From Androgen Dependence to Independence in Prostate Cancer: Unraveling Therapeutic Potential and Proteomic Landscape of Hydroxychloroquine as an Autophagy Inhibitor.

Prostate cancer is a major planetary health challenge wherein new ways of thinking drug discovery and therapeutics innovation are much needed. Numerous studies have shown that autophagy inhibition holds a significant role as an adjunctive intervention in prostate cancer. Hydroxychloroquine (HCQ) has gained considerable attention due to its established role as an autophagy inhibitor across diverse cancer types, but its proteomics landscape and systems biology in prostate cancer are currently lacking in the literature. This study reports the proteomic responses to HCQ in prostate cancer cells, namely, androgen-dependent LNCaP and androgen-independent PC3 cells. Differentially expressed proteins and proteome in HCQ-treated cells were determined by label-free quantification with nano-high-performance liquid chromatography and tandem mass spectrometry (nHPLC-MS/MS), and harnessing bioinformatics tools. In PC3 cells, there was a marked shift toward metabolic reprogramming, highlighted by an upregulation of mitochondrial proteins in oxidative phosphorylation and tricarboxylic acid cycle, suggesting an adaptive mechanism to maintain energy production under therapeutic stress. In contrast, LNCaP cells prioritized proteostasis and cell cycle regulation, indicating a more conservative adaptation strategy. To the best of our knowledge, this study is the first to demonstrate the differential responses of prostate cancer cells to autophagy inhibition by HCQ, suggesting that a combination therapy approach, targeting distinct pathways in androgen-independent and androgen-dependent cells, could represent a promising treatment strategy. Moreover, the varied proteomic responses observed between these cell lines underscore the importance of personalized medicine in cancer therapy. Future translational and clinical research on HCQ and prostate cancer are called for.

GFP Transfection Alters Protein Expression Patterns in Prostate Cancer Cells: A Proteomic Study.

PURPOSE: Green Fluorescent Protein is widely used as a cellular marker tool, but its potential influence on cells has been questioned. Although the potential off-target effects of GFP on tumor cells have been studied to some extent, the findings at the molecular level are insufficient to explain the effect of GFP expression on the tumorigenic capacity of cancer cells. Here, we aimed to investigate the effect of GFP expression on the tumorigenicity of PC3 prostate cancer cells. METHODS: Using GFP-expressing and wild-type PC-3 cells, xenograft models were generated in athymic BALB/C mice. To identify differentially expressed proteins, the change in cells proteome was investigated by label-free quantification with nano-high performance liquid chromatography to tandem mass spectrometry (nHPLC-MS/MS). Proteins that showed significantly altered expression levels were evaluated using the bioinformatics tools. RESULTS: Unlike the wild-type PC-3 cells, GFP-expressing cells failed to develop tumor. Comparative proteome analysis of GFP-expressing cells with WT PC-3 cells revealed a total of 216 differentially regulated proteins, of which 98 were upregulated and 117 were downregulated. CONCLUSION: Upon GFP expression, differential changes in several pathways including the immune system, translational machinery, energy metabolism, elements of cytoskeletal and VEGF signaling pathway were observed. Therefore, care should be taken into account to prevent reporting deceitful mechanisms generated from studies utilizing GFP.

Therapeutic Effects of AF219 on Interstitial Cystitis/Bladder Pain Syndrome Induced by Cyclophosphamide or Water Avoidance Stress in Rats.

