Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

Quantitative collagen analysis using second harmonic generation images for the detection of basal cell carcinoma with ex vivo multiphoton microscopy.

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.

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

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.

Supplemental estrogen and caloric restriction reduce obesity-induced periprostatic white adipose inflammation in mice.

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 17ß-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.

Multifunctional in vivo imaging of pancreatic islets during diabetes development.

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.

Multiphoton microscopy for rapid histopathological evaluation of kidney tumours.

OBJECTIVE: To explore the potential of multiphoton microscopy (MPM) for rapid evaluation and triaging of ex vivo kidney tissue. MATERIALS AND METHODS: Fresh neoplastic and non-neoplastic tissues from nephrectomy specimens (n = 40) were imaged with MPM and later submitted for routine histopathology. RESULTS: On MPM, normal kidney architecture was evident and clearly distinguishable from tumour. Forty malignant tumours (20 clear-cell renal cell carcinomas [RCCs], 10 papillary RCCs, five chromophobe RCCs and five papillary urothelial carcinomas [UCs], as diagnosed by haematoxylin and eosin staining) were imaged and subtyped as non-papillary and papillary, based on their architecture. Non-papillary tumours were further classified based on their unique cytoplasmic signatures. Clear-cell RCCs had a predominant population of cells with fat droplets in cytoplasm. Chromophobe RCCs had cells with non-fatty/homogeneous cytoplasm and distinct intra-cytoplasmic granules. Papillary RCCs had single-cell-lined papillae with often abundant histiocytes in their core, whereas PUC had multi-layered urothelium-lined papillae. The diagnostic accuracy of tumour subtyping by two independent uropathologists was 95%. CONCLUSIONS: We showed that MPM can reliably differentiate neoplastic from non-neoplastic kidney tissue and subtype kidney tumours in fresh, unprocessed tissue, MPM might therefore be useful as a rapid real-time diagnostic tool for the evaluation of kidney biopsies, and surgical margins in partial nephrectomies, to improve overall patient management.

Real-time in vivo periprostatic nerve tracking using multiphoton microscopy in a rat survival surgery model: a promising pre-clinical study for enhanced nerve-sparing surgery.

OBJECTIVES: To assess the ability of multiphoton microscopy (MPM) to visualise, differentiate and track periprostatic nerves in an in vivo rat model, mimicking real-time imaging in humans during RP and to investigate the tissue toxicity and reproducibility of in vivo MPM on prostatic glands in the rat after imaging and final histological correlation study. MATERIALS AND METHODS: In vivo prostatic rat imaging was carried out using a custom-built bench-top MPM system generating real-time three-dimensional histological images, after performing survival surgery consisting of mini-laparotomies under xylazine/ketamine anaesthesia exteriorising the right prostatic lobe. The acquisition time and the depth of anaesthesia were adjusted for collecting multiple images in order to track the periprostatic nerves in real-time. The rats were then monitored for 15 days before undergoing a new set of imaging under similar settings. After humanely killing the rats, their prostates were submitted for routine histology and correlation studies. RESULTS: In vivo MPM images distinguished periprostatic nerves within the capsule and the prostatic glands from fresh unprocessed prostatic tissue without the use of exogenous contrast agents or biopsy sample. Real-time nerve tracking outlining the prostate was feasible and acquisition was not disturbed by motion artefacts. No serious adverse event was reported during rat monitoring; no tissue damage due to laser was seen on the imaged lobe compared with the contralateral lobe (control) allowing comparison of their corresponding histology. CONCLUSIONS: For the first time, we have shown that in vivo tracking of periprostatic nerves using MPM is feasible in a rat model. Development of a multiphoton endoscope for intraoperative use in humans is currently in progress and must be assessed.

Microsurgical denervation of rat spermatic cord: safety and efficacy data.

