Intracytoplasmic Vesicles Research Articles

Abstract 1500: Breast cancer-derived extracellular vesicles modulate the endocytic metabolism and morphodynamics of the endothelial cells of the blood-brain barrier

Abstract Breast cancer, particularly the triple-negative subtype, is the primary cause of brain metastasis in women, affecting 10-20% of breast cancer patients. Breast cancer patients who develop brain metastasis have few treatment options and a dismal prognosis with a median survival time of 6-16 months. We demonstrated, for the first time, that EVs derived from brain-seeking breast cancer cells (BrEVs) breach the intact blood-brain barrier (BBB) via transcytosis and cause a significantly increased incidence of breast-to-brain metastasis. Within this context, we are now studying the interactions between BrEVs and the endothelial cells (ECs) of the BBB. BrEVs were isolated by ultracentrifugation, characterized following the latest guidelines published by the International Society of Extracellular Vesicles (MISEV 2018) and tested in vitro using primary human brain ECs and in vivo using our mouse model of breast-to-brain metastasis. ECs were treated with BrEVs and analyzed using antibody-based assays, impedance measurements, genetic engineering and fixed and live cell imaging. The imaging results were analyzed using novel machine learning approaches to detect and describe, for the first time, EV-mediated morphodynamic changes at the brain EC barrier level. In vivo, we administered BrEVs intravenously to our models of breast-to-brain metastasis, followed by the analysis of mouse brains and their cerebral microvessels to examine microscopic structure and protein expression. Our studies show that (1) BrEVs target BBB ECs both in vitro and in vivo, causing significant variations of multiple proteins involved in the intracytoplasmic vesicles' long-recycling loop, (2) BrEVs are responsible for specific EC barrier function and morphodynamic changes and (3) BrEVs mediate, at least in part, their effect(s) through the delivery of specific micro RNAs. Taken together, our studies demonstrate that BrEVs have a significant impact on brain EC endocytic metabolism and morphodynamics, promoting transcytosis and sustaining breast-to-brain metastasis. Our findings increase our understanding of the early events that facilitate brain metastasis and have the potential to identify key regulators of brain metastasis formation. These results also inform and contribute to the development of EV-based therapeutic and diagnostic strategies for brain metastasis. (This work was supported by NIH R21 CA253051-01, the Breast Cancer Research Foundation, NIH T32 5T32HL007917-22 and the Nile Albright Research Foundation.) Citation Format: Sara Busatto, Golnaz Morad, Marsha A Moses. Breast cancer-derived extracellular vesicles modulate the endocytic metabolism and morphodynamics of the endothelial cells of the blood-brain barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1500.

Cell death induction and intracellular vesicle formation in human colorectal cancer cells treated with Δ9-Tetrahydrocannabinol

BackgroundΔ9-Tetrahydrocannabinol (Δ9-THC) is a principal psychoactive extract of Cannabis sativa and has been traditionally used as palliative medicine for neuropathic pain. Cannabidiol (CBD), an extract of hemp species, has recently attracted increased attention as a cancer treatment, but Δ9-THC is also requiring explored pharmacological application.ObjectiveThis study evaluated the pharmacological effects of Δ9-THC in two human colorectal cancer cell lines. We investigated whether Δ9-THC treatment induces cell death in human colorectal cancer cells.MethodsWe performed an MTT assay to determine the pharmacological concentration of Δ9-THC. Annxein V and Western blot analysis confirmed that Δ9-THC induced apoptosis in colorectal cancer cells. Metabolic activity was evaluated using MitoTracker staining and ATP determination. We investigated vesicle formation by Δ9-THC treatment using GW9662, known as a PPARγ inhibitor.ResultsThe MTT assay showed that treatment with 40 μM Δ9-THC and above inhibited the proliferation of colorectal cancer cells. Multiple intracytoplasmic vesicles were detected upon microscopic observation, and fluorescence-activated cell sorting analysis showed cell death via G1 arrest. Δ9-THC treatment increased the expression of cell death marker proteins, including p53, cleaved PARP-1, RIP1, and RIP3, suggesting that Δ9-THC induced the death of colorectal cancer cells. Δ9-THC treatment also reduced ATP production via changes in Bax and Bcl-2. Δ9-THC regulated intracytoplasmic vesicle formation by modulating the expression of PPARγ and clathrin, adding that antiproliferative activity of Δ9-THC was also affected.ConclusionIn conclusion, Δ9-THC regulated two functional mechanisms, intracellular vesicle formation and cell death. These findings can help to determine how cannabinoids can be used most effectively to improve the efficacy of cancer treatment.

