Carnosine regulation of intracellular pH homeostasis promotes lysosome-dependent tumor immunoevasion.

Tumor cells and surrounding immune cells undergo metabolic reprogramming, leading to an acidic tumor microenvironment. However, it is unclear how tumor cells adapt to this acidic stress during tumor progression. Here we show that carnosine, a mobile buffering metabolite that accumulates under hypoxia in tumor cells, regulates intracellular pH homeostasis and drives lysosome-dependent tumor immune evasion. A previously unrecognized isoform of carnosine synthase, CARNS2, promotes carnosine synthesis under hypoxia. Carnosine maintains intracellular pH (pHi) homeostasis by functioning as a mobile proton carrier to accelerate cytosolic H+ mobility and release, which in turn controls lysosomal subcellular distribution, acidification and activity. Furthermore, by maintaining lysosomal activity, carnosine facilitates nuclear transcription factor X-box binding 1 (NFX1) degradation, triggering galectin-9 and T-cell-mediated immune escape and tumorigenesis. These findings indicate an unconventional mechanism for pHi regulation in cancer cells and demonstrate how lysosome contributes to immune evasion, thus providing a basis for development of combined therapeutic strategies against hepatocellular carcinoma that exploit disrupted pHi homeostasis with immune checkpoint blockade.

Histidine N1-position-specific methyltransferase CARNMT1 targets C3H zinc finger proteins and modulates RNA metabolism.

Histidine (His) residues are methylated in various proteins, but their roles and regulation mechanisms remain unknown. Here, we show that carnosine N-methyltransferase 1 (CARNMT1), a known His methyltransferase of dipeptide carnosine (ßAla-His), is a major His N1-position-specific methyltransferase. We found that 52 His sites in 20 proteins underwent CARNMT1-mediated methylation. The consensus methylation site for CARNMT1 was identified as Cx(F/Y)xH, a C3H zinc finger (C3H ZF) motif. CARNMT1-deficient and catalytically inactive mutant mice showed embryonic lethality. Among the CARNMT1 target C3H ZF proteins, RNA degradation mediated by Roquin and tristetraprolin (TTP) was affected by CARNMT1 and its enzymatic activity. Furthermore, the recognition of the 3' splice site of the CARNMT1 target C3H ZF protein U2AF1 was perturbed, and pre-mRNA alternative splicing (AS) was affected by CARNMT1 deficiency. These findings indicate that CARNMT1-mediated protein His methylation, which is essential for embryogenesis, plays roles in diverse aspects of RNA metabolism by targeting C3H ZF-type RNA-binding proteins and modulating their functions, including pre-mRNA AS and mRNA degradation regulation.

Anticancer actions of carnosine in cellular models of prostate cancer.

Treatments for organ-confined prostate cancer include external beam radiation therapy, radical prostatectomy, radiotherapy/brachytherapy, cryoablation and high-intensity focused ultrasound. None of these are cancer-specific and are commonly accompanied by side effects, including urinary incontinence and erectile dysfunction. Moreover, subsequent surgical treatments following biochemical recurrence after these interventions are either limited or affected by the scarring present in the surrounding tissue. Carnosine (ß-alanyl-L-histidine) is a histidine-containing naturally occurring dipeptide which has been shown to have an anti-tumorigenic role without any detrimental effect on healthy cells; however, its effect on prostate cancer cells has never been investigated. In this study, we investigated the effect of carnosine on cell proliferation and metabolism in both a primary cultured androgen-resistant human prostate cancer cell line, PC346Flu1 and murine TRAMP-C1 cells. Our results show that carnosine has a significant dose-dependent inhibitory effect in vitro on the proliferation of both human (PC346Flu1) and murine (TRAMP-C1) prostate cancer cells, which was confirmed in 3D-models of the same cells. Carnosine was also shown to decrease adenosine triphosphate content and reactive species which might have been caused in part by the increase in SIRT3 also shown after carnosine treatment. These encouraging results support the need for further human in vivo work to determine the potential use of carnosine, either alone or, most likely, as an adjunct therapy to surgical or other conventional treatments.

