Cell Growth Factor Research Articles

Safety, tolerability, and preliminary efficacy of tinodasertib as a monotherapy or in combination with pembrolizumab or irinotecan in metastatic colorectal cancer: Interim results from a phase II open-label, dose-finding, run-in and cohort expansion study.

183 Background: MNK inhibition has been shown to downregulate phosphorylation at serine 209 of eIF4E, a potent regulatory point of CAP-mediated translation of 5’ polyadenylated mRNA associated with growth factor and pro-oncogenic signals in cells. Elevated levels of eIF4E phosphorylation are observed in a broad range of tumors. MNK1/2 knock out mice are healthy and viable, as cellular house-keeping mRNA translation occurs readily using the IRES mechanism, whereas mRNA with more complex and longer 5’ untranslated ends such as those involved in growth factor and pro-oncogenic signaling, are MNK activated. Cells from MNK knockout animals become relatively resistant to subsequent oncogenic transformation. In a program of SAD and MAD Phase I studies conducted in normal healthy volunteers, Tinodasertib was found to be safe and well tolerated with no dose-limiting toxicity (DLT). The objectives of this ongoing Phase 2 study are to evaluate safety, and preliminary efficacy of Tinodasertib as monotherapy and in combination with either pembrolizumab or irinotecan in patients with advanced colorectal cancer (CRC). Methods: Patients had to have advanced CRC and previously received ≥2 lines of therapy. The dose escalation phase of the study was open to all patients with CRC. As of 23 July 2024, 22 patients were dosed in a modified 3X3 dose escalation design with Tinodasertib from 20 to 80 mg on alternate days. Out of 22 patients, 12 received monotherapy, 4 were treated with Tinodasertib and Irinotecan and 6 were treated with Tinodasertib and pembrolizumab. Majority (19) had MSS CRC and 3 had unknown status. Nine patients had KRAS-mut CRC. Results: No DLTs were observed and MTD was not reached. Grade 3 treatment-related adverse events (TRAEs) were observed in 2 (9%) patients and were related to irinotecan while no Grade 3 AEs were attributed to Tinodasertib by either the investigator or the sponsor. Most common TRAEs were related to gastrointestinal system organ class. There were no Grade 4-5 TRAEs. Of the 22 patients, 18 were evaluable for RECIST 1.1 response. No patient had an objective response to treatment, 12 had stable disease with disease control rate of 67% and a progression free survival of 2.99 months. Patients remained on therapy for up to 28 weeks. Overall survival (OS) at 52 weeks was 52% (CI 29 to 93). Conclusions: Tinodasertib either as monotherapy or combined with irinotecan or pembrolizumab, was well tolerated with no DLTs at the dose levels evaluated. Prolonged median time to progression and OS compared to historical controls were observed even during the dose escalation phase for the study population as a whole and for each of the monotherapy and combination arms. Enrolment in the dose escalation phase continues. Clinical trial information: NCT05462236 .

Effect of SHR-1701 on chemotherapy (chemo)-induced myelosuppression: Data from a phase 3 study in HER2-negative gastric/gastroesophageal junction adenocarcinoma (G/GEJA).

