Nifuroxazide (NFZ) is an antimicrobial drug, which has been found to be a promising antitumor agent in recent years. In addition to being a classic STAT3 inhibitor, NFZ can also act on IL-6 and exert an anti-tumor role through inflammatory factor pathways. It can also bind to target proteins of aldehyde dehydrogenase 1, one of the families of E-twenty-six transcription factors and ubiquitin-specific protease 21 to play an anti-tumor role in different pathways. NFZ is able to act on the tumor cell microenvironment to inhibit tumor angiogenesis and tumor cell migration, enhance tumor immune cells, increase the cytotoxicity of tumor cells and enhance the anti-tumor effect of other drugs. Furthermore, it has high safety with few toxic side effects. The anti-tumor mechanisms of NFZ were described in the current review, aiming to provide insight and a reference for future studies promoting the implementation of NFZ as an anti-tumor drug in the clinic.

Blood collection in mice is a common procedure in biomedical research. The choice of blood collection method and the need for analgesia and/or anaesthesia depend on multiple factors, including the experimental setup, animal welfare considerations and the intended downstream analyses. This minireview describes key non-surgical and surgical blood collection techniques, the appropriate use of analgesia and anaesthesia, and the best practice for documentation and adherence to reporting standards in animal studies. We here provide a table summarising collection procedures; a table listing animal welfare guidelines from multiple countries; a table describing the most common analgesics and anaesthetics, with doses and route of administration; and a table outlining key points for reporting blood collection, anaesthesia and analgesia protocols. A decision chart is also included to assist in selecting the most suitable method. Ultimately, with this minireview, we aim to promote standardised practices, improve data reproducibility, and support ethical animal research.

Per- and polyfluoroalkyl substances (PFAS) are a large group of persistent chemicals that are pervasive in the environment leading to widespread exposure for humans. Perfluorooctanoic acid (PFOA), one of the most commonly measured PFAS in people, disrupts liver and serum lipid homeostasis as shown in animal toxicity and human epidemiological studies. We tested the hypothesis that the effects of PFOA exposure in mice expressing mouse PPARα (mPPARα) are driven largely through PPARα-dependent mechanisms while non-PPARα dependent mechanisms will be more apparent in mice expressing human PPARα (hPPARα). Female and male mPPARα, hPPARα, and PPARα null mice were exposed to PFOA (0.5, 1.4 or 6.2 mg PFOA/L) via drinking water for 14 weeks. Concurrently, mice consumed an American diet containing human diet-relevant amounts of fat and cholesterol. Here, we focused on the effects in female mice, given the dearth of data reported on PFAS-induced effects in females. Increasing the duration of PFOA exposure reduced weight gain in all genotypes of female mice while end-of-study body fat was lower in PFOA exposed hPPARα and PPARα null mice. Serum cholesterol, but not triacylglyceride, concentrations were increased by PFOA exposure in a PPARα-dependent manner. Hepatic triacylglycerides were higher in vehicle-exposed mPPARα and PPARα null mice than hPPARα mice, and PFOA significantly reduced concentrations in mPPARα and PPARα null mice only. In contrast, PFOA increased hepatic cholesterol content in a PPARα-dependent manner. Changes in liver and serum cholesterol may be explained by a strong, PPARα-dependent downregulation of Cyp7a1 expression. PFOA significantly increased PPARα target gene expression in mPPARα mice. Other nuclear receptors were examined: CAR target gene expression was only induced by PFOA in hPPARα and PPARα null mice. PXR target gene expression was induced by PFOA in all genotypes. Results were similar in male mice with two exceptions: (1) vehicle-exposed male mice of all genotypes were equally susceptible to diet-induced hepatic steatosis; (2) male mice drank less water, resulting in lower serum PFOA levels, which may explain the less significant changes in lipid endpoints. Overall, our results show that PFOA modifies triacylglyceride and cholesterol homeostasis independently and that PPARα plays an important role in PFOA-induced increases in liver and serum cholesterol.

The integrity and functionality of the hepatic microcirculation are essential for maintaining liver health, which is influenced by sex and genetic background. Understanding these variations is crucial for addressing disparities in liver disease outcomes.

To investigate the sexual dimorphism and genetic heterogeneity of liver microcirculatory function in mice.

We assessed hepatic microhemodynamics in BALB/c, C57BL/6J, and KM mouse strains using laser Doppler flowmetry and wavelet analysis. We analyzed the serum levels of alanine transaminase, glutamic acid aminotransferase, total bile acid, total protein, alkaline phosphatase, and glucose. Histological and immunohistochemical staining were employed to quantify microvascular density and the expression levels of cluster of differentiation (CD) 31, and estrogen receptor α, and β. Statistical analyses, including the Mantel test and Pearson correlation, were conducted to determine the relationships among hepatic function, microcirculation, and marcocirculation between different sexes and across genetic backgrounds.

We identified sex-based disparities in hepatic microhemodynamics across all strains, with males exhibiting higher microvascular perfusion and erythrocyte concentration, but lower blood velocity. Strain-specific differences were evident, particularly in the endothelial oscillatory characteristics of the erythrocyte concentration. No sex-dependent differences in estrogen receptor expression were observed, while significant variations in CD31 expression and microvascular density were observed. The correlations highlighted relationships between hepatic microhemodynamics and liver function indicators.

Our findings indicate the influence of genetic and sex differences on hepatic microcirculation and liver function, highlighting the necessity of incorporating both genetic background and sex into hepatic physiology studies and potential liver disease management strategies.