The effects of residual dipolar coupling on carnosine in proton muscle spectra.

Carnosine, an MR-visible dipeptide in human muscle, is well characterized by two peaks at ~8 and ~7 ppm from C2 and C4 imidazole protons. Like creatine and other metabolites, carnosine is subject to residual dipolar coupling in the anisotropic environment of muscle fibers, but the effects have not been studied extensively. Single-voxel TE 30-32 PRESS spectra from three different 3T studies were acquired from gastrocnemius medialis and soleus muscles in the human lower leg. In these studies, carnosine T2 values were measured, and spectra were obtained at three different foot angles. LCModel was used to fit the carnosine peaks with a basis set that was generated using shaped RF pulses and included a range of dipolar couplings affecting the C4 peak. A seven-parameter analytic expression was used to fit the CH2 doublets of creatine. It incorporated an optimized "effective TE" value to model the effect of shaped RF pulses. The fits confirm that the triplet C4 peak of carnosine is dipolar coupled to a pair of CH2 protons, with no need to include a contribution from a separate pool of freely rotating uncoupled carnosine. Moreover, the couplings experienced by carnosine C4 protons and creatine CH2 protons are strongly correlated (R2 = 0.88, P<0.001), exhibiting a similar 3cos2 θ - 1 dependence on the angle θ between fiber orientation and B0. T2 values for the singlet C2 peak of gastrocnemius carnosine are inversely proportional to the C4 dipolar coupling strength (R2 = 0.97, P < 0.001), which in turn is a function of foot orientation. This dependence indicates that careful positioning of the foot while acquiring lower leg muscle spectra is important to obtain reproducible carnosine concentrations. As proton magnetic resonance spectroscopy of carnosine is currently used to non-invasively estimate the muscle fiber typology, these results have important implications in sport science.

PEGylation renders carnosine resistant to hydrolysis by serum carnosinase and increases renal carnosine levels.

Carnosine's protective effect in rodent models of glycoxidative stress have provided a rational for translation of these findings in therapeutic concepts in patient with diabetic kidney disease. In contrast to rodents however, carnosine is rapidly degraded by the carnosinase-1 enzyme. To overcome this hurdle, we sought to protect hydrolysis of carnosine by conjugation to Methoxypolyethylene glycol amine (mPEG-NH2). PEGylated carnosine (PEG-car) was used to study the hydrolysis of carnosine by human serum as well as to compare the pharmacokinetics of PEG-car and L-carnosine in mice after intravenous (IV) injection. While L-carnosine was rapidly hydrolyzed in human serum, PEG-car was highly resistant to hydrolysis. Addition of unconjugated PEG to carnosine or PEG-car did not influence hydrolysis of carnosine in serum. In mice PEG-car and L-carnosine exhibited similar pharmacokinetics in serum but differed in half-life time (t1/2) in kidney, with PEG-car showing a significantly higher t1/2 compared to L-carnosine. Hence, PEGylation of carnosine is an effective approach to prevent carnosine degradations and to achieve higher renal carnosine levels. However, further studies are warranted to test if the protective properties of carnosine are preserved after PEGylation.

Immobilized Dipeptidase in Manganese Ion-Loaded Polyethylenimine-Induced Calcium Phosphate Nanocrystals for Carnosine Synthesis.

Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.

Quantitative proteomics study of carnosine effect in an animal model of Western diet-induced nonalcoholic fatty liver disease.

