The effects of in vitro vitamin D treatment on glycolytic reprogramming of bone marrow-derived dendritic cells from Ldlr knock-out mouse.

Dendritic cells (DCs) undergo glycolytic reprogramming, a metabolic conversion process essential for their activation. Vitamin D has been reported to affect the function of DCs, but studies in metabolic diseases are insufficient. This study investigates the effects of in vitro 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment on glycolytic reprogramming of bone marrow-derived dendritic cells (BMDCs) from control, obese, and atherosclerosis mice. Six-week-old male C57BL/6J mice were fed a control diet (CON) or a Western diet (WD), and B6.129S7-Ldlrtm1Her/J mice were fed a Western diet (LDLR-/-) for 16 weeks. BMDCs were cultured in a medium containing 1,25(OH)2D3 (10 nM) for 7 days and stimulated with lipopolysaccharide (LPS, 50 ng/mL) for 24 h. In mature BMDCs, 1,25(OH)2D3 treatment decreased basal and compensatory glycolytic proton efflux rates (glycoPER), the expression of surface markers related to immune function of DCs (MHC class II, CD80, and CD86), and IL-12p70 production. In addition, mTORC1 activation and nitric oxide (NO) production were suppressed by 1,25(OH)2D3 treatment in mature BMDCs. The effect of 1,25(OH)2D3 treatment on IL-12p70 production and mTORC1 activity in the LDLR-/- group was greater than in the CON group. These findings suggest that vitamin D can affect the metabolic environment of BMDCs by regulating glycolytic reprogramming as well as by inducing tolerogenic phenotypes of DCs.

Longitudinal Genome-Wide Association Study of Cognitive Impairment after Subarachnoid Hemorrhage.

OBJECTIVES: The occurrence of cognitive deficits after subarachnoid hemorrhage (SAH) is highly possible, leading to vascular dementia. We performed a novel longitudinal genome-wide association study (GWAS) to identify genetic modifications associated with cognitive impairment following SAH in a long-term prospective cohort study. MATERIALS AND METHODS: This GWAS involved 153 patients with SAH sharing 5,971,372 markers after high-throughput imputation. Genome-wide Cox proportional hazard regression testing was performed to estimate the hazard ratio (HR) and 95% confidence interval (CI). Subsequently, a weighted polygenetic risk score (wPRS) was determined, based on GWAS-driven loci and risk stratification. RESULTS: Cognitive impairment was observed in 65 patients (42.5%) during a mean follow-up of 37.7 ± 12.4 months. Five genome-wide signals, including rs138753053 (PDCD6IP-LOC101928135, HR = 28.33, p = 3.4 × 10-8), rs56823384 (LINC00499, HR = 12.47, p = 2.8 × 10-9), rs145397166 (CASC15, HR = 11.16, p = 1.7 × 10-8), rs10503670 (LPL-SLC18A1, HR = 2.88, p = 4.0 × 10-8), and rs76507772 (IRS2, HR = 5.99, p = 3.5 × 10-8), were significantly associated with cognitive impairment following SAH. In addition, the well-constructed wPRS containing five markers showed nominal ability to predict cognitive impairment (AUROC = 0.745, 95% CI: 0.667-0.824). Tertile stratification showed a higher effectiveness in predicting cognitive impairment, especially in those with haptoglobin 2-1 (HR = 44.59, 95% CI: 8.61-231.08). CONCLUSIONS: Our study revealed novel susceptible loci for cognitive impairment, longitudinally measured in patients with SAH. The clinical utility of these loci will be evaluated in further follow-up studies.

Influence of Gelatin on Adhesion, Proliferation, and Adipogenic Differentiation of Adipose Tissue-Derived Stem Cells Cultured on Soy Protein-Agarose Scaffolds.

