Mucosal TLR5 activation controls healthspan and longevity.

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.

Design of an effective small expression tag to enhance GPCR production in E. coli-based cell-free and whole cell expression systems.

G protein-coupled receptors (GPCRs) play crucial roles in sensory, immune, and tumor metastasis processes, making them valuable targets for pharmacological and sensing applications in various industries. However, most GPCRs have low production yields in Escherichia coli (E. coli) expression systems. To overcome this limitation, we introduced AT10 tag, an effective fusion tag that could significantly enhance expression levels of various GPCRs in E. coli and its derived cell-free protein synthesis (CFPS) system. This AT10 tag consisted of an A/T-rich gene sequence designed via optimization of translation initiation rate. It is translated into a short peptide sequence of 10 amino acids at the N-terminus of GPCRs. Additionally, effector proteins could be utilized to suppress cytotoxicity caused by membrane protein expression, further boosting GPCR production in E. coli. Enhanced expression of various GPCRs using this AT10 tag is a promising approach for large-scale production of functional GPCRs in E. coli-based CFPS and whole cell systems, enabling their potential utilization across a wide range of industrial applications.

The Clinical Effects of Type 2 Diabetes Patient Management Using Digital Healthcare Technology: A Systematic Review and Meta-Analysis.

The disease control rate is very low (at less than 30%) for diabetes. The use of digital healthcare technology is increasing recently for continuous management in daily life. In this study, a meta-analysis was conducted to evaluate the clinical effects of digital healthcare technology for patients with type 2 diabetes management. For a review of the literature, databases such as PubMed, Embase, and Cochrane Library were searched using Medical Subject Heading (MeSH) terms published up to 9 August 2021. As a result, 2354 articles were identified, and 12 randomized controlled trial articles were finally included. Digital healthcare technology combined management for type 2 diabetes significantly decreased HbA1c (p < 0.00001, standardized mean difference (SMD) = −0.49) and marginally decreased triglyceride, compared with usual care (p = 0.06, SMD = −0.18). However, it did not significantly affect BMI (p = 0.20, SMD = −0.47), total cholesterol (p = 0.13, SMD = −0.19), HLD-C (p = 0.89, SMD = −0.01), LDL-C (p = 0.95, SMD = −0.01), systolic BP (p = 0.83, SMD = 0.03), or diastolic BP (p = 0.23, SMD = 0.65), compared with usual care. These results indicate that digital healthcare technology can improve HbA1c and triglyceride levels of type 2 diabetes patients. Further well-designed randomized controlled clinical trials are needed to confirm the clinical effect of digital healthcare technology.

Depletion of Mitochondrial Components from Extracellular Vesicles Secreted from Astrocytes in a Mouse Model of Fragile X Syndrome.

Mitochondrial dysfunction contributes to neurodegenerative diseases and developmental disorders such as Fragile X syndrome (FXS). The cross-talk between mitochondria and extracellular vesicles (EVs) suggests that EVs may transfer mitochondrial components as intermediators for intracellular communication under physiological and pathological conditions. In the present study, the ability of EVs to transfer mitochondrial components and their role in mitochondrial dysfunction in astrocytes were examined in the brains of Fmr1 knockout (KO) mice, a model of FXS. The amounts of mitochondrial transcription factor NRF-1, ATP synthases ATP5A and ATPB, and the mitochondrial membrane protein VDAC1 in EVs were reduced in cerebral cortex samples and astrocytes from Fmr1 KO mice. These reductions correspond to decreased mitochondrial biogenesis and transcriptional activities in Fmr1 KO brain, along with decreased mitochondrial membrane potential (MMP) with abnormal localization of vimentin intermediate filament (VIF) in Fmr1 KO astrocytes. Our results suggest that mitochondrial dysfunction in astrocytes is associated with the pathogenesis of FXS and can be monitored by depletion of components in EVs. These findings may improve the ability to diagnose developmental diseases associated with mitochondrial dysfunction, such as FXS and autism spectrum disorders (ASD).

Piperlongumine decreases cognitive impairment and improves hippocampal function in aged mice.

