Neuroserpin gene therapy inhibits retinal ganglion cell apoptosis and promotes functional preservation in glaucoma.

Our research has proven that the inhibitory activity of the serine protease inhibitor neuroserpin (NS) is impaired because of its oxidation deactivation in glaucoma. Using genetic NS knockout (NS-/-) and NS overexpression (NS+/+ Tg) animal models and antibody-based neutralization approaches, we demonstrate that NS loss is detrimental to retinal structure and function. NS ablation was associated with perturbations in autophagy and microglial and synaptic markers, leading to significantly enhanced IBA1, PSD95, beclin-1, and LC3-II/LC3-I ratio and reduced phosphorylated neurofilament heavy chain (pNFH) levels. On the other hand, NS upregulation promoted retinal ganglion cell (RGC) survival in wild-type and NS-/- glaucomatous mice and increased pNFH expression. NS+/+Tg mice demonstrated decreased PSD95, beclin-1, LC3-II/LC3-I ratio, and IBA1 following glaucoma induction, highlighting its protective role. We generated a novel reactive site NS variant (M363R-NS) resistant to oxidative deactivation. Intravitreal administration of M363R-NS was observed to rescue the RGC degenerative phenotype in NS-/- mice. These findings demonstrate that NS dysfunction plays a key role in the glaucoma inner retinal degenerative phenotype and that modulating NS imparts significant protection to the retina. NS upregulation protected RGC function and restored biochemical networks associated with autophagy and microglial and synaptic function in glaucoma.

Neuroserpin, a crucial regulator for axogenesis, synaptic modelling and cell-cell interactions in the pathophysiology of neurological disease.

Neuroserpin is an axonally secreted serpin that is involved in regulating plasminogen and its enzyme activators, such as tissue plasminogen activator (tPA). The protein has been increasingly shown to play key roles in neuronal development, plasticity, maturation and synaptic refinement. The proteinase inhibitor may function both independently and through tPA-dependent mechanisms. Herein, we discuss the recent evidence regarding the role of neuroserpin in healthy and diseased conditions and highlight the participation of the serpin in various cellular signalling pathways. Several polymorphisms and mutations have also been identified in the protein that may affect the serpin conformation, leading to polymer formation and its intracellular accumulation. The current understanding of the involvement of neuroserpin in Alzheimer's disease, cancer, glaucoma, stroke, neuropsychiatric disorders and familial encephalopathy with neuroserpin inclusion bodies (FENIB) is presented. To truly understand the detrimental consequences of neuroserpin dysfunction and the effective therapeutic targeting of this molecule in pathological conditions, a cross-disciplinary understanding of neuroserpin alterations and its cellular signaling networks is essential.

Worldwide Effects of Coronavirus Disease Pandemic on Tuberculosis Services, January-April 2020.

Coronavirus disease has disrupted tuberculosis services globally. Data from 33 centers in 16 countries on 5 continents showed that attendance at tuberculosis centers was lower during the first 4 months of the pandemic in 2020 than for the same period in 2019. Resources are needed to ensure tuberculosis care continuity during the pandemic.

Proteomics Reveals Cell-Surface Urokinase Plasminogen Activator Receptor Expression Impacts Most Hallmarks of Cancer.

While metastasis is the primary cause of colorectal cancer (CRC) mortality, the molecular mechanisms underpinning it remains elusive. Metastasis is propagated through driver oncogene/suppressor gene mutations, accompanied by passenger mutations and underlying genomic instability. To understand cancer biology, a unifying framework called the "hallmarks of cancer" (HoCs) has been developed, which organizes cell biological alterations under ten key hallmarks. Underlying these HoCs, genome instability generates mutational diversity that is amplified by inflammation. Recognizing how critical cancer cell-surface proteins influence, these HoCs have been proposed to accelerate precision medicine therapeutic development. A moderate decrease (43%↓) in HCT116 cell surface urokinase plasminogen activator receptor (uPAR) expression mitigates against many HoCs driven by these cell's KRAS and PIK3CA mutational signature. Comprehensive proteomics (whole cell lysis with two membrane protein enrichments) coupled with ingenuity pathway analysis (IPA) demonstrates that uPAR negates essential pathways across the HoC spectrum, particularly those associated with metastasis, resisting cell death, and sustaining proliferation, and parallels Cancer Hallmarks Analytics Tool analysis. Decreasing uPAR predominantly alters metastasis-related and uPAR-interactome protein expression (e.g., EGFR, caveolin, vitronectin, integrin ß4). Collectively, it is demonstrated that uPAR is a lynchpin protein capable of regulating several HoC pathways in a classical CRC mutational background.

