Prevention of Arthritis Markers in Experimental Animal and Inflammation Signalling in Macrophage by Karanjin Isolated from Pongamia pinnata Seed Extract

Karanjin, the furanoflavonoid reported to possess gastroprotective and anti‐diabetic properties, was investigated against experimental arthritis and its molecular signalling in inflammation was explored in macrophages. Karanjin was isolated from hexane extract of Pongamia pinnata seeds and was evaluated on arthritis markers in adjuvant induced arthritis model (AIA) in two doses (per oral; 10 mg/kg/day and 20 mg/kg/day). Karanjin dose dependently reduced collagen and cartilage breakdown markers viz. urinary hydroxyproline and glucosamine, respectively, serum lysosomal enzymes responsible for articular cartilage damage, and major proinflammatory cytokine TNFα, secreted by macrophages involved in articular inflammation and destruction. Karanjin also prevented joint damage as evidenced from arthritis score, radiographic and histopathological analysis. To delineate the molecular target of Karanjin, in vitro study on LPS induced macrophages were performed at calibrated non toxic doses (4 µg/mL and 6 µg/mL). Karanjin reduced TNFα production and also showed potent inhibitory effect on nitric oxide and reactive oxygen species production which is generally induced by TNFα from activated macrophages. NF‐κB, the key regulator of TNFα signalling during inflammation was significantly suppressed by Karanjin. Our study for the first time highlights the anti‐inflammatory role of Karanjin in experimental arthritis model as well as on macrophage signalling, thereby depicting its probable mechanism of action. Copyright © 2014 John Wiley & Sons, Ltd.     

Predictors of mortality in children due to severe and very severe pneumonia

Background:

Mortality due to pneumonia in children is more than any other illness. Limited data is available to predict mortality in children with pneumonia from central India.

Aim:

To study predictors of mortality in children aged 1-59 months hospitalised with severe and very severe pneumonia.

Materials and Methods:

Present study was observational longitudinal study that was done in a tertiary care hospital of central India. Two hundred and ninety children, aged 1-59 months, presented with severe and very severe pneumonia were enrolled in this study. Outcome and predictors of mortality were studied. Data was analysed with Chi-square test, univariate and multivariate regression analysis.

Results:

Out of 270 enrolled study subjects, maximum (108, 37.24%) were belonged to 1-6-months age group. Proportion of mortality was maximum (16, 64.00%) in that age group. Overall case fatality rate was 8.62%. Among significant variables, delayed hospital referral [adjusted odds ratio (OR)-52.09, 95% confidence interval (CI)- 6.74-402.39], incomplete immunisation (OR-12.28, 95% CI-2.15-69.93), severe malnutrition (Z score < −3) (OR-15.51, 95% CI- 2.04-117.83), refusal to feed (OR- 30.57, 95% CI- 2.47-378.26), and hypoglycaemia (OR- 6.98, 95% CI- 1.05-46.30) were found significant independently on multivariate regression analysis. Conclusion: Delayed hospital referral, incomplete immunisation, severe malnutrition, refusal to feed, and hypoglycaemia were independent predictors of mortality in children with severe and very severe pneumonia.     

Diagnostic Challenge with Nocardia Subretinal Abscess: A Case Report from Tuberculosis-Endemic Region

Purpose: To describe a case of Nocardia subretinal abscess, which posed a diagnostic challenge due to the presence of mycobacterial genome in sample obtained from fine needle aspiration biopsy (FNAB).

Methods: A retrospective chart review.

Results: A 25-year-old male presented with sudden, painless onset diminution of vision of left eye and found to have placoid patch of choroiditis just temporal to the macula in right eye and showed vitritis, subretinal abscess with exudative retinal detachment and vitreous hemorrhage in left eye. Both aqueous and vitreous samples were negative microbiologically and polymerase chain reaction for various genomes. Sample obtained from FNAB was positive for mycobacterial genome and yielded Nocardia arthritidis on culture. She was treated with antitubercular therapy and intravenous and intravitreal antimicrobials.

Conclusions: Nocardia subretinal abscess can be a diagnostic challenge in tuberculosis-endemic region, especially in conditions when there is co-infection with Mycobacterium tuberculosis.     

