Multiple Degenerative Diseases Research Articles

Hallmarks of aging in age-related macular degeneration and age-related neurological disorders: novel insights into common mechanisms and clinical relevance.

Age-related macular degeneration (AMD) and age-related neurological diseases (ANDs), such as Alzheimer's and Parkinson's Diseases, are increasingly prevalent conditions that significantly contribute to global morbidity, disability, and mortality. The retina, as an accessible part of the central nervous system (CNS), provides a unique window to study brain aging and neurodegeneration. By examining the associations between AMD and ANDs, this review aims to highlight novel insights into fundamental mechanisms of aging and their role in neurodegenerative disease progression. This review integrates knowledge from the emerging field of aging research, which identifies common denominators of biological aging, specifically loss of proteostasis, impaired macroautophagy, mitochondrial dysfunction, and inflammation. Finally, we emphasize the clinical relevance of these pathways and the potential for cross-disease therapies that target common aging hallmarks. Identifying these shared pathways could open avenues to develop therapeutic strategies targeting mechanisms common to multiple degenerative diseases, potentially attenuating disease progression and promoting the healthspan.

Prediction of Multiple Degenerative Diseases Based on DNA Methylation in a Co-Physiology Mechanisms Perspective.

Degenerative diseases oftentimes occur within the continuous process of aging, and the corresponding clinical manifestations may be neurodegeneration, neoplastic diseases, or various human complex diseases. DNA methylation provides the opportunity to explore aging and degenerative diseases as epigenetic traits. It has already been applied to age prediction and disease diagnosis. It has been shown that various degenerative diseases share co-physiology mechanisms with each other, clues of which may be gained from studying the aging process. Here, we endeavor to predict the risk of degenerative diseases in an aging-relevant comorbid mechanism perspective. Firstly, an epigenetic clock method was implemented based on a multi-scale convolutional neural network, and a Shapley feature attribution analysis was applied to discover the aging-related CpG sites. Then, these sites were further screened to a smaller subset composed of 196 sites by using biomics analysis according to their biological functions and mechanisms. Finally, we constructed a multilayer perceptron (MLP)-based degenerative disease risk prediction model, Mlp-DDR, which was well trained and tested to accurately classify nine degenerative diseases. Recent studies also suggest that DNA methylation plays a significant role in conditions like osteoporosis and osteoarthritis, broadening the potential applications of our model. This approach significantly advances the ability to understand degenerative diseases and represents a substantial shift from traditional diagnostic methods. Despite the promising results, limitations regarding model complexity and dataset diversity suggest directions for future research, including the development of tissue-specific epigenetic clocks and the inclusion of a wider range of diseases.

Toward understanding the role of genomic repeat elements in neurodegenerative diseases.

Neurodegenerative diseases cause great medical and economic burdens for both patients and society; however, the complex molecular mechanisms thereof are not yet well understood. With the development of high-coverage sequencing technology, researchers have started to notice that genomic repeat regions, previously neglected in search of disease culprits, are active contributors to multiple neurodegenerative diseases. In this review, we describe the association between repeat element variants and multiple degenerative diseases through genome-wide association studies and targeted sequencing. We discuss the identification of disease-relevant repeat element variants, further powered by the advancement of long-read sequencing technologies and their related tools, and summarize recent findings in the molecular mechanisms of repeat element variants in brain degeneration, such as those causing transcriptional silencing or RNA-mediated gain of toxic function. Furthermore, we describe how in silico predictions using innovative computational models, such as deep learning language models, could enhance and accelerate our understanding of the functional impact of repeat element variants. Finally, we discuss future directions to advance current findings for a better understanding of neurodegenerative diseases and the clinical applications of genomic repeat elements.

Ferroptosis biomarkers predict tumor mutation burden's impact on prognosis in HER2-positive breast cancer.