Piperlongumine (PL), a biologically active compound from the Piper species, has been shown to exert various pharmacological effects in a number of conditions, including tumours, diabetes, pain, psychiatric disorders and neurodegenerative disease. In this study, we evaluated the therapeutic effects of PL on hippocampal function and cognition decline in aged mice. PL (50 mg/kg/day) was intragastrically administrated to 23­month­old female C57BL/6J mice for 8 weeks. Novel object recognition and nest building behaviour tests were used to assess cognitive and social functions. Additionally, immunohistochemistry and western blot analysis were performed to examine the effects of PL on the hippocampus. We found that the oral administration of PL significantly improved novel object recognition and nest building behaviour in aged mice. Although neither the percentage area occupied by astrocytes and microglia nor the level of 4­hydroxynonenal protein, a specific marker of lipid peroxidation, were altered by PL treatment, the phosphorylation levels of N­methyl­D­aspartate receptor subtype 2B (NR2B), calmodulin­dependent protein kinase II alpha (CaMKIIα) and extracellular signal­regulated kinase 1/2 (ERK1/2) were markedly increased in the hippocampus of aged mice following the administration of PL. We also found that PL treatment resulted in a CA3­specific increase in the phosphorylation level of cyclic AMP response element binding protein, which is recognized as a potent marker of neuronal plasticity, learning and memory. Moreover, the number of doublecortin­positive cells, a specific marker of neurogenesis, was significantly increased following PL treatment in the dentate gyrus of the hippocampus. On the whole, these data demonstrate that PL treatment may be a potential novel approach in the treatment of age­related cognitive impairment and hippocampal changes.

Humulus japonicus inhibits the progression of Alzheimer's disease in a APP/PS1 transgenic mouse model.

Humulus japonicus Siebold & Zucc. (HJ) has traditionally been administered to patients with pulmonary disease, skin disease and hypertension in Korea, and it is considered to exert anti-inflammatory, antioxidant, antimicrobial and antimycobacterial effects. However, its effects against Alzheimer's disease (AD) have yet to be explored. Thus, this study was carried out to investigate whether HJ has a beneficial effect on the progression of AD in an animal model. A methanolic extract of HJ (500 mg/kg/day) was intragastrically administered to 5-month-old APP/PS1 transgenic (Tg-APP/PS1) mice for 2.5 months. Novel object recognition and Y-maze alteration tests were used to assess cognitive function, and an immunohistochemical assay was performed to assess amyloid ß (Aß)deposition, tau phosphorylation and gliosis. An in vitro assay using a microglial cell line was also performed to investigate the anti-inflammatory effects of HJ. Our results revealed that HJ significantly decreased the mRNA and protein expression levels of tumor necrosis factor-α (TNF­α), interleukin (IL)-1ß, IL-6 and inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide in the microglial cell line. The administration of HJ for 2 months improved the cognitive function of Tg-APP/PS1 mice. HJ notably reduced the area occupied by Aß and neurofibrillary tangles, and the number of activated astrocytes and microglia in the cortex of Tg-APP/PS1 mice. The findings of our study suggest that HJ has the therapeutic potential to inhibit the progression of AD and to improve cognitive deterioration in Tg-APP/PS1 mice.

Gadd45ß ameliorates L-DOPA-induced dyskinesia in a Parkinson's disease mouse model.

The dopamine precursor 3,4-dihydroxyphenyl-l-alanine (L-DOPA) is currently the most efficacious pharmacotherapy for Parkinson's disease (PD). However, long-term L-DOPA treatment leads to the development of abnormal involuntary movements (AIMs) in patients and animal models of PD. Recently, involvement of growth arrest and DNA damage-inducible 45ß (Gadd45ß) was reported in neurological and neurobehavioral dysfunctions. However, little is known about the role of Gadd45ß in the dopaminergic nigrostriatal pathway or L-DOPA-induced dyskinesia (LID). To address this issue, we prepared an animal model of PD using unilateral 6-hydroxydopamine (6-OHDA) lesions in the substantia nigra of Gadd45ß(+/+) and Gadd45ß(-/-) mice. Dyskinetic symptoms were triggered by repetitive administration of L-DOPA in these 6-OHDA-lesioned mice. Whereas dopamine denervation in the dorsal striatum decreased Gadd45ß mRNA, chronic L-DOPA treatment significantly increased Gadd45ß mRNA expression in the 6-OHDA-lesioned striatum of wild-type mice. Using unilaterally 6-OHDA-lesioned Gadd45ß(+/+) and Gadd45ß(-/-) mice, we found that mice lacking Gadd45ß exhibited long-lasting increases in AIMs following repeated administration of L-DOPA. By contrast, adeno-associated virus-mediated expression of Gadd45ß in the striatum reduced AIMs in Gadd45ß knockout mice. The deficiency of Gadd45ß in LID increased expression of ΔFosB and c-Fos in the lesioned striatum 90 min after the last administration of L-DOPA following 11days of daily L-DOPA treatments. These data suggest that the increased expression of Gadd45ß induced by repeated administration of L-DOPA may be beneficial in patients with PD.