OBJECTIVE: To describe a microsurgical technique for denervation of the spermatic cord and use of multiphoton microscopy (MPM) laser to identify and ablate residual nerves after microsurgical denervation. To evaluate structural and functional changes in the rat testis and vas deferens after denervation. MATERIALS AND METHODS: Nine Sprague-Dawley rats were divided into three experimental groups: sham, microsurgical denervation of the spermatic cord (MDSC), and MDSC immediately followed by laser ablation with MPM. At 2 months after surgery, we assessed testicular volume, functional circulation of the testicular artery with Doppler, patency of the vas deferens, and histology of the testis and vas deferens. RESULTS: There was a significant decrease in the median number of nerves remaining around the vas deferens with MDSC alone (3.5 nerves) or MDSC with MPM (1.5 nerves) compared with sham rats (15.5 nerves) (P = 0.003). Although, MDSC with MPM resulted in the fewest remaining nerves, this result was similar to MDSC alone (P = 0.29). No deleterious effects on spermatogenesis or vas patency were seen in the experimental groups when compared with the sham rats. CONCLUSION: A microsurgical approach can be used to effectively and safely denervate the rat spermatic cord with minimal changes to structure and function of the testis and vas deferens. MPM can be used as an adjunct to identify and ablate residual nerves after MDSC.

Correlation between non-alcoholic fatty liver disease and metabolic parameters in persons with newly diagnosed type 2 diabetes mellitus.

BACKGROUND: There are limited studies investigating the association between type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD) in the region of Bihar, India. AIM: To estimate the prevalence of NAFLD in persons with newly diagnosed T2DM in the population of North Bihar, India. METHODS: This single centre cross-sectional study was undertaken in the Research Centre for Diabetes Hypertension and Obesity, Samastipur, Bihar, India. Data were collected from persons newly diagnosed with T2DM or those diagnosed within 6 months of when the study was conducted between December 2022 to May 2023. RESULTS: A total of 148 people with newly diagnosed T2DM were included (median age 47 years, 46.6% female) and 109 patients with liver disease on ultrasound evaluation. The persons with liver disease consumed more fats and oils (88.1% vs 74.4%, P = 0.042) and they had significantly greater body mass index (27.4 vs 23.0, P < 0.001), waist circumference (37 vs 33, P < 0.001), and waist-to-hip ratio (1.00 vs 0.70, P = 0.025). Females were associated with greater liver disease [odds ratio (OR): 3.09, 95% confidence interval (CI): 1.09-8.80, P = 0.32]. Waist circumference (OR: 1.42, 95%CI: 1.22-1.66, P < 0.001) and low-density lipoprotein cholesterol (OR: 1.01, 95%CI: 1.01-1.02, P = 0.048) were associated with any liver disease. The factors most associated with grade 2/3 liver disease was right upper quadrant pain or heaviness (OR: 5.22, 95%CI: 1.40-19.41, P = 0.14), greater income (OR: 3.58, 95%CI: 1.28-10.04, P = 0.015) and waist circumference (OR: 1.31, 95%CI: 1.02-1.69, P = 0.036). CONCLUSION: NAFLD is common in new/recently diagnosed T2DM and disease burden is high and common among patients who are either high consumers of fats and oils or have obesity-associated markers.

Multiphoton imaging and laser ablation of rodent spermatic cord nerves: potential treatment for patients with chronic orchialgia.

PURPOSE: Microsurgical denervation of the spermatic cord has been done to treat chronic orchialgia. However, identifying the site of spermatic cord nerves is not feasible with an operating microscope or robotic stereoscope. We used multiphoton microscopy, a novel laser imaging technology, to identify and selectively ablate spermatic cord nerves in the rat. MATERIALS AND METHODS: The spermatic cords of adult male Sprague-Dawley® rats were initially imaged in vivo under a low power multiphoton microscopy laser. After assessing the number, diameter and site (vasal vs perivasal) of the nerves a higher power laser using the same objective was used to ablate the nerves. The precision of nerve ablation and the preservation of surrounding structures were determined by histological analysis. We assessed the heterogeneity of the number of nerves with the Wilcoxon signed rank test. RESULTS: The average number of nerves per spermatic cord was 10, which was similar bilaterally (p = 0.13). The vas and perivasal structures had a similar number of nerves (p = 0.4). The median diameter of all nerves was 32 µm. Confirmation of nerve ablation, and preservation of the vas deferens and vasculature were anatomically validated by histological analysis. CONCLUSIONS: Multiphoton microscopy can identify and ablate nerves selectively in vivo in the rat. It can potentially be used for spermatic cord denervation to treat chronic orchialgia. Such imaging may increase the efficacy of nerve ablation and can avoid the potential risks of testicular atrophy and hydrocele associated with spermatic cord microsurgical denervation.

Pilot study of the correlation of multiphoton tomography of ex vivo human testis with histology.