335 Background: Blocking TGF-β signaling has the potential to facilitate the recovery from chemo-induced myelosuppression. SHR-1701 is a bifunctional agent targeting PD-L1 and TGF-β. Methods: In the multicenter, randomized, double-blind phase 3 study (NCT04950322), SHR-1701 plus CAPOX demonstrated a statistically significant and clinically meaningful benefit in OS compared with placebo plus CAPOX in patients (pts) with previously untreated, unresectable locally advanced or metastatic HER2-negative G/GEJA (ESMO 2024, LBA60). Here, we reported the effect of SHR-1701 on chemo-associated myelosuppression. Results: In total, 364 and 366 pts in the SHR-1701 and placebo arm received assigned treatment. As of May 20, 2024, with a KM estimated median follow-up of 13.6 mo, all study medications showed similar exposure duration between the two arms (Table). Occurrence of treatment-related adverse events was generally comparable (any grade, 97.8% in SHR-1701 arm vs 98.4% in placebo arm; grade ≥3, 62.6% vs 59.0%). SHR-1701 reduced the incidence of any grade decreased platelet count, decreased neutrophil count, and decreased white blood cell count by 8.7%, 10.1%, and 11.2%, and for grade ≥3, by 9.1%, 4.3%, and 1.6%, respectively (Table). Compared with the placebo arm, less pts in the SHR-1701 arm used platelet growth factors (30.2% vs 42.9%) and white blood cell growth factors (32.7% vs 39.6%). Conclusions: SHR-1701 showed the capacity to suppress chemo-associated myelosuppression. Clinical trial information: NCT04950322 . Safety. SHR-1701 plus CAPOX(N=364) Placebo plus CAPOX(N=366) Treatment difference (SHR-1701 vs placebo) Treatment exposure (day), median (IQR) SHR-1701/placebo 137.0 (65.5-251.5) 127.5 (62.0-190.0) NA Capecitabine 122.0 (83.0-138.0) 122.0 (73.5-135.0) NA Oxaliplatin 107.0 (66.0-123.0) 108.5 (64.0-121.0) NA Treatment-related hematological toxicities, n (%) or % Any grade Grade ≥ 3 Any grade Grade ≥ 3 Any grade Grade ≥ 3 Platelet count decreased 217 (59.6%) 69 (19.0%) 250 (68.3%) 103 (28.1%) -8.7% -9.1% Neutrophil count decreased 163 (44.8%) 44 (12.1%) 201 (54.9%) 60 (16.4%) -10.1% -4.3% White blood cell count decreased 147 (40.4%) 13 (3.6%) 189 (51.6%) 19 (5.2%) -11.2% -1.6%

Exploring the role of inflammatory biomarkers in trigeminal neuralgia.

Trigeminal neuralgia (TN) is a severe facial pain disorder with complex etiology. Inflammation has been suggested as a contributing factor to TN pathogenesis. This study investigates the causal relationship between inflammatory biomarkers, including 41 circulating inflammatory cytokines, C-reactive protein (CRP), and procalcitonin (PCT), and TN using Mendelian randomization (MR) analysis. A two-sample MR approach was employed using genome-wide association study (GWAS) data from 8293 Finnish individuals for inflammatory cytokines and data from the FinnGen database for TN. Instrumental variables (IVs) were selected based on genome-wide significance and clumping thresholds to avoid linkage disequilibrium. Inverse variance weighting (IVW) was used as the primary method, complemented by MR Egger regression, weighted median, simple mode, and weighted mode methods. Additionally, Bayesian Weighted MR (BWMR) and Multivariable MR (MVMR) were utilized to validate the findings and explore potential confounders. The present MR analysis identified significant causal associations for three inflammatory cytokines with TN. Stem cell growth factor beta (SCGF-β) (OR=1.362, 95% CI=1.049-1.770, p=0.021) and Interleukin-4 (IL-4) (OR=1.533, 95% CI=1.014-2.316, p=0.043) were positively associated with TN, while Interleukin-16 (IL-16) (OR=0.720, 95% CI=0.563-0.921, p=0.009) had a protective effect. CRP levels were also linked to TN risk (OR=0.751, 95% CI=0.593-0.951, p=0.017). No significant causal effect of PCT on TN was observed. Sensitivity analyses confirmed the robustness of these findings, showing no evidence of horizontal pleiotropy or heterogeneity. This study highlights specific inflammatory biomarkers that may play pivotal roles in TN pathogenesis. SCGF-β and IL-4 are potential therapeutic targets due to their facilitative effects on TN, while IL-16 could offer protective benefits. CRP's association with TN further supports the involvement of systemic inflammation in this condition. These findings provide novel insights into TN's inflammatory mechanisms, suggesting new avenues for targeted interventions.

MHC Class II-Expressing Mucosal Mast Cells Promote Intestinal Mast Cell Hyperplasia in a Mouse Model of Food Allergy.

