Vehicle-treated Group Research Articles

Oral Administration of Lactilactobacillus curvatus LB-P9 Promotes Hair Regeneration in Mice.

This study was designed to examine the effect of Lactilactobacillus curvatus LB-P9 on hair regeneration. The treatment of LB-P9 conditioned medium increased the proliferation of both hair follicle dermal papilla cells and hair germinal matrix cells (hGMCs). Moreover, the expression levels of hair growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 7 were significantly elevated in hGMCs co-cultured with LB-P9. After time-synchronized depilation, mice were orally administered with either 4×107 colony forming unit (CFU) of LB-P9 (low dose) or 4×108 CFU of LB-P9 (high dose), once daily for 4 weeks. Compared with the vehicle (phosphate-buffered saline)-administrated group, the LB-P9-treated groups exhibited accelerated hair regrowth rate and enhanced hair thickness in a dose-dependent manner. Supporting this observation, both hair follicle numbers and the dermal thickness in skin tissues of the LB-P9-treated groups were increased, compared to those of the vehicle-treated group. These results might be explained by the increased level of β-catenin and number of hair follicle stem cells (CD34+CD49f+ cells) in the skin tissues of mice administered with LB-P9, compared to the vehicle-treated mice. Also, increased serum levels of hair growth factors such as VEGF and insulin-like growth factor-1, and superoxide dismutase were found in the LB-P9-treated groups, compared to those of the vehicle-treated group. Taken together, these results might demonstrate that the oral administration of LB-P9 promotes hair regeneration by the enhancement of dermal papilla proliferation through the stimulation of hair growth factor production.

Neuroprotective effect of berberine by amelioration of behavioural, biochemical and histological anomalies on valproate induced autistic features in experimental animals

ObjectiveThe goal of the current investigation was to establish the therapeutic prospective of berberine (BBR), the golden alkaloid of traditional Chinese medicine (TCM) for the treatment of valproic acid (VPA)-induced oxidative stress and neuronal damage in a postnatal model of autism, which is characterized by behavioural, biochemical, and histopathological changes. MethodsPups of the Wistar albino rat were divided into five categories of six at the age of 13 days: the vehicle-treated group (1 mg/mL normal saline), the VPA group (VPA 400 mg/kg, s.c.), the BBR per se, low (50 mg/kg, p.o.) and high dose (100 mg/kg, p.o.), marked as BBR I and BBR II. On postnatal day (PND) 14, everyone received 400 mg/kg (s.c.) of valproic acid (VPA), with the exception of the group that had received vehicle therapy. Berberine was given every day between PND 14 and 40. All the groups (II-V) getting routine therapy, were assessed for various neuro-behavioural indicators. Towards the conclusion of the study, animals were euthanized on PND 41 for antioxidant, inflammatory and histopathological computations. Results: Autism was effectively caused with a single dosage of 400 mg/kg s.c. of VPA. Treatment with berberine considerably improved neuro-behavioural activity, biochemical and inflammatory alterations, along with histological abnormalities in comparison to animals of autistic group. The results were more prominent at larger doses of berberine, providing proof that the deficiencies in autistic behaviour caused by valproic acid may be reversed. ConclusionBerberine exhibited neuro-protective benefits, perhaps as a result of its antioxidant and anti-inflammatory action, thus may be beneficial in the treatment of autism spectrum disorder (ASD). Future research is needed to fully understand berberine's mode of action and potential contribution to human autism spectrum disorders.

Resveratrol Administration Ameliorates Hepatotoxicity in Mercuric Chloride-‎Induced Liver Injury in Rats

Mercuric chloride (HgCl2) pollution and poisoning has been a worldwide health ‎concern for decades, especially after the industrial revolutions. The aim of this study ‎was to investigate the role of resveratrol in reversing the deleterious effects of ‎HgCl2 exposure to resume the normal functions of hepatocyte. To achieve the study, ‎mature Sprague Dawley rats were assigned to five groups. Negative control group ‎‎(C) kept without any treatment; vehicle-treated group (D) received dimethyl ‎sulfoxide (DMSO); resveratrol-treated group (R), received 100 mg/kg of resveratrol; ‎HgCl2-intoxicated group (HD), received i.p. injection of HgCl2 at a dose of 1 mg/kg ‎for 30 consecutive days along to oral gavage of DMSO; and finally HgCl2-‎intoxicated group treated with resveratrol (HR) as same treatment strategy of R-‎group. At the endpoint of the experiment, blood samples were collected for ‎biochemical liver function tests along with serum concentrations of ‎malondialdehyde (MDA), glutathione (GSH), body weight, as well as ‎histopathological investigation was done too. Study results revealed a significant ‎‎(P<0.05) elevation in serum AST, ALP, GGT, and MDA in HD group in comparison ‎with HR group. However, resveratrol treatment has led to a significant (P<0.05) ‎increase in serum levels of GSH in HR group in comparison with the HD group. ‎Histopathological sections showed vacuolar degeneration in HD hepatocytes while ‎resveratrol treatment protected the hepatocytes against the chemical injury. ‎Altogether, It is concluded that resveratrol administration has the ability to increase ‎the resistance of liver against the HgCl2-induced hepatotoxicity via increase the ‎antioxidant yields such as GSH resulted in reduction of hepatocellular texture ‎damage.