INTRODUCTION AND HYPOTHESIS: To evaluate the effect of AF219, a P2X3 receptor antagonist, in animal models of interstitial cystitis/bladder pain syndrome (IC/BPS) induced by cyclophosphamide (CYP) or water avoidance stress (WAS). METHODS: Thirty-two adult female Wistar albino rats were used in each IC/BPS model. Assessment of nociception and anxiety and severity of inflammation in the bladder were assessed by behavioral experiments and histopathological examinations respectively. The contraction responses of the bladder were evaluated in vitro and protein levels of P2X3, P2X7, Trk-A, TRPV1, and TRPA1 were analyzed by Western blot. RESULTS: The IC/BPS groups had shorter response times to noxious stimuli, exhibited more anxiety-like behavior, had higher inflammation-based histological scores, and showed greater increased contraction responses to carbachol, adenosine triphosphate, and electrical field stimulation in in vitro bladder strips than controls for both models (p < 0.05). The improvements in behavioral and bladder contraction responses and inflammation scores in the IC/BPS + AF219 groups were similar to control findings (p > 0.05). Exposure to WAS or CYP increased P2X3 expression in the bladder compared with the controls (p < 0.05). Apart from TRPA1, the levels of P2X7, Trk-A, and TRPV1 were also higher in the IC/BPS groups than in the controls (p < 0.05). No significant differences were observed between IC/BPS + AF219 and controls regarding P2X3, P2X7, Trk-A, and TRPV1 in the WAS model (p > 0.05). Moreover, P2X3 and P2X7 levels were significantly lower in IC/BPS + AF219 than in the AF219-untreated WAS model (p < 0.05). CONCLUSIONS: These findings suggest that P2X3 receptors play a significant role in bladder functional responses, nociception, and also the pathogenesis of IC/BPS. AF219 may be a promising therapeutic strategy for IC/BPS. Comparing AF219 with current IC/BPS treatment agents in future studies may yield valuable insights into its efficacy.

Streamlined Biotinylation, Enrichment and Analysis for Enhanced Plasma Membrane Protein Identification Using TurboID and TurboID-Start Biotin Ligases.

Plasma membrane proteins (PMPs) play pivotal roles in various cellular events and are crucial in disease pathogenesis, making their comprehensive characterization vital for biomedical research. However, the hydrophobic nature and low expression levels of PMPs pose challenges for conventional enrichment methods, hindering their identification and functional profiling. In this study, we presented a novel TurboID-based enrichment approach for PMPs that helped overcoming some of the existing limitations. We evaluated the efficacy of TurboID and its modified form, TurboID-START, in PMP enrichment, achieving efficient and targeted labelling of PMPs without the need for stable cell line generation. This approach resulted reduction in non-specific biotinylation events, leading to improved PMP enrichment and enabled assessment of the subcellular proteome associated with the plasma membrane. Our findings paved the way for studies targeting the dynamic nature of the plasma membrane proteome and aiming to capture transient associations of proteins with the plasma membrane. The novel TurboID-based enrichment approach presented here offers promising prospects for in-depth investigations into PMPs and their roles in cellular processes.

Is tear proteome profile a predictor of developing uveitis in ANA-positive patients with oligoarticular juvenile idiopathic arthritis?

PURPOSE: Although less than one-third of anti-nuclear antibody (ANA) positive patients with oJIA develop uveitis, ANA positivity is still the most well-known marker for assessing the risk of uveitis in oligoarticular JIA (oJIA). Therefore, novel biomarkers are needed to better assess the risk of developing uveitis. For this purpose, we performed a comparative tear proteome analysis of uveitis patients to reveal the identity of differentially regulated proteins. DESIGN: Tear samples were collected using the Schirmer strips in 7 oJIA and 7 oJIA patients with uveitis (oJIA-U). All oJIA-U patients had developed bilateral anterior uveitis and were inactive and topical treatment-free. METHODS: The nHPLC LC-MS/MS system was used for protein identification and label-free proteome comparisons. The PANTHER and STRING analyses were carried out using UniProt accession numbers of the identified proteins. RESULTS: Patient characteristics, e.g., age, gender, disease duration, and treatments were similar. For protein identification, three different databases were searched. Twenty-two, 147, and 258 database searches, respectively. Of these, 15 were common to all three proteome databases. Of these 15 proteins, 10 proteins were upregulated, and 2 were downregulated, based on the twofold regulation criteria. The upregulated proteins were, namely, cystatin-S, secretoglobin family 1D member, opiorphin prepropeptide, mammaglobin-B, lysozyme C, mesothelin, immunoglobulin kappa constant, extracellular glycoprotein lacritin, beta-2-microglobulin, and immunoglobulin J chain. The downregulated proteins were dermcidin and prolactin-inducible protein. Among the differentially regulated proteins, cystatin-S was the most regulated protein with an 18-fold upregulation ratio in tear samples from uveitis patients. CONCLUSION: Here, the identities and regulation ratios of several proteins were revealed when tear samples from uveitis patients were compared to patients without uveitis. These proteins are putative biomarkers for assessing uveitis risk and require further attention.