The nonalcoholic fatty liver disease (NAFLD), which is closely related to westernized dietary (WD) patterns, displays a rising epidemiological and economic burden. Since there is no pharmacological therapy approved for this disease, mechanistic studies are warranted. In this work, we investigated the action of carnosine (CAR), a natural dipeptide with several protection roles against oxidative stress in the liver of NAFLD rats. NAFLD was induced by WD-rich sugars and fat, verifying the histological evidence of steatosis. As intraperitoneal administration of CAR reversed liver steatosis, the protein profiles of NAFLD liver and CAR NAFLD liver were evaluated by label-free proteomics approach. A total of 2531 proteins were identified and the 230 and 276 were significantly up- and downregulated, respectively, by CAR treatment of NAFLD rats and involved in fundamental pathways such as oxidative stress and lipid metabolism. Perilipin 2 and apolipoprotein E, components of the plasma membrane of vesicle, resulted in highly downregulated in the CAR-treated NAFLD liver. The advanced bioanalytical approach demonstrated the efficacy of CAR in overcoming the main symptoms of NAFLD, ameliorating the steatosis in the liver.

Carnosine supplementation improves glucose control in adults with pre-diabetes and type 2 diabetes: A randomised controlled trial.

BACKGROUND AND AIMS: Type 2 diabetes (T2DM) is a major cause of morbidity and mortality globally. Carnosine, a naturally occurring dipeptide, has anti-inflammatory, antioxidant, and anti-glycating effects, with preliminary evidence suggesting it may improve important chronic disease risk factors in adults with cardiometabolic conditions. METHODS AND RESULTS: In this randomised controlled trial, 43 adults (30%F) living with prediabetes or T2DM consumed carnosine (2 g) or a matching placebo daily for 14 weeks to evaluate its effect on glucose metabolism assessed via a 2-h 75 g oral glucose tolerance test. Secondary outcomes included body composition analysis by dual energy x-ray absorptiometry (DEXA), calf muscle density by pQCT, and anthropometry. Carnosine supplementation decreased blood glucose at 90 min (-1.31 mmol/L; p = 0.02) and 120 min (-1.60 mmol/L, p = 0.02) and total glucose area under the curve (-3.30 mmol/L; p = 0.04) following an oral glucose tolerance test. There were no additional changes in secondary outcomes. The carnosine group results remained significant before and after adjustment for age, sex, and change in weight (all>0.05), and in further sensitivity analyses accounting for missing data. There were no significant changes in insulin levels. CONCLUSION: This study provides preliminary support for larger trials evaluating carnosine as a potential treatment for prediabetes and the initial stages of T2DM. Likely mechanisms may include changes to hepatic glucose output explaining the observed reduction in blood glucose without changes in insulin secretion following carnosine supplementation.

Efficacy of Normalising Serum Zinc Level for Patients with Olfactory Dysfunction and Zinc Deficiency.

INTRODUCTION: Zinc deficiency may worsen the severity of olfactory dysfunction; however, the relationship between serum zinc levels and therapeutic effects on olfactory dysfunction remains uncertain. This study investigated the relationship between normalising serum zinc levels and the therapeutic effects on olfactory dysfunction. METHODS: Forty-two patients diagnosed with post-infectious, post-traumatic, and idiopathic olfactory dysfunction, with serum zinc levels <70 µg/dL, were included in the study. All patients were treated with mecobalamin, tokishakuyakusan, and polaprezinc. The patients were divided into 2 groups: the zinc-normalised (≥70 µg/dL) and zinc-deficient (<70 µg/dL) groups, based on their post-treatment serum zinc levels. Olfactory test results were compared in each of the 2 groups. RESULTS: The patients were treated for a median of 133 days. The zinc-normalised group had significantly better results in all olfactory tests (detection/recognition thresholds of the T&T olfactometer, odour identification test (Open Essence), Visual Analogue Scale for olfactory dysfunction, and self-administered odour questionnaire). In contrast, only the self-administered odour questionnaire showed a significant improvement in the zinc-deficient group, with no significant differences observed in the other olfactory tests. When comparing the changes in the olfactory test scores between the 2 groups, significant differences were observed in the detection/recognition thresholds of the T&T olfactometer test and Open Essence results. CONCLUSION: These findings suggest that patients with olfactory dysfunction may have difficulty improving their olfactory function if they also have zinc deficiency. Furthermore, normalisation of zinc deficiency may contribute to the improvement of olfactory dysfunction with general treatment.