Ferroptosis has recently been associated with multiple degenerative diseases. Ferroptosis induction in cancer cells is a feasible method for treating neoplastic diseases. However, the association of iron proliferation-related genes with prognosis in HER2+ breast cancer (BC) patients is unclear. To identify and evaluate fresh ferroptosis-related biomarkers for HER2+ BC. First, we obtained the mRNA expression profiles and clinical information of HER2+ BC patients from the TCGA and METABRIC public databases. A four-gene prediction model comprising PROM2, SLC7A11, FANCD2, and FH was subsequently developed in the TCGA cohort and confirmed in the METABRIC cohort. Patients were stratified into high-risk and low-risk groups based on their median risk score, an independent predictor of overall survival (OS). Based on these findings, immune infiltration, mutations, and medication sensitivity were analyzed in various risk groupings. Additionally, we assessed patient prognosis by combining the tumor mutation burden (TMB) with risk score. Finally, we evaluated the expression of critical genes by analyzing single-cell RNA sequencing (scRNA-seq) data from malignant vs normal epithelial cells. We found that the higher the risk score was, the worse the prognosis was (P < 0.05). We also found that the immune cell infiltration, mutation, and drug sensitivity were different between the different risk groups. The high-risk subgroup was associated with lower immune scores and high TMB. Moreover, we found that the combination of the TMB and risk score could stratify patients into three groups with distinct prognoses. HRisk-HTMB patients had the worst prognosis, whereas LRisk-LTMB patients had the best prognosis (P < 0.0001). Analysis of the scRNA-seq data showed that PROM2, SLC7A11, and FANCD2 were significantly differentially expressed, whereas FH was not, suggesting that these genes are expressed mainly in cancer epithelial cells (P < 0.01). Our model helps guide the prognosis of HER2+ breast cancer patients, and its combination with the TMB can aid in more accurate assessment of patient prognosis and provide new ideas for further diagnosis and treatment.

The function of Bazhen decoction in rescuing progeroid cell senescence via facilitating G-quadruplex resolving and telomere elongation

Ethnopharmacological relevanceThe Bazhen decoction is one of the most extensively used Traditional Chinese medicine (TCM) prescriptions for treatment of aging related diseases. However, due to the complexity of the components, the pharmacological mechanism of Bazhen decoction is still limited. Aim of the studyIn this study, with the aim of helping the clinical precision medicine of TCM, we try out a systematic analysis for dissecting the molecular mechanism of complicated TCM prescription: Bazhen decoction. We identify the pharmacological mechanism of Bazhen decoction in telomere elongation as revealed by systematic analysis. Materials and methodsBy RNA sequencing and transcriptome analysis of Bazhen decoction treated wild type cells, we reveal the transcriptome profile induced by Bazhen decoction. We utilized the cells derived from Werner syndrome (WS) mice, which is known to be dysfunctional in telomere elongation due to the deficiency of DNA helicase Wrn. By Western blot, qPCR, Immunofluorescence, flow cytometry, telomere FISH, and SA-β-Gal staining, we verify the transcriptome data and confirm the pharmacological function of Bazhen decoction and its drug containing serum in telomere elongation and reversing progeroid cell senescence. ResultsWe reveal that Bazhen decoction may systematically regulate multiple anti-aging pathways, including stem cell regulation, protein homeostasis, cardiovascular function, neuronal function, anti-inflammation, anti-DNA damage induced stress, DNA helicase activity and telomere lengthening. We find that Bazhen decoction and its drug containing serum could up-regulate multiple DNA helicases and telomere regulating proteins. The increased DNA helicases promote the resolving of G-quadruplex (G4) structures, and facilitate DNA replication and telomere elongation. These improvements also endow the cellular resistance to DNA damages induced by replication stress, and rescue the WS caused cellular senescence. ConclusionsTogether these data suggest that Bazhen decoction up-regulate the expression of DNA helicases, thus facilitate G4 resolving and telomere maintenance, which rescue the progeroid cellular senescence and contribute to its anti-aging properties. Our data reveal a new molecular mechanism of Bazhen decoction in anti-aging related diseases via elongating telomere, this may shed light in the application of Bazhen decoction in multiple degenerative diseases caused by telomere erosion.

