Yeast Ataxin-2 Forms an Intracellular Condensate Required for the Inhibition of TORC1 Signaling during Respiratory Growth.

Yeast ataxin-2, also known as Pbp1 (polyA binding protein-binding protein 1), is an intrinsically disordered protein implicated in stress granule formation, RNA biology, and neurodegenerative disease. To understand the endogenous function of this protein, we identify Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region causes phase separation, forms reversible fibrils, and enables self-association into assemblies required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling.

P7C3 neuroprotective chemicals function by activating the rate-limiting enzyme in NAD salvage.

The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage.

Sulfur amino acids regulate translational capacity and metabolic homeostasis through modulation of tRNA thiolation.

Protein translation is an energetically demanding process that must be regulated in response to changes in nutrient availability. Herein, we report that intracellular methionine and cysteine availability directly controls the thiolation status of wobble-uridine (U34) nucleotides present on lysine, glutamine, or glutamate tRNAs to regulate cellular translational capacity and metabolic homeostasis. tRNA thiolation is important for growth under nutritionally challenging environments and required for efficient translation of genes enriched in lysine, glutamine, and glutamate codons, which are enriched in proteins important for translation and growth-specific processes. tRNA thiolation is downregulated during sulfur starvation in order to decrease sulfur consumption and growth, and its absence leads to a compensatory increase in enzymes involved in methionine, cysteine, and lysine biosynthesis. Thus, tRNA thiolation enables cells to modulate translational capacity according to the availability of sulfur amino acids, establishing a functional significance for this conserved tRNA nucleotide modification in cell growth control.

Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains.

The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.

A gain-of-function mutation in DHT synthesis in castration-resistant prostate cancer.

Growth of prostate cancer cells is dependent upon androgen stimulation of the androgen receptor (AR). Dihydrotestosterone (DHT), the most potent androgen, is usually synthesized in the prostate from testosterone secreted by the testis. Following chemical or surgical castration, prostate cancers usually shrink owing to testosterone deprivation. However, tumors often recur, forming castration-resistant prostate cancer (CRPC). Here, we show that CRPC sometimes expresses a gain-of-stability mutation that leads to a gain-of-function in 3ß-hydroxysteroid dehydrogenase type 1 (3ßHSD1), which catalyzes the initial rate-limiting step in conversion of the adrenal-derived steroid dehydroepiandrosterone to DHT. The mutation (N367T) does not affect catalytic function, but it renders the enzyme resistant to ubiquitination and degradation, leading to profound accumulation. Whereas dehydroepiandrosterone conversion to DHT is usually very limited, expression of 367T accelerates this conversion and provides the DHT necessary to activate the AR. We suggest that 3ßHSD1 is a valid target for the treatment of CRPC.

Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies.

Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.

Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

Applications of single-cell omics for chimeric antigen receptor T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment modality. The breakthroughs in CAR T cell therapy were, in part, possible with the help of cell analysis methods, such as single-cell analysis. Bulk analyses have provided invaluable information regarding the complex molecular dynamics of CAR T cells, but their results are an average of thousands of signals in CAR T or tumour cells. Since cancer is a heterogeneous disease where each minute detail of a subclone could change the outcome of the treatment, single-cell analysis could prove to be a powerful instrument in deciphering the secrets of tumour microenvironment for cancer immunotherapy. With the recent studies in all aspects of adoptive cell therapy making use of single-cell analysis, a comprehensive review of the recent preclinical and clinical findings in CAR T cell therapy was needed. Here, we categorized and summarized the key points of the studies in which single-cell analysis provided insights into the genomics, epigenomics, transcriptomics and proteomics as well as their respective multi-omics of CAR T cell therapy.

Sexual function index adaptation for breast cancer patients (FSFI-BC)- translation and psychometric properties of Persian version.

BACKGROUND: Effective interventions to improve sexual dysfunction in breast cancer survivors need screening of these dysfunctions with a suitable instrument. The aim of present study was translation and identifying psychometric properties of Female Sexual Function Index - Adapted for Breast Cancer (FSFI-BC) which has been specifically developed for breast cancer survivors. METHOD: This methodological study was performed between February 2017 and October 2018. 200 breast cancer survivors in stage 1 or 2 who were selected through convenience sampling method, completed the questionnaire. Reliability was assessed by Cronbach's alfa and test re-test analysis and construct validity was performed through confirmatory (CFA) and exploratory factor analysis( EFA). RESULTS: Six factors were extracted in exploratory factor analysis (EFA). These factors explained 74.6% of the total variance in in NSA group and 0.821 in SA group. Reliability evaluation indicated high internal consistency and good test re-test reliability. Cronbach's alpha coefficient in all areas of the tool was above 0.7 (the lowest and the highest measures were 0.885 and 0.945, respectively), which is a good indicator for reliability of an instrument. Confirmatory factor analysis showed an acceptable fitness for seven factors of FSFI-BC questionnaire (Normed Fit Index or NFI = 0.9 for both groups, Comparative of Fit Index or CFI = 0.93 and 0.92, χ 2/df = 1.68 and 1.71 for SA(Sexually Active) and NSA(No Sexually Active) individuals, respectively) . CONCLUSION: Study findings suggest that Persian version of FSFI-BC is a suitable instrument for sexual dysfunction screening in breast cancer survivors.

