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.

Retinal pathological features and proteome signatures of Alzheimer's disease.

Alzheimer's disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid ß-protein (Aß42) forms and novel intraneuronal Aß oligomers (AßOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aß uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aß42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aß pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aß42, far-peripheral AßOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.

An efficient one-pot synthesis and docking studies of bioactive new antiproliferative dispiro[oxindole/acenaphthylenone‒benzofuranone] pyrrolidine scaffolds.

A new and efficient method has been developed to synthesize dispiro[oxindole/acenaphthylenone-benzofuranone]pyrrolidine compounds. This is done by triggering the 1,3-dipolar cycloaddition reaction of azomethine ylides by reacting isatin/acenaphthoquinone with L-picolinic acid/L-proline/sarcosine/L-thioproline/tetrahydroisoquinolines, in a highly regioselective manner in an ionic liquid [DBU][Ac] with 4'-chloro-auron[2-(4-chlorobenzylidene)benzofuran-3(2H)-one]. Single-crystal X-ray diffraction data support the proposed structures of the new compounds. The heterocycles derived from amino acids such as L-picolinic acid, L-proline, and L-thioproline showed significant inhibitory effects against six human solid tumors, including lung, breast, cervix, colon, and others. These new structures were also tested in the active sites of the MDM2 receptor to further study their antiproliferative effects.

Thermal Adaptation Alters Response to Thermal Stress and Expression of Virulent Genes (eae, stx1, stx2, and hlyA) in Pathogenic Escherichia coli Isolated from Pork.

Escherichia coli encounter variety of environmental and processing stresses during their growth, survival, and infection. Herein, the thermotolerance behavior and transcription of virulent genes responsible for the pathogenicity in isolated strains of pathogenic E. coli were evaluated. Among 176 E. coli isolates, 4 isolates (2.27%) were confirmed to be pathogenic E. coli, out of which 2 isolates were positive for EHEC and 2 were positive for EPEC based on their virulence factors. Thermotolerance was induced under thermal adaptation at higher temperature, regardless of the pathotypes. Cells grown and adapted at 42 °C, exhibited highest transcription of genes associated with adhesion (eae), hemolysis (hlyA), and shiga toxin production (stx1). However, expression of these genes was downregulated in cells adapted at lower temperature of 4 °C and 25 °C compared to control. Further, transcription of stx2 was upregulated by 70% and 17% at 4 °C and 25 °C, respectively, while the transcription level was reduced by 44% relative to control at 42 °C. The findings indicate that expression of virulent genes in pathogenic E. coli at elevated temperature do not be depend on thermotolerance of the strain harboring these genes.

Retinal arterial Aß40 deposition is linked with tight junction loss and cerebral amyloid angiopathy in MCI and AD patients.

INTRODUCTION: Vascular amyloid beta (Aß) protein deposits were detected in retinas of mild cognitively impaired (MCI) and Alzheimer's disease (AD) patients. We tested the hypothesis that the retinal vascular tight junctions (TJs) were compromised and linked to disease status. METHODS: TJ components and Aß expression in capillaries and larger blood vessels were determined in post mortem retinas from 34 MCI or AD patients and 27 cognitively normal controls and correlated with neuropathology. RESULTS: Severe decreases in retinal vascular zonula occludens-1 (ZO-1) and claudin-5 correlating with abundant arteriolar Aß40 deposition were identified in MCI and AD patients. Retinal claudin-5 deficiency was closely associated with cerebral amyloid angiopathy, whereas ZO-1 defects correlated with cerebral pathology and cognitive deficits. DISCUSSION: We uncovered deficiencies in blood-retinal barrier markers for potential retinal imaging targets of AD screening and monitoring. Intense retinal arteriolar Aß40 deposition suggests a common pathogenic mechanism of failed Aß clearance via intramural periarterial drainage.

Disulfiram enhances meropenem activity against NDM- and IMP-producing carbapenem-resistant Acinetobacter baumannii infections.