Mechanism of action and therapeutic effects of oxidative stress and stem cell-based materials in skin aging: Current evidence and future perspectives.

Aging is associated with multiple degenerative diseases, including atherosclerosis, osteoporosis, and Alzheimer's disease. As the most intuitive manifestation of aging, skin aging has received the most significant attention. Skin aging results from various intrinsic and extrinsic factors. Aged skin is characterized by wrinkles, laxity, elastosis, telangiectasia, and aberrant pigmentation. The underlying mechanism is complex and may involve cellular senescence, DNA damage, oxidative stress (OS), inflammation, and genetic mutations, among other factors. Among them, OS plays an important role in skin aging, and multiple antioxidants (e.g., vitamin C, glutathione, and melatonin) are considered to promote skin rejuvenation. In addition, stem cells that exhibit self-replication, multi-directional differentiation, and a strong paracrine function can exert anti-aging effects by inhibiting OS. With the further development of stem cell technology, treatments related to OS mitigation and involving stem cell use may have a promising future in anti-skin aging therapy.

Naringin Inhibits Apoptosis Induced by Cyclic Stretch in Rat Annular Cells and Partially Attenuates Disc Degeneration by Inhibiting the ROS/NF-κB Pathway.

Oxidative stress and apoptosis play important roles in the pathogenesis of various degenerative diseases. Previous studies have shown that naringin can exert therapeutic effects in multiple degenerative diseases by resisting oxidative stress and inhibiting apoptosis. Although naringin is effective in treating degenerative disc disease, the underlying mechanism remains unclear. This study is aimed at investigating the effects of naringin on oxidative stress, apoptosis, and intervertebral disc degeneration (IVDD) induced by cyclic stretch and the underlying mechanisms in vitro and in vivo. Abnormal cyclic stretch was applied to rat annulus fibrosus cells, which were then treated with naringin, to observe the effects of naringin on apoptosis, oxidative stress, mitochondrial function, and the nuclear factor- (NF-) κB signaling pathway. Subsequently, a rat model of IVDD induced by dynamic and static imbalance was established to evaluate the effects of naringin on the degree of degeneration (using imaging and histology), apoptosis, and oxidative stress in the serum and the intervertebral disc. Naringin inhibited the cyclic stretch-induced apoptosis of annulus fibrosus cells, reduced oxidative stress, improved mitochondrial function, enhanced the antioxidant capacity, and suppressed the activation of the NF-κB signaling pathway. Additionally, it reduced the degree of IVDD (evaluated using magnetic resonance imaging) and the level of oxidative stress and inhibited apoptosis and p-P65 expression in the intervertebral discs of rats. Thus, naringin can inhibit cyclic stretch-induced apoptosis and delay IVDD, and the underlying mechanism may be related to the inhibition of oxidative stress and activation of the NF-κB signaling pathway. Naringin may be an effective drug for treating degenerative disc disease.

Methylsulfonylmethane Increases the Alveolar Bone Density of Mandibles in Aging Female Mice.

Methylsulfonylmethane (MSM) is a naturally occurring anti-inflammatory compound that effectively treats multiple degenerative diseases such as osteoarthritis and acute pancreatitis. Our previous studies have demonstrated the ability of MSM to differentiate stem cells from human exfoliated deciduous (SHED) teeth into osteoblast-like cells. This study examined the systemic effect of MSM in 36-week-old aging C57BL/6 female mice in vivo by injecting MSM for 13 weeks. Serum analyses showed an increase in expression levels of bone formation markers [osteocalcin (OCN) and procollagen type 1 intact N-terminal propeptide (P1NP)] and a reduction in bone resorption markers [tartrate-resistant acid phosphatase (TRAP) and C-terminal telopeptide of type I collag (CTX-I)] in MSM-injected animals. Micro-computed tomographic images demonstrated an increase in trabecular bone density in mandibles. The trabecular bone density tended to be higher in the femur, although the increase was not significantly different between the MSM- and phosphate-buffered saline (PBS)-injected mice. In mandibles, an increase in bone density with a corresponding decrease in the marrow cavity was observed in the MSM-injected mice. Furthermore, immunohistochemical analyses of the mandibles for the osteoblast-specific marker – OCN, and the mesenchymal stem cell-specific marker – CD105 showed a significant increase and decrease in OCN and CD105 positive cells, respectively. Areas of bone loss were observed in the inter-radicular region of mandibles in control mice. However, this loss was considerably decreased due to stimulation of bone formation in response to MSM injection. In conclusion, our study has demonstrated the ability of MSM to induce osteoblast formation and function in vivo, resulting in increased bone formation in the mandible. Hence, the application of MSM and stem cells of interest may be the right combination in alveolar bone regeneration under periodontal or other related diseases that demonstrate bone loss.