AdipoRon reduces TGFβ1-mediated collagen deposition in vitro and alleviates knee stiffness in vivo.

Arthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti-fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFβ1-mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFβ1-induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA-seq analysis corroborated the anti-myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle-treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.

Intracranial Assessment of Androgen Receptor Antagonists in Mice Bearing Human Glioblastoma Implants.

The median survival time of patients with an aggressive brain tumor, glioblastoma, is still poor due to ineffective treatment. The discovery of androgen receptor (AR) expression in 56% of cases offers a potential breakthrough. AR antagonists, including bicalutamide and enzalutamide, induce dose-dependent cell death in glioblastoma and glioblastoma-initiating cell lines (GIC). Oral enzalutamide at 20 mg/kg reduces subcutaneous human glioblastoma xenografts by 72% (p = 0.0027). We aimed to further investigate the efficacy of AR antagonists in intracranial models of human glioblastoma. In U87MG intracranial models, nude mice administered Xtandi (enzalutamide) at 20 mg/kg and 50 mg/kg demonstrated a significant improvement in survival compared to the control group (p = 0.24 and p < 0.001, respectively), confirming a dose-response relationship. Additionally, we developed a newly reformulated version of bicalutamide, named "soluble bicalutamide (Bic-sol)", with a remarkable 1000-fold increase in solubility. This reformulation significantly enhanced bicalutamide levels within brain tissue, reaching 176% of the control formulation's area under the curve. In the U87MG intracranial model, both 2 mg/kg and 4 mg/kg of Bic-sol exhibited significant efficacy compared to the vehicle-treated group (p = 0.0177 and p = 0.00364, respectively). Furthermore, combination therapy with 8 mg/kg Bic-sol and Temozolomide (TMZ) demonstrated superior efficacy compared to either Bic-sol or TMZ as monotherapies (p = 0.00706 and p = 0.0184, respectively). In the ZH-161 GIC mouse model, the group treated with 8 mg/kg Bic-sol as monotherapy had a significantly longer lifespan than the groups treated with TMZ or the vehicle (p < 0.001). Our study demonstrated the efficacy of androgen receptor antagonists in extending the lifespan of mice with intracranial human glioblastoma, suggesting a promising approach to enhance patient outcomes in the fight against this challenging disease.

Neurological Improvement via Lysophosphatidic Acid Administration in a Rodent Model of Cardiac Arrest-Induced Brain Injury.

Lysophosphatidic acid (LPA) serves as a fundamental constituent of phospholipids. While prior studies have shown detrimental effects of LPA in a range of pathological conditions, including brain ischemia, no studies have explored the impact of LPA in the context of cardiac arrest (CA). The aim of this study is to evaluate the effects of the intravenous administration of an LPA species containing oleic acid, LPA (18:1) on the neurological function of rats (male, Sprague Dawley) following 8 min of asphyxial CA. Baseline characteristics, including body weight, surgical procedure time, and vital signs before cardiac arrest, were similar between LPA (18:1)-treated (n = 10) and vehicle-treated (n = 10) groups. There was no statistically significant difference in 24 h survival between the two groups. However, LPA (18:1)-treated rats exhibited significantly improved neurological function at 24 h examination (LPA (18:1), 85.4% ± 3.1 vs. vehicle, 74.0% ± 3.3, p = 0.045). This difference was most apparent in the retention of coordination ability in the LPA (18:1) group (LPA (18:1), 71.9% ± 7.4 vs. vehicle, 25.0% ± 9.1, p < 0.001). Overall, LPA (18:1) administration in post-cardiac arrest rats significantly improved neurological function, especially coordination ability at 24 h after cardiac arrest. LPA (18:1) has the potential to serve as a novel therapeutic in cardiac arrest.

Modulation of Hematopoietic Injury by a Promising Radioprotector, Gamma-Tocotrienol, in Rhesus Macaques Exposed to Partial-Body Radiation.