Scaffolds play a key role in cultured meat production by providing an optimal environment for efficient cell attachment, growth, and development. This study investigated the effects of gelatin coating on the adhesion, proliferation, and adipogenic differentiation of adipose tissue-derived stem cells (ADSCs) cultured on soy protein-agarose scaffolds. Gelatin-coated scaffolds were prepared using 0.5% and 1.0% (w/v) gelatin solutions. The microstructure, water absorption rate, mechanical strength, cytotoxicity, cell adhesion, proliferation, and differentiation capabilities of the scaffolds were analyzed. Field emission scanning electron microscopy revealed the porous microstructure of the scaffolds, which was suitable for cell growth. Gelatin-coated scaffolds exhibited a significantly higher water absorption rate than that of non-coated scaffolds, indicating increased hydrophilicity. In addition, gelatin coating increased the mechanical strength of the scaffolds. Gelatin coating did not show cytotoxicity but significantly enhanced cell adhesion and proliferation. The gene expression levels of peroxisome proliferator-activated receptor gamma, CCAT/enhancer-binding protein alpha, and fatty acid-binding protein 4 were upregulated, and lipid accumulation was increased by gelatin coating. These findings suggest that gelatin-coated scaffolds provide a supportive microenvironment for ADSC growth and differentiation, highlighting their potential as a strategy for the improvement of cultured meat production and adipose tissue engineering.

Therapeutic Effect of Donepezil on Neuroinflammation and Cognitive Impairment after Moderate Traumatic Brain Injury.

BACKGROUND: Despite the important clinical issue of cognitive impairment after moderate traumatic brain injury (TBI), there is currently no suitable treatment. Here, we used in vitro and in vivo models to investigate the effect of Donepezil-an acetylcholinesterase (AChE) inhibitor-on cognitive impairment in the acute period following injury, while focusing on neuroinflammation and autophagy- and mitophagy-related markers. METHODS: The purpose of the in vitro study was to investigate potential neuroprotective effects in TBI-induced cells after donepezil treatment, and the in vivo study, the purpose was to investigate therapeutic effects on cognitive impairment in the acute period after injury by analyzing neuroinflammation and autophagy- and mitophagy-related markers. The in vitro TBI model involved injuring SH-SY5Y cells using a cell-injury controller and then investigating the effect of donepezil at a concentration of 80 µM. The in vivo TBI model was made using a stereotaxic impactor for male C57BL/6J mice. Immuno-histochemical markers and cognitive functions were compared after 7 days of donepezil treatment (1 mg/kg/day). Mice were divided into four groups: sham operation with saline treatment, sham operation with donepezil treatment, TBI with saline treatment, and TBI with donepezil treatment (18 mice in each group). Donepezil treatment was administered within 4 h post-TBI. RESULTS: In vitro, donepezil was found to lead to increased cell viability and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), along with decreased reactive oxygen species (ROS), lactate-dehydrogenase (LDH), 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA)-positive cells, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. The mRNA and protein expressions of neuroinflammation (Cyclooxygenase-2, COX-2; NOD-like receptor protein 3, NLRP3; Caspase-1; and Interleukin-1 beta, IL-1ß), as well as autophagy- and mitophagy-related markers (death-associated protein kinase 1, DAPK1; PTEN-induced kinase 1, PINK1; BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like, BNIP3L; Beclin-1, BECN1; BCL2-associated X protein, BAX; microtubule-associated protein 1A/1B-light chain 3B (LC3B); Sequestosome-1; and p62) were all found to decrease after donepezil treatment. The in vivo study also showed that donepezil treatment resulted in decreased levels of cortical tissue losses and brain swelling in TBI compared to the TBI group without donepezil treatment. Donepezil treatment was also shown to decrease the mRNA and Western blotting expressions of all markers, and especially COX-2 and BNIP3L, which showed the most significant decreases. Moreover, TBI mice showed an decreased escape latency, increased alteration rate, and improved preference index, altogether pointing to better cognitive performance after donepezil treatment. CONCLUSIONS: Donepezil treatment may be beneficial in improving cognitive impairment in the early phase of moderate traumatic brain injury by ameliorating neuroinflammation, as well as autophagy and mitophagy.