Downregulated GPD1 and MAGL protein levels as potential biomarkers for the metastasis of triple­negative breast tumors to axillary lymph nodes.

Glycerol-3-phosphate dehydrogenase (GPD1) and monoacylglycerol lipase (MAGL) levels are known to be significantly downregulated in both the tissue and serum samples of patients with triple-negative breast cancer (TNBC), compared with other BC subtypes and healthy controls. As such, the association between GPD1 and MAGL levels and lymph node metastasis was evaluated in the present study. Utilizing western blotting, lymph node protein extracts from metastasized BC subtypes were analyzed and a significant downregulation of GPD1 and MAGL protein expression levels in the lymph node metastases was demonstrated in the TNBC subtype, compared with healthy controls. This finding further highlighted the potential use of these two proteins in early BC onset and metastasis detection.

Comparative Proteomic Analysis of the Aqueous Humor from Patients with Pseudoexfoliation Syndrome.

Purpose: The goal of this study was to pinpoint potential molecular pathways that may have contributed to the onset of pseudoexfoliation syndrome (PEX), a systemic illness associated with aging that has no known cause and is brought on by the deposition of fibrillary white flaky debris in ocular tissues. Materials and methods: Protein pools representing each group were created using two-dimensional gel electrophoresis (2DE) in conjunction with a matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) mass spectrometer. Aqueous humor (AH) from patients with PEX and cataracts was also collected for a comprehensive study of the data; ingenuity pathway analysis (IPA) was used for the discovered proteins. Results: In comparison to controls, 2DE showed that 10 sites in PEX patients had differently altered gene expression. Two of these proteins, transthyretin (TTR) and apolipoprotein A4 (ApoA4) were significantly overexpressed in PEX patients, but the remaining proteins were only mildly altered. The liver X receptor (LXR) and the retinoid X receptors (RXR) may play a crucial role in the pathophysiology of PEX according to IPA employing these 10 proteins. Conclusion: The altered proteins, particularly ApoA4 and TTR, may be important in revealing the molecular process behind PEX, as anticipated by IPA. How to cite this article: Toprak M, Yuksel N, Akpinar G, et al. Comparative Proteomic Analysis of the Aqueous Humor from Patients with Pseudoexfoliation Syndrome. J Curr Glaucoma Pract 2023;17(3):118-125.

Using proteomics, q-PCR and biochemical methods complementing as a multiapproach to elicit the crucial responses of zebrafish liver exposed to neonicotinoid pesticide.

Pesticides enter the environment through runoff and leaching and this raises public concern about effects on non-target organisms. Imidacloprid (IMI) a synthetic pesticide, has an unstable half-life, metabolized in minutes to weeks in the water. To evaluate the effects of IMI on the zebrafish liver, we conducted proteomic, molecular and biochemical analysis in a multi-level approach, to highlight the complementary features regarding the results of each method. Adult zebrafish were exposed to 60 mg/L IMI for 48 h and were evaluated using nLC-MS/MS for proteins, q-PCR analysis for expression of cat, gpx, pxr, ache, along with CAT and AChE enzyme activities and GSH and MDA assays. Based on proteomics, the regulation of antioxidant and immune responses, as well as gene transcription were significant processes affected. Apoptosis and ER stress pathways were upregulated and there was a down-regulation of cat and gpx genes. There was also elevated CAT activity and GSH and decreased MDA. Additionally, elevated AChE activity and up regulation of ache expression was observed. The multi-approach results included regulators of antioxidant, xenobiotic response and neuro-protective related proteins (genes and enzymes), which overall reflected harmful effects of IMI. Consequently, this study highlights the effects of IMI on zebrafish liver and reveals new potential biomarkers. In this respect, evaluated outcomes reveal the complementary features emphasizing the importance of studying chemicals using several methods. Our study provides deeper insights for future work in ecotoxicological studies regarding IMI and contribute to existing toxicity literature.