Currently, no radioprotectors have been approved to mitigate hematopoietic injury after exposure to ionizing radiation. Acute ionizing radiation results in damage to both hematopoietic and immune system cells. Pre-exposure prophylactic agents are needed for first responders and military personnel. In this study, the ability of gamma-tocotrienol (GT3), a promising radioprotector and antioxidant, to ameliorate partial-body radiation-induced damage to the hematopoietic compartment was evaluated in a nonhuman primate (NHP) model. A total of 15 rhesus NHPs were divided into two groups, and were administered either GT3 or vehicle 24 h prior to 4 or 5.8 Gy partial-body irradiation (PBI), with 5% bone marrow (BM) sparing. Each group consisted of four NHPs, apart from the vehicle-treated group exposed to 5.8 Gy, which had only three NHPs. BM samples were collected 8 days prior to irradiation in addition to 2, 7, 14, and 30 days postirradiation. To assess the clonogenic ability of hematopoietic stem and progenitor cells (HSPCs), colony forming unit (CFU) assays were performed, and lymphoid cells were immunophenotyped using flow cytometry. As a result of GT3 treatment, an increase in HSPC function was evident by an increased recovery of CFU-granulocyte macrophages (CFU-GM). Additionally, GT3 treatment was shown to increase the percentage of CD34+ cells, including T and NK-cell subsets. Our data further affirm GT3's role in hematopoietic recovery and suggest the need for its further development as a prophylactic radiation medical countermeasure.

Intravenous transplantation of bone marrow-derived mesenchymal stem cells improved behavioral deficits and altered fecal microbiota composition of BTBR mice

AimsIt is recognized that autism spectrum disorder (ASD) is a highly complex neurodevelopmental disorder with communication deficits as well as multiple social barriers. The core symptoms of ASD are not treatable with current therapeutics. Therefore, finding new treatment strategies for ASD is urgently needed. Mesenchymal stem cells (MSC) have been shown to be a promising therapeutic approach in previous studies. However, the underlying mechanisms of MSC treatment for ASD through gut microbiota remain unclear and require further investigation. Main methodsBTBR mice were used as ASD model and then randomly assigned to the human bone marrow-derived mesenchymal stem cell (hBMMSC) intravenous treatment group or vehicle treatment group. C57BL/6J (C57) mice served as control. Multiple social behavioral tests were performed during the 6-week period and fecal samples were collected at different time points for 16 s rRNA sequencing analysis. Key findingsThe administration of hBMMSC improved social deficits of BTBR mice in the open field test (OFT), light-dark box test (LBT), novel object recognition (NOR), and free social test (FST), while also significantly reducing stereotypic behaviors. Additionally, hBMMSC administration notably reversed the alterations of microbiota abundance in BTBR mice, particularly the Firmicutes/Bacteroidetes ratio. Several specific differential taxa were further selected and showed a correlation with the prognosis and behavioral scores of ASD. SignificanceOverall, intravenous treatment with hBMMSC had a beneficial impact on ASD by ameliorating social deficits and modifying microbiota compositions. This outcome indicates that hBMMSC intravenous transplantation could be a promising therapeutic strategy for enhancing ASD symptoms improvements.

Evaluation of dermal toxicity and toxicokinetics of povidone‑iodine in Göttingen minipigs

BackgroundPovidone‑iodine (PVP-I) is an effective and commonly used broad-spectrum antiseptic; limited information exists around its long-term safety and impact on endocrine disruption. We assessed the dermal toxicity and toxicokinetics following a once-daily application of 7.5% (w/v) and 10% (w/v) PVP-I in Göttingen Minipigs® for up to 39 weeks. MethodsAn in vivo study was conducted in male (n = 27) and female (n = 27) minipigs. Animals were randomized into untreated control, 7.5% and 10% PVP-I, and matching vehicle treatment groups. Animals were assessed for general in-life measurements, including skin irritation and organ weights. Serum samples were analyzed for PVP, total iodine, triiodothyronine [T3], thyroxine [T4], thyroid stimulating hormone [TSH], and toxicokinetic parameters. ResultsNeither 7.5% nor 10% PVP-I affected general in-life measurements. Increased mean thyroid gland absolute weights were noted with 7.5% and 10% PVP-I. Serum levels of PVP, T3, T4, and TSH in the 7.5% and 10% PVP-I treatment group animals were similar to those in vehicle treatment group animals. Mean total serum iodine concentration was 52- and 13-fold higher with 7.5% and 10% PVP-I, respectively, vs respective vehicle treatments. There was no dose-dependent increase in mean maximum serum concentration and area under the curve from 0 to 24 h for PVP, T3, T4, and TSH, nor accumulation of PVP, T3, T4, or TSH in the study. ConclusionOnce-daily dermal application of 7.5% and 10% PVP-I for up to 39 weeks was safe and well tolerated in Göttingen Minipigs® and was not associated with skin irritation, thyroid dysfunction, or endocrine disruption. As the anatomy and physiology of the minipig skin closely resembles that of human skin, the findings of this study suggest that 7.5% and 10% PVP-I may be translated into antimicrobial benefits for humans without the risk of endocrine disruption.

The selective inhibition of the Syk tyrosine kinase ameliorates experimental autoimmune arthritis.