Laxative activity of Ethanolic extract of Capparis moonii W. fruit.

The growing value as potential preventive interventions of biologically active medicinal plants and their constituents has been a topic of intense research. Plant and plant derivatives have long been a source of medication in human culture. According to world health organizations, 80% of the population of developing countries relies for their primary health treatment on conventional medicines. In the future, safe and effective drugs are likely to be manufactured from medicinal plants to heal multiple degenerative diseases. Constipation has been identified as one of the daily health issues which has recently been increased due to increased intake of processed foods or foods deficient in natural dietary fibres. The present research was carried out to evaluate the laxative effects of ethanolic extract of Capparis moonii wight fruit was safe for administration in mice at the highest dose (2000mg/kg) tested. The Capparis moonii wight’s laxative activity was studied in mice. The result showed that an oral administration of Capparis moonii wight fruit extract produced a significant and dose-dependent increase in stool count in mice, as well as a dosedependent effect through distance traveled by charcoal meal. These effects were comparable with dulcoflex (bisacodyl) (standard drug) at dose of 5mg/kg. From the result it can be concluded that oral administration of Capparis moonii wight ethanolic extract indicates significant laxative activity in mice It can be reported that flavonoid, saponin, glycoside present in the extract may be responsible for an laxative effect and Thus the plant extract can explain the usefulness of Capparis moonii in constipation.

Longitudinal metabolic alterations in plasma of rats exposed to low doses of high linear energy transfer radiation

Astronauts embarking on deep space missions are at high risk of long-term exposure to low doses of high linear energy transfer (LET) radiation, which can contribute to the development of cancer and multiple degenerative diseases. However, long term effects of exposure to low doses of high LET radiation in plasma metabolite profiles have not been elucidated. We utilized an untargeted metabolomics and lipidomics approach to analyze plasma obtained from adult male Long Evans rats to determine the longitudinal effects of low-dose proton and low-dose oxygen ion whole-body irradiation on metabolic pathways. Our findings reveal that radiation exposure induced modest changes in the metabolic profiles in plasma, 7 months after exposure. Furthermore, we identified some common metabolite dysregulations between protons and oxygen ions, which may indicate a similar mechanism of action for both radiation types.

Exome-wide age-of-onset analysis reveals exonic variants in ERN1 and SPPL2C associated with Alzheimer\u2019s disease

