Prolactin Mediates Long-Term, Seasonal Rheostatic Regulation of Body Mass in Female Mammals.

A series of well-described anabolic and catabolic neuropeptides are known to provide short-term, homeostatic control of energy balance. The mechanisms that govern long-term, rheostatic control of regulated changes in energy balance are less well characterized. Using the robust and repeatable seasonal changes in body mass observed in Siberian hamsters, this report examined the role of prolactin in providing long-term rheostatic control of body mass and photoinduced changes in organ mass (ie, kidney, brown adipose tissue, uterine, and spleen). Endogenous circannual interval timing was observed after 4 months in a short photoperiod, indicated by a significant increase in body mass and prolactin mRNA expression in the pituitary gland. There was an inverse relationship between body mass and the expression of somatostatin (Sst) and cocaine- and amphetamine-regulated transcript (Cart). Pharmacological inhibition of prolactin release (via bromocriptine injection), reduced body mass of animals maintained in long photoperiods to winter-short photoperiod levels and was associated with a significant increase in hypothalamic Cart expression. Administration of ovine prolactin significantly increased body mass 24 hours after a single injection and the effect persisted after 3 consecutive daily injections. The data indicate that prolactin has pleiotropic effects on homeostatic sensors of energy balance (ie, Cart) and physiological effectors (ie, kidney, BAT). We propose that prolactin release from the pituitary gland acts as an output signal of the hypothalamic rheostat controller to regulate adaptive changes in body mass.

Exploring metabolic responses and pathway changes in CHO-K1 cells under varied aeration conditions and copper supplementations using 1 H NMR-based metabolomics.

The optimization of bioprocess for CHO cell culture involves careful consideration of factors such as nutrient consumption, metabolic byproduct accumulation, cell growth, and monoclonal antibody (mAb) production. Valuable insights can be obtained by understanding cellular physiology to ensure robust and efficient bioprocess. This study aims to improve our understanding of the CHO-K1 cell metabolism using 1 H NMR-based metabolomics. Initially, the variations in culture performance and metabolic profiles under varied aeration conditions and copper supplementations were thoroughly examined. Furthermore, a comprehensive metabolic pathway analysis was performed to assess the impact of these conditions on the implicated pathways. The results revealed substantial alterations in the pyruvate metabolism, histidine metabolism, as well as phenylalanine, tyrosine and tryptophan biosynthesis, which were especially evident in cultures subjected to copper deficiency conditions. Conclusively, significant metabolites governing cell growth and mAb titer were identified through orthogonal partial least square-discriminant analysis (OPLS-DA). Metabolites, including glycerol, alanine, formate, glutamate, phenylalanine, and valine, exhibited strong associations with distinct cell growth phases. Additionally, glycerol, acetate, lactate, formate, glycine, histidine, and aspartate emerged as metabolites influencing cell productivity. This study demonstrates the potential of employing 1 H NMR-based metabolomics technology in bioprocess research. It provides valuable guidance for feed medium development, feeding strategy design, bioprocess parameter adjustments, and ultimately the enhancement of cell proliferation and mAb yield.

Protective effect of oleic acid against very long-chain fatty acid-induced apoptosis in peroxisome-deficient CHO cells.

Very long-chain fatty acids (VLCFAs) are degraded exclusively in peroxisomes, as evidenced by the accumulation of VLCFAs in patients with certain peroxisomal disorders. Although accumulation of VLCFAs is considered to be associated with health issues, including neuronal degeneration, the mechanisms underlying VLCFAs-induced tissue degeneration remain unclear. Here, we report the toxic effect of VLCFA and protective effect of C18: 1 FA in peroxisome-deficient CHO cells. We examined the cytotoxicity of saturated and monounsaturated VLCFAs with chain-length at C20-C26, and found that longer and saturated VLCFA showed potent cytotoxicity at lower accumulation levels. Furthermore, the extent of VLCFA-induced toxicity was found to be associated with a decrease in cellular C18:1 FA levels. Notably, supplementation with C18:1 FA effectively rescued the cells from VLCFA-induced apoptosis without reducing the cellular VLCFAs levels, implying that peroxisome-deficient cells can survive in the presence of accumulated VLCFA, as long as the cells keep sufficient levels of cellular C18:1 FA. These results suggest a therapeutic potential of C18:1 FA in peroxisome disease and may provide new insights into the pharmacological effect of Lorenzo's oil, a 4:1 mixture of C18:1 and C22:1 FA.