Autoimmune arthritis - such as rheumatoid arthritis - affect a significant proportion of the population, which can cause everyday joint pain, decreased mobility and reduced quality of life. Despite having more and more therapeutic options available, there are still a lot of patients who cannot reach remission or low disease activity by current therapies. This causes an urgent need for the development of new treatment options. The Syk tyrosine kinase plays an essential role in B cell receptor, Fc receptor and integrin signaling. It has been shown that the hematopoietic cell-specific deletion of Syk resulted in a complete protection against autoantibody-induced experimental arthritis. This prompted us to test the effect of entospletinib, a second generation, Syk-selective inhibitor, which has a tolerable safety profile according to hematological clinical trials, in experimental autoimmune arthritis. We found that entospletinib dose-dependently decreased the macroscopic signs of joint inflammation, while it did not affect the health status of the animals. In line with these findings, local neutrophil accumulation and cytokine levels were reduced compared to the vehicle-treated group, while macrophage accumulation and synovial fibroblast numbers were not significantly altered. Meanwhile, entospletinib dose-dependently decreased the cell responses of immune complex- or integrin ligand-activated neutrophils. Overall, we found that selective Syk inhibition by entospletinib reduced the activity of autoantibody-induced experimental arthritis, which seems to be based mainly on the effect of the inhibitor on neutrophil functions. Our data raise the possibility that entospletinib could be a good drug candidate in the treatment of human autoimmune arthritis.

Abstract B006: High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC

Abstract KRASG12C inhibitors such as sotorasib and adagrasib have shown efficacy in patients with non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not respond. Therefore, there is a clear need for predictive biomarkers to guide treatment. The proximity ligation assay (PLA) is an immunofluorescent-based approach utilized to analyze endogenous protein-protein interactions. The method allows for the assessment of signaling-associated protein complexes, which can serve as potential targets for specific inhibitors. In particular, KRASG12C inhibitors can disrupt these complexes by interfering with the interaction between RAS and its effector RAF. By providing insights into the presence of these protein complexes, PLA offers a predictive readout for inhibitor sensitivity. We thus conducted a preclinical study to evaluate if a RAS-CRAF PLA could inform on the presence of RAS-RAF protein complexes and predict the response to KRASG12C inhibitors. To assess the specificity of the panRAS-CRAF PLA, we employed RNA interference in a KRASG12C-mutant H358 NSCLC cell line, wherein knockdown of either panRAS or CRAF resulted in a reduction in the panRAS-CRAF PLA signal. Pharmacological inhibition of RAS-RAF interaction with sotorasib also reduced the panRAS-CRAF PLA signal. We next performed panRAS-CRAF PLA in a panel of 11 KRASG12C-mutant NSCLC cell lines, revealing a higher number of panRAS-CRAF PLA spots in H358, LU65, and HCC1171. To further investigate the RAS-RAF interaction, we assessed the abundance of CRAF-bound RAS-GTP by quantifying the binding of RAS-GTP to the RAS-binding domain of CRAF. H358, LU65, and HCC1171 exhibited the highest levels of RAS-GTP among the 11 cell lines. We observed a strong correlation between the number of panRAS-CRAF PLA foci and the levels of CRAF-bound RAS-GTP (r = 0.87, P = 0.0002). These results indicated that the enhanced level of RAS-GTP for CRAF proteins facilitates the formation of RAS-CRAF complexes, thereby augmenting the panRAS-CRAF PLA signals. We then evaluated the in vitro antitumor activity of sotorasib across the G12C-mutated cell lines and found that the 50% inhibitory concentration values of sotorasib for H358, LU65, and HCC1171 were below 30 nmol/L. The panRAS-CRAF PLA was strongly associated with the sensitivity to sotorasib (r = 0.81, P = 0.002). We subsequently assessed the association of panRAS-CRAF PLA with efficacy of adagrasib in a patient-derived xenograft (PDX) cohort. PanRAS-CRAF PLA was assessed on sections of formalin-fixed paraffin-embedded tumors derived from vehicle-treated control groups at the end of efficacy studies in 18 NSCLC PDX models. Our findings revealed a significant association between high panRAS-CRAF PLA signal and increased sensitivity to adagrasib (r = 0.61, P = 0.008). Our results suggest that panRAS-CRAF PLA may serve as a predictive marker to identify patients who could derive optimal benefit from KRASG12C inhibitors, and may direct combination therapy approaches to those patients less likely to benefit from single-agent therapy. Citation Format: Ryoji Kato, Hitendra S. Solanki, Denis Imbody, Anurima Majumder, Yaakov Stern, Liznair Bridenstine, Harika Gundlapalli, Ida Aronchik, Joseph Johnson, Eric B. Haura. High RAS-RAF binding as assessed via proximity ligation assay is associated with sensitivity to KRASG12C inhibitors in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B006.