Despite recent discoveries in genome-wide association studies (GWAS) of genomic variants associated with Alzheimer’s disease (AD), its underlying biological mechanisms are still elusive. The discovery of novel AD-associated genetic variants, particularly in coding regions and from APOEε4 non-carriers, is critical for understanding the pathology of AD. In this study, we carried out an exome-wide association analysis of age-of-onset of AD with ~20,000 subjects and placed more emphasis on APOEε4 non-carriers. Using Cox mixed-effects models, we find that age-of-onset shows a stronger genetic signal than AD case-control status, capturing many known variants with stronger significance, and also revealing new variants. We identified two novel variants, rs56201815, a rare synonymous variant in ERN1, and rs12373123, a common missense variant in SPPL2C in the MAPT region in APOEε4 non-carriers. Besides, a rare missense variant rs144292455 in TACR3 showed the consistent direction of effect sizes across all studies with a suggestive significant level. In an attempt to unravel their regulatory and biological functions, we found that the minor allele of rs56201815 was associated with lower average FDG uptake across five brain regions in ADNI. Our eQTL analyses based on 6198 gene expression samples from ROSMAP and GTEx revealed that the minor allele of rs56201815 was potentially associated with elevated expression of ERN1, a key gene triggering unfolded protein response (UPR), in multiple brain regions, including the posterior cingulate cortex and nucleus accumbens. Our cell-type-specific eQTL analysis using ~80,000 single nuclei in the prefrontal cortex revealed that the protective minor allele of rs12373123 significantly increased the expression of GRN in microglia, and was associated with MAPT expression in astrocytes. These findings provide novel evidence supporting the hypothesis of the potential involvement of the UPR to ER stress in the pathological pathway of AD, and also give more insights into underlying regulatory mechanisms behind the pleiotropic effects of rs12373123 in multiple degenerative diseases including AD and Parkinson’s disease.

Mutant three-repeat tau expression initiates retinal ganglion cell death through Caspase-2

The microtubule-associated protein tau is implicated in multiple degenerative diseases including retinal diseases such as glaucoma; however, the way tau initiates retinopathy is unclear. Previous retinal assessments in mouse models of tauopathy suggest that mutations in four-repeat (4R) tau are associated with disease-induced retinal dysfunction, while shifting tau isoform ratio to favor three-repeat (3R) tau production enhanced photoreceptor function. To further understand how alterations in tau expression impact the retina, we analyzed the retinas of transgenic mice overexpressing mutant 3R tau (m3R tau-Tg), a model known to exhibit Pick's Disease pathology in the brain. Analysis of retinal cross-sections from young (3 month) and adult (9 month) mice detected asymmetric 3R tau immunoreactivity in m3R tau-Tg retina, concentrated in the retinal ganglion and amacrine cells of the dorsal retinal periphery. Accumulation of hyperphosphorylated tau was detected specifically in the detergent insoluble fraction of the adult m3R tau-Tg retina. RNA-seq analysis highlighted biological pathways associated with tauopathy that were uniquely altered in m3R tau-Tg retina. The upregulation of transcript encoding apoptotic protease caspase-2 coincided with increased immunostaining in predominantly 3R tau positive retinal regions. In adult m3R tau-Tg, the dorsal peripheral retina of the adult m3R tau-Tg exhibited decreased cell density in the ganglion cell layer (GCL) and reduced thickness of the inner plexiform layer (IPL) compared to the ventral peripheral retina. Together, these data indicate that mutant 3R tau may mediate toxicity in retinal ganglion cells (RGC) by promoting caspase-2 expression which results in RGC degeneration. The m3R tau-Tg line has the potential to be used to assess tau-mediated RGC degeneration and test novel therapeutics for degenerative diseases such as glaucoma.

Profiling the ‘deamidome’ of complex biosamples using mixed-mode chromatography-coupled tandem mass spectrometry

Deamidation is a spontaneous degenerative protein modification (DPM) that disrupts the structure and function of both endogenous proteins and various therapeutic agents. While deamidation has long been recognized as a critical event in human aging and multiple degenerative diseases, research progress in this field has been restricted by the technical challenges associated with studying this DPM in complex biological samples. Asparagine (Asn) deamidation generates L-aspartic acid (L-Asp), D-aspartic acid (D-Asp), L-isoaspartic acid (L-isoAsp) or D-isoaspartic acid (D-isoAsp) residues at the same position of Asn in the affected protein, but each of these amino acids displays similar hydrophobicity and cannot be effectively separated by reverse phase liquid chromatography. The Asp and isoAsp isoforms are also difficult to resolve using mass spectrometry since they have the same mass and fragmentation pattern in MS/MS. Moreover, the 13C peaks of the amidated peptide are often misassigned as monoisotopic peaks of the corresponding deamidated peptides in protein database searches. Furthermore, typical protein isolation and proteomic sample preparation methods induce artificial deamidation that cannot be distinguished from the physiological forms. To better understand the role of deamidation in biological aging and degenerative pathologies, new technologies are now being developed to address these analytical challenges, including mixed mode electrostatic-interaction modified hydrophilic interaction liquid chromatography (emHILIC). When coupled to high resolution, high accuracy tandem mass spectrometry this technology enables unprecedented, proteome-wide study of the 'deamidome' of complex samples. The current article therefore reviews recent advances in sample preparation methods, emHILIC-MS/MS technology, and MS instrumentation / data processing approaches to achieving accurate and reliable characterization of protein deamidation in complex biological and clinical samples.