In vitro and in vivo genotoxicity evaluation of ALP1018, a nanomineral food supplement.

The use of nano-based dietary supplements is increasing around the world, as nanotechnology can help enhance nutrient bioavailability. ALP1018 is a newly developed iron-zinc complex supplement designed as a nanoformulation to improve the efficacy of iron and zinc supplementation. However, safety concerns have been raised, as there is no clear evaluation of ALP1018 toxicity. The goal of this study was to determine the potential mutagenicity and genotoxicity of ALP1018 through three standard screenings: the Ames test, which evaluates bacterial reverse mutations; the in vitro test of chromosomal aberration in Chinese hamster lung cells; and the in vivo micronucleus assay using ICR mice. ALP1018 showed no mutagenic effect, as no increase was observed in the presence or absence of metabolic activation (S9 mix) in revertant colonies on all the bacterial strains used in the Ames test. No structural chromosomal abnormalities were observed in the presence or absence of the S9 mix in mammalian cells used in the chromosomal aberration assay. In the micronucleus test, the frequency of micronucleated polychromatic erythrocytes was not significantly increased in mouse bone marrow cells. Based on these findings, we can conclude that ALP1018 is safe to use and has no mutagenic or genotoxic potential.

Untargeted metabolomics reveals the regulatory effect of geniposidic acid on lipid accumulation in HepG2 cells and Caenorhabditis elegans and validation in hyperlipidemic hamsters.

BACKGROUND: Geniposidic acid (GPA) alleviates oxidative stress and inflammation in mice However, whether it can effectively regulate lipid accumulation and prevent hyperlipidemia requires further investigation. PURPOSE: This study combined the untargeted metabolomics of cells and a Caenorhabditis elegans model to evaluate the anti-hyperlipidemic potential of GPA by modulating oxidative stress and regulating lipid metabolism. A golden hamster model of hyperlipidemia was used to further validate the lipid-lowering effect and mechanism of action of GPA. METHODS: Chemical staining, immunofluorescence, and flow cytometry were performed to examine the effects of GPA on lipid accumulation and oxidative stress. Untargeted metabolomic analysis of cells and C. elegans was performed using ultra-performance liquid chromatography coupled with quadrupole electrostatic field Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap MS) to identify biomarkers altered by GPA action, analyze the affected metabolic pathways, and validate the mechanisms by which GPA regulates lipid metabolism and oxidative stress. A golden hamster model of hyperlipidemia was established to test the lipid-lowering effects of GPA. Body weight, biochemical markers, rate-limiting enzymes, and key proteins were assessed. Hematoxylin and eosin (H&E) and Oil Red O staining were performed. RESULTS: Phenotypic data showed that GPA decreased free fatty acid (FFA)-induced lipid buildup and high reactive oxygen species (ROS) levels, reversed the decrease in mitochondrial membrane potential (MMP), and increased the cellular reduced glutathione/oxidized glutathione disulfide (GSH/GSSG) ratio. GPA also reduces high glucose-induced lipid build-up and ROS production in C. elegans. Metabolomic analysis showed that GPA affected purine, lipid, and amino acid metabolism. Moreover, GPA inhibited xanthine oxidase (XOD), glutamate dehydrogenase (GLDH), fatty acid synthase (FAS), phosphorylation of P38 MAPK, and upregulated the expression of SIRT3 and CPT1A protein production to control lipid metabolism and produce antioxidant benefits in cells and golden hamsters. CONCLUSION: Current evidence suggests that GPA can effectively regulate lipid metabolism and the oxidative stress response, and has the potential to prevent hyperlipidemia. This study also provided an effective method for evaluating the mechanism of action of GPA.