Evolution of uracil based thymidine phosphorylase inhibitors,SAR and electronic correlation: revisit

Thymidine phosphorylase (TPase), a dimeric proteinaceous enzyme is responsible for elevation of drug resistance and cell apoptosis. Increased TPase levels lead to angiogenesis which is a major culprit for the metastasis and cancer in the body. Hence, an efficient approach for anticancer drug discovery is based up on inhibition of thymidine phosphorylase enzyme. Solving of the crystal structure of TPase made the drug discovery easier. Legion of drugs was designed and synthesized having a wide variety of structural moieties and different functionalities such as phosphonic acid, amines, arylamines, acetamides, benzoyl, esters, amides, and so forth. Among the structural designs and models engineered, uracil is one of the structural motifs utilized for drug design. In this review, uracil is chosen where in structural unit with its structural modifications were summarized. Uracil unit could be structurally modified at its N₁, N₃, C₅, and C₆ positions to afford innumerable uracil derivatives. In general consensus, N₁- and N₃-substituted uracil derivatives resulted in diminished/lower inhibitory activities which might be attributed to reduced acidic character. Whereas, substitution at 5- and 6-positions have yielded fruitful results and excellent thymidine phosphorylase inhibitors prevailed. Furthermore, uracil possessing nucleoside uridine/deoxyuridine was also derivatized to afford potential TPase inhibitors. 3′- & 5′-Hydroxyl groups of uridine/deoxyuridine were also exploited for derivative preparation and some of them were found to be potent TPase inhibitors.     

Genetic variation in the one-carbon transfer pathway and ovarian cancer risk

Dysfunction in enzymes involved in one-carbon (1-C) metabolism can lead to increased chromosomal strand breaking and abnormal methylation patterns, which are both associated with cancer risk. Availability of 1-C units may modify risk. We investigated the association of single-nucleotide polymorphisms (SNP) in 21 genes in the 1-C transfer pathway among 829 Caucasian cases with primary epithelial ovarian cancer and 941 frequency-matched unaffected controls enrolled at Mayo Clinic (Rochester, MN) and Duke University (Durham, NC) and examined risk modification by multivitamin supplement use. Multivariable-adjusted SNP-specific logistic regression and haplotype analyses were done for 180 SNPs and false positive report probabilities (FPRP) were calculated. Each copy of the minor allele in SHMT1 intron 5 A>G (rs9909104) was associated with epithelial ovarian cancer [odds ratio (OR), 1.2; 95% confidence interval (95% CI), 1.0-1.4; P trend = 0.02; FPRP = 0.16] and a 5-SNP SHMT1 haplotype was associated with decreased risk (P = 0.01; FPRP = 0.09). Three SNPs in DNMT3A were associated with risk among multivitamin supplement users: 3' untranslated region (UTR) C>G (rs13420827: OR, 0.8; 95% CI, 0.6-1.0; P interaction = 0.006; FPRP = 0.54), intron 6 G>A (rs11887120: OR, 0.8; 95% CI, 0.7-1.0; P interaction = 0.007; FPRP = 0.57), and intron 22 A>T (rs11695471: OR, 1.2; 95% CI, 1.0-1.5; P interaction = 0.01; FPRP = 0.66). These data extend previous findings from other cancers of a role for SHMT1 in ovarian cancer, and provide evidence that SNPs in methylation and DNA synthesis reactions are associated with risk of ovarian cancer. Interventions with modifiable factors such as multivitamin intake may reduce risk.     

Acquired, isolated third nerve palsies in infants with cerebrovascular malformations

PURPOSE: To report two infants with acquired, isolated third nerve palsies attributable to intracranial cerebrovascular malformations. DESIGN: Observational case report. METHODS: Two patients are described. Each was examined in a university-based pediatric ophthalmology and neuro-ophthalmology practice. RESULTS: An 8-month-old child presented with a pupil-involving partial left third nerve palsy because of a partially thrombosed fusiform aneurysm of the left internal carotid artery. A 3-month-old infant developed a right third nerve palsy from a giant arteriovenous fistula arising from an M2 branch of the right middle cerebral artery. CONCLUSION: Patients younger than 8 months and 3 months with acquired, isolated third nerve palsies resulting from intracranial cerebrovascular malformations could not be found in a MEDLINE search. In conclusion, at even this young age, acquired, isolated third nerve palsies may be the initial manifestation of an intracranial aneurysm or fistula. Magnetic resonance imaging (MRI) and MRI-angiography were adequate for detecting these processes.     

