Shear forces induce ICAM-1 nanoclustering on endothelial cells that impact on T-cell migration.

The leukocyte-specific ß2-integrin LFA-1 and its ligand ICAM-1, expressed on endothelial cells (ECs), are involved in the arrest, adhesion, and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1 has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear flow induces global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear forces caused actin rearrangements and promoted actin-dependent ICAM-1 nanoclustering before LFA-1 engagement. T cells adhered to mechanically prestimulated ECs or nanoclustered ICAM-1 substrates developed a promigratory phenotype, migrated faster, and exhibited shorter-lived interactions with ECs than when adhered to non mechanically stimulated ECs or to monomeric ICAM-1 substrates. Together, our results indicate that shear forces increase ICAM-1/LFA-1 bonds because of ICAM-1 nanoclustering, strengthening adhesion and allowing cells to exert higher traction forces required for faster migration. Our data also underscore the importance of mechanical forces regulating the nanoscale organization of membrane receptors and their contribution to cell adhesion regulation.

3D motion of vesicles along microtubules helps them to circumvent obstacles in cells.

Vesicle transport is regulated at multiple levels, including regulation by scaffolding proteins and the cytoskeleton. This tight regulation is essential, since slowing or stoppage of transport can cause accumulation of obstacles and has been linked to diseases. Understanding the mechanisms by which transport is regulated as well as how motor proteins overcome obstacles can give important clues as to how these mechanisms break down in disease states. Here, we describe that the cytoskeleton architecture impacts transport in a vesicle-size-dependent manner, leading to pausing of vesicles larger than the separation of the microtubules. We further develop methods capable of following 3D transport processes in living cells. Using these methods, we show that vesicles move using two different modes along the microtubule. Off-axis motion, which leads to repositioning of the vesicle in 3D along the microtubule, correlates with the presence of steric obstacles and may help in circumventing them.

Exhaled breath markers of alveolar macrophage activity in sarcoidosis.

OBJECTIVE: Granuloma formation in sarcoidosis is dependent upon the interaction between alveolar macrophages (AMs) and a CD4+-driven TH1 response. This study aimed to measure TNF-α and calcium ion concentrations as markers of AM activity, in addition to total protein as a non-specific inflammatory marker in the exhaled breath condensate (EBC) of patients with sarcoidosis as well as control subjects. METHODS: EBC was collected from 17 sarcoidosis patients and 23 healthy volunteers. Protein was measured by the bicinchoninic acid assay, TNF-α concentration was measured by ELISA and Ca(2+) concentration was measured by inductively coupled plasma-mass spectrometry. Conductivity of EBC was assessed using a conductivity probe. RESULTS: Total protein concentration was significantly elevated in EBC from patients with sarcoidosis compared to control subjects (19.51 ± 4.52 vs. 10.60 ± 1.31 µg/ml, p = 0.020), as was TNF-α (3.37 ± 0.38 vs. 2.59 ± 0.40 pg/ml, p = 0.037) and conductivity (66.68 ± 16.73 vs. 36.85 ± 3.070 µS/cm, p = 0.044). EBC Ca(2+) concentration was significantly higher in healthy controls compared to patients with sarcoidosis (116.50 ± 12.19 vs. 73.88 ± 13.35 µmol/l, p = 0.018), although this was in the context of normal serum Ca(2+) in the sarcoidosis cohort. CONCLUSIONS: Total protein and TNF-α concentrations were elevated in EBC from patients with sarcoidosis and could indicate disease activity. The reduction in EBC Ca(2+) concentrations could represent granulomatous activity in the lung.

Nanodiamond-Mediated Intercellular Transport of Proteins through Membrane Tunneling Nanotubes.

Recently discovered tunneling nanotubes (TNTs) are capable of creating intercellular communication pathways through which transport of proteins and other cytoplasmic components occurs. Intercellular transport is related to many diseases and nanotubes are potentially useful as drug-delivery channels for cancer therapy. Here, we apply fluorescent nanodiamond (FND) as a photostable tracker, as well as a protein carrier, to illustrate the transport events in TNTs of human cells. Proteins, including bovine serum albumin and green fluorescent protein, are first coated on 100-nm FNDs by physical adsorption and then single-particle tracking of the bioconjugates in the transient membrane connections is carried out by fluorescence microscopy. Stop-and-go and to-and-fro motions mediated by molecular motors are found for the active transport of protein-loaded FNDs trapped in the endosomal vehicles of human embryonic kidney cells (HEK293T). Quantitative analysis of the heterotypical transport between HEK293T and SH-SY5Y neuroblastoma cells by flow cytometry confirm the formation of open-ended nanotubes between them, despite that their TNTs differ in structural components. Our results demonstrate the promising applications of this novel carbon-based nanomaterial for intercellular delivery of biomolecular cargo down to the single-particle level.