Structure of the PUB Domain from Ubiquitin Regulatory X Domain Protein 1 (UBXD1) and Its Interaction with the p97 AAA+ ATPase.

AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing

MIST1, an Inductive Signal for Salivary Amylase in Mesenchymal Stem Cells.

Sjögren’s syndrome (SjS) is an autoimmune disease that destroys the salivary glands and results in severe dry mouth. Mesenchymal stem cell (MSC) transplantation has been recently proposed as a promising therapy for restoring cells in multiple degenerative diseases. We have recently utilized advanced proteomics biochemical assays to identify the key molecules involved in the mesenchymal-epithelial transition (MET) of co-cultured mouse bone-marrow-derived MSCs mMSCs with primary salivary gland cells. Among the multiple transcription factors (TFs) that were differentially expressed, two major TFs were selected: muscle, intestine, and stomach expression-1 (MIST1) and transcription factor E2a (TCF3). These factors were assessed in the current study for their ability to drive the expression of acinar cell marker, alpha-salivary amylase 1 (AMY1), and ductal cell marker, cytokeratin19 (CK19), in vitro. Overexpression of MIST1-induced AMY1 expression while it had little effect on CK19 expression. In contrast, TCF3 induced neither of those cellular markers. Furthermore, we have identified that mMSCs express muscarinic-type 3 receptor (M3R) mainly in the cytoplasm and aquaporin 5 (AQP5) in the nucleus. While MIST1 did not alter M3R levels in mMSCs, a TCF3 overexpression downregulated M3R expressions in mMSCs. The mechanisms for such differential regulation of glandular markers by these TFs warrant further investigation.

Genetic Rescue Reverses Microglial Activation in Preclinical Models of Retinitis Pigmentosa.

Microglia cells (MGCs) play a key role in scavenging pathogens and phagocytosing cellular debris in the central nervous system and retina. Their activation, however, contributes to the progression of multiple degenerative diseases. Given the potential damage created by MGCs, it is important to better understand their mechanism of activation. Here, we explored the role of MGCs in the context of retinitis pigmentosa (RP) by using four independent preclinical mouse models. For therapeutic modeling, tamoxifen-inducible CreER was introduced to explore changes in MGCs when RP progression halted. The phenotypes of the MGCs were observed using live optical coherence tomography, live autofluorescence, and immunohistochemistry. We found that, regardless of genetic background, MGCs were activated in neurodegenerative conditions and migrated beyond the layers where they are typically found to the inner and outer segments, where degeneration was ongoing. Genetic rescue not only halted degeneration but also deactivated MGCs, regardless of whether the intervention occurred at the early, middle, or late stage of the disease. These findings suggest that halting long-term disease progression may be more successful by downregulating MGC activity while co-administering the therapeutic intervention.

Abstract 2191: Multiple imaging reporter labeled acid-degradable dextran nanopolymer as a COX-2 siRNA carrier for COX-2 specific downregulation