Retinal prostheses aim to restore vision in individuals affected by degenerative conditions, such as age-related macular degeneration and retinitis pigmentosa. Traditional approaches, including implantable electrode arrays and optogenetics, often require invasive surgery or genetic modification and face limitations in spatial resolution and visual field size. While emerging nanoparticle-based methods offer minimally invasive solutions, some of them rely on intense visible light, which may interfere with residual vision. Plasmonic gold nanorods (AuNRs), tuned to absorb near-infrared (NIR) light, provide a promising alternative by enabling photothermal neuromodulation without affecting the remaining sight. However, effectively utilizing photothermal stimulation with patterned laser projection for precise neural activation remains underexplored. In this study, we introduce a less invasive approach using intravitreally injected anti-Thy1 antibody-conjugated AuNRs to primarily activate bipolar cells─a target traditionally reached through more invasive subretinal injections. This technique allows for extensive retinal coverage and facilitates high-resolution visual restoration via patterned NIR stimulation. Following injection, a scanning NIR laser beam projected in a square pattern with a spot size of 20 μm consistently triggered highly localized neuronal activation, specifically stimulating bipolar cells through temperature-sensitive ion channels. In vivo, this patterned stimulation evoked electrocorticogram responses in the visual cortex of both wild-type and fully blind mouse models without inducing systemic toxicity or significant retinal damage. Our innovative approach promises significant advancements in spatial resolution and broad applicability, offering a precise, customizable, and less invasive method to restore vision.

Well-characterised mouse models of disease may provide valuable insights into pathophysiology. This study characterises the Townes mouse model of sickle cell disease (SCD) and establishes a time window in which the disease is present but does not progress significantly in terms of severity. We examined Townes mice with the HbAA, HbAS, and HbSS genotypes from young (4 weeks) to mature (5 months) stages of life to assess the disease state at different ages and any progression. We conducted blood tests, histological organ damage evaluations, and metabolic assessments to identify a suitable time frame for study based on welfare considerations. Townes HbSS mice displayed key SCD features such as anaemia, haemolysis, thromboinflammation and organ pathology. Notably, these manifestations remained relatively stable over the study period, indicating a stable phase suitable for conducting intervention studies. Mice with HbAS and HbAA genotypes served as comparative controls, showing minimal to no pathology throughout. These findings are valuable for future research on SCD and may ultimately lead to the development of more effective treatments for this debilitating disease.

Anti-Activin Receptor Type IIA and Type IIB antibody (αActRIIA/IIB ab) is a recently developed drug class that targets the activin receptor signalling pathway. Inhibition of receptor ligands (activins, myostatin, growth differentiation factor 11, etc.) can lead to skeletal muscle hypertrophy, bone formation, and increased haematopoiesis. Despite the αActRIIA/IIB ab, bimagrumab, having progressed to clinical trials, two crucial questions about αActRIIA/IIB ab therapy remain: Does αActRIIA/IIB ab influence bone metabolism and bone strength similarly to its generic classmates (activin receptor-based ligand traps)? Does αActRIIA/IIB ab affect red blood cell parameters, thereby increasing the risk of thromboembolism, similar to its generic classmates? Therefore, the aim of the present study was to investigate the therapeutic potential of αActRIIA/IIB ab in a mouse model of concurrent sarcopenia and osteopenia and to investigate the effect on bone and haematopoiesis in more detail.

In C57BL/6JRj mice, combined sarcopenia and osteopenia were induced locally by injecting botulinum toxin A into the right hindlimb, resulting in acute muscle paresis. Immediately after immobilization, mice received twice-weekly intraperitoneal injections with αActRIIA/IIB ab (10 mg/kg) for 21 days, after which they were sacrificed. Muscle mass, skeletal muscle fibre size and Smad2 expression were analysed in the rectus femoris and gastrocnemius muscles. Bone mass and bone microstructure were analysed in the trabecular bone at the distal femoral metaphysis, while the cortical bone was analysed at the femoral mid-diaphysis. In a substudy, the effect on haematopoiesis was explored 2 and 7 days after a single αActRIIA/IIB ab (30 mg/kg) injection in C57BL/6JRj mice.

αActRIIA/IIB ab caused a large increase in muscle mass in both healthy (+21%) and immobilized (sarcopenic and osteopenic) (+12%) mice. Furthermore, αActRIIA/IIB ab increased trabecular bone (bone volume fraction) for both healthy (+65%) and immobilized (+44%) mice. For cortical bone, αActRIIA/IIB ab caused a small, but significant, increase in bone area (+6%) for immobilized mice, but not for healthy mice. Treatment with αActRIIA/IIB ab did not change red blood cell count, haemoglobin concentration or mean cell volume after either 2 or 7 days.

Treatment with αActRIIA/IIB ab caused a significant increase in both skeletal muscle mass and bone parameters in both healthy and immobilized mice, suggesting a potential in the treatment of concurrent osteopenia and sarcopenia. Interestingly, the bone anabolic effect of the treatment was much more pronounced on trabecular bone than on cortical bone. There was no pronounced effect of short-term treatment on haematopoiesis.

Ginsenosides, active compounds derived from Panax ginseng, exhibit promising potential in enhancing physical performance. This study investigates the impact of UG0712 (UG), a novel ginsenoside compound, on endurance capacity, body weight, organ weights, blood parameters, and specific transcriptomic changes in liver and muscle tissues using a C57BL/6N mouse model. The mice received UGs orally at three doses: UG50 (50 mg/kg), UG100 (100 mg/kg), and UG200 (200 mg/kg) for a specified duration. Endurance capacity, physiological parameters, and transcriptome signatures in liver and muscle tissues were assessed. UG administration significantly improved time to exhaustion, with UG50 and UG200 showing substantial enhancements. Body and organ weights exhibited no notable differences, suggesting a lack of adverse effects. Biochemical markers, except for decreased creatine kinase levels in the UG100 group, showed no significant variations. Transcriptome analysis revealed limited group separation and dose-dependent patterns. The UG100 group displayed significant enrichment in lipid metabolism and muscle-related terms. Identified dose-dependent improvements in endurance capacity highlight UGs' potential as supplements. The absence of adverse effects on body and organ weights, along with positive effects on biochemical markers, supports their safety. Despite limited dose-dependent patterns in transcriptomic analyses, the UG100 group showcased significant enrichment in pathways related to muscle and lipid metabolism. These findings offer valuable insights for athletes and aging individuals seeking to enhance physical performance, warranting further exploration into UG effects' on molecular mechanisms.