Coronary Stenosis and Cardiogenic Shock Secondary to Aortitis Following Aortic Root Support Procedure.

A woman with recent personalized external aortic root support implant presented in cardiogenic shock with bilateral coronary ostial occlusion and aortic inflammation requiring emergency coronary angioplasty. Subsequent computed tomography with positron emission tomography scanning demonstrated aortitis with extensive inflammation adjacent to the personalized external aortic root support mesh, the first report of this important complication.

Early Atopic Sensitization to House-Dust Mite in Children with Recurrent Wheeze-A Cross-Sectional Study.

OBJECTIVES: To determine sensitization to house-dust mite (HDM) antigen in under-five children with recurrent wheeze, compare it with nonwheezers, and assess atopic comorbidities in them. METHODS: A cross-sectional study was done in the Pediatric department of a teaching hospital in North India, in 190 children aged 1-5 y. Out of these, 127 had recurrent wheeze (RW), and 63 had no wheeze (NW). Sensitivity was done by skin prick test (SPT) for two dust mites antigens: Dermatophagoide farinae and Dermatophagoide pteronyssinus antigens. In addition, atopic comorbidities like atopic dermatitis and allergic rhinitis were assessed. RESULTS: Mean age of the study population was 34.52 ± 20.50 mo. SPT positivity for either of the dust mites was 97 (76.4%) in RW and 13 (20.6%) in NW which was significant (p < 0.001, aOR = 12.27). HDM species sensitization for D. pteronyssinus was 55.1% vs. 15.9% (p < 0.001 aOR = 7.81) and D. farinae was 39.4% vs. 9.5% (p < 0.001, aOR = 5.45) in groups, respectively. Mean wheal size in RW Group was also significantly higher than NW group for D. pteronyssinus (2.39 ± 1.44 vs. 0.52 ± 1.19 mm, median (IQR) 3 (1-3), p < 0.001), D. farinae (1.80 ± 1.39 vs. 0.32 ± 1.00 mm, median (IQR) 2 (0-3), p < 0.001). Allergic rhinitis was present in 55 (43.3%) vs. 7 (11.1%) (p < 0.001), atopic dermatitis in 28 (22%) vs. 2 (3.2%) (p = 0.001) in group 1 and 2, respectively. All children with allergic rhinitis had HDM sensitization in both groups. CONCLUSION: This study showed early sensitization to HDM in children with recurrent wheeze. Atopic comorbidities were also present in them.

Enterobacter cloacae Keratitis: Clinicomicrobiological Profiles, Risk Factors, and Outcomes.

PURPOSE: The purpose of the study was to report the clinical features, risk factors, antibiotic susceptibility, and treatment outcomes in a series of Enterobacter cloacae keratitis. METHODS: A retrospective analysis was performed of the electronic medical records of microbial keratitis caused by E. cloacae identified by the Vitek 2 system (BioMerieux, Craponne, France). We collected data pertaining to demographics, risk factors, ulcer characteristics, antibiogram, visual acuity at presentation and final follow-up, and management outcome. The main outcome measure was resolution of infection. The final visual acuity was the secondary outcome measure. RESULTS: Ten episodes of E. cloacae keratitis in 9 patients were identified between January 2009 and December 2019. Nine (90%) cases had undergone penetrating keratoplasty and 8 were failed grafts. Other risk factors included topical steroid use and irregular ocular surface due to epithelial bullae. The mean ulcer size was 17.55 ± 13.99 mm 2 . More than 80% of isolates were sensitive to chloramphenicol, gentamicin, and colistin. Nine (90%) cases healed on medical management within 56.55 ± 26.74 days (range 9-120 d), although almost all required adjunctive procedures: tissue adhesive application (n = 6) and/or tarsorrhaphy (n = 4). One case with a near total infiltrate had a mixed infection with Kocuria kristinae requiring therapeutic penetrating keratoplasty. One case developed endophthalmitis and phthisis after the corneal infiltrate resolved. CONCLUSIONS: E. cloacae keratitis is a rare clinical entity seen more often in immunocompromised host conditions such as failed corneal transplants with concomitant topical steroids. Most cases healed with medical management.

A case report of successful physiological pacing in a patient with lamin A/C cardiomyopathy.