IgE-mediated food allergy is accompanied by mucosal mast cell (MMC) hyperplasia in the intestinal mucosa. Intestinal MMC numbers correlate with the severity of food allergy symptoms. However, the mechanisms by which MMCs proliferate excessively are poorly understood. Here, we clarify the role of newly identified MHC class II (MHCII)-expressing MMCs in the effector phase of IgE-mediated food allergy. Mice reconstituted with MHCII-deficient or wild-type MMCs were used to generate a mouse mode of IgE-mediated food allergy. We assessed the extent of intestinal MMC hyperplasia and the severity of hypothermia in these mice. In addition, we performed invitro antigen presentation assay using induced MHCII-expressing MMCs generated from bone marrow cells to evaluate the effect of CD4+ T cell activation on MMC proliferation. In food-allergic mice, we identified the appearance of MHCII-expressing MMCs in the intestinal mucosa and showed that MMC hyperplasia was suppressed in mice with MHCII-deficient MMCs compared to mice with wild-type MMCs. Invitro assays demonstrated that MHCII-expressing MMCs incorporate food antigens directly and through the high-affinity IgE receptor FcεRI-mediated endocytosis and activate antigen-specific CD4+ T cells from food-allergic mice by antigen presentation. Activated CD4+ T cells secrete IL-4 and large amounts of IL-5, which enhance production of the mast cell growth factor IL-9 by IL-33-activated MMCs. Excess IL-9 causes excessive MMC proliferation, leading to the development of MMC hyperplasia. Antigen presentation to CD4+ T cells by MHCII-expressing MMCs triggers intestinal MMC hyperplasia and exacerbates IgE-mediated food allergy.

Pre-folding purification procedures for inclusion body-derived non-tagged cationic recombinant proteins with multiple disulfide bonds for efficient refolding.

The production of disulfide-containing recombinant proteins often requires refolding of inclusion bodies before purification. A pre-refolding purification step is crucial for effective refolding because impurities in the inclusion bodies interfere with refolding and subsequent purification. This study presents a new pre-refolding procedure using a reversible S-cationization technique for protein solubilization and purification by reversed-phase high performance liquid chromatography. This pre-folding purification step improves refolding yield by effectively removing the refolding inhibitors from contaminates from bacterial inclusion bodies, and reducing proteolytically degraded products. Because this procedure does not require a peptide tag for affinity purification, it is a superior technique to subsequently perform a simplified downstream process wherein the affinity tag needs to be removed. This study reports improved refolding and purification procedure to obtain the highly cationic (pI = 9.25) mouse vascular endothelial cell growth factor (188 amino acids form) that is used as a model protein in our study; this protein shows a homodimeric conformation and possesses multiple disulfides.

Immunohistochemical Expression of Vascular Endothelial Growth Factor (VEGF) in Primary Canine Mast Cell Tumors and Related Regional Lymph Node Metastasis.

Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and has been identified as an autocrine growth factor for neoplastic cells of several tumors. The aim of this study was to evaluate VEGF expression in canine mast cell tumors and their respective lymph node metastases. For this purpose, 28 patients with mast cell tumors and confirmed regional lymph node metastasis were selected. The samples were submitted for analysis with immunohistochemistry. VEGF was expressed in 14/28 patients (50%) and 35.7% of primary MCTs (10/28), and only 14.3% expressed VEGF in both the primary tumor and its respective metastasis (4/28), with fair agreement (Κ = 0.250). There was a weak correlation between VEGF and tumor size (p = 0.016, rs = 0.045). In this cohort, survival time was correlated with Kiupel grade, mitotic index, tumor necrosis, tumor location, and systemic treatment. VEGF immunolabeling had no influence on survival; however, patients with positive expression may benefit from specific therapy.

Distinct myeloid-derived suppressor cell populations in human glioblastoma.