Exploring the Effectiveness of Ajwain Cream in Treating Taxane-induced Peripheral Neuropathy in Cancer Patients: A Pilot, Randomised and Double-blind Clinical Trial.

Objectives: Chemotherapy-induced peripheral neuropathy is a common disorder among cancer patients receiving various chemotherapeutic protocols. The present study aimed to explore the feasibility of ajwain (Trachyspermum ammi [L.] Sprague) cream in treating peripheral neuropathy symptoms triggered by taxane chemotherapeutic agents. Materials and Methods: This was a pilot, double-blind, and randomised clinical trial on patients with peripheral neuropathy attributable to chemotherapy with taxane drugs during 2021-2022 in Tehran. Patients received ajwain or placebo cream for four weeks and filled out the chemotherapy-induced peripheral neuropathy assessment tool (CIPNAT) at the start and end finale of the trial. Side effects were also noted. Results: Thirty patients suffering from breast, lung, gastro-intestinal, or prostate cancer were allocated to each of the drug and placebo groups. The mean difference in CIPNAT score between the groups was 0.83, demonstrating the statistical ineffectiveness of the drug compared with the placebo (P = 0.372). The safety profile showed promising outcomes at the end of the trial. Conclusion: Although the effectiveness of ajwain cream was unacceptable in treating chemotherapy-induced peripheral neuropathy symptoms, multicentre controlled trials with ample sample size are mandatory for an all-inclusive inference.

Outsmarting trogocytosis to boost CAR NK/T cell therapy.

Chimeric antigen receptor (CAR) NK and T cell therapy are promising immunotherapeutic approaches for the treatment of cancer. However, the efficacy of CAR NK/T cell therapy is often hindered by various factors, including the phenomenon of trogocytosis, which involves the bidirectional exchange of membrane fragments between cells. In this review, we explore the role of trogocytosis in CAR NK/T cell therapy and highlight potential strategies for its modulation to improve therapeutic efficacy. We provide an in-depth analysis of trogocytosis as it relates to the fate and function of NK and T cells, focusing on its effects on cell activation, cytotoxicity, and antigen presentation. We discuss how trogocytosis can mediate transient antigen loss on cancer cells, thereby negatively affecting the effector function of CAR NK/T cells. Additionally, we address the phenomenon of fratricide and trogocytosis-associated exhaustion, which can limit the persistence and effectiveness of CAR-expressing cells. Furthermore, we explore how trogocytosis can impact CAR NK/T cell functionality, including the acquisition of target molecules and the modulation of signaling pathways. To overcome the negative effects of trogocytosis on cellular immunotherapy, we propose innovative approaches to modulate trogocytosis and augment CAR NK/T cell therapy. These strategies encompass targeting trogocytosis-related molecules, engineering CAR NK/T cells to resist trogocytosis-induced exhaustion and leveraging trogocytosis to enhance the function of CAR-expressing cells. By overcoming the limitations imposed by trogocytosis, it may be possible to unleash the full potential of CAR NK/T therapy against cancer. The knowledge and strategies presented in this review will guide future research and development, leading to improved therapeutic outcomes in the field of immunotherapy.

The inhibitory receptors PD1, Tim3, and A2aR are highly expressed during mesoCAR T cell manufacturing in advanced human epithelial ovarian cancer.