Background: Lamin A/C (LMNA) mutations account for 5-8% of familial dilated cardiomyopathies, and can manifest with conduction abnormalities and ventricular arrhythmias in 78% of patients. Therefore, when suspected, it is important to implant the correct type of device. Case summary: A 52-year-old gentleman with a family history of cardiomyopathy, presented with asymptomatic atrial fibrillation and complete atrioventricular block associated with a narrow QRS interval. Investigations confirmed dilated and severely impaired left ventricular systolic function. He underwent successful conduction system pacing in combination with a primary prevention defibrillator. Genetic screening confirmed LMNA cardiomyopathy. During 3 years follow up, his left ventricular function remained unchanged with stable conduction system capture and he received appropriate therapy from his device for ventricular tachycardia. Discussion: His-bundle pacing promotes rapid and synchronous activation of the ventricles via the intrinsic conduction system of the heart. In selected individuals with LMNA cardiomyopathy, conduction system pacing is viable alternative to conventional cardiac resynchronization therapy using coronary sinus tributaries.

Pediatric Microbial Keratitis: Identification of Clinical Biomarkers for Prognosis and Outcome of 218 Cases From 2009 to 2019.

PURPOSE: The aim of this study was to analyze the risk factors, microbiological profile, and treatment efficacy in pediatric microbial keratitis (MK) and to identify clinical biomarkers prognosticating outcome. METHODS: A retrospective analysis was conducted from patients younger than 16 years with MK-excluding viral, marginal, or interstitial keratitis. Data pertaining to predisposing factors, symptom duration, prior treatment, ulcer characteristics, microbiological profile, time to resolution, and final outcome were recorded. Statistical analysis was performed. The mixed-effects linear regression model with random intercept was used to evaluate factors affecting time to resolution. RESULTS: Among 218 episodes of 215 pediatric patients with MK, the geometric mean of central [median 3 mm, interquartile range (IQR) 1-4.3 mm] and peripheral ulcers (median 1 mm, IQR 1-2.5 mm) was significantly different ( P < 0.0001). Organisms identified were bacteria (56.9%), fungi (31.5%), and acanthamoebae (2.3%). Of 172 cases (78.8%), which resolved in a median resolution time of 22 days (IQR, 11-44 days), 107 (81.6%) with absent/negative microbiology healed on empirical therapy. On multivariate analysis, peripheral ulcers and geometric mean ulcer size affected time to resolution. Significantly higher percentage of eyes, which worsened or perforated, were on topical steroids compared with those which healed (31.8% vs. 9.2%, P = 0.0061). CONCLUSIONS: Good outcome even in culture negative cases suggests empirical therapy may be instituted for nonsevere peripheral pediatric MK; however, the importance of a microbiological workup cannot be underscored enough. Ulcer location and geometric mean size of ulcer may be used as clinical prognostic markers for resolution.

Polarization modulation spectroscopy of single fluorescent nanodiamonds with multiple nitrogen vacancy centers.

A technique based on polarization modulation spectroscopy (PMS) has been developed to determine quantitatively the number of fluorophores in nanoparticles at the single-molecule level. The technique involves rotation of the polarization of the excitation laser on a millisecond time scale, leading to fluorescence intensity modulation. By taking account of the heterogeneous orientation among the dipoles of the fluorophores and simulating the modulation depth distribution with Monte Carlo calculations, we show that it is possible to deduce the ensemble average and number distribution of the fluorophores. We apply the technique to fluorescent nanodiamonds (FNDs) containing multiple nitrogen vacancy (NV) centers. Comparing the experimental and simulated modulation depth distributions of 11 nm FNDs, we deduce an average number of = 3, which is in good agreement with independent photon correlation measurements. The method is general, rapid, and applicable to other nanoparticles, polymers, and molecular complexes containing multiple and randomly orientated fluorophores as well.

In vivo imaging and toxicity assessments of fluorescent nanodiamonds in Caenorhabditis elegans.