PURPOSE: Although microdissection testicular sperm extraction has become first line therapy for sperm retrieval in men with nonobstructive azoospermia, there are challenges to the procedure, including difficulty differentiating between seminiferous tubules with normal and abnormal spermatogenesis. Multiphoton microscopy illuminates tissue with a near infrared laser to elicit autofluorescence, which enables real-time imaging of unprocessed tissue without labels. We hypothesized that we could accurately characterize seminiferous tubular histology in humans using multiphoton microscopy. MATERIALS AND METHODS: Seven men with normal or abnormal spermatogenesis underwent testicular biopsies, which were imaged by multiphoton microscopy. We assessed these images in blinded fashion. The diagnosis rendered with multiphoton microscopy was then correlated with that of hematoxylin and eosin stained tissue. We evaluated the ability of multiphoton microscopy to differentiate normal from abnormal seminiferous tubules by examining autofluorescence characteristics and diameters, as imaged by multiphoton microscopy. Assessment was repeated with stained slides and results were compared. RESULTS: The overall concordance rate between multiphoton microscopy and stained slides was 86%. The seminiferous tubules of patients with nonobstructive azoospermia were smaller than those of controls when measured by multiphoton microscopy and staining (p <0.05). The proportion of normal tubules and the diameters obtained with multiphoton microscopy were not different from those obtained with hematoxylin and eosin (p >0.05). CONCLUSION: Multiphoton microscopy can be used to differentiate normal from abnormal spermatogenesis. Its characterization of seminiferous tubular architecture is similar to that provided by hematoxylin and eosin staining. Further investigation of the clinical applications of multiphoton microscopy may improve surgical sperm retrieval outcomes for patients with nonobstructive azoospermia.

COVID-19 management landscape: A need for an affordable platform to manufacture safe and efficacious biotherapeutics and prophylactics for the developing countries.

To gain world-wide control over COVID-19 pandemic, it is necessary to have affordable and accessible vaccine and monoclonal antibody technologies across the globe. In comparison to the western countries, Asian and African countries have less percentage of vaccination done which warrants urgent attention. Global manufacturer production capacities, dependency on advanced nations for the supply of vaccines or the raw material, national economy, limited research facilities, and logistics could be the factors. This review article elaborates the existing therapeutic and prophylactic strategies available for COVID-19, currently adopted vaccine and monoclonal antibody platforms for SARS-CoV-2 along with the approaches to bridge the gap prevailing in the challenges faced by low- and middle-income countries. We believe adoption of yeast-derived P. pastoris technology can help in developing safe, proven, easy to scale-up, and affordable recombinant vaccine or monoclonal antibodies against SARS-CoV-2. This platform has the advantage of not requiring a dedicated or specialized facility making it an affordable option using existing manufacturing facilities, without significant additional capital investments. Besides, the technology platform of multiantigen vaccine approach and monoclonal antibody cocktail will serve as effective weapons to combat the threat posed by the SARS-CoV-2 variants. Successful development of vaccines and monoclonal antibodies using such a technology will lead to self-sufficiency of these nations in terms of availability of vaccines and monoclonal antibodies.

Capacity Building for Vaccine Manufacturing Across Developing Countries: The Way Forward.

Approved vaccines prevent 2 to 3 million deaths per year. There is a lack of equitable access to vaccines in the low- and middle-income developing nations. Challenges in the life cycle of vaccine production include process development, lead time, intellectual property, and local vaccine production. A robust and stable manufacturing process and constant raw material supplies over decades is critical. In a continuously evolving vaccine landscape, the need of the hour for developing nations is to manufacture their own vaccines besides having supply security, control over production scheduling and sustainability, control of costs, socio-economic development, and rapid response to local epidemics. There is a need for capacity building of workforce development, technology transfer, and financial support. Technology transfer has improved vaccine access and reduced prices of vaccines. Capacity building for the manufacturing of vaccines in developing countries has always been an area of paramount importance and more so in a pandemic situation.

Identification of spermatogenesis with multiphoton microscopy: an evaluation in a rodent model.

