Lipid Droplet Motility Increases Following Viral Immune Stimulation

Lipid droplets (LDs) have traditionally been thought of as solely lipid storage compartments for cells; however, in the last decade, they have emerged as critical organelles in health and disease. LDs are highly dynamic within cells, and their movement is critical in organelle–organelle interactions. Their dynamics are known to change during cellular stress or nutrient deprivation; however, their movement during pathogen infections, especially at very early timepoints, is under-researched. This study aimed to track LD dynamics in vitro, in an astrocytic model of infection. Cells were either stimulated with a dsRNA viral mimic, poly I:C, or infected with the RNA virus, Zika virus. Individual LDs within infected cells were analysed to determine displacement and speed, and average LD characteristics for multiple individual cells calculated. Both LD displacement and mean speed were significantly enhanced in stimulated cells over a time course of infection with an increase seen as early as 2 h post-infection. With the emerging role for LDs during innate host responses, understanding their dynamics is critical to elucidate how these organelles influence the outcome of viral infection.     

Three-dimensional automated nanoparticle tracking using Mie scattering in an optical microscope

The forward scattering of light in a conventional inverted optical microscope by nanoparticles ranging in diameter from 10 to 50nm has been used to automatically and quantitatively identify and track their location in three-dimensions with a temporal resolution of 200ms. The standard deviation of the location of nominally stationary 50-nm-diameter nanoparticles was found to be about 50nm along the light path and about 5nm in the plane perpendicular to the light path. The method is based on oscillating the microscope objective along the light path using a piezo actuator and acquiring images with the condenser aperture closed to a minimum to enhance the effects of diffraction. Data processing in the time and spatial domains allowed the location of particles to be obtained automatically so that the technique has potential applications both in the processing of nanoparticles and in their use in a variety of fields including nanobiotechnology, pharmaceuticals and food processing where a simple optical microscope maybe preferred for a variety of reasons.     

Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression

Resolution of inflammation involves removal of recruited neutrophils from inflamed sites via a noninflammatory mechanism, possibly involving neutrophil apoptosis and engulfment/phagocytosis by macrophages. In this study, we describe the reduction in surface expression (> 90%) of the neutrophil molecule Fc gamma RIII (CD16) during in vitro culture at 37 degrees C, which was found to be temporally associated with the appearance of neutrophils with apoptotic morphology during in vitro culture and inhibitable by granulocyte-macrophage colony-stimulating factor (GM-CSF), which postpones apoptosis in the neutrophil. By using dual fluorescence analysis, CD16 "low" expressing neutrophils showed reduced staining with the DNA-binding dye propidium iodide, suggesting that CD16 low expressing neutrophils were apoptotic. Separation of CD16 "high" and CD16 "low" expressing neutrophils by fluorescence-activated cell sorting revealed that morphologically apoptotic cells exhibited the CD16 low phenotype. We did not observe similar marked changes in expression of other neutrophil surface molecules (including other phosphatidylinositol (PI)-linked molecules), indicating that generalized loss of surface molecules does not occur during apoptosis. We believe this to be the first reported cell type-specific membrane alteration in a surface glycoprotein associated with apoptosis, suggesting that the program of cell death in the neutrophil, in addition to morphologic and nuclear changes, includes alterations in expression of surface receptors.     

High-resolution crystal structures of human hemoglobin with mutations at tryptophan 37beta: structural basis for a high-affinity T-state,

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)beta is replaced with Tyr (betaW37Y), Ala (betaW37A), Glu (betaW37E), or Gly (betaW37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a "local Rfree" parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)beta results in (1) a change in quaternary structure at the alpha1beta2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3) beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-Nepsilon2His(F8) bond in the alpha and beta subunits of the betaW37G and betaW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order betaW37Y < betaW37A < betaW37E approximately betaW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.     

Extrarenal rhabdoid tumors of soft tissue: a clinicopathologic and immunohistochemical study of 18 cases

Rhabdoid tumor is a well-accepted clincopathologic entity among childhood renal neoplasms; similar tumors have been described in extrarenal locations. We present the clinicopathologic profile and the immunohistochemical features of a series of soft tissue rhabdoid tumors. Twenty-eight cases coded as extrarenal rhabdoid tumor (ERRT), RT, possible ERRT, and "large cell sarcoma" were retrieved from the Armed Forces Institute of Pathology soft tissue registry. The tumors were reclassified according to strict criteria by light microscopy, clinical information, immunohistochemistry, and, in some cases, electron microscopy. Soft tissue rhabdoid tumor (STRT) was defined as (1) a tumor composed of noncohesive single cells, clusters, or sheets of large tumor cells with abundant glassy eosinophilic cytoplasm, an eccentric vesicular nucleus, and an extremely large nucleolus; (2) positivity for vimentin and/or cytokeratin or other epithelial markers by immunostaining; and (3) exclusion of other tumor types with rhabdoid inclusions (melanoma, other sarcomas, carcinoma). Eighteen cases met our criteria for soft tissue rhabdoid tumors. The median patient age was 13 years (range, 6 months to 56 years). Ninety-four percent of STRT cases were positive for vimentin and 59% for pan-cytokeratin. Sixty-three percent and 60% were positive for CAM 5.2 and EMA, respectively. Seventy-nine percent stained for at least one epithelial marker; 76% stained for both vimentin and epithelial markers simultaneously. Forty-two percent stained for MSA, and 14% for CEA and SMA. CD99, synaptophysin, CD57 (Leu-7), NSE, and focal S100 protein were identified in 75%, 66%, 56%, 54%, and 31% of the STRT cases, respectively. All STRT cases examined were negative for HMB-45, chromogranin, BER-EP4, desmin, myoglobin, CD34, and GFAP. Follow-up examination in 61% of the STRT patients revealed that 64% of patients died of disease within a median follow-up interval of 19 months (range, 4 months to 5 years); 82% had metastases to lung, lymph nodes, or liver; 22% had local recurrences before metastasis; and 18% were alive without known disease status (median, 5.5 years). Soft tissue rhabdoid tumor is a highly aggressive sarcoma, predominantly of childhood. Besides having nearly consistent coexpression of vimentin and epithelial markers, STRTs show positivity for multiple neural/neuroectodermal markers that overlap with those of primitive neuroectodermal tumor.     