Micro-current stimulation could inhibit IL-1ß-induced inflammatory responses in chondrocytes and protect knee bone cartilage from osteoarthritis.

This study aimed to evaluate the inhibitory effects of micro-current stimulation (MCS) on inflammatory responses in chondrocytes and degradation of extracellular matrix (ECM) in osteoarthritis (OA). To determine the efficacy of MCS, IL-1ß-treated chondrocytes and monosodium iodoacetate (MIA)-induced OA rat model were used. To evaluate the cytotoxicity and nitric oxide (NO) production in SW1353 cells, the presence or absence of IL-1ß treatment or various levels of MCS were applied. Immunoblot analysis was conducted to evaluate whether MCS can modulate IL-1R1/MyD88/NF-κB signaling pathway and various indicators involved in ECM degradation. Additionally, to determine whether MCS alleviates subchondral bone structure destruction caused by OA, micro-CT analysis, immunoblot analysis, and ELISA were conducted using OA rat model. 25 and 50 µA levels of MCS showed effects in cell proliferation and NO production. The MCS group with IL-1ß treatment lead to significant inhibition of protein expression levels regarding IL-1R1/MyD88/NF-κB signaling and reduction of the nucleus translocation of NF-κB. In addition, the protein expression levels of MMP-1, MMP-3, MMP-13, and IL-1ß decreased, whereas collagen II and aggrecan increased. In animal results, morphological analysis of subchondral bone using micro-CT showed that MCS induced subchondral bone regeneration and improvement, as evidenced by increased thickness and bone mineral density of the subchondral bone. Furthermore, MCS-applied groups showed decreases in the protein expression of MMP-1 and MMP-3, while increases in collagen-II and aggrecan expressions. These findings suggest that MCS has the potential to be used as a non-pharmaceutical method to alleviate OA.

Fabrication of alginate composite hydrogel encapsulated retinoic acid and nano se doped CaP to augment in situ mineralization and osteoimmunomodulation for bone regeneration.

BACKGROUND: Bone tissue engineering endows alternates to support bone defects/injuries that are circumscribed to undergo orchestrated process of remodeling on its own. In this regard, hydrogels have emerged as a promising platform that can confront irregular defects and encourage in situ bone repair. METHODS: In this study, we aimed to develop a new approach for bone tissue regeneration by developing an alginate based composite hydrogel incorporating selenium doped biphasic calcium phosphate nanoparticles, and retinoic acid. The fabricated hydrogel was physiochemically evaluated for morphological, bonding, and mechanical behavior. Additionally, the biological response of the fabricated hydrogel was evaluated on MC3T3-E1 pre-osteoblast cells. RESULTS: The developed composite hydrogel confers excellent biocompatibility, and osteoconductivity owing to the presence of alginate, and biphasic calcium phosphate, while selenium presents pro osteogenic, antioxidative, and immunomodulatory properties. The hydrogels exhibited highly porous microstructure, superior mechanical attributes, with enhanced calcification, and biomineralization abilities in vitro. SIGNIFICANCE: By combining the osteoconductive properties of biphasic calcium phosphate with multifaceted benefits of selenium and retinoic acid, the fabricated composite hydrogel offers a potential transformation in the landscape of bone defect treatment. This strategy could direct a versatile and effective approach to tackle complex bone injuries/defects and present potential for clinical translation.

Assessment of hard and soft tissue thickness at mandibular symphysis in skeletal Class III patients with different vertical patterns.