The role of glioma-associated myeloid cells in tumor growth and immune evasion remains poorly understood. We performed single-cell RNA sequencing of immune and tumor cells from 33 gliomas, identifying two distinct myeloid-derived suppressor cell (MDSC) populations in isocitrate dehydrogenase-wild-type (IDT-WT) glioblastoma: an early progenitor MDSC (E-MDSC) population with up-regulation of metabolic and hypoxia pathways and a monocytic MDSC (M-MDSC) population. Spatial transcriptomics demonstrated that E-MDSCs geographically colocalize with metabolic stem-like tumor cells in the pseudopalisading region. Ligand-receptor analysis revealed cross-talk between these cells, where glioma stem-like cells produce chemokines attracting E-MDSCs, which in turn produce growth factors for the tumor cells. This interaction is absent in IDH-mutant gliomas, associated with hypermethylation and repressed gene expression of MDSC-attracting chemokines. Our study elucidates specific MDSCs that may facilitate glioblastoma progression and mediate tumor immunosuppression.

Mast cells: key players in digestive system tumors and their interactions with immune cells.

Mast cells (MCs) are critical components of both innate and adaptive immune processes. They play a significant role in protecting human health and in the pathophysiology of various illnesses, including allergies, cardiovascular diseases and autoimmune diseases. Recent studies in tumor-related research have demonstrated that mast cells exert a substantial influence on tumor cell behavior and the tumor microenvironment, exhibiting both pro- and anti-tumor effects. Specifically, mast cells not only secrete mediators related to pro-tumor function such as trypsin-like enzymes, chymotrypsin, vascular endothelial cell growth factor and histamine, but also mediators related to anti-tumor progression such as cystatin C and IL-17F. This dual role of mast cells renders them an under-recognized but very promising target for tumor immunotherapy. Digestive system tumors, characterized by high morbidity and associated mortality rates globally, are increasingly recognized as a significant healthcare burden. This paper examines the influence of mast cell-derived mediators on the development of tumors in the digestive system. It also explores the prognostic significance of mast cells in patients with various gastrointestinal cancers at different stages of the disease. Additionally, the article investigates the interactions between mast cells and immune cells, as well as the potential relationships among intratumoral bacteria, immune cells, and mast cell within digestive system microenvironment. The aim is to propose new strategies for the immunotherapy of digestive system tumors by targeting mast cells.

Novel design of potent anti-tumour activity of IL-2 prodrug by FAPα-mediated activation

Interleukin-2 (IL-2) is a T cell growth factor that is essential for the proliferation of T cells and the generation of effector and memory cells. The antitumor activity of high-dose IL-2 therapy requires maintaining the affinity between IL-2 and IL2-Rα, which can also bring serious toxic side effects. To address this issue, we designed ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine)2-IL-2-(K43C, K64C) based on the strategy of FAPα enzyme-activated prodrugs, and investigated their anti-tumour activity and side effects. In vitro FAPα enzyme cleavage results indicated that the side-chain modified ZGP-Cysteamine moiety could be precisely recognized and cleaved by FAPα, thereby restoring the activity of native IL-2 capable of binding to IL-2Rα in the tumour microenvironment, where it promotes the expansion of CD8+ T cells. Meanwhile, surface plasmon resonance analysis revealed that, compared to wt-IL-2, both ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine)2-IL-2-(K43C, K64C) exhibited significantly reduced affinity for IL-2Rα, while their affinity for IL-2Rβγ remained unchanged. Remarkably, ZGP-Cysteamine-IL-2-K64C and (ZGP-Cysteamine)2-IL-2-(K43C, K64C) almost completely eliminated the pulmonary edema and vascular permeability. Furthermore, the combination of ZGP-Cysteamine-IL-2-K64C and PD-1 blockade showed robust anti-tumour activity in mice tumour models. Our study provides new insights into the structural design of IL-2 prodrug with low side effect and robust anti-tumour efficacy.

CDK4/6 Inhibitor Resistance in ER+ Breast Cancer.