Investigation of the effect of pancreatic decellularized matrix on encapsulated Islets of Langerhans with mesenchymal stem cells.

OBJECTIVE: In this study, it was aimed to provide a therapeutic approach for T1DM by encapsulating the pancreatic islets with mesenchymal stem cells and decellularized pancreatic extracellular matrix to support the survival of islets while maintaining their cellular activity. METHOD: Pancreatic extracellular matrix was decellularized using different concentrations of detergent series. After the preparation of the protein-based tissue extracellular matrix was shown to be free of cells or any genetic material by molecular, immunofluorescence and histochemical techniques. Following the homogenization of the decellularized pancreatic extracellular matrix and the analysis of its protein composition by LC-MS, the matrix proteins were incorporated with pancreatic islets and rat adipose tissue-derived MSCs (rAT-MSCs) in alginate microcapsules. Glucose-stimulated insulin secretion property of the islet cells in the microbeads was evaluated by insulin ELISA. The gene expression profile of the encapsulated cells was analyzed by Real-Time PCR. RESULTS: Unlike the protein composition of whole pancreatic tissue, the decellularized pancreas matrix was free of histone proteins or proteins originated from mitochondria. The protein matrix derived from pancreatic tissue was shown to support the growth and maintenance of the islet cells. When compared to the non-encapsulated pancreatic islet, the encapsulated cells demonstrate to be more efficient in terms of insulin expression. CONCLUSION: The extracellular pancreatic matrix obtained in this study was directly used as supplementary in the alginate-based microcapsule enhancing the cell survival. The tissue matrix protein and alginate had a synergistic effect on total insulin secretion, which might have the potential to overcome the insulin deficiency. Despite the improvement in the cell viability and the number, the efficiency of the insulin secretion in response to glucose stimulation from the alginate microcapsules did not meet the expectation when compared with the non-encapsulated pancreatic islets.

An experimental workflow for enrichment of low abundant proteins from human serum for the discovery of serum biomarkers.

Serum contains proteins that possess important information about diseases and their progression. Unfortunately, these proteins, which carry the information in the serum are in low abundance and are masked by other serum proteins that are in high abundance. Such masking prevents their identification and quantification. Therefore, removal of high abundance proteins is required to enrich, identify, and quantify the low abundance proteins. Immunodepletion methods are often used for this purpose, but there are limitations in their use because of off-target effects and high costs. Here we presented a robust, reproducible and cost-effective experimental workflow to remove immunoglobulins and albumin from serum with high efficiency. The workflow did not suffer from such limitations and enabled identification of 681 low abundance proteins that were otherwise undetectable in the serum. The identified low abundance proteins belonged to 21 different protein classes, namely the immunity-related proteins, modulators of protein-binding activity, and protein-modifying enzymes. They also played roles in various metabolic events, such as integrin signalling, inflammation-mediated signalling, and cadherin signalling. The presented workflow can be adapted to remove abundant proteins from other types of biological material and to provide considerable enrichment for low-abundance proteins.

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival.

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.

Investigation of the effect of meclofenamic acid on the proteome of LNCaP cells reveals changes in alternative polyadenylation and splicing machinery.

Prostate cancer is the most common type of cancer among men, and there is still no definitively effective drug treatment. Thus, the search for novel drug agents that may be used for the effective treatment continues. Meclofenamic acid (MA), a non-steroidal anti-inflammatory drug, with anti-tumor effects in various types of cancers was used to investigate its effects on LNCaP cells, a prostate cancer cell line, at the proteome level. The cells were treated with 80 µM MA for 24 h and a comparative proteomic analysis was performed with their untreated control cells. Proteins were extracted from the cells and then were subjected to two-dimensional gel electrophoresis. Protein spots displaying changes in their regulation ratios for more than two-fold were excised from the gels and identified with MALDI-TOF/TOF mass spectrometry. Bioinformatics analysis of the differentially regulated proteins that we identified showed that they were all associated with and took part in related pathways. Glycolytic pathway, cytoskeletal formation, transport activity, protein metabolism, and most notably an mRNA processing pathway were affected by the MA treatment. In addition to presenting a detailed information for what is happening inside the cells upon MA treatment, the proteins affected by MA treatment hold the potential to be novel targets for prostate cancer treatment provided that further in vivo experiments are carried out.