OBJECTIVES: To evaluate the in vitro and in vivo efficacy of the FDA-approved drug disulfiram in combination with meropenem against MBL-expressing carbapenem-resistant Acinetobacter baumannii. METHODS: Chequerboard and antibiotic resistance reversal analysis were performed using 25 clinical isolates producing different MBLs. Three representative strains harbouring NDM, IMP or non-MBL genes were subjected to a time-kill assay to further evaluate this synergistic interaction. Dose-dependent inhibition by disulfiram was assessed to determine IC50 for NDM-1, IMP-7, VIM-2 and KPC-2. Further, to test the efficacy of meropenem monotherapy and meropenem in combination with disulfiram against NDM- and IMP-harbouring A. baumannii, an experimental model of systemic infection and pneumonia was developed using BALB/c female mice. RESULTS: Chequerboard and antibiotic reversal assay displayed a synergistic interaction against MBL-expressing A. baumannii strains with 4- to 32-fold reduction in MICs of meropenem. In time-kill analysis, meropenem and disulfiram exhibited synergy against NDM- and IMP-producing carbapenem-resistant A. baumannii (CRAb) isolates. In vitro dose-dependent inhibition analysis showed that disulfiram inhibits NDM-1 and IMP-7 with IC50 values of 1.5 ± 0.6 and 16.25 ± 1.6 µM, respectively, with slight or no inhibition of VIM-2 (<20%) and KPC-2. The combination performed better in the clearance of bacterial load from the liver and spleen of mice infected with IMP-expressing CRAb. In the pneumonia model, the combination significantly decreased the bacterial burden of NDM producers compared with monotherapy. CONCLUSIONS: These results strongly suggest that the combination of disulfiram and meropenem represents an effective treatment option for NDM- and IMP-associated CRAb infections.

Mouse model of Alzheimer's disease demonstrates differential effects of early disease pathology on various brain regions.

Different parts of the brain are affected distinctively in various stages of the Alzheimer's disease (AD) pathogenesis. Identifying the biochemical changes in specific brain regions is key to comprehend the neuropathological mechanisms in early pre-symptomatic phases of AD. Quantitative proteomics profiling of four distinct areas of the brain of young APP/PS1 mouse model of AD was performed followed by biochemical pathway enrichment analysis. Findings revealed fundamental compositional and functional shifts even in the early stages of the disease. This novel study highlights unique proteome and biochemical pathway alterations in specific regions of the brain that underlie the early stages of AD pathology and will provide a framework for future longitudinal studies. The proteomics data were deposited into the ProteomeXchange Consortium via PRIDE with the identifier PXD019192.

A Matter of Time: Does Gestational Age Affect the Duration of the Fetal Anatomic Survey?

OBJECTIVE: To assess the average duration of detailed fetal anatomic surveys in pregnancy in relation to gestational age (GA) and the maternal body mass index (BMI) to determine optimal timing of the examination. METHODS: This was a retrospective cohort study of gravidae presenting for detailed fetal anatomic examinations between January 1, 2010, and June 30, 2017. After excluding examinations expected to have longer duration (ie, multifetal, major fetal anomalies), there were a total of 6522 examinations performed between GAs of 18 weeks 0 days and 22 weeks 0 days. Women were grouped by BMI, and results were analyzed by logistic regression. RESULTS: Gravidae of normal weight (BMI, 18.5-24.9 kg/m2 ) had a decrease of 47.47 seconds of the examination time with each increasing week of gestation (P = .036). Overweight (BMI, 25-29.9 kg/m2 ) gravidae similarly had a decrease of 66.31 seconds of the examination time with each additional week of gestation (P = .017). Underweight (BMI, 8.5 kg/m2 ) and obese (BMI, ≥30 kg/m2 ) gravidae did not have differences in the examination time with increasing GA. Increases in suboptimal examinations were noted with an increasing BMI (P < .001). There was a decreased frequency of suboptimal examinations in obese gravidae with a BMI of 40 kg/m2 or higher with increasing GA (P = .037). CONCLUSIONS: The duration of detailed fetal anatomic examinations decreased with increasing GA in normal-weight and overweight gravidae but not in obese gravidae. Performing the anatomy scan earlier in class I and II obese gravidae (BMI, 30-40 kg/m2 ) may enable improved pregnancy management options without increasing the examination duration or likelihood of a suboptimal evaluation.

Retinal proteomics of experimental glaucoma model reveal intraocular pressure-induced mediators of neurodegenerative changes.