The cyclin-dependent kinases 4 and 6 inhibitors are the mainstay of treatment for patients with hormone receptor-positive and HER2-negative breast cancer. The ability of these drugs to improve the outcome of patients both in the metastatic and the early setting has been largely demonstrated. However, resistance, either de novo or acquired, represents a major clinical challenge. In the past years, efforts have been made to identify biomarkers that might help in a better selection of patients or to unravel the mechanisms leading to resistance in order to develop new therapeutic strategies to overcome it. Alterations of cell cycle-related genes and proteins are among the best characterized markers of resistance, and pathways impacting the cell cycle, including nuclear and growth factor receptors signaling, have been thoroughly investigated. Despite this, to date, cyclin-dependent kinases 4 and 6 inhibitors are administered based only on the hormone receptor and HER2 status of the tumor, and patients progressing on therapy are managed with currently available treatments. Here we summarize present knowledge on the cyclin-dependent kinases 4 and 6 inhibitors' mechanisms of action, efficacy data, and mechanisms of resistance.

Advances in the Development of Auricular Cartilage Bioimplants.

Conditions such as congenital abnormalities, cancer, infections, and trauma can severely impact the integrity of the auricular cartilage, resulting in the need for a replacement structure. Current implants, carved from the patient's rib, involve multiple surgeries and carry risks of adverse events such as contamination, rejection, and reabsorption. Tissue engineering aims to develop lifelong auricular bioimplants using different methods, different cell types, growth factors and maintenance media formulations, and scaffolding materials compatible with the host. This review aims to examine the progress in auricular bioengineering, focusing on improvements derived from invivo models and clinical trials, as well as the author's suggestions to enhance the methods. For this scope review, 30 articles were retrieved through Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, plus 6 manually selected articles. The methods reported in the articles were categorized into four levels according to the development phases: source of cells, cell media supplementation, scaffold, or scaffold-free methods, and experimental invivo or clinical approaches. Many methods have demonstrated potential for the development of bioimplants; four clinical trials reported a structure like the external ear that could be maintained after overcoming post-transplant inflammation. However, several challenges must be solved, such as obtaining a structure that accurately replicates the shape and size of the patient's healthy contralateral auricle and improvements to avoid immunological rejection and resorption of the bioimplant.

Effects of diabetes mellitus on osseointegration: a review of the available strategies for better outcomes

Diabetes mellitus, a metabolic disorder, is widely known to negatively affect bone healing, especially by interfering with dental implant osseointegration. This review aimed to explore the mechanisms of metabolic bone changes around dental implants in patients with diabetes and the available therapies to improve osseointegration. A literature search was conducted in the PubMed, Cochrane Library, and Embase electronic databases to identify relevant studies. A substantial body of evidence has demonstrated the negative impact of diabetes mellitus on bone health and osseointegration. This can be attributed to several factors, including the over-expression of reactive oxygen species, accumulation of advanced glycation end products, impaired angiogenesis, and altered expression of proteoglycans and bone-related biomarkers. Various therapeutic approaches have been proposed to enhance osseointegration of dental implants in patients with diabetes. These include insulin therapy, hypoglycemic agents, hyperbaric oxygen treatment, parathyroid hormone therapy, implant surface modification, natural substances, mesenchymal stem cell management, gene expression, and growth factor modulation. Despite the potential of numerous therapies, either for glycemic control or osseointegration improvement, additional preclinical and clinical investigations are necessary to validate their effectiveness and safety.

Innovative Biotherapies and Nanotechnology in Osteoarthritis: Advancements in Inflammation Control and Cartilage Regeneration.

Osteoarthritis (OA) is among the most prevalent degenerative joint disorders worldwide, particularly affecting the aging population and imposing significant disability and economic burdens. The disease is characterized by progressive degradation of articular cartilage and chronic inflammation, with no effective long-term treatments currently available to address the underlying causes of its progression. Conventional therapies primarily manage symptoms such as pain and inflammation but fail to repair damaged tissues. Emerging biotherapies and regenerative medicine approaches offer promising alternatives by addressing cartilage repair and inflammation control at the molecular level. This review explores the recent advancements in biotherapeutic strategies, including mesenchymal stem cell (MSC) therapy, growth factors, and tissue engineering, which hold the potential for promoting cartilage regeneration and modulating the inflammatory microenvironment. Additionally, the integration of nanotechnology has opened new avenues for targeted drug delivery systems and the development of innovative nanomaterials that can further enhance the efficacy of biotherapies by precisely targeting inflammation and cartilage damage. This article concludes by discussing the current clinical applications, the ongoing clinical trials, and the future research directions necessary to confirm the safety and efficacy of these advanced therapies for OA management. With these advancements, biotherapies combined with nanotechnology may revolutionize the future of OA treatment by offering precise and effective solutions for long-term disease management and improved patient outcomes.