INTRODUCTION: This study aimed to assess the bony and soft tissue parameters at mandibular symphysis among skeletal Class III patients with different vertical growth patterns, using cone-beam computed tomography (CBCT). MATERIALS AND METHODS: CBCT images of 60 skeletal Class III non-growing patients were evaluated (mean age 24.9 ± 8.4 years). Study samples were classified into three facial types based on the mandibular plane angle (SN-MP angle): low, normal, and high angle. The bony and soft tissue parameters at the mandibular symphysis were evaluated. RESULTS: Among hard tissue variables, symphysis and pogonion width were significantly narrower in the high-angle group (P < .05). The thickness of the buccal cortex at pogonion was also significantly thinner in subjects with high angles (P < .01). Symphysis height showed an increasing tendency from the low-angle to the high-angle group. However, no significant differences were found in chin width and height according to vertical patterns. Across all soft tissue measurements, the low-angle group exhibited the highest thickness, which gradually decreased in the high-angle group. Statistically significant differences in soft tissue thickness were observed at Menton (Me) and Gnathion (Gn) (P < .05). A significant negative correlation was observed between the SN-MP angle and the thickness of both hard and soft tissues. CONCLUSIONS: In skeletal Class III subjects, significant differences existed in both hard and soft tissues at the mandibular symphysis, depending on the vertical patterns. These results provide a comprehensive evaluation of symphyseal area, which can aid clinicians in identifying appropriate treatment approaches, especially for combined orthognathic and orthodontic treatment.

Deep learning application of vertebral compression fracture detection using mask R-CNN.

Vertebral compression fractures (VCFs) of the thoracolumbar spine are commonly caused by osteoporosis or result from traumatic events. Early diagnosis of vertebral compression fractures can prevent further damage to patients. When assessing these fractures, plain radiographs are used as the primary diagnostic modality. In this study, we developed a deep learning based fracture detection model that could be used as a tool for primary care in the orthopedic department. We constructed a VCF dataset using 487 lateral radiographs, which included 598 fractures in the L1-T11 vertebra. For detecting VCFs, Mask R-CNN model was trained and optimized, and was compared to three other popular models on instance segmentation, Cascade Mask R-CNN, YOLOACT, and YOLOv5. With Mask R-CNN we achieved highest mean average precision score of 0.58, and were able to locate each fracture pixel-wise. In addition, the model showed high overall sensitivity, specificity, and accuracy, indicating that it detected fractures accurately and without misdiagnosis. Our model can be a potential tool for detecting VCFs from a simple radiograph and assisting doctors in making appropriate decisions in initial diagnosis.

Clinical impact of pancreatic invasion in T1-stage distal bile duct cancer and prognostic factors associated with long-term survival: A multicenter study.

BACKGROUND/PURPOSE: The eighth edition of the AJCC staging system introduced a shift in the staging of distal bile duct cancer (DBC), emphasizing the depth of invasion over adjacent organ invasion. This study aimed to evaluate the clinical impact of pancreatic invasion in pT1-stage DBC and identify prognostic factors for long-term survival. METHODS: This multicenter retrospective analysis encompassed DBC patients who underwent pancreaticoduodenectomy between 2009 and 2019 in six Korean tertiary centers, specifically those with final pathology confirming AJCC eighth edition T1 stage and intrapancreatic bile duct tumor origin. Primary endpoints were five-year recurrence-free survival (RFS) and overall survival (OS). Secondary objectives included the identification of prognostic determinants. RESULTS: This study involved 287 patients, comprising 190 without and 97 with pancreatic invasion. Pancreatic invasion did not significantly influence five-year OS and RFS rates (OS: without pancreatic invasion 69.9% vs. with pancreatic invasion 54.1%, p = .25; RFS: 56.3% vs. 55.4%, p = .97). Multivariate analysis highlighted male gender, age, lymphovascular invasion, and N stage as significant OS determinants. Notably, male gender, ampulla of Vater invasion, lymphovascular invasion, and N1 stage were also associated with RFS. CONCLUSIONS: In pT1 DBC, pancreatic invasion demonstrates no substantial impact on long-term prognosis, in accordance with the depth-based paradigm of the eighth edition AJCC staging system. The prognostic factors influencing OS were identified as male gender, age, lymphovascular invasion, and nodal metastasis.

Bacteriophages: Natural antimicrobial bioadditives for food preservation in active packaging.