Current evidence suggests that exposure to chronically induced intraocular pressure (IOP) leads to neurodegenerative changes in the inner retina. This study aimed to determine retinal proteomic alterations in a rat model of glaucoma and compared findings with human retinal proteomics changes in glaucoma reported previously. We developed an experimental glaucoma rat model by subjecting the rats to increased IOP (9.3 ± 0.1 vs 20.8 ± 1.6 mm Hg) by weekly microbead injections into the eye (8 weeks). The retinal tissues were harvested from control and glaucomatous eyes and protein expression changes analysed using a multiplexed quantitative proteomics approach (TMT-MS3). Immunofluorescence was performed for selected protein markers for data validation. Our study identified 4304 proteins in the rat retinas. Out of these, 139 proteins were downregulated (≤0.83) while the expression of 109 proteins was upregulated (≥1.2-fold change) under glaucoma conditions (P ≤ .05). Computational analysis revealed reduced expression of proteins associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, cytoskeleton, and actin filament organisation, along with increased expression of proteins in coagulation cascade, apoptosis, oxidative stress, and RNA processing. Further functional network analysis highlighted the differential modulation of nuclear receptor signalling, cellular survival, protein synthesis, transport, and cellular assembly pathways. Alterations in crystallin family, glutathione metabolism, and mitochondrial dysfunction associated proteins shared similarities between the animal model of glaucoma and the human disease condition. In contrast, the activation of the classical complement pathway and upregulation of cholesterol transport proteins were exclusive to human glaucoma. These findings provide insights into the neurodegenerative mechanisms that are specifically affected in the retina in response to chronically elevated IOP.

Evolving geographic diversity in SARS-CoV2 and in silico analysis of replicating enzyme 3CLpro targeting repurposed drug candidates.

BACKGROUND: Severe acute respiratory syndrome (SARS) has been initiating pandemics since the beginning of the century. In December 2019, the world was hit again by a devastating SARS episode that has so far infected almost four million individuals worldwide, with over 200,000 fatalities having already occurred by mid-April 2020, and the infection rate continues to grow exponentially. SARS coronavirus 2 (SARS-CoV-2) is a single stranded RNA pathogen which is characterised by a high mutation rate. It is vital to explore the mutagenic capability of the viral genome that enables SARS-CoV-2 to rapidly jump from one host immunity to another and adapt to the genetic pool of local populations. METHODS: For this study, we analysed 2301 complete viral sequences reported from SARS-CoV-2 infected patients. SARS-CoV-2 host genomes were collected from The Global Initiative on Sharing All Influenza Data (GISAID) database containing 9 genomes from pangolin-CoV origin and 3 genomes from bat-CoV origin, Wuhan SARS-CoV2 reference genome was collected from GeneBank database. The Multiple sequence alignment tool, Clustal Omega was used for genomic sequence alignment. The viral replicating enzyme, 3-chymotrypsin-like cysteine protease (3CLpro) that plays a key role in its pathogenicity was used to assess its affinity with pharmacological inhibitors and repurposed drugs such as anti-viral flavones, biflavanoids, anti-malarial drugs and vitamin supplements. RESULTS: Our results demonstrate that bat-CoV shares > 96% similar identity, while pangolin-CoV shares 85.98% identity with Wuhan SARS-CoV-2 genome. This in-depth analysis has identified 12 novel recurrent mutations in South American and African viral genomes out of which 3 were unique in South America, 4 unique in Africa and 5 were present in-patient isolates from both populations. Using state of the art in silico approaches, this study further investigates the interaction of repurposed drugs with the SARS-CoV-2 3CLpro enzyme, which regulates viral replication machinery. CONCLUSIONS: Overall, this study provides insights into the evolving mutations, with implications to understand viral pathogenicity and possible new strategies for repurposing compounds to combat the nCovid-19 pandemic.

A Zn(II)-Coordination Polymer for the Instantaneous Cleavage of Csp3-Csp3 Bond and Simultaneous Reduction of Ketone to Alcohol.

Two coordination polymers of Zn(II) and Cu(II) with n-butylmalonic acid have been achieved in this work. The crystallographic structural descriptions along with the sedimentary rock-type microstructural morphology of these two coordination polymers (CPs) have been explored. The reactivity of ß-hydroxy ketones with these two CPs has also been investigated. The Zn(II)-CP shows a specific reactivity with ß-hydroxy ketone at room temperature and in open air conditions. Through a microcolumn-based filtration technique, the Zn(II)-CP shows the capability to break the Csp3-Csp3 σ bonds of ß-hydroxy ketone and simultaneously reduce the associated ketone to alcohol. Such conversion has been progressed without the use of any additional external reducing agent and any chemical workup or column chromatographic purification protocol. Other similar type CPs of Cu(II) and Mn(II) with n-butylmalonic acid completely failed to show similar reactivity with ß-hydroxy ketone. On the basis of much experimental evidence, the most possible mechanistic pathway of the reactivity between ß-hydroxy ketone and Zn(II)-CP has also been proposed through this work.