In Silico Peptide Repertoire of Human Olfactory Receptor Proteomes on High-Stringency Mass Spectrometry.

Human olfactory receptors (ORs) are seven-pass transmembrane G-protein coupled receptors (GPCR) involved in smell perception and many other signaling pathways. They are primarily expressed in the olfactory epithelium and ectopically expressed in several other organs and tissues. neXtProt contains 4 PE1 (protein existence 1, evidenced at the protein level) ORs, determined on the basis of either protein interaction data (i.e., OR1D4 and OR2AG1) or convincing genetic, haplotype, or biochemical data (i.e., OR1D2 and OR2J3). Not a single OR currently qualifies for neXtProt PE1 status based on mass spectrometry (MS) evidence. Many reasons for this absence of MS-based identification have been proposed, including (i) confined or spatiotemporal or developmental expression, (ii) low copy number, (iii) OR repertoire gene silencing, and (iv) limited tissue availability. OR transmembrane domains (TMDs) inherently limit MS identification because the hydrophobic nature restricts the access of trypsin to potential cleavage sites. Equally, the extremely low frequency or lack of accessible arginine and lysine residues in TMDs renders trypsin cleavage ineffective. Here, we demonstrate an analytical approach specifically focused on the hydrophilic (trypsin-accessible) domains of ORs [i.e., with all transmembrane segments and anchored peptides excluded). We predicted the ability of OR soluble (hydrophilic) domains to yield 2 or more >9 amino acids (aa) length unique mapping (unique to a protein only), non-nested (peptides with varying length at the N or C terminal but containing the same core sequence), leucine/isoleucine (I/L) switch examined (I and L have same mass and cannot be distinguished by MS) tryptic peptides. Our analysis showed that ∼58% of the human OR proteome could potentially generate tryptic peptides that satisfy current the Human Proteome Project data interpretation guidelines (version 2.1) when no missed cleavages are allowed and increases to ∼78% when one missed cleavage is allowed. The utilization of current biological data (adjuvant genomics, expression profile, transcriptomics, epigenome silencing data, etc.) and the adoption of a non-conventional proteomics approach (e.g., Confetti multiprotease digestion, CNBr cleavage of TMDs, and more-extreme chromatographic and MS methods) could aid in the detection of the remaining ORs.

Potential early clinical stage colorectal cancer diagnosis using a proteomics blood test panel.

BACKGROUND: One of the most significant challenges in colorectal cancer (CRC) management is the use of compliant early stage population-based diagnostic tests as adjuncts to confirmatory colonoscopy. Despite the near curative nature of early clinical stage surgical resection, mortality remains unacceptably high-as the majority of patients diagnosed by faecal haemoglobin followed by colonoscopy occur at latter stages. Additionally, current population-based screens reliant on fecal occult blood test (FOBT) have low compliance (~ 40%) and tests suffer low sensitivities. Therefore, blood-based diagnostic tests offer survival benefits from their higher compliance (≥ 97%), if they can at least match the sensitivity and specificity of FOBTs. However, discovery of low abundance plasma biomarkers is difficult due to occupancy of a high percentage of proteomic discovery space by many high abundance plasma proteins (e.g., human serum albumin). METHODS: A combination of high abundance protein ultradepletion (e.g., MARS-14 and an in-house IgY depletion columns) strategies, extensive peptide fractionation methods (SCX, SAX, High pH and SEC) and SWATH-MS were utilized to uncover protein biomarkers from a cohort of 100 plasma samples (i.e., pools of 20 healthy and 20 stages I-IV CRC plasmas). The differentially expressed proteins were analyzed using ANOVA and pairwise t-tests (p < 0.05; fold-change > 1.5), and further examined with a neural network classification method using in silico augmented 5000 patient datasets. RESULTS: Ultradepletion combined with peptide fractionation allowed for the identification of a total of 513 plasma proteins, 8 of which had not been previously reported in human plasma (based on PeptideAtlas database). SWATH-MS analysis revealed 37 protein biomarker candidates that exhibited differential expression across CRC stages compared to healthy controls. Of those, 7 candidates (CST3, GPX3, CFD, MRC1, COMP, PON1 and ADAMDEC1) were validated using Western blotting and/or ELISA. The neural network classification narrowed down candidate biomarkers to 5 proteins (SAA2, APCS, APOA4, F2 and AMBP) that had maintained accuracy which could discern early (I/II) from late (III/IV) stage CRC. CONCLUSION: MS-based proteomics in combination with ultradepletion strategies have an immense potential of identifying diagnostic protein biosignature.