Developing innovative films and coatings is paramount for extending the shelf life of numerous food products and augmenting the barrier and antimicrobial properties of food packaging materials. Many synthetic chemicals used in active packaging and food storage have the potential to leach into food, posing long-term health risks. It is imperative for active packaging materials to inherently possess biological protective properties to ensure food quality and safety throughout its storage. Bacteriophages, or simply phages, are bacteria-eating viruses that serve as promising natural biocontrol agents and antimicrobial bioadditives in food packaging materials, specifically targeting bacterial foodborne pathogens. These phages are generally recognized as safe (GRAS) by regulatory authorities for food safety applications. They exhibit targeted action against various Gram-positive and -negative foodborne pathogens, including Bacillus spp., Campylobacter spp., Escherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., and Vibrio spp., associated with foodborne spoilage and illness without affecting the beneficial microbes. Phage cocktails can be applied directly on food surfaces, incorporated into food packaging materials, or utilized during food processing treatments. Unlike chemical agents, phage activity increases proportionally with the rise in pathogenic bacterial populations. Researchers are exploring various packaging materials to deliver phages with broad host range, stability, and viability ensuring their effectiveness in safeguarding various food systems. The effectiveness of phage immobilization or encapsulation on active food packaging materials depends on various factors, including the characteristics of polymers, the choice of solvents, the type of phage, and its loading efficiency. Factors such as the orientation of phage immobilization on substrates, pH, temperature, exposure to carbohydrates and amino acids, exopolysaccharides, lipopolysaccharides, and metals can also influence phage activity. In this review, we comprehensively discuss the various active packaging systems utilizing bacteriophages as natural biocontrols and antimicrobial bioadditives to reduce the incidence of foodborne illness and enhance consumer confidence in the safety of food products.

Exploring advancements in early detection of Alzheimer's disease with molecular assays and animal models.

Alzheimer's Disease (AD) is a challenging neurodegenerative condition, with overwhelming implications for affected individuals and healthcare systems worldwide. Animal models have played a crucial role in studying AD pathogenesis and testing therapeutic interventions. Remarkably, studies on the genetic factors affecting AD risk, such as APOE and TREM2, have provided valuable insights into disease mechanisms. Early diagnosis has emerged as a crucial factor in effective AD management, as demonstrated by clinical studies emphasizing the benefits of initiating treatment at early stages. Novel diagnostic technologies, including RNA sequencing of microglia, offer promising avenues for early detection and monitoring of AD progression. Therapeutic strategies remain to evolve, with a focus on targeting amyloid beta (Aß) and tau pathology. Advances in animal models, such as APP-KI mice, and the advancement of anti-Aß drugs signify progress towards more effective treatments. Therapeutically, the focus has shifted towards intricate approaches targeting multiple pathological pathways simultaneously. Strategies aimed at reducing Aß plaque accumulation, inhibiting tau hyperphosphorylation, and modulating neuroinflammation are actively being explored, both in preclinical models and clinical trials. While challenges continue in developing validated animal models and translating preclinical findings to clinical success, the continuing efforts in understanding AD at molecular, cellular, and clinical levels offer hope for improved management and eventual prevention of this devastating disease.

Clipless internal mammary artery harvesting for minimally invasive coronary artery bypass grafting using the shear-tip harmonic scalpel.