Previously, the novel small molecule ISFP10 has been shown to inhibit fungal phosphoglucomutase (PGM) activity in Aspergillus fumigatus and Pneumocystis spp. With 50-fold selectivity over the human PGM molecule due to the presence of a unique yet conserved cysteine residue present in a number pathogenic fungal PGMs, use of this compound may provide a novel broad-spectrum approach to treating fungal infections. Accordingly, we sought to determine the tolerability in test animals receiving this compound, as well as the potential antifungal activity of ISFP10 on cultures of the common fungal pathogens Candida albicans and Candida glabrata .

C57BL6 mice received once daily intraperitoneal (IP) injections of 100 mL of vehicle control (DMSO) or ISFP10 at a concentration of 10 mg/kg. Body weights were recorded daily for 7 days of treatment. On the final day, mice were weighed and euthanized. Postmortem blood collection was conducted via cardiac puncture and distributed to EDTA and lithium heparin tubes for complete blood count (CBC) and comprehensive blood chemistry panels, respectively. Liver, kidney, and lung tissue were also harvested and placed in 10% formalin for H&E staining and blinded histopathologic scoring. Lung samples were further analyzed for proinflammatory cytokines using enzyme-linked immunosorbent assays (ELISA) and quantitative PCR (qPCR). Furthermore, ISPF10 was tested for antifungal activity via 8-hour growth curve analysis in a concentration-dependent fashion against Candida albicans and Candida glabrata .

There was no significant difference in the daily or final body weights of the mice receiving 10 mg/kg of ISFP10 compared to those of the vehicle control group. Extracellular matrix (ECM) transcripts for IL-6 and TNFα were statistically similar via qPCR. ELISA results of proinflammatory cytokines for IL-6 was not significant whereas TNFα levels in lung tissue from the ISFP10 treatment group were significantly reduced, indicating a potential anti-inflammatory effect of ISFP10 at this dosage. Overall, blood chemistry and CBC analysis revealed no overall significant differences between the two groups, except for increased neutrophil counts and decreased potassium levels in samples collected from ISFP10 treated animals compared to the vehicle control group. These laboratory abnormalities were not of clinical significance to the test animals. Blinded histopathological examination revealed no abnormalities or evidence of critical organ toxicity from all groups. Inhibition of C. albicans and C. glabrata culture growth by ISFP10 was concentration-dependent in YPD liquid media containing the ISFP10 compared to vehicle control.

Our preliminary testing of ISFP10 revealed no inherent safety or toxicology concerns within the observed parameters. These data further support significant culture suppressive activity against C. albicans and C. glabrata . Taken together, these observations of ISFP10 further indicate that targeting PGM might be a novel and viable therapeutic strategy for serious fungal infections.

An inhibitor specific to fungal PGM enzymes, termed ISFP10, was generally well tolerated when administered via intraperitoneal (IP) injection in mice. ISFP10 displays antifungal activity in a concentration-dependent manner against C. albicans and C. glabrata .

The development of cancer vaccines is at the forefront of cancer immunotherapy. Most existing strategies to induce an efficient anti-tumor immune response rely on molecular adjuvants and the incorporation of complex synthetic vectors into vaccine formulations. In contrast, this study introduces a one-step engineering technique to assemble the model antigen, Ovalbumin (OVA), into amyloid aggregates, leveraging biomimetic folding and aggregation to create non-fibrillar OVA globular aggregates and OVA amyloid-like fibrils as single-component, adjuvant-free vaccines. Notably, the OVA amyloid-like fibrils induced stronger immune responses compared to the native form, as evidenced by robust humoral immune reactions and the establishment of immune memory. These enhanced responses can be attributed to the self-adjuvant effect of the unique assembled structure, which preserves antigenic epitopes, improves antigen stability, facilitates antigen internalization, prolongs retention at the injection site, enhances antigen trafficking to the lymphoid organs, and promotes increased secretion of antibodies and cytokines. Furthermore, the efficacy of the vaccine was validated in a high OVA-expressing tumor model, demonstrating the potential of OVA amyloid-like fibrils as an effective vaccine for cancer immunoprevention. This minimalist self-adjuvant vaccine strategy holds promising implications for cancer immunotherapy and can inform the design of other protein antigen-based vaccines.

Korean Red Ginseng and ginsenosides have been studied for their efficacy against various diseases, including those related to aging. However, most aging studies use D-galactose to induce aging, which often does not accurately represent natural aging. This study aimed to verify improvements in auditory, cognitive, and liver function through administering red ginseng to an 18-month-old naturally aging mouse model.

Auditory function was assessed using Auditory Brainstem Response (ABR) and Auditory Middle Latency Response (AMLR). Cognitive function was evaluated electrophysiologically with P300 and mismatch negativity (MMN), and behaviorally using the Y-maze. Additionally, biochemical tests and histological analysis were conducted to assess liver function. The effects of red ginseng on gene expression regulation were also examined in the cochlea, auditory cortex, and liver, focusing on age-related disease processes.