Immunoadjuvant therapy in the regulation of cell death in sepsis: recent advances and future directions.

Sepsis is characterized by a concomitant early pro-inflammatory response by immune cells to an infection, and an opposing anti-inflammatory response that results in protracted immunosuppression. The primary pathological event in sepsis is widespread programmed cell death, or cellular self-sacrifice, of innate and adaptive immune cells, leading to profound immunological suppression. This severe immune dysfunction hampers effective primary pathogen clearance, thereby increasing the risk of secondary opportunistic infections, latent viral reactivation, multiple organ dysfunction, and elevated mortality. The types of cell death include apoptosis (type I programmed cell death), autophagy (type II programmed cell death), NETosis (a program for formation of neutrophil extracellular traps (NETs)) and other programmed cell deaths like pyroptosis, ferroptosis, necroptosis, each contributing to immunosuppression in distinct ways during the later phases of sepsis. Extensive apoptosis of lymphocytes, such as CD4+, CD8+ T cells, and B cells, is strongly associated with immunosuppression. Apoptosis of dendritic cells further compromises T and B cell survival and can induce T cell anergy or promote regulatory Treg cell proliferation. Moreover, delayed apoptosis and impaired neutrophil function contribute to nosocomial infections and immune dysfunction in sepsis. Interestingly, aberrant NETosis and the subsequent depletion of mature neutrophils also trigger immunosuppression, and neutrophil pyroptosis can positively regulate NETosis. The interaction between programmed cell death 1 (PD-1) or programmed cell death 1 ligand (PD-L1) plays a key role in T cell modulation and neutrophil apoptosis in sepsis. The dendritic cell growth factor, Fms-like tyrosine kinase (FLTEL), increases DC numbers, enhances CD 28 expression, attenuates PD-L1, and improves survival in sepsis. Recently, immunoadjuvant therapies have attracted attention for their potential to restore host physiological immunity and homeostasis in patients with sepsis. This review focuses on several potential immunotherapeutic agents designed to bolster suppressed innate and adaptive immune responses in the management of sepsis.

The involvement of T1R family receptors expressed outside the oral cavity in the regulation of metabolism

The membrane T1R taste receptor family interacts with sweet substances – carbohydrates, artificial sweeteners and some amino acids. An important result of research in the 21st century was the discovery of abundant expression of these receptors outside of the oral cavity, mainly in cells actively involved in metabolic processes: enteroendocrine cells of the intestine, pancreatic β-cells, adipose and bone tissue, etc. This review integrates and analyzes current data on the role of extraoral T1R receptors in the regulation of metabolism, cell growth and differentiation, which is achieved through modulation of hormone secretion (insulin, GLP-1, GIP), activity of membrane transporters and cell growth and proliferation factors. T1R mediated cellular responses to nutrients, mechanisms of signal transduction, effects on inositol triphosphate, cAMP and intracellular Ca2+ levels, stimulatory effects on glucose transporters SGLT1 and GLUT2, effects on mTOR and hormone secretion are described. The interaction of membrane receptor mechanisms and metabolic detection of glucose by the ATP/ADP ratio in the cell cytoplasm is also discussed. Putative evolutionary adaptation of metabolic processes related to nutrition and manifested in polymorphism of genes encoding T1R proteins is presented. It is suggested that extraoral taste receptors for sweet substances and amino acids may be a target for therapeutic interventions in obesity, hyperglycemia, insulin resistance, and hepatosteatosis.

Taxifolin-loaded cellulose/l-arginine-chitosan hydrogel promoting bone defect repair through osteogenesis and angiogenesis.