Background: The internal mammary artery (IMA) is the most commonly used graft in coronary artery bypass grafting (CABG) because of its superior long-term patency rate. However, its small diameter poses challenges in handling, and any vascular damage that may occur during harvesting can significantly affect surgical outcomes. The primary focus during IMA harvesting is to ensure safe and effective hemostasis without direct vascular injury, while ensuring secure and reliable ligation of the vascular branches. Various methods using multiple surgical instruments have been used for this purpose. Unlike traditional instruments, the shear-tip Harmonic scalpel offers more precise vessel branching control, while minimizing damage to surrounding tissues. In this study, we assessed the utility of the shear-tip Harmonic scalpel in patients undergoing minimally invasive coronary artery bypass grafting (MICABG). Methods: From April 2019 to May 2023, a total of 40 patients underwent MICABG. The IMA was harvested using the shear-tip Harmonic scalpel with a clipless skeletonized technique. In this cohort, 5 patients underwent complete endoscopic harvesting, while 34 patients underwent direct visualization harvesting through minimal thoracotomy. Graft patency was assessed by measuring a Doppler flowmeter in the bypass conduit. Results: Successful graft patency was achieved in all patients. The mean duration of IMA harvesting was 87 min. In total, 38 of the 40 patients underwent MICABG without the need for cardiopulmonary bypass, ensuring a stable procedure. There were no graft-related events or complications observed in any of the patients, and all were discharged without any issues. During a median follow-up period of 15.2 months, only one patient experienced graft occlusion necessitating intervention. Conclusions: The utilization of shear-tip Harmonic scalpel for IMA harvesting in MICABG is feasible and yields stable early results.

Lactobacillus brevis M2-Fermented Whey Protein Hydrolysate Increases Slow-Wave Sleep via GABAA Receptors in Rodent Models.

In this study, we investigated the effects of whey protein hydrolysate (WPH) fermented with Lactobacillus brevis on sleep behavior and GABAergic mechanisms in rodent models. Fermentation converted the glutamate in WPH to high (3.15 ± 0.21 mg/mL) levels of γ-aminobutyric acid (GABA). Fermented WPH (WP-SF) enhanced sleep duration in mice by increasing GABA content in the brain. The increase in sleep duration induced by WP-SF resulted from an increase in delta wave activity during non-rapid eye movement sleep, and its sleep-promoting effect in a caffeine-induced insomnia model was characterized by an increase in delta waves. WP-SF increased GABAergic receptors at both mRNA and protein levels. Cotreatment with GABAA receptor antagonists abolished the sleep-promoting effects of WP-SF, indicating that WP-SF shares binding sites with antagonists on GABAA receptors. Collectively, WP-SF effectively increased sleep duration by enhancing delta wave activity through GABAergic activation; thus, it is suggested as a functional food-grade ingredient for promoting sleep.

Lactitol Alleviates Loperamide-Induced Constipation in Sprague Dawley Rats by Regulating Serotonin, Short-Chain Fatty Acids, and Gut Microbiota.

The objective of this study was to examine the impact of lactitol on constipation caused by loperamide in Sprague Dawley rats, with a particular emphasis on its underlying mechanisms and potential health advantages. The lactitol effectively improved fecal parameters, intestinal tissue structure, and the expression of constipation-related gene expression and proteins. Lactitol alleviated fecal weight and water content altered by loperamide and enhanced gastrointestinal transit. The administration also restored mucosal and muscular layer thickness. Mechanistically, lactitol upregulated the mRNA expression and/or protein levels of mucins (MUC2 and MUC4), occludin, claudin-1, and zonula occludens, indicating improved intestinal barrier function. Lactitol positively regulated the composition of cecal microbiota, leading to an increased relative abundance of Bifidobacterium, Lactobacillus, and Romboutsia. Conversely, lactitol decreased the relative abundance of Prevotella, Aerococcus, Muribaculum, Blautia, and Ruminococcus. This study demonstrated the potential of lactitol to relieve constipation by modulating the gut microbiota. These findings suggest that lactitol is an alternative to traditional laxatives and has potential as a health-promoting food sweetener.

Yohimbine Inhibits PDGF-Induced Vascular Smooth Muscle Cell Proliferation and Migration via FOXO3a Factor.