Digital Measurement of the Diameter of the Collagen Fibers by Transmission Electron Microscopy for Diagnosis of Ehlers-Danlos Syndrome-Like in Small Animals

Background: Ehlers-Danlos-Like Syndrome (EDS) is a rare disease in small animals, whose diagnosis is based on the clinical findings and histopathological examination. The definitive diagnosis may require transmission electron microscopy. Despite this, the ultrastructural changes are poorly described in the literature. The aim of the present study is to describe the ultrastructural findings of collagen fibers and fibroblasts present in the dermis of two animals with EDS, and to evaluate the digital measurement of the diameter of the collagen fibers by transmission electron microscopic images.Cases: Two animals were evaluated with EDS by transmission electron microscopy. The first animal was an 1-year-old mixed breed female cat, due to spontaneous skin laceration, increased skin elasticity and an extensibility index corresponding to 25%. The second animal evaluated was a 5-month-old Golden Retriever female dog due to articular hypermobility, increased skin elasticity and an extensibility index corresponding to 16.6%. After a skin biopsy of the interscapular and lumbar regions, the samples were fixed in formalin 10% and glutaraldehyde for, respectively, histopathological examination by HE staining, and transmission electron microscopy. The histopathology of the affected cat revealed collagen fibers shortened and sometimes fragmented. The histopathology of the affected dog revealed disarranged and more eosinophilic staining collagen fibers. The collagen fibers were also of unequal sizes, shortened and slightly undulate. At the transmission electron microscopy of the affected cat was evidenced a greater spacing of the collagen fibers of variable diameters. Further this, the fibroblasts showed elongated nuclei with heterochromatic regions, which was surrounded externally by scant cytoplasm. The cytoplasm showed elongated and discrete organelles. At the transmission electron microscopy of the affected dog was evidenced a greater spacing of the collagen fibers of variable diameters. Further this, fibroblasts exhibited intense cytoplasmic vacuolization with similar appearance to that found in the dying process by autophagy. In addition, the images obtained by transmission electron microscopy were submitted to digital analysis to measure the diameter of the collagen fibers using the software Image J. For this purpose, it was obtained the average of the diameter of 10 collagen fibers in cross-section into four quadrants of 1μm each. The digital analysis of collagen fibers revealed significant alterations in the ultrastructure of collagen. In addition, it was verified cellular changes, such as the large amount of intracytoplasmic vesicles and the small amount of collagen fibers dispersed around the cells.Discussion: Microscopic abnormalities visualized by HE staining in this present study were compatible with the literature Transmission electron microscopy is fundamental to confirm the suspicion, since it revealed dermal alterations in the ultrastructure of collagen and fibroblasts. These findings indicate possible failure mechanisms of secretion and release of cellular products, as well as collagen. The digital measurement of the diameter of the collagen fibers contributed to the confirmation of the disease, since it was made randomly and reduced the subjectivity inherent of the evaluator. However, there are no studies using this method to allocate a range of significance for the diameter of collagen fibers that may be considered suggestive for the syndrome in small animals.

Appearances Can Be Deceiving - Viral-like Inclusions in COVID-19 Negative Renal Biopsies by Electron Microscopy.