A safer alternative approach to penetrating keratoplasty in opaque corneas

Background:This video demonstrates a useful technique of keratoplasty which can be routinely undertaken by all surgeons when imaging modalities such as anterior segment optical coherence tomography are not available and prior patient history is not forthcoming.Purpose:To demonstrate a technique of lamellar separation and layer by layer removal of host cornea when dealing with keratoplasty in perforated corneal ulcers, adherent leucomas, dense corneal opacities, which obscure visualization of the iris and anterior chamber details.Synopsis:In this video, we demonstrate penetrating keratoplasty in a failed opacified graft with iridocorneal adhesions, with no visualization of anterior chamber details. Lamellar dissection of the host cornea is done starting at its periphery and moving centrally, with gentle peeling of the superficial layers, the epithelium and bulk of stroma, following which, the deeper portion of the cornea is dissected and separated from underlying adherent iris tissue. Layer by layer separation allows better visualization through the remaining thin layers of the cornea. This permits fine dissection and layered removal of the cornea, thereby avoiding injury to iris and lens. Debulking of the host cornea decreases the force that is needed to be applied to separate adherent iris tissue from the host cornea, and reduces the chances of sudden entry into the anterior chamber and subsequent damage to the iris or lens. This also reduces the chance of iris tears, iridodialysis and bleeding from the iris and helps maintain iris integrity, which is essential intraoperatively for protection of lens and anterior chamber formation, and to avoid glare and photophobia postoperatively. Preventing iris damage also reduces the chances of formation of peripheral anterior synechiae (PAS), which can predispose to graft rejection, graft failure and secondary glaucoma.Highlights:Layer by layer corneal separation beginning inside the graft host junction, careful separation of iridocorneal adhesions and PAS is a helpful technique to optimally preserve the anterior segment anatomy during difficult cases of penetrating keratoplasty.Online Video Link: https://youtu.be/ZmQQhuOnAh4     

Subchronic Oral Cylindrospermopsin Exposure Alters the Host Gut Microbiome and Is Associated with Progressive Hepatic Inflammation,Stellate Cell Activation,and Mild Fibrosis in a Preclinical Study

Epidemiological studies have reported a strong association between liver injury and incidences of hepatocellular carcinoma in sections of humans globally. Several preclinical studies have shown a strong link between cyanotoxin exposure and the development of nonalcoholic steatohepatitis, a precursor of hepatocellular carcinoma. Among the emerging threats from cyanotoxins, new evidence shows cylindrospermopsin release in freshwater lakes. A known hepatotoxin in higher concentrations, we examined the possible role of cylindrospermopsin in causing host gut dysbiosis and its association with liver pathology in a mouse model of toxico-pharmacokinetics and hepatic pathology. The results showed that oral exposure to cylindrospermopsin caused decreased diversity of gut bacteria phyla accompanied by an increased abundance of Clostridioides difficile and decreased abundance of probiotic flora such as Roseburia, Akkermanssia, and Bacteroides thetaiotamicron, a signature most often associated with intestinal and hepatic pathology and underlying gastrointestinal disease. The altered gut dysbiosis was also associated with increased Claudin2 protein in the intestinal lumen, a marker of gut leaching and endotoxemia. The study of liver pathology showed marked liver inflammation, the release of damage-associated molecular patterns, and activation of toll-like receptors, a hallmark of consistent and progressive liver damage. Hepatic pathology was also linked to increased Kupffer cell activation and stellate cell activation, markers of progressive liver damage often linked to the development of liver fibrosis and carcinoma. In conclusion, the present study provides additional evidence of cylindrospermopsin-linked progressive liver pathology that may be very well-linked to gut dysbiosis, though definitive evidence involving this link needs to be studied further.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK’s multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