UV resonance Raman-selective amide vibrational enhancement: quantitative methodology for determining protein secondary structure

We have directly determined the amide band resonance Raman spectra of the "average" pure alpha-helix, beta-sheet, and unordered secondary structures by exciting within the amide pi-->pi* transitions at 206.5 nm. The Raman spectra are dominated by the amide bands of the peptide backbone. We have empirically determined the average pure alpha-helix, beta-sheet, and unordered resonance Raman spectra from the amide resonance Raman spectra of 13 proteins with well-known X-ray crystal structures. We demonstrate that we can simultaneously utilize the amide I, II, and III bands and the Calpha-H amide bending vibrations of these average secondary structure spectra to directly determine protein secondary structure. The UV Raman method appears to be complementary, and in some cases superior, to the existing methods, such as CD, VCD, and absorption spectroscopy. In addition, the spectra are immune to the light-scattering artifacts that plague CD, VCD, and IR absorption measurements. Thus, it will be possible to examine proteins in micelles and other scattering media.     

The rotator cuff-deficient arthritic shoulder: diagnosis and surgical management

The symptomatic rotator cuff-deficient, arthritic glenohumeral joint poses a complex problem for the orthopaedic surgeon. Surgical management can be facilitated by classifying the disorder in one of three diagnostic categories: (1) rotator cuff-tear arthropathy, (2) rheumatoid arthritic shoulder with cuff deficiency, or (3) degenerative arthritic (osteoarthritic) shoulder with cuff deficiency. If it is not possible to repair the cuff defect, surgical management may include prosthetic arthroplasty, with the recognition that only limited goals are attainable, particularly with respect to strength and active motion. Glenohumeral arthrodesis is a salvage procedure when other surgical measures have failed. Arthrodesis is also indicated in patients with deltoid muscle deficiency. Humeral hemiarthroplasty avoids the complications of glenoid loosening and is an attractive alternative to arthrodesis, resection arthroplasty, and total shoulder arthroplasty. The functionally intact coracoacromial arch should be preserved to reduce the risk of anterosuperior subluxation. Care should be taken not to "overstuff" the gleno-humeral joint with a prosthetic component. In cases of significant internal rotation contracture, subscapularis lengthening is necessary to restore anterior and posterior rotator cuff balance. If the less stringent criteria of Neer's "limited goals" rehabilitation are followed, approximately 80% to 90% of patients treated with humeral hemiarthroplasty can have satisfactory results.     

Prediction of indications for valve replacement among asymptomatic or minimally symptomatic patients with chronic aortic regurgitation and normal left ventricular performance

BACKGROUND: Optimal criteria for valve replacement are unclear in asymptomatic/minimally symptomatic patients with aortic regurgitation (AR) and normal left ventricular (LV) performance at rest. Moreover, previous studies have not assessed the prognostic capacity of load-adjusted LV performance ("contractility") variables, which may be fundamentally related to clinical state. Therefore, 18 years ago, we set out to test prospectively the hypothesis that objective noninvasive measures of LV size and performance and, specifically, of load-adjusted variables, assessed at rest and during exercise (ex), could predict the development of currently accepted indications for operation for AR. METHODS AND RESULTS: Clinical variables and measures of LV size, performance, and end-systolic wall stress (ESS) were assessed annually in 104 patients by radionuclide cineangiography at rest and maximal ex and by echocardiography at rest; ESS was derived during ex. During an average 7.3-year follow-up among patients who had not been operated on, 39 of 104 patients either died suddenly (n = 4) or developed operable symptoms only (n = 22) or subnormal LV performance with or without symptoms (n = 13) (progression rate=6.2%/y). By multivariate Cox model analysis, change (delta) in LV ejection fraction (EF) from rest to ex, normalized for deltaESS from rest to ex (deltaLVEF-deltaESS index), was the strongest predictor of progression to any end point or to sudden cardiac death alone. Unadjusted deltaLVEF was almost as efficient. Symptom status modified prediction on the basis of the deltaLVEF-deltaESS index. The population tercile at highest risk by deltaLVEF-deltaESS progressed to end points at a rate of 13.3%/y, and the lowest-risk tercile progressed at 1.8%/y. CONCLUSIONS: Currently accepted symptom and LV performance indications for valve replacement, as well as sudden cardiac death, can be predicted in asymptomatic/minimally symptomatic patients with AR by load-adjusted deltaLVEF-deltaESS index, which includes data obtained during exercise.