Proteomic Analysis of m.8296A>G Variation in the Mitochondrial tRNA Lys Gene.

Variation in the mitochondrial tRNA Lys gene at position 8296 was previously found to be associated with maternally inherited diabetes mellitus and deafness, hypertrophic cardiomyopathy, myoclonic epilepsy with ragged-red fibers and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. The pathogenicity of the m.8296A>G variation is unclear. In this study, we aimed to analyze the mitochondrial proteome in a patient with m.8296A>G variation to elucidate the effects of this mutation at the protein level. Whole-exome sequencing and mitochondrial genome analysis were performed in a patient with sensorineural hearing impairment, cognitive impairment, leukodystrophy, migraine-like headaches, and gastrointestinal dysmotility. Mitochondrial genome analysis identified a homoplasmic m.8296A>G variation in the mitochondrial tRNA Lys gene in the proband and unaffected mother. Global mitochondrial proteome analysis was carried out in the muscle mitochondria of the index patient and a control subject. Comparative muscle mitochondrial proteome analysis revealed a total of 13 nuclear-encoded mitochondrial proteins differently expressed with respect to the control. Ten of the 13 proteins were downregulated. Most of the proteins were involved in ATP synthesis and Krebs cycle and have strong interactions with each other. We considered the m.8296A>G variation to be pathogenic with variable penetrance for our patient's phenotype, and this variation led to different expressions of nuclear-encoded proteins involved in energy metabolism.

Tissue proteome analysis revealed an association between cancer, immune system response, and the idiopathic granulomatous mastitis.

Idiopathic Granulomatous Mastitis (IGM) is a disease that clinically mimics breast cancers with symptoms of pain, edema, erythema, nipple discharge, nipple retraction, and fistula. Although IGM is considered to be formed by autoimmune responses or infections, the molecular mechanism behind formation and progress is unknown. Therefore, in this study, we aimed to investigate molecular mechanisms underlying IGM formation, progress, and recurrence by monitoring the changes at the proteome level. Protein extracts prepared from IGM (n = 15) and within-control tissues (n = 15) were subjected to nHPLC followed by LC-MS/MS proteomic analysis. Label-free quantitation analysis revealed that sixty differentially regulated between the two groups. Those proteins were classified based on their role in metabolic pathways using bioinformatics tools. Based on DAVID analysis, 16 of the differently regulated proteins were associated with the immune system, while 17 proteins were involved in cancer metabolism. STRING analysis showed that five of the differentially regulated proteins were associated with combined immune deficiency which were PNP, TAP1, ITGAL, PRKDC, and PTPRC while the other proteins were involved in insulin response and neutrophil degranulation. This study is one of the very few studies that investigated the changes in protein expressions of IGM tissues compared to controls. For the first time, we have shown the relationship of IGM with the immune system at the protein level and also underlined the cancer-like behavior of the disease. Furthermore, the proteins that were pointed out as combined immune deficiency-related proteins may have value as diagnostic markers for idiopathic granulomatous mastitis although further studies are needed to shed more light on the pathogenesis of the disease.

Elucidation of the changes occurring at the proteome level in ovaries of high-fat diet-induced obese rats.

High-fat diet-induced obesity adversely affects the female reproductive system. The metabolic changes that the high-fat diet causes on the ovaries have not been elucidated. Herein, to understand the molecular and cellular mechanisms underlying the effects of long-term high-fat diet-fed, the changes in the global proteomic profile of the rat ovaries were investigated. The female rats were randomly divided into two groups based on their diets: the ones that were fed with the high-fat diet and the other ones that were fed with the control diet for 18 weeks. To identify differentially expressed proteins, the changes in ovary proteomes were investigated by two-dimensional electrophoresis coupled to matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight and label-free quantification with nano-high performance liquid chromatography to tandem mass spectrometry (nHPLC-MS/MS). A total of 80 proteins were differentially regulated. The upregulated proteins were involved in responses to chemical and organic substances, cytokines, external stimuli, and lipids. These proteins were particularly associated with vesicles, microbodies, and cell surface proteins. The downregulated proteins were involved in biological processes associated with cellular respiration. Those proteins created a network consisting of proteins involved in aerobic respiration and energy generation. Our results demonstrated that the mechanisms related to energy production in the ovary tissue were particularly affected by the high-fat diet.

