCa-Ag1. During chemical carcinogenesis, low levels of PaCa-Ag1 were first seen at the 2nd passage of the transforming cells (47%) suggesting PaCa-Ag1 as an early marker of transformation. An Ab (mAb3C4) -capture ELISA identified PaCa-Ag1 in spent media of pancreatic cancer cell cultures, in ascites and sera of mice transplanted with pancreatic cancer. Since mAb3C4 displayed potent complement-dependent cytotoxicity against rodent and human pancreatic carcinoma cells in vitro, the immunotherapeutic potential was evaluated by continuous infusion of mAb3C4 into athymic mice xeno transplanted with pancreatic carcinomas. The results showed a significant inhibition of tumor growth (p<0.0001) with a log of tumor-cell-kill of 2.34. Infusion of a non-specific but class identical mAb had no effect. Taken together, these results support the notion that the PaCa-Ag1 is a potentially valuable early pancreatic cancer-specific marker that together with mAb3C4 will allow the introduction of a novel diagnostic tool(s) and therapeutic approach(s) for this disease.