Multipronged quantitative proteomic analyses indicate modulation of various signal transduction pathways in human meningiomas.

Meningiomas (MGs) are frequent tumors of the CNS originating from the meningeal layers of the spinal cord and the brain. In this study, comparative tissue proteomic analysis of low and high grades of MGs was performed by using iTRAQ-based quantitative proteomics in combination with ESI-quadrupole-TOF and Q-Exactive MS, and results were validated by employing ELISA. Combining the results obtained from two MS platforms, we were able to identify overall 4308 proteins (1% false discover rate), among which 2367 exhibited differential expression (more than and equal to 2 peptide and ≥ 1.5-fold in at least one grade) in MGs. Several differentially expressed proteins were found to be associated with diverse signaling pathways, including integrin, Wnt, Ras, epidermal growth factor receptor, and FGR signaling. Proteins, such as vinculin or histones, which act as the signaling activators to initiate multiple signaling pathways, were found to be upregulated in MGs. Quite a few candidates, such as protein S-100A6, aldehyde dehydrogenase mitochondrial, AHNAK, cytoskeleton-associated protein 4, and caveolin, showed sequential increase in low- and high-grade MGs, whereas differential expressions of collagen alpha-1 (VI), protein S100-A9, 14 kDa phosphohistidine phosphatase, or transgelin-2 were found to be grade specific. Our findings provide new insights regarding the association of various signal transduction pathways in MG pathogenesis and may introduce new opportunities for the early detection and prognosis of MGs.

Computational refinement identifies functional destructive single nucleotide polymorphisms associated with human retinoid X receptor gene.

Alterations in the nuclear retinoid X receptor (RXRs) signalling have been implicated in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis and glaucoma. Single nucleotide polymorphisms (SNPs) are the main cause underlying single nucleic acid variations which in turn determine heterogeneity within various populations. These genetic polymorphisms have been suggested to associate with various degenerative disorders in population-wide analysis. This bioinformatics study was designed to investigate, search, retrieve and identify deleterious SNPs which may affect the structure and function of various RXR isoforms through a computational and molecular modelling approach. Amongst the 1,813 retrieved SNPs several were found to be deleterious with rs140464195_G139R, rs368400425_R358W and rs368586400_L383F RXRα mutant variants being the most detrimental ones causing changes in the interatomic interactions and decreasing the flexibility of the mutant proteins. Molecular genetics analysis identified seven missense mutations in RXRα/ß/γ isoforms. Two novel mutations SNP IDs (rs1588299621 and rs1057519958) were identified in RXRα isoform. We used several in silico prediction tools such as SIFT, PolyPhen, I-Mutant, Protein Variation Effect Analyzer (PROVEAN), PANTHER, SNP&Go, PhD-SNP and SNPeffect to predict pathogenicity and protein stability associated with RXR mutations. The structural assessment by DynaMut tool revealed that hydrogen bonds were affected along with hydrophobic and carbonyl interactions resulting in reduced flexibility at the mutated residue positions but ultimately stabilizing the molecule as a whole. Summarizing, analysis of the missense mutations in RXR isoforms showed a mix of conclusive and inconclusive genotype-phenotype correlations suggesting the use of sophisticated computational analysis tools for studying RXR variants.Communicated by Ramaswamy H. Sarma.