Yohimbine (YHB) has been reported to possess anti-inflammatory, anticancer, and cardiac function-enhancing properties. Additionally, it has been reported to inhibit the proliferation, migration, and neointimal formation of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor (PDGF) stimulation by suppressing the phospholipase C-gamma 1 pathway. However, the transcriptional regulatory mechanism of YHB controlling the behavior of VSMCs is not fully understood. In this study, YHB downregulated the expression of cell cycle regulatory proteins, such as proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4), and cyclin E, by modulating the transcription factor FOXO3a in VSMCs induced by PDGF. Furthermore, YHB decreased p-38 and mTOR phosphorylation in a dose-dependent manner. Notably, YHB significantly reduced the phosphorylation at Y397 and Y925 sites of focal adhesion kinase (FAK), and this effect was greater at the Y925 site than Y397. In addition, the expression of paxillin, a FAK-associated protein known to bind to the Y925 site of FAK, was significantly reduced by YHB treatment in a dose-dependent manner. A pronounced reduction in the migration and proliferation of VSMCs was observed following co-treatment of YHB with mTOR or p38 inhibitors. In conclusion, this study shows that YHB inhibits the PDGF-induced proliferation and migration of VSMCs by regulating the transcription factor FOXO3a and the mTOR/p38/FAK signaling pathway. Therefore, YHB may be a potential therapeutic candidate for preventing and treating cardiovascular diseases such as atherosclerosis and vascular restenosis.

Reassessing the genetic lineage tracing of lingual Lgr5+ and Lgr6+ cells in vivo.

Taste buds, the neuroepithelial organs responsible for the detection of gustatory stimuli in the oral cavity, arise from stem/progenitor cells among nearby basal keratinocytes. Using genetic lineage tracing, Lgr5 and Lgr6 were suggested as the specific markers for the stem/progenitor cells of taste buds, but recent evidence implied that taste buds may arise even in the absence of these markers. Thus, we wanted to verify the genetic lineage tracing of lingual Lgr5- and Lgr6-expressing cells. Unexpectedly, we found that antibody staining revealed more diverse Lgr5-expressing cells inside and outside the taste buds of circumvallate papillae than was previously suggested. We also found that, while tamoxifen-induced genetic recombination occurred only in cells expressing the Lgr5 reporter GFP, we did not see any increase in the number of recombined daughter cells induced by consecutive injections of tamoxifen. Similarly, we found that cells expressing Lgr6, another stem/progenitor cell marker candidate and an analog of Lgr5, also do not generate recombined clones. In contrast, Lgr5-expressing cells in fungiform papillae can transform into Lgr5-negative progeny. Together, our data indicate that lingual Lgr5- and Lgr6-expressing cells exhibit diversity in their capacity to transform into Lgr5- and Lgr6-negative cells, depending on their location. Our results complement previous findings that did not distinguish this diversity.

Presynaptic sensor and silencer of peptidergic transmission reveal neuropeptides as primary transmitters in pontine fear circuit.

Neurons produce and release neuropeptides to communicate with one another. Despite their importance in brain function, circuit-based mechanisms of peptidergic transmission are poorly understood, primarily due to the lack of tools for monitoring and manipulating neuropeptide release in vivo. Here, we report the development of two genetically encoded tools for investigating peptidergic transmission in behaving mice: a genetically encoded large dense core vesicle (LDCV) sensor that detects presynaptic neuropeptide release and a genetically encoded silencer that specifically degrades neuropeptides inside LDCVs. Using these tools, we show that neuropeptides, not glutamate, encode the unconditioned stimulus in the parabrachial-to-amygdalar threat pathway during Pavlovian threat learning. We also show that neuropeptides play important roles in encoding positive valence and suppressing conditioned threat response in the amygdala-to-parabrachial endogenous opioidergic circuit. These results show that our sensor and silencer for presynaptic peptidergic transmission are reliable tools to investigate neuropeptidergic systems in awake, behaving animals.

Dietary vitamin D3 supplementation enhances splenic NK cell activity in healthy and diabetic male mice.