Zinc-Carnosine Metallodrug Network as Dual Metabolism Inhibitor Overcoming Metabolic Reprogramming for Efficient Cancer Therapy.

The targeting of tumor metabolism as a novel strategy for cancer therapy has attracted tremendous attention. Herein, we develop a dual metabolism inhibitor, Zn-carnosine metallodrug network nanoparticles (Zn-Car MNs), which exhibits good Cu-depletion and Cu-responsive drug release, causing potent inhibition of both OXPHOS and glycolysis. Notably, Zn-Car MNs can decrease the activity of cytochrome c oxidase and the content of NAD+, so as to reduce ATP production in cancer cells. Thereby, energy deprivation, together with the depolarized mitochondrial membrane potential and increased oxidative stress, results in apoptosis of cancer cells. In result, Zn-Car MNs exerted more efficient metabolism-targeted therapy than the classic copper chelator, tetrathiomolybdate (TM), in both breast cancer (sensitive to copper depletion) and colon cancer (less sensitive to copper depletion) models. The efficacy and therapy of Zn-Car MNs suggest the possibility to overcome the drug resistance caused by metabolic reprogramming in tumors and has potential clinical relevance.

The role of diet and non-pharmacologic supplements in the treatment of chronic neuropathic pain: A systematic review.

BACKGROUND/IMPORTANCE: Dietary interventions, vitamins, and nutritional supplementation are playing an increasingly important role in the management of neuropathic pain. Current pharmacological treatments are poorly tolerated and ineffective in many cases. OBJECTIVE: This systematic review aims to study the efficacy of dietary interventions, vitamins, and nutritional supplementation in the management of chronic neuropathic pain in adults. EVIDENCE REVIEW: The review followed PRISMA guidelines and was registered with PROSPERO (#CRD42022300312). Ten databases and gray literature, including Embase.com, MEDLINE and Web of Science, were systematically searched using a combination of keywords and controlled vocabulary related to chronic neuropathic pain and oral non-pharmacological supplements. Studies on adult humans published between 2000 and 2021 were considered for inclusion. The Cochrane Handbook was used to assess risk of bias, and Grading of Recommendations Assessment, Development, and Evaluation was used to determine overall quality of evidence. FINDINGS: Forty studies were included in the final review, and results were categorized according to pain type including pain related to chemotherapy-induced peripheral neuropathy (CIPN, 22 studies, including 3 prospective cohorts), diabetic peripheral neuropathy (DPN, 13 studies, including 2 prospective), complex regional pain syndrome (CRPS-I, 3 studies, including 1 prospective), and other (2 studies, both RCT). The CIPN studies used various interventions including goshajinkigan (4 studies), vitamin E (5), vitamin B12 (3), glutamine (3), N-acetyl-cysteine (2), acetyl-l-carnitine (2), guilongtonluofang (1), ninjin'yoeito (1), alpha-lipoic acid (1), l-carnosine (1), magnesium and calcium (1), crocin (1), and antioxidants (1), with some studies involving multiple interventions. All CIPN studies involved varying cancers and/or chemotherapies, advising caution for generalizability of results. Interventions for DPN included alpha-lipoic acid (5 studies), vitamin B12 (3), acetyl-l-carnitine (3), vitamin E (1), vitamin D (2), and a low-fat plant-based diet (1). Vitamin C was studied to treat CRPS-I (3 studies, including 1 prospective). Magnesium (1) and St. John's wort (1) were studied for other or mixed neuropathologies. CONCLUSIONS: Based on the review, we cannot recommend any supplement use for the management of CIPN, although further research into N-acetyl-cysteine, l-carnosine, crocin, and magnesium is warranted. Acetyl-l-carnitine was found to be likely ineffective or harmful. Alpha-lipoic acid was not found effective. Studies with goshajinkigan, vitamin B12, vitamin E, and glutamine had conflicting results regarding efficacy, with one goshajinkigan study finding it harmful. Guilongtonluofang, ninjin'yoeito, and antioxidants showed various degrees of potential effectiveness. Regarding DPN, our review supports the use of alpha-lipoic acid, acetyl-l-carnitine, and vitamin D. The early use of vitamin C prophylaxis for the development of CRPS-I also seems promising. Further research is warranted to confirm these findings.