BACKGROUND: Chemotherapy and surgery have been the mainstays of epithelial ovarian cancer (EOC) treatment so far. Cellular immunotherapies such as CAR T cell therapy have recently given hope of a cure for solid tumors like EOC. However, extrinsic factors associated with the CAR T cell manufacturing process and/or intrinsic dysregulation of patient-derived T cells, which could be associated with cancer itself, cancer stage, and treatment regimen, may hamper the efficacy of CAR T cell therapy and promote their exhaustion or dysfunction. METHODS: To investigate the association of these factors with CAR T cell exhaustion, the frequency of T and CAR T cells expressing three immune inhibitory receptors (i.e., TIM3, PD1, A2aR) generated from T cells of EOC patients and healthy controls was measured during each stage of CAR T cell production. RESULTS: Our findings revealed that primary T cells from EOC patients show significantly elevated expression of immune inhibitory receptors, and this increase was more prominent in patients undergoing chemotherapy and those with advanced cancer. In addition, the CAR T cell manufacturing process itself was found to upregulate the expression of these inhibitory receptors and more importantly increase the population of exhausted mesoCAR T cells. CONCLUSIONS: Our observations suggest that intrinsic characteristics of patient-derived T cells and extrinsic factors in CAR T cell production protocols should be considered and properly counteracted during CAR T cell manufacturing process. In addition, mitigating the signaling of immune inhibitory receptors through pharmacological/genetic perturbation during CAR T cell manufacturing might profoundly improve CAR T cells function and their antitumor activity in EOC and other solid tumors.

Postoperative cosmetic outcome of intraoperative radiotherapy in comparison to whole breast radiotherapy in early stage breast cancer; a retrospective cohort study.

BACKGROUND: In this study, we aim to evaluate the cosmetic outcome differences between Intraoperative electron beam radiation therapy (IOERT) and whole breast radiotherapy (WBR) with further investigation of boosted IOERT. METHODS: This retrospective cohort study was conducted in two referral centers in Tehran, Iran. 116 women aged 30 to 79 with early-stage breast cancer (T0-2N0-1M0) eligible for breast conservation were divided into two groups of 58 based on the intervention they received, and further subgroups were defined based on receiving boosted IOERT. Patients in both groups underwent breast conservation surgery and those in the IOERT group received either a 21 Gy radical dose (radical IOERT) or 12 Gy boosted electron beam radiotherapy and a routine fractionated dose of 50 Gy in 25 sessions of WBR (boosted IOERT). Those in the WBR group were administered 50Gy in 32 sessions. Physician-assessed cosmetic outcome was defined as the primary result and incidence of fat necrosis and fibrosis and post-operative chronic pain were secondary outcomes. RESULTS: Post-operative cosmetic outcome scores and chronic pain, showed no significant difference between the two groups. The median cosmetic score in both groups was 9. Fat necrosis and fibrosis had significantly higher rates in the IOERT group (P. VALUE: 0.001). However, the majority (21/34 or 61.8%) of this complication was observed in the boosted IOERT subgroup and no statistical significance was recorded between the radical IOERT subgroup and the WBR group. CONCLUSIONS: In early-stage breast cancer treatment, radical IOERT has noninferiority compared to WBR in terms of cosmesis. Regarding fat necrosis and fibrosis, boosted IOERT was associated with higher rates in comparison to other groups. Therefore, radical IOERT seems to be a better treatment option for selected patients.

Control of selectivity in the preparation of 2-substituted benzoazoles by adjusting the surface hydrophobicity in two solid-based sulfonic acid catalysts.

A series of metal-free tandem reactions for the synthesis of pharmaceutically important 2-substituted benzoazoles from isothiocyanates and 2-aminothiophenol under catalyst-free conditions in the presence of Et-PMO-Me-PrSO3H (1a) and SBA-15-PrSO3H (1b) as solid acids were carried out in a highly selective way under solvent free conditions. A significant selectivity changeover toward either 2-mercaptobenzoxazole or 2-aminobenzoazole derivatives could be achieved by changing the employed catalyst from the relatively hydrophobic material 1a to the more hydrophilic catalyst 1b. This simple experimental procedure with a novel selective approach toward benzoazoles accompanied by green and reusable catalysts could be considered as an alternative to the existing methods for the synthesis of 2-substituted benzoazole derivatives.

Bcl-2 expression in cell lines breast cancer and death program.

Breast cancer is a hormone-dependence and heterogenic disease. Drug resistance is the main reason for the failure of breast cancer treatment. Combinatory medications are methods for treatment but they are not sufficient in action. However, new approaches like molecular therapy reveal a new insight into cancer treatment. Studies show that Bcl-2 gene family inhibitors and ER blockers cause the improvement of recovery. Interfering molecules such as antisense ones can inhibit the expression of Bcl-2 and push the cancer cells to apoptosis. Our team designed a new Antisense Oligonucleotide (ASO) based on Antisense oligo G3139. MCF-7 and MDA-MB-231 cell lines were used to evaluate cellular proliferation. Liposomes and cationic nano-complex (Niosome) are used to increase the cellular delivery of ASO and Tamoxifen. We also investigated the cytotoxicity and apoptotic effects of Tamoxifen, naked ASO and Nano-packed ASO. The results indicated significant down-regulation of the Bcl-2 gene and inhibition of MCF-7 and MDA-MB-231 cellular proliferation. Flow-cytometry showed early apoptosis in all cell groups. The newly designed ASO reduced the expression of the Bcl-2 gene. It also had a synergistic effect with the Tamoxifen. The cationic nano-complex (Niosome) was more efficient than the liposome in delivering designed oligo antisense Bcl-2 in the cancer cells.