Type 2 diabetes mellitus negatively affects the immune system, resulting in reduced natural killer (NK) cell activity. Vitamin D has been shown to regulate innate and adaptive immune cells. However, the effects of vitamin D on NK cells remain inconclusive, especially in the context of diabetes. We hypothesized that dietary vitamin D3 supplementation can enhance NK cell activity in diabetic mice. Therefore, we investigated the effects of dietary vitamin D3 on NK cell activity in control and diabetic mice and explored the mechanisms of NK cell activity modulation by vitamin D3. Control (CON) and diabetic mice (db/db) were randomly divided into 2 groups, then fed either a control diet (948 IU vitamin D3/kg diet, vDC) or a diet supplemented with vitamin D3 (9,477 IU vitamin D3/kg diet, vDS) for 8 weeks. Diabetic mice exhibited lower NK cell activity than control mice. The vDS group had significantly higher NK cell activity than the vDC group in both control and diabetic mice. The vDS group had a higher percentage of CD11b single-positive NK cells than the vDC group (CON-vDS 34%; db/db-vDS 30%; CON-vDC 27%; db/db-vDC 22%). The intracellular expression of splenic TGF-ß was significantly higher in the db/db group than in the CON group. Overall, vDS group had higher Bcl2 and Tbx21 mRNA expressions than the vDC group. In conclusion, the present study shows that NK cell activity is impaired under diabetic conditions, possibly due to the reduced percentage of mature NK cells. Moreover, NK activity is enhanced by dietary supplementation in both control and diabetic mice that may be associated with changes in the proportion of mature NK cells.

Formation of amygdalin/ß-cyclodextrin derivatives inclusion complexes for anticancer activity assessment in human cervical carcinoma HeLa cell line.

Nanoencapsulation has gained considerable attention because of its unique features and advantages in anticancer drug delivery. Amygdalin (AMY) is an anticancer compound, showing limitations in its applications by low stability. Herein, the inclusion complexes (ICs) of AMY with ß-cyclodextrin (ßCD), and its derivatives such as 2-hydroxypropyl-ßCD (HPßCD) and methyl-ßCD (MßCD) were fabricated. The fabricated AMY/CD-ICs were thoroughly evaluated using Fourier-transform infrared spectroscopy, powder X-ray diffraction, thermogravimetric/differential thermal analysis, proton nuclear magnetic resonance, ultraviolet-visible diffuse reflectance spectroscopy, and photoluminescence techniques. Double reciprocal profile study of the absorption and fluorescence spectra revealed that the AMY formed the ICs with ßCD derivatives at a guest/host stoichiometric ratio of 1/1. The thermal stability of AMY was enhanced as the IC formation aid observed by the shift of thermal degradation temperature of AMY from the range of âˆ¼ 220-250 °C to > 295 °C. Theoretical analyses of the energetic, electronic, and global reactivity parameters of the AMY/CD-ICs were evaluated using the PM3 method. Further assessment of the dissolution diagrams of AMY/CD-ICs revealed a burst release profile. In addition, cell toxicity was evaluated using the MTT assay, and the results showed that AMY/CD-ICs had significantly more efficacious in inhibiting HeLa cancer cells than AMY. These results proved that the IC formations with CDs significantly enhanced the anticancer activity of AMY.

Current status and challenges for cell-cultured milk technology: a systematic review.

Cellular agriculture is an innovative technology for manufacturing sustainable agricultural products as an alternative to traditional agriculture. While most cellular agriculture is predominantly centered on the production of cultured meat, there is a growing demand for an understanding of the production techniques involved in dairy products within cellular agriculture. This review focuses on the current status of cellular agriculture in the dairy sector and technical challenges for cell-cultured milk production. Cellular agriculture technology in the dairy sector has been classified into fermentation-based and animal cell culture-based cellular agriculture. Currently, various companies synthesize milk components through precision fermentation technology. Nevertheless, several startup companies are pursuing animal cell-based technology, driven by public concerns regarding genetically modified organisms in precision fermentation technology. Hence, this review offers an up-to-date exploration of animal cell-based cellular agriculture to produce milk components, specifically emphasizing the structural, functional, and productive aspects of mammary epithelial cells, providing new information for industry and academia.