Revisiting the therapeutic potential of gingerols against different pharmacological activities.

The rhizomes of ginger have been in use in many forms of traditional and alternative medicines. Besides being employed as condiment and flavoring agent, it is used in the treatment of nausea, osteoarthritis, muscle pain, menstrual pain, chronic indigestion, Alzheimer's disease, and cancer. Ginger rhizome contains volatile oils, phenolic compounds and resins, and characterization studies showed that [6]-gingerol, [6]-shogaol, and [6]-paradol are reported to be the pharmacologically active components. Gingerol is a major chemical constituent found as volatile oil in the rhizomes of ginger. It has several medicinal benefits and used for the treatment of rheumatoid arthritis, nausea, cancer, and diabetes. Many studies have been carried out in various parts of the world to isolate and standardize gingerol for their use as a complementary medicine. The present review summarizes wide range of research studies on gingerol and its pharmacological roles in various metabolic diseases.

Siponimod exerts neuroprotective effects on the retina and higher visual pathway through neuronal S1PR1 in experimental glaucoma.

Sphingosine-1-phosphate receptor (S1PR) signaling regulates diverse pathophysiological processes in the central nervous system. The role of S1PR signaling in neurodegenerative conditions is still largely unidentified. Siponimod is a specific modulator of S1P1 and S1P5 receptors, an immunosuppressant drug for managing secondary progressive multiple sclerosis. We investigated its neuroprotective properties in vivo on the retina and the brain in an optic nerve injury model induced by a chronic increase in intraocular pressure or acute N-methyl-D-aspartate excitotoxicity. Neuronal-specific deletion of sphingosine-1-phosphate receptor (S1PR1) was carried out by expressing AAV-PHP.eB-Cre recombinase under Syn1 promoter in S1PR1flox/flox mice to define the role of S1PR1 in neurons. Inner retinal electrophysiological responses, along with histological and immunofluorescence analysis of the retina and optic nerve tissues, indicated significant neuroprotective effects of siponimod when administered orally via diet in chronic and acute optic nerve injury models. Further, siponimod treatment showed significant protection against trans-neuronal degenerative changes in the higher visual center of the brain induced by optic nerve injury. Siponimod treatment also reduced microglial activation and reactive gliosis along the visual pathway. Our results showed that siponimod markedly upregulated neuroprotective Akt and Erk1/2 activation in the retina and the brain. Neuronal-specific deletion of S1PR1 enhanced retinal and dorsolateral geniculate nucleus degenerative changes in a chronic optic nerve injury condition and attenuated protective effects of siponimod. In summary, our data demonstrated that S1PR1 signaling plays a vital role in the retinal ganglion cell and dorsolateral geniculate nucleus neuronal survival in experimental glaucoma, and siponimod exerts direct neuroprotective effects through S1PR1 in neurons in the central nervous system independent of its peripheral immuno-modulatory effects. Our findings suggest that neuronal S1PR1 is a neuroprotective therapeutic target and its modulation by siponimod has positive implications in glaucoma conditions.

Retinoid X Receptor: Cellular and Biochemical Roles of Nuclear Receptor with a Focus on Neuropathological Involvement.

Retinoid X receptors (RXRs) present a subgroup of the nuclear receptor superfamily with particularly high evolutionary conservation of ligand binding domain. The receptor exists in α, ß, and γ isotypes that form homo-/heterodimeric complexes with other permissive and non-permissive receptors. While research has identified the biochemical roles of several nuclear receptor family members, the roles of RXRs in various neurological disorders remain relatively under-investigated. RXR acts as ligand-regulated transcription factor, modulating the expression of genes that plays a critical role in mediating several developmental, metabolic, and biochemical processes. Cumulative evidence indicates that abnormal RXR signalling affects neuronal stress and neuroinflammatory networks in several neuropathological conditions. Protective effects of targeting RXRs through pharmacological ligands have been established in various cell and animal models of neuronal injury including Alzheimer disease, Parkinson disease, glaucoma, multiple sclerosis, and stroke. This review summarises the existing knowledge about the roles of RXR, its interacting partners, and ligands in CNS disorders. Future research will determine the importance of structural and functional heterogeneity amongst various RXR isotypes as well as elucidate functional links between RXR homo- or heterodimers and specific physiological conditions to increase drug targeting efficiency in pathological conditions.