Since the discovery of the causative agent for the novel severe acute respiratory syndrome (SARS)–like pneumonia syndrome pandemic that started in China in 2019 (1,2), a coronavirus named SARS coronavirus 2 (SARS-CoV-2), electron microscopy images have populated the medical literature (2) and media outlets alike displaying the characteristic 60–140 nm round particles surrounded by a “corona” of 9–12 nm distinctive spikes (2). Although many of these images were obtained after “ in vitro ” infection of cultured cells with SARS-CoV-2 (2) and are thus likely a true representation of viral particles, we have observed morphologically indistinguishable inclusions within podocytes and tubular epithelial cells both in patients negative for coronavirus disease 2019 (COVID-19) as well as in renal biopsies from the pre–COVID-19 era. Although direct infection of the kidney is theoretically possible, given the presence of angiotensin-converting enzyme 2 (the receptor used by SARS-CoV-2 to gain access to cells) within proximal tubular epithelium (3) and podocytes (4), the virus has not been detected by real-time RT-PCR in urine samples from patients with COVID-19 (5⇓–7). Additionally, for the virus to have access to kidney parenchyma, viremia should occur, and this has only been detected in a minority of patients (6⇓–8). We would, therefore, like to issue a note of caution for inferring viral tissue infection by morphology alone using electron microscopy images from tissues obtained from biopsies or autopsy material in patients with COVID-19. Moreover, caution should be used when interpreting immunohistochemical results, especially within proximal tubules, which are prone to nonspecific staining by a variety of antibodies due to their intense reabsorptive capacity. Additionally, more specific techniques such as immunoelectron microscopy using specific viral antigens (9), or in situ hybridization for viral RNA, are likely necessary to undoubtedly confirm tissue infection in …

Foamy Gland Variant of Prostatic Adenocarcinoma (FGVPA), diagnostic pitfalls: A case report and literature review

A transurethral resection of prostate (TURP) specimen was received from a 72-year-old male who presented with a recent history of urinary tract infection and increased serum prostate specific antigen (PSA) of 15 ng/mL. There was an atypical glandular proliferation of which a predominant population demonstrated abundant pale xanthomatous cytoplasm with small hyperchromatic nuclei and inconspicuous nucleoli. A minor component of conventional prostatic acinar adenocarcinoma was identified focally in the background. Immunohistochemistry was used to confirm the diagnosis of adenocarcinoma. FGVPA is a rare variant, with foamy cytoplasm due to intracytoplasmic vesicles not lipid or neutral mucin, and usually with small pyknotic nuclei with minimal atypia. Gleason scoring is performed by the underlying architectural pattern. Immunohistochemistry of basal cell markers and AMACR are often used to confirm a diagnosis of prostatic adenocarcinoma; however, it is to be noted that variants of prostate cancer including FGVPA are labelled with AMACR in only 60–70% of cases. It is important for the pathologist to be aware of FGVPA as in its pure form can easily be missed especially on needle core biopsy. The differential diagnoses of benign mimics with clear cytoplasm include xanthoma, Cowper’s glands, clear cell cribriform hyperplasia and mucinous metaplasia.

Deformed Wing Virus Infection in Honey Bees (Apis mellifera L.).

Deformed wing virus (DWV) is a single-stranded RNA virus of honey bees (Apis mellifera L.) transmitted by the parasitic mite Varroa destructor. Although DWV represents a major threat to honey bee health worldwide, the pathological basis of DWV infection is not well documented. The objective of this study was to investigate clinicopathological and histological aspects of natural DWV infection in honey bee workers. Emergence of worker honey bees was observed in 5 colonies that were clinically affected with DWV and the newly emerged bees were collected for histopathology. DWV-affected bees were 2 times slower to emerge and had 30% higher mortality compared to clinically normal bees. Hypopharyngeal glands in bees with DWV were hypoplastic, with fewer intracytoplasmic secretory vesicles; cells affected by apoptosis were observed more frequently. Mandibular glands were hypoplastic and were lined by cuboidal epithelium in severely affected bees compared to tall columnar epithelium in nonaffected bees. The DWV load was on average 1.7 × 106 times higher (P < .001) in the severely affected workers compared to aged-matched sister honey bee workers that were not affected by deformed wing disease based on gross examination. Thus, DWV infection is associated with prolonged emergence, increased mortality during emergence, and hypoplasia of hypopharyngeal and mandibular glands in newly emerged worker honey bees in addition to previously reported deformed wing abnormalities.