Aurantio-obtusin alleviates allergic responses in ovalbumin-induced rhinitis

Allergic rhinitis (AR) is a growing global health concern. Despite being non-fatal, it has a detrimental effect on people's quality of life and poses a significant economic burden. In this study, we aimed to evaluate the anti-allergic effects of aurantio-obtusin against ovalbumin-induced murine model of allergic rhinitis. Aurantio-obtusin was administered to rats orally after sensitization to ovalbumin. Results of the study suggested that aurantio-obtusin causes a significant reduction in nose rubs and sneeze as compared to the vehicle-treated disease control group. The aurantio-obtusin treated group showed a dose-dependent significant reduction in serum TNF-α, IL-4, and IL-6 levels as well as histamine levels in the nasal mucosa. Additionally, it decreased neutrophil and eosinophil numbers in blood of AR rats'. In the nasal tissues of the vehicle-treated rats that were subjected to an OVA challenge, the histology of the nasal mucosa of AR rats revealed aberrant histological changes. A variety of histological abnormalities, including vascular congestion in the nasal mucosa, substantial inflammatory cell infiltration, and obvious alveolar wall oedema were present. In the rats treated with aurantio-obtusin, these characteristics were reversed. Collectively, the findings suggests that aurantio-obtusin could be used to enhance current AR treatment approaches.

Detection and Prevention of ADAR1p150-Induced Hematopoietic Stem and Progenitor Cell Aging

Introduction: Over the last decade, ADAR1p150 has been linked by our research team and others to malignant progenitor reprogramming and therapeutic resistance in a broad array of malignancies. However, ADAR1p150's role in accelerated hematopoietic stem and progenitor cell (HSPC) aging in response to inflammatory cytokines had not been clearly elucidated. Here, we investigate ADAR1 splice isoform switching and the ADAR1p150 inhibitory effects of Rebecsinib on human HSPC function in normal humanized aged normal bone marrow (aNBM) HSPC mouse models. Methods: Whole transcriptome RNA sequencing (RNA-seq) was utilized with splice isoform and editome analysis to detect ADAR1 splice isoform switching and adenosine to inosine RNA editing. Rebecsinib (17S-FD-895), a pre-IND drug candidate, has shown promising potential as an inhibitor of ADAR1p150-mediated RNA editing ( Crews, Ma...Jamieson. Cell Stem Cell 2023), which is an important therapeutic target in patients with myelofibrosis or acute myeloid leukemia that overexpress ADAR1p150 compared with ADAR1p110. Human CD34 + cells, isolated from aged normal bone marrow (aNBM) from patients undergoing elective hip replacement surgery, were transduced with a lentiviral ADAR1-nano-luciferase reporter and transplanted into NSG-SGM3 adult mice that secrete human SCF, IL-3, and GM-CSF cytokines that support normal hematopoiesis. After engraftment confirmation peripheral blood human CD45 flow cytometric assessment and IVIS 200 system imaging of ADAR1-nano-luc-GFP reporter expressing HSPC engraftment, the mice were randomly divided into vehicle-treated and Rebecsinib-treated groups (15mg/kg, twice a week for two weeks). Peripheral blood, bone marrow, and spleen were collected upon treatment completion for analysis. Results: By HSPC RNA-seq, we detected an imbalance between ADAR1p150 and ADAR1p110 expression together with increased RNA editing and splicing alterations using whole transcriptome RNA sequencing (RNA-seq) of aged compared with young bone marrow hematopoietic stem and progenitor cells (HSPCs). However, the functional relevance of ADAR1 splice isoform switching had not been evaluated in human-aged normal bone marrow HSPC xenograft (PDX) mouse models nor had this cytokine-induced HSPC aging been reversed. Flow cytometric analysis and immunohistochemistry staining revealed that Rebecsinib treatment at a dose of 10mg/kg, twice a week for two weeks, effectively spared normal human immune cells (CD3+, CD14+, and CD19+ cells). Remarkably, Rebecsinib treatment not only preserved Hematopoietic Stem (CD34+CD38-) cells and Progenitor (CD34+CD38+) cells in peripheral blood, bone marrow, and spleen but also promoted their expansion. These findings demonstrate the potential of Rebecsinib to enhance the retention of HSPC populations in the bone marrow niche. Conclusions: Our pre-clinical experiments utilizing RNA-seq, a novel ADAR1-nano-luciferase-GFP reporter, and aNBM PDX mouse models provide compelling evidence supporting the potential of Rebecsinib to restore the balance of ADAR1p150:p110 ratios and engraftment in humanized aNBM HSPC mouse models. These findings highlight Rebecsinib as a promising therapeutic candidate for targeting leukemia and myelofibrosis while preserving essential HSPC populations.