Hypothalamic tanycytes as mediators of maternally programmed seasonal plasticity.

In mammals, maternal photoperiodic programming (MPP) provides a means whereby juvenile development can be matched to forthcoming seasonal environmental conditions.1,2,3,4 This phenomenon is driven by in utero effects of maternal melatonin5,6,7 on the production of thyrotropin (TSH) in the fetal pars tuberalis (PT) and consequent TSH receptor-mediated effects on tanycytes lining the 3rd ventricle of the mediobasal hypothalamus (MBH).8,9,10 Here we use LASER capture microdissection and transcriptomic profiling to show that TSH-dependent MPP controls the attributes of the ependymal region of the MBH in juvenile animals. In Siberian hamster pups gestated and raised on a long photoperiod (LP) and thereby committed to a fast trajectory for growth and reproductive maturation, the ependymal region is enriched for tanycytes bearing sensory cilia and receptors implicated in metabolic sensing. Contrastingly, in pups gestated and raised on short photoperiod (SP) and therefore following an over-wintering developmental trajectory with delayed sexual maturation, the ependymal region has fewer sensory tanycytes. Post-weaning transfer of SP-gestated pups to an intermediate photoperiod (IP), which accelerates reproductive maturation, results in a pronounced shift toward a ciliated tanycytic profile and formation of tanycytic processes. We suggest that tanycytic plasticity constitutes a mechanism to tailor metabolic development for extended survival in variable overwintering environments.

Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support.

In recent years, serum-free medium for mammalian cell cultivation has attracted a lot of attention, considering the high cost of production and environmental load involved in developing the conventional animal sera. The use of alternative growth-promoting products in mammalian cell cultivation such as extracts from microalgae has proven to be quite beneficial and environmental-friendly. This research aims to cultivate mammalian cells with growth-promoting factors derived from Chlorococcum littorale. We have established a simple extraction using the ultrasonication method and applied the extract in place of serum on mammalian C2C12 cell lines, 3T3 cell lines, and CHO cell lines to compare and analyze the effectiveness of the extract. Cell passage was conducted in a suspended culture condition with the addition of the extract. The results indicate that the extract from microalgae shows a high proliferation rate in all cell lines without fetal bovine serum. Moreover, it is eco-friendly and has huge potential to replace the traditional cell culture system. It could be applied in the fields of regenerative medicine, gene/cell therapies, as well as cultured meat production.

Regulation of circulating thyroid hormone levels by hypothalamic tanycytes and hypophysial pars tuberalis-specific cells and their morphological and gene- and protein-expression changes under different photoperiods.

Thyroid hormone in the hypothalamus acts as a key determinant of seasonal transitions. Thyroid hormone-levels in the brain are mainly regulated by the hypothalamic tanycytes and pituitary pars tuberalis (PT)-specific cells. TSHß produced by the PT-specific cells stimulates Dio2 expression and decreases Dio3 expression of the tanycytes. Both tanycytes and PT-specific cells in photosensitive animals exhibit remarkable changes of morphological appearance and expressions of genes and proteins under different photoperiods. Long photoperiods induce increased gene- and protein-expressions and active features. Short photoperiods cause the decreased gene- and protein-expressions and inactive features. In the PT, expressions of TSHß, common α-subunit of glycoprotein hormones (α-GSU), and MT1 receptor of melatonin receptors and eyes absent 3 change under different photoperiods. Diurnal rhythms of α-GSU mRNA expression are observed in the PT of Djungarian hamsters. Hes1, Nkx2.1, and LIM homeodomain gene 2 (Lhx2) are involved in the differentiation of PT. In the hypothalamic tanycytes, expressions of Dio2, Dio3, vimentin, serine/threonine kinase 33, GPR50, Nestin, Retinoid signaling genes (retinaldehyde dehydrogenase 1, cellular retinol binding protein 1, and Stra6), monocarboxylate transporter 8, and neural cell adhesion molecule change under different photoperiods. Rax, Lhx2, Nfia/b/x, and fibroblast growth factor 10 are involved in the differentiation of tanycytes.