Trans-synaptic degeneration in the visual pathway: Neural connectivity, pathophysiology, and clinical implications in neurodegenerative disorders.

There is a strong interrelationship between eye and brain diseases. It has been shown that neurodegenerative changes can spread bidirectionally in the visual pathway along neuronal projections. For example, damage to retinal ganglion cells in the retina leads to degeneration of the visual cortex (anterograde degeneration) and vice versa (retrograde degeneration). The underlying mechanisms of this process, known as trans-synaptic degeneration (TSD), are unknown, but TSD contributes to the progression of numerous neurodegenerative disorders, leading to clinical and functional deterioration. The hierarchical structure of the visual system comprises of a strong topographic connectivity between the retina and the visual cortex and therefore serves as an ideal model to study the cellular effect, clinical manifestations, and deterioration extent of TSD. With this review we provide comprehensive information about the neural connectivity, synapse function, molecular changes, and pathophysiology of TSD in visual pathways. We then discuss its bidirectional nature and clinical implications in neurodegenerative diseases. A thorough understanding of TSD in the visual pathway can provide insights into progression of neurodegenerative disorders and its potential as a therapeutic target.

Neuroprotective Effects of Neuropeptide Y on Human Neuroblastoma SH-SY5Y Cells in Glutamate Excitotoxicity and ER Stress Conditions.

Neuropeptide Y (NPY), a sympathetic neurotransmitter, is involved in various physiological functions, and its dysregulation is implicated in several neurodegenerative diseases. Glutamate excitotoxicity, endoplasmic reticulum (ER) stress, and oxidative stress are the common mechanisms associated with numerous neurodegenerative illnesses. The present study aimed to elucidate the protective effects of NPY against glutamate toxicity and tunicamycin-induced ER stress in the human neuroblastoma SH-SY5Y cell line. We exposed the SH-SY5Y cells to glutamate and tunicamycin for two different time points and analyzed the protective effects of NPY at different concentrations. The protective effects of NPY treatments were assessed by cell viability assay, and the signalling pathway changes were evaluated by biochemical techniques such as Western blotting and immunofluorescence assays. Our results showed that treatment of SH-SY5Y cells with NPY significantly increased the viability of the cells in both glutamate toxicity and ER stress conditions. NPY treatments significantly attenuated the glutamate-induced pro-apoptotic activation of ERK1/2 and JNK/BAD pathways. The protective effects of NPY were further evident against tunicamycin-induced ER stress. NPY treatments significantly suppressed the ER stress activation by downregulating BiP, phospho-eIF2α, and CHOP expression. In addition, NPY alleviated the Akt/FoxO3a pathway in acute oxidative conditions caused by glutamate and tunicamycin in SH-SY5Y cells. Our results demonstrated that NPY is neuroprotective against glutamate-induced cell toxicity and tunicamycin-induced ER stress through anti-apoptotic actions.

Increased Moxifloxacin Dosing Among Patients With Multidrug-Resistant Tuberculosis With Low-Level Resistance to Moxifloxacin Did Not Improve Treatment Outcomes in a Tertiary Care Center in Mumbai, India.