Kv5.1 antibody in epilepsy patients with unknown etiology.

BACKGROUND: Neuronal autoantibodies and favorable response to immunosuppressive treatment have been described in patients with chronic epilepsy of unknown cause, suggesting autoimmune etiology. Our aim was to identify novel epilepsy-specific autoantibodies reactive with neuronal surface antigens. METHODS: Sera of 172 epilepsy patients with unknown cause and 30 healthy controls were screened with indirect immunofluorescence to identify IgG reacting with primary rat neuronal cultures. Putative target autoantigens were investigated with immunoprecipitation (IP) and liquid chromatography-mass/mass spectrometry (LC-MS/MS) studies using SH-SY5Y cells. Validation of LC-MS/MS results was carried out by IP and immunocytochemistry assays. RESULTS: Antibodies to neuronal cell surface antigens were detected in 18 epilepsy patients. LC-MS/MS analysis identified voltage-gated potassium channel modifier subfamily F member 1 (KCNF1, Kv5.1) as the single common cell surface antigen in 4 patients with Lennox-Gastaut syndrome (n = 2), focal epilepsy of unknown cause (n = 1) and mesial temporal lobe epilepsy with hippocampal sclerosis (n = 1). These patients had the common features of early seizure onset and treatment-resistance. IP assays and co-localization (serum IgG and commercial Kv5.1-antibody) studies done with non-fixed Kv5.1-transfected HEK293 cells and primary neuronal cultures confirmed the presence of Kv5.1-antibody in 4 epilepsy patients identified by LC-MS/MS. Similar findings were not obtained by sera of other patients with epilepsy, patients with autoimmune encephalitis and healthy controls. CONCLUSION: The herein described novel neuronal surface antibody to Kv5.1 appears to be associated with treatment-resistant epilepsy of unknown cause. Exact clinical and pathogenic significance of this antibody remains to be elucidated.

BEND4 as a Candidate Gene for an Infection-Induced Acute Encephalopathy Characterized by a Cyst and Calcification of the Pons and Cerebellar Atrophy.

Three siblings born to Turkish parents from the same village had normal brain development until acute neurological deterioration between 12 months and 8 years of age. Consequent loss of all acquired motor, social, and language functions following infections was associated with a pontine cyst, calcification, and cerebellar atrophy. Exome sequencing revealed a homozygous c.1297G>A (p.Gly433Ser) alteration in BEND4, which was predicted to be deleterious in in silico analysis tools and segregated in multiple affected individuals in the family. BEND4 has not been associated with any existing disease. Immunofluorescence microscopy analysis of wild-type and mutant BEND4 expressing Vero cells showed nuclear and cytoplasmic localization. Wild-type BEND4 displayed a network-like distribution, whereas mutant BEND4 showed a juxtanuclear distribution pattern. Differential proteome analysis of Vero cells expressing BEND4 revealed that mutant BEND4 expression caused selective increase in reticulocalbin-1 and endoplasmic reticulum resident protein-29. Both proteins are associated with the endoplasmic reticulum and are primarily involved in protein processing and folding pathways. Any defect or stress in protein folding creates stress on cells and may cause chronic damage. This is the first study showing that pathogenic BEND4 variants may lead to an infection-induced acute necrotizing encephalopathy as demonstrated in characteristic neuroimaging findings.

Comparison of before versus after intravitreal bevacizumab injection, growth factor levels and fibrotic markers in vitreous samples from patients with proliferative diabetic retinopathy.