New pyrazolyl-dibenzo[b,e][1,4]diazepinones: room temperature one-pot synthesis and biological evaluation.

Several new (5-aryloxy-pyrazolyl)- and (5-aryl/olefin-sulfanyl-pyrazolyl)-dibenzo[b,e] [1,4] diazepinone scaffolds have been synthesized, by assembling 5-substituted 3-methyl-1-phenyl-pyrazole-4-carbaldehydes of varied nature with different cyclic diketones and aromatic diamines successfully in the presence of indium chloride in acetonitrile, at room temperature. Desired products are excellent in the purity and isolated without chromatography. All new structures are confirmed, on the basis of single-crystal X-ray diffraction data of representative 29e. Compounds reported in the present work revealed good antioxidant, antimicrobial and antiproliferative activities with promising FRAP (ferric reducing antioxidant power), bacterial resistance and human solid tumor cell growth inhibitory values, respectively. Compounds 25c and 29e, overall, registered good to moderate activity against A549 (lung), HeLa (cervix), SW1573 (lung) T-47D (breast) and WiDr (colon) cell lines, with GI50 values in the 2.6-5.1 µM and 1.8-7.5 µM ranges, respectively. Molecular docking was carried out to elucidate the binding modes of the compounds (25c, 29e) to topoisomerase I and II.

Hepatoprotective effect of Aloe vera against cartap- and malathion-induced toxicity in Wistar rats.

Exposure to mixture of pesticides in agricultural practices pose a serious threat to the nontarget animals. In present work, we have evaluated the synergistic effect of cartap and malathion on rat liver followed by impact of Aloe vera leaves aqueous extract, which is not known. The animals in eight groups were used; each containing six rats: Group 1 acted as a control, Group 2-control with A. vera leaves aqueous extract, Group 3-with cartap, Group 4-with malathion, Group 5-with mixture of cartap and malathion, Group 6-cartap with the pretreatment of A. vera leaf extract, Group 7-malathion with the pretreatment of A. vera leaf extract, Group 8-mixture of cartap and malathion with the pretreatment of A. vera leaf extract . The animals treated for 15 days were killed after 24hr of last treatment. The biochemical studies in the rat liver demonstrated significant perturbations in the levels of nonenzymatic (glutathione and malondialdehyde) and enzymatic (superoxide dismutase, catalase, and glutathione- S-transferase) antioxidative indices. The histopathological examination of liver revealed serious congestion in central vein and the disorganization of hepatic cords due to pesticide treatment. The administration of A. vera leaves aqueous extract was able to markedly protect rat liver from the pesticides-induced toxicity. The data indicated that pesticides were able to significantly induce oxidative stress which was substantially reduced by the application of plant extract .

Iodine-NH4OAc mediated regioselective synthesis of 2-aroyl-3-arylimidazo[1,2-a]pyridines from 1,3-diaryl-prop-2-en-1-ones.

The present protocol describes an efficient, metal-free regioselective synthesis of 2-aroyl-3-arylimidazo[1,2-a]pyridines from 1,3-diaryl-prop-2-en-1-ones and 2-aminopyridine. The iodine-NH4OAc promoted reaction offers a novel route in the synthesis of 2-aroyl-3-arylimidazo[1,2-a]pyridines. This protocol offers significant flexibility in accessing medicinally important 2-aroyl-3-arylimidazo[1,2-a]pyridines with various substitution patterns.

Prioritization of natural compounds against mycobacterium tuberculosis 3-dehydroquinate dehydratase: A combined in-silico and in-vitro study.

Enormous efforts have been endeavored to develop inhibitors against the potential therapeutic target, mycobacterium tuberculosis 3-dehydroquinate dehydratase (MtbDHQase) to combat resistance. Over a dozen of small molecules have been crystallized to characterize the structural basis of the inhibition. However, the studies accomplished so far, have not incorporated all the essential interactions of these complexes simultaneously, to identify the novel inhibitors. Therefore, an attempt was made to construct the pharmacophore models and identify the essential features that can be employed to prioritize the molecules against this target. Based on validation and expertise, we have identified such complimentary features from the natural compounds that can be used as initial hits. Subsequently, these hits were tested for their inhibitory roles in reducing the mycobacterium tuberculosis (Mtb) culture growth. Moreover, the docking simulations were performed to seek the possible interactions accountable for the activity of these candidates against MtbDHQase.

Metal-free Cross-Dehydrogenative Coupling of HN-azoles with α-C(sp3)-H Amides via C-H Activation and Its Mechanistic and Application Studies.