Ultrastructural characteristics of primary renal epithelial tumours with granular oncocytic cytoplasm

Background/Aim. Ultrastructural analysis of tumours has shown many common characteristics of certain neoplasms, as well as their specificities. Primary renal epithelial tumours with granular oncocytic cytoplasm is a very heterogeneous group in their histological origin and biological behaviour, which results in a difference in treatment and prognosis of the disease, making accurate morphological diagnosis necessary. The aim of this study was to determine ultrastructural similarities and differences among primary renal epithelial tumours with granular oncocytic cytoplasm. Methods. The analysis of archival and routine material in the archives of the Department of Pathology, University Hospital in Plzen, Czech Republic, as well as archival and routine material in the Centre for Pathology and Histology, Clinical Centre of Vojvodina in Novi Sad, discovered 346 primary renal epithelial tumours with granular oncocytic cytoplasm and divided them into 5 groups: 1) renal oncocytoma (RO) (234 tumours), 2) oncocytic papillary renal cell carcinoma (OPRCC) (12 tumours), 3. sporadic renal hybrid oncocytic/ chromophobe tumour (HOCT) without evidence of Birt Hogg Dub? syndrome (BHD) (14 tumours), 4) chromophobe renal cell carcinoma (ChRCC) (21 tumours) and 5) granular renal cell carcinoma (RCC) [64 tumours + 1 clear cell RCC (CRCC) with hyaline globules]. Ultrastructural analysis of tumour cells at the subcellular level was done using electron microscope (Philips electron microscope TEM 208) at the Department of Pathology, University Hospital in Plzen, Czech Republic. Cellular organelles and pigments were evaluated in 5 tumours from each group according to the simple random sample principle with a total of 30 analysed tumours. Results. In all analysed primary renal epithelial tumours with granular oncocytic, cytoplasm dominant organelles were mitochondria. Specific ultrastructural characteristics of RO were round mitochondria with lamellar cristae, whereas ChRCC had numerous typical cytoplasmic microvesicles 100?700 nm large and mitochondria with tubulovesicular, lamellar and circular cristae. Ultrastructural specificity of hybrid tumours were rare microvesicles and numerous mitochondria of O-PRCC mitochondria with lamellar cristae and small intracytoplasmic vesicles, 100?200 nm large, and of granular RCC, in addition to mitochondria, also glassy hyaline globules (GHG). Conclusion. Ultrastructural analysis indicates mitochondria as the dominant organelle in the analysed tumours. Electron microscopy showed specificities, i.e., differences in appearance of cristae, presence and size of vesicles as well as deposition of pigment in and out of cytoplasm and glassy hyaline globules.

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56±6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.

Detection of Parapoxvirus in goats during contagious ecthyma outbreak in Ceará State, Brazil by transmission electron microscopy techniques