Red ginseng significantly decreased hearing thresholds and improved central auditory function. It also enhanced cognitive behavior and function in response to external stimulation. Furthermore, red ginseng regulated alkaline phosphatase (ALP), albumin (Alb), and total protein (TP) levels, notably decreasing aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Hematoxylin and eosin (H&E) staining of liver tissue showed significant improvement in fat droplets. These effects appear to be mediated by the regulation of aging-related genes Dec, c-Jun, Stat5b, and Lims2.

These results suggest that red ginseng improves auditory, cognitive, and liver functions in a naturally aged mouse model.

Mice are the most commonly used models of infectious disease, and disease in mice is similar to that of humans. As a consequence, standard hematology and biochemistry reference values in mice are essential to evaluate functional changes caused by experimental treatments, although very few data in the literature provide a comparative reference range. The aim of this investigation was to establish the reference intervals for major hematology and biochemistry analytes in 2 inbred mouse strains, BALB/c and C57BL/6, at 3 different age ranges. Parameters were assessed in 600 mice (300 male and 300 female) of BALB/c and C57BL/6 strains at 6 to 8 wk, 10 to 14 wk, and 6 to 9 mo of age. Reference intervals were calculated by nonparametric or robust methods according to sample size, and statistical analyses were performed to assess the changes in relation to sex, age, and strain. The data demonstrate that strain, sex, and age have significant effects on the hematologic and biochemical profiles of mice. Hemoglobin, Hct, MCH, MCHC, neutrophils, eosinophils, and ALP were found to be significantly greater in BALB/c mice. In contrast, WBC, lymphocytes, basophils, glucose, total protein, albumin, and urea were found to be significantly greater in C57BL/6 mice in all age ranges. Lymphocytes and ALP progressively decreased with age, while neutrophils increased with age in both strains. The study successfully defined and established reference intervals for hematologic and biochemical analytes in 2 inbred mouse strains at 3 different age ranges. The reference values reported here could be useful in characterizing the phenotype of experimental mice and assessing the changes caused by investigational treatments.

Consuming an unbalanced diet and being overweight represent a global health problem in young people and adults of both sexes, and may lead to metabolic syndrome. The diet-induced obesity (DIO) model in the C57BL/6J mouse substrain that mimics the gradual weight gain in humans consuming a "Western-type" (WD) diet is of great interest. This study aims to characterize this animal model, using high-frequency ultrasound imaging (HFUS) as a complementary tool to longitudinally monitor changes in the liver, heart and kidney. Long-term WD feeding increased mice body weight (BW), liver/BW ratio and body condition score (BCS), transaminases, glucose and insulin, and caused dyslipidemia and insulin resistance. Echocardiography revealed subtle cardiac remodeling in WD-fed mice, highlighting a significant age-diet interaction for some left ventricular morphofunctional parameters. Qualitative and parametric HFUS analyses of the liver in WD-fed mice showed a progressive increase in echogenicity and echotexture heterogeneity, and equal or higher brightness of the renal cortex. Furthermore, renal circulation was impaired in WD-fed female mice. The ultrasound and histopathological findings were concordant. Overall, HFUS can improve the translational value of preclinical DIO models through an integrated approach with conventional methods, enabling a comprehensive identification of early stages of diseases in vivo and non-invasively, according to the 3Rs.

The high stiffness of intravenous needles can cause tissue injury and increase the risk of transmission of blood-borne pathogens through accidental needlesticks. Here we describe the development and performance of an intravenous needle whose stiffness and shape depend on body temperature. The needle is sufficiently stiff for insertion into soft tissue yet becomes irreversibly flexible after insertion, adapting to the shape of the blood vessel and reducing the risk of needlestick injury on removal, as we show in vein phantoms and ex vivo porcine tissue. In mice, the needles had similar fluid-delivery performance and caused substantially less inflammation than commercial devices for intravenous access of similar size. We also show that an intravenous needle integrated with a thin-film temperature sensor can monitor core body temperature in mice and detect fluid leakage in porcine tissue ex vivo. Temperature-responsive intravenous needles may improve patient care.

Classic hereditary hemochromatosis (HH) is an autosomal recessive iron-overload disorder resulting from loss-of-function mutations of the HFE gene. Patients with HH exhibit excessive hepatic iron accumulation that predisposes these patients to liver disease, including the risk for developing liver cancer. Chronic iron overload also poses a risk for the development of metabolic disorders such as obesity, type 2 diabetes, and insulin resistance. We hypothesized that liraglutide, GLP1 receptor agonist, alters iron metabolism while also reducing body weight and glucose tolerance in a mouse model of HH (global HFE knockout, HFE KO) and diet-induced obesity and glucose intolerance. The total body HFE KO and wild-type control mice were fed high-fat diet for 8 weeks. Mice were subdivided into liraglutide and vehicle-treated groups and received daily subcutaneous administration of the respective treatment once daily for 18 weeks. Liraglutide improved glucose tolerance and hepatic lipid markers and reduced body weight in a mouse model of HH, the HFE KO mouse, similar to wild-type controls. Importantly, our data show that liraglutide alters iron metabolism in HFE KO mice, leading to decreased circulating and stored iron levels in HFE KO mice. These observations highlight the potential that GLP1 receptor agonist could be used to reduce iron overload in addition to reducing body weight and improving glucose regulation in HH patients.

Mutations in LMNA encoding nuclear envelope proteins lamin A/C cause dilated cardiomyopathy. Activation of the AKT/mTOR (RAC-α serine/threonine-protein kinase/mammalian target of rapamycin) pathway is implicated as a potential pathophysiologic mechanism. The aim of this study was to assess whether pharmacological inhibition of mTOR signaling has beneficial effects on heart function and prolongs survival in a mouse model of the disease, after onset of heart failure.