Totum-070, a Polyphenol-Rich Plant Extract, Prevents Hypercholesterolemia in High-Fat Diet-Fed Hamsters by Inhibiting Intestinal Cholesterol Absorption.

Atherosclerotic cardiovascular disease is the leading cause of mortality worldwide, and hypercholesterolemia is a central risk factor for atherosclerosis. This study evaluated the effects of Totum-070, a plant-based polyphenol-rich supplement, in hamsters with high-fat diet (HFD)-induced dyslipidemia. The molecular mechanisms of action were explored using human Caco2 enterocytes. Totum-070 supplementation reduced the total cholesterol (-41%), non-HDL cholesterol (-47%), and triglycerides (-46%) in a dose-dependent manner, compared with HFD. HFD-induced hepatic steatosis was also significantly decreased by Totum-070, an effect associated with the reduction in various lipid and inflammatory gene expression. Upon challenging with olive oil gavage, the post-prandial triglyceride levels were strongly reduced. The sterol excretion in the feces was increased in the HFD-Totum-070 groups compared with the HFD group and associated with reduction of intestinal cholesterol absorption. These effects were confirmed in the Caco2 cells, where incubation with Totum-070 inhibited cholesterol uptake and apolipoprotein B secretion. Furthermore, a microbiota composition analysis revealed a strong effect of Totum-070 on the alpha and beta diversity of bacterial species and a significant decrease in the Firmicutes to Bacteroidetes ratio. Altogether, our findings indicate that Totum-070 lowers hypercholesterolemia by reducing intestinal cholesterol absorption, suggesting that its use as dietary supplement may be explored as a new preventive strategy for cardiovascular diseases.

Risk assessment for biopharmaceutical single-use manufacturing: A case study of upstream continuous processing.

In the current transition to intensified upstream processing, the risks of adopting traditional single-use systems for high-titer, long-duration perfusion cultures, have thus far not been considered. This case study uses the Failure Modes and Effects Analysis (FMEA) method to evaluate the risks associated with implementing upstream single-use technology. The simulated model process was used to compare the risk level of single-use technology for a traditional fed-batch cell culture with that for perfusion culture, under the same annual protein production conditions. To provide a reasonable source of potential risk for FMEA, all single-use upstream operations for both fed-batch and perfusion processes were investigated using an analytical method developed to quantify the impact of process parameters and operating conditions on single-use system specifications and to ensure objectivity. Many of the risks and their levels, were similar in long-duration perfusion cultures and fed-batch cultures. However, differences were observed for high-risk components such as daily sampling and installation. The result of this analysis indicates that the reasons for risk are different for fed-batch cultures and perfusion cultures such as larger bioreactors in fed-batch and longer runs in perfusion, respectively. This risk assessment method could identify additional control measures and be part of a holistic contamination control strategy and help visualize their effectiveness.

Novel tetrahydrocurcumin integrated mucoadhesive nanocomposite κ-carrageenan/xanthan gum sponges: a strategy for effective local treatment of oral cancerous and precancerous lesions.