Skp2 Inhibition Diminishes Essential Cell Survival Markers c-Maf and c-Myc, Enhancing Therapeutic Efficacy Against Drug Resistant Multiple Myeloma

Multiple Myeloma (MM) presents challenges as a hematologic malignancy, with drug resistance leading to limited curative options and disease relapse despite advancements in therapy. Understanding the molecular mechanisms driving MM's development and progression is vital for identifying new treatment approaches. Clinical and experimental evidence supports the role of S-phase kinase-associated protein 2 (Skp2) in driving the progression of several cancers, including MM. We interrogated a public microarray dataset derived from clinical specimens of MM patients and observed a significantly higher expression of Skp2 within the relapsed patient cohort (N=28) compared to the newly diagnosed group (N=63) (P=0.0041). Additionally, we observed elevated Skp2 levels and decreased P27 Kip1 levels in drug-resistant MM cell lines (MM.1R and RPMI 8226.R5) compared to their parental cell lines (MM.1S and RPMI 8226). Hence, a comprehensive understanding of Skp2's molecular functions in drug-resistant MM becomes paramount for facilitating the development of precision-targeted interventions. We investigated Skp2's molecular interactions in MM by performing a targeted knockdown with siRNA in drug-resistant and sensitive cell lines (OPM1, RPMI.8226, MM.1S, and MM.1R). Immunoblot analysis showed reduced STAT3 activation and decreased levels of cell survival markers c-Maf and c-Myc. Treatment with 10uM of the small molecule inhibitor SkpinC1 also inhibited STAT3 activation and led to significant reductions in c-Maf and c-Myc protein levels compared to the vehicle-treated groups. These findings hold immense significance as c-Maf and c-Myc are intricately associated with MM progression and poorer outcomes, although there is limited information on their regulation by the Skp2/P27 axis. Functionally, treatment of the MM cell lines with increasing dosages of SkpinC1 showed a progressive decrease in MM cell line survival, with IC50 values spanning 5-15uM, signifying the broad applicability of the inhibitor among the tested cell lines. Moreover, flow cytometry-based analysis of the surviving cell population demonstrated that SkpinC1 based inhibition of the Skp2/P27 axis considerably decreased the percentage of proliferating M-phase cells, while elevating the proportion of quiescent and apoptotic cells relative to the control group. At the molecular level, targeted inhibition of the Skp2/P27 axis by SkpinC1 caused the accumulation of cell cycle inhibitors P27 Kip1, P21 (Cip1/Waf1), and P57 Kip2, activation of the tumor suppressive TP53 protein with a simultaneous decrease in phospho-MDM2 levels in addition to an increase in the levels of apoptotic markers cleaved PARP and Caspase 3. From a clinical perspective, we also probed the applicability of Skp2 inhibition using SkpinC1 as a potential strategy for managing therapeutic resistance in MM. We treated multiple MM cell lines (OPM1, RPMI.8226, MM.1S and MM.1R) using a combination of Lenalidomide (an immune modulator) or Bortezomib (a proteasomal inhibitor) with SkpinC1 and compared it with single agent treatments. Both Lenalidomide/SkpinC1 as well as Bortezomib/SkpinC1 showed a strong dose dependent decrease in the percentage of surviving cells relative to the single agent treatments, thereby highlighting the remarkable potential of targeting the Skp2/P27 axis for clinical therapy in MM. To gain deeper insights into the molecular roles of Skp2/P27 axis inhibition by SkpinC1, we subjected drug-resistant MM.1R cells to 10uM SkpinC1 treatment for 24 hours, followed by single-cell transcriptome sequencing. This approach illuminated considerable heterogeneity between the drug-treated and control cells, accompanied by global changes in various signaling pathways associated with cell survival. In conclusion, the inhibition of Skp2 in MM cells exerts a significant impact on crucial cell survival pathways, notably c-Maf and c-Myc, thereby promoting apoptosis and offering a promising therapeutic avenue to combat drug resistance in MM. The precise targeting of Skp2 using small molecule inhibitors, such as SkpinC1, demonstrates substantial therapeutic potential, enhancing the efficacy of combinatorial treatment approaches for more effective interventions in MM.

PKM2 Nuclear Translocation Promotes Glial Cell Activation and Aggravates the Brain Injury of Intracerebral Hemorrhage.

The purpose of this study was to investigate the potential involvement of pyruvate kinase M2 (PKM2), an enzyme acting as a rate-limiting enzyme in the final phase of glycolysis, in the regulation of glial activation and brain damage of intracerebral hemorrhage (ICH). Western blotting and immunofluorescence were performed to investigate PKM2 expression, terminal deoxynucleotidyl transferase deoxyurinary triphosphate (dUTP) nick end labeling staining, hematoxylin and eosin staining, and behavioral tests were employed to evaluate the brain damage of ICH mice, and RNA-seq and bioinformatic analyses were performed to detect gene expression changes in ICH mice treated with TEPP-46. Increased PKM2 levels in perihematomal brain tissue were found starting from 3 days following ICH and peaked at 5 and 7 days post ICH. The increased expression of PKM2 was mainly co-localized with glial fibrillary acidic protein (GFAP)+ astrocytes and ionized calcium binding adaptor molecule-1 (IBA-1)+ microglia. Furthermore, we observed a notable increase in the nuclear translocation of PKM2 in glial cells following ICH. TEPP-46 treatment significantly reduced PKM2 nuclear translocation, and effectively attenuated glial activation and brain injury, and improved functional recovery of mice with ICH. RNA-seq data indicated that 91.1% (205/225) of differentially expressed genes (DEGs) were down-regulated in the TEPP-46 treated groups compared with the vehicle-treated groups in ICH brains. Furthermore, bioinformatic analyses revealed that these down-regulated DEGs were involved in a variety of biological processes, including autophagy and metabolic processes. In addition, the majority of these downregulated DEGs had a primary high expression in neurons, with subsequent expression seen in endothelial cells, microglia, and astrocytes. These results indicate that increased PKM2 nuclear translocation promotes the activation of glial cells after ICH, hence aggravating ICH-induced brain damage, and aggravates the brain injury induced by ICH. This highlights a potential therapeutic target for inhibiting glial activation to attenuate brain injury after ICH.

