Cholesterol is most often found distributed nonrandomly in the plane of the bilayer, giving rise to cholesterol-rich and -poor domains. Many of these domains are thought to be crucial for the ...maintenance of membrane structure and function. However, such well-characterized domains generally occur in the membranes that contain relatively large amounts of cholesterol. Cholesterol organization in membranes containing very low amounts of cholesterol has not been investigated extensively. Recent evidence from differential-scanning calorimetric studies suggests that cholesterol may not form uniform monodisperse solutions, as assumed earlier, in the membranes even at very low concentrations. Fluorescent cholesterol analogues, when chosen carefully, offer a powerful approach for studying the distribution and organization of cholesterol in membranes at low concentrations. In this paper, we have studied the organization of cholesterol in membranes at very low concentrations (up to 5 mol % of the total lipid) using a fluorescent cholesterol analogue (NBD-cholesterol) which is labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group at the flexible acyl chain, without any alteration in the structural features necessary for proper membrane incorporation. Our results show that NBD-cholesterol exhibits local organization even at very low concentrations. This is consistent with the recently suggested model of cholesterol organization in membranes at low concentrations, involving the formation of transbilayer, tail-to-tail dimers Harris, J. S., Epps, D. E., Davio, S. R., & Kezdy, F. J. (1995) Biochemistry 34, 3851−3857. The implications of such local cholesterol organization in membranes that have very low cholesterol content in vivo, such as the endoplasmic reticulum and the inner mitochondrial membrane, open up interesting possibilities.
Background: Microdissection testicular sperm extraction (micro-TESE) has replaced conventional testis biopsies as a method of choice for obtaining sperm for in vitro fertilization for men with ...nonobstructive azoospermia. A technical challenge of micro-TESE is that the low magnification inspection of the tubules with a surgical microscope is insufficient to definitively identify sperm-containing tubules, necessitating tissue removal and cytologic assessment. Full field optical coherence tomography (FFOCT) uses white light interference microscopy to generate quick high-resolution tomographic images of fresh (unprocessed and unstained) tissue. Furthermore, by using a nonlaser safe light source (150 W halogen lamp) for tissue illumination, it ensures that the sperm extracted for in vitro fertilization are not photo-damaged or mutagenized. Materials and Methods: A focal Sertoli-cell only rodent model was created with busulfan injection in adult rats. Ex vivo testicular tissues from both normal and busulfan-treated rats were imaged with a commercial modified FFOCT system, Light-CT™, and the images were correlated with gold standard hematoxylin and eosin staining. Results: Light-CT™ identified spermatogenesis within the seminiferous tubules in freshly excised testicular tissue, without the use of exogenous contrast or fixation. Normal adult rats exhibited tubules with uniform size and shape (diameter 328 ±11 μm). The busulfan-treated animals showed marked heterogeneity in tubular size and shape (diameter 178 ± 35 μm) and only 10% contained sperm within the lumen. Conclusion: FFOCT has the potential to facilitate real-time visualization of spermatogenesis in humans, and aid in micro-TESE for men with infertility.
Pancreatic islet dysfunction leading to insufficient glucose-stimulated insulin secretion triggers the clinical onset of diabetes. How islet dysfunction develops is not well understood at the ...cellular level, partly owing to the lack of approaches to study single islets longitudinally in vivo Here, we present a noninvasive, high-resolution system to quantitatively image real-time glucose metabolism from single islets in vivo, currently not available with any other method. In addition, this multifunctional system simultaneously reports islet function, proliferation, vasculature and macrophage infiltration in vivo from the same set of images. Applying our method to a longitudinal high-fat diet study revealed changes in islet function as well as alternations in islet microenvironment. More importantly, this label-free system enabled us to image real-time glucose metabolism directly from single human islets in vivo for the first time, opening the door to noninvasive longitudinal in vivo studies of healthy and diabetic human islets.
Lipid and cholesterol trafficking in NPC Mukherjee, Sushmita; Maxfield, Frederick R.