Inhibition of adenylyl cyclase type 5 prevents L-DOPA-induced dyskinesia in an animal model of Parkinson's disease.

The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) is widely used as a therapeutic choice for the treatment of patients with Parkinson's disease. However, the long-term use of L-DOPA leads to the development of debilitating involuntary movements, called L-DOPA-induced dyskinesia (LID). The cAMP/protein kinase A (PKA) signaling in the striatum is known to play a role in LID. However, from among the nine known adenylyl cyclases (ACs) present in the striatum, the AC that mediates LID remains unknown. To address this issue, we prepared an animal model with unilateral 6-hydroxydopamine lesions in the substantia nigra in wild-type and AC5-knock-out (KO) mice, and examined behavioral responses to short-term or long-term treatment with L-DOPA. Compared with the behavioral responses of wild-type mice, LID was profoundly reduced in AC5-KO mice. The behavioral protection of long-term treatment with L-DOPA in AC5-KO mice was preceded by a decrease in the phosphorylation levels of PKA substrates ERK (extracellular signal-regulated kinase) 1/2, MSK1 (mitogen- and stress-activated protein kinase 1), and histone H3, levels of which were all increased in the lesioned striatum of wild-type mice. Consistently, FosB/ΔFosB expression, which was induced by long-term L-DOPA treatment in the lesioned striatum, was also decreased in AC5-KO mice. Moreover, suppression of AC5 in the dorsal striatum with lentivirus-shRNA-AC5 was sufficient to attenuate LID, suggesting that the AC5-regulated signaling cascade in the striatum mediates LID. These results identify the AC5/cAMP system in the dorsal striatum as a therapeutic target for the treatment of LID in patients with Parkinson's disease.

Pitx3 deficient mice as a genetic animal model of co-morbid depressive disorder and parkinsonism.

Approximately 40-50% of all patients with Parkinson׳s disease (PD) show symptoms and signs of depressive disorders, for which neither pathogenic understanding nor rational treatment are available. Using Pit3x-deficient mice, a model for selective nigrostriatal dopaminergic neurodegeneration, we tested depression-related behaviors and acute stress responses to better understand how a nigrostriatal dopaminergic deficit increases the prevalence of depressive disorders in PD patients. Pitx3-deficient mice showed decreased sucrose consumption and preference in the two-bottle free-choice test of anhedonia. Acute restraint stress increased c-Fos (known as a neuronal activity marker) expression levels in various brain regions, including the prefrontal cortex, striatum, nucleus accumbens, and paraventricular nucleus of the hypothalamus (PVN), in both Pitx3+/+ and -/- mice. However, the stress-induced increases in c-Fos levels in the cortex, dorsal striatum, and PVN were significantly greater in Pitx3-/- than +/+ mice, suggesting that signs of depressive disorders in parkinsonism are related to altered stress vulnerability. Based on these results, we propose that Pitx3-/- mice may serve as a useful genetic animal model for co-morbid depressive disorder and parkinsonism.

Detection and typing of HPV genotypes in various cervical lesions by HPV oligonucleotide microarray.