Contagious ecthyma or contagious pustular dermatitis, is a viral skin disease that occurs in sheep, goats and wild ruminants and is characterized by the formation of papules, nodules or vesicles that progress into thick crusts or heavy scabs on the lips, gingiva and tongue, caused by a member of the Parapoxvirus genus. Humans are occasionally affected constituting important zoonosis. The disease not only has an economic impact on farmers worldwide but also has a considerable negative effect on animal welfare. In this study, a contagious ecthyma outbreak which occurred in one flock with 90 goats located in the Ceara State, Brazil, was described. Twenty-two goats older than 6 months were affected. The animals presented crusted lesions on the buccal region, tongue, udder and teats, which began with swelling in the mouth area. Dried crusts and serum collected were processed for transmission electron microscopy utilizing, negative staining (rapid preparation), Immunocytochemistry (immunolabelling with colloidal gold particles) and resin embedding techniques. At the Philips EM 208 transmission electron microscopy all the samples were analyzed by negative staining technique and a great number of parapoxvirus particles ovoid or cylindrical, showing two morphological forms, a mulberry (M) form with a distinctive crisscross filament pattern derived from the superimposition of upper and lower virion surfaces and a capsular (C) form caused by stain penetration and distention of the virion core, measuring 300 x 180 nm was observed. Antigen antibody reaction was increased by the colloidal gold particles. In the ultrathin sections of crusts, we verified the presence of three types of intracytoplasmic inclusion bodies, type A or Bollinger inclusion bodies, outlined by membrane, presented in it is interior, oval, mature or complete viral particles, measuring on the average, 225nm x 130 nm, showing an inner dumbell-shaped core, two lateral bodies and an external envelope, or cigar shaped core. In the type B electron dense inclusions bodies, were visualized parapoxvirus particles budding of dense and amorphous material. Fibrillar intracytoplasmic inclusions were also found located between the virions, consisting of groups of fibrils, arranged in groups or concentrically in the middle of the granular material. Intracytoplasmic vesicles outlined by membranes, measuring 560 x 420 nm, containing granular material in its interior were also observed. The nuclei showed an aspect deformed.

The role of Aspartyl aminopeptidase (Ape4) in Cryptococcus neoformans virulence and authophagy.

In order to survive and cause disease, microbial pathogens must be able to proliferate at the temperature of their infected host. We identified novel microbial features associated with thermotolerance in the opportunistic fungal pathogen Cryptococcus neoformans using a random insertional mutagenesis strategy, screening for mutants with defective growth at 37°C. Among several thermosensitive mutants, we identified one bearing a disruption in a gene predicted to encode the Ape4 aspartyl aminopeptidase protein. Ape4 metalloproteases in other fungi, including Saccharomyces cerevisiae, are activated by nitrogen starvation, and they are required for autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway. However, none have been previously associated with altered growth at elevated temperatures. We demonstrated that the C. neoformans ape4 mutant does not grow at 37°C, and it also has defects in the expression of important virulence factors such as phospholipase production and capsule formation. C. neoformans Ape4 activity was required for this facultative intracellular pathogen to survive within macrophages, as well as for virulence in an animal model of cryptococcal infection. Similar to S. cerevisiae Ape4, the C. neoformans GFP-Ape4 fusion protein co-localized with intracytoplasmic vesicles during nitrogen depletion. APE4 expression was also induced by the combination of nutrient and thermal stress. Together these results suggest that autophagy is an important cellular process for this microbial pathogen to survive within the environment of the infected host.

A preliminary report on the characterization of epiretinal membranes excised from patients affected by macular pucker

Among the various pathologies of the vitreoretinal interface, idiopathic macular pucker (MP) is one of the most puzzling. MPs are characterized by the formation of an epiretinal membrane (ERM) that grows in front of the fovea and results in a major impairment of vision. MPs are also frequently complicated by the deformation of the regular macular anatomy, with stretching and deformation of all retinal layers and loss of the foveal pit. This result is usually referred to the presence of myofibroblast-like cells on the retinal surface that alter macular anatomy possibly exerting a significant traction on ERMs. In order to shed light on the physiopathological events that lead to the development of idiopathic MPs, we carried out an immunofluorescence study by confocal microscopy on ERMs excised from 32 eyes with diagnosis of MP using a panel of antibodies including anti-collagen I, anti-collagen IV, anti-collagen VI, anti-smooth muscle actin (SMA), anti-glial fibrillary acidic protein (GFAP) and anti-vimentin antibodies. Some samples were also challenged with anti-heat shock protein (HSP) 47, anti-HSP 90 and anti-receptor II of the transforming growth factor (TGFβRII) antibodies. ERMs broadly varied in thickness. Mostly, they were formed by a layer of collagen 1 adjacent the internal limiting membrane, collagen IV and a layer of vimentin+ cells. Cells also co-expressed SMA or GFAP. Collagen VI, in contrast, was almost always scanty, frequently within the intracytoplasmic vesicles. Some membranes showed a very high content of collagen IV, so abundant to resemble the distribution of interstitial collagens. Cells were almost always restricted to the vitreal side of the membrane; only rarely they could be seen embedded between layers of extracellular matrix. They were frequently HSP90+ and sometimes they contained collagen-immunoreactive materials in their cytoplasm. They also showed TGFβRII within intracytoplasmic vesicles. All in all, ERMs have many features resembling those characterizing fibrotic processes.