Nanoscale carbon materials hold great promise for biotechnological and biomedical applications. Fluorescent nanodiamond (FND) is a recent new addition to members of the nanocarbon family. Here, we report long-term in vivo imaging of FNDs in Caenorhabditis elegans (C. elegans) and explore the nano-biointeractions between this novel nanomaterial and the model organism. FNDs are introduced into wild-type C. elegans by either feeding them with colloidal FND solution or microinjecting FND suspension into the gonads of the worms. On feeding, bare FNDs stay in the intestinal lumen, while FNDs conjugated with biomolecules (such as dextran and bovine serum albumin) are absorbed into the intestinal cells. On microinjection, FNDs are dispersed in the gonad and delivered to the embryos and eventually into the hatched larvae in the next generation. The toxicity assessments, performed by employing longevity and reproductive potential as physiological indicators and measuring stress responses with use of reporter genes, show that FNDs are stable and nontoxic and do not cause any detectable stress to the worms. The high brightness, excellent photostability, and nontoxic nature of the nanomaterial have enabled continuous imaging of the whole digestive system and tracking of the cellular and developmental processes of the living organism for several days.

Improved Outcome of Pythium Keratitis With a Combined Triple Drug Regimen of Linezolid and Azithromycin.

PURPOSE: To describe the clinical features, microbiological profile, and outcome of a series of cases of Pythium keratitis treated with topical and oral linezolid and topical azithromycin eye drops. METHODS: This was a retrospective interventional case series of microbiologically and/or histopathologically proven cases of Pythium keratitis seen between October 2016 and December 2019. All patients received a combination of topical linezolid and/or azithromycin eye drops with oral linezolid. Analysis of demographic data, predisposing risk factors, microbiological results, treatment regimen, visual acuity, surgical intervention, and final outcome was performed. A subgroup analysis of cases >6 mm in size was performed. Success was defined as complete resolution on medical management. Failure was defined as worsening of infection necessitating therapeutic penetrating keratoplasty or evisceration. RESULTS: Of 21 cases, 2 were lost to follow up, 1 was diagnosed on histopathology, and 1 received only topical linezolid. Characteristic microbiological features were noted on 10% potassium hydroxide calcofluor white wet mount in 20/21 (95.23%) and Gram stain in 18/21 (85.71%). On triple drug regimen, 14/17 cases (82.35%) resolved. Average time to resolution was 87.64 ± 44.44 days. More than 60% infiltrates (13/21) were large, and 66.66% infiltrates resolved in 109.3 ± 57.06 days. Of the 5 failures, 4 needed therapeutic keratoplasty and 1 needed evisceration. All grafts failed. CONCLUSIONS: The dual topical drug regimen with oral linezolid has good cure rates (over 80%) for Pythium keratitis over prolonged duration. It is recommended to persevere with medical therapy even in large infiltrates because more than two thirds resolved.

Detyrosinated microtubules spatially constrain lysosomes facilitating lysosome-autophagosome fusion.

Microtubule post-translational modifications impart functional diversity to microtubules by affecting their dynamics, organization, and interaction with proteins. Using super-resolution microscopy, we show that only a small subpopulation of microtubules are detyrosinated in epithelial cells, while acetylated and tyrosinated microtubules comprise the majority of all microtubules. Surprisingly, lysosomes are enriched by approximately threefold on detyrosinated microtubules. Further, their motility on detyrosinated microtubules is impaired, showing shorter runs and more frequent and longer pauses. Lysosome enrichment is mediated through a kinesin-1-dependent mechanism, since knocking down this motor abolishes enrichment. Finally, correlative live-cell and super-resolution microscopy showed that lysosomes interact with autophagosomes on detyrosinated microtubules. Removal of detyrosinated microtubules or knockdown of kinesin-1 leads to a decrease in the percentage of autolysosomes, a fusion intermediate of autophagosomes and lysosomes. Taken together, our data reveal a new role of detyrosinated microtubules as hubs that spatially concentrate lysosomes on a small subset of microtubules and facilitate their interaction and fusion with autophagosomes to initiate autophagy.

Bacterial vaginosis and antibacterial susceptibility pattern of asymptomatic urinary tract infection in pregnant women at a tertiary care hospital, Visakhaptn, India.