PURPOSE: Microdissection testicular sperm extraction has replaced conventional testis biopsies for men with nonobstructive azoospermia and it has become first line treatment. The current problem is that the decision to retrieve tubules is based only on appearance and there is no guarantee that the tubules removed contain sperm. Multiphoton microscopy enables label-free immediate visualization of many biological processes in living tissue at subcellular resolution. MATERIALS AND METHODS: We used multiphoton microscopy to study the different developmental stages of spermatogenesis using neonatal, pubertal and adult rat testes. We used a testis hypothermia plus ischemia model to study different testicular histopathologies with multiphoton microscopy. To assess the risk of photo damage DNA fragmentation in testis biopsies imaged at different intensities was assessed by TUNEL assay. RESULTS: Multiphoton microscopy identified the stage of spermatogenesis in a seminiferous tubule in fresh tissue without using exogenous labels. We noted significant differences in fluorescence and spectroscopic characteristics between tubules with and without sperm. Sertoli's-cell only tubules had abundant autofluorescence in the 420 to 490 and 550 to 650 nm wavelength ranges while tubules containing sperm had autofluorescence only in the 420 to 490 nm range. On DNA fragmentation assay sperm from tubules imaged by multiphoton microscopy had minimal DNA fragmentation at the laser intensities needed to distinguish tubules with and without sperm. CONCLUSIONS: Multiphoton microscopy has the potential to facilitate real-time visualization of spermatogenesis in humans and aid in clinical applications, such as testicular sperm extraction for men with infertility.

Multiphoton microscopy for structure identification in human prostate and periprostatic tissue: implications in prostate cancer surgery.

OBJECTIVE: • To test whether multiphoton microscopy (MPM) might allow identification of prostatic and periprostatic structures with magnification and resolution similar to gold standard histopathology. MATERIAL AND METHODS: • The present study included 95 robotic radical prostatectomy patients who consented to participate in an Institutional Review Board-approved study starting in 2007. • The types of specimens used for imaging were excised surgical margins and biopsies, and sections obtained from the excised prostate. • The specimens were imaged with a custom-built MPM system. • All images were compared with haematoxylin/eosin histopathology of the same specimen. RESULTS: • MPM of freshly excised, unprocessed and unstained tissue can identify all relevant prostatic and periprostatic structures, such as nerves, blood vessels, capsule, underlying acini and also pathological changes, including prostate cancer. • Histological confirmation and correlation of these structures and pathologies have validated the findings of MPM. CONCLUSIONS: • MPM shows great promise as a tool for real-time intra-surgical histopathology without needing excision or administration of contrast agents. • The results will, however, need to be confirmed in true surgical settings using a miniaturized MPM microendoscope.

DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function.

The MRN complex, composed of MRE11, RAD50 and NBS, plays important roles in responding to DNA double-strand breaks (DSBs). In metazoans, functional studies of genes encoding these proteins have been challenging because complete loss-of-function mutations are lethal at the organismal level and because NBS has multiple functions in DNA damage responses. To study functions of Drosophila NBS in DNA damage responses, we used a separation-of-function mutation that causes loss of the forkhead-associated (FHA) domain. Loss of the FHA domain resulted in hypersensitivity to ionizing radiation and defects in gap repair by homologous recombination, but had only a small effect on the DNA damage checkpoint response and did not impair DSB repair by end joining. We also found that heterozygosity for an nbs null mutation caused reduced gap repair and loss of the checkpoint response to low-dose irradiation. These findings shed light on possible sources of the cancer predisposition found in human carriers of NBN mutations.

Facilitated self-assembly of a prevascularized dermal/epidermal collagen scaffold.

Introduction: Resurfacing complex full thickness wounds requires free tissue transfer which creates donor site morbidity. We describe a method to fabricate a skin flap equivalent with a hierarchical microvascular network. Materials & methods: We fabricated a flap of skin-like tissue containing a hierarchical vascular network by sacrificing Pluronic® F127 macrofibers and interwoven microfibers within collagen encapsulating human pericytes and fibroblasts. Channels were seeded with smooth muscle and endothelial cells. Constructs were topically seeded with keratinocytes. Results: After 28 days in culture, multiphoton microscopy revealed a hierarchical interconnected network of macro- and micro-vessels; larger vessels (>100 µm) were lined with a monolayer endothelial neointima and a subendothelial smooth muscle neomedia. Neoangiogenic sprouts formed in the collagen protodermis and pericytes self-assembled around both fabricated vessels and neoangiogenic sprouts. Conclusion: We fabricated a prevascularized scaffold containing a hierarchical 3D network of interconnected macro- and microchannels within a collagen protodermis subjacent to an overlying protoepidermis with the potential for recipient microvascular anastomosis.