OBJECTIVES: This study was conducted to evaluate a clinical efficacy of human papillomavirus (HPV) oligonucleotide microarray (Biomedlab Co., Seoul, South Korea) for the detection of HPVs in various cervical lesions. RESULTS: HPV DNAs from 234 patients were analysed by two methods. Among those, 212 patients were classified into 5 groups according to the histologic diagnosis: chronic cervicitis, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III, and invasive cervical carcinoma. PCR-RFLP could detect 7 types of high-risk HPVs (HPV 16/18/31/33/35/52/58) and HPV microarray could detect 15 types of high-risk HPVs (HPV 16/18/31/33/35/39/45/51/52/56/58/59/66/68/69) and 7 types of low-risk HPVs (HPV 6/11/34/40/42/43/44).HPV genotyping by HPV oligonucleotide microarray revealed that HPV16 was the most frequent type (30.2%) in all specimens tested and was significantly more frequent in CIN III and invasive carcinomas than other lesions. METHODS: HPV DNAs were detected in 158 and 174 of the 234 cervical samples by PCR-RFLP and HPV microarray, respectively. The correlation between the two methods was good in detecting HPVs in general (kappa index = 0.69) and HPVs 31 and 52 (kappa index = 0.70 and 0.70, respectively) and excellent in detecting HPVs 16, 18, 33, 35, and 58 (kappa index = 0.90, 0.88, 0.92, 0.77, and 0.84, respectively). Double HPV infection was detected in 10 cases and one triple infection was detected. By combining cytology and HPV testing, the sensitivity was improved to 87.5, 95.5, 96.1, and 97.2% in CIN I, CIN II, CIN III, and carcinoma, respectively. CONCLUSIONS: This results suggest that HPV oligonucleotide microarray is a highly comparable method to the previously used PCR-RFLP method for the detection of HPVs in cervical specimens. Genetic informations for HPV infection in cervical specimens may offer new strategies in manipulating the patients harboring cervical intraepithelial neoplasia and cervical carcinoma.

HPV oligonucleotide microarray-based detection of HPV genotypes in cervical neoplastic lesions.

BACKGROUND: In this study we examined the use of a new-human papillomavirus (HPV) detection method, the HPV oligonucleotide microarray system (Biomedlab Co., Korea), which we compared with the well-established HPV DNA detection system (Hybrid Capture II; HC-II, Digene Co.). This new method prompted us to develop a new HPV genotyping technique, using the oligonucleotide microarray, to detect the generic and type-specific sequence of HPV types. In particular, we undertook the evaluation of the clinical efficacy of the HPV oligonucleotide microarray for detecting HPV in cervical neoplastic lesions. METHODS: One hundred forty patients were involved and classified into three groups according to their histopathologic diagnoses: Group I (nonspecific chronic cervicitis; n = 61), Group II (low-grade squamous intraepithelial lesion (SIL); koilocytosis, and mild dysplasia; n = 39), and Group III (high-grade SIL; moderate, severe dysplasia and in situ carcinoma; n = 40). Cytological diagnoses were based on the Bethesda System and cervical samples were analyzed by the two methods. The HPV oligonucleotide microarray detected 15 types of high-risk HPV (HPV-16/-18/-31/-33/-35/-39/-45/-51/-52/-56/-58/-59/-66/-68/-69) and 7 types of low-risk HPV (HPV-6/-11/-34/-40/-42/-43/-44). RESULTS: In 105 of the 140 cervical samples (75%), HPV DNAs were examined using the HC-II method. HPV detection rates using the HPV microarray agreed with those of HC-II. One HC-II-positive, but HPV microarray-negative, case occurred in the low-grade SIL (Group II) and was later confirmed negative for HPV. The other HPV microarray-positive but HC-II-negative case was found to be HPV-18 by PCR. Low-risk types of HPV were detected in 3 of 39 low-grade SIL cases (Group II) using the HPV microarray. HPV-16 was the most frequent type (32.1%) in all specimens tested, and was significantly more frequent in low-grade or high-grade intraepithelial lesions (Groups II or III) than in normal controls (Group I) (P < 0.05). HPV-58 was the second most common type (17.5%) in Group III. The HPV microarray was found to have advantages in terms of identifying the HPV genotypes and cases of multiple HPV infection. Double HPV infections were detected in 12 cases and triple HPV infections in 7 cases. Two cases were positive for four types of HPV (HPV-16/18/33/35, HPV-16/18/58/68). The sensitivity of HPV testing (HC-II; 94.9%, HPV microarray; 93.7%) for identifying patients with squamous intraepithelial lesion was significantly better than the sensitivity of cytology (77.1%, P < 0.05). On using multiple logistic regression analysis to estimate the relative risk of SIL versus HPV type, HPV-16-positive cases were found to have a 7.5-fold risk of SIL (95% CI = 3.28-16.51; P < 0.01). HPV-33 and HPV-58 were found to be significantly related to high-grade SILs (P < 0.01). CONCLUSIONS: Our results suggest that the HPV oligonucleotide microarray is highly comparable to HC-II for detecting HPV in cervical specimens. The HPV oligonucleotide microarray provides useful information on viral genotype and multiple HPV infections in HPV-related cervical lesions. Genetic information on HPV in cervical specimens might be a particular benefit of the new procedure in the management of cervical neoplastic lesions