Oral cancer is one of the leading causes of death worldwide. Oral precancerous lesions (OPL) are the precursors of oral cancer, with varying degrees of progression. Tetrahydrocurcumin (THC) is a major metabolite of curcumin with superior anticancer properties against various types of cancer. However, THC's clinical outcome is limited by its poor aqueous solubility. Herein, we developed novel mucoadhesive biopolymer-based composite sponges for buccal delivery of THC, exploiting nanotechnology and mucoadhesion for efficient prevention and treatment of oral cancer. Firstly, THC-nanocrystals (THC-NC) were formulated and characterized for subsequent loading into mucoadhesive composite sponges. The anticancer activity of THC-NC was assessed on a human tongue squamous carcinoma cell line (SCC-4). Finally, the chemopreventive activity of THC-NC loaded sponges (THC-NC-S) was examined in DMBA-induced hamster OPL. The selected THC-NC exhibited a particle size of 532.68 ± 13.20 nm and a zeta potential of -46.08 ± 1.12 mV. Moreover, THC-NC enhanced the anticancer effect against SCC-4 with an IC50 value of 80 µg/mL. THC-NC-S exhibited good mucoadhesion properties (0.24 ± 0.02 N) with sustained drug release, where 90% of THC was released over 4 days. Furthermore, THC-NC-S had a magnificent potential for maintaining high chemopreventive activity, as demonstrated by significant regression in the dysplasia degree and a decline in cyclin D1 (control: 40.4 ± 12.5, THC-NC-S: 12.07 ± 5.2), culminating in significant amelioration after 25 days of treatment. Conclusively, novel THC-NC-S represent a promising platform for local therapy of OPL, preventing their malignant transformation into cancer.

Chemical characterization of Callingcard Vine (Entada polystachya (L.) DC. var. polystachya) aqueous seed extract and evaluation of its cytotoxic, genotoxic and mutagenic properties.

Callingcard Vine (Entada polystachya (L.) DC. var. polystachya - Fabaceae) is a common plant in coastal thickets from western Mexico through Central America to Colombia and Brazil, especially in Amazon biome. It has been popularly used as a urinary burning reliever and diuretic. However, the plant chemical constituents are poorly understood and Entada spp. genotoxic potential have not been previously investigated. In the present study we determined the chemical composition of the aqueous E. polystachya crude seed extract (EPCSE) and evaluated the cytotoxic, genotoxic and mutagenic properties of EPCSE in Salmonella typhimurium and Chinese hamster fibroblast (V79) cells. Cytotoxic activity was also evaluated in tumor cell lines (HT29, MCF7 and U87) and non-malignant cells (MRC5). The chemical analysis by High Resolution Mass Spectrometry (HRMS) of EPCSE indicated the presence of saponin and chalcone. The results of the MTT and clonal survival assays suggest that EPCSE is cytotoxic to V79 cells. Survival analysis showed higher IC50 in non-tumor compared with tumor cell lines. EPCSE showed induction of DNA strand breaks as revealed by the alkaline comet assay and micronucleus test. Using the modified comet assay, it was possible to detect the induction of oxidative DNA base damage by EPCSE in V79 cells. Consistently, the extract induced increase lipid peroxidation (TBARS), superoxide dismutase (SOD) and catalase (CAT) activities in V79 cells. In addition, EPCSE induced mutations in S. typhimurium TA98 and TA100 strains, confirming a mutagenic potential. Taken together, our results suggest that EPCSE is cytotoxic and genotoxic to V79 cells and mutagenic to S. typhimurium. These properties can be related to the pro-oxidant ability of the extract and induction of DNA lesions. Additionally, EPCSE could inhibit the growth of tumor cells, especially human colorectal adenocarcinoma (HT29) cell line, and can constitute a possible source of antitumor natural agents.

Cell-Free Synthesis and Electrophysiological Analysis of Multipass Voltage-Gated Ion Channels Tethered in Microsomal Membranes.

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) KV11.1 and the voltage-gated sodium channel hNaV1.5.

Automated Raman feed-back control of multiple supplemental feeds to enable an intensified high inoculation density fed-batch platform process.