B7-H3 as a Prognostic Biomarker and Therapeutic Target in Pediatric central nervous system Tumors.

B7-H3 (CD276), a member of the B7 superfamily, is an important factor in downregulating immune responses against tumors. It is also aberrantly expressed in many human malignancies. Beyond immune regulatory roles, its overexpression has been linked to invasive metastatic potential and poor prognosis in patients with cancer. Antibody-dependent cell-mediated cytotoxicity strategies targeting B7-H3 are currently in development, and early-phase clinical trials have shown encouraging preliminary results. To understand the role of B7-H3 in pediatric central nervous system (CNS) malignancies, a comprehensive panel of primary CNS tumors of childhood was examined by immunohistochemistry for levels and extent of B7-H3 expression. In addition, B7-H3 m-RNA expression status and association with overall survival in various pediatric CNS tumor types was accessed by curating publicly available patient gene expression data sets derived from bioinformatics analysis and visualization platforms (GlioVis). We demonstrate that B7-H3 is broadly expressed in pediatric glial and nonglial CNS tumors, and its aberrant expression, as determined by immunohistochemical staining intensity, correlates with tumor grade. Moreover, high B7-H3 m-RNA expression is significantly associated with worse survival and could potentially improve prognostication in various brain tumor types of childhood. B7-H3 can be used as a therapeutic target, given its tumor selectivity and the availability of targeted therapeutic agents to this antigen.

Adipsin preserves beta cells in diabetic mice and associates with protection from type 2 diabetes in humans.

Type 2 diabetes is characterized by insulin resistance and a gradual loss of pancreatic beta cell mass and function1,2. Currently, there are no therapies proven to prevent beta cell loss and some, namely insulin secretagogues, have been linked to accelerated beta cell failure, thereby limiting their use in type 2 diabetes3,4. The adipokine adipsin/complement factor D controls the alternative complement pathway and generation of complement component C3a, which acts to augment beta cell insulin secretion5. In contrast to other insulin secretagogues, we show that chronic replenishment of adipsin in diabetic db/db mice ameliorates hyperglycemia and increases insulin levels while preserving beta cells by blocking dedifferentiation and death. Mechanistically, we find that adipsin/C3a decreases the phosphatase Dusp26; forced expression of Dusp26 in beta cells decreases expression of core beta cell identity genes and sensitizes to cell death. In contrast, pharmacological inhibition of DUSP26 improves hyperglycemia in diabetic mice and protects human islet cells from cell death. Pertaining to human health, we show that higher concentrations of circulating adipsin are associated with a significantly lower risk of developing future diabetes among middle-aged adults after adjusting for body mass index (BMI). Collectively, these data suggest that adipsin/C3a and DUSP26-directed therapies may represent a novel approach to achieve beta cell health to treat and prevent type 2 diabetes.

Endocytic recycling compartments altered in cisplatin-resistant cancer cells.

The clinical utility of cisplatin to treat human malignancies is often limited by the development of drug resistance. We have previously shown that cisplatin-resistant human KB adenocarcinoma cells that are cross-resistant to methotrexate and heavy metals have altered endocytic recycling. In this work, we tracked lipids in the endocytic recycling compartment (ERC) and found that the distribution of the ERC is altered in KB-CP.5 cells compared with parental KB-3-1 cells. A tightly clustered ERC is located near the nucleus in parental KB-3-1 cells but it appears loosely arranged and widely dispersed throughout the cytoplasm in KB-CP.5 cells. The altered distribution of the ERC in KB-CP.5 cells is related to the amount and distribution of stable detyrosinated microtubules (Glu-alpha-tubulin), as previously shown in Chinese hamster ovary B104-5 cells that carry a temperature-sensitive Glu-alpha-tubulin allele. In addition, B104-5 cells with a dispersed ERC under nonpermissive conditions were more resistant to cisplatin compared with B104-5 cells with a clustered ERC under permissive conditions. We conclude that resistance to cisplatin might be due, in part, to reduced uptake of cisplatin resulting from an endocytic defect reflecting defective formation of the ERC, possibly related to a shift in the relative amounts and distributions of stable microtubules.