PURPOSE: In diabetic retinopathy patients, intravitreal bevacizumab (IVB) injections are widely used to facilitate dissection of retinal fibrovascular membranes during surgery, reduce the rate of perioperative hemorrhage, and prevent recurrent neovascularization. Previous studies have shown that IVB may worsen fibrosis and thereby impair vision. The aim of this study was to determine which markers are associated with fibrosis. METHODS: Twenty-three patients with proliferative diabetic retinopathy (PDR) underwent pars plana vitrectomy (PPV) with IVB pretreatment for intraocular hemorrhage (IOH) and/or tractional retinal detachment (TRD). Vitreous samples were obtained at the time of IVB injection and again at the beginning of PPV, about a week later. Using Western blot analysis, the concentrations of vascular endothelial growth factor (VEGF), placental growth factor (PIGF), insulin like growth factor-1 (IGF-1), angiogenin-1 (Ang-1), and vascular endothelial cadherin (VE-cadherin) were measured in vitreous samples. RESULTS: After treatment with IVB, VEGF, PIGF, and VE-cadherin concentrations in the vitreous significantly decreased (p < 0.001, p < 0.001, and p = 0.001, respectively), whereas the concentrations of IGF-1 increased (p = 0.001). There were no significant changes in Ang-1 concentrations in the vitreous after IVB injection (p = 0.732). There were no statistically significant differences in VEGF-A, PIGF, VE-cadherin, IGF, and Ang-1 levels before and after IVB injection when the IOH and TRD groups underwent subgroup analysis (p = 0.696, p = 0.516, p = 0.498, p = 0.188, and p = 0.243, respectively). CONCLUSION: The levels of VEGF and other cytokines changed in the vitreous after IVB. The adverse effects associated with IVB, such as fibrosis, may result from modulation of vitreous cytokine concentrations. In the treatment of PDR, drugs that optimize the effects of PIGF, IGF-1, and VE-cadherin to reduce these side effects may be useful.

Decreased serum levels of glycerol-3- phosphate dehydrogenase 1 and monoacylglycerol lipase act as diagnostic biomarkers for breast cancer.

BACKGROUND: Breast cancer (BC) is one of the most life-threatening cancer types among women. Despite major developments in medical sciences and technologies, the incidence and mortality rates of BC cases are still increasing. One of the reasons for this increase is the absence of an easy to perform early-diagnostic tool. Although there are defined BC biomarkers routinely used for diagnostic and prognostic purposes, none of these biomarkers is useful for early diagnosis. Therefore, early diagnosis of BC remains an important challenge and there is a great need for the early-diagnostic biomarker(s). OBJECTIVE: In this study, we aimed to evaluate the diagnostic and prognostic values of glycerol-3-phosphate dehydrogenase (GPD1) and monoacylglycerol lipase (MAGL) proteins as non-invasive serum biomarkers. METHODS: GPD1 and MAGL serum levels were determined by ELISA for BC patients (n= 100) from five different subtypes, and healthy controls (n= 20), and a comparative analysis was performed to determine statistically significant expression differences among the groups. RESULTS: The results provided evidence that GPD1 acted as a diagnostic biomarker in distinguishing triple-negative breast cancer (TNBC) patients from other subtypes, and MAGL acted as a diagnostic biomarker in distinguishing healthy individuals from BC patients. CONCLUSION: GPD1 and MAGL might be proposed as non-invasive diagnostic biomarkers for BC with high sensitivity and specificity.

Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice.

The SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A and smallpox proved to be reliable approaches for immunization for prolonged periods. In this study, a gamma-irradiated inactivated virus vaccine does not require an extra purification process, unlike the chemically inactivated vaccines. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral RNA copy per dose) of OZG-38.61.3 was initially determined in BALB/c mice. This was followed by testing the immunogenicity and protective efficacy of the vaccine. Human ACE2-encoding transgenic mice were immunized and then infected with the SARS-CoV-2 virus for the challenge test. This study shows that vaccinated mice have lowered SARS-CoV-2 viral RNA copy numbers both in oropharyngeal specimens and in the histological analysis of the lung tissues along with humoral and cellular immune responses, including the neutralizing antibodies similar to those shown in BALB/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.