Clathrin-mediated endocytosis (CME) regulates the uptake of nutrients, growth factors, adhesion molecules, transmembrane ion channels, transporters, signaling receptors, and other ligand–receptor complexes. Thus, CME plays a crucial role in cell homeostasis by constantly remodeling and controlling the composition of the plasma membrane (PM) in response to various extracellular and intracellular stimuli. Consequently, dysregulated CME has been extensively linked to disease (1, 2). CME is a multistep process involving 1) “priming,” i.e., the regulated and localized activation of clathrin assembly proteins, predominantly adaptor protein 2 (AP2) complexes at the PM; 2) initiation of clathrin assemblies; 3) stabilization of clathrin-coated pits (CCPs) in the form of a macromolecular complex; 4) productive CCP growth and maturation, which culminates in 5) fission and the release of newly formed clathrin-coated vesicles (CCVs) into the cytosol (3, 4). Many nascent CCPs fail to complete this multistep process and instead rapidly disassemble as early or late abortive pits (5–7). In addition to the major coat proteins, clathrin and AP2, successful completion of CME requires the activities of a myriad of endocytic accessory proteins (EAPs). These EAPs, many of which are multidomain proteins, encode multiple biochemical activities, including curvature generation and sensing, cargo recruitment, scaffolding, and lipid modification (1, 4, 8).The activities of EAPs, or of their individual functional domains, were largely identified through in vitro biochemical assays. The in vivo functions of EAPs in CME are frequently measured by cargo uptake, which scores the net accumulation of cargo inside cells, but lacks the temporal resolution and sensitivity to capture early steps or the regulation of CCP growth. Indeed, several publications showed that measurements of cargo uptake alone are unable to reveal alterations in the early kinetics of CCP maturation caused by the absence of one or several EAPs (9–11). An alternative approach to assess stage-specific EAP functions has been to measure the temporal hierarchy of their recruitment to CCPs. Using a pH-sensitive fluorescent cargo to mark scission events, Merrifield and colleagues (7) measured the recruitment profiles of 34 EAPs to CCPs with high temporal resolution, providing insight into their sequential roles in CME. The study also highlighted the nonuniform molecular composition of individual CCPs. However, its major limitation was that, prior to the advent of genome-editing technologies, fluorescently labeled EAPs were transiently overexpressed, which is especially problematic given the likely competition arising from widely shared protein interaction domains and binding motifs. Moreover, as individually tracked CCPs, referred to hereafter as intensity traces, were aligned to the terminal fission event, early molecular signatures of these EAPs at CCPs were missed, especially given the heterogeneity of CCP lifetimes (7, 12). Nonetheless, these and similar pioneering studies in yeast (13, 14) have led to the concept that the CME machinery is organized into functional modules that act sequentially, and in a stereotypic manner, during CCP maturation (1, 4).It has become increasingly evident that CME and cell surface receptor signaling are reciprocally regulated by feedback loops (15–20). This has led to the understanding that CME is not a passive process, but that it can respond and adapt to multiple inputs. Moreover, consistent with the essential role of CME in cellular physiology, the process is robust and exhibits plasticity, in that compensatory mechanisms can restore CME even when individual stages of CCP maturation are significantly perturbed (9, 19, 21). This robustness and plasticity likely derive from the overlapping functions, and thus redundancies, of EAPs regulating clathrin assembly and CCP maturation.Although the individual activities of many EAPs have been extensively studied, many controversies exist as to how, and at which stage(s), these activities contribute to the overall process of CME (22–29). In part, these controversies may reflect the wide range of cell types, experimental systems, and assays used to study EAP function. A more comprehensive analysis under identical experimental conditions has never been undertaken and could lead to a better understanding of the functional hierarchy and the role played by each EAP during CME. Such a systematic analysis requires a readout that directly measures discrete early stages of CCP nucleation, initiation, and maturation. Confocal and total internal reflection fluorescence microscopy (TIR-FM)-based live-cell imaging (5, 6, 30, 31) paired with unbiased and high-content image analyses (6, 9, 11, 32) are essential for the detection of these alterations in early regulatory stages of CCV formation.Equipped with live cell TIR-FM and computer vision tools to quantify several stage-specific parameters of CME, including rates of CCP initiation, stabilization, and maturation (9, 12, 33), we have quantified the knockdown (kd) effect (i.e., phenotype) of most known or suspected EAPs on CCP dynamics within a uniform and rigorous experimental framework. Based on previous studies and their biochemically defined activities, the 67 proteins studied can be assigned to functionally distinct modules (1, 4, 8). Here, we have clustered EAPs based on their phenotypic signatures. Interestingly, these phenotypically defined clusters do not overlap with the biochemically defined modules. Our results highlight the functional complexity of protein–protein interactions and EAP activity during CCV formation. The overlapping activities and hence functional redundancies of multidomain EAPs provide a mechanistic basis for the robustness of CME and the ability of cells to counter defects in CCP maturation by activation of compensatory mechanisms.     

Radiomics signature for temporal evolution and recurrence patterns of glioblastoma using multimodal magnetic resonance imaging

Glioblastoma is a highly infiltrative neoplasm with a high propensity of recurrence. The location of recurrence usually cannot be anticipated and depends on various factors, including the surgical resection margins. Currently, radiation planning utilizes the hyperintense signal from T2-FLAIR MRI and is delivered to a limited area defined by standardized guidelines. To this end, noninvasive early prediction and delineation of recurrence can aid in tailored targeted therapy, which may potentially delay the relapse, consequently improving overall survival. In this work, we hypothesize that radiomics-based phenotypic quantifiers may support the detection of recurrence before it is visualized on multimodal MRI. We employ retrospective longitudinal data from 29 subjects with a varying number of time points (three to 13) that includes glioblastoma recurrence. Voxelwise textural and intensity features are computed from multimodal MRI (T1-contrast enhanced [T1CE], FLAIR, and apparent diffusion coefficient), primarily to gain insights into longitudinal radiomic changes from preoperative MRI to recurrence and subsequently to predict the region of relapse from 143 ± 42 days before recurrence using machine learning. T1CE MRI first-order and gray-level co-occurrence matrix features are crucial in detecting local recurrence, while multimodal gray-level difference matrix and first-order features are highly predictive of the distant relapse, with a voxelwise test accuracy of 80.1% for distant recurrence and 71.4% for local recurrence. In summary, our work exemplifies a step forward in predicting glioblastoma recurrence using radiomics-based phenotypic changes that may potentially serve as MR-based biomarkers for customized therapeutic intervention.