Formation of intracellular lumina in human prostate carcinoma (DU145) cells, maturation into signet cells, and the cribriform morphology of tumors

ABSTRACTThe intracellular or intracytoplasmic lumen (IL) is an enigmatic histological structure that occurs in various tumor cells. A reassessment of diverse ILs fine-structure micrographs obtained out of previous studies encompassing the human prostate carcinoma (DU145) cell line and xenotransplanted carcinomas enabled us to propose aspects of ILs development in cancer cells: a combination of altered expressions in intercellular contacts and their cytoskeletal components would favor a disarray of self-apical polarity orientation; those defects, associated with a local, entwined enriched membranous structures growing as microvilli-like formations out of a disrupted endoplasm and trans-Golgi sorting, create ILs in cells’ perikarya. These misplaced intracytoplasmic domains can become enlarged through spaces made between the finger-like structures by accruing membranes of coalescent intracytoplasmic vesicles then adding microvilli and glycocalyx to constitute ILs. Cationic mucins added with or without a progressive or total loss of microvilli and content generate signet or ring cell, while ILs enlarge. Variable build-ups of these cells’ populations in carcinomas result in architectural mix-up of adjacent cells around these voids, misconstrued as new lumen, and establish a “cribriform” tumor pattern that often implies a poor cancer prognosis. Alternatively, cytotoxic changes caused by anticancer pro-oxidant treatment favor membrane alterations and exaggerate the ILs in xenotransplants into intracellular crypts that accompany other tumor degenerative changes.

Development and characterization of an in vitro model of colorectal adenocarcinoma with MDR phenotype.

The major cause of failure in cancer chemotherapy is the development of multidrug resistance (MDR), and the characterization of biological factors involved in this response to therapy is particularly needed. A doxorubicin‐resistant HCT‐8/R clone was selected from sensitive parental cells and characterized analyzing several parameters (cell cycle phase distribution, apoptotic activity, expression, distribution and functionality of the P‐gp efflux pump, the response to other chemotherapy agents, its ultrastructural features, invasiveness, and transcriptomic profile). HCT‐8/R cells showed a peculiar S phase distribution, characterized by a single pulse of proliferation, resistance to drug‐mediated apoptosis, increased expression and functionality of P‐gp and overexpression of stem cell markers (CD44 and aldehyde dehydrogenase 1A2). At the ultrastructural level, HCT‐8/R presented a greater cell volume and several intracytoplasmic vesicles respect to HCT‐8. Moreover, the resistant clone was characterized by cross resistance to other cytotoxic drugs and a greater capacity for migration and invasion, compared to parental cells. Our data reinforce the concept that the MDR phenotype in HCT‐8/R cells is multifactorial and involves multiple mechanisms, representing an interesting tool to understand the biological basis of MDR and to test strategies that overcome resistance to chemotherapy.

Defective hCNT1 transport contributes to gemcitabine chemoresistance in ovarian cancer subtypes: Overcoming transport defects using a nanoparticle approach