(E)-3-(4-(tert-Butyl)Phenyl)-N-Isobutyl-2-Methylacrylamide Suppresses Adipogenesis in High-Fat-Fed Mice

Obesity is caused by excessive adipogenesis and leads to metabolic diseases such as diabetes, hypertension, and degenerative arthritis. Therefore, there is an ever-increasing need to identify new anti-obesity compounds. In this study, the anti-obesity effects of (E)-3-(4-(tert-butyl)phenyl)-N-isobutyl-2-methylacrylamide (BPIMA) were evaluated using pre-adipocytes and a high-fat-fed C57BL/6J mouse model. As the results, BPIMA effectively inhibited adipocyte differentiation (64.3% at 40 μM) without exhibiting any cytotoxicity. Additionally, BPIMA decreased the protein levels of PPARγ, FAS, C/EBP, and SREBP in 3T3-L1 cells in a concentration-dependent manner. In C57BL/6J mice, a significant decrease in body weight, adipose tissue, and fatty liver was observed in the BPIMA-treated high-fat group compared to that observed in the vehicle-treated high-fat group; the serum levels of total cholesterol and glucose also significantly decreased in the BPIMA-treated high-fat group. Moreover, the weight and fat level of liver and serum AST, ALT activity in the BPIMA-treated high-fat group were similar to low fat diet-control group. These findings show that BPIMA may be a potential anti-obesity compound.

In Vitro and In Vivo Bone-Forming Effect of a Low-Molecular-Weight Collagen Peptide.

This study reveals that low-molecular-weight collagen peptide (LMWCP) can stimulate the differentiation and the mineralization of MC3T3-E1 cells in vitro and attenuate the bone remodeling process in ovariectomized (OVX) Sprague-Dawley rats in vivo. Moreover, the assessed LMWCP increased the activity of alkaline phosphatase (ALP), synthesis of collagen, and mineralization in MC3T3-E1 cells. Additionally, mRNA levels of bone metabolism-related factors such as the collagen type I alpha 1 chain, osteocalcin (OCN), osterix, bone sialoprotein, and the Runt family-associated transcription factor 2 were increased in cells treated with 1,000 μg/ml of LMWCP. Furthermore, we demonstrated that critical bone morphometric parameters exhibited significant differences between the LMWCP (400 mg/kg)-receiving and vehicle-treated rat groups. Moreover, the expression of type I collagen and the activity of ALP were found to be higher in both the femur and lumbar vertebrae of OVX rats treated with LMWCP. Finally, the administration of LMWCP managed to alleviate osteogenic parameters such as the ALP activity and the levels of the bone alkaline phosphatase, the OCN, and the procollagen type 1 N-terminal propeptide in OVX rats. Thus, our findings suggest that LMWCP is a promising candidate for the development of food-based prevention strategies against osteoporosis.

Aqueous remote loading of setmelanotide in poly(lactic-co-glycolic acid) microspheres for long-term obesity treatment

Setmelanotide (Imcivree™) was developed as a daily injectable therapeutic peptide for the treatment of rare forms of syndromic obesity, such as POMC deficiency and leptin receptor deficiency. The important option of poly(lactic-co-glycolic acid) (PLGA) controlled release microspheres has become more attractive for this class of drugs upon the discovery that net positively charged peptides can be remote-loaded rapidly from aqueous peptide solution into blank microspheres at high loading and encapsulation efficiency. Here we sought to remote-load setmelanotide in PLGA microspheres and examine its potential for long-term controlled release and body weight control. The influence of PLGA microsphere porosity was investigated with respect to morphology, drug loading, and in vitro release profiles. Increased density of the microspheres inhibited the progress of encapsulation of the dicationic peptide. A diet-induced obese murine model was then used to determine the pharmacokinetic profile and to evaluate long-term efficacy of an optimal formulation. Remote loaded PLGA formulations encapsulated setmelanotide as high as ∼63% (∼6.3% w/w loading) and exhibited slow and continuous peptide release over ∼6 weeks in vitro largely independent of microsphere porosity. The obtained in vivo release pattern from deconvolution of the pharmacokinetics after subcutaneous microsphere injection was consistent with the in vitro release profile but with a lower initial burst release and overall slightly faster release rate. After a single injection of remote-loaded setmelanotide, continuous long-term inhibition of food intake and body weight control was observed over 17 and 30 days, respectively. The improvement in body weight control over drug-free microsphere vehicle-treated control groups matched the observed PK profile. This study provides the first report of long-acting release formulation for 1-month controlled release of setmelanotide and body weight control in a diet induced obese murine model, and supports the further development of long-acting treatment options for obese patients.