Exploring the structural and dynamic differences between human carnosinase I (CN1) and II (CN2).

Human carnosinases (CNs) are dimeric dipeptidases in the metallopeptidase M20 family. Two isoforms of carnosinases (Zn2+ -containing carnosinase 1 (CN1) found in serum and Mn2+ -carnosinase 2 (CN2) in tissue) were identified. Both CNs cleave histidine-containing (Xaa-His) dipeptides such as carnosine where CN2 was found to accept a broader spectrum of substrates. A loss of CN function, resulting in a high carnosine concentration, reduces risk for diabetes and neurological disorders. Although several studies on CN activities and its Michaelis complex were conducted, all shed the light on CN1 activity where the CN2 data is limited. Also, the molecular details on CN1 and CN2 similarity and dissimilarity in structure and function remain unclear. Thus, in this work, molecular dynamics (MD) simulations were employed to study structure and dynamics of human CN1 and CN2 in comparison. The results show that the different catalytic ability of both CNs is due to their pocket size and environment. CN2 can accept a wider range of substrate due to the wider mouth of a binding pocket. The L1 loop seems to play a role in gating activity. Comparing to CN2, CN1 provides more electronegative entrance, more wettability, and higher stability of catalytic metal ion-pair in the active site which allow more efficient water-mediated catalysis. The microscopic understanding obtained here can serve as a basis for CN inhibition strategies resulting in higher carnosine levels and consequently mitigating complications associated with diseases such as diabetes and neurological disorder.

2-Oxo-imidazole dipeptides inhibit peroxynitrite-dependent tyrosine nitration.

Carnosine and anserine were reported to inhibit tyrosine nitration. However, there are no reports on the nitration inhibitory activities of balenine, 2-oxo-carnosine, 2-oxo-anserine, and 2-oxo-balenine. We demonstrated for the first time that these compounds exhibit inhibitory activities against peroxynitrite-dependent tyrosine nitration. 2-Oxo-imidazole dipeptides (2-oxo-IDPs) showed higher inhibitory activity than their precursor IDPs, thereby suggesting that 2-oxo-IDPs may be effective against nitrative stress-related diseases.

Effect of muscle fibre types and carnosine levels on the expression of carnosine-related genes in pig skeletal muscle.

It is generally accepted that carnosine (ß-alanyl-L-histidine) content is higher in glycolytic than in oxidative muscle fibres, but the underlying mechanisms responsible for this difference remain to be elucidated. A first study to better understand potential mechanisms involved was undertaken (1) to determine whether differences in the expression of carnosine-related enzymes (CARNS1, CNDP2) and transporters (SLC6A6, SLC15A3, SLC15A4, SLC36A1) exist between oxidative and glycolytic myofibres and (2) to study the effect of carnosine on myoblast proliferative growth and on carnosine-related gene expression in cultured myoblasts isolated from glycolytic and oxidative muscles. Immunohistochemistry analyses were conducted to determine the cellular localization of carnosine-related proteins. Laser-capture microdissection and qPCR analyses were performed to measure the expression of carnosine-related genes in different myofibres isolated from the longissimus dorsi muscle of ten crossbred pigs. Myogenic cells originating from glycolytic and oxidative muscles were cultured to assess the effect of carnosine (0, 10, 25 and 50 mM) on their proliferative growth and on carnosine-related gene expression. The mRNA abundance of CNDP2 and of the studied carnosine transporters was higher in oxidative than in glycolytic myofibres. Since carnosine synthase (CARNS1) mRNA abundance was not affected by either the fibre type or the addition of carnosine to myoblasts, its transcriptional regulation would not be the main process by which carnosine content differences are determined in oxidative and glycolytic muscles. The addition of carnosine to myoblasts leading to a dose-dependent increase in SLC15A3 transcripts, however, suggests a role for this transporter in carnosine uptake and/or efflux to maintain cellular homeostasis.