Nucleoside analogs are used as chemotherapeutic options for the treatment of platinum-resistant ovarian cancers. Human concentrative nucleoside transporter 1 (hCNT1) is implicated in sensitizing solid tumors to nucleoside analogs although its role in determining drug efficacy in ovarian cancers remains unclear. Here we examined the functional expression of hCNT1 and compared its contributions toward gemcitabine efficacy in histological subtypes of ovarian cancer. Radioactivity analysis identified hCNT1-mediated 3H-gemcitabine transport in ovarian cancer cells to be significantly reduced compared with that of normal ovarian surface epithelial cells. Biochemical and immunocytochemical analysis identified that unlike normal ovarian cells which expressed high levels of hCNT1 at the apical cell surface, the transporter was either diminished in expression and/or mislocalized in cell lines of various subtypes of ovarian cancer. Retroviral expression of hCNT1 selectively rescued gemcitabine transport in cell lines representing serous, teratocarcinoma, and endometrioid subtypes, but not clear cell carcinoma (CCC). In addition, exogenous hCNT1 predominantly accumulated in intracytoplasmic vesicles in CCC suggesting defective cellular trafficking of hCNT1 as a contributing factor to transport deficiency. Despite diminution of hCNT1 transport in the majority of ovarian cancers and apparent trafficking defects with CCC, the chemotherapeutic efficacy of gemcitabine was broadly enhanced in all subtypes when delivered via engineered nanoparticles (NPs). Additionally, by bypassing the transport requirement, the delivery of a gemcitabine-cisplatin combination in NP formulation increased their synergistic interactions. These findings uncover hCNT1 as a putative determinant for nucleoside analog chemoresistance in ovarian cancer and may help rationalize drug selection and delivery strategies for various histological subtypes of ovarian cancer.

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

Ciliary inclusion disease: report of a new primary ciliary dyskinesia variant.

Biopsies from 6 children with clinical presentations suggestive of primary ciliary dyskinesia (PCD) displayed respiratory epithelial cells with disorganized accumulations of basal bodies within the cytoplasm and large intracytoplasmic vesicles into which projected numerous microvilli and cilia. Microvilli, but few cilia, were present at the cell surface. Ultrastructural study revealed a variety of nonspecific abnormalities but demonstrated the cilia generally to be morphologically normal, suggesting that the cause of cilia malfunction was not any recognized primary cause or secondary effect. Repeat studies from 2 patients produced similar findings. It is proposed that this entity, termed ciliary inclusion disease, represents a variant form of PCD manifesting as a consequence of improper ciliogenesis caused by inhibited cytoskeleton-regulated migration of basal bodies to the luminal surface of the airway respiratory epithelial cells.

Macular Corneal Dystrophy: In Vivo Confocal and Structural Data

To demonstrate the corneal morphologic aspects obtained with invivo confocal microscopy (CM) and light and electron microscopy of specimens obtained from the same patients with macular corneal dystrophy (MCD). Case series. Five consecutive patients affected by MCD undergoing penetrating keratoplasty (PK) in 1 eye. The patients were examined with the slit-lamp, optical pachymetry, and CM before undergoing PK. The corneal buttons were processed for light, transmission, and scanning electron microscopy. Corneal invivo CM, corneal light, and electron microscopy. Confocal microscopy showed areas of altered reflectivity in basal epithelial cells, which appeared hyperreflective or completely white. In the anterior stroma, rectilinear hyperreflective areas were shown. The stroma was characterized by a granular appearance of both keratocytes and extracellular matrix. Dark striae of different length and orientation were present in the middle and posterior stroma. The corneal endothelium showed polymegethism and cells containing bright granules in their cytoplasm. The histopathologic study demonstrated areas of thickened Bowman's layer covered by an epithelium reduced in height. The Bowman's layer thickenings were due to the accumulation of free or vesiculated material of different electron density. The keratocytes showed intracytoplasmatic vesicles, whereas the extracellular matrix presented a large quantity of intercellular electron-lucent material and parallel lamellae with an undulated course. Occasional macrophages, filled with vesicles of granular-filamentous material and evident podosomes, were observed. Descemet's membrane was formed by a normal anterior banded zone and a posterior nonbanded zone of honeycombed aspect. The endothelial cells showed a large number of intracytoplasmic vesicles. The structural changes observed with the histopathologic methods give an account and provide an explanation for the pathologic changes demonstrated by CM in the course of MCD. This may contribute to the understanding of invivo imaging, allowing a better, noninvasive study of the disease evolution.