Abstract Cyclooxygenase-2 (COX-2) as a target has attracted interest for multiple degenerative diseases and cancers. COX-2 siRNA provides specific and effective down-regulation of COX-2. However, because artificial cationic polymers can induce proinflammatory signals that increase COX-2 expression, there are few if any reports about the use of artificial cationic polymers as siRNA carrier to downregulate COX-2 in cancer cells. Here we developed a method to efficiently synthesize a multiple imaging reporter labeled biodegradable dextran to use as an efficient cationic polymer carrier for COX-2 siRNA delivery. Amine function groups were conjugated to the dextran platform through acetal bonds. Acetal bonds were broken at acidic conditions that occur in cancer and endocytosis compartments. Rhodamine was labeled to the amine groups to detect degradation and removal of these groups from the cell, while the dextran scaffold was labeled with Cy5.5. The rapid cleavage and release of amine groups minimized the proinflammatory side effects of the positively charged amine groups.Colorimetric assay of Cy5.5 and rhodamine in different pH buffer indicated that the amine group was cleaved at pH 5.5 buffer but was stable in pH 7.4 buffer. Fluorescence imaging showed that the dextran siRNA nanoplex entered the cells through endocytosis that provided acidic conditions for the breaking of the amine group. For the first time, the intracellular breaking of acetal bonds was clearly visualized by multiple imaging reporters. Because the rapid cleavage and release of amine groups minimized the proinflammatory side-effects, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay indicated that this dextran COX-2 siRNA nanoplex could downregulate COX-2 expression efficiently. Prostaglandin E2 level decreased significantly with COX-2 siRNA/dextran treatment. In vivo imaging demonstrated that the COX-2 siRNA/dextran nanoplex accumulated in MDA-MB-231 tumors. QRT-PCR and western blot assays of COX-2 levels in tumor tissue demonstrated that this nanoplex significantly downregulated COX-2 expression in vivo efficiently and within 24h. This dextran nanopolymer can be used as a safe, reproducible, and biocompatible siRNA carrier to effectively reduce COX-2 expression in cancer cells and tumors. Citation Format: Zhihang Chen, Balaji Krishnamachary, Marie-France Penet, Zaver M. Bhujwalla. Multiple imaging reporter labeled acid-degradable dextran nanopolymer as a COX-2 siRNA carrier for COX-2 specific downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2191. doi:10.1158/1538-7445.AM2017-2191

Skin rejuvenation with non-invasive pulsed electric fields.

Degenerative skin diseases affect one third of individuals over the age of sixty. Current therapies use various physical and chemical methods to rejuvenate skin; but since the therapies affect many tissue components including cells and extracellular matrix, they may also induce significant side effects, such as scarring. Here we report on a new, non-invasive, non-thermal technique to rejuvenate skin with pulsed electric fields. The fields destroy cells while simultaneously completely preserving the extracellular matrix architecture and releasing multiple growth factors locally that induce new cells and tissue growth. We have identified the specific pulsed electric field parameters in rats that lead to prominent proliferation of the epidermis, formation of microvasculature, and secretion of new collagen at treated areas without scarring. Our results suggest that pulsed electric fields can improve skin function and thus can potentially serve as a novel non-invasive skin therapy for multiple degenerative skin diseases.

661. Assay Development and Qualification for Human Embryonic Stem Cell (hESC)-Derived Cardiomyocytes Intended for Clinical Studies

Human embryonic stem cell (hESC)-derived cellular products have been considered as promising candidates for cell replacement therapy for multiple degenerative diseases. At the Center of Biomedicine and Genetics (CBG), we have manufactured under cGMP numerous hESC-differentiated products including neuron stem cells (NSC), neuronal progenitor cells (NPC), retinal pigment epithelium (RPE) cells, dopaminergic neurons and cardiomyocytes intended for pre-clinical and clinical studies. Specifically, cardiomyocytes are produced through hESC differentiation procedure utilizing adherent and notably suspension culture systems. Prior to their release for clinical application, cardiomyocytes must be thoroughly tested for their identity and purity. We have developed a systematic approach to characterize hESC-derived cardiomyocytes using assays such as real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. The development and qualification of each assay involve the selection and banking of positive and negative controls, selection and banking of critical reagents, determination of assay conditions, qualification of assay, and generation of assay qualification report and standard operating procedure (SOP). We will also examine a number of critical factors including assay specificity, sensitivity, assay controls, and establishment of assay specifications.

Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening.

Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based shRNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, PFKFB4, drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.