We treated male LmnaH222P/H222P mice, after the onset of heart failure, with placebo or either of 2 orally bioavailable mTOR inhibitors: everolimus or NV-20494, a rapamycin analog highly selective against mTORC1. We examined left ventricular remodeling, and the cell biological, biochemical, and histopathologic features of cardiomyopathy, potential drug toxicity, and survival.

Everolimus treatment (n=17) significantly reduced left ventricular dilatation and increased contractility on echocardiography, with a 7% (P=0.018) reduction in left ventricular end-diastolic diameter and a 39% (P=0.0159) increase fractional shortening compared with placebo (n=17) after 6 weeks of treatment. NV-20494 treatment (n=15) yielded similar but more modest and nonsignificant changes. Neither drug prevented the development of cardiac fibrosis. Drug treatment reactivated suppressed autophagy and inhibited mTORC1 signaling in the heart, although everolimus was more potent. With regards to drug toxicity, everolimus alone led to a modest degree of glucose intolerance during glucose challenge. Everolimus (n=20) and NV-20494 (n=20) significantly prolonged median survival in LmnaH222P/H222P mice, by 9% (P=0.0348) and 11% (P=0.0206), respectively, compared with placebo (n=20).

These results suggest that mTOR inhibitors may be beneficial in patients with cardiomyopathy caused by LMNA mutations and that further study is warranted.

Rapid advances in nucleic acid therapies highlight the immense therapeutic potential of genetic therapeutics. Lipid nanoparticles (LNPs) are highly potent nonviral transfection agents that can encapsulate and deliver various nucleic acid therapeutics, including but not limited to messenger RNA (mRNA), silencing RNA (siRNA), and plasmid DNA (pDNA). However, a major challenge of targeted LNP-mediated systemic delivery is the nanoparticles' nonspecific uptake by the liver and the mononuclear phagocytic system, due partly to the adsorption of endogenous serum proteins onto LNP surfaces. Tunable LNP surface chemistries may enable efficacious delivery across a range of organs and cell types. Here, we describe a method to electrostatically adsorb bioactive polyelectrolytes onto LNPs to create layered LNPs (LLNPs). LNP cores varying in nucleic acid cargo and component lipids were stably layered with four biologically relevant polyanions: hyaluronate (HA), poly-L-aspartate (PLD), poly-L-glutamate (PLE), and polyacrylate (PAA). We further investigated the impact of the four surface polyanions on the transfection and uptake of mRNA- and pDNA-loaded LNPs in cell cultures. PLD- and PLE-LLNPs increased mRNA transfection twofold over unlayered LNPs in immune cells. HA-LLNPs increased pDNA transfection rates by more than twofold in epithelial and immune cells. In a healthy C57BL/6 murine model, PLE- and HA-LLNPs increased transfection by 1.8-fold to 2.5-fold over unlayered LNPs in the liver and spleen. These results suggest that LbL assembly is a generalizable, highly tunable platform to modify the targeting specificity, stability, and transfection efficacy of LNPs, as well as incorporate other charged targeting and therapeutic molecules into these systems.

Mild cognitive impairment (MCI) is an early stage that can result in dementia. MCI can be reversed, and diagnosis at an early stage is crucial to control the progression to dementia. Dementia is currently diagnosed based on interviews and screening tests; however, novel biomarkers must be identified to allow early MCI detection. Therefore, the present study aimed to identify novel biomarkers in the form of blood microRNAs (miRNAs/miRs) for the diagnosis of MCI or early dementia. Blood samples were collected from C57BL/6NJcl male mice at four time points, including 4-week-old (4W), 8-week-old (8W), 36-week-old (36W) and 58-week-old (58W), and serum was isolated. Body weight and blood total cholesterol levels were increased, and blood alkaline phosphatase was decreased with aging. The 8W mice exhibited the highest cognitive ability in the Morris water maze test, whereas the 58W mice demonstrated decreased cognitive ability. The serum RNA concentrations of the 4W, 8W, 36W and 58W mice demonstrated no significant differences. Furthermore, small RNA levels were detected in the serum of all mice. miRNA microarray analysis revealed a >1.5-fold increase in the serum expression of two miRNAs (miR-21a-5p and miR-92a-3p) and a >1.5-fold decrease in the serum expression of two other miRNAs (miR-6769b-5p and miR-709) in 58W mice compared with those in 8W mice. In the future, we aim to further analyze aged mice to discover novel MCI biomarkers.

Probiotics contribute to the integrity of the intestinal mucosa and preventing dysbiosis caused by opportunistic pathogens, such as intestinal helminths. Bacillus cereus GM obtained from Biovicerin® was cultured to obtain spores for in vivo evaluation on experimental schistosomiasis. The assay was performed for 90 days, where all animals were infected with 50 cercariae of Schistosoma mansoni on the 15th day. Three experimental groups were formed, as follows: G1-saline solution from the 1st until the 90th day; G2-B. cereus GM (105 spores in 300 μL of sterile saline) from the 1st until the 90th day; and G3-B. cereus GM 35th day (onset of oviposition) until the 90th day. G2 showed a significant reduction of 43.4% of total worms, 48.8% of female worms and 42.5% of eggs in the liver tissue. In G3, the reduction was 25.2%, 29.1%, and 44% of the total number of worms, female worms, and eggs in the liver tissue, respectively. G2 and G3 showed a 25% (p < 0.001) and 22% (p < 0.001) reduction in AST levels, respectively, but ALT levels did not change. ALP levels were reduced by 23% (p < 0.001) in the G2 group, but not in the G3. The average volume of granulomas reduced (p < 0.0001) 65.2% and 46.3% in the liver tissue and 83.0% and 53.2% in the intestine, respectively, in groups G2 and G3. Th1 profile cytokine (IFN-γ, TNF-α, and IL-6) and IL-17 were significantly increased (p < 0.001) stimulated with B. cereus GM in groups G2 and G3. IL-4 showed significant values when the stimulus was mediated by ConA. By modulating the immune response, B. cereus GM reduced the burden of worms, improved some markers of liver function, and reduced the granulomatous inflammatory reaction in mice infected with S. mansoni, especially when administered before infection.