Biologics manufacturing is increasingly moving toward intensified processes that require novel control strategies in order to achieve higher titers in shorter periods of time compared to traditional fed-batch cultures. In order to implement these strategies for intensified processes, continuous process monitoring is often required. To this end, inline Raman spectroscopy was used to develop partial least squares models to monitor changes in residual concentrations of glucose, phenylalanine and methionine during the culture of five different glutamine synthetase piggyBac® Chinese hamster ovary clones cultured using an intensified high inoculation density fed-batch platform process. Continuous monitoring of residual metabolite concentrations facilitated automated feed-rate adjustment of three supplemental feeds to maintain glucose, phenylalanine, and methionine at desired setpoints, while maintaining other nutrient concentrations at acceptable levels across all clones cultured on the high inoculation density platform process. Furthermore, all clones cultured on this process achieved high viable cell concentrations over the course of culture, indicating no detrimental impacts from the proposed feeding strategy. Finally, the automated control strategy sustained cultures inoculated at high cell densities to achieve product concentrations between 5 and 8.3 g/L over the course of 12 days of culture.

Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia.

Antibody-drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg-1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

Toxin type and domain sequence affect accumulation of recombinant immunotoxins.Transient expression in plant cell packs and intact plants correlates well.IC₅₀ values of toxicity correlate with the cell surface receptor concentration.

Trace metal optimization in CHO cell culture through statistical design of experiments.

A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high-producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three-level factorial experimental design was performed in fed-batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter-influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late-stage ROS activity in a dose-dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity.

Marine Chitosan-Oligosaccharide Ameliorated Plasma Cholesterol in Hypercholesterolemic Hamsters by Modifying the Gut Microflora, Bile Acids, and Short-Chain Fatty Acids.

This study evaluated the cholesterol-alleviating effect and underlying mechanisms of chitosan-oligosaccharide (COS) in hypercholesterolemic hamsters. Male hamsters (n = 24) were divided into three groups in a random fashion, and each group was fed one particular diet, namely a non-cholesterol diet (NCD), a high-cholesterol diet (HCD), and an HCD diet substituting 5% of the COS diet for six weeks. Subsequently, alterations in fecal bile acids (BAs), short-chain fatty acids (SCFAs), and gut microflora (GM) were investigated. COS intervention significantly reduced and increased the plasma total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) levels in hypercholesteremic hamsters. Furthermore, Non-HDL-C and total triacylglycerols (TG) levels were also reduced by COS supplementation. Additionally, COS could reduce and increase food intake and fecal SCFAs (acetate), respectively. Moreover, COS had beneficial effects on levels of BAs and GM related to cholesterol metabolism. This study provides novel evidence for the cholesterol-lowering activity of COS.

Evaluation of the topical gel and oral administration of Punica Granatum Var Pleniflora on oral mucositis induced by 5-Fluorouracil in golden hamsters.

BACKGROUND: Oral mucositis (OM), an acute inflammation of the oral cavity, is a common complication in patients undergoing invasive myeloblastic chemotherapy or radiation therapy. 5-fluorouracil (5-FU) is one of the most effective therapeutic drugs, but one of the common side effects of 5-FU administration is OM. Unfortunately, no suitable treatment has been found, so far to control its side effects. Studies showed that herbal medicine like Punica granatum var pleniflora (PGP) has medicinal properties such as anti-inflammatory and antibacterial and can be an alternative for the treatment of fungal infection. Accordingly, we decided to investigate the therapeutic effect of PGP in the treatment of OM caused by 5-FU in golden hamsters. METHODS: Sixty male golden hamsters were divided into six main group. Chemotherapy with 5-FU at dose of 60 mg/kg was performed at a ten-day duration. Then, cheek pouches of the hamsters were scratched with an 18-gauge sterile needle to induce oral mucositis in animals. On the twelfth day, as a day of intensification of OM, treatment with PGP including topical gel with concentrations of 5% and 10% and oral administration of hydro-alcoholic extract with doses of 125 mg/kg and 250 mg/kg for three- and five-day therapeutic duration were separately started. Finally, samples of cheek pouches in hamsters were collected on 14th and 17th days and histopathologic score (HPS), malondialdehyde (MDA), and myeloperoxidase (MPO) levels were assayed. RESULTS: A significant (p < 0.05) decrease in histopathologic score was observed in G10%-, P125-treated groups in comparison to the Ctrl group. Our data showed that treatment with G10% is more potent than P125-treated group. In contrast, histopathologic score in G10%, P125, and P250 treated groups demonstrated almost similar values On the 17th day. However, the levels of MDA and MPO in the treatment groups were enhanced compared with control group (p < 0.05). CONCLUSIONS: It is possible that PGP can play protective role in the healing of tissue damage caused by chemotherapy with 5-FU due to the presence of its natural compounds and antioxidant properties.