Podocyte-specific deletion of NDST1, a key enzyme in the sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo

Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to- and pedicel organization on- the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1-/-) was developed, having podocyte-specific deletion of NDST1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1-/- mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

Fluorescence Micro-Spectroscopy Study of Individual Photosynthetic Membrane Vesicles and Light-Harvesting Complexes

Single-molecule spectroscopy, by getting rid of unwanted ensemble averaging effects, has proved to be a very valuable tool in the research of individual photosynthetic light-harvesting (LH) complexes. However, to learn about real photosynthetic processes the minimal unit to study is a single photosynthetic membrane complete with all elements of its machinery. In the present work, the fluorescence spectra of excitons in lone intracytoplasmic (IC) photosynthetic membrane vesicles of the wild type purple bacterium Rhodobacter sphaeroides that involve peripheral (LH2) and core (RC-LH1-PufX) antenna pigment-protein complexes were investigated at ambient temperature under continuous-wave laser excitation into the Q(x) absorption band of the bacteriochlorophyll-a (BChl) chromophores at 594 nm. In parallel, the spectra of mutant membrane vesicles occupied by just one type of complexes (either LH2 or RC-LH1-PufX) and the spectra of individual purified LH2 and RC-LH1-PufX complexes were measured. The fluorescence from full IC membranes shows a high sensitivity to excitation intensity, being varied over more than four orders of magnitude between 0.1 W/cm(2) and 2 kW/cm(2). At low to moderate excitation intensities, the spectra of IC membranes could be well reproduced by its component spectra, the ratio of the spectra related to peripheral and core complexes being the only adjustable parameter. The spectra of both intact chromatophores and individual membrane components recorded over 1-50 s experimental time frames are robust, strongly suggesting that large spectral fluctuations hardly play a role in the functional photosynthetic process. The significant, up to 14 times, variation of the LH2 and LH1 emission ratio observed in individual IC membranes could be related to variations in the stoichiometric ratio of the peripheral and core complexes. Evidence was found for the presence of LH2 parts that are detached from efficient energy transfer pathways. Upon strong and prolonged illumination, the membrane spectra reveal significant permanent modifications. These alterations, which mostly concern peripheral antenna complexes, were shown to be due to photo-oxidation of various numbers of BChl molecules in the B850 compartment of LH2.

Functional Interfacing of Rhodospirillum rubrum Chromatophores to a Conducting Support for Capture and Conversion of Solar Energy

Owing to the considerable current interest in replacing fossil fuels with solar radiation as a clean, renewable, and secure energy source, light-driven electron transport in natural photosynthetic systems offers a valuable blueprint for conversion of sunlight to useful energy forms. In particular, intracytoplasmic membrane vesicles (chromatophores) from the purple bacterium Rhodospirillum rubrum provide a fully functional and robust photosynthetic apparatus, ideal for biophysical investigations of energy transduction and incorporation into biohybrid photoelectrochemical devices. These vesicular organelles, which arise by invagination of the cytoplasmic membrane, are the sites of the photochemical reaction centers and the light harvesting 1 (LH1) complex. The LH1 protein is responsible for collecting visible and near-IR radiant energy and funneling these excitations to the reaction center for conversion into a transmembrane charge separation. Here, we have investigated the morphology, fluorescence kinetics and photocurrent generation of chromatophores from Rsp. rubrum deposited directly onto gold surfaces in the absence of chemical surface modifications. Atomic force microscopy showed a significant coverage of the gold electrode surface by Rsp. rubrum chromatophores. By in situ fluorescence induction/relaxation measurements, a high retention of the quantum yield of photochemistry was demonstrated in the photoactive films. Chronoamperometric measurements showed that the assembled bioelectrodes were capable of generating sustained photocurrent under white light illumination at 220 mW/cm(2) with a maximum current of 1.5 μA/cm(2), which slowly declines in about 1 week. This study demonstrates the possibility of photoelectrochemical control of robust chromatophore preparations from Rsp. rubrum that paves the way for future incorporation into functional solar cells.