Biochimica et biophysica acta,
10/2004, Volume:
1685, Issue:
1
Journal Article
Peer reviewed
Niemann–Pick type C, or NPC for short, is an early childhood disease exhibiting progressive neurological degeneration, associated with hepatosplenomegaly in some cases. The disease, at the cellular ...level, is a result of improper trafficking of lipids such as cholesterol and glycosphingolipids (GSLs) to lysosome-like storage organelles (LSOs), which become engorged with these lipids. It is believed that the initial defect in trafficking, whether of cholesterol or a GSL, results in an eventual traffic jam in these LSOs. This leads to the retention of not only other lipids, but also of transmembrane proteins that transiently associate with the late endosomes (LE) in normal cells, on their way to other cellular destinations such as the trans-Golgi network (TGN). In this review, we discuss the biophysical properties of lipids and cholesterol that might determine their intracellular itineraries, and how these itineraries are altered in NPC cells, which have defects in the proteins NPC1 or NPC2. We also discuss some potential therapeutic directions being suggested by recent research.
Basal cell carcinoma (BCC) is the most common skin cancer, and its incidence is rising. Millions of benign biopsies are performed annually for BCC diagnosis, increasing morbidity, and healthcare ...costs. Non‐invasive in vivo technologies such as multiphoton microscopy (MPM) can aid in diagnosing BCC, reducing the need for biopsies. Furthermore, the second harmonic generation (SHG) signal generated from MPM can classify and prognosticate cancers based on extracellular matrix changes, especially collagen type I. We explored the potential of MPM to differentiate collagen changes associated with different BCC subtypes compared to normal skin structures and benign lesions. Quantitative analysis such as frequency band energy analysis in Fourier domain, CurveAlign and CT‐FIRE fibre analysis was performed on SHG images from 52 BCC and 12 benign lesions samples. Our results showed that collagen distribution is more aligned surrounding BCCs nests compared to the skin's normal structures (p < 0.001) and benign lesions (p < 0.001). Also, collagen was orientated more parallelly surrounding indolent BCC subtypes (superficial and nodular) versus those with more aggressive behaviour (infiltrative BCC) (p = 0.021). In conclusion, SHG signal from type I collagen can aid not only in the diagnosis of BCC but could be useful for prognosticating these tumors. Our initial results are limited to a small number of samples, requiring large‐scale studies to validate them. These findings represent the groundwork for future in vivo MPM for diagnosis and prognosis of BCC.
Tuberculosis (TB) is a global health problem with 480000 new cases being reported annually, 9 percent of which exhibit extensive drug resistant TB. With 9.6 cases per lac population early detection ...of drug resistance at peripheral levels is must.
The study was planned to use Fluorescent microscopy (FM) with Fluorescein Diacetate Ethidium Bromide (FDA/EB) stain to evaluate the viability of the mycobacteria to predict the drug resistance.
This cross-sectional analytical study was carried out on all sputum smear positive patients after obtaining the Institutional Ethics Committee clearance over a period of 3 months. Samples were obtained on day 0, 3, 7 and 14. After staining with the working solution, observation was made by two independent observers for viable verses non-viable mycobacteria. FDA will stain the live bacilli and fluoresce green whereas dead bacilli lack the esterase activity and are counterstained by ethidium bromide and appear red. Data was maintained in MS Excel and analysed using tests of proportion and significance.
A total of 30 participants were included based on the inclusion criteria. There was a loss of follow up from 14 patients. 4/16 (25%) patients were found be harbor drug resistant mycobacteria strains bases on decreasing ratio of viable to non-viable bacteria on followup. Results when compared with gold standard of Line Probe Assay (LiPA) was found to be highly significant with Chi square value of 11.73 and p value <0.001
This study establishes the utility of fluorescent microscopy using FDA and EB to detect drug resistance in patients of pulmonary tuberculosis by evaluation of viable and non-viable at peripheral microscopic centers
Fluorescent Microscopy with FDA/EB staining holds a potential to predict drug resistance in sputum smear positive patients at peripheral designated microscopic centers which will reduce financial as well as human resource burden.