Renal cyst progression in autosomal dominant polycystic kidney disease (ADPKD) is highly dependent on agents circulating in blood. We have previously shown, using different in vitro models, that one of these agents is the hormone ouabain. By binding to Na+-K+-ATPase (NKA), ouabain triggers a cascade of signal transduction events that enhance ADPKD cyst progression by stimulating cell proliferation, fluid secretion, and dedifferentiation of the renal tubular epithelial cells. Here, we determined the effects of ouabain in vivo. We show that daily administration of ouabain to Pkd1RC/RC ADPKD mice for 1-5 mo, at physiological levels, augmented kidney cyst area and number compared with saline-injected controls. Also, ouabain favored renal fibrosis; however, renal function was not significantly altered as determined by blood urea nitrogen levels. Ouabain did not have a sex preferential effect, with male and female mice being affected equally. By contrast, ouabain had no significant effect on wild-type mice. In addition, the actions of ouabain on Pkd1RC/RC mice were exacerbated when another mutation that increased the affinity of NKA for ouabain was introduced to the mice (Pkd1RC/RCNKAα1OS/OS mice). Altogether, this work highlights the role of ouabain as a procystogenic factor in the development of ADPKD in vivo, that the ouabain affinity site on NKA is critical for this effect, and that circulating ouabain is an epigenetic factor that worsens the ADPKD phenotype.NEW & NOTEWORTHY This work shows that the hormone ouabain enhances the progression of autosomal dominant polycystic kidney disease (ADPKD) in vivo. Ouabain augments the size and number of renal cysts, the kidney weight to body weight ratio, and kidney fibrosis in an ADPKD mouse model. The Na+-K+-ATPase affinity for ouabain plays a critical role in these effects. In addition, these outcomes are independent of the sex of the mice.

Adriamycin (ADR) nephropathy is the most widely used nephropathy model to study the pathophysiological mechanisms of chronic kidney disease (CKD) in mice. However, its application is limited to a few mouse strains such as the BALB/c strain; the standard strain, C57BL/6J (B6J), does not develop ADR nephropathy. Nevertheless, Arif et al. reported that C57BL/6N (B6N), another standard strain, is ADR-susceptible. Since then, no follow-up reports or other studies have been published on ADR nephropathy in B6N mice. Therefore, the goal of this study was to determine whether B6N mice are indeed susceptible to ADR nephropathy and whether there are differences in ADR susceptibility among the substrains of C57BL/6NCrl (NCrl) and C57BL/6NJcl (NJcl). NCrl mice showed marked albuminuria and mesangial cell proliferation, which are associated with mild ADR nephropathy, confirming that NCrl mice were susceptible to ADR nephropathy. On the other hand, NJcl mice did not exhibit these symptoms. ADR nephropathy models are usually generated by administering ADR through the tail vein, but Arif et al. administered ADR through the orbital vein. Therefore, we investigated the effect of the route of administration on ADR nephropathy. The degree of ADR nephropathy was found to vary based on the route of administration: more severe nephropathy was observed upon administration through the tail vein than through the orbital vein. Therefore, we conclude that NCrl mice are susceptible to ADR nephropathy, and the severity of ADR-induced nephropathy through orbital vein administration is relatively lower than that through the tail vein.

Metastatic non-small cell lung cancer (NSCLC) is refractory with a very poor prognosis. Docetaxel (DTX) injection (Taxotere®) has been approved for the treatment of locally advanced or metastatic NSCLC. However, its clinical application is restricted by severe adverse effects and non-selective tissue distribution. In this study, we successfully developed DTX-loaded human serum albumin (HSA) nanoparticles (DNPs) with modified Nab technology, by introducing medium-chain triglyceride (MCT) as a stabilizer. The optimized formulation had a particle size of approximately 130 nm and a favorable stabilization time of more than 24 h. DNPs dissociated in circulation in a concentration-dependent manner and slowly released DTX. Compared with DTX injection, DNPs were more effectively taken up by NSCLC cells, thus exerting stronger inhibitory effects on their proliferation, adhesion, migration, and invasion. In addition, DNPs showed prolonged blood retention and increased tumor accumulation relative to DTX injection. Ultimately, DNPs produced more potent inhibitory effects on primary or metastatic tumor foci than DTX injections but caused markedly lower organ toxicity and hematotoxicity. Overall, these results support that DNPs hold great potential for the treatment of metastatic NSCLC in clinical.

Adenylosuccinic acid (ASA) is a small molecule dicarboxylate that could be a strong clinical development candidate for inherited myopathies involving dysregulated purine nucleotide metabolism. Currently, there are no published pharmacokinetic/dynamic or toxicology data available, although 10-year clinical trial data on Duchenne muscular dystrophy patients suggests it is a chronically safe drug. In this study, we tested the toxicity of ASA to cultured myoblasts in vitro and its acute systemic toxicity in mice. ASA is a non-toxic small molecule with an LD50 > 5000 mg/kg. Some background necrotic foci in the liver, kidney and gastrointestinal tract were shown that are likely incidental but warrant follow-up sub-/chronic oral exposure studies.