BACKGROUND: Mycobacterium tuberculosis (Mtb) strains resistant to isoniazid and rifampin (multidrug-resistant tuberculosis [MDR-TB]) are increasingly reported worldwide, requiring renewed focus on the nuances of drug resistance. Patients with low-level moxifloxacin resistance may benefit from higher doses, but limited clinical data on this strategy are available. METHODS: We conducted a 5-year observational cohort study of MDR-TB patients at a tertiary care center in India. Participants with Mtb isolates resistant to isoniazid, rifampin, and moxifloxacin (at the 0.5 µg/mL threshold) were analyzed according to receipt of high-dose moxifloxacin (600 mg daily) as part of a susceptibility-guided treatment regimen. Univariable and multivariable Cox proportional hazard models assessed the relationship between high-dose moxifloxacin and unfavorable treatment outcomes. RESULTS: Of 354 participants with MDR-TB resistant to moxifloxacin, 291 (82.2%) received high-dose moxifloxacin. The majority experienced good treatment outcomes (200 [56.5%]), which was similar between groups (56.7% vs 54.0%, P = .74). Unfavorable outcomes were associated with greater extent of radiographic disease, lower initial body mass index, and concurrent treatment with fewer drugs with confirmed phenotypic susceptibility. Treatment with high-dose moxifloxacin was not associated with improved outcomes in either unadjusted (hazard ratio [HR], 1.2 [95% confidence interval {CI}, .6-2.4]) or adjusted (HR, 0.8 [95% CI, .5-1.4]) models but was associated with joint pain (HR, 3.2 [95% CI, 1.2-8.8]). CONCLUSIONS: In a large observational cohort, adding high-dose (600 mg) moxifloxacin to a drug susceptibility test-based treatment regimen for MDR-TB was associated with increased treatment-associated side effects without improving overall outcomes and should be avoided for empiric treatment of moxifloxacin-resistant MDR-TB.

Antibiotic procurement and ABC analysis for a comprehensive primary health care clinic in the Eastern Cape province, South Africa.

BACKGROUND: Antimicrobial resistance (AMR), a major threat to global public health, can be addressed using a managed care approach. This includes timely analysis of antibiotic consumption and procurement data to drive evidence-based policies and practices in healthcare facilities. 'ABC analysis' presents an opportunity for this. METHODS: ABC analysis data for a comprehensive Primary Health Care (PHC) clinic in the Eastern Cape province of South Africa was obtained from the Provincial Department of Health for 01 April 2015 to 31 March 2018. Procured antibiotics were analysed on the quantities purchased, total cost, route of administration and spectrum of activity. Antibiotic categorization was also carried out according to the World Health Organization Model List of Essential Medicines (WHO EML) 2017. RESULTS: Antibiotics made up approximately 7% of the total annual pharmaceutical expenditure. A total of 31, 35 and 34 antibiotics were procured in the first, second and third years, respectively. The most procured antibiotics were: (1) isoniazid, (2) flucloxacillin, (3) azithromycin, (4) a combination of rifampicin, isoniazid, pyrazinamide, and ethambutol and (5) amoxicillin. Overall, 55%, 2% and 15% of antibiotics accounted for the 'Access', 'Watch' and 'Access and Watch' categories, respectively, of the WHO EML. No 'Reserve' antibiotics were procured. The remaining 28% were antituberculosis medicines. Altogether, 89%, 8% and 3% of the antibiotics were respectively administered orally, systemically, and topically. A total of 58% were broad-spectrum and 42% were narrow-spectrum antibiotics. CONCLUSION: Oral antibiotics in the 'Access' category presented favourable usage of antibiotics. Decreasing the use of broad-spectrum antibiotics requires consideration.

Retinoid x receptor modulation protects against ER stress response and rescues glaucoma phenotypes in adult mice.

Retinoid X receptors (RXRs) play an important role in transcription, are involved in numerous cellular networks from cell proliferation to lipid metabolism and are essential for normal eye development. RXRs form homo or heterodimers with other nuclear receptors, bind to DNA response elements and regulate several biological processes including neurogenesis. Mounting evidence suggests that RXR activation by selective RXR modulators (sRXRms) may be neuroprotective in the central nervous system. However, their potential neuroprotective role in the retina and specifically in glaucoma remains unexplored. This study investigated changes in RXR expression in the human and mouse retina under glaucomatous stress conditions and investigated the effect of RXR modulation on the RGCs using pharmacological approaches. RXR protein levels in retina were downregulated in both human glaucoma and experimental RGC injury models while RXR agonist, bexarotene treatment resulted in upregulation of RXR expression particularly in the inner retinal layers. Retinal electrophysiological recordings and histological analysis indicated that inner retinal function and retinal laminar structure were preserved upon treatment with bexarotene. These protective effects were associated with downregulation of ER stress marker response upon bexarotene treatment under glaucoma conditions. Overall, retinal RXR modulation by bexarotene significantly protected RGCs in vivo in both acute and chronic glaucoma models.