Nanobodies with cross-neutralizing activity provide prominent therapeutic efficacy in mild and severe COVID-19 rodent models.

The weakened protective efficacy of COVID-19 vaccines and antibodies caused by SARS-CoV-2 variants presents a global health emergency, which underscores the urgent need for universal therapeutic antibody intervention for clinical patients. Here, we screened three alpacas-derived nanobodies (Nbs) with neutralizing activity from twenty RBD-specific Nbs. The three Nbs were fused with the Fc domain of human IgG, namely aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc, which could specifically bind RBD protein and competitively inhibit the binding of ACE2 receptor to RBD. They effectively neutralized SARS-CoV-2 pseudoviruses D614G, Alpha, Beta, Gamma, Delta, and Omicron sub-lineages BA.1, BA.2, BA.4, and BA.5 and authentic SARS-CoV-2 prototype, Delta, and Omicron BA.1, BA.2 strains. In mice-adapted COVID-19 severe model, intranasal administration of aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc effectively protected mice from lethal challenges and reduced viral loads in both the upper and lower respiratory tracts. In the COVID-19 mild model, aVHH-13-Fc, which represents the optimal neutralizing activity among the above three Nbs, effectively protected hamsters from the challenge of SARS-CoV-2 prototype, Delta, Omicron BA.1 and BA.2 by significantly reducing viral replication and pathological alterations in the lungs. In structural modeling of aVHH-13 and RBD, aVHH-13 binds to the receptor-binding motif region of RBD and interacts with some highly conserved epitopes. Taken together, our study illustrated that alpaca-derived Nbs offered a therapeutic countermeasure against SARS-CoV-2, including those Delta and Omicron variants which have evolved into global pandemic strains.

16-Hydroxy-ent-halima-5(10),13-dien-15,16-olide from Polyalthia longifolia targets adipogenesis by inhibiting mitotic clonal expansion and ameliorates dyslipidemia.

Obesity-related metabolic disorders are increasing at an alarming rate worldwide. The FDA has approved many molecules for weight loss therapy; most of them act on the gut level by inhibiting lipid uptake or on the central nervous system by controlling appetite. Limitations and drawbacks have propelled the search for new pharmacophores exhibiting favourable metabolic alteration at adipocytes, and natural products have always been there to prove their worth. In our efforts, we have identified 16-hydroxy-ent-halima-5(10),13-dien-15,16-olide (PLH), a halimane diterpene isolated from Polyalthia longifolia, demonstrating anti-adipogenic and anti-dyslipidemic activity. It inhibited adipogenesis in 3T3-L1 preadipocyte and C3H10T1/2 mesenchymal stem cell lines. Furthermore, it decreased set of adipogenic markers at transcript and protein levels. Cell cycle studies indicated that PLH halts the mitotic clonal expansion. Mechanistic studies shows that PLH activate Wnt/ß-catenin signaling pathway to inhibit the adipogenesis. The study suggested that PLH inhibited adipogenesis during the early phase of differentiation by targeting mitotic clonal expansion and arresting the cell cycle in the G1 phase of the cell cycle. It improved the dyslipidemic condition in HFD-fed hamsters by decreasing the body weight, fat mass, eWAT weight and improving the serum lipid profile. Overall, PLH has been found as a potential drug candidate and a pharmacophore for combating metabolic disorders including obesity and dyslipidemia.