Bone defects have always been a difficult problem in clinical practice. Taxifolin (TAX) is beneficial to bone regeneration. In order to obtain more attractive biomaterials, we extracted cellulose (LC) from larch sawdust and prepared a TAX-loaded hydrogel (DCT) together with L-arginine chitosan (CA). The hydrogel had a uniform porous structure and good mechanical properties. After drug loading, the adhesion, antibacterial, and antioxidant properties of the hydrogel were improved. In addition, DCT has good biocompatibility. In vitro cell experiment results showed that DCT can promote cell proliferation and migration, and DCT has a certain ability to promote the osteogenic activity and enhance the expression of osteoblastic factors (OCN and Osx) and vascular endothelial growth factor (VEGF) in MC3T3-E1 cells. In addition, in vivo studies showed that the DCT treatment could promote bone regeneration by increasing the levels of osteogenic markers (OPN, OCN, and Osx) and VEGF. Transcriptome analysis showed that DCT intervention affected the hematopoiesis function in bone tissue. Meanwhile, KEGG analysis showed that DCT promoted bone regeneration mainly by activating the PI3K/AKT signaling pathway, which was verified by a western blot. Therefore, DCT is a good material for bone repair.

Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans.

Cryptococcus neoformans is a globally distributed pathogenic fungus posing a significant threat to immunocompromised individuals, particularly those with HIV/AIDS. Effective genetic manipulation tools are essential for understanding its biology and developing new therapies. However, current genetic tools, including the variation of versatile selectable markers, are limited. This study develops and validates the phosphite dehydrogenase gene (ptxD)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in C. neoformans. A codon-optimized ptxD gene from Pseudomonas stutzeri was cloned under the TEF promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the ptxD gene into the C. neoformans genome and assessed the impact of ptxD integration on cell growth and virulence factors. The ptxD/Phi system effectively selected transformed cells on Phi-containing media. Growth assays showed that ptxD integration did not adversely affect cell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the ADE2 gene using this system, confirming its applicability for gene deletion. Taken together, the ptxD/Phi system provides a robust and versatile tool for genetic manipulation in C. neoformans, facilitating further research into its biology and pathogenicity.IMPORTANCECryptococcus neoformans is a type of fungus that can cause serious illnesses in people who have weakened immune systems, like those with HIV/AIDS. To better study this fungus and find new treatments, scientists need tools to change its genes in precise ways. However, the current tools available for this are somewhat limited. This research introduces a new tool called the phosphite dehydrogenase gene/phosphite system, which does not rely on antibiotics to work. It uses a gene from a different bacterium that helps select and grow only the fungus cells that have successfully incorporated new genetic information. This is particularly useful because it does not interfere with the normal growth of the fungus or the features that make it harmful (like its ability to change shape or produce protective coatings). By making it easier and more effective to manipulate the genetics of C. neoformans, this tool opens up new possibilities for understanding how this fungus operates and for developing therapies to combat its infections. This is crucial for improving the treatment of infections in vulnerable populations.

Exploring therapeutical targets and innovative treatments for ischemic stroke: a comprehensive review

This review focuses on the current advances in the field of therapeutic targets and treatments for stroke. Stroke is a major health problem worldwide, with significant impacts on morbidity and mortality, and a considerable burden on the medical and socio-economic systems. This review provides a comprehensive overview of the current state of knowledge on acute treatments and therapeutic targets. Current stroke treatments like recanalization therapies focus mainly on restoring blood flow to the brain, reducing cell death, and preventing further damage, but have limitations in terms of efficacy and long-term outcomes. Besides acute treatments (mobile stroke units, telerehabilitation) and acute therapeutic targets, the review focuses on longer-term therapeutic targets, such as neuroprotection and neuroregeneration. Neuroprotective strategies target the mechanisms underlying energy failure, cellular acidosis, mitochondrial dysfunction, endoplasmic reticulum stress, excitotoxicity, calcium channels dysregulation, oxidative stress, neuroinflammation, blood-brain barrier disruption, apoptosis, and ischemia-reperfusion injury. Neuroregenerative approaches include stem cell therapy, gene therapy, growth factors, and rehabilitation techniques that promote the rewiring of neuronal circuits in the brain. Non-pharmacological treatments like neurostimulation and bioengineering are also presented. Additionally, we highlight the challenges and future directions in translating these therapies into clinical practice. Overall, the treatment of ischemic stroke is a complex and multifaceted process that requires a combination of acute measures as well as longer-term strategies to promote brain repair and recovery. The treatment of ischemic stroke has made significant progress in recent years with the development of new treatments and ongoing research to improve outcomes for stroke patients. However, before these therapies can be successfully integrated into routine clinical practise, further research is needed to establish standardised protocols, overcome methodological limitations, and overcome clinical challenges. By further deepening our understanding of the pathophysiology of ischemic stroke and developing innovative treatments, we can improve outcomes and quality of life for stroke survivors.