The growing significance of messenger RNA (mRNA) therapeutics in diverse medical applications, such as cancer, infectious diseases, and genetic disorders, highlighted the need for efficient and safe delivery systems. Lipid nanoparticles (LNPs) have shown great promise for mRNA delivery, but challenges such as toxicity and immunogenicity still remain to be addressed. In this study, we aimed to compare the performance of polyplex nanomicelles, our original cationic polymer-based carrier, and LNPs in various aspects, including delivery efficiency, organ toxicity, muscle damage, immune reaction, and pain. Our results showed that nanomicelles (PEG-PAsp(DET)) and LNPs (SM-102) exhibited distinct characteristics, with the former demonstrating relatively sustained protein production and reduced inflammation, making them suitable for therapeutic purposes. On the other hand, LNPs displayed desirable properties for vaccines, such as rapid mRNA expression and potent immune response. Taken together, these results suggest the different potentials of nanomicelles and LNPs, supporting further optimization of mRNA delivery systems tailored for specific purposes.

There is a major need for the development of new therapeutics to combat antibiotic-resistant Staphylococcus aureus. Recently, gallium (Ga)-based complexes have shown promising antimicrobial effects against various bacteria, including multidrug-resistant organisms, by targeting multiple heme/iron-dependent metabolic pathways. Among these, Ga protoporphyrin (GaPP) inhibits bacterial growth by targeting heme pathways, including aerobic respiration. Ga(NO3)3, an iron mimetic, disrupts elemental iron pathways. Here, we demonstrate the enhanced antimicrobial activity of the combination of GaPP and Ga(NO3)3 against methicillin-resistant S. aureus (MRSA) under iron-limited conditions, including small colony variants (SCV). This therapy demonstrated significant antimicrobial activity without inducing slow-growing SCV. We also observed that the combination of GaPP and Ga(NO3)3 inhibited the MRSA catalase but not above that seen with Ga(NO3)3 alone. Neither GaPP nor Ga(NO3)3 alone or their combination inhibited the dominant superoxide dismutase expressed (SodA) under the iron-limited conditions examined. Intranasal administration of the combination of the two compounds improved drug biodistribution in the lungs compared to intraperitoneal administration. In a murine MRSA lung infection model, we observed a significant increase in survival and decrease in MRSA lung CFUs in mice that received combination therapy with intranasal GaPP and Ga(NO3)3 compared to untreated control or mice receiving GaPP or Ga(NO3)3 alone. No drug-related toxicity was observed as assessed histologically in the spleen, lung, nasal cavity, and kidney for both single and repeated doses of 10 mg Ga /Kg of mice over 13 days. Our results strongly suggest that GaPP and Ga(NO3)3 in combination have excellent synergism and potential to be developed as a novel therapy for infections with S. aureus.

Desertification and desert sandstorms caused by the worsening global warming pose increasing risks to human health. In particular, Asian sand dust (ASD) exposure has been related to an increase in mortality and hospital admissions for respiratory diseases. In this study, we investigated the effects of ASD on metabolic tissues in comparison to diesel particulate matter (DPM) that is known to cause adverse health effects. We found that larger lipid droplets were accumulated in the brown adipose tissues (BAT) of ASD-administered but not DPM-administered mice. Thermogenic gene expression was decreased in these mice as well. When ASD-administered mice were exposed to the cold, they failed to maintain their body temperature, suggesting that the ASD administration had led to impairments in cold-induced adaptive thermogenesis. However, impaired thermogenesis was not observed in DPM-administered mice. Furthermore, mice fed a high-fat diet that were chronically administered ASD demonstrated unexplained weight loss, indicating that chronic administration of ASD could be lethal in obese mice. We further identified that ASD-induced lung inflammation was not exacerbated in uncoupling protein 1 knockout mice, whose thermogenic capacity is impaired. Collectively, ASD exposure can impair cold-induced adaptive thermogenic responses in mice and increase the risk of mortality in obese mice.

We aimed to develop a new biocompatible gastrin-releasing peptide receptor (GRPR) targeted optical probe, IRDye800-RM26, for fluorescence image-guided surgery (FGS) of brain malignancies in near-infrared window II (NIR-II) imaging. We developed a novel GRPR targeting probe using a nine-amino-acid bombesin antagonist analog RM26 combined with IRDye800CW, and explored the fluorescent probe according to optical properties. Fluorescence imaging characterization in NIR-I/II region was performed in vitro and in vivo. Following simulated NIR-II image-guided surgery, we obtained time-fluorescent intensity curves and time-signal and background ratio curves. Further, we used histological sections of brain from tumor-beating mice model to compare imaging specificity between 5-aminolevulinic acid (5-ALA) and IRDye800-RM26, and evaluated biodistribution and biocompatibility. IRDye800-RM26 had broad emission ranging from 800 to 1200 nm, showing considerable fluorescent intensity in NIR-II region. High-resolution NIR-II imaging of IRDye800-RM26 can enhance the advantages of NIR-I imaging. Dynamic and real time fluorescence imaging in NIR-II region showed that the probe can be used to treat brain malignancies in mice between 12 and 24 h post injection. Its specificity in targeting glioblastoma was superior to 5-ALA. Biodistribution analysis indicated IRDye800-RM26 excretion in the kidney and liver. Histological and blood test analyses did not reveal acute severe toxicities in mice treated with effective dose (40 μg) of the probe for NIR-II imaging. Because of the considerable fluorescent intensity in NIR-II region and high spatial resolution, biocompatible and excretable IRDye800-RM26 holds great potentials for FGS, and is essential for translation into human use.

Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins (ACDMPV) is a fatal congenital disease resulting from a pulmonary vascular endothelial deficiency of FOXF1, producing abnormal morphogenesis of alveolar capillaries, malpositioned pulmonary veins and disordered development of lung lobes. Affected neonates suffer from cyanosis, severe breathing insufficiency, pulmonary hypertension, and death typically within days to weeks after birth. Currently, no treatment exists for ACDMPV, although recent murine research in the Kalinichenko lab demonstrates nanoparticle delivery improves survival and reconstitutes normal alveolar-capillary architecture. The aim of the present study is to investigate the safety of intravenous administration of FOXF1-expressing PEI-PEG nanoparticles (npFOXF1), our pioneering treatment for ACDMPV.

npFOXF1 was constructed, validated, and subsequently administered in a single dose to postnatal day 14 (P14) mice via retro-orbital injection. Biochemical, serologic, and histologic safety were monitored at postnatal day 16 (P16) and postnatal day 21 (P21).

With treatment we observed no lethality, and the general condition of mice revealed no obvious abnormalities. Serum chemistry, whole blood, and histologic toxicity was assayed on P16 and P21 and revealed no abnormality.

In conclusion, npFOXF1 has a very good safety profile and combined with preceding studies showing therapeutic efficacy, npFOXF1 can be considered as a good candidate therapy for ACDMPV in human neonates.

Fusion of the E-26 transformation-specific (ETS)-related gene (ERG) with transmembrane serine protease 2 (TMPRSS2) is a crucial step in the occurrence and progression of approximately 50% of prostate cancers. Despite significant progress in drug discovery, ERG inhibitors have yet to be approved for the clinical treatment of prostate cancer. In this study, we used computer-aided drug design (CADD)-based virtual screening to screen for potential inhibitors of ERG. In vivo and in vitro methods revealed that nifuroxazide (NFZ) inhibited the proliferation of a TMPRSS2:ERG fusion-positive prostate cancer cell line (VCaP) with an IC50 lower than that of ERG-negative prostate cancer cell lines (LNCaP, DU145, and WPMY cells). Poly [ADP-ribose] polymerase 1, the critical mediator of parthanatos, is known to bind ERG and is required for ERG-mediated transcription. NFZ blocked this interaction and overly activated PARP1, leading to cell death that was reduced by olaparib, a PARP1 inhibitor. These results show that NFZ inhibits ERG, leading to parthanatic cell death.

D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8+ T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6+CD8+ T cells with a progenitor phenotype and decreased terminally exhausted CD8+ T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8+ T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established CD40 up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.

MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl^-/- and Ripk3^-/- mice on a congenic C57BL/6 J genetic background. We show that genetic deletion of Mlkl in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. -Seventeen-month-old Mlkl^-/- female mice were also protected against age-related chronic sterile inflammation in connective tissue and skeletal muscle relative to wild-type littermate controls, exhibiting a reduced number of immune cell infiltrates in these sites and fewer regenerating myocytes. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans.

We have recently developed probiotics that can express bovine, human, or porcine lactoferrin (LF), and the present study evaluated the effect of these probiotics in improving non-alcoholic fatty liver disease (NAFLD). Three kinds of probiotic supplements, including lactic acid bacteria (LAB), LAB/LF, and inactivated LAB/LF, were prepared. The LAB supplement was prepared from 10 viable LAB without recombinant LF-expression, the LAB/LF supplement was prepared from 10 viable probiotics expressing LF, and the inactivated LAB/LF supplement was prepared from 10 inactivated probiotics expressing LF. A model of obese/NAFLD mice induced by a high-fat diet was established, and the mice were randomly divided into four groups and fed with a placebo, LAB, LAB/LF, or inactivated LAB daily for four weeks via oral gavage. The body weight, food intake, organ weight, biochemistry, and hepatic histopathological alterations and severity scoring were measured. The results revealed that the obese mice fed with any one of the three probiotic mixtures prepared from recombinant probiotics for four weeks exhibited considerably improved hepatic steatosis. These findings confirmed the assumption that specific probiotic strains or LF supplements could help to control NAFLD, as suggested in previous reports. Our data also suggest that the probiotics and LFs in probiotic mixtures contribute differently to improving the efficacy against NAFLD, and the expressed LF content in probiotics may help to boost their efficacy in comparison with the original probiotic mixtures. Moreover, when these LF-expressing probiotics were further inactivated by sonication, they displayed better efficacies than the viable probiotics against NAFLD. This study has provided intriguing data supporting the potential of recombinant probiotics in improving hepatic steatosis.

Multimodal imaging is attractive in biomedical research because it can provide multidimensional information about objects that individual techniques cannot accomplish. In particular, combining over one-thousand-nanometer near-infrared (OTN-NIR) fluorescence and magnetic resonance (MR) imaging is promising for detecting lesions with high sensitivity and structural information. Herein, we describe the development of a bimodal OTN-NIR/MRI probe from gadolinium-tetraazacyclododecanetetraacetic acid (Gd-DOTA) conjugated poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) copolymer (PLGA-b-PEG) micelle encapsulated IR-1061 at two different locations. One configuration contains Gd-DOTA at the end of the PEG of the hydrophilic shell and the other contains Gd-DOTA at the border of PLGA/PEG. The two structures show remarkable differences in fluorescence and R₁ relaxation rates in biological environments; the structure with Gd-DOTA at the border of PLGA/PEG exhibits stable fluorescence and T₁ signal distribution in live mice. The introduction ratio of Gd-DOTA to PEG is significant for controlling the properties of both structures; a higher Gd-DOTA ratio is preferable for the contrast enhancement effect. We found that Gd-DOTA ratios higher than 10% degraded the fluorescence intensity when Gd-DOTA was bound to the end of PEG. In contrast, the introduction of 70% Gd-DOTA at the border of PLGA/PEG did not exhibit a degraded signal, and the structural stability was enhanced with higher ratios of Gd-DOTA. In conclusion, we confirmed that the location of Gd-DOTA is a crucial factor in designing high-performance probes. The overall properties improve when Gd-DOTA is set on the border of PLGA/PEG. These improvements in the properties by controlling the probe structures are promising for future biomedical applications.