A metal-free one step coupling reaction between various N-azole rings and diverse α-C(sp3)-H containing amides has been developed under oxidative reaction conditions. Commercially available tetrabutyl ammonium iodide (TBAI) in the presence of terbutylhydroperoxide (TBHP), under neat reaction condition, efficiently catalyzed the coupling. Various azole types, such as 1H-benzotriazoles, 1H-1,2,3-triazoles, 1H-1,2,4-triazoles, 1H-tetrazoles, 1H-pyrazoles, and 1H-benzimidazoles, and α-C(sp3)-H containing amides, such as N,N-dimethylacetamide, N,N-dimethylbenzamide, N-methylacetamide, N,N-diethylacetamide, N-methylpyrrolidine, and pyrrolidine-2-one, were successfully employed for the coupling. A series of designed and controlled experiments were also performed in order to study the involvement of the different intermediates. Based on the evidence, a plausible mechanism is also proposed. These novel, simple, rapid, attractive, and straightforward transformations open the way of the construction of novel highly functionalized N-azoles via direct covalent N-H bond transformations onto N-C bonds. This approach allows to the synthesis of complex molecules requiring number of steps using classical synthetic ways. In addition, the range of α-C(sp3)-H containing amide substrates is virtually unlimited highlighting the potential value of this simple system for the construction of complex heterocyclic molecules, such as fused azoles derivatives.

Efficient synthesis and biological evaluation of new benzopyran-annulated pyrano[2,3-c]pyrazole derivatives.

A one-pot method has been described to synthesize benzopyran-annulated pyrano[2,3-c]pyrazoles, effectively by combining O-alkenyloxy/alkynyloxy-acetophenones with various pyrazolones in triethylammonium acetate (TEAA) under microwave irradiation. While combination of O-allyloxy- or O-prenyloxy-acetophenones with pyrazolones occurred efficiently, that of O-propargyloxy-acetophenones was found effective in the presence of ZnO catalyst, via a domino Knoevenagel-hetero-Diels-Alder (DKHDA) reaction. Aminobenzopyran frameworks were also synthesized, after nitro-containing products were reduced in tandem with iron(II) in an acidic medium. The in vitro antiproliferative activity of these compounds was measured and discussed against gram-positive, gram-negative and M. tuberculosis bacteria, fungi, and various representative human solid tumor cell lines, in addition to their ferric reducing antioxidant capability.

Travel During Pregnancy: Results From an Ultrasound Unit-Based Questionnaire.

BACKGROUND: The frequency of domestic and international travel among women residing in the United States, and specifically Wisconsin, during pregnancy is not known. Given the recent epidemic of Zika virus disease, clinicians should be aware of the frequency of travel during pregnancy and should inquire about travel by pregnant women, women of reproductive age, and their sexual partners. METHODS: Due to the Zika epidemic, our obstetric ultrasound center added questions about international and domestic travel to a general health form that is routinely distributed to all patients presenting for anatomic ultrasounds. The forms were then collected and recorded in order to provide an estimate of the frequency of travel during the first half of pregnancy. RESULTS: Of 1,256 women screened, 64 (5.1%) traveled internationally and 498 (39.6%) traveled domestically prior to their anatomic ultrasound. Additionally, 77 (6.1%) women screened reported international travel by their sexual partner. Among international travelers, 20 (28.1%) traveled to destinations with active ongoing transmission of Zika virus disease, and 16 (25%) traveled after the Centers for Disease Control and Prevention (CDC) issued a travel alert for the area. Among domestic travelers, Florida was the sixth most common destination, and Texas was the 10th most common. CONCLUSIONS: In the population of women screened by this questionnaire, 5.1% traveled internationally and 39.6% traveled domestically prior to their anatomic ultrasound. Notably, Florida and Texas are common travel destinations among women at this clinic, and both have had active local transmission of Zika virus.

A Base-Catalyzed, Domino Aldol/hetero-Diels-Alder Synthesis of Tricyclic Pyrano[3,4-c]chromenes in Glycerol.

The domino aldol/hetero-Diels-Alder synthesis of some new tricyclic pyrano[3,4-c]chromene derivatives has been achieved successfully after assembling a variety of acyclic or cyclic monoketones with prenyl ether-tethered aldehydes in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene in glycerol at 120 °C. The hitherto unreported stereochemical outcome of this synthetic sequence was studied and established on the basis of single-crystal X-ray diffraction data and 2D NMR NOESY spectroscopy along with the isolation and characterization of the intermediate Aldol condensation product.