The role of cellular senescence in aortic dissection

Abstract Background Aortic dissection (AD) is a catastrophic disease with high mortality. Survived patients frequently suffer from serious complications due to progressive destruction of the aortic walls. Recently we have reported that cell proliferation precedes the inflammatory response which is important in AD development and progression. Since cell proliferation promotes cellular senescence that can induce inflammatory response through SASP (senescence-associated secretary phenotype), we hypothesized that cellular senescence plays a critical role in AD pathogenesis. Purpose To investigate if cellular senescence contributes to AD pathogenesis in a mouse AD model. Methods and Results A mouse AD model was created by continuous infusion of beta-aminopropionitrile and angiotensin II (BAPN+AngII), where AD occurs to most of the mice in 14 days. BAPN+AngII challenge resulted in the appearance of senescent cells in the aortic walls before AD onset, which persisted for 14 days. Senescent cells, as demonstrated by the expression of senescence-associated beta-galactosidase, were evident in multiple types including endothelial cells, medial smooth muscle cells, adventitial macrophages and fibroblasts. Rapamycin, an inhibitor of mTOR pathway, suppressed the cell proliferation, the cellular senescence, and the development and progression of AD. To examine the role of cellular senescence in AD pathogenesis, ABT263, which is a senolytic drug that eliminates senescent cells, was administered orally to AD model mice. ABT263 treatment reduced the expression of the senescence markers. In the ABT263-treated group, the AD mortality was significantly decreased compared to that in vehicle-treated group (35% vs 67%, P &amp;lt; 0.05 by log-rank test). The severity of AD, as assessed by the lesion length, was also attenuated in ABT263 group (24.6 ± 1.8 mm vs 33.2 ± 3.1 mm, P &amp;lt; 0.05). Transcriptome analysis revealed that ABT263 treatment suppressed the aortic immune and inflammatory response induced by BAPN+AngII. Quantitative RT-PCR of aortic tissue confirmed that ABT263 treatment prevented the induction of p21, interleukin-6, several chemokines and M1 macrophage marker (CD80). ABT263 treatment suppressed CD80 expression also in cultured macrophages. Conclusions These results indicated that cell proliferation and senescence via mTOR pathway are critically involved in AD pathogenesis. Cellular senescence contributes to the inflammatory response including M1 differentiation of macrophages, and to the severity and the mortality in AD. Cellular senescence could be a new therapeutic target for AD.

The effects of Src tyrosine kinase inhibitor, saracatinib, on the markers of epileptogenesis in a mixed-sex cohort of adult rats in the kainic acid model of epilepsy.

Neurodegeneration and neuroinflammation are key processes of epileptogenesis in temporal lobe epilepsy (TLE). A considerable number (∼30%) of patients with epilepsy are resistant to currently available antiseizure drugs and thus there is a need to develop adjunct therapies to modify disease progression. A vast majority of interventional strategies to treat TLE have utilized males which limits the translational nature of the studies. In this study, we investigated the effects of repeated low-dose kainic acid (KA) injection on the initial status epilepticus (SE) and the effects of Src kinase inhibitor, saracatinib (SAR/AZD0530; 20 mg/kg, oral, daily for 7 days), in a mixed-sex cohort of adult Sprague Dawley rats during early epileptogenesis. There were no sex differences in response to KA-induced SE, and neither did the stage of estrus influence SE severity. KA-induced SE caused significant astrogliosis and microgliosis across the hippocampus, piriform cortex, and amygdala. SAR treatment resulted in a significant reduction of microgliosis across brain regions. Microglial morphometrics such as branch length and the endpoints strongly correlated with CD68 expression in the vehicle-treated group but not in the SAR-treated group, indicating mitigation by SAR. KA-induced SE caused significant neuronal loss, including parvalbumin-positive inhibitory neurons, in both vehicle (VEH) and SAR-treated groups. SAR treatment significantly mitigated FJB-positive neuronal counts as compared to the VEH group. There was an increase in C3-positive reactive astrocytes in the VEH-treated group, and SAR treatment significantly reduced the increase in the piriform cortex. C3-positive astrogliosis significantly correlated with CD68 expression in the amygdala (AMY) of VEH-treated rats, and SAR treatment mitigated this relationship. There was a significant increase of pSrc(Y419)-positive microglia in both KA-treated groups with a statistically insignificant reduction by SAR. KA-induced SE caused the development of classical glial scars in the piriform cortex (PIR) in both KA-treated groups, while SAR treatment led to a 42.17% reduction in the size of glial scars. We did not observe sex differences in any of the parameters in this study. SAR, at the dose tested in the rat kainate model for a week in this study mitigated some of the markers of epileptogenesis in both sexes.