BACKGROUND AND OBJECTIVES: The association between bacterial vaginosis and urinary tract infection (UTI) in pregnant women is at a greater risk comparatively than patients with bacterial vaginosis or UTI. Bacterial vaginosis and asymptomatic UTI both pose risk for mother and fetus. Early diagnosis and treatment can save the life of both. The present investigation was aimed to find out the magnitude of asymptomatic bacteriuria in pregnant women with noticeable bacterial vaginitis attending antenatal outpatient and inpatient of a tertiary care hospital and to identify the organisms causing it. MATERIALS AND METHODS: A total of 117 antenatal women from different age and parity groups with different gestational ages were included in the study. The samples were subjected to standard microbiological techniques for identification of microorganisms. While performing Per speculum examination, vaginal secretions were collected from the posterior fornix. Swabs from the posterior fornix were tested for pH using litmus paper. A wet mount and Gram smear was made and examined for the presence of bacteria, polymorphs and clue cells indicating bacterial vaginosis. Amsel's criteria and Nugent scoring system were applied for diagnosis of bacterial vaginosis. Antibiotic susceptibility of the isolated bacteria was performed using Kirby-Bauer method. RESULTS: Bacterial vaginosis infection rate (62.3%) was common in the present study followed by asymptomatic UTI (n=60, 51%). It was also observed that asymptomatic urinary tract infection (UTI) with Bacterial vaginosis prevalent rate was 49 (41.8%) in the current study. CONCLUSION: Bacterial vaginosis was more common than asymptomatic bacteriuria in pregnant women. It is recommended that antenatal health care facilities should incorporate screening of vaginitis among pregnant women to prevent the complications of pregnancy. And those women with Bacterial vaginosis should be screened for UTI. Proper use of antibiotics should be encouraged, abuse of antibiotics should be in check.

Endophthalmitis caused by gram-positive bacteria resistant to vancomycin: Clinical settings, causative organisms, antimicrobial susceptibilities, and treatment outcomes.

PURPOSE: To report the clinical settings, causative organisms, antimicrobial susceptibilities, and treatment outcomes of patients with endophthalmitis caused by gram-positive bacteria resistant to vancomycin. METHODS: Retrospective case series of all patients with culture-proven endophthalmitis caused by gram-positive bacteria resistant to vancomycin between January 2010 and December 2016 in LV Prasad Eye Institute, Visakhapatnam, India. RESULTS: The current study included 14 patients. The clinical settings were post-cataract surgery in 8/14 (57.1%) and open globe injury in 6/14 (42.8%). Primary intervention for all patients included tap and intravitreal antibiotic injection. During subsequent follow-up, pars plana vitrectomy was performed in 6 patients and one patient underwent penetrating keratoplasty. Mean number of intravitreal antibiotic injections performed were 3.4 per patient. The most common organisms isolated were coagulase-negative Staphylococci in 6/14 (42.8%), Staphylococcus aureus in 5/14 (35.7%), Streptococcus sp in 2/14 (14.2%) and Bacillus sp in 1/14 (7.14%). In addition to vancomycin, resistance to multiple drugs (three or more groups of antibiotics) was found in all 14 cases. Antimicrobial susceptibility results showed susceptibility to amikacin in 7/14 (50.0%), gatifloxacin in 6/14 (42.8%), moxifloxacin in 3/13 (23.0%), cefazoline in 5/14 (35.7%), cefuroxime in 3/14 (21.4%), ciprofloxacin in 2/14 (14.2%) and linezolid in 5/5 (100%). The mean duration of follow-up was 30.7 weeks (6 weeks-90 weeks). At last follow-up, visual acuity (VA) of 20/200 or better was recorded in 7/14 (50%) and VA < 5/200 occurred in 7/14 (50%). CONCLUSION AND IMPORTANCE: Antimicrobial susceptibility testing may help in selection of suitable antimicrobial agents for repeat intravitreal injection. Inspite of retreatment with intravitreal antibiotics, these patients generally had poor VA outcomes.

Fluorescent nanodiamond tracking reveals intraneuronal transport abnormalities induced by brain-disease-related genetic risk factors.

Brain diseases such as autism and Alzheimer's disease (each inflicting >1% of the world population) involve a large network of genes displaying subtle changes in their expression. Abnormalities in intraneuronal transport have been linked to genetic risk factors found in patients, suggesting the relevance of measuring this key biological process. However, current techniques are not sensitive enough to detect minor abnormalities. Here we report a sensitive method to measure the changes in intraneuronal transport induced by brain-disease-related genetic risk factors using fluorescent nanodiamonds (FNDs). We show that the high brightness, photostability and absence of cytotoxicity allow FNDs to be tracked inside the branches of dissociated neurons with a spatial resolution of 12 nm and a temporal resolution of 50 ms. As proof of principle, we applied the FND tracking assay on two transgenic mouse lines that mimic the slight changes in protein concentration (∼30%) found in the brains of patients. In both cases, we show that the FND assay is sufficiently sensitive to detect these changes.

Meningococcal group A, C, Y and W-135 conjugate vaccine.

In February 2010, a quadrivalent conjugate vaccine (Menveo; Novartis Vaccines and Diagnostics) was approved by the US FDA to prevent invasive meningococcal disease caused by Neisseria meningitidis serogroups A, C, Y and W-135 in people aged 11-55 years.