Macromolecular crowding agent dependent extracellular matrix deposition and growth factor retention in human corneal fibroblast cultures

The major obstacle in the commercialisation and clinical translation of tissue engineered medicines is the required for the development of implantable tissue surrogates prolonged in vitro culture. Macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition, thus offering an opportunity to bridge the gap between research and development in tissue engineered substitutes. However, the optimal MMC agent is still elusive. Herein, we first assessed the biophysical properties of the most widely used MMC agents [κλ carrageenan (κλ CR), λ carrageenan (λ CR) and Ficoll™ cocktail (FC)] and then assessed their effect in basic cell function, ECM deposition and growth factor retention in human corneal fibroblast (hCF) cultures. Dynamic light scattering analysis revealed that both CR macromolecules had significantly lower and higher zeta potential and hydrodynamic radius, respectively, than the FC. None of the MMC agents affected hCF morphology and all induced similar hCF viability, proliferation and metabolic activity. Electrophoresis and immunofluorescence analyses made apparent that at day 10 (longest time point assessed), the FC brought about the highest fibronectin and collagen types I, III, IV, V and VI deposition. Deposited ECM pattern analysis showed that at day 10, the FC induced the lowest lacunarity and normalised end points and the highest fractal dimension and % high density matrix. Further immunofluorescence analysis revealed no significant differences between the groups in vimentin, aldehyde dehydrogenase 3 family member A1, keratocan, paired box protein 6 and α-smooth muscle actin. Importantly, at day 10, the FC resulted in the highest growth factor retention (20 molecules). Our data clearly illustrate a MMC agent dependent cell response, with the FC having the highest positive effect in hCF cultures.

TERT mutations and aggressive histopathologic characteristics of radioiodine-refractory papillary thyroid cancer.

Radioiodine (RI) ablation following thyroid-stimulating hormone suppression is an effective treatment for papillary thyroid cancer (PTC), typically leading to favorable outcomes. However, RI-refractory tumors exhibit aggressive behavior and poor prognoses. Recent studies highlight the role of genetic abnormalities in PTC signaling pathways, including the activation of telomerase reverse transcriptase (TERT), and the correlation of mutations with adverse outcomes. This study analyzed mutations in BRAF V600E and the TERT-promoter genes, comparing clinicopathological features between RI-refractory and RI-responsive PTCs. Among 82 RI-refractory patients, formalin-fixed, paraffin-embedded tissues from initial surgeries were available for 26. Another 89 without distant metastasis over 5 years formed a matched RI-responsive control group. Histopathologically, RI-refractory PTCs showed increased frequencies of small tumor clusters without fibrovascular cores, hobnail features, and a high height-to-width ratio of tumor cells. These tumors were more likely to exhibit necrosis, mitosis, lymph node metastasis, extrathyroidal extension, and involvement of resection margins. TERT-promoter mutations were statistically significantly associated with these aggressive clinicopathologic features. Immunohistochemically, decreased expression of sodium iodide symporter and thyroglobulin stimulating hormone receptor proteins was common in RI-refractory PTCs, along with lower levels of oncogenic proteins such as vascular endothelial cell growth factor, vascular endothelial cell growth factor receptor 2, and nuclear factor kappa-light-chain-enhancer of activated B cells. Total loss of PTEN expression was occasionally observed. In contrast, all cases tested positive for cytoplasmic β-catenin. RI-refractory PTCs are linked to TERT mutations and exhibit specific aggressive histopathologic features, particularly in tumor centers.