Background: Here, we report the first use of a commercial prototype of full-field optical coherence tomography called Light-CTTM. Based on the principle of white light interferometry, Light-CT™ ...generates quick high-resolution three-dimensional tomographic images from unprocessed tissues. Its advantage over the current intra-surgical diagnostic standard, i.e. frozen section analysis, lies in the absence of freezing artifacts, which allows real-time diagnostic impressions, and/or for the tissues to be triaged for subsequent conventional histopathology. Materials and Methods: In this study, we recapitulate known normal histology in nine formalin fixed ex vivo rat organs (skin, heart, lung, liver, stomach, kidney, prostate, urinary bladder, and testis). Large surface and virtually sectioned stacks of images at varying depths were acquired by a pair of 10x/0.3 numerical aperture water immersion objectives, processed and visualized in real time. Results: Normal histology of the following organs was recapitulated by identifying various tissue microstructures. Skin: epidermis, dermal-epidermal junction and hair follicles with surrounding sebaceous glands in the dermis. Stomach: mucosa with surface pits, submucosa, muscularis propria and serosa. Liver: hepatocytes separated by sinusoidal spaces, central veins and portal triad. Kidney: convoluted tubules, medullary rays (straight tubules) and collecting ducts. Prostate: acini and fibro-muscular stroma. Lung: bronchi, bronchioles, alveolar ducts, alveoli and pleura. Urinary bladder: urothelium, lamina propria, muscularis propria, and serosa. Testis: seminiferous tubules with intra-tubular sperms. Conclusion: Light-CTTM is a powerful imaging tool to perform fast histology on fresh and fixed tissues, without introducing artifacts. Its compact size, ease of handling, fast image acquisition and safe incident light levels makes it well-suited for various intra-operative and intra-procedural triaging and decision making applications.
Obesity is associated with an increased incidence of high-grade prostate cancer (PC) and worse prognosis for PC patients. Recently, we showed in men that obesity-related periprostatic white adipose ...tissue (WAT) inflammation, characterized by macrophages surrounding dead or dying adipocytes forming crown-like structures, was associated with high-grade PC. Possibly, interventions that suppress periprostatic WAT inflammation will improve outcomes for men with PC. Here, we tested the hypothesis that supplemental 17β-estradiol (E2) could decrease periprostatic WAT inflammation in obese male mice. Mice were fed a high-fat diet to induce periprostatic WAT inflammation before being treated with supplemental E2. E2 supplementation suppressed caloric intake, induced weight loss, decreased periprostatic WAT inflammation and downregulated the expression of genes linked to inflammation including Cd68, Mcp1 and Tnf. Similar to the effects of E2 supplementation, treatment with diethylstilbestrol, a synthetic estrogen, also suppressed caloric intake and reduced periprostatic WAT inflammation. To determine whether the observed effects of supplemental estrogen could be reproduced by caloric restriction (CR) alone, obese mice were put on a 30% CR diet. Like estrogen treatment, CR was effective in reducing body weight, periprostatic WAT inflammation and the expression of pro-inflammatory genes. Transcriptomic analyses of periprostatic fat showed that obesity was associated with enrichment in inflammatory response pathways, which were normalized by both supplemental E2 and CR. Taken together, these findings strengthen the rationale for future efforts to determine whether either CR or supplemental estrogen will decrease periprostatic WAT inflammation and thereby improve outcomes for men with PC.
Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to ...understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.
Background: Full-field optical coherence tomography (FFOCT) is a real-time imaging technique that generates high-resolution three-dimensional tomographic images from unprocessed and unstained ...tissues. Lack of tissue processing and associated artifacts, along with the ability to generate large-field images quickly, warrants its exploration as an alternative diagnostic tool. Materials and Methods: One section each from the tumor and from adjacent non-neoplastic tissue was collected from 13 human lobectomy specimens. They were imaged fresh with FFOCT and then submitted for routine histopathology. Two blinded pathologists independently rendered diagnoses based on FFOCT images. Results: Normal lung architecture (alveoli, bronchi, pleura and blood vessels) was readily identified with FFOCT. Using FFOCT images alone, the study pathologists were able to correctly identify all tumor specimens and in many cases, the histological subtype of tumor (e.g., adenocarcinomas with various patterns). However, benign diagnosis was provided with high confidence in roughly half the tumor-free specimens (others were diagnosed as equivocal or false positive). Further analysis of these images revealed two major confounding features: (a) Extensive lung collapse and (b) presence of smoker’s macrophages. On a closer inspection, however, the smoker’s macrophages could often be identified as distinct from tumor cells based on their relative location in the alveoli, size and presence of anthracosis. We posit that greater pathologist experience, complemented with morphometric analysis and color-coding of image components, may help minimize the contribution of these confounders in the future. Conclusion: Our study provides evidence for the potential utility of FFOCT in identifying and differentiating lung tumors from non-neoplastic lung tissue. We foresee its potential as an adjunct to intra-surgical frozen section analysis for margin assessment, especially in limited lung resections.
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