Trehalose-Carnosine Prevents the Effects of Spinal Cord Injury Through Regulating Acute Inflammation and Zinc(II) Ion Homeostasis.

Spinal cord injury (SCI) leads to long-term and permanent motor dysfunctions, and nervous system abnormalities. Injury to the spinal cord triggers a signaling cascade that results in activation of the inflammatory cascade, apoptosis, and Zn(II) ion homeostasis. Trehalose (Tre), a nonreducing disaccharide, and L-carnosine (Car), (ß-alanyl-L-histidine), one of the endogenous histidine dipeptides have been recognized to suppress early inflammatory effects, oxidative stress and to possess neuroprotective effects. We report on the effects of the conjugation of Tre with Car (Tre-car) in reducing inflammation in in vitro and in vivo models. The in vitro study was performed using rat pheochromocytoma cells (PC12 cell line). After 24 h, Tre-car, Car, Tre, and Tre + Car mixture treatments, cells were collected and used to investigate Zn2+ homeostasis. The in vivo model of SCI was induced by extradural compression of the spinal cord at the T6-T8 levels. After treatments with Tre, Car and Tre-Car conjugate 1 and 6 h after SCI, spinal cord tissue was collected for analysis. In vitro results demonstrated the ionophore effect and chelating features of L-carnosine and its conjugate. In vivo, the Tre-car conjugate treatment counteracted the activation of the early inflammatory cascade, oxidative stress and apoptosis after SCI. The Tre-car conjugate stimulated neurotrophic factors release, and influenced Zn2+ homeostasis. We demonstrated that Tre-car, Tre and Car treatments improved tissue recovery after SCI. Tre-car decreased proinflammatory, oxidative stress mediators release, upregulated neurotrophic factors and restored Zn2+ homeostasis, suggesting that Tre-car may represent a promising therapeutic agent for counteracting the consequences of SCI.

Carnosine increases insulin-stimulated glucose uptake and reduces methylglyoxal-modified proteins in type-2 diabetic human skeletal muscle cells.

Type-2 diabetes (T2D) is characterised by a dysregulation of metabolism, including skeletal muscle insulin resistance, mitochondrial dysfunction, and oxidative stress. Reactive species, such as methylglyoxal (MGO) and 4-hydroxynonenal (4-HNE), positively associate with T2D disease severity and can directly interfere with insulin signalling and glucose uptake in skeletal muscle by modifying cellular proteins. The multifunctional dipeptide carnosine, and its rate-limiting precursor ß-alanine, have recently been shown to improve glycaemic control in humans and rodents with diabetes. However, the precise mechanisms are unclear and research in human skeletal muscle is limited. Herein, we present novel findings in primary human T2D and lean healthy control (LHC) skeletal muscle cells. Cells were differentiated to myotubes, and treated with 10 mM carnosine, 10 mM ß-alanine, or control for 4-days. T2D cells had reduced ATP-linked and maximal respiration compared with LHC cells (p = 0.016 and p = 0.005). Treatment with 10 mM carnosine significantly increased insulin-stimulated glucose uptake in T2D cells (p = 0.047); with no effect in LHC cells. Insulin-stimulation increased MGO-modified proteins in T2D cells by 47%; treatment with carnosine attenuated this increase to 9.7% (p = 0.011). There was no effect treatment on cell viability or expression of other proteins. These findings suggest that the beneficial effects of carnosine on glycaemic control may be explained by its scavenging actions in human skeletal muscle.

Zinc Carnosine Metal-Organic Coordination Polymer as a Potent Broadly Active Influenza Vaccine Platform with In Vitro Shelf-Stability.