Development of deep eutectic solvent-based microwave-assisted extraction combined with temperature controlled ionic liquid-based liquid phase microextraction for extraction of aflatoxins from cheese samples.

In this study, for the first time, a deep eutectic solvent-based microwave-assisted extraction was combined with ionic liquid-based temperature controlled liquid phase microextraction for the extraction of several aflatoxins from cheese samples. Briefly, the analytes are extracted from cheese sample (3 g) into a mixture of 1.5 mL choline chloride:ethylene glycol deep eutectic solvent and 3.5 mL deionized water by exposing to microwave irradiations for 60 s at 180 W. The liquid phase was taken and mixed with 55 µL 1-hexyl-3-methylimidazolium hexafluorophosphate. By cooling the solution in the refrigerator centrifuge, a turbid state was obtained and the analytes were extracted into the ionic liquid droplets. The analytes were determined by high-performance liquid chromatography equipped with fluorescence detector. Low limits of detection (9-23 ng kg-1 ) and quantification (30-77 ng kg-1 ), high extraction recovery (66%-83%), acceptable enrichment factor (40-50), and good precision (relative standard deviations ≤ 5.2%) were obtained using the offered approach. These results reveal the high extraction capability of the method for determination of aflatoxins in the cheese samples. In this method, there was no need for organic solvents and it can be considered as green extraction method.

Glyco-nanoparticles: New drug delivery systems in cancer therapy.

Cancer is known as one of the most common diseases that are associated with high mobility and mortality in the world. Despite several efforts, current cancer treatment modalities often are highly toxic and lack efficacy and specificity. However, the application of nanotechnology has led to the development of effective nanosized drug delivery systems which are highly selective for tumors and allow a slow release of active anticancer agents. Different Nanoparticles (NPs) such as the silicon-based nano-materials, polymers, liposomes and metal NPs have been designed to deliver anti-cancer drugs to tumor sites. Among different drug delivery systems, carbohydrate-functionalized nanomaterials, specially based on their multi-valent binding capacities and desirable bio-compatibility, have attracted considerable attention as an excellent candidate for controlled release of therapeutic agents. In addition, these carbohydrate functionalized nano-carriers are more compatible with construction of the intracellular delivery platforms like the carbohydrate-modified metal NPs, quantum dots, and magnetic nano-materials. In this review, we discuss recent research in the field of multifunctional glycol-nanoparticles (GNPs) intended for cancer drug delivery applications.

Restricting tumor lactic acid metabolism using dichloroacetate improves T cell functions.

BACKGROUND: Lactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism. In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells. RESULT: We found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis. CONCLUSION: Taken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells.

Leukocytoclastic vasculitis presenting clinically as bullous pyoderma gangrenosum following leucovorin, fluorouracil and oxaliplatin chemotherapy: a rare case report and literature review.

There are no published cases about bullous pyoderma gangrenosum induced by leucovorin, fluorouracil and oxaliplatin (FOLFOX) chemotherapy. With the increasing incidence of gastric and colorectal cancers and the increased usage of targeted therapies, some cutaneous adverse effects may become common. An 84-year-old male presented to our clinic with multiple ulcerative plaques covered with hemorrhagic crusts on both extremities after several FOLFOX chemotherapy sessions for gastric cancer and liver metastasis. Two weeks later, multiple bullae also appeared, especially on the acral areas. The histopathology examination was compatible with acute leukocytoclastic vasculitis. The FOLFOX chemotherapy regimen is increasingly administered considering the rising incidence of gastrointestinal cancers. Hence, our understanding of its possible side effects and complications must be heightened.

Epigenetic strategies to boost CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has led to a paradigm shift in cancer immunotherapy, but still several obstacles limit CAR T cell efficacy in cancers. Advances in high-throughput technologies revealed new insights into the role that epigenetic reprogramming plays in T cells. Mechanistic studies as well as comprehensive epigenome maps revealed an important role for epigenetic remodeling in T cell differentiation. These modifications shape the overall immune response through alterations in T cell phenotype and function. Here, we outline how epigenetic modifications in CAR T cells can overcome barriers limiting CAR T cell effectiveness, particularly in immunosuppressive tumor microenvironments. We also offer our perspective on how selected epigenetic modifications can boost CAR T cells to ultimately improve the efficacy of CAR T cell therapy.