Current seasonal influenza vaccines are limited in that they need to be reformulated every year in order to account for the constant mutation of the virus. Hemagglutinin (HA) immunogens have been developed using a computationally optimized broadly reactive antigen (COBRA) methodology, which are able to elicit an antibody response that neutralizes antigenically distinct influenza strains; however, subunit proteins are not immunogenic enough on their own to generate a substantial immune response. Due to this, different delivery strategies and adjuvants can be used to improve immunogenicity. Recently, we reported a new coordination polymer composed of the dipeptide carnosine and zinc (ZnCar) that is able to deliver protein antigens along with CpG to generate a potent immune response. In the present work, ZnCar was used to deliver the COBRA HA immunogen Y2 and the adjuvant CpG. We incorporated Y2 into ZnCar using two different methods to assess which would be the most immunogenic. Mice vaccinated with Y2 and CpG complexed with ZnCar showed an improved humoral and cellular response when compared to mice vaccinated with soluble Y2 and CpG. Further, we demonstrate in vitro that when Y2 and CpG are coordinated with ZnCar, they are protected from degradation at 40 °C for 3 months or 24 °C for 6 months. Overall, ZnCar shows promise as a delivery vehicle for subunit vaccines, given its superior immunogenicity and in vitro storage stability.

Carnosine-Based Reversal of Diabetes-Associated Cognitive Decline via Activation of the Akt/mTOR Pathway and Modulation of Autophagy in a Rat Model of Type 2 Diabetes Mellitus.

INTRODUCTION: Carnosine can suppress secondary complications in diabetes and show robust neuroprotective activity against neurodegenerative diseases. Here, we report that carnosine ameliorates diabetes-associated cognitive decline in vivo through the modulation of autophagy. METHODS: A high-fat diet (HFD) and one intraperitoneal injection of 30 mg/kg streptozotocin (STZ) were used to induce type 2 diabetes mellitus in Sprague-Dawley rats. The rats were randomly divided into five groups: control (CON), HFD/STZ, and three intragastric carnosine treatment groups receiving low (100 mg/kg), medium (300 mg/kg), and high (900 mg/kg) doses over 12 weeks. Body weight, blood glucose levels, and cognitive function were continuously monitored. From excised rat hippocampi, we determined superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels; carnosine concentration; protein expressions of Akt, mTOR and the autophagy markers LC3B and P62 and performed histopathological evaluations of the cornu ammonis 1 region. RESULTS: The HFD/STZ group showed increased blood glucose levels and decreased body weight compared to the CON group. However, there were no significant differences in body weight and blood glucose levels between carnosine-treated and -untreated HFD-STZ-induced diabetic rats. Diabetic animals showed obvious learning and memory impairments in the Morris water maze test compared to the CON group. Compared to those in the HFD/STZ group, carnosine increased SOD activity and decreased MDA levels, increased hippocampal carnosine concentration, increased p-Akt and p-mTOR expression, decreased LC3B and P62 expression, alleviated neuronal injuries, and improved cognitive performance in a dose-dependent manner. CONCLUSION: Independent of any hyperglycemic effect, carnosine may improve mild cognitive impairments by mitigating oxidative stress, activating the Akt/mTOR pathway, and modulating autophagy in the hippocampus of type 2 diabetic rats.

Reduction of transient carnosine radicals depends on ß-alanyl amino group charge.

Reduction of transient carnosine (ß-alanyl-L-histidine) radicals by L-tryptophan, N-acetyl tryptophan, and the Trp-Gly peptide in neutral and basic aqueous solutions was studied using the technique of time-resolved chemically induced dynamic nuclear polarization (TR CIDNP). Carnosine radicals were generated in the photoinduced reaction with triplet excited 3,3',4,4'-tetracarboxy benzophenone. In this reaction, carnosine radicals with their radical center at the histidine residue are formed. Modeling of CIDNP kinetic data allowed for the determination of pH-dependent rate constants of the reduction reaction. It was shown that the protonation state of the amino group of the non-reacting ß-alanine residue of the carnosine radical affects the rate constant of the reduction reaction. The results were compared to those obtained previously for the reduction of histidine and N-acetyl histidine free radicals and to newly obtained results for the reduction of radicals derived from Gly-His, a homologue of carnosine. Clear differences were demonstrated.